Cheese

Chemistry, Physics and

Microbiology
Volume 1 General Aspects
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 Cheese
Chemistry, Physics and
Microbiology
Volume 1 General Aspects

Third edition

Edited by
Patrick F. Fox, Paul L.H. McSweeney, Timothy M. Cogan
and Timothy P. Guinee

AMSTERDAM • BOSTON • HEIDELBERG • LONDON • NEW YORK • OXFORD

PARIS • SAN DIEGO • SAN FRANCISCO • SINGAPORE • SYDNEY • TOKYO
This book is printed on acid-free paper
First published 1987 by Elsevier Applied Science
Second edition 1993 by Chapman & Hall
Third edition 2004

Copyright © 2004, Elsevier Ltd. All rights reserved.

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British Library Cataloguing in Publication Data

A catalogue record for this book is available from the British Library

Library of Congress Control Number: 2004105782

Volume 1 ISBN 0-1226-3652-X

Volume 2 ISBN 0-1226-3653-8
Set ISBN 0-1226-3651-1

Cover images:
Bacteria (detail): reprinted with permission from Donald J. McMahon,
Food Structure, 1993, Vol. 12, pp. 251–258, Fig. 3A.
Sensory Circle: adapted with permission from Pierre Lavanchy et al.,
A Guide to the Sensory Evaluation of Texture of Hard and Semi-Hard Cheeses, 1994, Institut National
de la Recherché Agronomique, Paris.
Electropheresis gel: published with kind permission of Vivek Upadhyay, University College Cork,
Republic of Ireland.

Typeset by Integra Software Services Pvt. Ltd, Pondicherry, India

Printed and bound in Italy
04 05 06 07 08 9 8 7 6 5 4 3 2 1
Contents

Foreword vii
List of Contributors ix
Preface to the First Edition xiii
Preface to the Second Edition xv
Preface to the Third Edition xvii

Cheese: An Overview 1
P.F. Fox and P.L.H. McSweeney
Rennets: General and Molecular Aspects 19
M.J.C. Crabbe
Rennet-induced Coagulation of Milk 47
D.S. Horne and J.M. Banks
The Syneresis of Rennet-coagulated Curd 71
P. Dejmek and P. Walstra
Formation, Structural Properties and Rheology of Acid-coagulated Milk Gels 105
J.A. Lucey
Starter Cultures: General Aspects 123
E. Parente and T.M. Cogan
Starter Cultures: Genetics 149
M.J. Callanan and R.P. Ross
Starter Cultures: Bacteriophage 163
S. McGrath, G.F. Fitzgerald and D. van Sinderen
Secondary and Adjunct Cultures 191
J.-F. Chamba and F. Irlinger
Salt in Cheese: Physical, Chemical and Biological Aspects 207
T.P. Guinee and P.F. Fox
Application of Membrane Separation Technology to Cheese Production 261
V.V. Mistry and J.-L. Maubois
The Microbiology of Cheese Ripening 287
T. Beresford and A. Williams
Raw Milk Cheeses 319
E. Beuvier and S. Buchin
Biochemistry of Cheese Ripening: Introduction and Overview 347
P.L.H. McSweeney
Metabolism of Residual Lactose and of Lactate and Citrate 361
P.L.H. McSweeney and P.F. Fox
Lipolysis and Catabolism of Fatty Acids in Cheese 373
Y.F. Collins, P.L.H. McSweeney and M.G. Wilkinson
vi Contents

Proteolysis in Cheese during Ripening 391

V.K. Upadhyay, P.L.H. McSweeney, A.A.A. Magboul and P.F. Fox
Catabolism of Amino Acids in Cheese during Ripening 435
Á.C. Curtin and P.L.H. McSweeney
Sensory Character of Cheese and its Evaluation 455
C.M. Delahunty and M.A. Drake
Cheese Flavour: Instrumental Techniques 489
J.-L. Le Quéré
Rheology and Texture of Cheese 511
D.J. O’Callaghan and T.P. Guinee
Growth and Survival of Microbial Pathogens in Cheese 541
C.W. Donnelly
Toxins in Cheese 561
N.M. O’Brien, T.P. O’Connor, J. O’Callaghan and A.D.W. Dobson
Nutritional Aspects of Cheese 573
N.M. O’Brien and T.P. O’Connor
Factors that Affect the Quality of Cheese 583
P.F. Fox and T.M. Cogan
Index 609
Foreword

The art of cheesemaking has been augmented steadily by greater knowledge on the science of cheesemaking. This
evolution has resulted from basic and applied research and from the increased need to understand and control the
characteristics of milk, the microorganisms used in the manufacture and maturation of cheese, the manufacturing
technologies, and the physical properties and flavour of cheese. Traditional methods of cheese manufacture have
been modified by the need for greater efficiencies in the manufacture and maturation of cheese and by changes
in the marketing channels for cheese. Accommodating these changes while maintaining the characteristics of a
given cheese variety has been accomplished by the application of scientific principles. The need for greater under-
standing of the characteristics of cheese has also been driven by the increased use of cheese as an ingredient in
other foods. This has required specific control of selected properties of cheese to impart the desired properties to
the food, and to retain characteristics of the cheese during various food processing technologies.
The successive editions of Cheese: Chemistry, Physics and Microbiology have documented the application of
science to the art of cheesemaking. Certain characteristics are common in all editions: a thorough description
and evaluation of scientific and technological advances, prodigious referencing to direct readers to more in-depth
discussion of topics, and careful editing to impart consistency of discussion and a smooth transition between
chapters. However, each edition has been revised to incorporate new information and to reflect recent trends in
describing the science of cheesemaking and maturation and in the use of cheese as a food ingredient. Scientific
principles emphasised in Volume 1 cover microbiological, chemical and physical attributes of cheese as in previous
editions. Greater emphasis is given to the genetics and metabolic activity of lactic starters and on the secondary
microflora in the third edition. Conversion of components (lactose, lactate, citrate, lipids, proteins) by microbial
metabolism and enzymatic action is discussed in several chapters. Inclusion of modern sensory evaluation tech-
niques and instrumental identification of flavour compounds recognises the relationship between these areas. A new
chapter on acid gels provides the basic background for discussion in Volume 2 on cheese varieties made by acid
or heat plus acid coagulation that are becoming more important as food ingredients. Volume 2, as in previous
editions, focuses on various types of cheese, but the cheeses have been grouped into more logical categories
based upon characteristics rather than geographical regions of production. The first chapter of Volume 2 pro-
vides an overview of the diversity of cheese varieties and systems of categorising varieties. A similar approach in
the second chapter familiarises the reader with the general aspects of cheese technology to emphasise that there
are common elements in cheesemaking and maturation and that cheese varieties result from specific deviations
from or additions to these common elements. The last chapter is appropriately a discussion of cheese as an
ingredient, which recognises recent trends in the science of cheese.
A substantial bank of knowledge has been accumulated on cheese and this has been rigorously incorporated
into the two volumes. It is inevitable that this bank of knowledge will be revised and expanded. The third edition
of Cheese: Chemistry, Physics and Microbiology provides the base upon which these revisions and expansions can
be undertaken objectively.
N.F. Olson
Department of Food Science, University of Wisconsin, Madison
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List of Contributors

Dr J.M. Banks Ms Y.F. Collins

CHARIS Food Research Dairy Products Research Centre
Hannah Research Institute Teagasc, Moorepark
Ayr KA6 5HL Fermoy
Scotland Cork
Ireland
Dr T. Beresford
Dairy Products Research Centre Professor M.J.C. Crabbe
Teagasc, Moorepark Division of Cell and Molecular Biology
Fermoy School of Animal and Microbial Sciences
Cork The University of Reading
Ireland Whiteknights
Reading RG6 6AJ
Dr E. Beuvier UK
Station de Recherches en Technologie et Analyses
Laitières Dr Á.C. Curtin
Institut National de La Recherche Agronomique Department of Food and Nutritional Sciences
F-39801 Poligny Cedex University College
France Cork
Ireland
Dr S. Buchin
Professor P. Dejmek
Station de Recherches en Technologie et Analyses
Department of Food Engineering
Laitières
Lund University
Institut National de La Recherche Agronomique
Box 124, 221 00 Lund
F-39801 Poligny Cedex
Sweden
France
Dr C.M. Delahunty
Dr M.J. Callanan Department of Food and Nutritional Sciences
Dairy Products Research Centre University College
Teagasc, Moorepark Cork
Fermoy Ireland
Cork
Ireland Dr A.D.W. Dobson
Department of Microbiology
Dr J.-F. Chamba University College
Institut Technique Francais de Fromages Cork
74801 La Roche sur Foron Cedex Ireland
France
Dr C.W. Donnelly
Professor T.M. Cogan Department of Nutrition and Food Sciences
Dairy Products Research Centre University of Vermont
Teagasc, Moorepark 200 Carrigan Building
Fermoy Burlington
Cork VT 05405-0044
Ireland USA
x List of Contributors

Dr M.A. Drake Dr A.A.A. Magboul

Department of Food Science DAL Food Industries
North Carolina State University Industrial Area
Campus Box 7624 No. 1/15 Block 4F
Raleigh Khartoum North, PO Box 708
NC 27695-7624 Sudan
USA
Professor J.-L. Maubois
Dr G.F. Fitzgerald Laboratoire de Recherches Laitières
National Food Biotechnology Centre Institut National de la Recherche
Departments of Microbiology and Food & Nutritional Agronomique
Sciences 35012 Rennes Cedex
University College France
Cork Dr S. McGrath
Ireland National Food Biotechnology Centre
Professor P.F. Fox Department of Microbiology
Department of Food and Nutritional Sciences University College
University College Cork
Cork Ireland
Ireland Dr P.L.H. McSweeney
Department of Food and
Dr T.P. Guinee
Nutritional Sciences
Dairy Products Research Centre
University College
Teagasc, Moorepark
Cork
Fermoy
Ireland
Cork
Ireland Professor V.V. Mistry
Dairy Science Department
Dr D.S. Horne
South Dakota State University
CHARIS Food Research
Brookings
Hannah Research Institute
SD 57007
Ayr KA6 5HL
USA
Scotland
Dr N.M. O’Brien
Dr F. Irlinger Department of Food and
Institut National de La Recherche Agronomique Nutritional Sciences
78850 Thiverval-Grignon Cedex University College
France Cork
Dr J.-L. Le Quéré Ireland
Institut National de la Recherche Agronomique Dr D.J. O’Callaghan
Unité Mixte de Recherche sur les Aromes Dairy Products Research Centre
17 rue Sully Teagasc, Moorepark
F-21065, Dijon Fermoy
France Cork
Ireland
Dr J.A. Lucey
Department of Food Science Dr J. O’Callaghan
University of Wisconsin-Madison Department of Microbiology
1605 Linden Drive University College
Madison, WI 53706-1565 Cork
USA Ireland
 List of Contributors xi

Dr T.P. O’Connor Mr V.K. Upadhyay

Department of Food and Nutritional Sciences Department of Food and Nutritional Sciences
University College University College
Cork Cork
Ireland Ireland
Dr E. Parente Professor P. Walstra
Dipartimenta Biologia Department of Food Science
Università della Basilicata The Argricultural University
Campus di Macchia Romana 6703 HD Wageningen
85100 Potenza The Netherlands
Italy
Dr R.P. Ross Dr M.G. Wilkinson
Dairy Products Research Centre Department of Life Sciences
Teagasc, Moorepark University of Limerick
Fermoy Castletroy
Cork Limerick
Ireland Ireland

Dr D. van Sinderen Professor A. Williams

Department of Microbiology CHARIS Food Research
University College Hannah Research Institute
Cork Ayr KA6 5HL
Ireland Scotland
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Preface to the First Edition

Cheese manufacture is one of the classical examples of food preservation, dating from 6000–7000 BC. Preserva-
tion of the most important constituents of milk (i.e. fat and protein) as cheese exploits two of the classical prin-
ciples of food preservation, i.e.: lactic acid fermentation, and reduction of water activity through removal of
water and addition of NaCl. Establishment of a low redox potential and secretion of antibiotics by starter
microorganisms contribute to the storage stability of cheese.
About 500 varieties of cheese are now produced throughout the world; present production is ⬃107 tonnes
per annum and is increasing at a rate of ⬃4% per annum. Cheese manufacture essentially involves gelation of
the casein via iso-electric (acid) or enzymatic (rennet) coagulation; a few cheeses are produced by a combination
of heat and acid and still fewer by thermal evaporation. Developments in ultrafiltration facilitate the production
of a new family of cheeses. Cheeses produced by acid or heat/acid coagulation are usually consumed fresh, and
hence their production is relatively simple and they are not particularly interesting from the biochemical view-
point although they may have interesting physico-chemical features. Rennet cheeses are almost always ripened
(matured) before consumption through the action of a complex battery of enzymes. Consequently they are in a
dynamic state and provide fascinating subjects for enzymologists and microbiologists, as well as physical
chemists.
Researchers on cheese have created a very substantial literature, including several texts dealing mainly with
the technological aspects of cheese production. Although certain chemical, physical and microbiological aspects
of cheese have been reviewed extensively, this is probably the first attempt to review comprehensively the scien-
tific aspects of cheese manufacture and ripening. The topics applicable to most cheese varieties, i.e. rennets,
starters, primary and secondary phases of rennet coagulation, gel formation, gel syneresis, salting, proteolysis,
rheology and nutrition, are reviewed in Volume 1. Volume 2 is devoted to the more specific aspects of the nine
major cheese families: Cheddar, Dutch, Swiss, Iberian, Italian, Balkan, Middle Eastern, Mould-ripened and
Smear-ripened. A chapter is devoted to non-European cheeses, many of which are ill-defined; it is hoped that the
review will stimulate scientific interest in these minor, but locally important, varieties. The final chapter is
devoted to processed cheeses.
It is hoped that the book will provide an up-to-date reference on the scientific aspects of this fascinating
group of ancient, yet ultramodern, foods; each chapter is extensively referenced. It will be clear that a consider-
ably body of scientific knowledge on the manufacture and ripening of cheese is currently available but it will be
apparent also that many major gaps exist in our knowledge; it is hoped that this book will serve to stimulate sci-
entists to fill these gaps.
I wish to thank sincerely the other 26 authors who contributed to the text and whose co-operation made my
task as editor a pleasure.
P.F. Fox
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Preface to the Second Edition

The first edition of this book was very well received by the various groups (lecturers, students, researchers and
industrialists) interested in the scientific and technological aspects of cheese. The initial printing was sold out
faster than anticipated and created an opportunity to revise and extend the book.
The second edition retains all 21 subjects from the first edition, generally revised by the same authors and in
some cases expanded considerably. In addition, 10 new chapters have been added: Cheese: Methods of chemical
analysis; Biochemistry of cheese ripening; Water activity and the composition of cheese; Growth and survival of
pathogenic and other undesirable microorganisms in cheese; Membrane processes in cheese technology, in
Volume 1 and North-European varieties; Cheeses of the former USSR; Mozzarella and Pizza cheese; Acid-coagulated
cheeses and Cheeses from sheep’s and goats’ milk in Volume 2. These new chapters were included mainly to fill
perceived deficiencies in the first edition.
The book provides an in-depth coverage of the principal scientific and technological aspects of cheese. While
it is intended primarily for lecturers, senior students and researchers, production management and quality con-
trol personnel should find it to be a very valuable reference book. Although cheese production has become
increasingly scientific in recent years, the quality of the final product is still not totally predictable. It is not
claimed that this book will provide all the answers for the cheese scientist/technologist but it does provide the
most comprehensive compendium of scientific knowledge on cheese available.
Each of the 31 chapters is extensively referenced to facilitate further exploration of the extensive literature on
cheese. It will be apparent that while cheese manufacture is now firmly based on sound scientific principles,
many questions remain unanswered. It is hoped that this book will serve to stimulate further scientific study on
the chemical, physical and biological aspects of cheese.
I wish to thank sincerely all the authors who contributed to the two volumes of this book and whose co-
operation made my task as editor a pleasure.

P.F. Fox
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Preface to the Third Edition

Very considerable progress has been made on the scientific aspects of cheese since the second edition of this
book was published in 1993. This is especially true for the Microbiology of Cheese and the Biochemistry of
Cheese Ripening; consequently those sections have been expanded very considerably. The general structure of
the book is similar to that of the earlier editions, with the more general aspects being treated in Volume 1 and
the more applied, variety-related aspects in Volume 2. The book contains 36 chapters. Reflecting the very exten-
sive research on cheese starters in recent years, four chapters have been devoted to this topic in the third edition.
Another new feature is the inclusion of two chapters on cheese flavour; one on sensory aspects, the other on
instrumental methods.
In Volume 2 of the second edition, cheese varieties were treated mainly on a geographical basis. While some
elements of the geographical distribution remain, cheese varieties are now treated mainly based on the charac-
teristic features of their ripening. Obviously, it is not possible to treat all 1000 or so cheese varieties, but the
10 variety-related chapters in Volume 2 cover at least 90% of world cheese production and it is very likely that
your favourite cheese is included in one of those 10 chapters.
Cheese is the quintessential convenience food and is widely used as an ingredient in other foods and in the
USA approximately 70% of all cheese is used as a food ingredient. The use of cheese as a food ingredient is a
major growth area; consequently, a chapter has been devoted to the important features of cheese as an ingredi-
ent, including a section on Enzyme-modified Cheese.
Each chapter is extensively referenced to facilitate further exploration of the extensive literature on cheese.
While the book is intended for primarily lecturers, senior students and researchers, production management and
quality control personnel should find it to be a very useful reference book.
We wish to thank sincerely all authors who contributed to the two volumes of this book and whose co-
operation made our task as editors a pleasure. Special thanks are due to Ms Anne Cahalane for very valuable
assistance.
P.F. Fox
P.L.H. McSweeney
T.M. Cogan
T.P. Guinee
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 Cheese: An Overview
P.F. Fox and P.L.H. McSweeney, Department of Food and Nutritional Sciences,
University College, Cork, Ireland
Historical
Cheese is the generic name for a group of fermented
milk-based food products, produced in a wide range of
flavours and forms throughout the world. Although the
primary objective of cheesemaking is to conserve the
principal constituents of milk, cheese has evolved to
become a food of haute cuisine with epicurean qualities,
as well as being highly nutritious. Sandine and Elliker
(1970) suggested that there are more than 1000 var-
ieties of cheese. Walter and Hargrove (1972) described
more than 400 varieties and listed the names of a further
400, while Burkhalter (1981) classified 510 varieties
(although some are listed more than once). Jim Path
(University of Wisconsin) has compiled a list of 1400
varieties (visit www.cdr.wisc.edu). As discussed in detail
in ‘Diversity of cheese varieties: An Overview’, Volume 2,
a number of attempts have been made to classify cheese
varieties into meaningful groups. The most common
criterion for the classification is texture (very hard,
hard, semi-hard, semi-soft, soft) which is related mainly
to the moisture content of the cheese. Various attempts
have been made to improve on this basis of classifica-
tion, for example, by including the milk-producing
species, moisture to protein ratio, method of coagula-
tion, cooking temperature, microflora. These classifica-
tion schemes are discussed in ‘Diversity of cheese
varieties: An Overview’, Volume 2. However, no classifi-
cation scheme developed to date is completely satisfac-
tory; the inclusion of chemical indices of ripening
would be useful.
It is commonly believed that cheese evolved in a
region known as the ‘Fertile Crescent’, i.e., from the
Tigris and Euphratres rivers, through what is now
southern Turkey to the Mediterranean coast, some
8000 years ago. The so-called ‘Agricultural Revolu-
tion’ occurred in this region with the domestication of
plants and animals. Presumably, humans soon recog-
nized the nutritive value of milk produced by domes-
ticated animals and contrived to share the mother’s
milk with her offspring. Apparently, goats and sheep,
which are gregarious and docile, were the first dairy
animals domesticated, but cattle have become the
dominant dairy species in most parts of the world
(c. 85% of the total world supply of milk is obtained
from cows).
Cheese: Chemistry, Physics and Microbiology, Third edition – Volume 1: General Aspects
ISBN: 0-1226-3652-X
Set ISBN: 0-1226-3651-1
Milk is also a rich source of nutrients for bacteria
which contaminate the milk, some species of which
utilize milk sugar, lactose, as a source of energy, produc-
ing lactic acid. Bacterial growth and acid production
would have occurred during storage or during attempts
to dry milk in the prevailing warm, dry climate to pro-
duce a more stable product – air-drying of meat, fruits
and vegetables appears to have been practised as a primi-
tive form of food preservation at this period in the devel-
opment of civilization. When sufficient acid has been
produced, the principal proteins of milk, the caseins,
coagulate, i.e., at their isoelectric points – ⬃pH 4.6, to
form a gel in which the fat is entrapped. The rate of acid-
ification by the adventitious microflora would usually be
slow, allowing the (unhomogenized) fat globules to form
a cream layer. This layer of sour cream could be blended
into the lower protein gel or scooped off for the produc-
tion of butter. Thus originated three of our classical
fermented dairy products: fermented milks, sour cream
and lactic butter, all of which are still produced widely,
sometimes depending on the adventitious microflora for
acidification, but now usually through the growth of cul-
tures of lactic acid bacteria.
The first fermented dairy foods were produced by
a fortuitous combination of events – the ability of a
group of bacteria, the lactic acid bacteria (LAB), to grow
in milk and to produce enough acid to reduce the
pH of milk to the isoelectric point of the caseins, at
which these proteins coagulate. Neither the LAB nor the
caseins were designed for this outcome. The caseins
were ‘designed’ to coagulate following limited proteoly-
sis in the stomach of neonatal mammals, the gastric
pH of which is around 6, i.e., very much higher than
the isoelectric point of the caseins. The ability of Lacto-
coccus lactis to ferment lactose, a sugar specific to milk,
is plasmid-encoded, suggesting that this characteristic
was acquired relatively recently in the evolution of these
bacteria. Their natural habitats are vegetation and/or
the intestine, from which they presumably colonized
the teats of dairy animals, contaminated with lactose-
containing milk; it is likely that through evolutionary
pressure, these bacteria acquired the ability to ferment
lactose.
When an acid-coagulated milk gel is broken, e.g., acci-
dentally by movement of the storage vessel or intentionally
Copyright © 2004 Elsevier Ltd
All rights reserved
2 Cheese: An Overview
by breaking or cutting, it separates into curds and whey. It
would have been realized quickly that the acid whey is a
pleasant, refreshing drink for immediate consumption
while the curds could be consumed fresh or stored for
future use. In fact, whey was long considered to have
medicinal benefits (see Hoffmann, 1761). It was probably
soon realized that the shelf-life of the curds could be
extended by dehydration and/or by adding salt; heavily
salted cheese varieties are still widespread throughout the
Middle East and small quantities of a number of dehy-
drated cheeses are produced in North Africa and the Mid-
dle East, e.g., Tikammart and Aoules (Algeria), Djamid
(Jordan), Ekt (Saudi Arabia) and Madraffarah (Syria) (see
Phelan et al., 1993).
It is presumed that one of the principal families of
cheese, the acid cheeses, modern members of which
include Cottage cheese, Cream cheese and Quarg, origin-
ated in this way. While lactic acid, produced in situ, is
believed to have been the original milk coagulant, an
alternative mechanism was also recognized from an
early date. Many proteolytic enzymes can modify the
casein system in milk, causing it to coagulate under cer-
tain circumstances. Enzymes capable of causing this
transformation are widespread in nature, e.g., bacteria,
moulds, plant and animal tissues, but an obvious source
would have been animal stomachs. It would have been
observed that the stomach of young mammals after
slaughter contained curds, especially if the animals had
suckled shortly before slaughter; curds would also have
been observed in the vomit of human infants. Before the
development of pottery (⬃5000 BC), storage of milk in
bags made from animal skins was probably common (as
it still is in many countries). Stomachs of slaughtered
animals provided ready-made, easily sealed containers;
under such circumstances, milk would extract enzymes
(chymosin and some pepsin) from the stomach tissue,
leading to its coagulation during storage. The properties
of rennet-coagulated curds are very different from those
produced by isoelectric (acid) precipitation, e.g., they
have better syneresis properties which makes it possible
to produce low-moisture cheese curd without harden-
ing. Rennet-coagulated curds can, therefore, be con-
verted to a more stable product than acid curds and
rennet coagulation has become predominant in cheese
manufacture, being exploited for c. 75% of total world
production.
Although animal rennets were used from early times,
rennets produced from a range of plant species, e.g., fig
and thistle, also appear to have been common in ancient
times. However, plant rennets are not suitable for the
manufacture of long-ripened cheese varieties and gastric
proteinases from young animals became the standard
rennets until a recent shortage of supply made it neces-
sary to introduce ‘rennet substitutes’.
While the coagulation of milk by the in situ produc-
tion of lactic acid was, presumably, accidental, the use
of rennets to coagulate milk was intentional. It was, in
fact, quite an ingenous invention – if the conversion of
milk to cheese by the use of rennets was discovered
today, it would be hailed as a major biotechnological
discovery!
The advantages accruing from the ability to convert
the principal constituents of milk to cheese would
have been apparent from the viewpoints of storage sta-
bility, ease of transport and, presumably, as a means of
diversifying the human diet and cheese manufacture
became well established in the ancient civilizations of
the Middle East, Egypt, Greece and Rome. There are
numerous references to cheese and other foods in the
Bible (see MacAlister, 1904). Milk and dairy products
formed an important part of the diet of peoples of
the Near East during Biblical times; indeed Palestine
was praised as ‘a land flowing with milk and honey’
(Exodus 3.8). Animals herded during Biblical times
for milk production included goats (e.g., Proverbs
27.27), sheep (e.g., Deuteronomy 14.4) and possibly
camels (Genesis 32.15). Cows’ milk is rarely specified
in the Old Testament, presumably because of the
unsuitability of the terrain of the Holy Land for cow
pasture. In addition to milk, other foods of dairy ori-
gin mentioned in the Bible include curds (perhaps fer-
mented milk: Genesis 18.8; Isaiah 7.22) and butter
(Psalms 55.21). There are several clear references in
the Old Testament to cheese, e.g., Job (1520 BC, where
Job remarks to God ‘did Thou not pour me out like
milk and curdle me like cheese’; Job 10.10) and
Samuel (1170–1017 BC; as a delicacy sent by Jesse to
his sons (I Samuel 17.18) and as a gift presented to
David (II Samuel 17.29)).
Cheese is represented in the tomb art of Ancient
Egypt and in Greek literature. Vegetable rennets are
mentioned in the first work of European literature;
Homer (c. eighth century BC) implies the use of fig
rennet in the Iliad (‘. . . as when fig juice is added to
white milk and rapidly coagulates, and the milk
quickly curdles as it is stirred, so speedy was his heal-
ing of raging Ares.’ Iliad 5) and describes the Cyclops,
Polyphemus, making ewes’ milk cheese in the Odyssey
(Book 9) using ‘well made dairy vessels’ and ‘pails
swimming with whey’. Other Greek authors who men-
tion cheese include the Father of History, Herodotus
(484–408 BC), who referred to ‘Scythian cheese’ and
the philosopher, Aristotle (384–322 BC), who noted
that ‘Phrygian’ cheese was made from the milk of
mares and asses. Apparently, cheese was prescribed in
the diet for Spartan wrestlers in training.
Cheese manufacture was well established in the
Roman Empire and was a standard item in the rations

issued to Roman soldiers. Cheese must have been popu-
lar with Roman civilians also and demand exceeded
supply, forcing an emperor, Diocletian (AD 284–305),
to fix a maximum price for cheese. Many Roman
writers, e.g., Cato the Elder (234–149 BC), Varro,
Columella and Pliny the Elder, described cheese
manufacture and quality and the culinary uses of
cheese. Pliny the Elder (AD 23–79) mentioned cheese
in his encyclopedia, Historia Naturalis (Book 28) and
described its uses in the diet and in medicinal applica-
tions. Varro (c. 116–27 BC; De Agricultura 2.3–2.6)
distinguished between ‘soft and new cheese’ and that
which is ‘old and dry’ and described the Roman
cheesemaking season in the spring and summer. Varro
briefly described cheese manufacture: to about 2 congii
(c. 5.7 L) of milk was added a piece of rennet from
the hare or kid (in preference to that from the lamb).
Varro described the quantity of rennet to be added as
‘the size of an olive’, implying that the rennet was
solid, perhaps a piece of stomach tissue. If so, then
this rennet may be analogous to rennet paste, which
is used today for the manufacture of certain Italian
cheese varieties (see ‘Biochemistry of Cheese Ripen-
ing: Introduction and Overview’, ‘Lipolysis and
Catabolism of Fatty Acids in Cheese’, Volume 1). Fig
latex and vinegar were mentioned by Varro as an
alternative rennet and vinegar is also mentioned as a
means for coagulating milk (as practised today in the
manufacture of some forms of Queso Blanco and
Ricotta).
However, the most complete ancient description of
cheesemaking is given by Lucius Junius Moderatus Col-
umella, a Roman soldier and author from Gades (mod-
ern Cadiz), in his treatise on agriculture, De Re Rustica
(c. AD 50). A manufacturing procedure for Roman cheese,
based on the description of Columella, is given in Fig. 1,
which includes many observations and practices recog-
nizable by modern cheesemakers. He recommends that
the (raw) milk be held at ‘some degree of heat’ but warns
against over-heating by placing the pail on the flames of
a fire. Columella distinguished between cheese with a
‘thin consistency’ (soft?) which must be sold quickly
‘while it is still fresh and retains its moisture’ and that
with a ‘rich and thick consistency’ (hard?) which may be
held for a long period. Since the concept of pH and the
existence of bacteria were unknown in antiquity, no
mention is made of starter; the cheese curd was acidified
using the adventitious microflora of the raw milk. How-
ever, Columella did discuss different types of rennets
in some detail. He recommended coagulation using
rennet from lamb or kid but states that milk can also
be coagulated using flowers of certain thistles (perhaps
Cynara cardunculus), seeds of the safflower (Carthamus
tinctorius), or sap from the fig tree. Interestingly, Col-
Cheese: An Overview 3
PAIL OF MILK
(Sheep or goat)
Rennet, > weight of
‘Some degree of heat’ denarius (c. 3.4 g)
(Stand not far from the fire) (Lamb, kid or other)
Coagulum
Drain whey quickly when milk
coagulated using wicker baskets
or moulds. Aid whey drainage
WHEY
using weights
Curds
Place cheese in a cool shady place
Surface application of dry salt
Rind formation
Pressing using weights
Further application of dry salt
Repeat for 9 days
Wash cheeses using water
Place cheeses in rows on wickerwork trays
Allow them to become ‘moderately dry’
Pack closely on shelves in an enclosed place
not exposed to the wind
Cheese becomes ‘more tender’
Cheese can be ‘exported beyond the sea’
CHEESE
Figure 1 Flow diagram for the manufacture of a type of Roman
cheese based on the description of Columella (De Re Rustica,
7.8.1–7.8.7).
umella recommended that the smallest amount of rennet
possible be used to ensure high quality cheese. This may
be related to the excessive proteolytic activity of plant
proteinases used as rennets which often produce bitter
cheese. Whey drainage was through wicker baskets,
perhaps analogous to the drainage of whey through
moulds in the manufacture of certain soft cheeses (e.g.,
Camembert). No mention was made by Columella of
cooking the curds/whey mixture prior to whey drainage;
moisture control seems to have been by pressing the
curds during whey drainage or pressing the cheese after
salting. Salting was by means of the repeated application
of dry salt to the cheese surface (which is still practiced,
e.g., in the manufacture of Blue cheese), which encour-
aged further loss of moisture (‘acid liquid’). However,
Columella also mentioned brine salting as a method
of ‘hardening’ cheese. The cheeses were washed with
water, allowed to form a rind and placed on shelves in
an enclosed place ‘so that the cheese may remain more
4 Cheese: An Overview
tender’. Interestingly, the comparative form of the adjec-
tive used in the Latin text (tenerior) can also be trans-
lated as ‘more soft’; if this is the intended meaning, it is
the first recorded mention of the changes which occur in
cheese during ripening. Columella also discussed defects
which may occur in cheese, including being ‘full of
holes’ (perhaps mechanical openings as the remedy rec-
ommended is increased pressing), too salty or too dry.
According to Columella, cheeses were flavoured with
herbs and coloured with smoke, practices which persist
to a certain extent today. He also described briefly the
manufacture of ‘hand-pressed’ (manu pressum) cheese in
which hot water is poured over the curds which are
then shaped by hand, a practice perhaps related to the
kneading and stretching steps for pasta-filata varieties.
Thus, cheesemaking practice appears to have changed
little from the time of Columella until the nineteenth
century!
The great migrations of peoples throughout Europe
immediately before and after the fall of the Western
Roman Empire must have promoted the further spread
of cheese manufacture, as did the Crusaders and other
pilgrims of the Middle Ages. Probably, the most import-
ant agents contributing to the development of cheese
‘technology’ and to the evolution of cheese varieties
were monasteries and feudal estates. In addition to their
roles in the spread of Christianity and in the preserva-
tion and expansion of knowledge during the Dark Ages,
the monasteries made considerable contributions to the
advancement of agriculture in Europe and to the devel-
opment and improvement of food commodities, notably
wine, beer and cheese. Many of our current well-known
cheese varieties were developed in monasteries, e.g.,
Wenslydale (Rievaulx Abbey, Yorkshire), Port du Salut
or Saint Paulin (Monastery de Notre Dame du Port
du Salut, Laval, France), Fromage de Tamie (Abbey of
Tamie, Lac d’Annecy, Geneva), Maroilles (Abbey
Moroilles, Avesnes, France) and Trappist (Maria Stern
Monastery, Banja Luka, Bosnia). The inter-monastery
movement of monks would have contributed to the
spread of cheese varieties and probably to the develop-
ment of new hybrid varieties.
The great feudal estates of the Middle Ages were
self-contained communities. The conservation of sur-
plus food produced in summer for use during winter
was a major activity on such estates and undoubtedly
cheese represented one of the more important of these
conserved products, along with cereals, dried and
salted meats, dried fruits, dried and fermented vegeta-
bles, beer and wine. Cheese probably represented an
item of trade when amounts surplus to local require-
ments were available. Within these estates, individuals
acquired special skills which were passed on to suc-
ceeding generations. The feudal estates evolved into
villages and some into larger communities. Because
monasteries and feudal estates were essentially self-
contained communities, it is readily apparent how sev-
eral hundred distinct varieties of cheese evolved from
essentially the same raw material, milk or rennet-coagu-
lated curds, especially under conditions of limited
communication. Traditionally, many cheese varieties
were produced in quite limited geographical regions,
especially in mountainous areas, where communities
are isolated. The localized production of certain vari-
eties is still apparent and indeed is preserved for those
varieties with controlled designations of origin (Appela-
tion d’Origine Contrôlée). Regionalization of certain
cheese varieties is particularly marked in Spain, Portu-
gal and Italy, where the production of many varieties
is restricted to very limited region. Almost certainly,
most cheese varieties evolved by accident because of
a particular set of local circumstances, e.g., a peculiar-
ity of the local milk supply, either with respect to
chemical composition or microflora, an ‘accident’ dur-
ing storage of the cheese, e.g., growth of mould or
other microorganisms. Presumably, those accidents that
led to desirable changes in the quality of the cheese
were incorporated into the manufacturing protocol;
each variety thus underwent a series of evolutionary
changes and refinements.
The final chapter in the spread of cheese through-
out the world resulted from the colonization of north
and south America, Oceania and Africa by European
settlers who carried their cheesemaking skills with
them. Cheese has become an item of major economic
importance in some of these ‘new’ countries, notably
the US, Canada, Australia and New Zealand, but the
varieties produced are mainly of European origin,
modified in some cases to meet local requirements.
Cheese was not manufactured in these regions before
colonization by Europeans; in fact, there were no
cattle, sheep or goats in Australia, North or South
America and there were no land mammals in New
Zealand before the arrival of Europeans.
For further information on the history of cheese,
the reader is referred to Squire (1937), Cheke (1959),
Davis (1965), Kosikowski (1977), Scott (1986), Kosi-
kowski and Mistry (1997) and Robinson and Wilbey
(1998). For references on Roman agriculture, see White
(1970).
Cheesemaking remained an art rather than a science
until relatively recently. With the gradual acquisition of
knowledge on the chemistry and microbiology of milk
and cheese, it became possible to direct the changes
involved in cheesemaking in a more controlled fashion.
Although few new varieties have evolved as a result of
this improved knowledge, the existing varieties have
become better defined and their quality more consistent.

Considering the long history of cheesemaking, one
might be inclined to the idea that what have come to
be regarded as standard varieties have been so for a
long time. However, although the names of many cur-
rent varieties were introduced several hundred years
ago (Table 1), these cheeses were not standardized; for
example, the first attempt to standardize the well-
known English varieties, Cheddar and Cheshire, was
made by John Harding in the mid-nineteenth century.
Prior to that, ‘Cheddar cheese’ was that produced in
a particular area in England around the village of
Cheddar, Somerset, and probably varied considerably
depending on the manufacturer and other factors.
Cheese manufacture was a farmstead enterprise until
the mid-nineteenth century – the first cheese factory in
the US was established near Rome, NY, in 1851 and the
first in Britain at Longford, Derbyshire, in 1870. Thus,
there were thousands of cheese manufacturers and
there must have been great variation within any one
general type. This situation persists in a modified form
today in Switzerland and Italy where there are a large
number of small cheese factories, often grouped
together into consortia for the purposes of marketing
and quality control. When one considers the very
considerable inter-factory, and indeed intra-factory,
variations in quality and characteristics which occur
today in well-defined varieties, e.g., Cheddar, in spite
of the very considerable scientific and technological
advances, one can readily appreciate the variations that
must have existed in earlier times.
Some major new varieties, notably Jarlsberg and
Maasdamer, have been developed recently as a conse-
quence of scientific research. Many other varieties have
evolved very considerably, even to the extent of becom-
ing new varieties, as a consequence of scientific research
and the development of new technology – notable exam-
ples are (US) Queso Blanco, various cheeses produced
by ultrafiltration and various forms of Quarg. There has
been a marked resurgance of farmhouse cheesemaking
in recent years; many of the cheeses being produced on
farms are not standard varieties and some of these may
evolve to become new varieties.
Table 1 First recorded date for some major cheese varieties
(Scott, 1986)
Goronzola 897 Cheddar 1500
Schabzieger 1000 Parmesan 1579
Roquefort 1070 Gouda 1697
Maroilles 1174 Gloucester 1783
Schwangenkäse 1178 Stilton 1785
Grana 1200 Camembert 1791
Taleggio 1282 St Paulin 1816
Cheese: An Overview 5
A major cause of differences in the characteristics of
cheese is the inter-species differences in the composition
and physico-chemical characteristics of the milk used.
Although milks from several species are used in cheese
manufacture, the cow is by far the most important;
sheep, goat and buffalo are commercially important in
certain areas. Approximately 85, 11, 2 and 2% of total
milk is produced from cows, buffalo, sheep and goats,
respectively. However, most sheep’s and goats’ milk is
used for cheese manufacture and therefore are dispro-
portionately important; many famous cheese varieties
are made from sheep’s milk, e.g., Roquefort, Manchego,
Feta and all the various Pecorino and Canestrato var-
ieties. There are very significant inter-species differences
in the composition of milk which are reflected in the
characteristics of the cheeses produced from them.
Major inter-species differences of importance in cheese-
making are the concentration and types of caseins, con-
centration of fat and especially the fatty acid profile,
concentration of salts, especially of calcium. There are
also significant differences in milk composition between
breeds of cattle and these also influence cheese quality,
as do variations due to seasonal, lactational and nutri-
tional factors and of course the methods of milk produc-
tion, storage and collection.
Cheese Science and Technology
Cheese is the most diverse group of dairy products and
is, arguably, the most academically interesting and chal-
lenging. While many dairy products, if properly manu-
factured and stored, are biologically, biochemically,
chemically and physically very stable, cheeses are, in
contrast, biologically and biochemically dynamic, and,
consequently, are inherently unstable. Throughout
manufacture and ripening, cheese production represents
a finely orchestrated series of consecutive and concomit-
ant biochemical events which, if synchronized and
balanced, lead to products with highly desirable aromas
and flavours but when unbalanced, result in off-flavours
and odours. Considering that, in general terms, a basic-
ally similar raw material (milk from a very limited
number of species) is subjected to a manufacturing
protocol, the general principles of which are common
to most cheese varieties, it is fascinating that such a
diverse range of products can be produced. No two
batches of the same variety, indeed probably no two
cheeses, are identical.
A further important aspect of cheese is the range of
scientific disciplines involved: study of cheese manu-
facture and ripening involves the chemistry and
biochemistry of milk constituents, fractionation and
chemical characterization of cheese constituents,
microbiology, enzymology, molecular genetics, flavour
6 Cheese: An Overview

chemistry, nutrition, toxicology, rheology and chem- in the first volume. The second volume deals with spe-
ical engineering. It is not surprising, therefore, that cific aspects of the principal families of cheese. The
many scientists have become involved in the study of principal objective of this introductory chapter is to
cheese manufacture and ripening. A voluminous sci- provide an integrated overview of cheese manufacture
entific and technological literature has accumulated, and to provide some general background for the more
including a range of books (e.g., Sammis, 1948; Van detailed later chapters that follow.
Slyke and Price, 1949; Kosikowski and Mocquot,
1958; Davis, 1965, 1967; Kosikowski, 1977; Davies
and Law, 1984; Eck, 1984; Scott, 1986; Fox, 1987, Outline of Cheese Manufacture
1993; Buch Kristensen, 1995; Kosikowski and Mistry,
Almost all acid-coagulated and a little rennet-coagu-
1997; Law, 1997, 1999; Robinson and Wilbey, 1998; Eck
lated cheese is consumed fresh, i.e., the flavour, texture
and Gilles, 2000; Fox et al., 2000) and chapters in
and appearance of the cheese are in their final form at
many others.
the end of curd production and the curds are not sub-
In addition, there are numerous encyclopedias or
jected to a period of maturation/ripening. The produc-
pictorial books, with brief descriptions of cheese,
tion of acid-coagulated cheeses can be summarized as:
e.g., Simon (1956), Layton (1973), Mair-Waldburg
(1974), Cantin (1976), Eekhof-Stork (1976), Christian
(1984), Robinson (1995), Jenkins (1996) and
Harbutt (1999, 2002). There are also a number of Acidification Cut/break
country-specific or variety-specific books, e.g., Squire Milk Coagulum Curds and whey
(biological or
(1937), Cheke (1959), Fraser (1960), Meyer (1973), chemical) Cook
Separate
Montandon (1981), Rance (1982), Gonzalez and del Wash
Cerro (1988), Berger et al. (1989), Anifantakis (1991),
Robinson and Tamime (1991), Zehren and Nusbaum Cold-pack cheese Curds

(1992), Resmini et al. (1992), Masui and Yamada Optional

flavours/dressings
(1996), Vizzardi and Maffeis (1999), Ottogalli (2001) heat
and Kammerlehner (2003). homogenize
Package
Most of the above books deal mainly with cheese
technology; the present book concentrates on the Hot-pack cheese
more scientific aspects of cheese. The book is in two
volumes. The more general aspects of cheese manufac-
ture, i.e., molecular properties of rennets, coagulation
mechanism, curd syneresis, starters, salting, rheol- However, the production of the majority of rennet-
ogy, the biochemistry of ripening, pre-concentration coagulated cheese varieties can be sub-divided into
by ultrafiltration and nutritional aspects, which apply, two well-defined phases, manufacture and ripening,
more or less, to most cheese varieties, are considered both of which involve a number of processes:

Manufacture (5–24 h) Cheese Ripening (2 week–2 year)

Milk Mature cheese
curd
Preparation of milk Development of characteristic
Selection microflora
Standardization Metabolism of residual lactose
Pasteurization Citrate metabolism
*Others Proteolysis
Acidification Lipolysis
Coagulation Secondary reactions
Syneresis (dehydration) Fatty acid catabolism
Cut Amino acid catabolism
Cook Lactate metabolism
Agitation
Other operations, e.g.,
Cheddaring
Kneading/stretching
Pressing
Salting
* e.g., bactofugation, microfiltration, addition of colour (annato)

The manufacturing phase might be defined as
those operations performed during the first 24 h,
although some of these operations, e.g., salting and
dehydration, may continue over a longer period.
Although the manufacturing protocol for individual
varieties differs in detail, the basic steps are common
to most varieties; these are: acidification, coagulation,
dehydration (cutting the coagulum, cooking, stirring,
pressing, salting and other operations that promote
gel syneresis), shaping (moulding and pressing) and
salting.
Cheese manufacture is essentially a dehydration
process in which the fat and casein in milk are concen-
trated between 6- and 12-fold, depending on the vari-
ety. The degree of dehydration is regulated by the
extent and combination of the above five operations,
in addition to the chemical composition of the milk.
In turn, the levels of moisture and salt, the pH and the
cheese microflora regulate and control the biochemical
changes that occur during ripening and hence deter-
mine the flavour, aroma and texture of the finished
product. Thus, the nature and quality of the finished
cheese are determined largely by the manufacturing
steps. However, it is during the ripening phase that the
characteristic flavour and texture of the individual
cheese varieties develop.
Selection and pre-treatment of cheese milk
Cheese manufacture commences with the selection of
milk of high microbiological and chemical quality. The
adventitious microflora of milk is normally heterogen-
eous. Some of these microorganisms, especially the
LAB, may be beneficial. Previously, and still for some
minor artisanal cheeses, the indigenous LAB were
responsible for acid production but selected starter
LAB cultures are used for acidification in most cases.
Non-starter LAB (NSLAB) probably contribute posi-
tively to the ripening of raw milk cheese (see ‘Microbio-
logical changes during ripening’, ‘Biochemistry of
Cheese Ripening: Introduction and Overview’, ‘Metab-
olism of Residual Lactose and of Lactate and Citrate’,
‘Lipolysis and Catabolism of Fatty Acids in Cheese’,
‘Proteolysis in Cheese during Ripening’ and ‘Catab-
olism of Amino Acids in Cheese During Ripening’,
Volume 1) but they are variable and uncontrolled and
may be responsible for some of the variability in raw
milk cheese. For large-scale cheesemaking operations,
it is preferable to kill the NSLAB by pasteurization
(although this is not the primary objective of pasteur-
ization). There is increasing interest in inoculating
pasteurized milk with selected lactobacilli as an
adjunct culture (see ‘Secondary and Adjunct Cultures’,
Volume 1).
Cheese: An Overview 7
Some members of the adventitious microflora are
undesirable. The most important are number of
pathogens, the killing of which is the primary objective
of pasteurization (see ‘Growth and Survival of Microbial
Pathogens in Cheese’, Volume 1). Raw milk may also
contain several spoilage microorganisms, e.g., coliforms
(which are unlikely to be a problem today), psy-
chrotrophs (especially if the milk is cold-stored for a long
period) and Clostridium tyrobutyricium. Growth of this
sporeforming organism during ripening of most cheese
varieties results in a defect known as late gas blowing
caused by an anaerobic metabolism of lactate to butyrate
and H2. Contamination with Cl. tyrobutyricum is mini-
mized by good on-farm hygiene, contaminants may be
removed by bactofugation or microfiltration, or their
growth may be prevented by NaNO3 or lysozyme.
Cheesemilk must be free from antibiotics, which
totally, or partially, inhibit the starter bacteria; delayed
acidification results in an abnormal composition and
microflora and consequently in flavour and textural
defects and perhaps very significantly in the growth of
harmful, pathogenic or food-poisoning microorganisms.
All aspects of cheese curd production (rennet coagu-
lation, gel firmness, syneresis) are affected by the
chemical composition of the cheesemilk, especially the
concentrations of casein, calcium and pH. The specific
effects of compositional parameters on various aspects
of curd formation will be described in detail in several
subsequent chapters. For a comprehensive description
of the chemistry of milk and milk constituents, the
reader is refered to Fox (1982, 1983, 1985, 1987, 1992,
1995, 1997) and Fox and McSweeney (1998, 2003).
In modern commercial practice, milk for cheese is
normally cooled to 4 °C immediately after milking and
may be held at about this temperature for several days
on the farm and at the factory. Apart from being selective
for the development of an undesirable psychrotrophic
microflora, cold-storage causes physico-chemical changes
(e.g., shifts in calcium phosphate equilibrium and disso-
ciation of some micellar caseins) which have undesirable
effects on the cheesemaking properties of milk; these
changes are reversed on heating, e.g., at 50 °C for
10–20 min or under HTST pasteurization conditions
and hence are of no practical significance. However, cold
storage after heat treatment aggravates the adverse
effects of heating on the rennet coagulation of milk; this
effect is known as rennet hysteresis.
The composition of most cheeses falls within certain
compositional ranges, sometimes with legal status. The
most important compositional factors are fat-in-dry
matter (FDM), moisture in non-fat substances (MNFS;
which is, in effect, the ratio of moisture to protein),
moisture, salt (best expressed as salt-in-moisture, S/M)
and pH. The values for FDM and MNFS are determined
8 Cheese: An Overview
mainly by the composition of the cheesemilk and
extent of syneresis, respectively. The composition of
milk should be adjusted to give the desired values of
fat and protein. Previously, the ratio of fat:protein
was altered by natural creaming (which is still used for
Parmigiano-Reggiano, Grana Padano and some other
Italian cheeses) or by the addition of cream or skim
milk. It is now possible, and commercially practiced to
an increasing extent, to adjust the concentrations, as
well as the ratio, of fat and protein in the cheesemilk by
manipulating the fat content of the milk together with
protein standardization using low concentration factor
ultrafiltration. These operations improve the cheese-
making characteristics of the milk and increase and
standardize the yield of curd.
Owing to the importance of Ca2 in various aspects
of cheese manufacture and quality (see ‘Rennets: Gen-
eral and Molecular Aspects’, ‘Rennet-induced Coagula-
tion of Milk’ and ‘The Syneresis of Rennet-coagulated
Curd’, Volume 1), it is common practice to supple-
ment cheesemilk with CaCl2. pH is also a critical fac-
tor in cheesemaking and since the pH of milk varies
(e.g., increases with advancing lactation and during
mastitic infection), it is recommended that the pH
should be standardized, preferably using the acidogen,
gluconic acid--lactone.
Although raw milk is still used in both industrial
and farmhouse cheesemaking, most cheesemilk is pas-
teurized, usually immediately before use. Pasteuriza-
tion alters the indigenous microflora and facilitates the
manufacture of cheese of more uniform quality, but
unless due care is exercised, it may damage the rennet
coagulability and curd-forming properties of the milk,
as will be discussed in ‘Rennet-induced Coagulation of
Milk’, Volume 1.
Even when milk is properly pasteurized, the result-
ing cheese develops a less intense flavour and ripens
more slowly than raw milk cheese. Several heat-
induced changes, e.g., killing of indigenous microor-
ganisms, inactivation of indigenous milk enzymes and
partial denaturation of whey proteins and their inter-
action with micellar -casein, could be responsible
for these changes. The relative contribution of these
factors to the differences between cheeses made from
raw or pasteurized milk has been an active area of
research in recent years (see Fox et al., 2000; ‘The
Microbiology of Cheese Ripening’, ‘Raw Milk Cheeses’
and ‘Biochemistry of Cheese Ripening: Introduction
and Overview’, Volume 1).
A number of approaches have been used to render
cheesemilk free from its indigenous microflora or
to inhibit the growth of NSLAB in order to study
their contribution to ripening. Non-starter lactic acid
bacteria have been removed physically from raw
skim milk by microfiltration (e.g., McSweeney et al.,
1993; Beuvier et al., 1997), environmental contamin-
ation has been minimized by manufacturing cheese
under strictly controlled microbiological conditions
(McSweeney et al., 1994), ripening cheese at a low (c.
1 °C) temperature to reduce the growth rate of NSLAB
(Shakeel-Ur-Rehman et al., 2000b,c) and the use of
antibiotics to inhibit the growth of NSLAB (Shakeel-
Ur-Rehman et al., 1999). Attempts have been made to
mimic the NSLAB microflora of raw milk cheese by
adding selected strains of NSLAB (see Lynch et al.,
1999) to pasteurized cheesemilk or by inoculating
pasteurized cheesemilk with raw milk (by blending a
low level, e.g., 1%, of raw milk with pasteurized milk;
Shakeel-Ur-Rehman et al., 2000a). The results of these
studies suggest that heat-induced changes to the
microflora of raw milk are the major cause of the dif-
ferences between raw and pasteurized milk cheeses.
However, denaturation of certain indigenous enzymes,
particularly lipoprotein lipase, may contribute to the
observed differences.
Pasteurization of cheesemilk minimizes the risk of
cheese serving as a vector for food-poisoning or patho-
genic microorganisms, so that even high-quality raw
milk may be unacceptable for cheese manufacture. In
addition to rendering milk safe from a public health
viewpoint, pasteurization renders good quality raw
milk almost free of bacteria and improves the consist-
ency of cheese. Pasteurization of milk is essential for
the production of cheese of consistent quality in the
large, highly mechanized factories that are common
today. Although more consistent than cheese made
from raw milk, it is also less highly flavoured. To
increase the intensity of the flavour of cheese made
from pasteurized milk, it is becoming increasingly
common to inoculate pasteurized milk with selected
organisms, usually lactobacilli, isolated from good
quality raw milk cheese (see Lynch et al., 1999;
‘Secondary and Adjunct Cultures’ and ‘The Microbiol-
ogy of Cheese Ripening’, Volume 1). Thermization
(⬃65 °C  15 s) of cheesemilk on arrival at the fac-
tory is common or standard practice in some coun-
tries. The objective of thermization is to control
psychrotrophs and the milk is normally pasteurized
before cheesemaking. Microfiltration and bactofuga-
tion may be used to remove spores from milk to avoid
the defect known as late gas blowing (see ‘The Micro-
biology of Cheese Ripening’, Volume 1).
Not more than 75% of the total protein in milk is
recovered in rennet-coagulated cheeses. Obviously, a
considerable economic advantage would accrue if
some or all of the whey proteins could be incorpor-
ated into the cheese. Ultrafiltration (UF) offers a
means for accomplishing this, with considerable

success in the case of semi-soft or soft cheeses, espe-
cially Feta and Quarg, but with less success for hard
and semi-hard varieties. The application, and associ-
ated problems, of UF in cheese manufacture is com-
prehensively reviewed in ‘Application of Membrane
Separation Technology to Cheese Production’, Vol-
ume 1. An alternative approach is to heat denature
the whey proteins (e.g., 90 °C  1 min) to induce
their interaction with the casein micelles. Normally,
such severe heat treatments are detrimental to the
renneting properties of milk but the effects can be
off-set by acidification or supplementation with
calcium (see ‘Rennet-induced Coagulation of Milk’
and ‘The Syneresis of Rennet-coagulated Curd’, Vol-
ume 1). In the authors’ experience, yield increases of
up to 8% can be achieved by this approach, while
retaining acceptable quality. However, to the authors’
knowledge, the technique is not used commercially
except for Quarg, e.g., the thermo-Quarg process (see
‘Acid- and Acid-Rennet-Curd Cheeses: Part A Quark,
Cream Cheese and Related Varieties’, Part B Cottage
Cheese’, and Part C ‘Acid-heat Coagulated Cheeses’,
Volume 2).
Acidification
One of the basic operations in the manufacture of
most, if not all, cheese varieties is a progressive acidifi-
cation throughout the manufacturing stage, i.e., up to
24 h, and, for some varieties, during the early stages of
ripening also, i.e., acidification commences before and
transcends the other manufacturing operations. Acid-
ification is normally via in situ production of lactic
acid, although pre-formed acid or acidogen (usually
gluconic acid--lactone) is now used to directly acidify
curd for some varieties, e.g., Mozzarella, UF Feta-type
and Cottage cheese. Until relatively recently, and still
in some cases, especially in artisanal varieties, the
indigenous microflora of milk was relied upon for
acid production. Since this was probably a mixed
microflora, the rate of acid production was unpre-
dictable and the growth of undesirable bacteria led to
the production of gas and off-flavours. It is now almost
universal practice in industrial cheesemaking to add
a culture (starter) of selected lactic acid-producing
bacteria to raw or pasteurized cheesemilk to achieve a
uniform and predictable rate of acid production. For
cheese varieties that are cooked to not more than
40 °C, a starter consisting of Lactococcus lactis subsp.
lactis and/or Lc. lactis subsp. cremoris is normally used
while cultures of Streptococcus thermophilus and a
Lactobacillus spp. (Lb. delbrueckii subsp. bulgaricus,
Lb. delbrueckii subsp. casei, Lb. delbrueckii subsp. lactis
or Lb. helveticus) or a Lactobacillus culture alone is
Cheese: An Overview 9
used for varieties that are ‘cooked’ to higher tempera-
tures, e.g., Swiss and hard Italian varieties.
Probably the earliest form of starters were ‘slop-
back’ cultures – a sample of whey from one day’s
cheesemaking was incubated overnight and used as a
starter culture on the following day. Such starters are
still used for some high-cook cheese varieties (e.g.,
Parmigiano-Reggiano and Grana Padano). Incubation
of hot whey is selective for thermophilic microorgan-
isms and although slop-back cultures are very hetero-
geneous, they work well if managed carefully.
Originally, and still for many varieties, mixed-strain
mesophilic starters were used for low-cook cheese.
Because the bacterial strains in these starters may be
phage-related (i.e., subject to infection by a single
strain of bacteriophage) and also because the strains in
the mixture may be incompatible, thereby leading to
the dominance of one or a few strains, the rate of acid
production by mixed-strain starters is variable and
unpredictable, even when the utmost care in their
selection and handling is exercised. To overcome these
problems, single-strain mesophilic starters were intro-
duced in New Zealand about 1935. Unfortunately,
many of the fast acid-producing, single-strain starters
produced bitter cheese, the cause(s) of which will
be discussed in ‘Starter Cultures: General Aspects’,
Volume 1. This problem was resolved by using selected
pairs of fast and slow acid producers. Defined-strain
mesophilic starters are widely used in many countries,
frequently consisting of a combination of 2–6 selected,
phage-unrelated strains which give very reproducible
rates of acid production if properly selected and main-
tained. The use of defined-strain thermophilic starters
is becoming more common.
The science and technology of starters have
become highly developed and specialized; ‘Starter Cul-
tures: General Aspects’, Volume 1, is devoted to these
developments.
Acid production at the appropriate rate and time is
the key step in the manufacture of good quality cheese
(excluding the enzymatic coagulation of the milk,
which is a sine qua non for rennet-coagulated cheese
varieties). Acid production affects several aspects of
cheese manufacture, many of which will be discussed
in more detail in later chapters, i.e.:
• Coagulant activity during coagulation.
• Denaturation and retention of the coagulant in the
curd during manufacture and hence the level of
residual coagulant in the curd; this influences the
rate of proteolysis during ripening, and may affect
cheese quality.
• Strength of the coagulum, which influences cheese
yield.
10 Cheese: An Overview
• Gel syneresis, which controls cheese moisture and
hence regulates the growth of bacteria and the activ-
ity of enzymes in the cheese; consequently, it
strongly influences the rate and pattern of ripening
and the quality of the finished cheese.
• The rate of pH decrease determines the extent of
dissolution of colloidal calcium phosphate which
modifies the susceptibility of the caseins to proteo-
lysis during manufacture, influences the rheological
properties of the cheese, e.g., compare the texture of
Emmental, Gouda, Cheddar and Cheshire cheese,
and determines the meltability and stretchability of
cheese curd (e.g., Mozzarella and Pizza cheese).
• Acidification controls the growth of many species of
bacteria in cheese, especially pathogenic, food poi-
soning and gas-producing microorganisms; in fact,
properly made cheese is a very safe product from
the public health viewpoint. In addition to produc-
ing acid, many starter bacteria produce bacteriocins
that also restrict or inhibit the growth of non-starter
microorganisms.
Mesophilic Lactococcus spp. are capable of reducing
the pH of cheese to ⬃4.6 and Lactobacillus spp. to
somewhat lower values, perhaps 3.8. The natural ultim-
ate pH of cheese curd falls within the range 4.6–5.1.
However, the period required to attain the ultimate pH
varies from ⬃5 h for Cheddar to 6–12 h for Blue, Dutch
and Swiss varieties. The differences arise from the
amount of starter added to the cheesemilk (0.2–5%),
the cooking temperature and schedule which may
retard the growth of the starter microorganisms and the
rate of subsequent cooling of the curd.
The level and method of salting have a major influ-
ence on pH changes in cheese. The concentration of
NaCl in cheese (commonly 0.7–4%, i.e., 2–10% salt in
the moisture phase) is sufficient to halt the growth of
starter bacteria. Some varieties, mostly of British ori-
gin, are salted by mixing dry salt with the curd
towards the end of manufacture and hence the pH of
curd for these varieties must be close to the ultimate
value (⬃pH 5.1) at salting. However, most varieties
are salted after moulding by immersion in brine or by
application of dry salt on the surface; as discussed in
‘Salt in Cheese: Physical, Chemical and Biological
Aspects’, Volume 1, salt diffusion in cheese moisture is
a slow process and thus there is ample time for the pH
to decrease to ⬃5.0 before the salt concentration
becomes inhibitory. The pH of the curd for most
cheese varieties, e.g., Swiss, Dutch, Tilsit, Blue, etc., is
6.2–6.5 at moulding and pressing but decreases to ⬃5
during or shortly after pressing and before salting. The
significance of various aspects of the concentration
and distribution of NaCl in cheese will be discussed in
‘Salt in Cheese: Physical, Chemical and Biological
Aspects’, Volume 1.
In a few special cases, e.g., Domiati, a high level of
NaCl (10–12%) is added to the cheesemilk, traditionally
to control the growth of the indigenous microflora. This
concentration of NaCl has a major influence, not only
on acid development, but also on rennet coagulation, gel
strength and syneresis (cf., ‘Rennet-induced Coagulation
of Milk’ and ‘The Syneresis of Rennet-coagulated Curd’,
Volume 1).
Coagulation
The essential characteristic step in the manufacture of all
cheese varieties is coagulation of the casein component
of the milk protein system to form a gel which entraps
the fat, if present. Coagulation may be achieved by:
• limited proteolysis by selected proteinases;
• acidification to pH ⬃4.6;
• acidification to about pH 5.2 in combination with
heating to ⬃90 °C.
The majority of cheeses are produced by enzymatic
(rennet) coagulation. With a few exceptions (e.g.,
Serra da Estrêla (Portugal) in which acid proteinases
from the flowers of the cardoon thistle, Cynara
cardunculus, are used), acid (aspartyl) proteinases of
animal or fungal origin are used. Rennet from the
stomachs of young animals (calves, kids, lambs, buf-
falo) was used traditionally. The principal enzyme in
rennet prepared from young animal stomachs is chy-
mosin (⬃95% of total milk-clotting activity), with a
little pepsin. However, limited supplies of such ren-
nets (due to the birth of fewer calves and an increas-
ing trend in many countries to slaughter calves at an
older age than previously), concomitant with a world-
wide increase in cheese production, have led to a
shortage of calf rennet and consequently rennet sub-
stitutes (usually bovine or porcine pepsins and less
frequently, chicken pepsin, and the acid proteinases
from Rhizomucor miehei and less frequently R. pusillus
or Cryphonectria parasitica) are now used widely for
cheese manufacture in many countries with more
or less satisfactory results. The calf chymosin gene
has been cloned in Kbryveromyces lactis, E. coli and
Aspergillus niger and chymosin from these organisms
is now widely used. Reviews on rennet substitutes
include Sardinas (1972), Ernstrom and Wong (1974),
Nelson (1975), Sternberg (1976), Green (1977),
De Koning (1979), Phelan (1985), Fox and McSweeney
(1997). The molecular and enzymatic properties of
calf chymosin and other acid proteinases used as ren-
nets are reviewed in detail in ‘Rennets: General and
Molecular Aspects’, Volume 1.

Although it appears to have been recognized since
1917 (see Berridge, 1942) that milk is not coagulated by
rennet at low temperatures, Berridge (1942) is usually
credited with clearly demonstrating that the rennet-
catalysed coagulation of milk occurs in two phases:
a primary enzymatic phase and a secondary non-
enzymatic phase. The primary phase has a temperature
coefficient (Q10) of ⬃2 and occurs in the range 0–50 °C,
while the secondary phase has a Q10 of ⬃16 and occurs
very slowly or not all at temperatures ⬃18 °C. The
two phases can thus be readily separated by performing
the primary phase at a low temperature, e.g., ⬃10 °C;
when cold-renneted milk is warmed, coagulation occurs
very quickly. Cold renneting, followed by rapid warm-
ing, forms the basis of attempts to develop methods for
the continuous coagulation of milk but such approaches
have not been successful commercially. Normally, the
two phases of rennet coagulation overlap to some
extent, the magnitude of overlap being quite exten-
sive at low pH, high temperature and in milk concen-
trated by ultrafiltration.
The primary phase of rennet action appears to have
been recognized, in general terms, by Hammersten,
during the period 1880–1890, who reported the for-
mation of small peptides during renneting. Richmond
(1899) described the action of rennet as follows: ‘the
action of rennet is to split the casein into a dyscaseose,
the calcium salt of which is insoluble and which forms
a curd, and a soluble caseose; the insoluble curd car-
ries down with it a large portion of the fat.’ The coagu-
lation of milk attracted quite a lot of interest during
the early part of the twentieth century. For example,
Alexander (1910, 1912) proposed that the casein in
milk exists as an unstable colloid which is protected
and stabilized by the whey protein, lactalbumin (He
was probably the first to use the idea of a ‘protective
colloid’ in casein chemistry); he proposed that rennet
coagulated milk by hydrolysing (destroying) the pro-
tective colloid. The protective colloid (Schutzcolloid)
hypothesis of the colloidal stability of casein and the
rennet coagulation of milk was supported by Marui
(1926) and Linderstrøm-Lang (1929) but not by
Palmer and Richardson (1925) and Palmer (1935)
who claimed that increased sensitivity of rennet-
altered casein was responsible for the rennet-induced
coagulation of milk rather than the destruction of a
protective colloid. The early literature on the rennet
coagulation of milk was reviewed by Palmer and
Richardson (1925) and Palmer (1935) and in a long
series of articles in Le Lait by Porcher (1929, 1930,
1931). However, a full explanation of the process had
to await the isolation of the casein micelle-protective
protein, -casein, by Waugh and von Hippel (1956).
These workers showed that the protective capacity of
Cheese: An Overview 11
-casein was destroyed on renneting and Wake (1959)
demonstrated that -casein is the only milk protein
hydrolysed during the primary phase of rennet action.
Only one peptide bond, Phe1059Met106, is hydrolysed
(Delfour et al., 1965), resulting in the release of the
hydrophilic C-terminal segment of -casein (the
(caseino)macropeptides, some of which are glycosyl-
ated). The unique sensitivity of the Phe9Met bond of
-casein, hydrolysis of which occurs optimally at
pH 5.1–5.5 has been the subject of extensive study
since 1965 and this work is reviewed in ‘Rennet-
induced Coagulation of Milk’, Volume 1.
The visual coagulation of milk is really only the start
of the gelation process which continues for a consider-
able period thereafter. Although these post-coagulation
changes determine many of the critical cheesemaking
properties of the gel, e.g., curd tension (which influences
cheese yield) and syneresis properties (which determine
the moisture content and hence the ripening profile of
the cheese), it is perhaps the least well-understood phase
of the cheesemaking process. The recent literature on
aspects of the post-visual coagulation phase is reviewed in
‘Rennet-induced Coagulation of Milk’ and ‘The Syneresis
of Rennet-coagulated Curd’, Volume 1.
Post-coagulation operations
A rennet-coagulated milk gel is quite stable if main-
tained under quiescent conditions but if it is cut or
broken, it syneresis rapidly, expelling whey. The rate
and extent of syneresis are influenced, inter alia, by
how finely the coagulum is cut (small pieces promote
syneresis; the coagulum for high-moisture cheeses is
not cut but is ladeled into moulds), milk composition,
especially [Ca2] and [casein], pH, cooking tempera-
ture, rate of stirring of the curd–whey mixture and of
course, time (see ‘The Syneresis of Rennet-coagulated
Curd’, Volume 1). The composition of the finished
cheese is to a very large degree determined by the
extent of syneresis and since this is under the control
of the cheesemaker, the differentiation of the individ-
ual cheese varieties really begins at this stage, although
the composition of cheesemilk, the amount and type
of starter and the amount and type of rennet are also
significant in this regard.
The temperature to which the curds are cooked
varies from ⬃30 °C (i.e., no cooking) for high-moisture
cheeses (e.g., Camembert) to ⬃55 °C for low-moisture
cheese (e.g., Parmigiano-Reggiano).
After cooking, the curds and whey are separated by
various, variety-specific techniques. The curds for
most varieties are transferred to moulds where further
drainage and acidification occur. Curds that have
undergone extensive syneresis in the vat (i.e., have a
12 Cheese: An Overview
low moisture content) are pressed in the moulds,
sometimes according to a programmed increase in
pressure, with the objective of fusing the curds and
rendering the cheeses free from mechanical openings
and reducing the moisture content further. The curds
for two families of cheese, Cheddar and pasta-filata,
are subjected to special treatments prior to moulding.
Cheddar-type cheese undergoes a process called ‘ched-
daring’. In the traditional process, the drained curds
are piled into two beds in the vat, separated by a chan-
nel for whey drainage. The beds of curd are cut into
blocks, ⬃10 cm side, which are inverted every 15 min
and later piled two or three blocks high. This process
continues for ⬃2 h until the pH decreases to ⬃5.4.
During cheddaring, the blocks of curd flow slightly
and the cheese acquires a fibrous texture similar to
that of cooked chicken breast meat. In the modern
mechanized process, the drained curds are transferred
pneumatically to a tower (⬃10 m tall) or to a moving
belt; in the tower or on the belt, the mass of curds flows
slightly but much less than in the traditional process.
Previously, it was believed that the flow during ched-
daring was essential for the texture of Cheddar, but it
is likely that the most important change during ched-
daring is acidification which dissolves the CCP – when
the Ca:protein ratio decreases to a certain value, the
texture assumes the characteristics of Cheddar cheese.
The manufacture of Mozzarella curd is similar to that
for Cheddar up to the point at which the pH decreases
to ⬃5.4. The acidified curds are then heated in hot water
to 60–65 °C, kneaded and stretched. It is claimed that
the kneading and stretching are essential for the charac-
teristic fibrous texture and stretchability of Mozzarella.
However, it may be that the function of heating and
kneading is simply to inactivate enzymes and kill bac-
teria and, in effect, to stabilize the characteristics of the
cheese. Heating and kneading were probably introduced
originally to control the microflora of cheese curd pro-
duced from milk of poor microbiological quality.
The unique manufacturing schedule for the specific
varieties is not considered in this book and the inter-
ested reader is referred to appropriate texts (e.g., Van
Slyke and Price, 1949; Davis, 1965, 1967; Kosikowski,
1977; Eck, 1984; Scott, 1986; Robinson, 1995;
Kosikowski and Mistry, 1997; Robinson and Wilbey,
1998; Eck and Gilles, 2000). Some chemical and
physico-chemical aspects of the manufacture of the
major cheese families are discussed in Volume 2. Flow
diagrams for some important cheese varieties are
shown in Fig. 2.
The last manufacturing operation is salting. While
salting contributes to syneresis (⬃2 kg H2O are lost per
kg NaCl taken up), it should not be used as a means of
controlling the moisture content of cheese. Salt has
several functions in cheese which are described in ‘Salt
in Cheese: Physical, Chemical and Biological Aspects’,
Volume 1. Although salting should be a very simple
operation, quite frequently it is not performed properly,
with consequent adverse effects on cheese quality.
As indicated previously, cheese manufacture is essen-
tially a dehydration process. With the development of
ultrafiltration as a concentration process, it was obvious
that this process would have applications in cheese
manufacture, e.g., for standardizing cheesemilk with
respect to fat and casein, or for the preparation of a con-
centrate with the composition of the finished cheese,
commonly referred to as ‘pre-cheese’. Standardization of
cheesemilk by adding UF concentrate (retentate) is now
common in some countries and the manufacture of pre-
cheese has been successful commercially for a range of
soft and semi-soft cheese varieties (see ‘Application of
Membrane Separation Technology to Cheese Produc-
tion’, Volume 1).
Ripening
Some cheeses, mainly acid-coagulated varieties, are
consumed fresh and such cheeses constitute a major
proportion of the cheese consumed in some coun-
tries; the principal acid-curd cheeses are described
in ‘Acid- and Acid-Rennet-Curd Cheeses: Part A
Quark, Cream Cheese and Related Varieties’, Part B
Cottage Cheese’ and Part C ‘Acid-heat Coagulated
Cheeses’, Volume 2. However, most cheese varieties
undergo a period of ripening (curing, maturation)
which varies from ⬃2 weeks (e.g., for Mozzarella)
to 2 years (e.g., Parmigiano-Reggiano or extra-
mature Cheddar), the duration of ripening being
generally inversely related to the moisture content
of the cheese. Many varieties may be consumed at
any of the several stages of maturity, depending on
the flavour preferences of consumers and economic
factors.
Although curds for different cheese varieties are
recognizably different at the end of manufacture (mainly
as a result of compositional and textural differences aris-
ing from differences in milk compositional and process-
ing factors), the unique characteristics of the individual
cheeses develop during ripening, although in most cases
the biochemical changes that occur during ripening, and
hence the flavour, aroma and texture of the mature
cheese, are largely pre-determined by the manufacturing
process, i.e., by composition, especially moisture, NaCl
and pH, by the type of starter and in many cases by sec-
ondary inocula added to, or gaining access to, the
cheesemilk or curd.
 (a) Cows’ milk (b) Cows’ milk (c) (d) Raw cows’ milk
Cows’ milk
Standardize casein:fat to 0.7:1.0 Standardize, pasteurize, cool to 30 °C
Pasteurize (HTST) Rennet, Evening milk
Cool to 31°C 30–32 °C thermophilic starter,
±propionibacteria Rennet Gravity creaming overnight
Cream
Starter (1–2%, v/v): CaCl2, 0.02%, w/v Coagulum (mesophilic) DL starter
Lactococcus lactis subsp. cremoris nitrate Raw cows’ milk
Rennet (1:15,000) Semi-skimmed milk
and/or Lc. lactis subsp. lactis
Morning milk
Cut coagulum,
Cook to c. 55 °C Coagulum
Coagulum Copper-plated conical vat
Hot water
Whey Calf rennet Acidification by natural whey cultures
Cut [~1 cm cubes] Pitch curds-whey into moulds c. 36 °C

Stir for c. 30 min Coagulum

Whey
Curds and whey
Press
Break coagulum
Press under whey
Cook [Raise temperature: 30 °C to 37–39 °C
over ~30 min; hold for ~1 h]
Dry cheese surface
Cook to 53–55 °C
Apply salt to the surface
Drain [~pH 6.1] Place curds in moulds
1–2 days Whey
Whey 5– 6 h
Place curds in mould
Brine salt
Curds Brine salt Further whey drainage, light pressure
2 days, 8–10 °C
3– 5 days
Cheddar Cool storage Brine salt
Dry rind forms
Wax
10–14 days
Mill [pH ~5.4]
10–15 °C, 90% ERH
Fresh cheese
Hot room Fresh cheese
Dry salt [NaCl, ~2%, w/w] 20–24 °C, 80–83% erh
3 weeks–2 months
Mould, press and package Eye development Ripen
Ripen
Cool storage ~2 years
2–3 months at ~5 °C
1–2 months
Fresh cheese Parmigiano-Reggiano cheese
7 °C
Gouda cheese

Ripen [3–24 months; 4–12 °C] Emmental cheese

Mature Cheddar cheese

Figure 2 Flow diagrams for the manufacture of (a) Cheddar, (b) Emmental, (c) Gouda and (d) Parmigiano Reggiano cheeses.
13
14 Cheese: An Overview
During ripening, an extremely complex set of bio-
chemical changes occur through the catalytic action of
the following agents:
• coagulant;
• indigenous milk enzymes, especially plasmin and
lipoprotein lipase, which are particularly important
in cheese made from raw milk;
• starter bacteria and their enzymes;
• secondary microflora and their enzymes.
This secondary microflora may arise from the adventi-
tious microorganisms in milk that survive pasteuriza-
tion or gain entry to the milk after pasteurization, e.g.,
Lactobacillus, Pediococcus, Micrococcus, or they may be
inoculated as secondary starter, e.g., Propionibacterium
in Swiss cheese, Penicillium roqueforti in Blue varieties,
P. camemberti in Camembert or Brie, or the cheese may
acquire a surface microflora from the environment dur-
ing ripening, e.g., the complex Gram-positive micro-
flora of smear-ripened cheeses such as Tilsit, Limburger,
etc. In many cases, the characteristics of the finished
cheese are dominated by the metabolic activity of these
microorganisms.
The primary biochemical changes which occur
during ripening involve the metabolism of residual
lactose and of lactate and citrate, lipolysis and prote-
olysis which are described in ‘Biochemistry of Cheese
Ripening: Introduction and Overview’, ‘Metabolism of
Residual Lactose and of Lactate and Citrate’, ‘Lipolysis
and Catabolism of Fatty Acids in Cheese’, ‘Proteolysis
in Cheese during Ripening’, Volume 1. These primary
changes are followed and overlapped by a host of
secondary catabolic changes, including the various
reactions involving amino acid catabolism (transamina-
tion, deamination, decarboxylation and various lyase
activities ‘Catabolism of Amino Acids in Cheese Dur-
ing Ripening’, Volume 1), fatty acid catabolism and
related reactions (-oxidation of fatty acids, esterifi-
cation, formation of thioesters) and the catabolism of
lactic acid to CO2 and H2O or the propionic, acetic or
butyric acids and CO2 or H2.
While it is not possible to review the biochemistry
involved in the ripening of all individual cheese var-
ieties, an overview of the principal ripening reactions
is presented in ‘Biochemistry of Cheese Ripening:
Introduction and Overview’, ‘Metabolism of Residual
Lactose and of Lactate and Citrate’, ‘Lipolysis and
Catabolism of Fatty Acids in Cheese’, ‘Proteolysis in
Cheese during Ripening’, ‘Catabolism of Amino Acids
in Cheese During Ripening’, Volume 1. The rheolog-
ical properties of cheese are reviewed in ‘Rheology
and Texture of Cheese’, Volume 1. Microbiological
changes which occur during ripening are discussed
in ‘The Microbiology of Cheese Ripening’, Volume 1
and the volatile flavour compounds in cheese and
sensory properties of cheese are discussed in ‘Sensory
Character of Cheese and its Evaluation’ and ‘Instru-
mental Techniques’, respectively, of Volume 1.
More detailed discussions of specific aspects of the
ripening of the principal families of cheese, extra-hard
varieties, Cheddar, Gouda, Swiss-type cheese, mould
ripened varieties, pasta-filata cheeses and sheep’s and
goats’ milk are given in Volume 2 together with discus-
sion of processed cheese products and the uses of
cheese as a food ingredient.
While most people consume cheese principally for
its organoleptic qualities, it must be remembered that
cheese is a very valuable source of nutrients, especially
protein, calcium and phosphorus; see ‘Nutritional
Aspects of Cheese’, Volume 1.
Cheese is the quintessential convenience food
which can be consumed in many forms without prepar-
ation. In addition, a large proportion of cheese
(50–70%) is used as an ingredient (see ‘Cheese as an
Ingredient’, Volume 2) or converted to more stable,
more convenient products by heat treatment to yield
processed cheeses which are discussed in ‘Pasteurized
Processed Cheese and Substitute/Imitation Cheese
Products’, Volume 2.
Cheese Production and Consumption
World cheese production was ⬃16.5  106 tonnes
in 2002 and has increased at an average annual rate
of ⬃3% over the past 20 years. Europe, with a produc-
tion of ⬃8.7  106 tonnes per annum (⬃53% of world
production) is the largest producing region; North and
Central America produces ⬃28% of world cheese. The
production of cheese by country and region are shown
in Table 2.
Cheese consumption varies widely between
countries, even within Europe; it is noteworthy that
with the exceptions of Israel and the Dutch Antilles,
no Asian, African or South American country is
listed among the top 23 cheese-consuming countries
(Table 3). Cheese consumption in most countries for
which data are available has increased considerably
since 1970.
Thus, while cheese manufacture is practised world-
wide, it is apparent from Tables 2 and 3 that cheese is
primarily a product of European countries and those
populated by European emigrants. However, cheese in
some form is produced in most countries throughout
the world and some interesting minor varieties are
produced in ‘non-dairying’ countries (see Phelan et al.,
1993).
 Cheese: An Overview 15

Table 2 Production of all types of cheese (tonnes) in 2001 (source: www.FAO.org)

World 16507068 Ireland 93750

Israel 102029
Africa 704227
Italy 1020712
Asia 1039789
Japan 123000
Europe (total) 8674772
Jordan 3662
European Union (15) 6834006
Kazakhstan 6750
North and Central America 4653978
Kenya 210
South America 709686
Kyrgyzstan 3500
Oceania 724615
Latvia 12400
Developed countries 14145817 Lebanon 21564
Developing countries 2361251 Lithuania 57900
Albania 12050 Macedonia,The Fmr Yug Rp 1540
Algeria 1540 Malta 282
Angola 1230 Mauritania 2058
Argentina 420000 Mexico 153861
Armenia 2616 Moldova, Republic of 5250
Australia 444000 Mongolia 1260
Austria 145320 Morocco 7716
Azerbaijan, Republic of 10750 Myanmar 31976
Bangladesh 1000 Namibia 70
Belarus 54497 Netherlands, The 660000
Belgium-Luxembourg 65000 New Zealand 280615
Bhutan 44 Nicaragua 13195
Bolivia 6834 Niger 14655
Bosnia and Herzegovina 8700 Nigeria 6955
Botswana 2214 Norway 81700
Brazil 38500 Oman 824
Bulgaria 46150 Panama 7866
Canada 359720 Peru 8934
Chile 57184 Poland 460100
China 217250 Portugal 72800
Colombia 52500 Romania 37500
Costa Rica 6861 Russian Federation 433000
Croatia 21879 Slovakia 54660
Cuba 14500 Slovenia 21684
Cyprus 5030 South Africa 36000
Czech Republic 139074 Spain 180374
Denmark 300000 Sudan 151000
Dominican Republic 2500 Sweden 132000
Ecuador 7265 Switzerland 162300
Egypt 465000 Syrian Arab Republic 93475
El Salvador 2400 Tajikistan 6715
Eritrea 312 Tanzania, United Rep of 2000
Estonia 15500 Tunisia 6420
Ethiopia 3975 Turkey 126156
Finland 106000 Turkmenistan 1600
France 1666850 Ukraine 109000
Georgia 75 United Kingdom 382000
Germany 1773000 United States of America 4073000
Greece 236200 Uruguay 29320
Guatemala 11100 Uzbekistan 20675
Honduras 8975 Venezuela, Boliv Rep of 89150
Hungary 89240 Yemen 11185
Iceland 4860 Yugoslavia, Fed Rep of 11500
Iran, Islamic Rep of 199168 Zambia 773
Iraq 30586 Zimbabwe 2100
16 Cheese: An Overview

Table 3 Supply of cheese (kg per caput per annum) in 2000 (source: www.FAO.org)

World 2.6 Iran, Islamic Rep of 3.0 Brazil 0.3

Greece 25.4 Russian Federation 2.9 Gabon 0.3
France 23.6 Saudi Arabia 2.8 Turkmenistan 0.3
Netherlands, The 22.5 Uruguay 2.8 Vanuatu 0.3
Italy 20.5 Botswana 2.7 China 0.2
Austria 19.2 Japan 2.6 Malaysia 0.2
Germany 18.9 Antigua and Barbuda 2.3 Philippines 0.2
Sweden 17.4 Swaziland 2.3 Zimbabwe 0.2
Israel 16.7 Jordan 2.2 Angola 0.1
Netherlands Antilles 16.2 Mauritius 2.2 Djibouti 0.1
Iceland 15.5 Mexico 2.0 Eritrea 0.1
Denmark 15.1 Turkey 2.0 Ethiopia 0.1
Norway 15.1 Macedonia,The Fmr Yug Rp 1.9 Gambia 0.1
United States of America 14.9 Dominica 1.8 Georgia 0.1
Switzerland 14.8 Romania 1.8 Haiti 0.1
Finland 14.1 Saint Vincent/Grenadines 1.8 Kiribati 0.1
Belgium-Luxembourg 13.7 El Salvador 1.7 Lesotho 0.1
Czech Republic 13.5 Cuba 1.6 Liberia 0.1
Estonia 13.1 Honduras 1.6 Nigeria 0.1
Malta 13.0 Libyan Arab Jamahiriya 1.6 Senegal 0.1
Argentina 12.2 Jamaica 1.5 Tanzania, United Rep of 0.1
Canada 11.8 Costa Rica 1.4 Zambia 0.1
Poland 11.1 Guyana 1.4 Bangladesh 0.0
New Zealand 10.2 Iraq 1.4 Benin 0.0
Lebanon 9.7 Ukraine 1.4 Burkina Faso 0.0
Slovakia 9.4 Yugoslavia, Fed Rep of 1.4 Burundi 0.0
Australia 9.2 Azerbaijan, Republic of 1.3 Cambodia 0.0
United Kingdom 9.2 Niger 1.3 Cameroon 0.0
Hungary 8.9 Colombia 1.2 Central African Republic 0.0
Portugal 8.8 Guatemala 1.2 Chad 0.0
Ireland 8.5 Moldova, Republic of 1.2 Comoros 0.0
Slovenia 8.1 Saint Kitts and Nevis 1.2 Congo, Dem Republic of 0.0
Egypt 7.0 Suriname 1.1 Congo, Republic of 0.0
Kuwait 6.7 Tajikistan 1.1 Côte d’Ivoire 0.0
Spain 6.3 Uzbekistan 1.1 Ghana 0.0
Bermuda 6.0 Seychelles 1.0 Guinea 0.0
Barbados 5.8 South Africa 0.9 Guinea-Bissau 0.0
Cyprus 5.7 Tunisia 0.9 India 0.0
Saint Lucia 5.5 Yemen 0.9 Indonesia 0.0
Syrian Arab Republic 5.4 Bolivia 0.8 Kenya 0.0
Grenada 5.3 Mauritania 0.8 Madagascar 0.0
Croatia 5.2 Algeria 0.7 Malawi 0.0
Lithuania 5.0 Armenia 0.7 Mali 0.0
French Polynesia 4.8 Dominican Republic 0.7 Mozambique 0.0
New Caledonia 4.8 Kyrgyzstan 0.7 Namibia 0.0
Sudan 4.8 Myanmar 0.7 Nepal 0.0
Bulgaria 4.5 Ecuador 0.6 Pakistan 0.0
Trinidad and Tobago 4.3 Korea, Republic of 0.6 Papua New Guinea 0.0
Venezuela, Boliv Rep of 4.2 Nicaragua 0.6 Sao Tome and Principe 0.0
Albania 4.1 Cape Verde 0.5 Sierra Leone 0.0
Panama 4.1 Kazakhstan 0.5 Solomon Islands 0.0
Latvia 3.9 Mongolia 0.5 Sri Lanka 0.0
United Arab Emirates 3.9 Brunei Darussalam 0.4 Thailand 0.0
Bahamas 3.8 Fiji Islands 0.4 Togo 0.0
Belarus 3.8 Maldives 0.4 Uganda 0.0
Belize 3.8 Morocco 0.4 Viet Nam 0.0
Bosnia and Herzegovina 3.7 Paraguay 0.4
Chile 3.7 Peru 0.4

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 Rennets: General and Molecular
Aspects
M.J.C. Crabbe, Division of Cell and Molecular Biology, School of Animal and Microbial
Sciences, The University of Reading, UK
Introduction
Natural chymosin may consist of up to six molecular
species, corresponding to genetic variants A and B,
each of which is a mixture of three forms differing at
the N-terminus, with one being three residues longer,
and the other two residues shorter, than the mature
chymosin (Lilla et al., 2001). The function of chy-
mosin is to coagulate milk in the stomach. Rennet may
be considered as a functional enzyme preparation that
is effectively and naturally adapted to the purposes of
cheesemaking (Ye et al., 2000).
Proteolytic enzymes can be classified on the basis of
their catalytic activity into one of the four groups –
serine, cysteine, metallo and aspartic proteinases (Kay,
1985). Chymosin (rennin; EC 3.4.23.4) is a neonatal
gastric aspartic proteinase and is of commercial import-
ance in cheesemaking. It belongs to the aspartic pro-
teinase family which is widely distributed in many
organisms and tissues with different physiological and
functional properties (Chitpinityol and Crabbe, 1998).
The nucleotide and amino acid sequences and the
three-dimensional structures of several aspartic pro-
teinases are available and provide information for the
protein engineering design of this protein family.
Enzymes can now be produced recombinantly in vari-
ous expression systems in sufficient amounts for struc-
tural and functional studies.
Chymosin and Other Aspartic Proteinases
Aspartic proteinases contain two aspartyl residues
(Asp32 and Asp215, pepsin numbering) at the active
site (Tang et al., 1973). They are susceptible to inhibi-
tion by pepstatin, a pentapeptide naturally produced
by Streptomyces strains (Umezawa et al., 1970), and to
affinity labelling at the catalytic aspartates using either
diazoacetylnorleucinemethyl ester (DAN) in the pres-
ence of cupric ions (Rajagopalan et al., 1966) or 1,2-
epoxy-3-(p-nitrophenoxy) propane (EPNP).
Natural sources
Aspartic proteinases can be found throughout nature
from viruses to higher plants and mammals. They are
Cheese: Chemistry, Physics and Microbiology, Third edition – Volume 1: General Aspects
ISBN: 0-1226-3652-X
Set ISBN: 0-1226-3651-1
generally divided into two major groups – pepsin-like
and retroviral enzymes. These enzymes have been isol-
ated from five major sources:
a. The stomach. Several types of gastric enzyme, pepsin
(EC 3.4.23.1), pepsin B (EC 3.4.23.2), gastricsin (EC
3.4.23.3) and chymosin (EC 3.4.23.4), are produced
in the abomasal mucosa as inactive precursors,
zymogens. Pepsin is the predominant proteinase in
adult mammals (Tang et al., 1973). Gastricsins are
found in all parts of the mammalian stomach, -cells
of pancreatic islet, prostate gland and seminal ves-
icles. Chymosin is produced early on during gesta-
tion (in utero) in the abomasal mucosa of newborn
mammals, including calf (Foltmann, 1970), piglet
(Foltmann et al., 1978), kitten (Jensen et al., 1982),
seal (Shamsuzzaman and Haard, 1984) and lamb
(Baudys et al., 1988; Pungercar et al., 1991). The pro-
duction of these enzymes varies, depending on the
age of the animal and the feeding regime (Andrén
and Björck, 1986).
b. Lysosomes of many cell types contain cathepsin D
(Hurley et al., 2000) and cathepsin E. Cathepsin E
is found in gastric mucosa, thymus, spleen and
blood cells (Kageyama, 1995). Human cathepsin D
is possibly involved in the degradation of intracel-
lular and endocytosed proteins, and is a prognostic
indicator of breast tumour invasiveness. There
appears to be a role for this enzyme during proteolysis
in cheese ripening, most clearly in cheese where
rennet activity is low, such as Swiss cheese, Quarg
and Feta.
c. Tissues such as kidney and sub-maxillary gland
produce renin (Kay, 1985).
d. Plants, including squash, cucumber, tomato, barley,
rice, wheat, sorghum and lotus (Doi et al., 1980;
Morris et al., 1985; Polanowski et al., 1985; Belozersky
et al., 1989).
e. Micro-organisms. Several aspartyl proteinases are
secreted by fungi, including Cryphonectria parasitica
(Sardinas, 1968), Penicillium janthinellum (Hofmann
and Shaw, 1964), Rhizomucor pusillus (Arima et al.,
1970), Rhizomucor miehei (Sternberg, 1971), Rhizopus
chinensis (Fumamoto et al., 1967), Aspergillus awamori
Copyright © 2004 Elsevier Ltd
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20 Rennets: General and Molecular Aspects
(Ostoslavskaya et al., 1986), Aspergillus niger (Koaze,
et al., 1964) and Trichoderma reesei (Pitts, 1992). Pro-
teinases have been found in the yeasts Saccharomyces
cerevisiae (MacKay et al., 1988), Candida tropicalis
(Togni et al., 1991) and Yarrowia lipolytica (Yamada
and Ogrydziak, 1983). Thermopsin is secreted by
Sulfolobus acidocaldarius, a thermophilic archaebac-
terium (Lin and Tang, 1990).
Retroviral aspartic proteinases are dimeric, and each
monomer is about half the size of a eukaryotic aspartic
proteinase and carries only one catalytic aspartic residue.
Retropepsins have been found in several viruses, includ-
ing human immunodeficiency virus (HIV), Rous sar-
coma virus, avian myeloblastosis virus and simian
immunodeficiency virus (SIV) (Toh et al., 1985; Kotler
et al., 1989). These proteinases are required for process-
ing for RNA dimerization within the virion, and hence
for infectivity.
Physical properties and stability of aspartyl
proteinases
Molecular weight and isoelectric point
Chymosin and aspartic proteinases have a molecular
weight in the range 32–39 kDa, with a multiplicity
of isoelectric points corresponding to a number of
isozymes, auto-degradation and post-translationally
modified products. N-linked glycosylation has been
found in several proteinases such as cathepsin D (N67
and N183), S. cerevisiae proteinase A (N67 and N263),
rhizomucor protease (N173) and human renin (N67).
Specific receptors for phosphorylation have been
found in porcine pepsin at S68 (Tang et al., 1973),
bovine pepsin and human cathepsin D (Martin and
Corre, 1984; Metcalf and Fusek, 1993). Transgenic
sheep chymosin appears to be identical to calf chy-
mosin (Mezina et al., 2001).
Enzyme stability
Chymosin is most stable at pH values between 5.3 and
6.3. However, even at pH 2, chymosin is relatively stable
(Foltmann, 1959a). Under acidic conditions (pH 3–4),
the enzyme loses its activity rapidly, probably caused by
auto-degradation, while at alkaline pH values (above
9.8), loss is due to an irreversible conformational change
(Cheeseman, 1965). Loss of activity of chymosin A is
higher than for chymosin B (Foltmann, 1966). Chy-
mosin is more stable at 2 °C than at room temperature
(Foltmann, 1959b). Kawaguchi et al. (1987) reported
the rapid loss of the activity of chymosin when the tem-
perature is increased from 45 to 55 °C. Photo-oxidation
of histidine, as well as modification of the -amino
group of lysine, slightly affects the activity of chymosin
(Hill and Laing, 1965; Smith et al., 1991b,c). Chymosin
loses approximately half of its activity after incubation in
4.6 mol/l urea at 37 °C for 30 min (Sugrue et al., 1990).
It has been shown that both pro-part and cysteine
residues are essential for refolding of chymosin after
denaturation (Sugrue et al., 1990; Huang et al., 1992).
Chymosin in a crystalline form appears to be very stable
(Foltmann, 1992).
Prochymosin is more stable than chymosin at neutral
pH (Foltmann, 1966). At pH values below 5.0, prochy-
mosin is converted to chymosin whereas at pH above
11.0 the stability of prochymosin is lost due to a confor-
mational change. Pseudochymosin is stable at acidic pH
for days but is quickly converted to chymosin if the pH
is increased above 4.5 (Barkholt et al., 1979).
Rhizomucor protease, cryphonectria protease and
S. cerevisiae proteinase A are stable at pH 3.5–7.0
(Sardinas, 1968; Dreyer et al., 1986; Bailey and
Siika-aho, 1988). Pepsin shows greater general stability
than chymosin; for example, after incubation in 6 mol/l
urea at 37 °C for 30 min, only 10% of the original
activity is lost (Cheeseman, 1965). The thermostability
of pepsin is reduced in solution at high pH, in the pres-
ence of urea or salt solution, but is increased in the
presence of pepstatin (Privalov et al., 1981). At pH 6.0,
pepsin is more stable than pepsinogen. At pH values
between 8.5 and 10.5, pepsinogen is less stable than
prochymosin and cannot be converted to the active
form in an acidic environment (McPhile, 1975). Inactiv-
ation of pepsin can be initiated by dissociation of the
N-fragment, and the sequence of this portion is a major
determinant of enzyme stability (Tanaka and Yada,
2001). Prochymosin can be efficiently refolded in high
yields by controlled air oxidation (Menzella et al.,
2002).
Aspartic proteinases containing carbohydrate are more
stable towards high temperature, denaturants and degrad-
ation than proteins without carbohydrate (Aikawa et al.,
1990; Berka et al., 1991; Brown and Yada, 1991). Glyco-
sylation of rhizomucor protease by either chemical or
genetic modification resulted in a loss of stability and an
increase in the ratio of milk-clotting activity to proteo-
lytic activity (C/P ratio) (Brown and Yada, 1991; Aikawa
et al., 1992). The stability of rhizomucor proteinase was
reduced by pre-treatment with acid, oxidation of methio-
nine or modification of the -amino group of lysine
(Hubble and Mann, 1984; Smith et al., 1991b).
Enzyme solubility
The solubility of chymosin is affected by pH, tempera-
ture and ionic strength of the solution (Foltmann,
1959b). Non-crystallized chymosin is soluble in solution
containing 1 mol/l NaCl and at pH 5.5. In a solution
of 2 mol/l NaCl, chymosin appears to be insoluble.
Crystallized chymosin shows higher solubility at 25 °C

than at 2 °C (Foltmann, 1970); however, amorphous
precipitates of chymosin are more stable at 2 °C than at
25 °C. At pH values close to the isoelectric point, chy-
mosin is very insoluble at an ionic strength of 0.005; its
solubility is increased by increasing its ionic strength.
Structure of chymosin and other aspartic
proteinases
Gene sequence and primary structure
The genomic DNA of avian and mammalian aspartic
proteinases, chicken embryonic pepsinogen (Hayashi
et al., 1988), human renin (Miyazaki et al., 1984), bovine
chymosin (Hidaka et al., 1986) and human pepsinogen
(Sogawa et al., 1983), is comprised of nine exons
separated by eight introns, and all exon–intron junc-
tion points are highly conserved. These results support
the belief that the genes for these enzymes have
evolved from a common ancestral gene. Conversely, in
several microbial aspartic proteinases, including those
of S. cerevisiae (Ammerer et al., 1986), C. tropicalis
(Togni et al., 1991), R. pusillus (Tonouchi et al., 1986)
and R. miehei (Gray et al., 1986), no intron was found
in the genes for these enzymes. However, in the genes
for the aspartic proteinases of R. niveus (Horiuchi et al.,
1988) and A. awamori (Berka et al., 1990), one and
three short introns, respectively, were found, but their
exon–intron junctions were at different positions from
those in the genes for mammalian and avian aspartic
proteinases.
Calf chymosin is found in two major forms, A and B,
chymosin B being more abundant. Chymosins A and B
differ at only one amino acid position: chymosin A has
an aspartate residue at position 243 (pepsin numbering),
whereas this is a glycine residue in chymosin B. A third
form, chymosin C, appears to be a degradation product
of chymosin A that lacks three residues, D244–F246
(Danley and Geoghegan, 1988). It is likely that chy-
mosins A and B are synthesized from different alleles of
the same polymorphic gene, rather than a multiple gene
family, as only one locus of the chymosin gene was
found from the hybridization of the calf genome with
the chymosin gene (Donnelly et al., 1986). Fig. 1 illus-
trates the nucleotide (cDNA) and amino acid sequences
of calf chymosin B. The secretion signals of aspartic pro-
teinases are approximately 15–24 residues long with low
sequence homology (Fig. 2). These secretion sequences
tend to be rich in hydrophobic amino acids.
The known pro-regions of aspartic proteinases are
shown in Fig. 3. The pro-peptides are 38–54 amino
acid residues in length and are rich in basic residues.
Although sequence identity is high among the closely
related enzymes, there are variables in the cleavage
site between the pro-segment and the mature enzyme.
Rennets: General and Molecular Aspects 21
A lysine residue (K36P; pepsinogen numbering) is con-
served in all proteinases, except lamb prochymosin
and barley aspartic proteinase, and this residue has
been postulated to interact with the catalytic aspartate
residues in the zymogen molecule (James and Sielecki,
1986; Foltmann, 1988). The pro-segments are probably
important for correct folding, targeting and control of
the activation of zymogens (Koelsch et al., 1994).
Chymosin is a single polypeptide chain enzyme of
323 amino acid residues with a low content of basic
residues, and rich in dicarboxylic and -hydroxy amino
acid residues (Foltmann et al., 1977, 1979; Harris et al.,
1982; Moir et al., 1982; Hidaka et al., 1986). The
sequence alignment of calf chymosin with those of
lamb chymosin, porcine pepsin, penicillopepsin, rhi-
zopus protease and S. cerevisiae proteinase A is illus-
trated in Fig. 4.
There are variable numbers of cysteine residues in
their sequences but their positions, when present, are
conserved. Therefore, there is the potential for two disul-
fide bridges in the Rhizomucor and Rhizopus enzymes, a
single disulfide bridge in the Cryphonectria, Penicillium
and Aspergillus enzymes, and no disulfide bridges in
the Irpex aspartic proteinase.
Secondary structure
The secondary structure of chymosin consists mainly
of -sheets with a few small -helical segments. The
secondary structure of chymosin is illustrated in Fig. 5.
The sheets and helices are named by analogy to the
scheme adapted for cryphonectria protease (Blundell
et al., 1985, 1990). The strands are named aN, bN, cN,
dN, a N, b N, c N, d N, qN and rN in the N-terminal
domain and aC, bC, cC, dC, a C, b C, c C, d C, qC and rC
in the C-terminal domain. The helices are named hN
and hC in the N- and the C-terminal domains, respect-
ively. The antiparallel -strands form three well-
defined sheets (Newman et al., 1991). The sheets, 1N
and 1C, are formed by seven or eight strands in a simi-
lar pattern in both lobes and are related by a topolog-
ical two-fold axis. The b, c, b and c strands form
sheets 2N and 2C which occur beneath 1N and 1C,
respectively. Sheet 3 is formed by six -strands, aN, rN,
qN, qC, rC and aC, all of which are antiparallel. This
sheet resides beneath the strands forming the base of
the active site cleft. In each lobe, strands labelled a, b,
c, d are related to a , b , c , d by the intra-lobe diad
and these strands are related to their equivalents in the
opposite lobe by the inter-lobe diad. The helices hN,
h N, hC and h C occur in topological intra- and inter-
domain two-fold symmetry in that they all occur after
the d strands. The fifth helix occurs between the c N
and d N strands and the sixth occurs at a large inser-
tion in the C-terminal domain.
22 Rennets: General and Molecular Aspects

CCC AGA TCC AAG ATG AGG TGT CTC GTG GTG CTA CTT GCT GTC TTC GCT CTC TCC CAA GGC GCT
M R C L V V L L A V F A L S Q G A
PP1 P1

GAG ATC ACC AGG ATC CCT CTG TAC AAA GGC AAG TCT CTG AGG AAG GCG CTG AAG GAG CAT GGG
G I T R I P L Y K G K S L R K A L K E H G
P10 P20

CTT CTG GAG GAC TTC CTG CAG AAA CAG CAG TAT GGC ATC AGC AGC AAG TAC TCC GGC TTC * GGG
L L E N F L E K E E Y G I S S K Y S G F G
P30 P40 1

GAG GTG GCC AGC GTG CCC CTG ACC AAC TAC CTG GAT AGT CAG TAC TTT GGG AAG ATC TAC CTC
E V A S V P L T N Y L D S Q Y F G K I Y L
10 20

GGG ACC CCG CCC CAG GAG TTC ACC GTG CTG TTT GAC ACT GGC TCC TCT GAC TTC TGG GTA CCC
G T P P N E F T V L F D T G S S D F W V P
30 40

TCT ATC TAC TGC AAG AGC AAT GCC TGC AAA AAC CAC CAG CGC TTC GAC CCG AGA AAG TCG TCC
S I Y C K S N A C K N H Q R F D P R K S S
50 60

ACC TTC CAG AAC CTG GGC AAG CCC CTG TCT ATC CAC TAC GGG ACA GGC AGC ATG CAG GGC ATC
T F Q N L G K P L S I H Y G T G S M Q G I
70 80

CTA GGC TAT GAC ACC GTC ACT GTC TCC AAC ATT GTG GAC ATC CAG CAG ACA GTA GGC CTG AGC
L G Y D T V T V S N I V D I Q Q T V G L S
90 100

ACC CAG GAG CCC GGG GCA GTC TTC ACC TAT GCC GAA TTC GAC GGG ATC CTG GGG ATG GCC TAC
T Q E P G D V F T Y A E F D G I L G M A Y
110 120

CCC TCG CTC GCC TCA GAG TAC TCG ATA CCC GTG TTT GAC AAC ATG ATG AAC AGG CAC CTG GTG
P S L A S E Y S I P V F D N M M N R H L V
130 140

GCC CAA GAC CTG TTC TCG GTT TAC ATG GAC AGG AAT GGC CAG GAG AGC ATG CTC ACG CTG GGG
A Q D L F S V Y M D R N G Q E S M L T L G
150 160

GCC ATC AAC CCG TCC TAC TAC ACA GGG TCC CTG CAC TGG GTG CCC GTG ACA GTG CAG CAG TAC
A I N P S Y Y T G S L H W V P V T V Q Q Y
170 180 190

TGG CAG TTC ACT GTG GAC AGT GTC ACC ATC AGC GGT GTG GTT GTG GCC TGT GAG GGT GGC TGT
W Q F T V D S V T I S G V V V A C E G G C
200 210

CAG GCC ATC TTG GAC ACG GGC ACC TCC AAG CTG GTC GGG CCC AGC AGC GAC ATC CTC AAC ATC
Q A I L D T G T S K L V G P S S D I L N I
220 230

CAG CAG GCC ATT GGA GCC ACA CAG AAC CAG TAC GGT GAG TTT GAC ATC GAC TGC GAC AAC CTG
Q Q A I G A T Q N Q Y G E F D I D C D N L
240 250

AGC TAC ATG CCC ACT GTG GTC TTT GAG ATC AAT GGC AAA ATG TAC CCA CTG ACC CCC TCC GCC
S Y M P T V V F E I N G K M Y P L T P S A
260 270

TAT ACC AGC CAA GAC CAG GGC TTC TGT ACC AGT GGC TTC CAG AGT GAA AAT CAT TCC CAG AAA
Y T S Q D Q G F C T S G F Q S E N H S Q K
280 290

TGG ATC CTG GGG GAT GTT TTC ATC CGA GAG TAT TAC AGC GTC TTT GAC AGG GCC AAC AAC CTC
W I L G D V F I R E Y Y S V F D R A N N L
300 310

GTG GGG CTG GCC AAA GCC ATC TGA TCACATCGCTGACCA...........

V G L A K A I
320 323

Figure 1 Nucleotide and amino acid sequence of calf chymosin B cDNA (adapted from Moir et al., 1982).
 Rennets: General and Molecular Aspects 23

Secretion signal peptides The structure of chymosin B described below was

Fungi
A. awamori 1 MVVFSKTAALVLGLSSAVSA*A
A. oryzae 2 MVILSKVAAVAVGLSTVASA*L
MLFSQITSAILLTAASLSLTTA*R
R. miehei 3
R. pusillus 4 MLFSKISSAILLTAASFALTSA*R
MKFTLISSCVALAAMTLAVEAA*P
R. niveus 5
R. chinensis 6 MTFTLNSSCIAIAALAVAVNAA*P
Mammals
Bovine chymosin7 MRCTVVLLAVFALSQG*A
Lamb chymosin8 MRCLVVLLAVFALSQG*A
Porcine pepsin9 MKWLLLLSLVVLSEC*L
Human pepsin10 MKWLLLLGLVALSE*C
Rat pepsin11 MKWMVVALLCLPLLEA*S
Rat cathepsin D12 MQTPGVLLLILGLLDASS*S
Yeast
S. cerevisiae (YPA)13 MFSLKALLPLALLLVSANQVAA*K
S. cerevisiae (BAR1)14 MSAINHLCLYLILASFAIINTITA*L
C. tropicalis15 MATIFLFTKNVFIA.LA.FA.L
Plant
Barley APR16 MGTRGLALALLAAVLLQTVPAASEA*E
Figure 2 Alignment of the secretion signal peptides of aspartic
proteinases. The junctions between putative signal sequence and
proenzyme are indicated by (*) and possible sites are indicated
by (•). References: (1) Berka et al. (1990); (2) Ward and Kodama
(1991); (3) Gray et al. (1986) and Boel et al. (1986); (4) Tonouchi
et al. (1986); (5) Chen et al. (1991); (6) Horiuchi et al. (1988);
(7) Harris et al. (1982) and Moir et al. (1982); (8) Pungercar et al.
(1991); (9) Lin et al. (1989); (10) Hayano et al. (1988); (11) Ishihara
et al. (1989); (12) Birch and Loh (1990); (13) Ammerer et al.
(1986); (14) MacKay et al. (1988); (15) Togni et al. (1991);
(16) Runeberg-Roos et al. (1991) (adapted from Orprayoon, 1994).
Tertiary structure
The three-dimensional structure of several aspartic
proteinases has been solved by X-ray crystallography
(Fig. 6). These include porcine pepsin (Andreeva et al.,
1984; Abad-Zapatero et al., 1990; Cooper et al., 1990;
Sielecki et al., 1990), pepsinogen ( James and Sielecki,
1986; Hartsuck and Remington, 1988), human renin
(Sielecki et al., 1989), cryphonectria protease (Blundell
et al., 1990), penicillopepsin ( James and Sielecki, 1983),
rhizopus protease (Suguna et al., 1987) and retroviral
proteinases (Lapatto et al., 1989; Miller et al., 1989;
Wlodawer et al., 1989).
Crystals of chymosin obtained by Bunn et al. (1971)
showed that the space group was either I222 or I212121,
with one molecule in the asymmetric unit. The structure
of recombinant bovine chymosin has been independ-
ently solved and refined at 2.3 Å resolution (Gilliland
et al., 1990) and at 2.2 Å resolution (Newman et al.,
1991). Crystallographic studies at 2.0 Å resolution have
also been performed on a site-specific mutant of chy-
mosin, in which V111 was replaced by phenylalanine,
and the structure has been refined to an R-factor of
19.5% (Strop et al., 1990). All the molecules display very
similar secondary and tertiary structures.
solved by Gilliland et al. (1990) and by Newman et al.
(1991). The crystals of chymosin have the space group
of 1222 with approximate overall dimensions of 40 
60  65 Å (Gilliland et al., 1990). The protein has a
bilobal folding pattern formed by the N- and the
C-terminal domains divided by a deep active-site cleft.
A 2.5-Å extended cleft contains the catalytic aspartates
and the substrate-binding pockets. These two lobes are
related by an approximately 2-fold axis which passes
between the two catalytic aspartate residues, 32 and
215, and forms the approximate intra-molecular sym-
metry (Fig. 7). High symmetry between the N- and the
C-lobes is found inside the active site and the core of
the enzyme (Newman et al., 1991). Intra-domain
pseudo-diad axes in the N- and the C-domains of chy-
mosin have rotations of 180° and 177°, respectively,
with negligible translations (Newman et al., 1991).
There are three disulphide bridges at positions 45 . . .
50, 206 . . . 210 and 249 . . . 282. In addition, ion-pairs
are found between R59 . . . D57, R157 . . . E308, R157 . . .
I326 (COO ), R307 . . . D11 and R315 . . . D138 (Gilliland
et al., 1990; Newman et al., 1991). Chymosin also con-
tains a single cis-proline, P23, on the -turn connecting
strand bN to cN (Gilliland et al., 1990; Newman et al.,
1991). In rhizomucor protease, cryphonectria protease
and porcine pepsin, a cis-proline is found at an identical
position to that in chymosin (Blundell et al., 1990;
Cooper et al., 1990; Newman et al., 1993) while two
cis-proline residues are found at positions 23 and 324 in
rhizopus protease (Suguna et al., 1987) and three cis-
proline residues are found at positions 111, 194 and 297
in human renin (Dhanaraj et al., 1992).
The active site of aspartic proteinases is highly con-
served and consists of residues, Asp9Thr9Gly, from
each domain of the enzyme. Nine per cent sequence
identity is observed between the N- and the C-terminal
lobes of chymosin (Newman et al., 1991).
A comparison of chymosin structure with that of
other aspartic proteinases reveals a high degree of struc-
tural homology (Gilliand et al., 1990). Chymosin has
the closest structural agreement with porcine pepsin. Of
the fungal proteinases, the rhizopus protease molecule
has higher structural homology with chymosin than
with penicillopepsin or cryphonectria protease.
The structural superposition of aspartic proteinases
reveals that the N-terminal domain has greater structural
similarity than the C-terminal domain (Gilliland et al.,
1990). The C-terminal domain is more separated from
the rest of the molecule than the N-terminal domain,
and the rigid body movement appears in the C-terminal
domain (residues 190–302) (Sali et al., 1992). The great-
est differences between these proteinases are in the
surface loop regions. One remarkable difference is the
24 Rennets: General and Molecular Aspects

Propeptide
Fungi
A. awamori APR1 APAPRTRKGFTINQIARPANKTRTINLPGMYARS-------LA-KFGGTVPQSVKEA-A*SK
A. oryzae APR2 LPTGPSHSPHARRGFTINQITRQTARVGPKTASFPAIYSRALA-KYGGTVPAKLKSAVA*GH
A. miehei APR3 RPVSKQSESKDKLLALPLTSVSRKFSQTKFGQQQ-------LAEKLAG-----LKPFSE*AA
M. pusillus APR4 RPVSKQSDADDKLLALPLTSVNRKYSQTKHGQQ--------AAEKLGG-----IK-A-F*AE
R. niveus APR5 PNGKKINIPLAKNN----SY-KPSA--KNALNKA------LA-KYNRRKVGSGGITTE*AS
R. chinensis APR6 PGEKKISIPLAKNP----NY-KPSA--KNAIQKA------IA-KYNKHKINTSTGGIV*AG
Mammals
Bovine prochymosin7 AEITRIPLYKGKSLRKAL-KEHGLLE-DFLQKQQYG-ISSKYS-------GF*GE
Lamb prochymosin8 AEITRIPLYKGKPLRKAL-KERGLLE-DFLQKQQYG-ISSEYS-------GF*GE
Chicken pepsinogen9 SIHRVPLKK GKSLRKQL-KDHGLLE-DFLKKHPYN-PASKYHPV------L*TA
Porcine pepsinogen10 LVKVPLVRKKSLRQNLIKD-GKLK-DFLKTHKHN-PASKYFPE---AAAL*IG
Human pepsinogen11 IMYKVPLIRKKSLRRTL-SERGLLK-DFLKKHNLN-PARKYFPQWE-APTL*VD
Human progastricsin12 AVVKVPLKKFKSIRETM-KEKGLLG-EFLRTHKYD-PASKYRFGD-----L*SV
Mouse prorenin13 TFSLPTRTATFERIPLKKMPSVREIL-EERG--V-DMIRLSAEWGVFTK----------R*PS
Human prorenin14 TFGLPTDTTTFKRIFLKRMPSIRESL-KERG--V-DMARLGPEWSQPMK----------R*LT
Human procathepsin D15 SALVRIPLHKFTSIRRTM-SEVGGSVEDLIAK----GPVSKYSQAV-PAVTE*GP
Rat procathepsin D16 SALIRIPLRKFTSIRRTM-TEVGGSVGDLI----LKGPITKYSMQSSPRTKE*PV
Yeast
S. cerevisiae APR17 KVHKAKIYKHELSDEMKEVTFEQHLAHLGQKYLTQFEKANPEVVFSREHPFFTE*GG
C. tropicalis APR18 LAFALFAQGLTIPD-----GIEKRTDKVVSLDFTVIRKPFNATAHR---LIQKR*SD
Plant
Barley APR19 EGLVRIALKKRP-IDRNSRVATGLSGGEEQP---LLSG------AN---PLR*SE

Figure 3 Alignment of the propeptides of aspartic proteinases. The junctions between proenzyme and mature enzyme are indi-
cated by (*). References: (1) Berka et al. (1990); (2) Ward and Kodama (1991); (3) Gray et al. (1986) and Boel et al. (1986); (4)
Tonouchi et al. (1986); (5) Horiuchi et al. (1988); (6) Chen et al. (1991); (7) Harris et al. (1982) and Moir et al. (1982); (8) Pungercar
et al. (1991); (9) Baudys and Kostka (1983); (10) Lin et al. (1989); (11) Sogawa et al. (1983); (12) Wong and Tang (1986); (13) Holm
et al. (1984); (14) Imai et al. (1983); (15) Faust et al. (1985); (16) Birch and Loh (1990); (17) Ammerer et al. (1986); (18) Togni et al.
(1991); (19) Runeberg-Roos et al. (1991) (adapted from Orprayoon, 1994).

position of the flap (residues 73–85 in chymosin). This
region participates in the substrate-binding specificity. In
chymosin, the position of Y77 is stabilized by interaction
with hydrophobic residues F119 and L32 (Gilliland et al.,
1990). In other aspartic proteinases, Y77 hydrogen-
bonds to W39. In pepsin, the location of the hydroxyl
group of Y77 is occupied by a water molecule, w424, in
the chymosin crystal. This water molecule forms two
hydrogen bonds with the hydroxyl group of Y75 and
with the conserved water molecule, w403. In the V111F
mutant chymosin, the flap appears to occupy two differ-
ent conformations corresponding to that found in native
chymosin and pepsin (Strop et al., 1990). This suggests
that chymosin can exist in two alternative structural
forms: the active form in which S1 and S3 binding pock-
ets are free for substrate binding and the self-inhibited
form in which these pockets are occluded by its own Y77
residue (Andreeva et al., 1992; Gustchina et al., 1996).
The structure of S. cerevisiae proteinase A agrees overall
with other uninhibited aspartic proteinases, although
the conformation of Y75 occupying the S1 substrate-
binding pocket is similar to that in chymosin, suggest-
ing a functional significance for this conformation
(Gustchina et al., 2002). The conversion of chymosin
from the self-inhibited to the active form can be pro-
moted by an allosteric activator, the histidine–proline
cluster (9His9Pro9His9Pro9His9) of -casein,
thereby explaining the catalytic specificity of chymosin
towards -casein.
Three-dimensional structures of homodimer retroviral
proteinases are to a large extent similar and bear close
resemblance to the structure of bilobal fungal and mam-
malian aspartic proteinases (Lapatto et al., 1989; Miller
et al., 1989; Navia et al., 1989; Wlodawer et al., 1989).
The structural superpositions between the retroviral
enzymes and the eukaryotic aspartic proteinases appear
to be similar. It is not clear whether the eukaryotic pro-
teinases are derived from a homodimer enzyme by gene
duplication and fusion (Tang et al., 1978) or evolved
from a cellular gene by one or more deletion events (Rao
et al., 1991). Nevertheless, an engineered homodimer of
the pepsin N-terminal lobe, which exhibits a general pro-
teolytic activity, reveals the close relationship between
these two aspartic proteinase families (Lin et al., 1992).
The active site
The active-site aspartates, D32 and D215, are situated on
the corners of the two extended loops (-structures
 Rennets: General and Molecular Aspects 25

10 20 30 40 50
4CMS GEVASVPLTNY-LDSQYFGKIYLGTPPNEFTVLFDTGSSDFWVPSIYCKSNAC-KNHQR
4PEP IGDEPLENY-LDTEYFGTIGIGTPAQDFTVIFDTGSSNLWVPSVYCSSLAC-TNHNL
2APP AASGVATNTPTANDIEEYIPVTIG--GTTLNLNFDTGSSDLWVFSTELP-ASQQSGHSV
2APR AGVGTVPMTDYGNDIEYYGQVTIGTPGKKFNLDFDTGSSDLWIASTLCT--NCGSGQTK
4APE STYSATTTPIDSLDDAYITPVQIGTPAQTLNLDFDTGSSDLWVFSSETTASE-VDGQTI
YPE GGH-DVPLTNYLNA-QYYTDITLGTPPQNFKVILDTGSSNLWVPSNECGSLAC-FLHSK

Strand a´ Strand b´ Strand c´

60 70 80 90 100 110
4CMS FDPRKSSTFQNL-GKPLSIHYGT-GSMQGILGYDTVTVSNIVDIQQTVGLSTQEPGDVFTY
4PEP FNPQDSSTYQST-SGELSITYGT-GSMTGILGYDTVQVGGISDTNQIFGLSETEPGSFLYY
2APP YNPSA--TGKELSGYTWSISYGDGSSASGNVFTDSVTVGGVTAHGQAVQAAQQISAQFQQD
2APR YDPNQSSTYQAD-GRTWSISYGDGSSASGILAKDNVNLGGLLIKGQTIELAKREAASFASG
4APE YTPSKSTSTKLLSGATWSISYGDGSSSSSDVYTDTVSVGGLTVTGQAVESAKKVSSSFTED
YPE YDHEASSSYKAN-GTEFAIQYGTG-SLEGYISQDTLSIGDLTIPKQDFAEATSEPGLTFAF

Strand d´ Strand q Strand r

120 130 140 150 160
4CMS AEFDGILGMAYPSLASE---YSIPVFDNMMNRHLVAQDLFSVYMDRNG----QESMLTLG
4PEP APFDGILGLAYPSISAS---GATPVFDNLWDQGLVSQDLFSVYLSSNG---DSGSVVLLG
2APP TNNDGLLGLAFSSINTVQPQSQTTFFDTVKSS-L-AQPLFAVALKHQ-----QPGVYDFG
2APR -PNDGLLGLGFDTITTVR--GVKTPMDNLISQGLISRPIFGVYLGKAKN--GGGGEYIFG
4APE STIDGLLGLAFSTLNTVSPTSQQTFFDNAKA-S-LDSPVFTADLGY-----HAPGTYNFG
YPA GKFDGILGLGYDTISVD---KVVPPFYNAIQQDLLDEKRFAFYLGDTSKDTENGGEATFG

Strand a Strand b Strand c Strand d

170 180 190 200 210 220
4CMS AIDPSYYTGSLHWVPVTV-QQYWQFTVDSVTISGVVVACEGGCQAILDTGTSKLVGPSSD
4PEP GIDSSYYTGSLNWVPVSV-EGYWQITLDSITMDGETIACSGGCQAIVDTGTSLLTGPTSA
2APP FIDSSKYTGSLTYTGVDNSQGFWSFNVDSYTAGSQ-SG-DG-FSGIADTGTTLLLDDSVV
2APR GYDSTKFKGSLTTVPIDNSRGWWGITVDRATVGTSTVA-SS-FDGILDTGTTLLILPNNI
4APE FIDTTAYTGGITYTAVSTLQHFWEWTSTGYAVGSGTFKSTS-IDGIADTGTTLLYLPATV
YPA GIDESKFKGDITWLPVRRK-AYWEVKFEGIGLGDEYAELES-HGAAIDTGTSLITLPSGL

Strand a´ Strand b´ Strand c´

230 240 250 260 270 280
4CMS ILNIQQAI-GATQNQ-YGEFDIDCDNLSYMPTVVFEINGKMYPLTPSAYTSQD---QGFC
4PEP IANIQADI-GASENS-DGEMVISCSSIDSLPDIVFTIDGVQYPLSPSAYILQD---DDSC
2APP VSQYYSQVSGAQQDSNAGGYVFDCST--NLPDFSVSISGYTATVPGSLINYGPSGDGSTC
2APR AASVARAY-GASDNS-DGTYTISCDT-SAFKPLVFSINGASFQVSPDSLVFEEF--QGQC
4APE VSAYWAQVSGAKSSSS-VGYVFPCSAT--LPSFTFGVGSARIVIPGDYIDFGPISTGSSC
YPA AEMINAEI-GAKKGW-TGQYTLDCNTRDNLPDLIFNFNGYNFTIGPYDYTLEV---SGSC

Strand d´ Strand q Strand r

290 300 310 320
4CMS TSGFQSENHS----QKWILGDVFIREYYSVFDRANNLVGLAKAI
4PEP TSGFEGMDVPTSSGELWILGDVFIRQYYTVFDRANNKVGLAPVA
2APP LGGIQSNSGI----GFSIFGDIFLKSQYVV
FDSDGPQLGFAPQA
2APR IAGFGYG-NW----GFAIIGDTFLKNNYVVFNQGVPEVQIAPVAE
4APE FGGIQSSAGIG----INIFGDVALKAAFVVFNGATTPTLGFASK
YPA ISAITPMDFPEPVGPLAIVGAFLRKYYSIYDLGNNAVGLAKAI

Figure 4 The sequence alignment of calf chymosin (4CMS, Newman et al., 1991) with other aspartic proteinases based on three-
dimensional structures. References: 4PEP: porcine pepsin (Sielecki et al., 1990); 2APP: penicillopepsin (James and Sielecki, 1983);
2APR: rhizopuspepsin (Suguna et al., 1987); 4APE: endothiapepsin (Pearl and Blundell, 1984); YPA: S. cerevisiae proteinase A
(Dreyer et al., 1986) (adapted from Orprayoon, 1994).

within sheets cNdN and cCdC) in the N- and the
C-terminal domains. The side chains of these two aspar-
tates are oriented towards each other around the
pseudo-interlobe diad axis in a complicated hydrogen-
bonding network, known as the ‘fireman’s grip’ (Pearl
and Blundell, 1984) shown in Fig. 8. This network is
formed by the interaction of two loops (residues 31–35
and residues 214–218) and a central water molecule.
The side chain of T33 and its symmetry-related T126 form
hydrogen bonds across the diad axis to the carbonyl
oxygens of L214 and F31, respectively, and to the peptide
N atoms of T216 and T33, respectively. The carboxyl
26 Rennets: General and Molecular Aspects

Figure 5 A schematic diagram of the secondary structure of chymosin. The directions of the strands are indicated by the large
arrows. The inter- and intra-lobe two-fold axes are shown as large and small diad markers. The main hydrogen bonds are indicated
by arrows in the direction of donor to acceptor (adapted from Newman et al., 1991).

oxygens of D32 and D215 are hydrogen-bonded with
nitrogen atoms of the conserved G34 and G217, respect-
ively. In addition, the side chains of S35 and T218
also form hydrogen bonds with the outer oxygen atoms
of D32 and D215, respectively. There are some con-
served glycine residues in eukaryotic aspartic proteinases
which are believed to be important; among them, G34
and G217 are conserved in all aspartic proteinases. Side
chains at these positions would interfere sterically with
the catalytic aspartates. Residue D303 is conserved
among all proteinases with an acidic pH optimum.
However, in renins, which have a more neutral pH opti-
mum, this residue is replaced by an alanine. The effect
of the side chain at this position on the pKa has
 Rennets: General and Molecular Aspects 27

Figure 6 Three-dimensional structures of aspartic proteinases showing the high degree of structure homology among these pro-
teinases (adapted from Pitts et al., 1992).

been revealed by site-directed mutagenesis of renin
(Yamauchi et al., 1988) and chymosin (Mantafounis
and Pitts, 1990). The hydrogen bond between D303 and
T216 may affect the pKa of D215 via the peptide dipole of
T216–G217 (Pearl and Blundell, 1984).
Catalytic mechanisms
The catalytic mechanism of aspartic proteinases has
been modelled based on the structural analysis of
several aspartic proteinase–inhibitor complexes. Early
mechanisms (James et al., 1977, 1982; James and
28 Rennets: General and Molecular Aspects
Figure 7 A plot of the C position of chymosin. The approx-
imate molecular symmetry axes are shown as follows: (i) the
inter-lobe non-crystallographic 2-fold screw axis relating the
N- and C-terminal lobes; (ii) the intra-domain axis for the N-terminal
domain; (iii) the intra-domain axis for the C-terminal domain
(adapted from Newman et al., 1991).
Sielecki, 1985) proposed that catalysis was initiated by
protonation of the carbonyl oxygen of the substrate by
a proton from D215, followed by nucleophilic attack on
the carbonyl carbon of the substrate aspartate residue
by a hydroxide ion generated from water after dona-
tion of its proton to D32. These protonation events
lead to the formation of the tetrahedral intermediate.
The breakdown of the intermediate is generated by
Figure 8 The ‘fireman’s grip’ at the active site of chymosin. Hydrogen bonds (broken lines) involved are: T216N . . . T33O1 (2.8 Å),
T33O1 . . . K214O (2.7 Å), T33N . . . T216O1 (2.9 Å) and T216O1 . . . F31O (2.8 Å). Other hydrogen bonds contributing to the stability
of D32 and D215 are also shown (adapted from Newman et al., 1991).
protonation of the nitrogen atom either from bulk sol-
vent or from the catalytic carboxyl group of D215. Pro-
tonation of the substrate carbonyl and nucleophilic
attack may appear simultaneously during the forma-
tion of the tetrahedral intermediate. Similarly, proton
transfer from the intermediate to the diad may occur
at the same time as the protonation of the nitrogen
atom of the substrate during the cleavage of the result-
ant intermediate (Polgár, 1987). Pearl (1987) sug-
gested that the distortion of the scissile bond towards
the enzyme–substrate binding may facilitate the col-
lapse of the intermediate by generating lone pair
orbitals antiperiplanar to the C9N bond but not to
the hydroxyl C9O bond. Therefore, the leaving prod-
uct is a free amine rather than the original nucle-
ophile. In addition, the charged oxygen of a solvent
molecule forms hydrogen bonds with residues D32 and
S35 (Suguna et al., 1987) or residues G76, D77 or Y75
on the flap (Blundell et al., 1987; Pearl, 1987).
Veerapandian et al. (1990) have proposed the cata-
lytic mechanistic model outlined in Fig. 9. The pro-
R(statine-like) hydroxyl of the tetrahedral carbonyl
hydrate is hydrogen-bonded to the outer oxygen of D32
and D215. The second hydroxyl oxygen of the hydrate is
hydrogen-bonded only to the carboxyl oxygen of D32.
The scissile bond carbonyl is protoned by D32 and is
simultaneously attacked by a water molecule polarized
into a nucleophilic state by D215. The rigid movement in
the enzyme–substrate complex may impel distortion of
the amide bond and facilitate the attack of nucleophilic
water on the polarized carbonyl. Thus, in the tetrahedral
intermediate I, the negatively charged D31 is stabilized
by extensive hydrogen bonding. The amide nitrogen will
have been pyramidalized with the new arrangement,

Figure 9 A proposed catalytic mechanism for aspartic proteinases (Veerapandian et al., 1990).
favouring protonation. A proton can be transferred from
bulk solvent or from D215. A similar mechanistic pro-
posal has been described by James et al. (1992).
Pepsin and chymosin have been shown to catalyse
peptide synthesis (Fruton, 1982; Jakubke, 1987; Abdel
Malak, 1992). Formation of a peptide is catalysed by
chymosin optimally at pH 4–5 which is similar for
peptide hydrolysis (Abdel Malak, 1992). The pH opti-
mum for peptide synthesis catalysed by pepsin is fur-
ther from that for peptide hydrolysis. The catalytic
ability of the enzyme is sensitive to the amino acid
residues flanking the bond to be formed or hydrolysed
as well as the nature of adjacent amino acid residues.
Zymogen activation
The structure of porcine pepsinogen has been refined
at high resolution (James and Sielecki, 1986; Sielecki
et al., 1991; Hartsuck et al., 1992). Structural compar-
isons between pepsin and pepsinogen suggest that the
enzyme and proenzyme structures are very similar.
Most of the differences occur in the proximity of the
cleft which, in pepsinogen, is covered and filled by the
pro-part (1P–44P) and the first 13 residues of pepsin.
Rennets: General and Molecular Aspects 29
The extension of 13 residues adopts completely differ-
ent conformations in the active and the zymogen
forms (James and Sielecki, 1986).
The secondary structure of the zymogen consists
mainly of -sheet, with an approximate 2-fold axis of
symmetry ( James and Sielecki, 1986). The activation
peptide packs into the active site cleft, and the
N-terminus (2P–9P) occupies the position of the mature
N-terminus (2–9) since the first ten amino acids of the
pro-part form -strand aN of pepsinogen. Therefore,
changes upon activation include excision of the acti-
vation peptide and proper relocation of the mature
N-terminus. At neutral or alkaline pH, the pro-segment
of pepsin binds and is stabilized across the active site
between the two lobes by electrostatic, hydrogen-
bonding and hydrophobic interactions which con-
tribute to the binding between the pro-segment and
the rest of the protein (Sielecki et al., 1991). Lowering
of pH protonates acidic residues on the mature
enzyme portion of the molecule, thereby disrupting
favourable electrostatic interactions with positively
charged amino acid residues on the pro-segment. Sub-
sequent conformational change of the zymogen leads
to intramolecular proteolytic cleavage that liberates
30 Rennets: General and Molecular Aspects
the pro-segment from the zymogen (McPhile, 1972;
Nielsen and Foltmann, 1993).
The mechanisms of activation of zymogens of the
aspartic proteinase are different and depend on the
pH. At pH below 2.5, conversion of pepsinogen is
primarily by an intramolecular mechanism. The
propeptide is cleaved monomolecularly at position
M16P–E17P, resulting in an active pseudo-enzyme
which is enzymatically active and may form a com-
plex with the released pro-segment. At pH values
below 4.0, the L44P9I1 bond is not susceptible to
proteolytic cleavage but becomes susceptible at
higher pH. At low pH, the cleavage sites differ among
the aspartic proteinases – F27P9L28P for calf prochy-
mosin, human progastricsin and chicken pepsinogen,
M16P9E17P for porcine pepsinogen B and L26P9I27P
for procathepsin D (Barkholt and Foltmann, 1975;
Barkholt et al., 1979; Truk et al., 1985; Foltmann,
1993; Larsen et al., 1993). Removal of the entire pro-
peptide predominately occurs at pH 3–4 through an
intermolecular mechanism. It has been suggested
that cleavage of the F42P9G1 bond of prochymosin is
faster at pH 2 than at pH 4.5 (Barkholt et al., 1979).
The recombinant pepsinogen originally from Rhizo-
pus and produced in E. coli can convert to the active
enzyme in an acidic medium by a similar mechanism
as for pepsinogen (Chen et al., 1991). The pseudo-
rhizopus protease and rhizopus protease are generated
by the cleavage at N38P9T39P and V45P9A1, respect-
ively (p  prochymosin). Moore et al. (1995) have
studied the crystal and the molecular structures of
human progastricsin at 1.62 Å resolution and suggest
that human progastricsin has a conformational struc-
ture and mechanism of activation analogous to those
for pepsinogen.
Site-directed mutagenesis at the two sites for autopro-
teolysis of prochymosin suggests that these processing
sites can function independent of one another (McCa-
man and Cummings, 1986, 1988). Changing the prochy-
mosin sequence from F27P9L28P9Q29P9K30P9Q31P to
F27P9P28P9R29P9Q30P9Q31P resulted in the partially
activated zymogen at pH 2, while at pH 4.5, normal acti-
vation processing and proteolytic processing occurred
(McCaman and Cummings, 1986). Conversely, when the
seven residues including the processing site at pH 4.5
were removed, a new cleavage site (S37P9V38P) was gen-
erated at pH 4.5 while the processing site at pH 2 was
not affected (McCaman and Cummings, 1988).
The activation reactions are dependent on pH, salt
concentration and temperature. At pH 5 and ⬃20 °C,
activation is completed in two or three days (Rand and
Ernstrom, 1964), while at pH 2, ⬃20 °C and an ionic
strength of 0.1, activation is completed in 5–10 min
(Foltmann, 1962). However, autoproteolysis alone may
not be able to generate the mature form of the enzymes
as shown in procathepsin D, which cannot autoactivate
to the mature enzyme at acidic pH (Larsen et al., 1993).
Prochymosin is also activated by proteolytic enzymes,
including plasmin, Legionella pneumophila metallopro-
teinase and Aspergillus oryzae thermolysin (Stepanov
et al., 1990).
Position 36p in the propeptides of gastric aspartic
proteinases is generally occupied by lysine or arginine.
This has led to the conclusion that a basic residue at
this position, which interacts with the active-site aspar-
tates, is essential for folding and activation of the
zymogen. Lamb prochymosin has been shown by
cDNA cloning to possess glutamic acid at position 36p.
To investigate the effect of this natural mutation which
appears to contradict the proposed role of this residue,
calf and lamb prochymosins and their two reciprocal
mutants, K36pE and E36pK, respectively, were expressed
in E. coli, refolded in vitro and autoactivated at pH
2 and 4.7 (Francky et al., 2001). All four zymogens
could be activated to active chymosin and, at both pH
values, the two proteins with E36p showed higher acti-
vation rates than the two K36p forms. E36p was also
demonstrated in natural prochymosin isolated from the
fourth stomach of lamb, as well as being encoded in
the genomes of sheep, goat and mouflon, which belong
to the subfamily Caprinae. A conserved basic residue at
position 36p of prochymosin is thus not obligatory for
its folding or autocatalytic activation. The apparently
contradictory results for porcine pepsinogen A (Richter
et al., 1999) can be reconciled with those for prochy-
mosin. K/R36p is involved in stabilizing the propep-
tide–enzyme interaction, along with residues nearer the
N-terminus of the propeptide, the sequence of which
varies between species. The relative contribution of
residue 36p to stability differs between pepsinogen and
prochymosin, being larger in the former (Francky
et al., 2001).
B-Crystallin, the small heat shock protein (Plater
et al., 1996; Crabbe and Hepburne-Scott, 2001; Derham
et al., 2001) can form a complex with prochymosin.
After activation, once chymosin is recovered without
bound B-crystallin, the yield of activity is increased
(Chitpinityol et al., 1998b).
Substrate-binding pockets and specificity
A high concentration of NaCl or (NH4)2SO4
increases the hydrolytic activity of pepsin and retro-
viral proteinases, in addition to broadening their
specificities (Kotler et al., 1989; Tropea et al., 1992).
Aspartic proteinases have an extended substrate-
binding pocket that can accommodate at least seven
amino acid residues. Detailed structural studies of
 Rennets: General and Molecular Aspects 31

aspartic proteinase–inhibitor complexes have been
used to identify the amino acid residues in each sub-
site (Bott et al., 1982; Andreeva et al., 1984; James
et al., 1985; Blundell et al., 1987; Cooper et al.,
1987; Foundling et al., 1987; James and Sielecki,
1987; Suguna et al., 1987). In chymosin, the subsites
S1 and S1 are shallow pockets within the active site
cleft. The S1 subsite (for binding of F105) has greater
specificity than S1 , and is blocked by Y75 (Gilliland
et al., 1990). Therefore, a significant movement of
the flap is essential to allow binding of the substrate.
The S1 is quite hydrophobic compared to S1 in
which an additional charged residue, E290, is near to
the -casein M106 side chain. The S2 pocket has low
specificity and allows the peptide side-chains to
adopt a range of conformations whereas at subsites
S1 and S3, the conformation of the side chains is
strongly restricted (Dhanaraj et al., 1992). Chymosin
residues involved in the interactions with the corres-
ponding residues of the substrate are shown
together with a sequence identical to that of the
-casein cleavage site in Table 1. There are two differ-
ences in the S1 subsites of chymosin and the fungal
proteinases that promote more hydrophobic S1
subsites (Gilliland et al., 1990). The first is the posi-
tion of the flap region which is due to the reorienta-
tion of Y75 and a deletion of one amino acid residue
in this loop. Another difference is the substitution
of L30 in chymosin for the D30 or the N30 of the
rhizopus protease and penicillopepsin, respectively.
In human cathepsin E, the important specificity-
determining interactions are found in the S3 (E13)
and S2 (T222, E287, L289, I300) subsites (Raonaik
et al., 1995). Figure 10 summarizes the results of
cleavage of the B chain of oxidized insulin by chy-
mosin and some related acid proteinases. In chy-
mosin, the S1 subsite has favourable interactions
with aromatic amino acids at P1 whereas the S1 sub-
site is less specific (Bang-Jensen et al., 1964; Folt-
mann, 1964; Guillou et al., 1991; Nedjar et al.,
1991).
The fungal and yeast proteinases have an S1 subsite
with a deeper pocket and broader specificity. There-
fore, the S1 pocket can accomodate lysine as well as
hydrophobic residues at P1 (Oka et al., 1973; Hofmann
et al., 1984; Newman et al., 1993). However, in rhi-
zomucor protease, specificity for lysine at P1 was not
observed due to the absence of polar residues at posi-
tions 30 and 111. In retroviral aspartic proteinases, the
primary specificities for HIV-1 and HIV-2 aspartic pro-
teinases at P1 are L, M, Y and F, and at P1 are P, M, F
and A (Poorman et al., 1991).
Among the isozymes of chymosin, chymosin A has
a significantly higher specific activity than chymosin B
(Foltmann, 1960) which may be the result of the
enhanced binding affinity of -casein through, possi-
bly, the stronger electrostatic interactions between the
substrate and chymosin A. In addition, these two
isozymes have different pH optima, 4.2 and 3.7 for
chymosin A and B, respectively. These different values
may be the result of an extensive hydrogen-bonding
network near the two catalytic aspartates. The opti-
mum pH for proteolysis by aspartic proteinases
depends upon the species from which the enzyme is
produced, and the substrate used (Table 2). HIV-1
proteinase and renin have a high pH optimum among
aspartic proteinases. The residues S35, T218 and D303
have been postulated to play a role in the pH profile of
aspartic proteinases. In vitro mutagenesis of A35S of
HIV-1 proteinase (A28S in HIV-1 numbering) showed a
lowering of pKa2 (compared to wild type) by 1.2 units
but no effect was found in the pKa1 value (Ido et al.,
1991). In contrast, mutation of S35A in porcine pepsin
lowered pKa1 and pKa2 but raised it for rhizopuspro-
tease. Site-directed mutagenesis of T218A in porcine
pepsin, chymosin and rhizopus protease shifted the
pH optimum by 0.2–0.5 units (Mantafounis and Pitts,
1990; Tang et al., 1992). Mutation of A303D in renin
lowered the pH optimum by 0.5 units (Yamauchi
et al., 1988). Similarly, mutation of D303A in chymosin
raised the optimum pH by 0.6 units (Mantafounis
and Pitts, 1990). The double mutations, T218A/D303A

Table 1 The substrate-binding pockets of chymosin. Chymosin residues involved in the interactions with the corresponding residues
of the substrate are shown together with residues at the -casein cleavage site (adapted from Gilliland et al., 1990; Newman et al.,
1991)

Subsite -Casein residues Chymosin residues

S4 His102 Ser219, Lys220, Gln288

S3 Leu103 Ser12, Gln13, Tyr75, Phe117, Gly217, Thr218, Ser219
S2 Ser104 Gly76, Thr77, Gly217, Thr218, Lys220
S1 Phe105 Leu30, Asp32, Gly34, Tyr75, Gly76, Phe117, Ile120, Asp215, Gly217, Thr219
S1 Met106 Gly34, Tyr189, Asp215, Thr218, Glu289, Ile301
S2 Ala107 Gly34, Ser35, Tyr189
S3 Ile108 Tyr189
32 Rennets: General and Molecular Aspects

1 10 20 30
F V N Q H L C G S H L V E A L Y L V C G E R G F F Y T P K A
Chymosin1 ↑ ↑ ⇑ ↑ ↑ ↑ ⇑ ↑ ↑
Pepsin2 ↑ ⇑ ⇑ ↑ ↑ ↑ ⇑ ↑ ⇑ ⇑
Rhizopuspepsin3 ↑ ↑ ⇑ ↑ ↑ ⇑ ⇑ ↑ ↑ ⇑ ⇑
Penicillopepsin3 ↑ ↑ ⇑ ↑ ↑ ↑ ↑ ⇑ ⇑ ⇑ ↑ ⇑ ↑ ⇑ ⇑
Endothiapepsin4 ↑ ↑ ⇑ ↑ ⇑ ⇑ ⇑
Proteinase A5 ⇑ ⇑ ⇑
Cathepsin E6 ↑ ⇑ ⇑ ↑ ⇑ ⇑ ↑ ⇑ ⇑
R. miehei APR7 ⇑ ⇑ ⇑ ⇑ ⇑ ↑
R. pusillus APR8 ⇑ ⇑ ⇑ ⇑ ⇑

Figure 10 Comparison of the cleavage specificity of chymosin towards the B-chain of oxidized insulin with those of some other
aspartic proteinases. References: (1) Foltman (1964); (2) Sanger and Tuppy (1981); (3) Oka et al. (1973); (4) William et al. (1972); (5)
Takahashi (1995); (6) Athauda et al. (1991) (pH 3.0); (7) Rickert (1971); (8) McCullough and Whitaker (1971). Legends: (y) Main
cleavage site and (q) other sites of action.

in chymosin affected the pH optimum similarly to that
of D303A mutatagenesis (Pitts et al., 1993).
The substrate specificity of aspartic proteinases is
affected by the operating pH and the presence of salts
(Kotler et al., 1989; Athauda et al., 1991; Tropea et al.,
1992). The pH dependence of hydrolysis of synthetic
substrates demonstrates that secondary specificity
occurs at subsite S3 of mammalian aspartic proteinases
whereas lower specificity is found in microbial pro-
teinases (Dunn et al., 1986). In chymosin, isoleucine or
valine is favoured at P3, and tyrosine, valine or serine
at P2 (Guillou et al., 1991). The favourable interaction
between K220 (NH3) of chymosin and glutamate
(COO ) in P2 of the substrate is suggested to cause the
effects of pH on hydrolysis (Dunn et al., 1987). The
specificity at P2 towards both K220 and Q288 has been
determined by in vitro mutagenesis (Suzuki et al., 1990;
Quinn et al., 1991).
We have studied the effect of replacing threonine 77
of chymosin by aspartate (mutant T77D), as well as the
addition of two residues (9H9G) (mutant PC  2) to
the C-terminus of the protein, on the activity of
the enzyme on a synthetic hexapeptide, L9S9
F(NO2)9NI9A9L9OMe, as substrate (Chitpinityol
et al., 1996, 1998a). For the recombinant wild type, the
optimum pH was 3.7, similar to that reported for the
authentic chymosin B using the same substrate (Martin
et al., 1980). The PC  2 mutant had an optimum pH

Table 2 pH optimum for general proteolysis by chymosin and other aspartic proteinases

Optimum
Enzymes Substrates pH References

Chymosin Acid-denatured haemoglobin 3.7 Berridge (1945); Fish

(1957)
Bovine serum albumin 3.4 Foltmann (1959a)
Oxidized B-chain of insulin 3.5 Fish (1957)
-,-Caseins 4.5 Lindqvist and Storgads
(1960)
Milk-clotting activity 6–6.3 Okigbo et al. (1985a)
Synthetic peptides 4.7 Raymond et al. (1972)
-Casein 5.5 van Hooydonk et al. (1984)
H9P9H9P9H9L9S9F9M9A9I9P9P9K9K 5.4 Visser et al. (1976, 1987)
Pepsin Oxidized B-chain of insulin 2.0 Fish (1957)
Penicillopepsin Trypsinogen 3–4 Hofmann and Shaw (1964)
Endothiapepsin Haemoglobin 2–2.5 William et al. (1972)
Rhizomucorpepsin Haemoglobin 4.0 Arima et al. (1970)
-Casein 4.5 Arima et al. (1970)
Milk clotting 5.5 Arima et al. (1970)
Hammerten casein 3.5 Arima et al. (1970)
z9Phe9Leu9Ala9Ala 3–4 Oka et al. (1973)
S. cerevisiae Acid-denatured haemoglobin 3.2 Dreyer et al. (1986)
proteinase A
A. niger proteinase A Haemoglobin 1.1 Takahashi (1995)

similar to the native enzyme. The optimum pH of T77D
mutant chymosin was shifted towards neutrality by 1 pH
unit, to pH 4.7 from 3.7. The optimum temperature for
the activity of the T77D mutant was increased relative
to the wild-type enzyme, from approximately 45 °C for
the wild type and PC  2 mutant, to 55 °C for the
T77D mutant. These changes may be due to the
increased negative charge at the ‘flap’ region that may
have altered the network hydrogen bonding and influ-
enced the substrate recognition of the enzyme.
Inhibitors
All aspartic proteinases are inhibited by pepstatin, by
the binding of the hydroxyl group of statine to the two
catalytic aspartates (Marciniszyn et al., 1976a,b). The
inhibition constant (Ki) of pepstatin for chymosin
determined at pH 6.0 and 3.2 is 2.2  10 7 mol/l and
3.2  10 8 mol/l, respectively (Powell et al., 1985).
Pepsin and cathepsin also show pH-dependency of the
inhibitory effect (Knight and Barrett, 1976; Baxter
et al., 1990), and psuedochymosin is more sensitive to
pepstatin than chymosin (McCaman et al., 1985).
As pepstatin is relatively ineffective towards calf chy-
mosin, analogue inhibitors have been developed. A series
of inhibitors have been designed by Powell et al. (1985),
including R(CO)NH9L9S9Sta9A9I9P9P9K9K
(R  acyl group) which has a Ki value for chymosin
almost 20-fold better than pepstatin at pH 6.0 and
approximately 10-fold better at pH 3.1 than pepstatin.
Chymosin is inhibited by the pro-part of chicken
pepsinogen (Ki value of 8  10 8 mol/l at pH 5.6) but
not by its own pro-segment (Strop et al., 1990).
Mechanism of Milk Clotting
In milk, the primary soluble proteins are the whey pro-
teins, -lactalbumin and -lactoglobulin. The insoluble
proteins are found in large colloidal particles, called
casein micelles. -Casein is a calcium-insensitive pro-
tein which forms a protective layer around the calcium-
sensitive caseins ( S1-, S2-, - and -), resulting in
stable casein micelles. In the presence of chymosin,
milk clotting occurs in two separate steps.
The first phase starts with the cleavage of -casein
at the F1059M106 bond which results in the release of
a hydrophilic glycopeptide (residues 106–169) that
passes into the whey, and para--casein that remains
in the micelles. para--Casein is positively charged at
neutral pH and causes a decrease of electric repulsive
forces between casein micelles (Green, 1973).
Hydrolysis of other proteins in milk, including S1-,
S2- and -caseins and -lactalbumin monomer, by
chymosin has been reported with a much slower rate
Rennets: General and Molecular Aspects 33
of proteolysis (Carles and Dumas, 1985; Miranda
et al., 1989). The proteolytic action of microbial pro-
teases on -casein has been reported (de Koning,
1967; Yu et al., 1968; Larson and Whitaker, 1970).
Porcine pepsin A and C, and R. miehei proteinase
cleave the same bond as chymosin (F1059M106), but
C. parasitica proteinase cleaves the S1049F105 bond
(Drønse and Foltmann, 1989). Chymosin causes lim-
ited hydrolysis of -casein, with the formation of only
macropeptide and para--casein, while fungal pro-
teinases cause extensive non-specific hydrolysis of
both -casein and para--casein (Shammet et al.,
1992).
Visser et al. (1980) suggested that other residues near
the cleaved bond are also involved in the hydrolytic
reaction. From studies with synthetic peptides, two
additional residues at both sides of the hydrolysable bond
are required for appreciable reaction (Raymond et al.,
1972). The peptide corresponding to residues 98–111
of -casein (H9P9H9P9H9L9S9F9M9A9I9
P9P9K) was found to provide a complete requirement
for hydrolysis (Visser et al., 1987, 1988).
Initially, the stability of the micelle is destroyed by
the action of chymosin. This is followed by a non-
enzymatic secondary phase in which the aggregation of
para--casein and other caseins occurs under the influ-
ence of Ca2 and eventually results in gel formation
(Bringe and Kinsella, 1986a; Merin et al., 1989). The
formation of a clot is Ca2 dependent. The primary
and the secondary phases of milk clotting overlap as
the aggregation of micelles begins before the enzymatic
process is complete (Brown and Collinge, 1986; Bringe
and Kinsella, 1986b).
Several factors influence the milk-clotting process,
including pH, temperature, ionic strength, enzyme con-
centration and salts (Foltmann, 1970; Okigbo et al.,
1985a,b; Bringe and Kinsella, 1986a,b). The reaction is
pH dependent; at high pH (6.6–6.7), the clotting time
and the curd firmness are reduced (Okigbo et al.,
1985a), while at low pH (3–4), the hydrolytic activity
is high and a decrease in curd yield occurs. Generally,
milk clotting is performed at pH 6.3–6.6; only when
direct acidification is used does rennet coagula-
tion occur at a pH value down to 5.6. The rate of milk
clotting increases with temperature as long as the enzyme
is stable (Berridge, 1942). Increasing the tempera-
ture above 30–32 °C or reducing the pH from 6.6 per-
mits flocculation at a lower percentage of -casein
hydrolysis (Dalgleish, 1982). However, the induction of
gel formation at 35 °C requires approximately 65%
hydrolysis of -casein (Carlson et al., 1986). The differ-
ences in milk constituents (both proteins and other
chemicals) as well as the pre-treatment process can
affect the rate of the primary enzymatic stage. The time
34 Rennets: General and Molecular Aspects
taken to coagulate milk decreases with increasing
enzyme concentration, but the formation and firmness
of the gel is not altered (Bringe and Kinsella, 1986a).
Milk-clotting activity is also dependent on the source
of chymosin; for example, porcine chymosin is eight
times more active on porcine milk than on bovine milk;
calf chymosin is only half as active on porcine milk as
on bovine milk and the activity of lamb chymosin is
about 20% higher on ovine milk than on bovine milk
(Foltmann, 1992). Calcium ion concentration affects
milk clotting by forming bridges between micelles to
form the coagulum and minimizes variability arising
from inconsistency in milk composition (Berridge,
1952; Bringe and Kinsella, 1986b). However, Pyne
(1955) reported that other ions, such as strontium,
magnesium and barium, could affect the Ca2 require-
ment for coagulation. Milk clotting is inhibited by
anions (Bringe and Kinsella, 1986b).
While synthetic substrates have been used to reveal
the hydrolytic mechanism of chymosin and other aspar-
tic proteinases (Raymond et al., 1972; Martin et al.,
1980; Visser et al., 1987, 1988), milk clotting is much
more complicated than the hydrolysis of a synthetic
substrate. For cheesemaking, the appropriate enzyme
should have a high ratio of milk-clotting activity to pro-
teolytic activity (C/P) (Dalgleish, 1982). The C/P ratio
of chymosin is higher than for other enzymes; over 2
times higher than rhizomucor protease, 4 times higher
than cryphonectria protease and over 25 times higher
than pepsin, trypsin and papain (Martin et al., 1980;
Yada and Nakai, 1986).
Recombinant Calf Chymosin
Chymosin has been used as the milk-clotting enzyme for
the industrial production of cheese. Several rennet sub-
stitutes have been used, including bovine pepsin (from
adult cows), fungal proteinases and other proteolytic
enzymes. However, they have a much greater level of
non-specific proteolytic activity, and in some cases
higher thermostability that causes more degradation of
milk proteins to peptides, leading to a reduction in yield
and poor flavour development in some types of cheese.
Consequently, there have been numerous attempts to
produce chymosin in micro-organisms.
Prokaryotic expression
The first report of an attempt to produce chymosin in
E. coli was that of Uchiyama et al. (1980). Efforts to
express prochymosin cDNA in E. coli led to intracellu-
lar accumulation of inactive chymosin in the form of
inclusion bodies (Emtage et al., 1983; Nishimori et al.,
1984; McCaman et al., 1985; Kawaguchi et al., 1987;
Chitpinityol et al., 1998a). Generally, chymosin was
synthesized in the form of M-prochymosin or N-terminal
fusion proteins under the control of E. coli lac pro-
moter (Nishimori et al., 1984; McCaman et al., 1985),
trp promoter (Beppu, 1983; Emtage et al., 1983;
Kawaguchi et al., 1984; Marston et al., 1984; Nishimori
et al., 1984), tac promoter (McCaman et al., 1985;
Strop et al., 1990), PR promoter (Caulcott et al.,
1985), pho A promoter (Little et al., 1989) or T7 pro-
moter (Chitpinityol et al., 1998a).
In E. coli expression systems, the recombinant prochy-
mosin was expressed at a high level which resulted in
the accumulation of highly refractive inclusion bodies
(Emtage et al., 1983; Kawaguchi et al., 1984; Shoemaker
et al., 1985). The inclusion bodies produced were up
to 40% of the total cell mass and were organized
in an irregular mass without any obvious membrane-
like boundary, with an average diameter of 0.5–1 m
(Marston et al., 1984; McCaman et al., 1985; Strop et al.,
1990; Kaprálek et al., 1991). The synthesis of prochy-
mosin as intracellular inclusion bodies causes a fragilility
of cell membranes, and the loss of cell respiratory activ-
ity and their ability to multiply (Marston et al., 1985;
Kaprálek et al., 1991). The production of inclusion bod-
ies can be improved by the plasmid construction, plas-
mid stability, host strain, composition of the cultivation
medium and growth temperature (Caulcott et al., 1985;
Kawaguchi et al., 1986, 1987; Kaprálek et al., 1991).
The N-terminal methionine of Met-prochymosin can be
removed together with pro-part during acid activation.
The insoluble form of prochymosin requires denatur-
ing condition (8 mol/l urea or 6 mol/l guanidine HCl)
to solubilize prochymosin, followed by renaturation to
generate correctly folded protein that can be activated
(Shoemaker et al., 1985). The deletion of disulfide
bonds from prochymosin showed that the presence of
disulfide bonds was not reponsible for inclusion body
formation (McCaman, 1989).
Improvements in the production of recombinant chy-
mosin in E. coli have been successively developed by
selection of host strain, the modification of plasmids and
the optimization of cultivation conditions (Kawaguchi
et al., 1986; Kaprálek et al., 1991). It has also been found
that a high yield of active recombinant calf chymosin
can be achieved by optimizing solubilization and renat-
uration conditions (Tichy et al., 1993; Yonezawa et al.,
1993; Chitpinityol et al., 1996; Chitpinityol et al.,
1998a,b). In our experiments (Chitpinityol et al., 1998a),
the recombinant enzyme was refolded by a modified pro-
cedure based on that of Marston et al. (1984). Table 3
shows that the yield of chymosin was maximal
when the urea mixture was diluted 25-fold (0.32 mol/l
final urea concentration). If the solubilization mixture
was diluted over 25-fold, the yield of chymosin was
 Rennets: General and Molecular Aspects 35

Table 3 Effect of dilution ratios on the yield of chymosin recovered by refolding. Washed inclusion pellets (protein concentration,
7.9 mg/ml) were solubilized in 8 mol/l urea buffer, pH 8. The urea mixture was incubated at 25 °C for 1 h before the insoluble mole-
cules were removed by centrifugation. The urea solution was then diluted in a high pH buffer (pH 10.7) for renaturation of prochy-
mosin. The protein concentration was determined by using a BCA Protein Assay Reagent

Dilution ratio of urea mixture Final urea Initial concentration Final amount of activated
in buffer pH 10.7 concentration (M) of protein in buffer (mg/ml) chymosin (mg)

1:10 0.80 0.79 0.46

1:20 0.40 0.39 0.49
1:25 0.32 0.32 0.50
1:30 0.27 0.26 0.40
1:40 0.20 0.20 0.26

dependent on the protein concentration in the alkaline
buffer. Table 4 shows that 0.25 mg/ml protein was opti-
mal under the refolding conditions used. This optimized
procedure improves the yield of recombinant enzyme
nearly three-fold.
Since insoluble proteins require a further refolding
process prior to regaining enzymatic activity, there have
been attempts to produce prochymosin extracellularly.
The N-terminal sequence of prochymosin was fused to
a signal peptide of the outer membrane protein A; this
resulted in cell lysis after induction (Elliott et al.,
1989). Holland et al. (1990) reported that the fusion of
hemolysin Hly A8 signal sequence to the C-terminal of
prochymosin resulted in the production of hybrid pro-
tein up to 25% of the total cell proteins, of which 0.8%
was a soluble hybrid product. An expression system
for the production of soluble porcine pepsinogen A has
been developed by fusing the pepsinogen and the
thioredoxin genes and then expressing the fused prod-
uct in E. coli (Tanaka and Yada, 1996).
Other bacterial expression systems used to produce
prochymosin include Lc. lactis, Bacillus subtilis and L
forms of Proteus mirabilis (Kaprálek et al., 1991; Parente
et al., 1991; Simons et al., 1991). In Lc. lactis, the cDNA
for prochymosin was expressed under the control of
proteinase prtP promoter by fusion with various lengths
of Lc. lactis cell envelope-located protease (Simons et al.,
1991). Under the control of the T5 phage promoter and
the induction of a two-cistron sequence at the 5 region
of the gene, prochymosin was synthesized as insoluble
aggregates in B. subtilis cells but the yield was still low
(Parente et al., 1991). The extracellular production of
prochymosin in B. subtilis can be achieved by fusing the
prochymosin gene to the B. subtilis subtilisin signal
sequence and production reached up to 100 mg/l (Par-
ente et al., 1991). Using L forms of a P. mirabilis expres-
sion system, the fusion of prochymosin cDNA minus
codons 1–4 to streptococcal pyrogenic exotoxin type A
gene (speA ) sequences resulted in the secretion of
fusion prochymosin up to 40 g/ml of cell-free culture
fluid (Kaprálek et al., 1991).
Eukaryotic expression
Several eukaryotes, including yeast, fungi, insect and
mammalian cells, have been used for the production of
prochymosin and chymosin. In S. cerevisiae, the cDNA
coding for preprochymosin, prochymosin or chymosin
has been expressed under the control of phosphoglycer-
ate kinase (pgk), galactosidase (gal 1 and gal 10) and
triosephosphate isomerase (tpi). The proteins are syn-
thesized mainly as insoluble forms which accumulate in
the cells and are difficult to activate (Mellor et al., 1983;
Goff et al., 1984; Moir and Davidow, 1991). Expression
of preprochymosin cDNA did not allow the secretion of
chymosin, while substituting yeast invertase signal

Table 4 Effect of protein concentration on the refolding of recombinant prochymosin. Inclusion bodies solubilized in 8 M urea were
diluted to various protein concentrations in phosphate buffer, pH 10.7. The urea final concentrations were kept at 0.32 mol/l. The pro-
tein concentrations were determined by using a BCA protein assay reagent

Initial protein Initial amount of Final amount of activated

concentration (mg/ml) protein (mg) chymosin (mg) % of refolding

0.32 1.58 0.33 20.86

0.28 1.42 0.31 21.85
0.25 1.26 0.37 28.99
0.22 1.11 0.28 25.54
0.19 0.95 0.18 18.66
36 Rennets: General and Molecular Aspects
peptide for the chymosin secretion signal peptide led to
the secretion of approximately 10% of the total prochy-
mosin made (Moir et al., 1985). The secretion of prochy-
mosin is critical for obtaining soluble activatable
proteins. The failure to form or the incorrect formation
of disulfide bonds is characterized by insoluble prochy-
mosin produced in the cytoplasm of both yeast (Smith
et al., 1985) and E. coli (Shoemaker et al., 1985). Using
yeast secretion signals, integration of the transcriptal
units into yeast genome and mutations of the host
genome, the secretion of prochymosin increased at least
80-fold which allowed the production of activatable
prochymosin to a level of 20 mg/l of culture medium
(Smith et al., 1985; Moir and Davidow, 1991).
Kluyveromyces lactis has been developed as an alterna-
tive host to S. cerevisiae in the expression of recombinant
proteins. It has been used successfully to secrete prochy-
mosin under various signal sequences. Efficient synthe-
sis and secretion of prochymosin to more than 95% of
the theoretical levels was achieved by using the K. lactis
lactase gene (Lac4) (van den Berg et al., 1990). Commer-
cially viable yields have been obtained from this species
by DSM Food Specialities, Delft, the Netherlands. The
yeast, Yarrowia lipolytica, has also expressed prochymosin
using either the Leu2 or the alkaline protease XPR2
promoters (Franke et al., 1988). All of the prochymosin
produced by these systems was readily activated to
mature chymosin.
Filamentous fungi have also been used as hosts for
the production of chymosin. In Aspergillus nidulans, chy-
mosin was synthesized as an active extracellular enzyme
using the glucoamylase (glaA) promoter from A. niger
(Cullen et al., 1987). A commercial strain of A. niger
var. awamori has been used to express prochymosin
cDNA under different expression cassettes (Ward, 1989;
Ward et al., 1990). The level of active extracellular chy-
mosin was 250 mg/l when prochymosin cDNA was
fused with the entire coding sequence for glucoamylase
and expressed in the host which has been deleted of
the aspergillopepsin A gene (pepA) (Ward et al., 1990).
The introduction of an N-linked glycosylation site on
the flap region resulted in a 10-fold increase in the
production of secreted glycosylated chymosin over the
wild-type chymosin, possibly as a result of improved
secretion efficiency. The milk-clotting activity of glycosy-
lated chymosin was reduced to about 20% of the native
enzyme. However, almost all of the activity was recov-
ered after endoglycosidase H treatment (Berka et al.,
1991). The production of chymosin by A. niger var.
awamori has been obtained up to 1.3 g/l by combining a
mutagenesis and an efficient screening programme
(Dunn-Coleman et al., 1991).
About 10 mg/l chymosin has been produced by
A. oryzae using a host -amylase promoter (Boel et al.,
1987) whereas in the same expression system, a more
than 3 g/l of rhizomucor protease was obtained (Chris-
tensen et al., 1988).
The production of chymosin by Trichoderma reesei
has also been reported, using chymosin signal peptide,
cellobiohydrolase I (cbh I) sequence or the fusion of cbh
I-chymosin signal sequence (Harkki et al., 1989). Chy-
mosin A was produced at a level of 40 mg/l (Harkki
et al., 1989). A number of chymosin mutants cloned in
T. reesei have been reported to exhibit novel properties,
including mutants with a shift in the pH optimum,
substrate-specificity pocket and an altered surface loop
(Pitts et al., 1991, 1993). These mutants might be of
interest in commercial investigations. The prochymosin
secreted by T. reesei was readily activated to chymosin.
In HeLa cells, calf preprochymosin cDNA has been
expressed under the CMV-SV promoter (Kolmer et al.,
1991). The product was processed to prochymosin prior
to secretion into the cultivation medium at a level of
10–20 mg/l and readily activated to chymosin by acid
treatment.
Recombinant chymosin is now produced in large-
scale commercial operations using E. coli (California Bio-
technology and DSM Food Specialities, the Netherlands),
Kluyveromyces lactis (DSM Food Specialities, the
Netherlands) or mammalian cells (Upjohn, USA) as the
hosts (Hodgson, 1993). Many firms, including Genen-
cor/Genentech, Celltech, Hansen and Novo, produce
recombinant enzymes for laboratory use. Varieties of
cheese have been made using recombinant chymosin
and evaluated in comparison to cheese produced using
the natural enzyme. No significant differences could
be detected between them, regarding recovery of milk
solids, rate of proteolysis during ripening, as well as in
the characteristics of the final cheese products (Green
et al., 1985; Kawaguchi et al., 1987; Hicks et al., 1988;
Bines et al., 1989; Flamm, 1991; Ward and Kodama,
1991). Recombinant lamb chymosin has been used as
an alternative coagulant in cheese production, and the
overall quality was at least comparable to cheese made
with recombinant calf chymosin, and was scored better
than cheese made using bovine rennet (Rogelj et al.,
2001).
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 Rennet-induced Coagulation of Milk
D.S. Horne and J.M. Banks, Charis Food Research, Hannah Research Institute,
Ayr KA6 5HL, Scotland
Introduction
The first stage of cheese manufacture is the conversion
of liquid milk to cheese curd. Traditionally, this was
achieved by the addition of rennet to coagulate the milk
and by the subsequent expulsion of the whey by synere-
sis. In this chapter, we will be concerned with the first
of these steps, the enzyme-induced coagulation. Later
chapters will review syneresis and curd-handling proced-
ures. We will consider the basic chemistry and physics
underlying aggregation and gel formation, and the tech-
nological factors (milk composition, processing) that
influence the coagulation process. In doing so, we will
be covering ground treated in two separate consecutive
chapters on enzymatic coagulation of milk (Dalgleish,
1993) and on post-coagulation phenomena (Green and
Grandison, 1993) in the earlier editions, but updating
and broadening those reviews. Attention is also drawn
to more recent work by Lomholt and Qvist (1999).
Milk can also be clotted by acidification or a combin-
ation of significant acidification and minor enzymic
action. These aspects form the subject of ‘Formation,
Structural Properties and Rheology of Acid-coagulated
Milk Gels’, Volume 1.
After the addition of chymosin or other milk-clotting
enzyme to the milk, nothing apparently happens for
some time until the milk coagulates rapidly. During
this lag phase, the enzyme hydrolyses the -casein
which stabilizes the casein micelles. When sufficient
destabilization has been brought about, an aggrega-
tion reaction is set in train and this eventually leads
to a three-dimensional, space-filling gel, the cheese
curd. Previously, this overall coagulation reaction has
been envisaged as occurring in three stages. The enzym-
atic proteolysis forms the first or primary phase, and
leads to the activation of the aggregating species. In
the secondary phase, which overlaps the first, since the
milk may begin to clot before the enzymatic cleavage of
-casein is complete (Green et al., 1978; Dalgleish, 1979;
Chaplin and Green, 1980), the destabilized micelles
begin to aggregate and most of the previous discussions
stop with this stage (Dalgleish, 1992, 1993; Hyslop,
2003). Those authors recognize the possibility of a
third stage but consider this to involve changes largely
in the curd structure once it has formed. Just as the
first and the second stage overlap, it will be our con-
Cheese: Chemistry, Physics and Microbiology, Third edition – Volume 1: General Aspects
ISBN: 0-1226-3652-X
Set ISBN: 0-1226-3651-1
tention that so too does the aggregation phase overlap
the development of curd structure and properties.
Indeed conceptually, together they comprise the gela-
tion process. We therefore view this partitioning of
second and third stages as wholly (or largely?) artifi-
cial and aim to treat the formation of the coagulum,
at least to the cutting stage, in a single mechanistic
framework. The drastic changes, post-cutting, take the
curd into a completely different environment, and
their treatment is beyond the remit of this chapter.
Milk Properties
Cheesemaking capitalizes on the curdling of milk. To
understand the coagulation reaction, we must look
more closely at the individual components of milk to
discern their role, particularly the fat, the protein and
the minerals.
Fat
Fat exists in natural milk as small globules surrounded
by membrane proteins and in a size range dependent
on breed, lactational status and diet of the cow. The fat
in milk helps to produce flavour, aroma and body in
mature cheese. Unless the milk is homogenized, the
fat globules are physically trapped in the protein net-
work created in gel formation. Hence, their size and
the network mesh size interact in determining the
overall yield of cheese. Otherwise, the fat globules play
the part of an inert filler in influencing curd rheolog-
ical properties but no active role in gel formation.
Homogenization of the milk, which creates many more
smaller fat droplets, stabilizes these droplets by utiliza-
tion of adsorbed whey and casein proteins. Where
these are caseins, they can be involved in gel forma-
tion and influence its development. These aspects are
considered in later sections.
Protein
Two types of protein are found in milk: the globular
whey proteins, which are soluble in the serum phase,
and the caseins which exist in a stable colloidal suspen-
sion of aggregates known as casein micelles. Cheese-
making exploits the destabilizing mechanisms nature
Copyright © 2004 Elsevier Ltd
All rights reserved
48 Rennet-induced Coagulation of Milk
has built into this colloidal system by using the natural
enzyme, chymosin, originally extracted from the stom-
ach of the calf but nowadays available in cloned form,
to hydrolyse the -casein and induce the destabilization
of the casein micelle system to form a gel. The proper-
ties of the caseins and their micellar form play a major
role in defining that reaction and its final outcome.
Casein chemistry
The caseins are a family of phosphoproteins. In bovine
milk, the family consists of four distinct gene prod-
ucts, designated s1-, s2-, - and -caseins. Together,
they constitute around 80% of bovine milk protein
and are found in the approximate proportions 4:1:4:1,
respectively (Davies and Law, 1980; Walstra and Van
Vliet, 1986). Two post-translational modifications of
the proteins, newly synthesized in the mammary
gland, have a major impact on the physico-chemical,
functional and assembly properties of the proteins.
These reactions are glycosylation and phosphorylation.
In bovine casein, only -casein is found glycosylated
with several threonine, and occasionally serine, residues
in the hydrophilic C-terminal end of the -casein mole-
cule carrying relatively short sugar chains (Zevaco
and Ribadeau-Dumas, 1984; Vreeman et al., 1986).
Glycosylation is not complete, however, and the non-
glycosylated form is still the major component (Vreeman
et al., 1986). These oligosaccharide chains increase the
negative charge (through inclusion of sialic acid), the
hydrodynamic bulk and the hydrophilic character of
the C-terminal end of the -casein molecule.
The second post-translational reaction is phosphory-
lation. All the caseins are phosphorylated at serine, or
rarely threonine, residues to varying extents. The phos-
phorylation reaction requires a particular sequence
template, 9Ser9X9A, where X is any amino acid and
A is Glu, SerP or, rarely, Asp (Mercier, 1981). The pat-
tern of serine residues along the amino acid sequences of
s1-, s2- and -caseins ensures that most of the phos-
phorylated residues are found in clusters in these mole-
cules, one in -casein, two in s1-casein and three in
s2-casein (Swaisgood, 1992). -Casein is unique
amongst the caseins in the absence of phosphoseryl
clusters along its sequence. Most -casein molecules
contain only one phosphoseryl residue but some evidence
indicates a minor amount of doubly phosphorylated pro-
teins but still only as singlets (Vreeman et al., 1977).
Bovine caseins are almost always fully phosphorylated
to the level of their potential. At most only one of the
template serines is found without its expected phos-
phate residue in s1- and -caseins. Most gaps are
found with s2-casein where the variability ranges from
10 to 13 moles P per mole protein (Whitney, 1988).
The influence of the varying degrees of phosphoryl-
ation of the caseins is mirrored in the sensitivity of
these molecules to calcium-induced precipitation.
Thus, s2-casein is the most calcium sensitive, precipi-
tating at Ca2 concentrations less than 2 mM (Aoki
et al., 1985), while s1-casein precipitates in the range
3–8 mM (Parker and Dalgleish, 1981; Aoki et al., 1985;
Farrell et al., 1988) and -casein precipitates in the
range 8–15 mM Ca2 at 37 °C but remains in solution
at 1 °C (Parker and Dalgleish, 1981; Farrell et al.,
1988). -Casein remains soluble at all these calcium
concentrations and prevents the precipitation reaction
when present with the other casein types, producing
instead a colloidal suspension.
Casein structures
Controversy still exists over the level of secondary
structure present in the caseins. Previously, much of
this was designated to the random coil in line with the
open, highly hydrated state presented by the mole-
cules in solution. Because of this, the caseins have
been described as rheomorphic proteins, indicating
that their conformational structure is dictated by, and
is responsive to, the molecular environment (Holt and
Sawyer, 1993). Views have shifted, however, and cur-
rent opinion suggests that parts of - and -caseins
might adopt the polyproline II-helix structural motif
(Farrell et al., 2001; Syme et al., 2002).
From the point of view of their self-association and
micellar assembly, the amphiphilicity of the caseins
may play a more crucial role than recognizable sec-
ondary structural elements (Horne, 2002). The clus-
tering of the phosphoseryl residues has already been
mentioned, and these groupings are further flanked by
polar and charged residues making these regions very
hydrophilic. Other regions of the casein molecules
have a high concentration of hydrophobic residues,
conferring on the molecules an almost block copolymer-
like structure. Thus, the N-terminal peptide of -casein
with the phosphoseryl cluster is very hydrophilic and
the C-terminal is very hydrophobic. The behaviour
of this protein on adsorption at a hydrophobic inter-
face reflects this segregation, with the hydrophobic
C-terminus adsorbing strongly and the hydrophilic
N-terminal sticking out into solution (Horne and Leaver,
1995). Ample experimental evidence from dynamic light
scattering, neutron reflectivity, enzyme proteolysis and
surface force measurements confirm this view (Horne
and Leaver, 1995). Further support comes from self-
consistent field calculations to determine the segment
density function of a polymer model of -casein, normal
to the adsorbing surface (Dickinson et al., 1997a,b).
Similar calculations carried out on s1-casein suggest that
it can be sub-divided into three blocks, a hydrophobic

N-terminal region, a hydrophilic central loop, con-
taining the phosphoseryl clusters which extends out
into the aqueous phase on adsorption of the molecule
to a hydrophobic surface, and a hydrophobic C-terminal
peptide (Dickinson et al., 1997a). Calculations suggest
that these hydrophobic regions are entrained close to
the adsorbing surface. Such block structures reflect the
general distribution of hydrophilic and hydrophobic
residues along these casein protein sequences. By anal-
ogy, a block polymer structure can be drawn for s2-
casein, depicting it as having four blocks. Moving
from the N- to the C-terminus, these are a hydrophilic
N-terminal tail with a phosphoseryl cluster, a hydropho-
bic train, a hydrophilic loop, containing further clus-
ters of phosphoseryl residues, and finally a second
hydrophobic train at its C-terminus. -Casein is seen
to be a mirror image of -casein, with a hydrophilic
C-terminus, the caseinomacropeptide cleaved off by
chymosin, and a hydrophobic N-terminal block pre-
ceding the Phe1059Met106 bond. Importantly, however,
the macropeptide has no phosphoseryl cluster.
Casein self-assembly
Individual caseins in solution exhibit strong ten-
dencies to self-associate, and the shape and topogra-
phy of the structure adopted reflects the distributions
of hydrophobic/hydrophilic residues just described.
Thus, -casein, which resembles a detergent molecule
with a hydrophilic head and a hydrophobic tail, forms
detergent-like micelles with the tails forming a central
core and the hydrophilic heads sticking out into the
aqueous solution like the bristles on a hedgehog
(Payens et al., 1969). In like fashion, s1-casein self-
associates in solution to form a worm-like chain poly-
mer with the hydrophobic ends of one molecule
interacting with those of different molecules (Schmidt,
1970). -Casein also self-associates in solution, inter-
acting via its hydrophobic C-terminal, exhibiting a
monomer 4 micelle equilibrium but, here, polymer
growth is also influenced by the intermolecular disul-
phide linkages produced by reaction of its cysteine
residues (Vreeman et al., 1977; Vreeman, 1979).
For s1- and -caseins, it has been demonstrated that
the size of the s1-casein polymer or the -casein
micelle produced by self-association is dependent on
pH and ionic strength, and also sensitively on tempera-
ture in the case of -casein. Temperature is important
for the strength of hydrophobic attraction, but pH and
ionic strength govern electrostatic repulsion and its
range. The balance of these attractive and repulsive
components in the overall interaction free-energy equa-
tion thus controls the aggregate size and, more import-
antly in a local situation, the strength of individual
intermolecular bonds.
Rennet-induced Coagulation of Milk 49
Casein micelle assembly
From these concepts, Horne (1998) devised a poly-
merization scheme for the assembly of casein micelles.
Cross-linking of the molecules is envisaged as pro-
ceeding via two routes, hydrophobic interactions
between groups on different molecules forming one
pathway, with more than two molecules possibly joining
at such junctions, and a second pathway where chain
extension is through calcium phosphate nanoclusters,
small calcium phosphate crystallites, the precipitation
of which is regulated by the presence of the caseins. A
calcium phosphate nanocluster acts as a neutralizing
bridge between two phosphoseryl clusters on different
molecules of s1-, s2- or -casein. Again, more than
two casein molecules could be involved with any one
calcium phosphate nanocluster. If the casein molecule
is -casein, further extension of this chain is through a
hydrophobic linkage. Both routes permit branching
and hence lead to a three-dimensional network struc-
ture. -Casein can link only to a hydrophobic region
on another molecule. Because it has no phosphoseryl
cluster on the opposite end of the molecule to permit
further extension, the polymer chain ends there. No
further growth occurs beyond this point. This occurs
for each growing chain and hence the proportion of
-casein limits the micelle size. In consequence, the
micelle acquires an external coat of -casein which
acts as a steric stabilizer for the micelle.
In devising this mechanism for micellar assembly,
no new features are ascribed to the casein molecules.
The ability to bond and the strength of those bonds is
the resultant of a localized favourable balance of attract-
ive hydrophobic interaction and electrostatic repulsion.
Reducing that hydrophobic interaction by lowering the
temperature, or increasing electrostatic repulsion by
dissolving out calcium phosphate but maintaining pH,
weakens those bonds and causes (partial) disintegration
of the micelle.
Casein micelle properties
Almost all the casein proteins present in bovine milk
are incorporated into the casein micelles, together
with a high proportion of the available calcium and
inorganic phosphate. These micelles have an average
molecular weight of ⬃108 Da and a mean diameter of
⬃100 nm (range 50–600 nm). The micelles are very
open, highly hydrated structures with typical hydra-
tion values of 2–4 g H2O g 1 protein, depending on
the method of measurement. The structure is not
rigidly fixed but dynamic. Cooling the milk from the
37 °C of the udder to storage at refrigeration tempera-
tures brings about solubilization of a significant
fraction of -casein, some -casein and much lower
levels of s1- and s2-caseins from the micelles, and all
50 Rennet-induced Coagulation of Milk
of this is pH-dependent also (Dalgleish and Law, 1988).
Raising the temperature back to 37 °C reverses this
process. Almost complete disintegration of the micelles
can be achieved either through the addition of a strong
calcium sequestrant such as EDTA (Griffin et al., 1988)
or through the addition of high concentrations of urea
(McGann and Fox, 1974). Dissociation to molecular
level is not achieved, and the dissociated species have
average diameters of the order of 10–15 nm and are
also of variable composition (Aoki et al., 1985).
All of these experimentally observed properties, tem-
perature- or reagent-dependent dissociation, variable
composition with size, location of -casein, inverse
relationship of micellar size and -casein content are
predicted or are manifest as a consequence of the dual-
binding assembly model described above.
As far as the mechanism of chymosin-induced aggre-
gation of casein micelles is concerned, the proposed
theories largely neglect internal micellar structure or at
least regard it as of no consequence to the outcome of
the reaction. Whilst we do not deviate from this view, as
described below, we feel that internal micellar structure
and the modifications to it as a consequence of pH, salt
and temperature changes occurring during curd manu-
facture should also be considered and that these must
impact on curd properties.
Micelle stability
The casein micelle system is an excellent example of
colloidal dispersion. Repulsive forces hold the micelles
in suspension until removed by some external influ-
ence. Because the casein micelles were negatively
charged, resulting in a zeta potential of about 20 mV,
and this charge is reduced by ⬃50% on rennet treat-
ment (Green and Crutchfield, 1971; Pearse, 1976;
Darling and Dickson, 1979; Dalgleish, 1984), attempts
were made to explain the stability of the casein micelle
using the DLVO (Derjaguin-Lamdau-Verwey-Overbeek)
theory of the stability of lyophobic colloids (Verwey
and Overbeek, 1948). Such concepts envisage stability
as arising from the presence of a repulsive energy bar-
rier, the resultant of ubiquitous attractive Van der
Waals forces and repulsive electrostatic forces. Unfor-
tunately, as Payens (1979) calculated, this energy bar-
rier is located at such a short inter-surface distance
(⬃0.1 nm) as to be physically meaningless, lying well
within the orbit of surface roughness, the loops and
the tails of the protein molecules in the outer micellar
regions. Though the complete DLVO theory is rendered
inapplicable by the above and other failures related to
ionic strength changes, the general concept of micellar
stability being due to the presence of a repulsive energy
barrier is still valid. It is now accepted that micellar sta-
bility arises from the presence of a sterically stabilizing
outer layer of -casein molecules, the C-terminal por-
tion of which extends out into the solution (Holt,
1975; Walstra, 1979; Holt and Horne, 1996). Repulsion
arises due to the increase in free energy brought about
when the protein layer of one micelle is brought into
contact with (or overlaps) the layer of another micelle.
The role of chymosin is to proteolyze -casein,
splitting it at the Phe1059Met106 bond and thus shave
off the hairy layer, so that the subsequently exposed
micelle cores begin to aggregate, once sufficient of
their -casein has been hydrolysed. The overall milk
clotting process is shown diagrammatically in Fig. 1.
Of these stages, only the proteolytic cleavage can be
monitored totally independently of the others by fol-
lowing the release of the glycomacropeptide, -CNf
106–169, or the formation of para--casein, residues
1–105. The aggregation reaction of the destabilized
micelles is a consequence of this proteolysis. Its rate
cannot be separated easily from that of the proteolysis
reaction. The aggregation overlaps the proteolysis
reaction; the latter is certainly not complete before the
aggregation begins. Aggregation leads to bigger and
bigger clusters until eventually the system acquires the
solid-like nature of the gel. Again, there is a smooth
continuum through this point and beyond as the gel
matures. As we discuss in greater detail later, the separ-
ation into aggregation and gelation stages is largely
artificial, driven in most instances by the requirements
of the experimental technique or the mechanistic
model.
Primary Enzymatic Phase
The -casein molecules provide a steric stabilizing layer
with their hydrophilic C-terminal peptides protruding
into the aqueous phase. Gel formation is initiated by the
proteolysis of the -casein molecules which is accompan-
ied by the release of a hydrophilic peptide, termed the
caseinomacropeptide, into the serum (whey) phase.
The remaining N-terminal region of the -casein,
termed the para--casein remains bound in the casein
network. Gradual loss of the caseinomacropeptide is
accompanied by a decrease in the micellar zeta potential
which results in destabilization of the micelles and
aggregation into a gel.
Proteases capable of initiating the required proteoly-
sis of -casein are aspartic proteinases (EC 3.4.23).
Milk clotting enzymes were obtained originally by
extraction from the stomachs of ruminants, and calf
and adult bovine rennets are widely used in cheese
manufacture today. Concerns in the 1960s that world
cheese production had increased to such an extent
that the production of rennet products derived from
 Rennet-induced Coagulation of Milk 51

(A) Micelles (O) + enzyme ( )

(B) Partially renneted micelles

(C) Aggregating micelles in small clusters

(D) Percolating clusters

Figure 1 A schematic description of the various stages envisaged in the enzymatic coagulation of milk, starting from the initial
mixture of casein micelles and enzyme (A) and proceeding through proteolysis (B), initial aggregation into small clusters (C) and
reaching a gel point at percolation (D).

animal tissue would be insufficient to meet future
demand led to the development of alternative prod-
ucts. Rhizomucor miehei, R. pusillus and Cryphonectria
parasitica were used to produce aspartic proteinases by
fermentation, and these new coagulants were success-
fully introduced to the market. In the late 1980s,
recombinant DNA technology was used to clone the
gene for chymosin, the main clotting component of
calf rennet. E.coli, Aspergillus niger and Kluveromyces
lactis were used as host organisms (Teuber, 1990; Harboe,
1992). The chymosin products generated are now
referred to as fermentation-produced chymosin (FPC).
A wide range of clotting agents are now available for
cheese manufacture and the use of these coagulants in
cheese manufacture has been reviewed extensively
(Guinee and Wilkinson, 1992; Wigley, 1996; Fox and
McSweeney, 1997; Harboe and Budtz, 1999). Calf ren-
net and adult bovine rennet still dominate in cheese
52 Rennet-induced Coagulation of Milk
production, but market share for FPC continues to
increase, and microbial coagulants derived from R. miehi
are the third most commonly used coagulants (Harboe
and Budtz, 1999).
Chymosin (EC 3.4.23.4) is a gastric proteinase
which is secreted in the abomasal mucosa of new-born
ruminants and other mammals during the first days of
life (Foltmann, 1992). It is the main clotting enzyme in
calf rennet. The activity of chymosin differs markedly
from that of other gastric aspartic proteinases in that it
exhibits low general proteolytic activity but is particularly
active in hydrolysis of the Phe1059Met106 of -casein.
Milk clotting enzymes differ in the rate at which they
continue to degrade casein following the hydrolysis to
initiate gel formation. Only those enzymes with a high
ratio of milk-clotting activity to general proteolytic
activity are considered suitable for cheese manufac-
ture. A high level of non-specific proteolysis can lead
to a weak gel structure, high losses of protein and fat
in the whey and reduced cheese yield. The higher the
level of proteolysis, the greater is the reduction in
cheese yield. Chymosin activity on -casein is limited
with only formation of the caseinomacrpeptide and
para--casein, while in the case of fungal proteinases,
extensive non-specific hydrolysis of both -casein and
para--casein occurs (Shammet et al., 1992). The use
of microbial rennets is generally considered to result in
reduced cheese yield, compared with calf rennet
(Olson, 1977; Emmons et al., 1990a; Lucey and Kelly,
1994). The fermentation-produced chymosins have a
high ratio of milk clotting to general proteolytic activ-
ity and no significant differences in cheese yield have
been reported between recombinant chymosin and calf
rennet (Green et al., 1985; Hicks et al., 1988; Ustinol
and Hicks, 1990; Emmons et al., 1990b; Banks, 1992;
van den Berg, 1992).
The properties of chymosin and other aspartic
proteinases have been reviewed comprehensively by
Chitpinityol and Crabbe (1998) (see ‘Rennets: General
and Molecular Aspects’, Volume 1). Rennet preparations
are generally prepared from multiple calf stomachs and
are heterogeneous in their chymosin content. Calf
chymosin occurs in three forms, A, B and C, chymosin
B being the most abundant in natural rennet. Chy-
mosins A and B are allelic variants which differ at only
one amino acid position; Asp243 in chymosin A is
replaced by Gly243 in chymosin B. Chymosin C
appears to be a degradation product of chymosin A
which lacks three residues, Asp244–Phe246 (Danley and
Geoghegan, 1988). The three variants show differ-
ences in clotting activity, and of the three forms, chy-
mosin A has the highest specific clotting activity and
chymosin C the lowest. The A and B forms are equally
efficient in cheese manufacture (Harboe and Budtz,
1999). The cloned chymosins derived from Aspergillus
niger and Kluveromyces lactis are B variants (Harboe
and Budtz, 1999).
The specificity of chymosin for the Phe9Met bond
has been studied extensively (see Fox and McSweeney,
1997). The length of the peptide and the sequence
around the sectile bond are important determinants of
enzyme–substrate interactions. Observations that syn-
thetic di-, tri- or tetra-peptides containing a Phe9Met
bond were not susceptible to hydrolysis by chymosin
suggested that other residues close to the cleaved bond
are also required for the hydrolytic reaction (Visser
et al., 1980). Kinetic studies on synthetic peptides
indicated that two additional residues at either side of
the hydrolysable bond are required for appreciable
reaction (Hill, 1968, 1969; Raymond et al., 1972). The
sequence of His989Lys111 includes all the necessary
determinants (Visser et al., 1980), and this tetrade-
capeptide is hydrolysed with a kcat/Km of ca 2M 1 s 1,
which is similar to that of intact -casein (see Fox and
McSweeney, 1997).
The Phe and Met residues in the chymosin-sensitive
bond of -caseins are not essential for chymosin action
on caseins. The residues in the chymosin-sensitive
bond differ across species which suggests that it is the
amino acid sequence surrounding this bond rather
than the residues in the bond itself, which contain the
important determinants of hydrolysis (see Fox and
McSweeney, 1997). Porcine pepsin (A and C) and
R. miehei proteinase cleave the Phe9Met bond in a simi-
lar fashion to chymosin but the C. parasitica proteinase
cleaves the Ser1049Phe105 bond (Dronse and Foltmann,
1989).
Calf rennet is the standard product against which all
other coagulants are assessed. Adult bovine rennet con-
tains a higher proportion of pepsin and therefore a more
general proteolytic activity. Rennets extracted from
ovine, caprine and porcine stomachs are the most effi-
cient at clotting milk of their own species (Foltmann,
1992).
Coagulants extracted from the flowers of the thistle
Cynara cardunculus are used in the production of
artisanal cheeses in the Iberian peninsula (Sousa et al.,
2001). The coagulants are aspartic proteinases and
comprise of two enzymes, cardosins A and B. Both
enzymes hydrolyse the Phe1059Met106 bond of
-casein (Esteves et al., 1995). Kinetic parameters of
cardosin A are similar to those of chymosin while
those for cardosin B are similar to pepsin (Verissimo
et al., 1995). The ratio of clotting to proteolytic activ-
ity is low compared to chymosin, and non-specific
casein hydrolysis results in lower gel firmness com-
pared to that obtained with chymosin (Esteves et al.,
2002).

Measurement of Clotting Time and
Curd-Cutting Time
The most easily detected outcome of chymosin proteoly-
sis and rennet clotting is the visible observation of the
presence of flocs in a milk sample in a rotating tube.
The time taken for their appearance is defined as the
rennet coagulation time, and for the cheesemaker inter-
ested in the activity of an enzyme preparation, this may
be the only quantity of interest. The importance of its
determination is reflected in the number of techniques
tested over the years (see Lucey, 2002; O’Callaghan
et al., 2002, for reviews of these methods).
Since the coagulum is cut sometime after the coagu-
lation point when it has attained sufficient firmness,
the more technically successful techniques are those
which continuously monitor the development of
the coagulum with time by measuring changes in
some particular physical attribute, such as viscosity
(Scott Blair and Oosthuizen, 1961), reflectivity (Hardy
and Fanni, 1981; Ustinol et al., 1991), thermal con-
ductivity (Hori, 1985) or ultrasound transmission
(Benguigui et al., 1994) to name only a few. Few of
these techniques have entered commercial practice for
in-vat applications not only because the instruments
are often difficult to clean and maintain properly but
also because the changing processing conditions and
schedules in response to such instrumental readings
are not always an attractive option to a large modern
cheese factory. In such cases, the standardization of milk
protein is the preferred approach, since this minimizes
differences in coagulation and it has been observed
that cheese yield does not seem to be very sensitive to
small changes in gel firmness, at cutting, in such stand-
ardized circumstances.
Many of the techniques described by Lucey (2002)
and O’Callaghan et al. (2002) have also been devel-
oped from research tools used to study the influence of
reaction variables such as temperature, pH and milk
composition and pre-treatment on gel development.
The most useful of these techniques are those where
the behaviour of the variable of interest can be pre-
dicted by a mathematical model based on a mechanis-
tic description of the reaction. Few, if any, techniques
provide direct relationships applicable over the entire
course of the reaction from micelle to gel. Indeed, this
is perhaps one reason why gel formation has been split
into primary and secondary phases, since the early
aggregation phase can be followed readily by turbidity
or light scattering (Payens et al., 1977; Dalgleish et al.,
1981a,b; Dalgleish, 1983; Bauer et al., 1995; Lomholt
et al., 1998) whereas the gel formation and development
is most easily monitored in the laboratory by rheometry
(Tokita et al., 1982; Bohlin et al., 1984; Van Hooydonk
Rennet-induced Coagulation of Milk 53
and Walstra, 1987; Zoon et al., 1988a,b,c, 1989a,b;
Van Vliet et al., 1991; Horne, 1995, 1996; Lopez et al.,
1998; Mellema et al., 2002). Each technique suffers
from limitations. Light scattering requires a dilute dis-
persion of particles so that only singly scattered pho-
tons are collected at the detector. Direct conversion to
molecular weight and/or size is also limited by the
ratio of particle size to light wavelength. Studies using
light scattering are thus limited to early stages of aggre-
gation, where growth of molecular weight or degree of
polymerization is obtained as a function of reaction time.
Rheological measurements suffer from the opposite fail-
ing. There, the limitation is instrument-sensitivity and
a detectable signal is realized only after the reaction has
progressed to a significant extent. The relationship
between measured viscoelasticity and gel structure and
bonding is also highly model-dependent, as we shall
see, and interpretations are often controversial.
Kinetic Models of Rennet Coagulation
The earliest attempt to describe the kinetics of the
clotting process was made in the 1870s by Storch and
Segeleke (see Foltmann, 1959, 1971). This simply
stated that the clotting time was inversely related to
the concentration of rennet used to clot the milk. A
further refinement was postulated by Holter (1932)
and rearranged by Foltmann (1959) to give the famil-
iar equation:
k
RCT  A (1)
[E]
where k and A are constants and [E] is the enzyme
concentration, RCT being the rennet coagulation time.
This relationship is purely empirical, but it is an
important relationship which has to be satisfied by any
more descriptive mechanistic model, even if only over
a restricted range of enzyme concentration and RCT
values.
The Holter modification separated the coagulation
time into two components, an enzyme proteolysis
stage and a secondary coagulation phase. The equation
assumes that there is no overlap between the proteoly-
sis and the coagulation phases and that the extent of
proteolysis is always the same at RCT (Foltmann,
1971). Experimental evidence suggests that the prote-
olyzed fraction of -casein is very high. Dalgleish
(1979) suggested 60–80% of the -casein must be
hydrolysed, though his plot shows no significant
aggregation below 90% proteolysis. Green et al. (1978)
found that aggregation did not start until after about
60–80% of the RCT had passed, by which time the
54 Rennet-induced Coagulation of Milk
enzyme action was almost complete. In a separate
experiment, Green et al. (1978) found that the viscos-
ity of milk rose sharply when the enzyme reaction was
⬃86% complete. Other groups have found that viscos-
ity increases well before the visible coagulation time
indicated by the presence of flocs (Guthy and Novak,
1977), confirming an inescapable fact that the detec-
tion of an aggregation point is determined by the sen-
sitivity of the measuring technique to the presence of
aggregates and also that the two processes, proteolysis
and subsequent aggregation, overlap in time. The
extent of overlap, or the percentage of -casein prote-
olyzed before aggregation becomes detectable, is also
dependent on milk pH and ionic calcium content,
decreasing as pH is decreased (Van Hooydonk et al.,
1986; Carlson et al., 1987a,b) or as Ca2 content
increased (De Kruif, 1999; Horne and McCreight,
unpublished observations).
The functional description of the overall kinetics of
coagulation must therefore take both proteolysis and
aggregation reactions into account. The kinetics of the
proteolysis reaction has been discussed by Dalgleish
(1993) and Hyslop (2003). In milk, the reaction
appears to be of first order, but whether the reaction is
truly first order throughout or follows standard
Michaelis–Menten kinetics for a single-step enzyme-
catalysed reaction with a relatively high value for the
dissociation constant of the chymosin–-casein com-
plex, where the Michaelis–Menten equation approxi-
mates to a first-order picture, is still undecided (Hyslop,
2003).
Many attempts have been made to model the aggre-
gation reaction, the models differing in how the aggre-
gation rate constant is modelled and how it depends
on the enzymatic proteolysis of -casein. Beginning
with the model calculations of Payens (1976, 1977,
1989), Payens et al. (1977) Payens and Brinkhuis
(1986), and Hyslop (2003) has outlined the nuances
of the various schemes, highlighting their differences
and critically pointing out their shortcomings. Depend-
ing on the assumptions made and the experimental
circumstances involved, he concluded that three
models, step-function (Dalgleish, 1980a,b), energy bar-
rier (Darling and Van Hooydonk, 1981) and function-
ality theory (Hyslop, 1993; Hyslop and Qvist, 1996)
may be used to describe the aggregation reaction.
The step-function model (Dalgleish, 1980a,b) is
based on the idea of a critical level of proteolysis before
aggregation is possible, but does not explain why this
should be necessary. A plausible explanation arises if
an energy barrier is gradually reduced by rennet prote-
olysis, leading to a gradually increasing probability
of reaction on collision in the aggregation reaction.
Introduced by Darling and Van Hooydonk (1981), the
energy barrier model has been tested extensively (Van
Hooydonk and Walstra, 1987; Dalgleish, 1988; Hyslop,
1989; Payens, 1989; Hyslop and Qvist, 1996).
Energy barrier models are mean-field models. They
imply the existence of a uniform repulsive force that
decreases gradually with time, and therefore uniform
micellar surfaces. Since the -casein molecules are
hydrolysed individually, a more realistic approach might
be the creation of attractive patches on the micelle sur-
face by the removal of sufficient macropeptide hairs, as
envisaged in the geometric model of Dalgleish and Holt
(1988). Continuing removal of -casein hairs would
lead to multiple patches and the creation of conditions
necessary for the operation of a polyfunctional model
of the Flory–Stockmayer type (Stockmayer, 1943),
which gives the rate constant as:
kij  K{4  2(f 2) (i  j)  (f 2)2 ij} (2)
where K  proportionality factor; f  number of func-
tional sites (functionality); i, j  number of particles of
type i, j.
If f  1, only dimers are possible, if f  2, linear poly-
mers are predicted and if f 2, chain-branching occurs
and gelation is possible. In the beginning at t  0, f  0,
the micelles have no reactivity or inclination to aggre-
gate, and a realistic model has to account for the growth
of f during the course of the reaction. This is generally
done by proposing that f is some function of the degree
of proteolysis of -casein, most frequently linear. A fur-
ther refinement in this model is to allow the proportion-
ality factor to depend on the energy barrier height
(Bauer et al., 1995).
All the various models describe essentially the
growth in average molar mass of the micellar aggregate
with time of reaction. Average molecular weight is most
readily measurable by static light-scattering techniques.
Due to problems encountered with multiple light scat-
tering, where the detected photon has encountered
more than one scatterer in its passage through the sus-
pension, these techniques are applicable only in highly
diluted suspensions (Dalgleish et al., 1981a,b; Brinkhuis
and Payens, 1984; Bauer et al., 1995), or over very
short path lengths, as in the turbidity measurements of
Lomholt et al. (1998).
Increasing complexity in the models increases the
number of parameters the value of which can be varied
to fit experimental data. Possibly their most stringent
test so far has been carried out by Lomholt et al. (1998)
who considered most variations involving energy barri-
ers. They found that they could obtain good representa-
tions of the initial stages of renneting, up to aggregates
of ⬃5–10 micelles, with plausible values for the vari-
able parameters, energy barrier height for native casein

micelles and after completion of rennet proteolysis.
They reproduced the growth of aggregate size with
time, adequately accounting for the effect of enzyme
concentration and to some extent micellar casein con-
centration. They were, however, unable to differentiate
between the different model expressions, all giving
equally acceptable fits, and thus were unable to reliably
relate experimental data to any particular mechanistic
picture.
A major disadvantage of these experiments is that
they are concerned only with the initial stages of the
coagulation reaction, largely because of constraints
relating to the experimental techniques. Early light-
scattering studies (Dalgleish et al., 1981a,b; Brinkhuis
and Payens, 1984) operated in dilute solution because
of this but even in the case of the later work of
Lomholt et al. (1998) the effects of multiple scattering
by large aggregates produced an upper limit to the size
which can be extracted from such data (Worning et al.,
1998). Consequently, although they operated in a
concentration regime where coagulation eventually
occurred, Lomholt et al. (1998) were unable to derive
any information on the progress of the reaction in this
region.
Adhesive Sphere Models and Viscosity
Viscometry was one of the techniques used in early
attempts to monitor the course of the rennet coagula-
tion reaction (Scott Blair and Oosthuizen, 1961). The
relationship between viscosity and molecular weight is
rather complex and the results therefore do not lend
themselves to ready testing of the theoretical models
outlined above. On addition of chymosin to milk, there
is an initial decrease in the viscosity which then passes
through a minimum before increasing sharply as the
coagulation progresses. Rather than interpreting this rise
as due to the formation of aggregates and from them a
gelling network, De Kruif and coworkers (De Kruif
et al., 1992; De Kruif, 1999) have proposed a different
mechanism based on treating casein micelles as ster-
ically stabilized hard spheres which become sticky, or
adhesive, as the -casein is proteolyzed. They write the
relative viscosity of skim milk as:
冢
r  1  2.5  5.9 
 冣
1.9 2
 (3)
where  is the micellar volume fraction and  a sticki-
ness parameter related through the second virial coeffi-
cient (B2) to the depth ( ) of an attractive square well
potential created as the -casein hairs are proteolyzed.
The relevant equations are:
Rennet-induced Coagulation of Milk 55
1
B2  4 (4)


冢 冢 kT 冣冣
B2
 4  12 1 exp (5)
VHS 
 hkT ln 冢 [P][P] [P] 冣


(6)
where [P] is the concentration of macropeptide released at
time t, and the other parameters are defined in Fig. 2, VHS
being the hard sphere volume 4/3 a3, where a is the effect-
ive micelle radius. The only fitting parameter is h, which
is of order 2 (De Kruif, 1999). The initial decrease in the
viscosity arises because of the decrease in hydrodynamic
volume fraction, , as the -casein hairs are sheared. This
decrease in hydrodynamic size has been measured experi-
mentally using dynamic light scattering techniques in both
dilute (Walstra et al., 1981) and concentrated (Horne and
Davidson, 1993) micellar suspensions.
In the adhesive sphere model, however, the loss of
the -casein-stabilizing hairs also causes the attractive
well depth to increase in proportion to the logarithm
of the normalized hair loss. This stickiness then pro-
duces the observed increase in viscosity in this model.
At later times, however, the viscosity also increases
due to aggregation and network formation. Passage to
this status is seamless, and with no indication of its
occurrence, making realistic assessment of the validity
of the model at least problematical. Nevertheless, on
its basis, De Kruif (1999) has demonstrated that the
micellar system can be flocculated at higher hair den-
sity (lower levels of proteolysis) when ionic calcium
level is increased and that renneting time can be short-
ened by the inclusion of increasing amounts of
ethanol. A major disadvantage of the adhesive sphere
model is that its time dependence relates only to the
proteolysis reaction. Indeed, it is confined purely to
Figure 2 A schematic defining the terminology of the adhesive
sphere model. The micelle of diameter () has a hairy layer of
thickness () equivalent to the width of the square well potential.
The depth of this attractive potential ( ) deepens as the hairs
are proteolyzed by chymosin.
56 Rennet-induced Coagulation of Milk

pre-aggregation events and can say nothing of the
kinetics of aggregation and gel formation, highly sig-
nificant events in the definition of gel properties.
Development of Rheological Properties
during Rennet Coagulation
Possibly the most direct way to measure gel formation is
to monitor the evolution of rheological properties (Bohlin
et al., 1984; Dejmek, 1987; Zoon et al., 1988a; Horne,
1995, 1996). Dynamic rheology applies an oscillatory
shear stress (0 ) or strain (0) and measures the response
from the developing gel. The measurement yields the
elastic or storage modulus (G ), which is a measure of
the energy stored per oscillation cycle and reflects how
the sample behaves as an elastic solid, and the viscous or
loss modulus (G) which is a measure of the energy dissi-
pated per cycle and indicates how much the sample
behaves as a viscous liquid. Their ratio is tan , the tan-
gent of the phase angle of the response to the applied
stress or strain. The shear moduli are defined as follows:
G  冢  冣cos 
0
Thus, when 45°, the viscous component domin-
ates, whereas when 45°, the sample appears more
like an elastic solid.
Ideally, measurements should be made in the linear
viscoelastic region, i.e., deformation should be propor-
tional to the applied stress. When the gel is very weak
in the earliest stages of reaction, it is debatable whether
this situation is achievable. Minimum forces must be
applied for the instrument to produce a discernible
movement in its geometry, or in the case of a constant
strain rheometer, the movement has to be detectable
but a minimum force will be required to overcome
inertia, friction in bearings, etc. This minimum move-
ment or stress may be sufficient to damage the gel at
this point in its development. Fortunately, the rennet-
induced gel develops viscoelasticity rapidly and so
moves into linear response quickly but, nevertheless,
the gel point may vary between instruments or instru-
ment settings, and too large applied stresses or strains
should be avoided.
Figure 3 shows an example of the development of
viscoelasticity in a skim milk sample during renneting.
The sharp decrease in the phase angle, from close to
the 90° of a Newtonian liquid to around 20° coincides

0
(7) approximately with the visible coagulation time. In the
same time frame, gel firmness as indicated by the
growth of shear moduli, becomes apparent. Although
not visible on the scale plotted, G initially lags on
G  冢  冣sin 
0

0
(8) growth of viscosity or viscous modulus, G, but
quickly crosses and dominates as gel elasticity rapidly
develops, the transition point occurring at   45°.
G Plots of shear moduli versus reaction time evolve as
tan   (9)
G sigmoidal curves which tend to approach a constant

90 90

80 80
Shear moduli, G ′, G ″ (Pa)

70 70
Phase angle (deg)

60 δ 60
G′
50 50

40 40

30 30

20 20

10 G″ 10

0 0
0 0.5 1.0 1.5 2.0 2.5 3

Time (ks)

Figure 3 A typical example of the changes in viscoelastic parameters observed during gel formation, showing increases in shear
moduli (G , G) and decreases in phase angle () with time after enzyme addition.

value appropriate to each milk sample at very long
times. In practice, of course, the gel is generally cut
at a fixed time after rennet addition or after visible
coagulation is noted, perhaps after a period of no more
than 30 min. In either instance, the gel firmness is
likely to be of the order of 30 Pa and possibly has not
passed its maximum rate of firming.
A rennet-induced milk gel is described as a visco-
elastic solid. In rheological terms, the critical gelation
point is often taken as the time at which the elastic
modulus exceeds the viscous modulus (Ross Murphy,
1995). Others demand compliance with more strin-
gent conditions, such as the Winter and Chambon
(1986) criterion which gives the gelation as occurring
at the time when both G and G show power-law
dependence on oscillation frequency, both dependen-
cies with the same positive exponent. Derived for
chemically cross-linked polymers, this time does not
necessarily coincide with that for G and G cross-over
in a single frequency experiment. Depending on the
instrument sensitivity and the reaction, it may not
even be possible to detect this cross-over. In some con-
centrated systems, the value of G may have already
exceeded G, whilst in others it may occur early in the
reaction below the instrument detection threshold. In
the latter circumstance, an acceptable working defin-
ition of the gel point would be when instrument
response rises above the background noise level. What
physically defines the gel point and, beyond that crit-
ical point, what defines the dynamic evolution of the
viscoelasticity of the gel in terms of a mechanism,
describing network, creation and growth are the sub-
jects of the next section.
Theoretical Basis of Viscoelasticity
Continuity of structure and permanency of that struc-
ture are general features of a gel. We continue to visual-
ize the rennet-induced milk gel as a particle gel. The
spatial distribution of the casein micelles (the particles)
in the gel network and the strength of the bonding
between those elements define the existence and the
rheology of the gel. By considering the deformation of
the network following application of a shearing force,
Van Vliet and Walstra (1985) derived the following
equation for the modulus of the network:
d2F
G  CN (10)
dx2
where N is the number of stress-bearing strands per
unit area in a cross-section perpendicular to x, the
direction of the external force or stress, C is a charac-
Rennet-induced Coagulation of Milk 57
teristic length determining the geometry of the net-
work, dF is the change in Gibbs free energy when the
elements are moved apart over a distance dx, and is
therefore related to the bond strength in systems
where enthalphic effects dominate over entropic, as
argued by Van Vliet and Walstra (1985). In the case of
particles which are homogeneously distributed over
the available space, or at least homogeneously on the
length scales of the experimental measuring device, all
particles involved in the network will contribute to the
network modulus to the same extent. N will then be
directly proportional to the volume fraction of parti-
cles in the network.
In describing the formation of the rennet-induced
milk gel, G will be a function of reaction time, and
thus the growth of the shear modulus with time will
depend on the rate of incorporation of particles into
the network and the rate of change of bond strength
(or increase in well depth) with time. Two possible
scenarios present themselves. In the first, only the
well depth is considered as a function of time. This is
akin to the adhesive sphere concept of De Kruif and
coworkers (1992); De Kruif (1999) and De Kruif and
Holt (2003) would assume that all of the casein
micelles would be considered as a network from time
zero, the point at which -casein proteolysis is initiated
and the attractive well begins to deepen. Dickinson
(2003) has emphasized that the hard sphere model
operates within the framework of equilibrium statistical
mechanics. It therefore describes the gelation for rela-
tively weak, short-range attractions under reversible
conditions. That is, the particles are free to dissociate,
as well as to associate, the dynamic equilibrium con-
stant or bond lifetime being a function of well depth.
With well depth increasing as proteolysis progresses,
the bonds become increasingly stronger and could
become effectively irreversible within the time-span of
the experiment. This bond lifetime or relaxation time
has an important bearing on the response of the net-
work in the rheometer geometry, where the lifetime of
a bond must be correlated with the oscillation time of
the applied deformation.
Strong bonds with a high energy content will gener-
ally have a long relaxation time. They establish the
permanent or elastic character of the gel. Weak bonds
generally break and reform spontaneously over much
shorter timescales. They contribute to the temporary
character of the gel network, appearing as the viscous
component. Thus, non-relaxing bonds only contribute
to G whereas rapidly relaxing bonds only contribute to
G. Bonds with relaxation times in the timescale of the
measurement contribute to both G and G. Because
bond lifetime changes smoothly during proteolysis
and gel formation, in this model from very short to
58 Rennet-induced Coagulation of Milk

very long, the gelling system passes monotonically
from a viscous liquid to a firm elastic solid and the
phase angle, , passes through the critical 45° value.
Changes in phase angle may therefore more sensitively
reflect changes in the nature of the bonds (Roefs,
1985). As Fig. 3 shows, however, during the formation
of a rennet-induced milk gel, the phase angle decreases
rapidly in the early stages through the gel point but
remains substantially constant throughout the main
growth period in the shear moduli, as was also observed
by Dejmek (1987) and Lopez et al. (1998). This indi-
cates that the nature and strength of the bonds does
not change significantly during this growth. It is there-
fore likely that the increase in gel firmness is caused
by an increase in the number of bonds with time, the
other time-dependent factor in our theoretical expres-
sion for the modulus, G, in equation 10.
In the second scenario, the micelles aggregate into
clusters and these clusters eventually form the gel net-
work. The kinetics of this aggregation can be viewed
as an increase in the number of bonds with time. What
point in the time course of the progress of this reac-
tion can be identified as the gel point, and can this be
detected experimentally? Again, two extreme scenar-
ios can be envisaged (Fig. 4). The first is a strong gela-
tion where bond energy is kT and bond formation
is virtually irreversible. As depicted in Fig. 4, gelation
corresponds to the achievement of a percolation clus-
ter spanning the container (Stauffer, 1976; DeGennes,
1979). This critical percolation cluster is present only
for a very short moment. Gel curing continues as the
remaining free particles and clusters are incorporated
into the network, creating more stress-carrying strands.
This is what is observed as the growth of shear modu-
lus with time.
In the second scenario in aggregate growth (Fig. 4b),
bonds are weak, aggregation is reversible, and clusters
break up as readily as they form. The critical gelation
condition is reached when the clusters achieve a pseudo-
close-packed arrangement as depicted in Fig. 4b.
The majority of potential bonds are satisfied at this junc-
ture, and increases in shear moduli occur as the system
rearranges itself in its quest for an equilibrium structure.
Can the measured kinetics of gel formation assist
in differentiating these two extremes? As is shown in
Fig. 5a, gel cure curves, plotted as complex modulus,
G*, versus t, are typical for each milk. In the set shown,
the same reaction conditions, rheometer settings and

(a)

(b)

Figure 4 Two different scenarios defining the occurrence of the gel point. (a) The percolation cluster model where gelation is
sensed by the measuring device whenever clusters extend across the container. Note the number of clusters and monomers which
have still to be incorporated into the gel, creating more links and strands to carry applied stress. (b) The formation of a weak gel
where most particles are weakly linked into the network but reversibility allows rearrangements. The larger circles depict the fractal
‘blob’ concept where the system is fractal within but closed packed outside.
 Rennet-induced Coagulation of Milk 59

1.5
150
(a) (b)
7
1.25
125 4
5 1
1.00
Shear modulus, G ′ (Pa)
100
3

G ′/G ′ at 3*tg
75 0.75
6

50 8 0.50

25 0.25
2

0 0
0 2.0 4.0 6.0 8.0 10.0 12.0 14 0.5 1.0 1.5 2.0 2.5 3.0 3.5

Time from rennet addition (ks) Reduced time, t/tg

Figure 5 Depiction of the derivation of the master curve plot from a series of gel cure curves obtained by renneting individual milks
(1–7) under the same reaction conditions (left-hand plot). The data in the right-hand plot were obtained by dividing the reaction time
(time after enzyme addition) by the clotting time (tg) and then normalizing each individual curve against its particular value at 3tg.

chymosin concentration were used in each case but
the individual milks exhibit their own gelation times,
tg, rates of curd firming and ultimate gel firmness.
When these curves are re-plotted against a reduced
time, t/tg, and each normalized against its particular
value at a low multiple of tg, usually two or three, all
curves collapse onto a master curve (Fig. 5b) (Horne,
1995). Such behaviour is known as ‘scaling’.
The fact that the curves can be scaled has several
implications. It implies that the mathematical expres-
sion describing the time-dependent growth of the
shear modulus of the curve can be factorized into the
product of two terms and written as:
G(t)  G f 冢 tt 冣
g
(11)
where G is a simple constant, the asymptotic value of
the shear modulus at infinite reaction time for a particu-
lar milk system. As a constant, it embodies all the
static attributes of the gel. The second term, f(t/tg), is
more complicated, but it is nevertheless a function of a
single variable, the reduced time, t/tg, and all data sets
follow this master function which describes the
dynamics of the gel-formation process.
It is important to realize the significance of the
dependence on the critical gel time, tg. It is defined as
the time required to form the critical gel network and
is the rate parameter controlling the aggregation reac-
tion leading to that network. Its presence as the
parameter also controlling the rate of gel cure indi-
cates that essentially the same kinetics is dominating
that reaction. In essence, this is a pointer to the first,
the percolation, scenario as being the more true pic-
ture, with the further incorporation of micelles and
clusters being simply another part of the ongoing
aggregation reaction throughout all phases of gel
development. The gel-development kinetics clearly
depends on the rate of the proteolysis reaction, the
rate controlling the achievement of the gel time.
Lomholt and Qvist (1997) have raised doubts about
this scaling approach, showing that multiplying reac-
tion time by the rate constant for proteolysis, ke, does
not produce a master cure curve in a series of experi-
ments conducted with varying enzyme and protein
concentrations. Our results use tg 1 as the reducing
factor, and other groups have found that the product
of tg and ke is not always unity, as attempting to con-
sider them equivalent would imply (Tokita et al.,
1982). Perhaps the answer lies in the mode of action
of the enzyme and how the activation of the micelle
towards aggregation comes about. Proteolysis estim-
ates give a global figure for the suspension as a whole
which may not relate to the status of individual
micelles, whereas gel times are a direct measure of the
aggregation behaviour. Further work in this area is
undoubtedly required.
A further advantage of the scaling approach is the
observation that f(t/tg) is a single-valued function, in
the sense that any one value of the ratio t/tg leads to a
single value for f(t/tg). That being the case, the value of
G at a defined value of the reduced time, say t/tg  3,
60 Rennet-induced Coagulation of Milk
will always be the same fraction of G. Thus, whilst G
is essentially unattainable, all information on its behav-
iour is accessible through the value of the shear modulus
as the multiple of tg selected for comparison purposes.
Based on this accessibility to G, Horne (1996) has
developed a mechanical model for the gel. If it is
assumed that each micelle has the potential for forming
an average number of bonds, k, with its neighbours,
then these will all be fully linked into the network
attained at infinite time and will therefore define the
value of G. With all of the micelles incorporated into
the network, the number of bonds will be proportional
to the number of micelles present. Hence, according to
equation 10, G will be proportional to the number of
micelles, assuming that all bonds are of equal strength.
If the average micelle is of diameter, d, then at a given
concentration of c g/ml, the number of micelles is pro-
portional to c/d3. Hence, this simple model predicts:
c basis which makes them less useful as predictive tools.
G  (12)
d3
and since G at 3tg is a fixed fraction of G by the scaling
requirements, then
c to describe the increase in shear modulus (G) with
Gat 3tg  (13)
d3
Horne (1996) fractionated casein micelles according
to size from individual cow milks using a sequence
of eight consecutive centrifugation steps, applying
the centrifugation to the supernatant of the previous
step, as described in detail by Dalgleish et al. (1989).
These pellets were re-suspended in milk ultrafiltrate
to the same casein concentration and their average
size was measured independently by dynamic light
scattering. The micellar suspensions were then ren-
neted under the same incubation conditions. Growth
of the elastic modulus was monitored as a function of
time. G* at 3tg was found to be inversely proportional
to the cube of micelle size, as predicted by equation
13. In a separate experiment, milk from a single source
was concentrated by ultrafiltration and then diluted
back to give a series of milks of the same micellar size
distribution but differing in protein concentration.
When these milks were renneted, the value of G* at
2tg was found to be a linear function of the concentra-
tion factor, again as predicted by Horne (1996).
Niki et al. (1994) also prepared sized fractions separ-
ated from skim milk by differential centrifugation. They
confirmed that smaller micelles gave firmer gels but not
as strongly as the inverse cubic dependence predicted.
Their micellar fractions were, however, used as prepared
and not adjusted to a constant protein concentration.
Obviously, those fractions from the wings of the size dis-
tribution have a lower protein content than those
obtained around the mean size. Niki et al. (1994) do not
give details of the protein content or composition of their
fractions, making it impossible to recalculate their data
on the basis of the behaviour predicted by equation 13.
Modelling the Gel-Firming Kinetics
Many attempts have been made to produce mathemat-
ical equations to predict the growth of gel firmness with
time. These range from the purely empirical (Scott
Blair and Burnett, 1958) to those rooted in some
kinetic mechanism (Tuszynski, 1971; Douillard, 1973;
Carlson et al., 1987a; Clark and Amici, 2003). Tested
simply on their ability to fit the observed growth
curves, some are more successful than others which fail
to reproduce salient features. Others have no theoretical
Scott Blair and Burnett (1958) proposed an empirical
model of the form:
G(t)  Gexp 冢 (t 
tg) 冣 (14)
time beyond the gel point occurring at tg,  being a
constant characteristic of the sample and determined
by fitting. Dejmek (1987) demonstrated that this
model provides a good fit to experimentally obtained
cure curves but has non-random residuals, indicating
that the function may not be appropriate for its
intended purpose. Dejmek (1987) also proposed
equations relating the parameters of the Scott Blair
equation to the value of the experimental cure curve
at its inflexion point, obviating the need for measure-
ments at extended times, but relying perhaps too
heavily on a few points in that particular region of the
cure curve. In favour of this model is the observation
that this function shows an acceleratory growth phase
immediately following the gel point, that it evinces a
maximum rate of firmness and tends to a plateau as t
tends to infinity. Against, it is its purely empirical
nature which makes prediction of the dependence of
its parameters on reaction variables such as tempera-
ture, pH or enzyme concentration all but impossible.
Arguing that the shape of the growth curve was
similar to that for simple autocatalytic reactions,
Tuszynski (1971) proposed that the growth kinetics be
fitted by the model:
dG
 kG(G G) (15)
dt*

where t*  t tg, G is the value of the shear modulus
at t   and k is the rate constant for the process but
essentially a fitting parameter. Again, the model pro-
vides no indication how changes in reaction variables
will influence the gel-firming process. Like the Scott
Blair model, it too gives sigmoidal behaviour but it is a
symmetric function, predicting that the inflexion occurs
when G  0.5G, and this is not observed in practice.
Douillard (1973) proposed a model in which the
rate of change of shear modulus with time followed
first-order kinetics:
dG
 k(G G) t*  0 or t  tg (16)
dt*
This equation can be integrated to give:
G  G[1 exp ( kt*)] (17)
with the initial condition that G  0 at t  tg.
This equation has recurred several times in the
history of studies on the rennet coagulation of milk.
Tokita et al. (1982) fitted their cure curves to an
nth-order reaction equation and determined that the
first-order form, the Douillard equation above, gave the
best fit. They further demonstrated that the gel time, tg,
obtained in these studies varied inversely with enzyme
concentration, and that the rate parameter, k, had a
power-law dependence on enzyme concentration with
an exponent of 0.8. In their discussion, Tokita et al.
(1982) extended the consideration of the Smol-
uchowski equation and its use in the kinetics of polymer
gelation by Ziff (1980) and Ziff and Stell (1982) to ren-
net gel formation. Assuming that ‘gel’ reacts with ‘poly-
mer’ in the sol phase and that ‘gel’ does not cross-link
by itself, they show that the concentration of gel is pro-
portional to 1 exp( t). With the further assump-
tion that is proportional to the rate constant of the
Smoluchowski equation and that the elastic modulus is
proportional to the concentration of gel, the Douillard
equation emerges. Tokita et al. (1982) thus reached the
important conclusion that beyond the gel point, most
of the growth in gel firmness arises as a result of aggre-
gation between the infinite cluster and the smaller
clusters and micelles in the sol phase.
In a later paper, Tokita (1989) considered gel forma-
tion as a percolation process. In percolation, the bond
formation probability is defined by N/Ntotal where N is
the number of bonds formed up to that point and Ntotal
is the total number of bonds possible in the system.
Ntotal is the product of the number of lattice sites mul-
tiplied by their functionality, Z, the number of bonds
Rennet-induced Coagulation of Milk 61
allowed for each site. The percolation probability, P(p),
is defined as the probability that a site chosen at ran-
dom will belong to the infinite cluster. With the
assumption that P(p)  1 exp( Zp) for p  pc, the
critical percolation probability, he equated G to P(p)
and went on to derive the Douillard equation. Fitting
his latest data to this equation, Tokita (1989) found
that the reaction rate constant was best described as a
linear function of the enzyme concentration used,
slightly different from their earlier result.
The Douillard equation also emerges as a limiting
case of the Carlson model (Carlson et al., 1987a) dis-
cussed below. Whilst it is thus widely applied, the
Douillard equation does not reproduce one of the most
significant features experimentally observed in the gel
cure curves obtained with modern more sensitive
rheometers, namely the acceleratory phase immedi-
ately after the gel point, and hence further refinements
to it are required. Some of these are to be found in the
Carlson model.
Carlson et al. (1987a) derived the rather complex
model given by:
G  G 1冤 冢 k k k 冣exp(
l
l
f
kf t*)
 冢 k ⴚk k 冣exp(
l
f
f
冥
klt*) (18)
where t*  t tg, kl is the rate constant for the creation
of ‘active’ sites and kf is the rate constant for the
destruction of these sites as they are incorporated into
the gel network. Essentially, the model envisages ‘active’
sites being created on micelles, which then go on to
react with one another in forming bonds in the gel net-
work. Both reactions, activation and destruction, are
considered as first-order processes. Activation requires
the enzymatic hydrolysis of -casein, and therefore kl
emerges as proportional to the enzyme concentration.
That the site destruction reaction should also be a first-
order process is an empirical observation which fits in
well with the idea of the gel network mopping-up
smaller clusters and micelles still requiring to be acti-
vated beyond the gel point. When the enzyme concen-
tration is large, -casein hydrolysis is rapid compared to
the removal of activated micelles and the Douillard
equation is recovered with a rate constant now equal to
that for the activation reaction and therefore governed
by the enzymatic hydrolysis process.
In the hands of the present authors, application of
the Carlson model to gel firming curves gave excellent
fits with very low standard errors (Horne, unpublished
observations). This confirms the views of Esteves et al.
(2001) who compared its performance to those of the
62 Rennet-induced Coagulation of Milk
Scott Blair and Douillard models, although they finally
considered the Scott Blair model superior because it
gave a smaller standard error and lower fluctuations in
the systematic oscillations of the residuals. Unfortun-
ately, as we commented above, the Scott Blair model
has no basis in theory which would permit predictive
use of its parameters. Further efforts should therefore
be directed to more extensive tests of the Carlson
model with perhaps extensions to incorporate the
refinements of aggregation models now extant. More
quantitative testing of the model would also discover
whether the many parameters involved possess realis-
tic values or whether they are merely ‘best-fits’.
Along yet another avenue, Clark and Amici (2003)
have compared the predictions of cascade theory, a ran-
dom cross-linking polymerization theory, with experi-
mental biopolymer gelation curves. The comparison was
made of log (G/G) versus tg/t, a linear transform of the
Scott Blair equation, for calculated and experimental data.
For rennet-induced milk gels they obtained reasonable fits
when the critical gelling concentration (C0) was set much
less than the micellar concentration (C). The calculations
require that the ratio C/C0 be set, so the experimental data
were compared to a series of theoretical curves calculated
for a range of these values. Accepting that the fits are not
outstanding, Clark and Amici (2003) point out that the
cascade model used does not contain any pre-gelation
kinetic terms and, in other studies of polymer gelation
carried out with this theoretical approach, their inclusion
markedly influenced later events beyond the gel point.
These theoretical approaches are very interesting and tan-
talizing but more work is needed to fully explore the
implications of their results before a definitive model of
the cure curve can be achieved.
Fractal Models of Rennet-Induced Milk Gels
and Rearrangements
Fractal aggregation theories have been applied to the
flocculation of casein particles by Bremer and cowork-
ers (Bremer et al., 1989; Bremer, 1992). Aggregates can
be considered fractal if their geometry is scale invariant
which implies that their structure is similar when
viewed over a reasonably large range of length scales or
magnifications. The emphasis of the fractal concept is
therefore on structure. It is a mathematical description
of the distribution of a particle cluster or network in
space. Various models are then used to predict gel or
cluster properties based on that structural organization.
The number of particles in an aggregate or cluster
(Np) is given by:
冢 Ra 冣
Df
Np  (19)
where R is the radius of the floc, a is the primary parti-
cle size and Df is the fractal dimension. The latter is
usually a non-integer and is always less than the geo-
metric or Euclidean dimension of three. This equation
implies that the cluster becomes ever more tenuous as
it grows, as verified in computer simulations of aggre-
gation reactions (Kolb et al., 1983; Meakin, 1983) and
experimental measurements on dilute colloidal sys-
tems (Lin et al., 1990). These results demonstrate that
extremes of reaction probability give rise to different
fractal dimensions, ranging from 1.7 for a diffusion-
limited cluster–cluster aggregation to 2.5 for a reac-
tion-limited particle–cluster process.
Since the number of particles that could be present
in a close-packed cluster is given by:
冢 Ra 冣 ,
3
Nc  (20)
the volume fraction of the cluster is given by:
冢 aR 冣
Np Df
cluster   (21)
Nc
The average volume fraction therefore decreases as the
cluster grows. When it reduces to the volume fraction
of particles in the system, 0, the clusters fill the total
space available and the gel is formed. Bremer and
coworkers (Bremer et al., 1989; Bremer, 1992) define
the gel point by this event which implies that all parti-
cles present in the system are incorporated in the clus-
ters. The real question is whether this can be equated
to the rheological gel point recognized experimentally
but this appears to be the assumption made.
The decrease in density can be accomplished only if
the growing cluster develops holes or voids of ever-
increasing size as the cluster grows. This is the mean-
ing of scale invariance. When such a cluster grows to
macroscopic size, it should have macroscopic holes on
that length scale. No such holes are seen in particle
gels, of which rennet-induced milk gels are our exam-
ples. Instead, they appear as a homogeneous, solid-like
mass. Brown (1987) circumvented this difficulty by
introducing the concept of the fractal blob, suggesting
that clusters grow to a size, Rblob, and these then close-
pack homogeneously to give a uniform volume frac-
tion defined by that of the blob at that point. The
picture of the network is then similar to that depicted
in Fig. 4b, and at the gel point the volume fraction
achieved is again that originally in the suspension, 0.
With all of the particles (micelles) already bonded
into the network at the gel point, the only way in
which gel firmness can grow with time thereafter is
 Rennet-induced Coagulation of Milk 63

through rearrangements of the bonds already in the
structure, a phenomenon referred to as ageing by
Mellema et al. (2002). Various models have been elab-
orated relating the elastic modulus of the gel to the
volume fraction through a power-law equation, with
the exponent of this equation written as a simple func-
tion of the fractal dimension and other possible
parameters (Bremer, 1992). Such models allow the
stress-carrying strands to be straight or curved. Fur-
ther, in the models of Shih et al. (1990), the elasticity
of the gels may be determined by the elasticity of the
flocs or blobs (strong links between blobs) or domin-
ated by the elastic content of the inter-floc links
(weak-link regime). It perhaps should be mentioned
here that Shih et al. (1990) categorically state that
their models apply well above the gelation threshold.
This creates a total of four possible expressions for the
exponent, yet the experimentally observed power-law
dependence of G on volume fraction gives a single
value. In some instances, model candidates can be
eliminated because they yield unphysical values for
the fractal dimension. In others, no choice can be
selected without other independent information. In a
summarizing expression, Mellema (2000) has written
the exponent as:
With this background, Mellema (2000) went on to
consider four levels of rearrangement in rennet-
induced gels, operating on different length scales.
These were intra-micellar rearrangement, individual
micelle shifts, strand rearrangements and in the
whole gel (syneresis), the latter as a result (mainly)
of the previous three categories listed. These possibil-
ities are depicted in Fig. 6. Applying this picture to
the cure curves, Mellema (2000) accommodated
increases in elastic modulus with time by postulating
changes in the parameter , as the gel aged. This
necessitated assuming that the fractal dimension, Df,
was constant throughout, or that its variation with
time was measured independently in separate experi-
ments (Mellema et al., 2000). Gel cure was therefore
interpreted as arising from changes in strand thick-
ness, strand conformation and number of linkages
depending on how was modified but no kinetic
mechanism was derived to predict the dynamics of
these changes and directly test the speculations, no
matter how reasonable.
Milk Processing and Gel Formation
Rennet clotting activity as influenced by milk
processing

 where 2 1 (22) Milk coagulation by rennet can be influenced by a

3 Df
 is the number of junctions or links per strand (0, 1
or 2), and the value of is set by the dominant type of
macroscopic deformation: bending (  1) or stretch-
ing (  0).
number of processing treatments applied to the milk
(Harboe and Budtz, 1999). The gel formation charac-
teristics of high-pressure and heat-treated milks for
cheesemaking have been studied extensively in recent
years. Interest in these areas will be sustained as both
treatments can be used to maximize cheese yield.

Figure 6 Diagrams of the various pathways open in rearrangements of the network. These can occur on several length-scales, at
internal micellar links where the individual proteins rearrange themselves and allow more and more links to be formed between the
original particles, along the chains where particles can detach and reattach forming new links (centre drawing) and the detachment
of chains either at one or both ends leaving them to find a new home elsewhere on the network. All three processes contribute to
macroscopic syneresis of the maturing curd (adapted from Mellema et al., 2002).
64 Rennet-induced Coagulation of Milk
High pressure
High pressure treatment influences the coagulation and
cheesemaking properties of milk indirectly through
a number of effects on milk proteins, including a
reduction in the size of casein micelles, denaturation
of -lactoglobulin and possible interaction of -
lactoglobulin with micellar -casein (Trujillo et al.,
2000, 2002; O’Reilly et al., 2001; Huppertz et al., 2002).
Gel firmness and cheese yield can be improved by high-
pressure treatment of milk through an increased recov-
ery of whey proteins and increased moisture content.
Treatment of milk at pressures of up to 200 MPa for
30 min reduces the RCT while higher pressures, up to
600 MPa, result in RCT values similar to those of
untreated milk (Lopez-Fandino et al., 1996, 1997;
Needs et al., 2000). Changes in RCT observed are
associated with changes in both the enzymatic primary
phase of coagulation and the secondary phase of
aggregation. These changes would be expected to be
associated with changes in micelle size resulting from
high-pressure treatment.
The average casein micelle size is unchanged in
reconstituted skim milk treated at pressures of
150–250 MPa (Desobry-Banon et al., 1994; Gaucheron
et al., 1997), although one report (Needs et al., 2000)
suggests a small increase of 9% in micelle size in raw
skim milk using a pressure of 200 MPa. At pressures
between 250 and 600 MPa, micelle size is reduced by
40–50% in reconstituted skim milk (Desobry-Banon
et al., 1994) or raw skim milk (Needs et al., 2000).
The effect of pressure treatment on micelle size in
reconstituted skim milk is temperature-dependent
(Gaucheron et al., 1997). Pressure treatment of milk at
4 °C reduced micelle size, at 20 °C resulted in no
change and at 40 °C causes an increase in micelle size
which may be associated with interactions between
fragments of casein micelles and denatured whey pro-
teins (Buchheim et al., 1996). Transfer of individual
caseins from the colloidal to the soluble phase has
been observed at pressures of 100–400 Mpa (Law
et al., 1998; Lopez-Fandino et al., 1998).
Treatment of raw milk at a pressure of up to 200
MPa for 30 min reduced the RCT, while further
increases in pressures up to 400 MPa resulted in RCT
values close to those for untreated milk (Lopez-
Fandino et al., 1996, 1997; Needs et al., 2000). The
RCT of pressure-treated milk is affected by both the
temperature of treatment and the pH of the milk. Treat-
ment at 200 MPa at 60 °C or 300 MPa at 50 °C
inhibits the rennet coagulation of milk (Lopez-Fandino
and Olano, 1998). Acidification of milk to pH 5.5 prior
to high pressure treatment reduced its RCT whereas
increasing pH to 7.0 had the opposite effect (Arias
et al., 2000).
The reduced release of CMP during the primary
phase for samples treated at 400 MPa or at 300 MPa
at 40 °C has been associated with the interaction of
high-pressure-denatured -lactoglobulin with glyco-
sylated -casein, which would hinder the action of
chymosin on -casein (Lopez-Fandino et al., 1997;
Lopez-Fandino and Olano, 1998). Blocking agents
have been used to show that high pressure effects
observed on RCT are associated with sulphydryl inter-
actions which cause the -lactoglobulin to bind to the
surface of micelles via interaction with -casein (Needs
et al., 2000). However, Needs et al. (2000) reported
that the release of glycosylated CMP was unaffected by
high-pressure treatment, and only the second phase of
rennet coagulation (rate of micelle aggregation) was
affected. Rates of aggregation and gel formation of
milk treated at 200 MPa were considerably higher than
for untreated milk, but these rates decreased at higher
pressures. Samples treated at 400 or 600 MPa pro-
duced higher gel strengths than samples treated at 200
MPa or untreated samples. The authors concluded that
two opposing mechanisms operate to control the rate
of aggregation – there was a direct effect of pressure on
the properties of the micelles, which resulted in their
rapid aggregation (following the hydrolysis of -casein),
while increasing -lactoglobulin denaturation reduced
the rate of aggregation. Coagulation time was not
related to the degree of -casein hydrolysis, which sug-
gested that pressure favoured the aggregation stage.
Heat treatment
Heat treatment of milk results in a number of changes
in physico-chemical properties. These include the
denaturation of whey proteins, the interactions between
the denatured whey proteins and the casein micelles
and the conversion of soluble calcium to the colloidal
state. High heat treatment of milk for cheesemaking
provides a potential route for maximizing cheese yield
by the inclusion of whey proteins in curd (Singh
and Waungana, 2001). However, milk which has been
heated at a temperature in excess of pasteurization
has poor renneting and gel formation characteristics
(Morrissey, 1969; Dalgleish, 1992), and a number of
studies have explored the extent to which the primary
enzymatic and the secondary phases of aggregation are
influenced by heat treatment (Van Hooydonk et al.,
1987; Dalgleish, 1990; Leaver et al., 1995; Waungana
et al., 1996).
Thermal denaturation of -lactoglobulin is known
to affect the cheesemaking properties of milk. It has
been claimed that heating milk affects the clotting
process by slowing or inhibiting the primary phase of
rennet action as -casein--lactoglobulin cross-linking

reduces the susceptibility of -casein to hydrolysis by
chymosin (Van Hooydonk et al., 1987; Leaver et al.,
1995). The decrease in the rate of gel formation and
final gel firmness in heated milks can also be attrib-
uted to the association of whey protein aggregates
with casein micelle surfaces through the formation of
a -Lg–-casein complex which may protrude from
the micelle surface (Singh and Waungana, 2001). This
association would affect the close approach of the
reactive sites formed on the micelles by the action of
rennet. Following the hydrolysis of -casein to para-
-casein, aggregation would occur mostly between
micelles not fully covered with -Lg, resulting in the for-
mation of fewer bridges with fewer and weaker bonds.
The severity of heat treatment will determine the
extent of inhibition of either the primary enzymatic
phase or the secondary aggregation phase (Singh and
Waungana, 2001).
The rennet coagulation properties of heated milk
can be partially restored either by (i) acidification of
heated milks to pH values below 6.2, (ii) acidification
of heated milk to low pH values (⬃5.5) followed by
reneutralization to 6.7, which is termed pH cycling or
(iii) heating at elevated pH combined with pH cycling
and CaCl2 addition (see Singh and Waungana, 2001,
for a review).
Acidification or pH cycling has been used in the
manufacture of Cheddar cheese from severely heated
milks (Banks et al., 1987, 1993; Banks, 1988; Imafidon
and Farkye, 1993). Improvements in cheese yield of
up to 4.0% on a dry solids basis were achieved (Banks
et al., 1993).
Conclusions
Despite intensive research effort, now spanning many
decades, there is still no definitive overall description
of the kinetics of gel formation which would allow
prediction of the cutting time from a knowledge of
milk composition and treatment. Even now, we are
perhaps only realizing that we can treat the reaction
as a continuum and that we have largely forgotten
that it was mainly the constraints of earlier theories
and methodologies that artificially divided the process
and confined studies to particular stages of aggrega-
tion and curd development. In rheometry, we now
have the instrumentation to directly measure cure
curves and as these instruments become ever more
sensitive, the effects of possible gel damage in the early
stages of the reaction lessen, allowing aggregation and
gelation to come seamlessly together instrumentally.
Exciting new developments in the fields of
microscopy and image analysis allow the potential to
Rennet-induced Coagulation of Milk 65
follow the mobility of particles through the gel point
as they are confined and incorporated into a gel net-
work. This will perhaps settle the question as to whether
gel cure is the result of the firming up of a percolated
initial structure for the gel as sol material is included
into the network or whether the rearrangement process
dominates beyond the gel point, as the fractal models
demand.
The fractal picture is important, however, because
it forces us to confront the role of rearrangement in
determining gel firmness, particularly the shifts in
bonding within the micelle, which we would argue
should be considered within the context of the dual-
binding model of the micelle described earlier.
Whilst the majority of model studies of rennet-
induced gelation have been carried out (fortuitously)
at high pH where micellar integrity seems assured,
many cheesemaking procedures involve a lowering
of the pH by straightforward adjustment or by fer-
mentative growth of starter cultures. Lowering the
pH leads to a solubilization of calcium phosphate, a
consequent decrease in the number of bonds pre-
serving micellar integrity and an increased propen-
sity for rearrangements of protein molecules within
and between aggregated micelles. Horne (2001,
2003) has demonstrated the influence of such break-
down in micellar integrity in the context of gel
development in studies of model yoghurt systems.
The rate at which such processes occur will impact
on the rate of increase of the elastic modulus of the
gel with time but this aspect is yet to be considered
in the context of the dynamics of rennet-induced gel
development. Further work in this area is required
to assess that impact, perhaps by looking for devia-
tions in the scaling behaviour as the gel cures, perhaps
by including terms in the Carlson (1987a,b) model to
accommodate reversibility in the removal of activated
micelles, or perhaps by considering altogether new
models for the network structure and the manner in
which the elastic modulus is related to that structure.
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A novel approach to turbidimetry of dense systems. An
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Zevaco, C. and Ribadeau-Dumas, B. (1984). A study of the
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Ziff, R.M. and Stell, G. (1982). Kinetics of polymer gelation.
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 The Syneresis of
Rennet-coagulated Curd
P. Dejmek, Department of Food Engineering, Lund University, Lund, Sweden
P. Walstra, Department of Agrotechnology and Food Sciences, Wageningen University,
Wageningen, The Netherlands

Introduction (Note: throughout this chapter we will use the word

‘moisture’ for any liquid that may move through curd
Gels formed from milk by renneting or acidification
or cheese; it is thus generally an aqueous solution and
under quiescent conditions may subsequently show
not just water).
syneresis, i.e., expel liquid (whey), because the gel
Thus, the importance of syneresis is obvious.
(curd) contracts. Under quiescent conditions, a rennet-
Accordingly, numerous research reports have been
induced milk gel may lose two-thirds of its volume,
published, providing many important data on the
and up to 90%, or even more, if external pressure is
influence of various factors on the rate, and sometimes
applied. Often, syneresis is undesired, e.g., during stor-
on the end-point, of syneresis. However, the results
age of products like yoghurt, sour cream, cream cheese
vary considerably according to the conditions during
or quark; hence, it is useful to know under what condi-
the test method employed and are difficult to inter-
tions syneresis can be (largely) prevented. In making
pret. Grounds to a deeper understanding of syneresis
cheese from renneted or acidified milk, syneresis is an
were laid in the late 1980s and early 1990s (van Dijk,
essential step. Consequently, it is useful to understand
1982; van den Bijgaart, 1988; Akkerman, 1992; Walstra
and quantitatively describe syneresis as a function of
et al., 1985). Recent overviews were given by van Vliet
milk properties and process conditions, particularly
and Walstra (1994) and Lucey (2001).
when new methods or process steps are introduced in
cheesemaking. This concerns several aspects:
Gel Formation and Properties
• regulation of the water content of the cheese implies
controlling syneresis; The casein micelle
• the rate of syneresis affects the method of process-
As shown in ‘Rennet-induced Coagulation of Milk’, the
ing, and thereby the equipment and time needed,
caseins of milk occur under physiological conditions as
and the losses of fat and protein in the whey;
large polydisperse aggregates, i.e., casein micelles, up to
• rate of syneresis in relation to other changes (e.g.,
0.5 m. Details of the internal structure of the micelles
acidification, proteolysis, inactivation of rennet
are still being discussed, but there is little doubt that
enzymes) affects cheese composition and properties;
the existence of this aggregated state is dependent on
• the way in which syneresis of curd grains proceeds
hydrophobic interactions and on calcium phosphate
may affect the propensity of the grains to fuse into a
nanoclusters connected to the phosphoserines of the
continuous mass during shaping and/or pressing;
individual casein molecules. The solution stability of
• differences in syneresis throughout a mass of curd
the micelles is dependent on the presence of charged
cause differences in the composition of the cheese
groups and steric stabilization (Walstra, 1990). Both of
between loaves of one batch and between sites in
these can be manipulated in dairy processing with the
one loaf;
aim of destabilizing the micelles and promoting further
• after a cheese loaf has been formed, it may still
aggregation of the caseins. The aggregation may lead to
show syneresis and hence loss of moisture.
a gel and then to gel shrinkage, syneresis.
Most of the -casein of the micelles is at the surface
and the strongly hydrophilic C-terminal part of these
(based on Chapter 5 in the 2nd edition of ‘Cheese: Chem- molecules apparently sticks out from the micelle surface as
istry, Physics and Microbiology’, P.F. Fox, ed., Chapman & a flexible chain that perpetually changes its conform-
Hall, London 1993, by P. Walstra, revised and updated by ation by Brownian motion (Walstra and Jenness, 1984),
P. Dejmek). thereby causing steric repulsion, though only a third of
Cheese: Chemistry, Physics and Microbiology, Third edition – Volume 1: General Aspects Copyright © 2004 Elsevier Ltd
ISBN: 0-1226-3652-X All rights reserved
Set ISBN: 0-1226-3651-1
72 The Syneresis of Rennet-coagulated Curd
the surface appears to be covered by -casein (Dalgleish,
1998). The micelles are thus said to be ‘hairy’. They also
have a negative charge, causing electrostatic repulsion
between them. Steric and electrostatic repulsion provide
complete stability of the micelles against aggregation
under physiological conditions.
There are multiple dynamic equilibria between
caseins, and different forms of calcium and phosphate in
the micelles and in the solution. The micelles may
change considerably due to changes in their environ-
ment. At low temperatures, a part of the casein, espe-
cially -casein, goes into solution and additional ‘hairs’
of partly protruding -casein molecules are presumably
formed. A small part of the micellar calcium phosphate
also goes into solution. The micelles attain a higher
voluminosity (i.e., they swell). These changes are
reversible, although it is not quite certain that the
micelles regain exactly their native structure after cool-
ing and rewarming. At high temperatures, the amount of
micellar calcium phosphate increases somewhat. At tem-
peratures high enough for serum proteins to denature,
association of denatured serum proteins with the
micelles occurs, to an extent greatly dependent on pH –
the lower the pH, the stronger the association.
Lowering the pH causes considerable change. Some
trends are illustrated in Fig. 1; in as far as it concerns
properties of a rennet gel, these are discussed later. The
main change is that micellar calcium and phosphate go
into solution, thereby loosening the bonds keeping the
micelles together. This leads to dissolution of casein,
especially at low temperature. At still lower pH, electro-
static bonds between positive and negative groups on
the caseins keep the micelles together, and at the isoelec-
tric pH, these bonds are quite strong, again. In fact, the
casein particles at this pH are very different from the
micelles at physiological conditions, although their size
distribution has not changed greatly (Roefs et al., 1985).
It should also be noted that a lower pH in milk leads to a
higher calcium ion activity, which also lowers the nega-
tive charge on the micelles. Starting at about pH 5
(Vasbinder et al., 2001) and at not too low a tempera-
ture, the casein particles begin to aggregate; electrostatic
repulsion is now absent and the -casein hairs, which
provide steric repulsion, are also lost (they are presum-
ably ‘curled up’). Addition of calcium at a constant pH to
milk reduces the negative charge on the micelles and
increases the amount of micellar phosphate. This
reduces the stability of the micelles and high levels of
added calcium cause their aggregation.
Renneting
During the renneting of milk, the proteolytic enzymes
in the rennet (mainly chymosin) hydrolyse the -casein
molecules to para--casein and soluble caseino-
40
% Ca
Pin
20
30 °C
0
–ζ (mV)
10
20 °C
0
500 20 °C
G ′ (Pa)
250
0
0.4
tan δ
0.2 20 °C
0
4.5 5.5 6.5
pH
Figure 1 The proportion of calcium (Ca) and inorganic phos-
phate (Pin) and the electro-kinetic potential () of casein micelles,
as well as the dynamic shear modulus (G , frequency 1 s 1) and
the loss tangent (tan , frequency 0.01 s 1) of rennet-induced
skim milk gels, as a function of pH (from Walstra, 1990).
macropeptides (the C-terminal region), thereby largely
removing the hairs and greatly reducing steric and
electrostatic repulsion. The micelles can now approach
one another closely and it is observed that they
flocculate, i.e., remain close together. The kinetics
of renneting is intricate since two reactions are
involved. The enzymic reaction is essentially first order
and the flocculation can be described, in principle, by
Smoluchowski kinetics (van Hooydonk and Walstra,
1987). The caseinomacropeptide segments are removed
from the micelles one by one (a micelle contains in the
order of 1000 -casein molecules, and the number of
micelles is roughly 100 times the number of chymosin
molecules normally added to cheese milk). Conse-
quently, the reactivity of the micelles, i.e., the prob-
ability that micelles which encounter each other will
become flocculated, at first remains low but strongly
increases as a greater proportion of the -casein has
been hydrolysed (see also Fig. 2). The reactivity is
roughly an inverse exponential function of the con-
centration of unhydrolysed -casein molecules on the

100
50
S G
V
0
0 20 40
Time (min)
Figure 2 Approximate example of the changes that occur in
milk after adding rennet. Degree of hydrolysis of -casein (S ),
aggregation of para-casein micelles as measured by viscosity
(V ) and shear modulus (G) of the gel formed as a percentage of
the values after 40 min, as a function of time.
micelles. As long as less than about 70% is hydrolysed,
the flocculation rate is virtual1y zero, at least at
physiological pH and 30 °C. If the pH is lowered, the
enzymic reaction becomes much faster and, moreover,
flocculation starts at a lower proportion of hydrolysed
-casein molecules (van Hooydonk et al., 1986). It
appears that at low pH, the chymosin becomes
adsorbed onto the micel1es and this causes the hydro-
lysis of the -casein to be not quite random any more.
Presumably, a chymosin molecule now often makes a
‘bare’ patch on the micelle before becoming desorbed
and diffusing away, to find another (or possibly the
same) micel1e on which to act. At such a bare spot,
the micel1e is reactive. This implies that at a lower pH,
flocculation starts at a stage where less -casein has
been hydrolysed. The reactivity of ful1y renneted
micel1es, i.e., those that are ful1y converted into para-
casein micel1es, depends little on pH, increases with
Ca2 concentration, decreases with increasing ionic
strength (NaCl) and increases markedly with tempera-
ture, especial1y from 15 to 30 °C (Dalgleish, 1983).
Above 50 °C, the flocculation rate becomes almost
independent of temperature, being roughly equal to
that predicted by Smoluchowski’s equation for diffu-
sion-controlled coagulation (Dalgleish, 1983). The
temperature dependence is often taken as indicative of
hydrophobic interactions being responsible for the
reaction between the para-casein micelles (Dalgleish,
1983). An alternative explanation is that with decreas-
ing temperature only the activation free energy for
flocculation increases, presumably because of protru-
sion of -casein chains.
Gel formation
After a while, flocculation leads to the formation of a
gel (see Fig. 2). Microscopically, one can observe that
aggregates are formed, at first irregular, but often
The Syneresis of Rennet-coagulated Curd 73
somewhat thread-like; these grow to form large tenu-
ous flocs, until they start to touch and form a continu-
ous network (Mulder et al., 1966; Henstra and
Schmidt, 1970; Walstra et al., 1985). Electron
microscopy reveals (e.g., Kalab and Harwalkar, 1973;
Knoop and Peters, 1975a; Green et al., 1978) that the
network can be described as consisting of strands of
micelles, 1–4 micelles thick and some 10 micelles
long, alternated by thicker nodes of micelles and leav-
ing openings up to 10 m in diameter.
The essential requirement for gel formation is of
course a thermodynamic instability of the system, i.e.,
an attraction between the particles high enough (rela-
tive to thermal energy) to bring about the formation of
a condensed phase at the existing particle volume frac-
tion. Gels are typically formed in systems where the
range of the interparticle attraction is short compared
to particle size. In addition to short range, the attrac-
tion needs to be sufficiently strong. A weak attraction
allows the particles to asociate and dissociate until
they find a position binding with many neighbours,
and thus form a compact aggregate. When the attrac-
tion is sufficiently large, particles will stick at first con-
tact, and a ramified structure may span the entire
system, provided that the kinetics of aggregation is
faster than the sedimentation of the aggregates formed.
The topology of the resulting network can readily
be described by the theory of ‘fractal’ aggregation
(Family and Landau, 1984; Meakin, 1988). For parti-
cle gel formation, the fractal mechanism was pointed
out in a qualitative sense by Walstra et al. (1985) and
quantitatively applied to the flocculation of casein par-
ticles by Bremer and coworkers (Bremer et al., 1989,
1990; Walstra et al., 1990; Bremer, 1992). The current
view of fractal particle gels in foods was summarized
by Walstra (2000).
Assuming random aggregation of particles and of
aggregates already formed (called cluster–cluster
aggregation), computer simulations show the aggre-
gates formed to be stochastic fractals, i.e., structures
that are on average scale-invariant at scales larger than
that of the primary particles (radius a). The number of
particles in an aggregate or floc is given by:
冢 Ra 冣
D
Na  (1)
where R is the radius of the floc and D the fractal
dimensionality, which is always smaller than three.
This implies that the floc becomes ever more tenuous
as it becomes larger; computer simulations show rare-
fied structures, consisting mainly of long irregular
strands of particles, which are in most places only one
particle thick. Equation (1) has been shown to hold
74 The Syneresis of Rennet-coagulated Curd
remarkably well over a wide range of R and under
many conditions, both in simulations and experi-
ments; colloidal interaction forces and geometrical
constraints determine the value of D. The number of
particles that could be present in a floc if the particles
were closely packed, obviously is:
冢 Ra 冣
3 potentials can affect the properties of the gel network
Na  (2)
This implies that the average volume fraction of parti-
cles in a floc is given by:
冢 Ra 冣
NP D 3
floc   (3)
Na
The average volume fraction of the flocs thus
decreases during flocculation, and when it has reached
the volume fraction of particles in the system,  (for
para-casein micelles at 30 °C, about 0.09), the flocs fill
the total space available and a gel has formed. It also
follows that the average radius of the flocs at the
moment of gelation is given by:
Rgel  a1/(D 3) (4)
In the above derivation, it has been implicitly assumed
that flocculation proceeds undisturbed. But if the liq-
uid is stirred during flocculation, gel formation may be
hindered. Another disturbance may be appreciable
sedimentation of the flocs occurring before a gel can
be formed. The casein micelles in milk are small
enough, and differ little enough in density from the
milk serum, for sedimentation to be negligible. It may
thus be assumed that under normal renneting condi-
tions, gel formation occurs unhindered.
If equal-sized spherical particles flocculate in
Brownian motion and if each encounter leads to last-
ing contact (so-called diffusion-limited cluster–clus-
ter aggregation), the fractal dimensionality turns out
to be about 1.8. Several deviations from this simplest
model, for instance a situation (as during renneting)
in which only a certain small proportion of the
encounters of particles leads to their lasting contact
(so-called chemically limited aggregation), or
rearrangements occurring in the floc structure, lead
to higher D values. Moreover, during gel formation,
the flocs interpenetrate to some extent and this also
causes a higher dimensionality. One type of change
that certainly does occur in the flocs is a rearrange-
ment of just-flocculated particles in such a way that
each particle will touch more than two other parti-
cles; this leads, in principle, to strands of thickness of
about three particles rather than one (Meakin, 1988).
This is in agreement with microscopical observations
on casein gels (Bremer, 1992). Such a rearrangement
does not detract from the initial fractal nature of the
flocs or the gel formed from the flocs.
Brownian dynamics is a tool which has been used to
probe theoretically how different choices of interaction
(Bijsterbosch et al., 1995; Bos and van Opheusden,
1996; Mellema et al., 1999; Dickinson, 2000; Rzepiela
et al., 2001). The findings modulate the simple irre-
versible fractal description; the low cut-off of the fractal
regime, i.e., the size of the building block typically
increases in time, and the fractal dimension may
depend on both the interactions and the volume frac-
tion. For low-capture efficiency, the fractal dimensional-
ity tends to 2.35 (Walstra, 2000; Mellema et al., 2002b).
For real casein gels, volume fractal dimensions have
been determined experimentally by a variety of meth-
ods, including wavelength dependence of turbidity,
angle dependence of light scattering and analysis of
electron or confocal microscopy images. The values
most commonly found are D  2.2 2.4 (Bremer et al.,
1989, 1990; de Kruif et al., 1995; Mellema et al., 2000).
The experimental values of D obtained are, however,
much dependent on the evaluation procedure (Mellema
et al., 2000) and the assumptions involved in interpre-
tation of the experimental data (Bushell et al., 2002).
Worning et al. (1998) questioned the validity of the
turbidity approach, and the same group found by light
scattering, D  2 (Lehner et al., 1999).
Assuming the radius of para-casein micelles to be
55 nm and their volume fraction in milk to be 0.09, it
is calculated that the average radius of the flocs at the
onset of gelation is about 2.5 m and that these flocs
contain several thousand para-casein micelles. There
is, however, considerable spread in these values within
one gel, and the gel is thus fairly inhomogeneous (see
Fig. 3). The average pore size in the gel is of the order
of Rgel but some pores are larger. Average pore size is
related to the permeability, B, in the equation of Darcy:
v 冢 B 冣p (5)
which relates the superficial velocity, v, of a liquid of
viscosity, , flowing through the gel due to a pressure
gradient p. The permeability of a ‘fractal’ gel is, under
some assumptions, given by:
B  const. a22/(D 3) (6)
The constant is not easily calculated; it is much smaller
than unity. For D  2.3, the power of  is about 2.9
 The Syneresis of Rennet-coagulated Curd 75

Figure 3 Optical sections, made by confocal scanning laser microscopy in fluorescent mode, of rennet-induced skim milk gels,
aged for 1 h (top) or 18 h at 30 °C. The bars indicate 10 m (from Bremer, 1992).

(in agreement with experiments), which implies that
the permeability of the gel depends strongly on the
initial  and thus on casein concentration. A similar
strong dependence on  holds for some other
properties and for the size of the flocs at the onset of
flocculation.
Above, it has been tacitly assumed that skim milk is
renneted. In the presence of fat globules, flocculation
and gel formation proceed somewhat differently, but
not greatly. The pores in the gel of para-casein micelles
are roughly large enough (about 4 m) and sufficient
in number (about 2.1016 m 3) to accommodate the fat
globules (average diameter – about 3.4 m; number of
globules larger than 1 m – 3.1015 m 3; Walstra and
Jenness, 1984). Nevertheless, the pore size distribu-
tion in the gel is, of course, somewhat influenced by
the presence of the fat globules, and most fat globules
are entrapped in the gel.
Rheological characteristics
The discussion will be based mainly on the extensive
results of Zoon et al. (1988a,c, 1989a,b). A convenient
and easily observed characteristic of a gel is its small
76 The Syneresis of Rennet-coagulated Curd
deformation modulus, i.e., the ratio of the applied
stress over the resulting strain (relative deformation).
Mostly, the dynamic shear modulus, G, is determined
(which implies that the deformation type is simple
shear) as a function of the frequency of deformation, .
Most gels are visco-elastic materials and these are char-
acterized by two parameters. The storage modulus, G ,
is a measure of the true elastic property of the gel, the
loss modulus, G, of the viscous property; G/ can be
seen as a viscosity. We further have G2  G 2  G2. In
these dynamic measurements, the material is brought
under an oscillating small strain, and G and G can be
determined separately, each as a function of ; the
timescale of the deformation is about  1. Values of G
are shown in Fig. 1. The moduli were observed to
depend generally on  and to increase steeply with ,
in agreement with the theory of fractal gels (Bremer
et al., 1990; Bremer and van Vliet, 1991). The model
predictions linking fractal dimensionality to rheological
properties need to take into account the topology and
the connectivity of the network, information which is
not contained in the fractal dimensionality (Roberts
and Knackstedt, 1996; Mellema et al., 2002a).
An important parameter is the loss tangent (tan  
G/G ), as it is a measure of the preponderance of vis-
cous (or liquid-like) or over-elastic (or solid-like)
properties of the gel. It is related to the relaxation of
bonds in the gel during its deformation, and therefore
it mostly increases with increasing timescale (decreas-
ing oscillation frequency); this is because, in general, a
greater proportion of the bonds that are under stress
can relax when the timescale is longer. For rennet milk
gels at physiological pH and 30 °C, tan   0.45 0.6
at   10 3 s 1, i.e., under conditions relevant for
syneresis. This implies that a rennet milk gel has a sig-
nificant viscous component in its rheological behav-
iour. In accordance with this, it is observed that its
relaxation time, i.e., the time needed for the stress to
decrease to 1/e of its initial value if a certain small
deformation is applied to the material, is of the order
of only 1 min. The loss tangent does not depend on
casein concentration and is virtually independent of
the age of the gel once formed.
The modulus of the gel strongly increases after it is
formed (see Fig. 2). Potentially, the increase could be
a b
Figure 4 Schematic picture of the change in conformation of flocculated para-casein micelles during ageing of the gel (from
Walstra and van Vliet, 1986).
due to two phenomena. One is that additional junc-
tions are formed between casein particles, partly
because there are strands of particles that are attached
to the gel at only one end, partly because additional
casein particles and small clusters thereof become
incorporated into the gel. The latter situation will
always occur to some extent during the formation of a
particulate gel, but more strongly during normal ren-
neting, since at the moment of gel formation not all
casein micelles have been fully transformed into para-
casein micelles. However, only a negligible amount of
free casein was found in the whey from a rennet milk
gel quite early in the renneting process, at G less than
10% of its ultimate value (Mellema et al., 2002b), and
similar values were found in simulations (Mellema
et al., 1999).
The other phenomenon is illustrated in Fig. 4,
which is derived from electron microscopical studies
(Knoop and Peters, 1975b). Any ‘junction’, by which
is meant a contact region between two original
micelles, must contain several bonds, and the number
of bonds per junction increases on ageing. One may
say that the micelles more or less fuse, and after some
hours the original particles making up the gel can no
longer be distinguished. If no starter is added and the
proteolytic enzymes of milk have been inactivated,
the increase in modulus continues for about 24 h
(Zoon et al., 1988a). The lower the temperature, the
slower and the longer-lasting is the increase in modu-
lus. As mentioned, the increase in the number of
bonds does not lead to a significant change in the loss
tangent.
For deformations (in shear) larger than about 3%,
the rheological behaviour of developed rennet milk gels
becomes non-linear; during the early stages of gel devel-
opment, the linear range is wider. In curd-making prac-
tice, the stresses applied are often too large for linear
behaviour. Figure 5 shows what happens when a rela-
tively large stress is applied (Zoon et al., 1989b). After
the instantaneous (elastic) response, the deformation
soon becomes virtually viscous, i.e., d/dt is constant.
After some, often fairly long, time, the deformation rate
increases and eventually becomes infinite – the gel frac-
tures. Fracture does not mean falling into pieces, but
rupture of the gel matrix only – the cleft formed fills
c d

238 Pa
Deformation (γ)
68 Pa
1
35 Pa
0
0 100
Time (s)
Figure 5 Deformation in shear () of a gel of renneted skim milk as a function of time, applying a constant stress. Temperature:
30 °C, pH: 6.65, gel aged for 3.5 h. The stress applied is indicated near the curves. At 35 Pa, fracture occurred after 1350 s (from
results of Zoon et al., 1989b).
with whey immediately. Presumably, local fracture
occurs already at an early stage, as soon as the linear
deformation range is exceeded; the small cracks formed
increase slowly in size and number, and coalesce until a
fracture plane throughout the whole test piece has
formed. This implies that long before macroscopic frac-
ture, the gel structure has been altered markedly, which
has been confirmed in loading–unloading experiments
(Zoon et al., 1989b). Note that the shear at fracture is
very large; values between 1 and 5 have been obtained
(van Dijk, 1982; Zoon et al., 1989b), according to con-
ditions. It is seen (Fig. 5) that a higher stress leads to
smaller deformation at fracture and to a much shorter
time than needed for fracture to occur. In other words,
at a shorter timescale, the fracture stress is higher. Like
the modulus, the fracture stress increases with ageing of
the gel. The results of experiments at large deformations
depend on the type of test applied (e.g., creep or
dynamic), but the same trends are observed.
Attempts to gain insights into the causal relation-
ships between the range and the strength of forces
between particles and network flow behaviour are
being made by simulations (Whittle and Dickinson,
1998; Dickinson, 2000; Rzepiela et al., 2002).
Temperature has a big effect on gel properties (Zoon
et al., 1988b, 1989b). One should, however, distinguish
between temperature of renneting and of measuring rhe-
ological properties. If renneting is at a lower tempera-
ture, gel formation is much slower and the modulus of
the gel may consequently be smaller when measured at
the same time, but this is not a true representation of the
effect of temperature on gel properties. Lowering the
The Syneresis of Rennet-coagulated Curd 77
200 300
temperature of a formed rennet milk gel generally causes
a very brief decrease in modulus, but the latter subse-
quently starts to increase to reach a constant higher level
after, say, 1 h. At   10 3 s 1, the storage modulus at
20 °C is about 2.4 times that at 30 °C. From the effect of
temperature on the loss tangent it is seen that a rennet
milk gel is much more solid-like at lower temperatures;
similar behaviour could be seen even in a non-renneted,
centrifuged pellet of casein micelles which gels at a low
temperature (Horne, 1998).
The permeability tends to be higher at higher tem-
peratures; this will be considered later. At large
deformations, a higher temperature causes a larger
deformation and a lower stress at fracture. Some
effects of acidity (Zoon et al., 1989a; Roefs et al.,
1990) are shown in Fig. 1. Again, one should distin-
guish the pH of renneting from that at measurement,
since renneting at a lower pH causes faster gelation.
Figure 1 gives results obtained several hours after ren-
neting at the pH values indicated, and it is seen that
the storage modulus at first increases with decreasing
pH, to decrease again at still lower pH values; the loss
tangent continues to increase, until the pH range
where a ‘rennet reinforced acidic gel’ (Tranchant et al.,
2001) begins to develop. At large deformations, the
effects of moderate acidity are not great (Zoon et al.,
1989b); the fracture stress is somewhat higher for a
lower pH, if determined at the same timescale.
Acid gels
The casein particles at pH 4.6 are rather different from
those at physiological pH, as is illustrated in Fig. 1. They
78 The Syneresis of Rennet-coagulated Curd

are very prone to aggregation (except at low tempera- ‘Formation, Structural Properties and Rheology of Acid-
ture), they contain no undissolved inorganic phosphate coagulated Milk Gels’, Volume 1.
and they have a (presumed) voluminosity at 30 °C of
about 3.4 ml g 1 (leading to   0.08 in skim milk).
Casein particles from strongly heat-treated milk differ Syneresis
significantly in their properties, and due to their associ- Mechanism of syneresis
ated whey proteins may behave as -lactoglobulin during
acidification (Vasbinder et al., 2001) and recently it was Various mechanisms have been held responsible for
recognized that thiol cross-linking occurs in acid gels syneresis (for an early review, see Walstra et al., 1985).
from heat-treated milk (Vasbinder et al., 2003). Unmodi- Summarizing, the following types of mechanism were
fied caseins form a gel at temperatures above about distinguished:
10 °C. Gel properties have been studied extensively • A decrease in solvation or water binding of the
(Roefs and van Vliet, 1990; Roefs et al., 1990a; Lucey material making up the gel. For a particulate gel,
et al., 1997a,b) including those of gels made by the com- this way of explaining syneresis does not appear
bined action of acid and rennet (Roefs et al., 1990b; suitable and there is no indication that an ongoing
Lucey et al., 1998, 2000, 2001; Tranchant et al., 2001) change in solvation is involved.
and gels from transglutaminase-cross-linked micelles • Shrinkage of the building blocks of the gel, i.e., the
(Schorsch et al., 2000). The gels are in many respects para-casein micelles in our case. This may happen
quite comparable to renneted milk gels (Table 1). They when the pH is lowered or the temperature
are also of a fractal nature and have roughly the same increased, but syneresis also occurs under constant
fractal dimensionality and thereby about the same conditions.
dependence of modulus and of permeability on casein • Rearrangement of the network of para-casein
concentration (Bremer et al., 1989, 1990). The absolute micelles. This is the main cause of syneresis.
value of the permeability is also roughly the same, as is
the pore size distribution. The rheological properties are, The para-casein particles in the gel form junctions
however, rather different. They are treated separately in with a limited number (mostly 2–4) of others. (Strictly
speaking, this is not true. As mentioned earlier, there
is a rapid rearrangement into thicker strands, leading
Table 1 Properties of skim milk gels obtained by renneting to a higher coordination number. However, one may
(aged for about 1 h) or by acidification (aged for 6–16 h). Acid use the same arguments by considering the ‘particles’
gels are of type 1 (obtained by cold acidification and subsequent to be aggregates of, on average, three micelles.) How-
warming) or of type 2 (obtained by slow acidification with glucono-
-lactone at 30 °C). Approximate results at 30 °C (from various
ever, the particles are expected to be reactive over their
sources) entire surface (or to contain numerous reactive sites
smeared out over their surface), and in the initial frac-
Acid gel tal network, by far the greater part of the surface of
each particle does not touch (form bonds with)
Property Rennet gel Type 1 Type 2
another one. Rearrangement of the particles into a
pH 6.65 4.6 4.6 more compact network would thus increase the num-
G at   ber of bonds and hence decrease the total free energy
0.01 rad s 1 (Pa) 32 180 20 (the counteracting loss in mixing entropy is very
Tan  at  
small). But the particles cannot easily attain a more
0.01 rad s 1 0.55 0.27 0.27
Fracture compact configuration because they are almost immobil-
stressa (Pa) 10 100 100 ized in the network. In other words, the network has
Fracture to be deformed locally to form new junctions.
straina ( ) 3.0 0.5 1.1 Thermal motion of the strands may occasionally
Permeability
bring two particles in different strands close to each
B (m2) 0.25 0.15 0.15
Fractal other so that a new junction is formed, especially
dimensionalityb 2.23 2.39 2.36 shortly after renneting. This would lead to the build-up
dB/dt (nm2 s 1) 20 1 – of a tensile stress in at least some of the strands. The
Initial syneresis fusion process illustrated in Fig. 4 may also cause such
ratec 15 1 1
a stress to develop. As a result, strands may occasionally
a Loading time 1000 s.
break, providing a possibility for the formation of more
b From the relation between concentration and B. new junctions, thereby tending to make the network
c Arbitrary units. contract. These events are illustrated in Fig. 6. Even if

Figure 6 Schematic representation of strands of para-casein micelles forming new cross-links, leading to breaking of one of the
strands (from van Dijk, 1982).
no syneresis would follow, the changes mentioned
would cause the strands of particles to become straight-
ened. This is indeed in agreement with the relation
found between the modulus and the volume fraction of
particles making up the gel (Bremer et al., 1990).
The propensity of the strands to break has been
carefully studied by van Vliet et al. (1991). They con-
cluded that spontaneous breakage is possible if (1) the
bonds in a junction can relax, and (2) the number of
bonds in a junction is not too high. If the first condi-
tion is met, this is reflected in the loss tangent being
fairly high on the timescale considered; the second is
met if the strands are (locally) only one-particle thick
and the junction zones fairly small (small particles, lit-
tle fusion). For normal para-casein micelle gels, the
critical loss tangent appears to be about 0.4, syneresis
being stronger at a higher tan . To say it in other
words, the activation free energy for the breaking of
bonds should be fairly low for syneresis to be possible.
But also the activation free energy for bond formation
should be fairly low, since otherwise no new junctions
will be formed.
Attempts to unify the approach to gel formation and
syneresis
It has been earlier recognized that casein gels are trans-
ient networks (Bremer, 1992; Bijsterbosch et al., 1995).
In the last decade, a unified theoretical framework for
the formation of more or less transient particle gels has
been proposed (Poon and Haw, 1997; Tanaka, 1999;
Prasad et al., 2003), not least due to the convenient
experimental system of colloid particles in a non-
interacting polymer solution. In such a system, because
the centre of gravity of the dissolved polymer is steric-
ally excluded from the region close to the particle sur-
face, i.e., the surface region is depleted of the polymer,
an effective attractive short range potential between the
particles is created. Both the strength of the attraction
(via osmotic pressure of the solution) and its range (via
the polymer size) can be manipulated easily.
The Syneresis of Rennet-coagulated Curd 79
The framework attempts to unify the description of
all phase separation phenomena, including gel form-
ation. The gel is considered as a possible transient
phenomenon on the way to full phase separation or to
a final arrest of the phase separation process through
glass transition. A general theory of viscoelastic phase
separation has been developed by Tanaka and collabor-
ators (recently reviewed by Tanaka, 2000). The theory
proposes the formation of a transient gel as a possibil-
ity in any system in which one of the emerging sepa-
rating phases has dynamics much slower than the
other, covering both polymers and colloidal systems.
In this approach, after the initial diffusion-
controlled spinodal decomposition, diffusion is hin-
dered by the viscoelasticity, or more specifically the
bulk and the shear moduli of the emerging ‘slow’
phase. The bulk modulus of the gel phase, which is
assumed to include contributions from the excluded
volume, the network topology and the particle binding
energy, and which need not necessarily scale the same
way as the shear modulus, is also the origin of gel con-
traction, i.e., microscopic syneresis. In very late stages
of the process, it becomes once again dominated by
the, by then slow, diffusional processes. In simula-
tions, the model correctly predicts the morphology of
the resulting gels both in polymers (Araki and Tanaka,
2001) and in colloids (Tanaka et al., 2003). The tran-
sient gel model clearly emphasizes that the apparent
‘equilibrium’ properties of the gel are the consequence
of a drastic slowing down of an ongoing process and
can thus not be expected to correlate neatly with the
thermodynamic state variables such as composition,
temperature and pH.
There are several attractive features in the viscoelas-
tic phase separation model. One is that it offers a
predictive dynamic model which relates the thermo-
dynamic driving force for separation on a molecular
level to the relaxation bulk and the shear moduli of
the gel. Another is that it allows in the same general
framework modelling of both the early stages of the
aggregation, where the casein micelles can be treated
80 The Syneresis of Rennet-coagulated Curd
as individual colloid particles, and the late stages
where a fused casein phase may be better modelled as
a viscoelastic fluid.
The thermodynamical driving force, the gradient of
free energy of mixing, can be related to independently
measurable properties of the system. For renneted
casein, the attractive energy has been derived from
measurements of viscosity and light scattering, evalu-
ated with the adhesive hard sphere model pioneered in
the dairy context by de Kruif (de Kruif et al., 1992,
1995; Mellema et al., 1999) and it should even be
accessible to direct measurement in AFM (atomic force
microscope). AFM could also give a direct answer to
the much-discussed issue of whether a renneted or
acidified casein micelle can be regarded as uniformly
attractive or as possessing ‘hot spots’.
As mentioned earlier, attempts have been made to
relate the rheological properties of the network to
observables such as fractal dimensionality. It has
become clear that the fractal dimensionality and volume
fraction of the gel phase do not completely determine
the rheological properties of the network; further topo-
logical assumptions or facts are needed (Roberts and
Knackstedt, 1996; Mellema et al., 2002a,b). Confocal
scaning microscopy (CSM) can provide topographical
information which can then be transformed directly
into more model-independent rheological properties
(Mellema et al., 2000), or, with time resolved CSM it is
possible to evaluate directly the rheological properties
of the network based on the observed movement of the
network components under thermal (Brownian) excita-
tion (Dinsmore and Weitz, 2002).
The above-mentioned hopes for more general pre-
dictive theories may be overoptimistic (Walstra, 2003);
gels are not homogeneous and relevant phenomena
occur on many length scales, involving different types
of bonds, and are therefore not easily interpretable by
localized studies such as microscopy, and pose formid-
able problems to Brownian simulations.
Syneresis of renneted milk
Under typical cheesemaking conditions, if the gel is
formed undisturbed and sticks completely to the wall of
the vessel in which it is formed (e.g., clean glass), it usu-
ally shows no apparent syneresis, at least if the vessel is
not too large and has vertical walls, and if the tempera-
ture is not too high (e.g., 30 °C) (van Dijk, 1982).
Apparently, the gel is now constrained and cannot
shrink. Spontaneous syneresis is observed if the milk is
renneted in a conical flask; presumably, the gel tears
loose from the glass wall by gravity before it is fully set.
Similarly, spontaneous syneresis may occur in a cylindr-
ical glass if it is tilted slightly for a moment during setting.
After the gel has become firmer, it can withstand a
greater disturbance without exhibiting spontaneous
syneresis. Usually, it does not show syneresis at the milk
surface, either. The composition of the milk surface is
not known with certainty. It may be lipid-rich or may be
covered by protein, presumably -casein (Holt and
White, 1999), oriented with its hydrophobic chains
towards the air. If this surface layer is bonded to the
para-casein matrix, the hydrophobic air interface must
be wetted to allow the serum to leave the matrix, and
thus the matrix to shrink, thus it is essentially the capil-
lary forces that prevent spontaneous syneresis. As soon
as the gel is cut or the surface (locally) wetted, syneresis
occurs. This effect permits experimentally starting
syneresis at any desired moment after a gel has formed.
These observations imply that in a constrained milk
gel, no syneresis occurs. However, the processes
depicted in Fig. 6 will nevertheless occur; there is no
reason to suppose they would not. This implies that
on a local scale, the gel network becomes more dense;
this has been called microsyneresis (van Dijk, 1982;
Walstra et al., 1985). At the same time, the network
will become less dense at other sites; these changes are
shown in Fig. 3. The surface-weighted average pore
size will thus increase and it is indeed observed that
the permeability of a constrained gel keeps increasing
(see Fig. 7). It may be argued that the rate of change
of the permeability, dB/dt, is a measure of the tendency
of the gel to exhibit syneresis.
Until now, only the inherent or endogenous tendency
of a gel to show syneresis has been considered. Exerting
a stress on the gel may be expected to speed up greatly
the expulsion of whey, because of the increased pressure
(see equation 7). Moreover, it may enhance syneresis by
1.5 5.35
5.75
1.0
B (μm2)
5.97
0.5 6.33
6.68
0
0 1 2 3
Time (h)
Figure 7 Permeability, B, of rennet-induced skim milk gels of vari-
ous pH (indicated near the curves) as a function of time after ren-
neting. Temperature 30 °C (from results by van den Bijgaart, 1988).

bringing strands of the network closer to each other and,
perhaps more importantly, it will enhance breaking of
strands, thereby providing a greater possibility for the
number of junctions to increase. As was discussed above
in relation to Fig. 5, deformation of the gel also causes
local rupture of the network, thereby increasing its per-
meability. Indeed, local densification of the structure and
the formation of empty holes was predicted in simula-
tions which specifically included imposed deformation
(Rzepiela et al., 2002).
One-dimensional syneresis at constant conditons
In this section, the detailed investigations by van Dijk,
van den Bijgaart and coworkers (van Dijk, 1982; van
Dijk et al., 1984; van Dijk and Walstra, 1986; van den
Bijgaart, 1988), as complemented by the recent work
by Lodaite (2002), Lodaite et al. (2000, 2002), Unger
Grundelius (2004) and Unger Grundelius et al. (2000),
will be discussed. They studied horizontal slabs of ren-
neted milk, the top of which was moistened at a prede-
termined time after renneting, after which syneresis
was followed by measuring the change in height, h, of
the slab; whey could flow out only at the top. The milk
was brought to the desired pH and the apparatus was
kept at a constant temperature. Examples of results are
shown in Fig. 8. The diameter of the cylindrical slabs
was much larger than their thickness (mostly 5 mm).
In this way, one-dimensional syneresis under constant
conditions could be determined. This is, of course, an
over-simplification of the situation during actual curd-
making, but it allowed precise and unequivocal deter-
mination of syneresis under various conditions,
providing insight into the processes occurring, and per-
mitting the development and the testing of a simple
mathematical model.
10
8
6
h (mm)
4 since the rate of syneresis, v, B, l and  can be meas-
2 thereby ps, because pg can also be calculated. The cal-
0
0 2 4 6 8 10 ∞ • permeability increases with time (Fig. 7);
Time (h)
Figure 8 The height of slabs of renneted skim milk of various
initial height, h, as a function of time after initiation of syneresis.
Temperature 30 °C, pH 6.7. The values at infinite time are from
extrapolation of log–log plots (from results by van Dijk, 1982).
The Syneresis of Rennet-coagulated Curd 81
In parallel experiments, the permeability, B, and its
change with time, dB/dt, were determined. Unless stated
otherwise, the results pertain to renneted skim milk.
Modelling the process
The gel can be considered as two inter-penetrating
continuous spaces, one consisting of the para-casein
matrix and the other of whey. If there is to be macro-
scopic syneresis, the para-casein matrix must contract
and the whey move in the opposite direction. This rela-
tive movement is accompanied by a friction force,
proportional to the relative velocity between the
matrix and the whey. This frictional resistance against
flow can be described by the equation of Darcy (equa-
tion 5), which is conveniently written as:
v 冢 B 冣 " 冢 pl 冣 (7)
where v is the relative superficial velocity of the liquid
in the direction of l, the distance over which the liquid
has to flow. The pressure causing the syneresis can, in
general, be written as:
p  ps  pg  pc (7a)
which terms are, respectively, the endogenous synere-
sis pressure, the pressure exerted by the network itself
due to gravity and any external pressure applied to the
network. Results for pc  0 will be discussed first;
note that pg varies from zero at the top of the slab to a
maximum of g h ! at the bottom, e.g., 1 Pa for a 1 cm
slab (!  density difference between the para-casein
network itself and the interstitial whey).
Attempts to directly measure ps failed; its value was
too small. Only the order of magnitude could be esti-
mated, and it was 1 Pa (van Dijk et al., 1979; van Dijk,
1982). This is a very small pressure – it corresponds to
the pressure exerted by a water ‘column’ of 0.1 mm
and, as seen in Fig. 8, unaided syneresis is indeed very
slow – it takes 7 h at 30 °C for a 6-mm slab to be
reduced to 3 mm. Since equation (7) must hold, and
ured, it is possible to determine p indirectly and
culation is, however, very intricate, because:
• permeability becomes smaller because of syneresis;
• most likely, endogenous syneresis pressure also
varies with ongoing syneresis;
• pressure due to gravity changes as well;
• the coordinates change with syneresis.
82 The Syneresis of Rennet-coagulated Curd
Consequently, most variables vary with time and
location. Syneresis will start in the uppermost layer,
thereby altering its permeability, etc., and progressively
reach deeper layers. A finite difference model was devel-
oped (van Dijk et al., 1984), in which the slab was
divided into parallel thin slices, to each of which equa-
tion (7) and the equation of continuity were applied to
calculate the outflow of liquid in small time intervals. By
inserting various values for ps and comparing the com-
puted results with the observed h as a function of time,
the endogenous syneresis pressure could be derived.
By assuming p and B to be constant, which may be
assumed to be the case at the very beginning of synere-
sis, an analytical solution can be found (Biot, 1941),
which is mathematically equivalent to the solution of
the diffusion equation (Tanaka and Fillmore, 1979).
This would imply that h changes proportionally to the
square root of time and directly yields the initial endoge-
nous syneresis pressure, ps0. This proved not to be the
case. In careful experiments, an initial proportionality
with time t, the power of about 3/4 was obtained by van
den Bijgaart (1988) and about 1 by Lodaite et al. (2000).
The explanation probably is as follows (van den
Bijgaart, 1988) – an implicit assumption in the applica-
tion of equation (7) is that the network can without sig-
nificant resistance comply with the outflow of whey. But
the initial shrinkage rate of the outermost layer would
then be very high (the proportionality with square root
of time even implies an infinite rate at t  0) and that is
clearly not possible.
A significant effort went into the refinement of the
model and the numerical fitting to calculate the intrin-
sic syneresis pressure as a function of time and process
conditions, such as in Fig. 9. The observed trends in
the intrinsic pressure could be qualitatively explained
2 6.33
ps (Pa)
6.48
1 ity of that layer becomes very low and its modulus high,
6.68
0
0 1 2 gel is firm enough to allow the estimation of B.
Time (h)
Figure 9 The endogenous syneresis pressure (ps) of rennet-
induced skim milk gels as a function of the time elapsed after
renneting when syneresis was initiated, at various pH (indicated
near the curves). Temperature, 30 °C. The broken lines are
assumed (from results by van den Bijgaart, 1988).
a posteriori, however, the prediction power of the model
remained poor.
A possible explanation could lie in the fact that
equation (7) is formulated for the liquid space of the
network. This means that the forces needed for the
deformation of the matrix are not considered explicitly.
The intrinic syneresis pressure is thus the observable
difference between the balance of the thermodynamic
forces attempting to reach a new equilibrium, the even-
tual external forces and the forces caused by the vis-
coelastic properties of the matrix. The magnitude of
the latter depends on the deformation of the matrix,
the deformation rate and the deformation history at
each point in time and space. An order of magnitude
estimate can be based on Fig. 10. Some 8 Pa external
pressure doubled the initial rate of syneresis; thus, the
viscoelastic resistance of the matrix would seem to be
almost an order of magnitude larger than the observed
intrinsic syneresis pressure.
While the derived intrinsic pressure is not easily
interpreted, one can argue that the same balance of
thermodynamic and viscoelastic forces that causes the
macrosyneresis is also reflected in microsyneresis, and
thus the rate of change of permeability should be a
direct index to the intrinsic rate of syneresis.
Even in the measurement of permeability there are
some caveats. While the observed constrained perme-
ability and its rate of change, measured by the stand-
ard tube method was found to be independent of curd
column length and driving pressure difference by van
Dijk (1982), it was found to depend on both the size
of the tube and the driving pressure difference by
Unger Grundelius (2004).
Some important results are given in Fig. 11. The
variables, temperature and pH, affect dB/dt in a similar
way, in accordance with the ideas outlined above. All
these trends would cause faster syneresis at a higher
temperature and lower pH; this is indeed observed. An
additional complication, which will lead to syneresis
being given an additional dependency on the physical
dimensions of the synerising sample, is that rapid
syneresis also leads to the rapid formation of a highly
shrunken outer layer, which implies that the permeabil-
thereby slowing down further syneresis. The close cor-
relation between B and dB/dt is presumably due to the
latter factor already causing an increase in B before the
Temperature has a very large effect – below 20 °C,
endogenous syneresis is virtually zero. The relations for
the effect of concentrating the milk by ultrafiltration are
different. Naturally, B decreases with increasing concen-
tration, and the network becomes denser. The bonds
remain of the same type, however, which is reflected in
 The Syneresis of Rennet-coagulated Curd 83

0.4
B (μm2)
0.2

dB/dt

syn

0
20 40 5.5 6.5 0 0.1 1 2 3

T (°C) pH CaCl2 (%) UF

Figure 10 Properties of rennet-induced skim milk gels. Permeability (B), rate of change of permeability (dB/dt), and approximate
initial syneresis rate (syn, arbitrary scale) as a function of temperature (T ), pH, added quantity of CaCl2 and preconcentration by
ultrafiltration (UF, degree of concentration) (mostly from results by van Dijk, 1982; van den Bijgaart, 1988).

tan  remaining constant (Zoon et al., 1988a). Never-
theless, dB/dt decreases with increasing concentration.
The overall result is that the rate of syneresis decreases
with increasing concentration. The relative shrinkage
rate, i.e., relative to one minus the volume fraction of
para-casein particles in the gel, increases somewhat
with increasing concentration (van den Bijgaart, 1988).
It also appears that the rate of syneresis of a gel from
pre-concentrated milk (by UF) is higher than that of a
gel of the same concentration but caused by syneresis;
at least part of the explanation is presumably that in the
latter case, considerable relaxation of the internal
stresses in the network has occurred, implying a lower
syneresis pressure.
The effect of adding CaCl2 is somewhat puzzling – B
and dB/dt are not affected and neither is tan  (Zoon
et al., 1988b), whereas syneresis rate increases. It should
be noticed, however, that the effect of adding CaCl2 is
rather variable and that the time elapsed between addition
and doing the experiments also affects the results (van
den Bijgaart, 1988). It would require painstaking investi-
gations to settle these fine points. Some other variables
also (slightly) affect endogenous syneresis. The quantity
of rennet added has very little effect, provided that the
time elapsed after rennet addition has been sufficient to
ensure almost complete hydrolysis of the -casein.
Adding NaCl has very little effect, unless a large
quantity is added. Comparison of renneted milk with

62

6.33
Initial rate of syneresis

62
6.68
27
8

0
34 °C

0
20 30 0 2 4 6 8
Temperature (°C) pe/Pa

Figure 11 Effects of temperature and external pressure (pe in Pa, indicated near the curves in the left-hand graph) on syneresis of
renneted skim milk. pH was 6.68 (filled circles) or 6.33 (open circles) (from results by van den Bijgaart, 1988).
84 The Syneresis of Rennet-coagulated Curd
skim milk shows that the presence of fat globules causes
a somewhat lower permeability; dB/dt is not affected,
and syneresis is a little slower. As an example, after 5 h a
slab had shrunk to 53% of its height, compared to 48%
in the case of skim milk (van den Bijgaart, 1988).
The effect of ethanol, a less-polar solvent than
water, was investigated by Renault et al. (1997).
Syneresis decreased in the presence of ethanol, which
may suggest that para-casein was more strongly sol-
vated; micellar casein was found to dissociate on heat-
ing in the presence of ethanol (O’Connell et al., 2001).
Experiments on the influence of an external pres-
sure, pe, were performed by placing a porous disc on
top of the syneresing slab. Some results are shown in
Fig. 11. It is seen that the effect is considerable and is
about proportional to the square root of pressure. The
effect of external pressure cannot be seen as an ampli-
fication of syneresis – it is about additive to the
endogenous syneresis. These results are in accordance
with those of model calculations. Figure 11 also sug-
gests that in the presence of an external pressure, the
lowest temperature at which syneresis can occur is
lower, the more so for a higher pressure. Although this
has not been verified by experiments, the effect must
exist at least to some extent.
Presumably, syneresis has an endpoint. Eventually,
the system will be close-packed. Such an end-point
has, however, not been observed. Figure 12 gives
some results up to 50-h syneresis. It is seen also that
after a long time, the shrinkage is greater for a higher
temperature, a lower pH and a higher external pres-
sure. A higher fat content also causes somewhat less
shrinkage after long time. It would be of great import-
ance for theory formation to establish the apparent
0.4
T pe
(°C) (Pa)
0.3
30 0 6.67
0.2 34 0 6.67
i
30 8 6.67
30 8 6.33
0.1
30 62
0 (determined by oven drying) as a function of time after renneting.
0 20 30 40 50
t (h)
Figure 12 Effects of temperature (T), external pressure (pe)
and pH on the shrinkage of renneted skim milk. Relative remain-
ing volume (i ) as a function of time (t) after renneting. Initial slab
height 5 mm (from results by van den Bijgaart, 1988).
equilibrium voluminosity of para-casein micelles as a
function of pH, temperature and salt content; however,
no such work has been reported.
Syneresis during curdmaking
After renneting has led to a gel of sufficient firmness,
it is usually cut into pieces to promote whey release.
For most types of cheese, the mixture of curds and
whey is then stirred, often, part of the whey is
removed, and it is fairly common to increase the tem-
perature of the mixture after some time (scalding or
cooking), all of which are measures aimed at enhanc-
ing syneresis. Moreover, during this process of curd-
making, the pH decreases, again enhancing syneresis.
An example of the water content of the curd during
the course of the process is given in Fig. 13. Note how
time, temperature, acidity and pressure affect the
water content; the effect of pressure is seen when the
curd is taken out of the whey for moulding, by which
action the pressure due to gravity increases by a factor
of about 30. All these effects are in qualitative agree-
ment with the above results.
In this section, the effect of several variables under
conditions during actual curdmaking, or conditions
more or less mimicking these, will be considered. This
is because most published experiments were done in
such a way, and methods for estimating syneresis will
be reviewed briefly. Some effects of milk composition
and pre-treatment will also be discussed.
90
70
% Water
pH
50
0 2 4 6 8
Time (h)
6.33
Figure 13 Examples of changes in the water content of curd
The gel was cut after 0.5 h and the curd and whey mixture was
stirred continually. At two moments (indicated by arrows), curd
was removed from the whey and put into a cheese mould. Experi-
ments with (filled line) and without (dashed line) adding starter.
Temperature of the whey was 32 °C throughout, temperature in
the mould gradually fell to 20 °C (recalculated from van de
Grootevheen and Geurts, 1977; Kwant et al., unpublished).

It goes without saying that curdmaking is aimed at
other things besides regulating the rate and the
extent of syneresis. The main aspect is that a higher
moisture content goes along with a higher sugar con-
tent of the curd, which, in turn, leads to a lower pH.
This can be modified by ‘washing’ the curd. A lower
pH at the moment of separating curds and whey
causes the cheese to contain less calcium phosphate.
Other aspects are the limitation of the loss of curd
fines, the inclusion and the activity of rennet in the
cheese and, in some types, the killing of undesired
micro-organisms (caused by scalding).
Methods for estimating syneresis
The ultimate result of syneresis is reflected in the water
content of the cheese after pressing. Determining
only this quantity yields, however, little understanding.
It is much more interesting to follow syneresis during
the curdmaking process, but it is not easy to do this
unequivocally. The various methods and their pros and
cons have been reviewed extensively (Walstra et al.,
1985); only the salient points will be described here. The
methods may be classified as follows:
1. Measuring the shrinkage of the curd, either the
height of a slab (as discussed earlier) or the volume
or mass of a slab or pieces of curd (in air or in
whey). These methods are typically applied in labor-
atory experiments.
2. Determining the amount of whey expelled. This
can be done in two ways:
a. determination of the volume of whey drained off.
The results strongly depend on conditions, espe-
cially the often imprecisely known external pres-
sure. It also may be fairly uncertain how much
interstitial whey is left between the curd grains.
b. determination of the degree of dilution of an
added tracer. This method has an inherent uncer-
tainty, in as much as the tracer may adhere onto
or diffuse into the curd.
3. Determination of the dry matter content of curd
pieces taken out of the whey. The main uncertainty
is the unknown quantity of whey adhering to the
curd particles; trying to remove the adhering whey
may introduce the opposite error.
4. Determining the density of the curd grains by putting
them in solutions of various density. This method is
fairly crude, but is hardly biased if carefully executed.
In addition to the classical methods, the use of
low-resolution NMR was introduced by Tellier et al.
(1993). The method showed great promise, as it was
able to monitor both the degree of syneresis and give
a quantitative measure of pore size distribution in
The Syneresis of Rennet-coagulated Curd 85
the curd, but was not further pursued for syneresis
studies.
All these methods can, of course, be executed
under various conditions that affect syneresis, e.g.,
temperature, pH and effective pressure, and at various
times after renneting or cutting. Most authors have
used method 2a, but methods 2b and 3 have also been
fairly popular, especially in experiments involving stir-
ring of the curds–whey mixture.
Hardly ever have different methods been com-
pared on the same curd. Figure 14 gives an example,
and it is seen that the difference is considerable. It
may be concluded that absolute values are hard to
obtain and that most methods provide only trends.
Even then, one has to be careful, since the method
may not be linear. In relation to this, it should be
realized that at the beginning of syneresis a large
amount of whey has to be removed for the moisture
content of the curd to become appreciably lower,
whereas at the end of the process the opposite is true
(see Fig. 15).
Rate equations
As was discussed above, even for a very simple model
for the case of one-dimensional syneresis under constant
conditions, solution of equation (7) in conjunction with
the equation of continuity leads to complicated rela-
tions. This will be even more so for the situations con-
sidered here, where the geometric boundary conditions
are more complicated and changing, and where the
physico-chemical conditions affecting syneresis are not
constant either. Nevertheless, some authors have tried to
give simple analytical expressions for the process.
90
Water content (%)
80
0 20 40 60 80
Time (min)
Figure 14 Water content of curd from skim milk renneted and
kept at 31 °C as a function of time after cutting, determined from
the concentration in the whey of added polyvinyl alcohol (filled
circles) and by oven drying of pieces of curd strained off (open
circles) (from Kwant et al., unpublished).
86 The Syneresis of Rennet-coagulated Curd
80
% Water in curd
40
0 20 60
100
% Whey removed
Figure 15 Calculated relation between the water content of
curd (from whole milk of 12.3% dry matter) and the quantity of
whey (6.8% dry matter) expelled as % (w/w) of the original milk.
Kirchmeier (1972) reported that the change in volume,
V, of a piece of curd is, under ‘constant conditions’,
given by:
V  Vo exp( Kt)
where Vo  original volume, t  time after starting
syneresis and K would be a first-order rate constant,
linearly dependent on temperature. A similar relation,
albeit with some ‘extra’ syneresis immediately after
cutting, was observed by Marshall (1982). Apart from
the lack of theoretical justification for equation (8), it
predicts that V approaches zero for very high t, which
is clearly impossible. Weber (1984), therefore, modi-
fied equation (8) to:
V  Vo [0.15  0.85 exp( Kt)]
where it was assumed that the curd eventually shrinks
to 0.15 times its original volume (actually, Weber used
mass rather than volume). A further modification was
made by Peri et al. (1985) who introduced the final
(relative) volume, Vinf, as a variable and obtained:
V Vinf  [(Vo Vinf) exp( Kt)]
This would be a correct equation for a simple relaxation
process where 1/K is the relaxation time. As we have
seen, syneresis can certainly not be considered such a
process; nevertheless, Peri et al. (1985) found a good
agreement with their results, obtained under a fairly wide
range of conditions. The good fit may have been due to
equation (10) containing two adjustable parameters.
Caron et al. (2001) chose yet another asymptotic two-
parameter fit, of the same form as the Michaelis–Menten
equation. Daviau et al. (2000c) used two exponential
relaxation times, and thus five adjustable parameters.
Several workers (Koestler and Petermann, 1936; Stoll,
1966; Lawrence and Hill, 1974) found that the amount of
whey expel1ed (Vo V) from pieces of curd was propor-
tional to t1/2, and concluded that ‘rate of syneresis is sub-
stantially diffusion-controlled’. Such a conclusion had
also been reached for syneresis in cross-linked polymer
gels (Beltman, 1975).
The results quoted above are incompatible; for
Kt  1, equations (8–10) predict that (Vo V) is
proportional to t, not t l/2; more generally, different
authors find different relations. The only conclusion
can be that, under constant conditions, the rate of
syneresis ( dV/dt) decreases as syneresis proceeds.
This need not always be true for the relative rate of
syneresis ( d ln V/dt), although this quantity will also
eventually approach zero.
Effects of curd grain size
Cutting the renneted milk gel into pieces creates a free
surface through which syneresis can occur. Before cut-
ting, the gel mostly sticks to the wall and its top sur-
(8) face does not show syneresis, unless it is wetted.
Moreover, the distance over which the whey has to
flow through the curd is greatly reduced.
Empirically, cutting strongly enhances syneresis.
Tests using the standard cheesemaking procedures,
where a body of curd is cut into pieces, compress the
curd and can thus induce structural changes or even
cracks in the gel. They thus do not allow to differenti-
ate between the effect of size of the curd grains on one
hand and the cutting-induced effects on the other.
Unger Grundelius et al. (2000) used curd cylinders of
different sizes, produced by rennetting in plastic
(9) syringes, but even with the best care, initial whey
expulsion could not be avoided.
An indication of the effect of grain size is given in
Fig. 16; the trends agree with the corresponding one-
dimensional syneresis data. The time for a given (low)
10000
Time [s] to 10% (slab) or 20%
pH 6.4
(10)
(grain) shrinkage
pH 6.0
1000
100
1 10 100
Slab thickness or 1/2 grain size (mm)
Figure 16 Initial one-dimensional syneresis in curd slabs
(lines) and three-dimensional syneresis in curd grains (symbols)
as a function of size (from Unger Grundelius et al., 2000 and
Lodaite et al., 2000).

level of initial syneresis scaled more or less propor-
tionally to curd grain size at pH 6.4, but at a power
less than one at a lower pH. Small pieces of curd
shrink more than large ones. The latter implies that
uneven cutting will cause local variations in moisture
content and acidity in the fresh cheese.
Stirring
Stirring enhances syneresis (see, for example, Fig. 17).
The main factor may be the prevention of sedimenta-
tion of the curd particles. Although in a sedimented
layer the pressure on the curd may be higher, the pos-
sibility of the whey flowing out of the curd layer soon
becomes small, thereby strongly impeding syneresis
(see further below).
Another factor is that stirring causes some pressure
to be exerted on the curd grains, and external pressure
has a large effect (see below). van den Bijgaart (1988)
has made some rough calculations. Stirring causes
velocity gradients and consequently, according to
Bernoulli’s law, pressure differences. In laminar flow,
these remain fairly small; they may amount to several
Pa during curd-making. Mostly, flow will be turbulent
and pressures up to 160 Pa were calculated, although
these exist only for short times. Collision of curd par-
ticles with each other or with the stirrer gives rise to
brief pressure bursts of the order of 100 Pa, although
the average external pressure will probably be about
10 Pa. It has indeed been observed that more vigorous
stirring (Patel et al., 1972) or removing more whey
(Lawrence, 1959; Birkkjaer et al., 1961), which causes
more frequent collisions between curd grains, hence a
higher average pressure, leads to somewhat more whey
expulsion.
The intermittent deformation of the curd grains
occurring during stirring may have another effect. As
discussed in relation to Fig. 5, large deformation of a
80
Stirring
% Whey
40
No stirring
0 1 2 3
Time (h) after cutting
Figure 17 The volume of whey expelled (as % of the original
milk volume) from curd kept in the whey at 38 °C as a function of
time after cutting, with or without stirring (from Lawrence, 1959).
The Syneresis of Rennet-coagulated Curd 87
renneted milk gel causes cracks to be formed in it.
Experiments in which an amount of renneted milk gel
between two concentric cylinders was brought tem-
porarily under shear and the permeability determined
before and afterwards (van Dijk and Walstra, 1986),
yielded the following results. Up to a shear of 0.35, B
had altered little, a shear of about 0.7 caused an
increase by, on average, 20%, and a larger shear could
cause a much higher permeability. Unger Grundelius
(2004) observed enhanced permeability with time at
constant pressure 7.5 kPa/m curd height. It has also
been observed (Akkerman, 1992) that an external
pressure of the order of 100 Pa can, under certain, not
very well-known, conditions cause several small
cracks to appear at the outside of shrunken curd
grains. (Perhaps the cracks are always formed if the
local pressure is high enough, but are often sealed
again.) To what extent these phenomena mitigate the
strong inhibition of further syneresis due to the form-
ation of a dense outer layer on the curd grains is
unknown.
Practical conditions of modern curd-making usually
allow few opportunities to markedly affect syneresis
by varying cutting, stirring, etc. For instance, the size
to which the renneted milk gel is cut certainly has an
effect on the water content of the cheese, but the effect
is small (Sammis et al., 1910; Wurster, 1934; Thomé
et al., 1958; Kammerlehner, 1974), at the most some
1% water in the cheese (Straatsma and Heijnekamp,
1988). The main reason presumably is that curd size
cannot be varied greatly. If the initial particles are very
large, they will inevitably be broken into smaller ones
during stirring as long as they are still soft. If one tries
to make very small particles, a considerable loss of
curd fines occurs. Stirring for a longer time causes a
lower moisture content (see, e.g., Fig. 13), but a cer-
tain minimum duration of stirring is needed to give
the particles sufficient firmness. After that, any longer
stirring leads to a slope of, for instance, 0.04% water
in the cheese per minute stirring for semi-hard cheese
(Straatsma and Heijnekamp, 1988). Consequently,
other measures should be taken to influence the water
content, especially varying the temperature.
After stirring, the curd particles are usually allowed
to sediment. If they are sufficiently rigid (which
implies mainly after they have lost sufficient whey),
they will deform and fuse only to a limited extent in
the sedimented layer, implying that any additional
external pressure leads to a considerable loss of whey.
This is illustrated in Fig. 18; the lower pressures in
this graph were due to stirring curds and whey, the
higher ones due to pressure exerted on the sedimented
curd layer, either by the curd itself or by the perforated
plates lying on top.
88 The Syneresis of Rennet-coagulated Curd
100
% Whey
50
0 50 100 104
External pressure (Pa)
Figure 18 The amount of whey expelled from curd (as % of
the original milk volume) after 2 h at 30 °C as a function of the
external pressure applied to the curd (approximate results,
recalculated from van Dijk et al. (1979) (100 Pa  10 3 bar)).
Effects of other process variables
Numerous authors have studied the effects of product
and process variables on syneresis rate, beginning with
Sammis et al. (1910). Other extensive studies were by,
successively, Wurster (1934), Koestler and Petermann
(1936), van der Waarden (1947), Thomé et al. (1958),
Stoll (1966) and Daviau et al. (2000a,b,c,d). Several
others have studied one or a few variables.
As will be seen below, the results often vary some-
what. Results that are obviously in error in view of our
present understanding have generally been omitted.
But even then, differences in the individual milk sam-
ples, in the methods used and in the conditions
employed, cause variation. Particularly, the stage at
which syneresis is measured affects the results. More-
over, the effect of one variable may be influenced
greatly by the level of another, and altering one factor
often causes other conditions to change also.
Heat treatment of the milk
Heat treatment of milk to such an extent that serum
proteins are denatured, increasingly diminishes the
syneresis rate of renneted milk, according to many
authors (Wurster, 1934; van der Waarden, 1947;
Dimov and Mineva, 1962; Stoll, 1966; Kammerlehner,
1974; Nilsen, 1982; Pearse et al., 1985; Daviau et al.,
2000c). Some found even a slight decrease caused by
mild heat treatments (Siegenthaler and Flückiger,
1964; Stoll, 1966; Nilsen, 1982), but the others did
not. Pearse et al. (1985) found the decrease in synere-
sis to be almost linearly correlated with denaturation
of -lactoglobulin. Heat treatment of synthetic milk
free of serum proteins hardly affected syneresis. Add-
ition of -casein to milk diminished the detrimental
effect of heating, presumably because -lactoglobulin
now reacted primarily with -casein in the serum dur-
ing heating, thus affecting the casein micelles less.
Ovine milk was less sensitive to heat treatment, and
caprine even less so (Calvo and Balcones, 2000).
Homogenization of the milk
Homogenization or recombination of milk signifi-
cantly decreases syneresis rate (Vaikus et al., 1970;
Kammerlehner, 1974; Emmons et al., 1980; Humbert
et al., 1980; Green et al., 1983; Storry et al., 1983;
Ghosh et al., 1994). This is related to the incorpor-
ation of micellar casein in the surface coat of the fat
globules, which causes the fat globules to become part
of the para-casein network, which, in turn, may hin-
der shrinking of the network. A comparable effect on
syneresis was observed if milk had been concentrated
by evaporation and diluted again before clotting
(Cheeseman and Mabbitt, 1968) which has a similar
consequence for the fat globules (Mulder and Walstra,
1974). If fat is homogenized into whey, so that the fat
globules do not contain much casein on their surface
layers, the detrimental effect of homogenization on
syneresis is clearly less (Emmons et al., 1980).
Various additions to the milk
Additions meant to modify specific residues of the milk
proteins, in order to study the clotting reaction, will
not be considered here. Adding sugars, which are fairly
unreactive, has been reported to cause no effect (Stoll,
1966; Grandison et al., 1984a), a slight decrease (van
der Waarden, 1947) or a slight increase (Cheeseman,
1962) in syneresis rate. About the same holds for add-
ition of up to 10% urea (van der Waarden, 1947;
Cheeseman, 1962).
In cheesemaking, some CaCl2 is frequently added
to enhance coagulation. Most authors report that small
additions (e.g., up to 10 mM) of CaCl2 enhanced
syneresis somewhat (Wurster, 1934; van der Waarden,
1947; Stoll, 1966; Kammerlehner, 1974; Lelievre and
Creamer, 1978) while others found little or no effect
(Lawrence, 1959; Cheeseman, 1962; Emmons et al.,
1980); larger additions were generally found to reduce
syneresis (Wurster, 1934; Gyr, 1944; Tarodo de la
Fuente and Alais, 1975). van der Waarden (1947)
clearly showed that the main enhancing effect of
CaCl2 is due to its lowering the pH; if the pH was kept
constant, addition of CaCl2 caused syneresis to
decrease, while MgCl2 caused a marked increase (van
der Waarden, 1947; Stoll, 1966; Kovalenko and
Bocharova, 1973). Presumably, one has to consider
two points – increasing of the calcium ion activity
(enhancing syneresis) and of the colloidal calcium
phosphate (diminishing syneresis). Presumably, Mg2

act much the same as Ca2, whereas Mg-phosphates
are much more soluble than Ca-phosphates (addition
of MgC12 may thus cause some dissolution of col-
loidal phosphate). Lowering the pH causes, of course,
a dissolution of colloidal phosphate and an increase in
Ca2 activity. Addition of phosphate, citrate, oxalate
or EDTA (van der Waarden, 1947; Stoll, 1966) at con-
stant pH, all reduce syneresis; these additions consider-
ably reduce Ca2 activity and adding phosphate also
increases colloidal phosphate content. The salt equi-
libria in milk are intricate, depend on several condi-
tions and often exhibit slow changes, as discussed by,
for instance, by Walstra and Jenness (1984).
Increasing the ionic strength of milk with univalent
ions (e.g., NaCl) has been reported to cause at first no
change (Cheeseman, 1962) or a slight increase in
syneresis (Stoll, 1966); it tends to reduce the amount
of colloidal phosphate and possibly the Ca2 activity.
A large increase in ionic strength causes a decrease in
syneresis (van der Waarden, 1947; Cheeseman, 1962;
Stoll, 1966), but then, milk with added salt coagulates
very poorly on renneting. Decreased ionic strength
increased syneresis (Daviau et al., 2000c). Addition of
AlCl3 reduces syneresis (Stoll, 1966).
Coagulation
Most authors agree that rennet concentration has no
effect on syneresis (Sammis et al., 1910; Wurster, 1934;
Stoll, 1966; Lelievre, 1977). Others found that more
rennet gave a slight increase (Gyr, 1944; Kammerlehner,
1974; Lelievre and Creamer, 1978; Kaytanli et al., 1994)
or decrease (Kovalenko and Bocharova, 1973) in
syneresis, or observed an optimum concentration
(Weber, 1984). These effects should be considered in
relation to the time of cutting (Weber, 1984; Stoll, 1966;
Lelievre, 1977). As made clear by, for instance, Weber
(1984), it is the stage of the coagulation process or the
firmness of the curd at the moment of cutting that is the
variable; if cutting is very late, syneresis may be some-
what less. It has also been observed that a higher coagu-
lation temperature leads to slightly less syneresis
(Straatsma and Heijnekamp, 1988); this may be due to
the cutting starting at an effectively later stage.
Whether milk is renneted by chymosin or pepsin
makes no significant difference (Andersson and Andrén,
1990). Renneting with proteolytic enzymes from Rhizo-
mucor miehei or Gyphonectria parasitica caused some-
what slower syneresis, but curd firming was slower also,
and when cutting 45 rather than 30 min after adding the
rennet, the syneresis rate was observed to be normal
(Gouda and El-Shabrawy, 1987).
Disturbance of the gel during setting may consider-
ably enhance syneresis rate (Wurster, 1934). This was
ascribed to the increase of free surface, but also when
The Syneresis of Rennet-coagulated Curd 89
the free surface was kept constant, such disturbance
caused syneresis rate to increase by 20–30% in some
experiments (Cheeseman and Chapman, 1966). This
may have been due to the disturbance affecting the
structure of the network. Curd formed solely by acidi-
fication shows very little syneresis if left undisturbed.
In milk clotted below about pH 5, the presence of ren-
net was found to enhance syneresis considerably, the
more so when the amount of added rennet was
increased (Emmons et al., 1959). Presumably, this sig-
nifies a gradual change from an acid to a rennet-
induced gel and is of importance in the production of
fresh cheese types (Weber, 1984).
Temperature
Temperature greatly affects syneresis rate of rennet
curd; some results are summarized in Fig. 19. All
authors agree as to the trend and all results show that
the rate of change of syneresis with temperature (Q10
or d ln V/dT) decreases with increasing temperature,
but otherwise the results are fairly different. At 25 °C,
reported values of Q10 vary from about 2.5 to 15, at 45 °C
from about 1.1 to 1.5.
100
80
60
% Whey
40
20
0
20 40 60
Temp. (°C)
Figure 19 The volume of whey expelled (as % of the original milk
volume) from curd set, kept and treated at different (constant) tem-
peratures. Most results were obtained 1 h after cutting (recalculated
from  Sammis et al. (1910),  Wurster (1934),  Koestler and
Petermann (1936),  Gyr (1944),  Lawrence (1959),  Stoll
(1966),  Kirchmeier (1972),  Kammerlehner (1974),  Marshall
(1982)).
90 The Syneresis of Rennet-coagulated Curd
The initial rate is increased, but the final amount of
syneresis may even decrease, as temperature is raised
above 45 °C (Huber et al., 2001). It appears that the rate
at which temperature is changed (dT/dt) does not, as
such, affect syneresis (Wurster, 1934; Patel et al., 1972).
Keeping the milk for some time at a low temperature
before renneting has been reported to have no effect
(Johnston et al., 1983), a small detrimental effect on
syneresis (Nilsen, 1982) or a considerable effect –
holding for 20 h at 5 °C reduced syneresis by about 30%
(Kammerlehner, 1974). Raynal and Remeuf (2000)
observed a similar decrease for bovine milk, but no effect
on caprine and ovine milk. Any detrimental effect of pre-
cooling is probably reversed by prewarming the milk to a
fairly high temperature before renneting, as is commonly
done in cheesemaking to ensure normal setting.
Acidity
If milk has been acidified to a lower pH before rennet-
ing, syneresis rate is faster. Some observations are sum-
marized in Fig. 20; other authors have reported similar
results (Sammis et al., 1910; Koestler and Petermann,
100
80
60
% Whey
40
20
0 tent of the moisture (liquid) in the curd. Hence, the
5 6 7
pH
Figure 20 The volume of whey expelled (as % of the original
milk volume) from curd set, kept and treated at different pH; in
some cases, pH decreased slightly during the experiment. Most
results were obtained 1 h after cutting (recalculated from Wurster
(1934), heavy line  average of several experiments;  Gyr (1944);
 Cheeseman (1962);  Stoll (1966);  Berridge (1970);  Patel
et al. (1972);  Marshall (1982);  Pearse et al. (1984);  Weber
(1984). See text for the results of  Emmons et al. (1959)).
1936; van der Waarden, 1947; Tarodo de la Fuente and
Alais, 1975; Lelievre and Creamer, 1978). Although the
observed trends were mostly the same, there were,
again, considerable quantitative differences. The deviat-
ing relation found by Berridge (1970) is not due to inac-
curacy but may be related to the different experimental
set-up (syneresis of a cylinder of curd attached to a grid,
in air). The inflection points in the curves near pH 6 are
also realistic. Stoll (1966) observed that the effect of pH
was relatively greater at lower temperature and in the
absence of stirring, i.e., if syneresis was slower. There
were no appreciable differences according to the acid
used (van der Waarden, 1947).
If the pH falls during syneresis this may enhance
syneresis rate to a greater extent than is found when
the pH is previously brought to the same value (Stoll,
1966; van de Grootevheen and Geurts, 1977) because
the building blocks of the protein network tend to
shrink due to the change in pH. This is also exempli-
fied by some results by Emmons et al. (1959) shown
in Fig. 20. Here, the milk contained variable numbers
of starter bacteria, and the pH values indicated in the
figure are those at the moment of cutting. A higher
pH at that stage implies a greater drop in pH after cut-
ting, hence more syneresis. Milk that has already been
soured to a very low pH (e.g., 4.5) exhibits only weak
syneresis, even after renneting (Sammis et al., 1910).
Washing of the curd
Washing, i.e., adding water after part of the whey has
been removed, has been reported to enhance syneresis
(Kammerlehner, 1974), and to give a slightly, possibly
insignificantly, lower water content (Casiraghi et al.,
1987). However, washing may coincide with a change
in temperature and a difference in the effectiveness of
stirring, both of which affect syneresis. In studies by
van de Grootevheen and Geurts (1977), either water
or an equal quantity of whey at the same temperature
was added at a certain stage during cheesemaking and
the water content of the curd determined at various
times. The water content of the curd to which water
had been added was up to two percentage units
higher, but the difference could be fully explained by
taking into account the difference in dry matter con-
osmotic effects of washing are negligible.
Ultrafiltration
Ultrafiltration of cheese milk and renneting the
retentate allows the manufacture of curd in such a
way that less, or even no, syneresis occurs. The latter,
i.e., concentrating the milk to a composition roughly
equal to that of the (unsalted) cheese to be made, is
feasible only for soft-type cheese; it usually involves

diafiltration also. For harder cheeses, partial ultrafil-
tration can be applied and an important point then is
to what extent syneresis is affected. Some results
were already given in Fig. 11. The comparison of the
conclusions of different studies may be sometimes
misleading, as the authors may relate their syneresis
rates to the original amount of milk, or the amount
of whey to be expelled.
Extensive studies were made by Peri et al. (1985),
applying equation (10). They concentrated the milk
up to 5.2-fold. The rate constant of the first-order
equation, which is thus a measure of the rate relative
to the amount of whey yet to be removed, varied little
with the degree of concentration; clear correlations
with pH or extent of diafiltration were not observed
either. The extrapolated proportion of whey eventu-
ally expelled (Vo Vinf) varied roughly linearly with
the reciprocal of the degree of concentration. A lower
pH resulted in a lower (extrapolated) final moisture
content. The effects of diafiltration, pre-acidification
and sequestering of Ca were also studied (Casiraghi
et al., 1987). When adjusting casein cocentration with
UF or MF retentate powders, Caron et al. (2001)
found faster syneresis for milks adjusted with MF
retentate of preacidified milk.
Other workers obtained slightly different results
(Green et al., 1983; Storry et al., 1983). This may have
been due to variation in the time elapsed after renneting
before cutting. When renneting normal milk, about 2%
of the -casein is still unhydrolysed at the moment of
cutting, whereas this may be about 12% for a milk con-
centrated two-fold by ultrafiltration (van Hooydonk
and van den Berg, 1988); this proportion is higher for a
more concentrated mi1k. Consequently, the early stages
of gel formation and syneresis probably proceed some-
what differently, depending on the moment of cutting.
High-pressure treatment
The effects of high-pressure treatment are twofold, the
breakdown of the casein micelles at pressures of about
400 MPa, and denaturation of -lactoglobulin at higher
pressures, similar to the effects of heating. Casein
micelle disruption causes faster aggregation and a finer
gel structure with stiffer gels, but syneresis was only
affected at pressures above 400 MPa (Needs et al., 2000).
Effect of milk composition
Milk composition may clearly affect syneresis, but the
effect is usually not large. A higher fat content in the
milk on average is accompanied by somewhat slower
syneresis (Beeby, 1959; Feagan et al., 1965; Stoll, 1966;
Kammerlehner, 1974; Emmons et al., 1980; Storry et al.,
1983; Weber, 1984; Grandison et al., 1984a). In practice,
The Syneresis of Rennet-coagulated Curd 91
milk is usually standardized as to fat content. A higher
casein content goes along with a slower absolute rate of
syneresis, but a hardly different relative rate (see under
Ultrafiltration).
Minor components may have a larger influence
and it must be presumed that the calcium ion activ-
ity is an especially important variable. For instance,
separate milkings of individual cows may vary by
a factor of three in syneresis rate (Koestler and
Petermann, 1936; Thomé et al., 1958; Kammerlehner,
1974; Grandison et al., 1984a,b), but addition of
some CaCl2 greatly reduces the variation. Minor
variation has been observed with the stage of lacta-
tion (Kammerlehner, 1974; Grandison et al., 1984b)
and this may possibly be related to the calcium ion
activity also. Milk from cows suffering severe masti-
tis exhibits poor clotting by rennet and somewhat
diminished syneresis (Thomé and Liljegren, 1959;
Kiermeier and Keis, 1964; Kiermeier et al., 1967).
Extensive growth of pseudomonads in milk was
shown to reduce syneresis markedly (Lelievre et al.,
1978). On the other hand, considerable proteolysis
caused by plasmin activity hardly affected whey
expulsion (Pearse et al., 1986b).
The effect of casein composition has been studied.
Pearse et al. (1986a) made milk with synthetic
micelles of variable casein composition. The propor-
tions of - and -caseins clearly affected clotting time,
but syneresis far less. Dephosphorylation of -casein
caused the clotting time to increase and the syneresis
rate to decrease. Interpretation of these results is very
difficult without knowing such variables as micelle
size and voluminosity, or loss tangent and permeabil-
ity of the renneted milk. There also appears to be
some correlation between syneresis and genetic vari-
ants of milk proteins, especially with the -lactoglobu-
lin variant (McLean and Schaar, 1989). This may,
again, be due to differences in the calcium ion activity,
which correlates with the genetic variants.
Other conditions being equal, renneted goats’
milk exhibited greater syneresis than cows’ milk, and
ewes’ milk syneresed less (Storry et al., 1983). It may
be noted that the latter usually has a clearly higher
casein content (Walstra and Jenness, 1984). The
effect of several variables on syneresis of renneted
cows’ milk, as discussed above, was often different
for either ewes’ (Raynal and Remeuf, 2000) or buf-
faloes’ milk (Dimov and Mineva, 1962).
Concluding Remarks
The results on syneresis during practical cheesemak-
ing presented here (see also a review by Pearse and
Mackinlay, 1989) generally agree with experiments on
92 The Syneresis of Rennet-coagulated Curd

undisturbed curd, although only in a qualitative sense.
In a practical situation, quantitative predictions on
syneresis rate can hardly be made. Nevertheless, it may
be concluded that the main variables affecting synere-
sis rate are:
• the geometrical constraints (dimension of the curd
grain);
• pressure applied to the curd (grains), where the rela-
tive effect is greatest in the low pressure range;
• pH;
• temperature, where the relative effect is the greatest
in the low temperature range.
The effect of the other variables is generally small
(with the exception of intense heat treatment) and
tends to be relatively smaller when overall syneresis
rate is higher. Stoll (1966) observed, for instance,
that stirring the curd–whey mixture almost elimin-
ated differences caused by some variables observed
when studying syneresis under quiescent conditions.
This was explained by van den Bijgaart (1988) from
the over-riding effect of the permeability of the outer
layer of the curd grains. Any condition leading to
very rapid syneresis also causes rapid development of
a poorly permeable layer, which then markedly slows
down any further syneresis. In a qualitative sense,
this has been observed before, e.g., by Koestler and
Petermann (1936). Cheesemakers speak of a ‘skin’
around the curd grains, and it is even assumed that
very rapid initial syneresis may lead to an ultimately
higher water content in the curd, as compared to a
situation where syneresis proceeds more slowly; cf.
the high-temperature syneresis results of Huber et al.
(2001).
Behaviour of Curd during Processing
When the curd grains are sufficiently dry, they are usu-
ally allowed to sediment into a ‘bed’ in the cheese vat
or in a drainage pipe. The layer of curd grains com-
pacts, more whey is expelled from the grains and the
grains partly fuse to form a coherent mass. Com-
paction may be due to pressure exerted by the layer
itself or by perforated plates laid on top. Effective pres-
sure ranges from about 100–500 Pa. The compaction
is either allowed to proceed for a considerable time,
after which the curd mass is cut into small pieces
(‘milling’, as for Cheddar types), or, after a short while,
blocks of curd are cut from it, which then are sub-
jected to moulding and pressing.
The phenomena that occur during compaction and
drainage are complicated. At first, compaction occurs
due to sedimentation and reorientation of the curd
grains. Further events are summarized in Table 2. The
different processes are mutually dependent, and espe-
cially the deformation of the grains, and the expulsion
of whey from them are coupled. Moreover, fusion of
curd grains is greatly enhanced when they are
deformed. By and large, processes 1 and 2 lead to a
lower moisture content, whereas 3 impedes the loss of
moisture. Fusion may proceed until the pores between
the grains are no longer interconnected, when further
drainage virtually stops. On the other hand, some ini-
tial fusion will promote drainage, as it can prevent fur-
ther reorientation of the grains into a denser packing.
These conclusions may be true enough, but they are
only qualitative and thus not very helpful. To arrive at
quantitative relations, the processes were studied in
some detail by Akkerman (1992) and Akkerman et al.
(1994) for Dutch-type cheese. Lodaite (2002) and
Lodaite et al. (2002) studied curd deformation and
fusion for the conditions corresponding to soft cheeses.
The Akkerman group used whole milk, mostly without
added starter, and made curd grains that had been left
to synerese to roughly a quarter of their initial volume
while Lodaite and collaborators used reconstituted skim
milk, and allowed little or no syneresis to occur prior to
deformation. The temperature was mostly 35 °C.
Expression of single grains, fusion of a collection of
grains, compaction of a column and change in pore size
(distribution) in a column of grains were studied separ-
ately. Because of the many variables of importance, the

Table 2 Processes occurring during compaction and drainage of a curd–whey column

Process Results in Depends on

Expulsion of whey from grains
Drainage of whey from column
Deformation and fusion of grains
Lower whey content of grains
Grains less deformable
Closer packing of grains
Narrower pores
Narrower pores
Smaller free surface area
Degree of concentration, temperature, pH, effective
pressure, free surface area of grains
External pressure, pore size distribution (hence,
grain size distribution and shape), geometrical
constraints
Deformability of grains (hence degree of
concentration, temperature, pH), external
pressure, duration

intricacy of the processes and the experimental difficul-
ties, the results are to some extent uncertain, but they
clearly show quantitative trends.
Syneresis under pressure
Some results of Akkerman (1992) on uniaxial expres-
sion of single grains are shown in Fig. 21. The situa-
tion was quite different from that leading to the results
in Figs 11 and 12. A synerized curd grain was now
involved, which implied that the outer layer was much
more dense than the centre. Moreover, when the pres-
sures were generally higher, the curd grain could
deform sideways and most of the outflow of whey was
in directions perpendicular to the applied force. It is
seen that the grain showed an immediate, i.e., elastic,
deformation, followed by a viscous one that became
ever slower. If the pressure was released within a few
seconds, the grain more or less regained its original
shape, but after some minutes the deformation was
permanent; this agrees with the average relaxation
time of renneted milk of about 1 min found earlier
(Zoon et al., 1989b, 1990). Analysis of the results led
to the conclusion that the deformability of the grain,
more precisely its effective biaxial elongational viscos-
ity, was rate-determining, the elongational viscosity
markedly depending on (decrease with) the stress
applied. Akkerman (1992) defined a pseudo Poisson
number, , as:
  0.5 1 冢 d ln
V
d
H冣 (11)
where V is grain volume and H is the relative deform-
ation expressed as the Hencky strain. He found, as an
average over the first 15 min,   0.27, almost inde-
pendent of conditions. This implies that a (nearly)
constant part of the decrease in height of the grain is
due to shrinkage. The constancy of  agrees with the
deformability (i.e., the rheological properties) of the
6 0.30
pe = 1200 Pa t = 900 s
h (mm)
3 0.15
i
0 0
0 500 1000 0 2 4 fracture stress.
Time (s) pe (kPa)
Figure 21 Uniaxial compression of curd grains at constant
stress. h is the height of the curd grain, t duration of pressing, i
the relative remaining volume and pe the pressure applied (from
Akkerman, 1992).
The Syneresis of Rennet-coagulated Curd 93
curd now being rate-determining. As shown in Fig. 21,
the deformation, and thereby the expression of whey,
depends on the remaining volume, i, pressure and
time. It is little dependent on the grain size. The
expression reasonably followed the relation:
(it iinf)
 exp( Kpe sqrt(t)) (12)
(i0 iinf)
with iinf  0.1 and where the rate constant K  4 
10 5 Pa l s 0.5.
Whereas Akkerman (1992) followed compression
at various constant pressures, Lodaite (2002) followed
the (non-lubricated) compression of non-synerized
curd at different constant (Cauchy) deformation rates.
Some results are given in Fig. 22. The pressure was
seen to increase more or less proportionally with
deformation, but not in proportion with the deform-
ation rate. The pressures observed were much lower
than what would correspond to the permeability of the
curd; this presumably means that during deformation,
minute cracks appeared in the curd, vastly increasing
its permeability. An empirical formula of the form of
Hooke’s law, with a compression rate-dependent modu-
lus, described the data:
pe  E (1 i) (13)
with E  K (d(1 i)/dt)3/4 and K  40 kPa s 3/4.
The effect of pH was not studied, but in view of the
effect of pH on the rheological properties of renneted
milk (see e.g., Fig. 1) and cheese, it must be significant.
Curd fusion
Akkerman (1992) and Akkerman et al. (1993) evalu-
ated the fusion of curd grains in a curd–whey column
by determining the fracture stress when pulling two
parts of the column, separated by a perforated plate,
away from each other. The force divided by the contact
area of the grains was taken as the fracture stress. The
pressure and time during which it was applied had a
strong effect. At 34 °C, the fracture stress obtained was
about 60% higher than at 32 °C, whereas at 36 °C it
was, maybe, somewhat lower again. The less the grains
had shrunk prior to the experiment, the higher the
Lodaite (2002) and Lodaite et al. (2002) devised a
method for measuring fusion on a single grain pressed
against a thin layer of curd, allowing the simultaneous
measurement of deformation. Fusion pressure, fusion
time and syneresis time, temperature, pH (6.0 and 6.4)
94 The Syneresis of Rennet-coagulated Curd

25 Compaction of a curd column

The compaction of a curd column was studied (Akker-

20 man, 1992; Akkerman et al., 1994) for radial drainage.
The results depended strongly on the geometrical con-
15 straints, especially the radius of the column. The curd
particles tended to stick to the wall, and leakage along
the wall was also of importance. The total pressure
10 exerted was, following Schwartzberg et al. (1985), pre-
sumed composed of three terms:
5
pe  pc  pl  pw (14)
u = 0.1 mm min–1
0
A part of the pressure is lost by friction to the wall
(subscript w), and the remainder may not only be on
500
Compressive stress (Pa)

the network of curd particles (subscript c), but also on

the liquid (subscript l). As soon as the outflow of
400
whey is hindered by a lack of interconnected pores,
the pressure on the liquid increases rapidly. If the
300
pores become completely disconnected, all the pres-
sure is exerted on the liquid (except for pw) or, in
200
other words, the pressure is isotropic, and expression
from the curd grains stops.
100
Some results are shown in Fig. 24. The total pres-
u = 1 mm min–1
sure exerted appears to be the dominant variable. For
0
a low pe, the expression increases strongly with pres-
1500
sure, roughly following equation (12). But above a cer-
tain pe, called the threshold pressure, any higher
1200 pressure leads to a progressively increasing pl. Also,
the pressure loss at the wall, pw, markedly increases
900 with total pressure, being, for instance, proportional to
pe2.5. Altogether, at a high pe the effective pressure on
600 the grains, pc, soon becomes very small and expression
(almost) stops. The other results in Fig. 24 speak for
300 themselves; note the very strong decrease of the
u = 10 mm min–1
0
0.0 0.1 0.2 0.3 0.4 0.5 0.6 2.5

Relative deformation
2 0–2 kPa, 500 s
Fracture stress [kPa]

Figure 22 Uniaxial compression of a curd slab at different con- 1 kPa, 0 –1500 s

stant Cauchy strain rates. The initial slab height  5 mm, u  linear
compression rate (from Lodaite, 2002). 1.5

and casein and rennet concentrations were varied. It

was concluded that the total deformation (which is of 0.5
course dependent on fusion pressure and time) was
the best predictor of fusion, and for a given total 0
deformation, the fusion was independent of pH and 0 0.2 0.4 0.6 0.8 1
rennet concentration. The fusion–deformation relation- Relative deformation
ship appears to hold even for the results of Akkerman
Figure 23 Strength of fusion of curd grains in a column
(1992) and Akkerman et al. (1993) (see Fig. 23). Lodaite expressed as the fracture stress f after pressing for various
et al. (2002) confirmed the strong effect of tempera- times at various pressures as a function of deformation (recalcu-
ture in the studied range 28–33 °C. lated from Equation 12 from results by Akkerman, 1992).
 The Syneresis of Rennet-coagulated Curd 95

drainage rate, as the fine particles tend to block the

10 pores between the grains; the threshold pressure now
30 is much lower. It must be assumed that the curd parti-
cle size (average and spread) has some influence, but
h (cm)

450 not a lot within the range studied. The effect of pH

5
2100 was not estimated.
Akkerman et al. (1995) further compared the
results obtained for single grains with the compaction
0
of a curd column by developing a computer model of
the process. Up to pressures of a few hundred Pa, the
calculated results agreed reasonably well with the
experimental data. He also concluded that at a high pe
Φv

0.1
the curd grains can, in principle, initially be expressed
530
quite fast, without the pressure on the liquid, pl
becoming substantial. This opens up possibilities for
0
improving the drainage process.
In the Wageningen laboratory, some preliminary
studies on axial drainage in a curd column were per-
2
530 formed under a wider range of conditions (Heerink
d (mm)

and Geurts, 1981); actually there may have been con-

siderable radial drainage as well. Curd was made from
1
skim milk, without a starter. After cutting and stirring
(and removing some whey), a column of curd and
0 whey, 30 cm high, was taken; the curd sedimented
almost immediately to a height of about 20 cm, after
which pressure was applied via a perforated disc, and
the curd column gradually compressed to a height of,
B* (m2)

10–10 for instance, 5 cm; the compression was allowed to

820
10–12
0 0.5 1 means liquid containing dissolved substances, i.e.,
Time (ks)
Figure 24 Compression of a curd–whey column (radius 6 cm)
under uniaxial compression and with radial drainage. Column
height (h), volume fraction of pores (v), volume- average appar-
ent pore diameter (d ) and average radial permeability (B*) cal-
culated as a function of time after applying pressure (indicated
near the curves, Pa) (from results by Akkerman, 1992).
permeability of the curd column with time, despite pe
being fairly small.
Other variables affecting drainage are the degree of
concentration of the curd grains at the beginning,
given as i0, and the temperature. For a higher i0 and a
higher temperature, the initial rate of compaction is
higher, but the threshold pressure mentioned above is
lower; in other words, the highest pressure that can be
applied for the drainage to proceed satisfactorily is
smaller (Akkerman et al., 1996). Threshold pressures
are mostly somewhere between 800 and 2000 Pa; they
depend considerably on the geometry of the system.
The presence of curd fines may strongly lower the
proceed for 90 min. The final moisture content of the
curd column was determined and the earlier values
were calculated from the change in height and
expressed as the mass fraction of moisture (moisture
whey in this case). The proportion of moisture
between the grains was originally about 40%. Also
here, very high pressures gave little improvement. At
20 °C, very little whey was expelled from the curd
grains, in accordance with the strong dependence of
the apparent curd viscosity (Zoon et al., 1988b) and of
syneresis on temperature.
Some of the conclusions drawn in this section may
in a sense be derived from earlier observations (Vas,
1931; Tarodo de la Fuente and Alais, 1975; Lelievre,
1977; Lelievre and Creamer, 1978; Johnston and Mur-
phy, 1984; Grandison et al., 1984a). Especially inter-
esting is the work of Scott Blair and Coppen (1940),
who reasoned that firmer curd grains would permit
faster drainage of whey from a mass of grains, and
made use of this in devising a test method to deter-
mine the ‘pitching point’ of the curd, i.e., the moment
at which the grains have lost sufficient moisture, and
stirring can cease. A volume of curds and whey is put
into a perforated cylinder and allowed to drain for a
fixed time; now the ‘superficial density’ is determined,
96 The Syneresis of Rennet-coagulated Curd
i.e., the weight of curd divided by the height of the
curd column. They found a fairly good positive correl-
ation between the water content and the superficial
density, indicating that high moisture, and, thus, soft
grains deformed rapidly to close the channels between
them, thereby greatly hindering further drainage. Firm
(i.e., ‘dry’) grains permitted ongoing drainage, leading
to a low superficial density, because the voids between
particles now become filled with air. As is to be
expected, other factors affect the draining rate. Scott
Blair and Coppen (1940) found for the same value of
superficial density, a range in water content of about
14 percentage units. There was a tendency for rapid
initial syneresis, hence presumably the presence of a
more or less rigid ‘skin’ around the curd grains, to lead
to a lower superficial density. Likewise, curd at a lower
pH (Cheshire as compared to Cheddar) tended to have
a lower superficial density. It would be useful to study
these and other variables in greater detail.
The water content of cheese
There obviously is a lowest possible water content of
(freshly made) cheese – the para-casein particles have
a given voluminosity. This aspect was reviewed by
Walstra et al. (1985) and it was concluded that few
hard conclusions can be drawn. The equilibrium volu-
minosity of para-casein micelles at room temperature
and physiological pH was roughly estimated to corres-
pond to 1.4 g water per g protein; this would come
down to a water content of an unsalted full-cream
cheese of about 40%. The voluminosity would be
lower for a lower pH and a higher temperature. The
latter effect is considerable. It is also known that a
cheese or curd may take up moisture when the tem-
perature is lowered (e.g., Delbeke and Naudts (1970),
on Herve cheese; results obtained in the Wageningen
laboratory on renneted milk ultrafiltration retentate at
pH 5.2; observations in the Lund laboratory on ren-
neted microfiltration retentates at pH 6, observations
in practice on Feta cheese kept in brine). The depend-
ence is corrobated by the results of Teo et al. (1996)
on reconstituted renneted casein.
Moderate NaCl concentrations increase the volumin-
osity of both native (Famelart et al., 1999) and ren-
neted (Creamer, 1985) casein micelles. Electron
microscopy observations indicated that the volume of
the casein matrix increased, and the volume of the
interstitial dilute phase decreased in salt-injected
Muenster cheese (Pastorino et al., 2003) and salted
non-fat Mozzarella (Paulson et al., 1998). This would
appear to be in contradicton to a large number of stud-
ies showing that a higher salt content correlates with a
lower level of moisture in cheese, but during salting in
brine or in dry salt, cheeses lose water by pseudo-
osmosis to the highly concentrated brine (Geurts et al.,
1974), and thus other things being equal, the more
heavily salted the cheese is the more drier it will be.
In a cheese, the lowest possible water to protein
ratio may be slightly lower for a lower fat content; in
practice, a lower fat in dry matter content always goes
along with a distinctly lower water to protein ratio,
but this presumably is due to faster syneresis. Alto-
gether, the final moisture content of most cheeses is
determined primarily by the rate and the duration of
the processes causing whey expulsion, rather than by
the equilibrium-swelling state of the para-casein.
After curd-making and drainage, one of the follow-
ing procedures is usually applied.
• Moulding the curd, followed by further drainage
under its own weight; this is applied only for fairly
soft cheese.
• Moulding and pressing the curd; this is the common
method for semi-hard and several hard cheeses.
• Letting the curd rest for a considerable time to
develop sufficient acidity (often while allowing the
curd to flow, e.g., cheddaring) after which the
coherent curd mass is cut into fairly small pieces
(milling), salted, moulded and pressed.
• Intensively working the already acidified curd (pH
5.3) at a quite high temperature, as is done in mak-
ing pasta-filata cheeses.
During several of these process steps, the curd may
lose considerable moisture. Merely taking the curd out of
the whey, allowing further whey to leak out, has already a
marked effect; see e.g., Figs 13 and 25. Sometimes, the
curd grains are worked after removal from the whey,
which leads to a much drier cheese with an open texture
(numerous small, irregularly shaped holes). The pasta-
filata treatment also causes appreciable loss of moisture
(high temperature and pressure), although the fairly large
size of the lumps of curd formed has a mitigating effect
(long distance and relatively small surface area).
Pressing of the curd mass is aimed at obtaining a
coherent mass with a closed rind. The formation of a
rind, i.e., an outer layer in which all the curd grains are
fully fused with their neighbours, is greatly favoured by
the possibility of rapid removal of moisture from the
outer layer, for instance by application of a cloth around
the curd mass (Mulder et al., 1966). The closed rind
greatly reduces further expression of moisture. Figure 24
shows that the effective permeability of a drained mass
of curd is about 10 12 m2, which still allows consider-
able flow of moisture under a pressure of 10–100 kPa,
which are common in practice. The permeability in the
outer layer may be as low as 10 16 m2 or less, and even
a layer of a few mm then makes a substantial barrier.

The effects of moulding, pressing and resting on the
moisture content have been carefully studied by
Geurts (1978) and some results are shown in Fig. 25.
It is seen that the lower the moisture content before
pressing, the less the further loss of moisture. The ini-
tial water content has other important consequences.
First, consider the situation where it is high, say about
55% at the beginning of pressing. Now, pressing at an
earlier stage or at a higher pressure leads to a higher
water content (more precisely, a less reduced water
content); the difference is of the order of 1% water.
60
% Water
50
S ture in unsalted cheese. Some results are shown in
40
0 edly due to the heat generated by the growing starter
0 5
Time (h)
Figure 25 Water content of a loaf of curd as a function of time
after moulding. The dotted lines give the assumed course during
pressing. Spherical loaves of about 22 cm diameter, except for
one of 12 cm (designated S) (from Geurts, 1978).
49
Moisture content of cheese (%)
1-kg CHEESE
45
41
0 40 80
Figure 26 Water distributions in unsalted spherical cheeses (1 and 6 kg), moulded from the same curd, lightly pressed and kept for
a few days. The broken lines indicate the average water content (from Geurts, 1978).
The Syneresis of Rennet-coagulated Curd 97
The explanation is that a closed rind is formed at an
earlier stage or of a greater thickness. Pressing at a
higher initial temperature or having a larger loaf of
curd led to a lower water content. Although a higher
temperature implies a softer curd, and thereby pre-
sumably easier rind formation, the overriding effect
seems to be the effect of temperature itself on syneresis
(see e.g., Fig. 10). A smaller loaf will cool faster and
thereby lose less moisture (see Fig. 25).
These relations are rather different if the curd mass
has a low water content at the beginning of pressing,
say 40%. (Such a low water content can be obtained
only by prolonged stirring at a high temperature and
letting the pH decrease appreciably.) A higher pres-
sure, a smaller loaf and a lower temperature all lead to
a lower water content. Presumably, it takes a longer
time to obtain a closed rind, the more so for a lower
temperature, whereby more moisture can be pressed
out of the loaf before the rind is formed.
Geurts (1978) also studied the distribution of mois-
Fig. 26. Apart from a thin outer layer, i.e., the rind,
which has a slightly reduced water content, the lowest
water content is at the centre. This is the region where
the temperature has remained highest, especially in a
large loaf. It was even observed that the temperature
increased in the greater part of a large loaf, undoubt-
10 bacteria. If an unsalted cheese is left to rest, the water
content tends to become somewhat lower at the bot-
tom side. It may thus be concluded that the moisture
moves away from regions where the temperature is
higher and/or the pressure higher than elsewhere.
Soon, however, the process of fusion of curd grains
6-kg CHEESE
120 160 200
mm
98 The Syneresis of Rennet-coagulated Curd
becomes complete, say after two days (Luyten, 1988),
and the permeability of the cheese mass becomes too
low to permit appreciable transport of moisture.
In several types of cheese, the drained mass of curd is
allowed to spread laterally for a considerable time (‘ched-
daring’). Olson and Price (1970) found that this led to a
higher moisture content (1–2% more water), compared
to curd kept for the same time but which was prevented
from spreading. Although cheddaring may have caused a
slightly lower average temperature, the main cause for
the differences was presumably that the flow of curd pro-
moted deformation of the curd grains; hence, closing of
pores between grains and hindering drainage of any
moisture, still leaving the grains due to syneresis.
The water content of the cheese must, to a consid-
erable extent, depend on the amount of syneresis dur-
ing curd preparation, and the results for syneresis as
given earlier, indeed qualitatively, agree with results on
the water content of cheese (e.g., Sammis et al., 1910;
Whitehead, 1948; Whitehead and Harkness, 1954;
Birkkjaer et al., 1961; Feagan et al., 1965; Straatsma
and Heijnekamp, 1988). Whether there is exact agree-
ment is uncertain. The water content of cheese always
shows considerable random variation (e.g., Straatsma
et al., 1984) and this makes exact comparisons diffi-
cult. During cheesemaking, conditions usually change,
for instance pH, temperature and effective pressure
acting on the curd, so that one has to take some kind
of average. Moreover, the factors are inter-related; for
instance, temperature affects the rate of acidification.
The latter is also affected by oxygen content (Gillies,
+2
Change in water content (% units)
–2
60 70 80
Past. temp. (°C)
+1
–1
40 60 80
Time (min)
Figure 27 The effect of some variables in treatment of milk and in curd-making on the water content of unsalted Gouda type
cheese, 5.5 h after renneting, other conditions being equal; time means time after cutting. The water content under standard condi-
tions was about 46% (from Straatsma and Heijnekamp, 1988).
1959) and this may explain why some authors found a
negative correlation between stirring rate of the
curds–whey mixture and the final water content of the
cheese (Kiermeier and von Wüllerstorf, 1963); pre-
sumably, faster stirring caused a higher oxygen con-
tent, hence inhibition of starter bacteria, slower
acidification, and consequently less syneresis.
The main causes of discrepancy may be, however, the
considerable effects of the conditions of curd drainage and
further treatment, such as pressing. If these processes are
kept constant, as is nearly always more or less the case
during modern cheesemaking, the correlation between
syneresis and final water content may be fairly good; one
should then take into account the remark made earlier
about the inhibiting effect of the formation of a dense
outer layer around the curd grains. Some interesting prac-
tical results for the case of semi-hard brine-salted cheese
were obtained by Straatsma and Heijnekamp (1988), and
some of these are summarized in Fig. 27. It is seen that
most variables have, within the variation that can reason-
ably be applied in practice, a fairly small effect. Only the
scalding (cooking) temperature and the acidity of the
curd had a significant influence. The acidity depends
primarily on type and quantity of starter added, any
preacidification applied and temperature and duration of
acid development. The rate at which the acidity was
reached appeared to make little difference.
It may finally be mentioned that the water content of
cheese also depends, of course, on salting (by pseudo-
osmosis, Geurts et al., 1974), drying and proteolysis
(which causes water to be converted into dry matter).
5.5 6.0 6.5 33 35 37
pH after 4 h Scalding temp. (°C)
5 6 0 2 4
Grain size (mm) CaCl2 (mmolar)

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This Page Intentionally Left Blank
 Formation, Structural Properties
and Rheology of Acid-coagulated
Milk Gels
J.A. Lucey, Department of Food Science, University of Wisconsin-Madison,
Madison, USA

Introduction micelles. The stability of casein micelles of milk is

attributed to their net negative charge and steric repul-
Fresh acid-coagulated cheese varieties include Cream
sion by the flexible macropeptide region of -casein
cheese, Cottage cheese and Quarg and other cheeses
(the so-called ‘hairs’). Different types of interaction are
where the coagulation of milk occurs by acid rather
responsible for micelle integrity, including Ca-induced
than by rennet, as in most other cheese varieties
interactions between protein molecules, electrostatic,
(e.g., Cheddar). Fresh acid cheeses differ from fer-
hydrophobic and hydrogen bonding. These inter-
mented milk products in having a significant amount
actions are probably also involved in the formation
of the moisture (whey) removed after coagulation.
and structural properties of acid casein gels.
Whey removal methods, such as centrifugal separ-
Various models for the structure of casein micelles
ation and ultrafiltration (UF), are used for Quarg and
have been proposed, and it has been the source of con-
Cream cheese whereas cutting of the coagulum into
troversy over the years. The latest model by Horne
granules and a high cook temperature are used for
(1998) envisages a polymerization scheme (dual-binding
Cottage cheese. Cultures of mesophilic lactic acid bac-
model) for the assembly of casein micelles. Cross-linking
teria (i.e., usually Lactococcus spp. and Leuconostoc
of the molecules proceeds via two routes, hydrophobic
spp.) and sometimes probiotic species are used as
interactions between groups on different molecules
cultures for most fresh acid curd cheeses. A common
forming one pathway, with more than two molecules
factor in all of these acid cheese products is that the
possibly joining at such junctions, and a second path-
initial step involves the formation of an acid-induced
way where chain extension is through a CCP nanoclus-
gel, which is then further processed. The formation
ter acting as a neutralizing bridge between two
and physical properties of acidified milk gels have
phosphoseryl clusters on separate molecules of s1-,
been reviewed recently (Lucey and Singh, 1997,
s2- or -casein. Both routes permit branching and
2003; Horne, 1999; Lucey, 2002a). There has been
hence lead to a three-dimensional network. However,
considerable research on acid milk gels made with
-CN can link only to hydrophobic residues on another
thermophilic cultures for the production of yogurt
CN molecule. Because it has no phosphoseryl cluster to
(e.g., Tamime and Robinson, 1999). The manufacture
permit further extension, the polymer chain ends there.
and technologies involved in the production of fresh
As a consequence, the -CN acquires an external sur-
acid cheeses have also been reviewed (Guinee et al.,
face position where it acts as a steric stabilizer.
1993; Puhan et al., 1994; Kosikowski and Mistry,
As the pH of milk is reduced, CCP dissolves and
1997; Fox et al., 2000; Lucey, 2002b). This chapter
the caseins are liberated into the serum phase (Dalgleish
focuses primarily on the formation of these acid-milk
and Law, 1988). The extent of liberation of caseins
gels and their physical, rheological and microstruc-
depends on the temperature at acidification (Dalgleish
tural properties.
and Law, 1988), which has little effect on the solubil-
ization of CCP. Apparently, little change in the average
Casein micelles
hydrodynamic diameter of casein micelles occurs dur-
Caseins constitute approximately 80% of the protein ing acidification of (unheated) milk to pH ⬃5.0
in bovine milk, with four main types ( s1-, s2-, (Roefs et al., 1985; de Kruif, 1997), although the
- and -caseins (CN)) in combination with appre- internal structure of the casein micelles is altered due
ciable quantities of micellar or colloidal calcium phos- to the loss of CCP (Walstra, 1993). Aggregation of
phate (CCP) in the form of aggregates called casein casein occurs as the isoelectric point (pH ⬃4.6) is
Cheese: Chemistry, Physics and Microbiology, Third edition – Volume 1: General Aspects Copyright © 2004 Elsevier Ltd
ISBN: 0-1226-3652-X All rights reserved
Set ISBN: 0-1226-3651-1
106 Formation, Structural Properties and Rheology of Acid-coagulated Milk Gels
0.25
Casein in solution (%)
0.20
0.15
0.10
0.05
0.00
1 2 3 4 5 6 7 8
pH
Figure 1 Solubility of whole casein in water as a function of pH
(figure replotted with permission from Strange et al., 1994).
approached (Fig. 1); under conditions of rapid acidifi-
cation and/or agitation, the casein aggregates precipi-
tate from solution and this is the basis of acid casein
manufacture.
Coagulation Mechanisms
Theoretical models
Acid milk gels are examples of particle gels and at least
three theoretical models, namely fractal, adhesive hard
spheres and percolation models, have been used to
model the formation of acidified milk gels (Horne,
1999; Lucey and Singh, 2003). Only a brief overview
is given here and interested readers can refer to these
review articles.
Fractal aggregation theory has been applied to the
formation of various casein gels (Bremer et al., 1989,
1990, 1993; Vetier et al., 2000). From the fractal
approach, a number of scaling laws have been used to
derive relations between the physical properties of
gels and the fractal dimensionality (Bremer, 1992).
The fractal approach has successfully described semi-
quantitative features of casein gels (e.g., rheological
properties), but appears to have some deficiencies,
including the lack of any allowance for aggregate
rearrangement or interpenetration, and the assump-
tion that all aggregates have the same size at the gel
point (Dickinson, 1997). If there are only limited
rearrangements, the fractal dimensionality probably
increases, but after severe rearrangements a fractal
description of the clusters will no longer hold (van
Vliet, 1999). Horne (1999) has also questioned the
fractal definition of the gelation point (i.e., the sug-
gestion that all casein particles become part of the gel
matrix at the point of gelation).
Casein aggregation during the acidification of milk
has also been modelled using the adhesive hard
sphere theory (de Kruif et al., 1995; de Kruif and
Roefs, 1996; de Kruif, 1997, 1999). In this model, it is
proposed that the caseinomacropeptide (CMP) part of
-casein sterically stabilizes casein micelles and is
considered as a polyelectrolyte brush, which collapses
on the surface of the micelle as the pH of the system
approaches the pKa of the charged (carboxylic acid)
groups on the brush. Horne (1999, 2003) pointed out
that this model assumes that only the surface features
of casein particles have any bearing on the structural
properties of acid milk gels. However, it has been
shown recently that the loss of CCP from casein
micelles dramatically influences the properties of
casein gels (Lucey et al., 1998c; Horne, 2001, 2003).
Horne (1999) reviewed the suitability of percolation
models for acid milk gels and suggested that such
models may be suitable only at the gel point and that
it is difficult to use this theory to model the mechan-
ical properties of acid milk gels.
Physico-chemical mechanisms involved in the
formation of gels from unheated milk
Native casein micelles (in milk of normal pH) are
stabilized by a negative charge and steric repulsion
(Walstra, 1990; Mulvihill and Grufferty, 1995). Some
of the techniques that have been used to study the
acid coagulation process are listed in Table 1.
The surface charge of casein micelles can be
approximated from the zeta potential and a plot of the
changes in zeta potential as a function of pH is shown
in Fig. 2. Casein micelles exhibit some unusual zeta
potential behaviour. There is a minimum at pH 5.4
(negative) and a maximum at pH 5.1 (Schmidt and
Poll, 1986; Anema and Klostermeyer, 1996). It has been
suggested that the shape of the zeta potential–pH profile
is due to subtle dissociation and association phenomena
of the caseins in this pH region (Heertje et al., 1985).
Table 1 Some of the various techniques used to study the acid
coagulation process
Viscometry
Rheometry
Thrombelastography
Texture analysis
Dynamic light scattering
Diffusing wave spectroscopy
Turbidity
Colorimetry
Confocal laser scanning microscopy
Electron microscopy
Permeability
 Formation, Structural Properties and Rheology of Acid-coagulated Milk Gels 107
–12
–10
–8
Zeta potential (mV)
–6
–4
–2
0
4.4
Figure 2 Dependence on pH of the zeta-potential of washed unheated casein micelles on pH (from Schmidt and Poll, 1986 reproduced
with permission from Elsevier).
They proposed that at pH ⬃5.5, there is preferential
dissociation of -casein and that at pH ⬃5.2 it reasso-
ciates with the micelles and this coincides with a ‘stage
of contraction and rearrangement’. However, recent
studies (Law, 1996; Singh et al., 1996) have shown
that at temperatures 20 °C, which are commonly
used for the formation of acid milk gels, no preferen-
tial dissociation of -casein from the micelles occurs
during the acidification of milk. It is more likely that
this unusual behaviour of the zeta potential is caused
by the solubilization of CCP, which modifies the ionic
environment around casein micelles.
Three pH regions in the acidification of milk
from pH 6.7 to 4.6 (which is the pH range of interest
for the various types of acid-type cheeses) can be
distinguished:
1. pH 6.7 to ⬃6.0. The decrease in pH causes a
decrease in the net negative charge on the casein
micelles, thereby reducing electrostatic repulsion.
Only a relatively small amount of CCP is dissolved
above pH 6.0, so the structural features of the
micelles are relatively unchanged (e.g., size). As a
consequence of this reduced repulsion (as the pH is
5.0 6.0 7.0 8.0
pH
lowered) there is a decrease in the gelation time and
an increase in gel firmness if rennet is used to coagu-
late milk (Zoon et al., 1989).
2. pH ⬃6.0 to ⬃5.0. The decrease in pH causes a
decrease in the net negative charge on the casein
micelles, thereby reducing electrostatic repulsion.
The -casein ‘hairs’ on the micelle surface are
charged, so their charged ‘hairs’ may shrink as the
pH decreases. The net result is a decrease in both
electrostatic repulsion and steric stabilization, the
two factors that are primarily responsible for micelle
stability. The CCP within casein micelles is dissolved
completely by pH ⬃5.0 in the case of milk, but a
considerable proportion of CCP remains intact in the
manufacture of natural, rennet-coagulated cheese
(Lucey and Fox, 1993), presumably due to a protect-
ive effect of the higher solids. The dissociation of
casein from the micelle is very dependent on
temperature and pH. The pH of maximum dissociation
(at temperatures #20 °C) is 5.2–5.4 (Dalgleish and
Law, 1988), presumably due to loosening of the molecu-
lar interactions between caseins due to the loss of
CCP, which causes increased electrostatic repulsion
between the newly exposed phosphoserine groups. At
108 Formation, Structural Properties and Rheology of Acid-coagulated Milk Gels

low temperatures, for e.g., 5 °C, considerable dissoci-
ation occurs, especially at pH 5.4–5.2; some disso-
ciation occurs at 20 °C, but dissociation decreases
rapidly 20 °C, and at 30 °C there is virtually no
liberation of caseins (Dalgleish and Law, 1988).
-Casein dissociates to a greater extent than the
other caseins during acidification at low tempera-
tures (Dalgleish and Law, 1988); since the -casein
‘hairs’ provide a stabilizing layer (both sterically
and electrostatically), any reduction in this stabil-
ization should render the micelles more sensitive to
aggregation.
3. pH #5.0. The net negative charge on the
casein micelles declines with the approach of the
isoelectric point and there are increased electrostatic
interactions and reduced electrostatic repulsion,
which allow increased hydrophobic interactions
(Horne, 1998). In unheated milk gels, in which
acidification is the only coagulation method, gela-
tion occurs around pH 4.9 unless acidification is
performed at a very high temperature when a higher
gelation pH is observed.
On acidification, casein particles aggregate as a result
of (mainly) charge neutralization, the main titratable
groups in milk are shown in Table 2. Acidification even-
tually leads to the formation of chains and clusters that
are linked together to form a three-dimensional network
(Mulvihill and Grufferty, 1995). Acid casein gels can be
formed from sodium caseinate and gelation also occurs at
pH ⬃5.0 (Lucey et al., 1997b,c). Direct acidification of
milk at a low temperature may allow solubilization of
CCP prior to gelation and therefore these gels may
undergo less change in their mechanical properties (e.g.,
syneresis) than traditional cultured products. Glucono--
lactone (GDL) is also used to acidify milk but these acid-
induced gels probably have different rheological and
structural properties from gels produced by in situ acid
production by bacterial cultures (Lucey et al., 1998d).
Hydrophobic interactions are unlikely to play a direct
role in the strength of acid gels as the G of these gels
increases with decreasing assay temperature. Cooling
such gels results in an increase in G , probably due to
swelling of casein particles (caused by the weaker
hydrophobic interactions) and an increase in the contact

Table 2 Main titratable groups in milk (reprinted with permission from Singh et al., 1997)

Approximate
concentration
Group (mM)a,b Expected pKac,d pKa (in milk)a

Salts
Inorganic phosphate 21.0e 2.1, 7.2, 12.3 3, 5.8, 6.6f
Citrate 9.0–9.2 3.1, 4.7, 5.4 3, 4.1, 4.8f
Organic phosphate esters 2.5–3.5 1.4, 6.6? 1.7, 5.9f
Carbonate 2.0 6.4, 10.1 6.4, 10.1
Lactic acid 0.4 3.9 3.9
Formic acid 0.2–1.8 3.6 3.6
Acetic acid 0.05–0.8 4.7 4.8
Various amines 1.5 ⬃7.6 7.6

Ionizable groups of proteins Concentration (mM)a Expected pKaa,c,d,g pKa (in milk)a
Aspartic acid (-COOH) 19 4.6 4.1
Glutamic acid (-COOH) 50 4.6 4.6
Histidine (imidazole) 6 7.0 6.5
Tyrosine (phenol) 12 9.6
Lysine ( -NH3) 20 10.2 10.5
Phosphoserine (phosphate) 7 1.5, 6.5 2.6
N-acetylneuraminic acid (COOH) 0.5 2.6 5.0
Terminal carboxyl ( -COOH) 3.7 3.7
Terminal amino ( -NH3) } 1.5 7.9 7.9

a Data from Walstra and Jenness (1984).

b Data from Jenness (1988).
c Data from Tanford (1962).
d Data from Edsall and Wyman (1958).
e About 10 mM colloidal phosphate, 11 mM in solution (at pH 6.6).
f pKa values from titration with Ca(OH)2.
g Data from Damodaran (1996).
 Formation, Structural Properties and Rheology of Acid-coagulated Milk Gels 109
area between particles. With increasing ionic strength,
charged groups on casein would be screened, thereby
weakening interactions between casein particles.
Physical Properties of Acid-Induced Gels
Rheological properties of acid milk gels
Acid milk gels are viscoelastic and exhibit shear thin-
ning when sheared and slow recovery after shearing is
stopped. The textural and rheological properties of
acid milk gels can be assessed by a range of fundamen-
tal and empirical methods such as small amplitude
oscillatory rheology (SAOR), large amplitude oscilla-
tory shear, penetration, texture profile analysis, rota-
tional viscometry and flow through an orifice such as a
Posthumus funnel (Benezech and Maingonnat, 1994;
Velez-Ruiz and Barbosa Canovas, 1997). A combin-
ation of techniques should ideally be used to monitor
the gel formation phase, as well as the impact of fur-
ther processing steps on the gel properties (e.g., stir-
ring). The rheological parameters characterizing acid
casein gels depend on the number and strength of
bonds between the casein particles, on the structure of
the latter and the spatial distribution of the strands
making up these particles (Roefs et al., 1990a).
Some techniques (e.g., texture profile analysis) may
give only a single-point measurement and damage the
sample. The initial step in the manufacture of most acid-
type cheeses is gelation, so dynamic non-destructive
techniques, such as SAOR, are needed to study this
process. The SAOR technique involves the application of
an oscillating strain or stress that is within the linear vis-
coelastic region for that material (usually less than 5%
strain for most milk gels). Some of the main parameters
determined from these tests include the elastic or storage
modulus (G ), which is a measure of the energy stored
per oscillation cycle, the viscous or loss modulus (G ),
which is a measure of the energy dissipated as heat per
cycle, and the loss tangent (tan ), which is the ratio of
the viscous to the elastic properties (Lopes da Silva and
Rao, 1999). These parameters are defined as follows:
G'  冢  冣cos 


(1)
G"  冢  冣sin 


(2)
G
tan   (3)
G observed due to the high incubation temperature (32 °C)
where 0 is the amplitude of the shear stress, 0 is the
amplitude of the strain and  is the phase angle.
The rheological properties of acid milk gels have
been studied extensively over the past 15 years or so
(see reviews by Benezech and Maingonnat, 1994;
Lucey and Singh, 1997). In general, unheated skim
milk forms a weak gel (G 50 Pa), and the pH at
gelation is generally ⬃4.8–5.0. An example of some
rheological properties of a high-fat acid milk gel (simi-
lar to cream cheese) is shown in Fig. 3. After gelation,
G increases rapidly and only starts to plateau during
ageing of the gel (in the region of pH ⬃4.6), tan 
decreases to 0.4 soon after gelation and decreases to
⬃0.2–0.3 during the ageing of acid milk gels. Roefs
(1986) demonstrated that for acid gels, G could con-
tinue to increase for up to several days, due, presum-
ably, to slow ongoing fusion/rearrangements of casein
particles.
An unusual rheological phenomenon is observed
soon after the formation of an acid-induced gel from
heated milk; tan  decreases initially but then
increases to a maximum value before decreasing
again (e.g., Biliaderis et al., 1992). A high tan  indi-
cates an increased susceptibility of bonds and
strands in the gel to break or relax, thus facilitating
more rearrangements of the gel (van Vliet et al.,
1991). The maximum in tan  may be a conse-
quence of a partial loosening of the weak initial gel
network due to the solubilization of CCP, while at
lower pH values there are increased protein–protein
attractions between casein particles as the net charge
decreases with the approach of the isoelectric point
(Lucey et al., 1998c). The maximum in the value of
tan  occurs in acid gels that have a high gelation
pH, e.g., gels made from heated or unheated milk to
which some rennet is added (Lucey et al., 1998c,
2000).
An example of some rheological properties of an
acid skim milk gel made from severely heated milk
(similar to quarg cheese) is shown in Fig. 4. The very
low incubation temperature (23 °C) and the slow
acidification and gelation processes result in a
smaller maximum for tan  (more of a flattening of
the curve) compared to gels made at a higher tempera-
ture (e.g., 30 °C). Presumably, under those gelation
conditions there is a slower and less dramatic impact
of solubilization of CCP on the mechanical properties
of casein gels (more CCP is solubilized pre-gelation).
An example of some rheological properties of an acid
skim milk gel made from unheated milk with a small
amount of rennet added (similar to Cottage cheese) is
shown in Fig. 5. Gelation occurs at a high pH due to the
action of rennet and a clear maximum in tan  is
110 Formation, Structural Properties and Rheology of Acid-coagulated Milk Gels

0.50 250 7.0

0.45 200 6.5

Storage and loss modulus, Pa

0.40
150 6.0
Loss tangent

0.35

pH
100 5.5
0.30

50 5.0
0.25

0 4.5
0.20

0.15 4.0
0 100 200 300 400 500 600 700 800 900 1000

Time (min)

Figure 3 Rheological changes during the formation of the initial cream cheese gel. Storage modulus (), loss modulus (), loss
tangent () and pH changes (solid line) as a function of time during the incubation of 12% fat milk with 2% of a mesophilic starter
culture at 23 °C. Milk was homogenized at 17.5 and 5 MPa double stage at ⬃60 °C. The applied frequency was 0.1 Hz and the strain
was 1% (unpublished data of Lucey et al., 2003).

and the fast acidification rate (5% starter culture added).
The G increases rapidly initially but the profile flat-
tens in the vicinity of the maximum in tan . These
trends are similar to that observed for model gels made
with a combination of rennet and acid (GDL) (Lucey
et al., 2000).
Horne (1999) reported that the rheological properties
of acidified milk gels exhibit a form of scaling behaviour.
For acid gels made from unheated or heated milk, there
are two distinct ‘master curves’, which implies that there
are fundamental differences in the kinetics and dynamics
of the gel formation process in these two types of gels.

0.50 160 7.0

140
0.45
6.5
Storage and loss modulus, Pa

120

0.40 100
Loss tangent

6.0
80
pH

0.35
60
5.5
0.30 40

20 5.0
0.25
0

0.20 4.5
0 100 200 300 400 500 600 700 800 900 1000 1100
Time (min)

Figure 4 Rheological changes during the formation of the initial quarg cheese gel. Storage modulus (), loss modulus (), loss
tangent () and pH changes (solid line) as a function of time during the incubation of skim milk with 1% of a mesophilic starter cul-
ture at 22 °C. Milk was pre-heated at 90 °C for 5 min. The applied frequency was 0.1 Hz and the strain was 1% (unpublished data of
Lucey et al., 2003).
 Formation, Structural Properties and Rheology of Acid-coagulated Milk Gels 111
1.0
0.9
0.8
Loss tangent
0.7
0.6
0.5
0.4
0.3
0 50 100 150
Time (min)
Figure 5 Rheological changes during the formation of the initial cottage cheese gel. Storage modulus (), loss modulus (), loss
tangent () and pH changes (solid line) as a function of time during the incubation of skim milk with 5% of a mesophilic starter cul-
ture at 32 °C. Approximately 1 ml of standard strength rennet was added per 450 kg milk just after culture addition. The applied fre-
quency was 0.1 Hz and the strain was 1% (unpublished data of Lucey et al., 2003).
In experiments where the time-scale of the applied
deformation was varied (frequency sweeps), log G
versus log angular frequency gave linear curves with a
slope of ⬃0.15 for various types of acid casein gels
(Roefs and van Vliet, 1990; Lucey and Singh, 1997).
This suggests that similar (fundamental) structural
components (bonds) are present in all types of (aged)
acid casein gels.
Acid casein gels are very brittle and fragile com-
pared with rennet-coagulated milk gels. It is difficult
to form a gel suitable for cutting and this approach is
used for only a few cheeses (e.g., Cottage). Most acid
gels when stirred or mixed have a smooth, non-curdy
texture. Improper equipment design and excessive
pumping can damage or shatter this fragile gel and
result in yield losses. There is little published informa-
tion on the fundamental large deformation properties
of acid milk gels although this would provide useful
information on properties that may be related to the
consistency of the gel during consumption, cutting or
shearing.
Mixing and stirring of acid milk gels prior to rheo-
logical testing means that many reported ‘fracture’
(yield) properties are not those of the original ‘set’ gel
(Lucey and Singh, 1997). Another problem that can
affect viscometric measurements of acid milk gels is
slip when using flow curves (Suwonsichon and Peleg,
1999). Unrealistically low values (0.5) that have
been reported for the flow index (n) of stirred acid
milk gels could be due to these problems (Suwonsichon
and Peleg, 1999). Acid milk gels exhibit time-dependent
80 6.6
6.4
60 6.2
Storage and loss modulus, Pa
6.0
40
5.8
pH
5.6
20
5.4
5.2
0
5.0
4.8
200 250
flow behaviour (Benezech and Maingonnat, 1994; Velez-
Ruiz and Barbosa Canovas, 1997).
Fundamental large deformation rheological proper-
ties of acid casein gels have been reported (Bremer et al.,
1990; van Vliet et al., 1991; van Vliet and Keetels, 1995;
Lucey et al., 1997a,b, 2000). Gross fracture of acid
casein gels made with GDL was observed at a strain of
0.5–0.6. The shear stress at fracture increases with
decreasing gelation temperature and with ageing of the
gel. The strain at fracture decreases with ageing of the
gel. Heat treatment of milk prior to acidification (with
GDL) results in a large reduction in the strain at fracture,
from ⬃1.5 for gels made from unheated milk to 0.5–0.8
for gels made from milk samples heated at a temperature
80 °C. Partial rebodying (structural recovery) of acid
milk gels occurs after the structure has been disrupted
by shearing (Arshad et al., 1993), which presumably
reflects reforming of some of the weak (electrostatic,
hydrophobic) interactions between casein particles.
Texture and sensory properties
The microstructure of acid milk gels has a marked
effect on their texture and sensory attributes (Langton
et al., 1996). An excessively firm texture can be
caused by factors such as a very high total solids
content of the mix (both fat and casein) or an exces-
sive amount of added stabilizers. A weak body can
be caused by factors such as a low solids (fat) con-
tent of the mix, insufficient heat treatment of the
milk, low acidity (high pH) and a too low gelation
temperature.
112 Formation, Structural Properties and Rheology of Acid-coagulated Milk Gels
For Cream cheese it is considered that if the pH of
the cheese is too high (i.e., 4.7) the texture will be
soft and the cheese will lack flavour. At a very low
pH (4.6), Cream cheese may become too grainy
and the flavour too acid. Defects in Cream cheese
include whey separation from the product during
storage, lack of spreadability and a grainy chalky tex-
ture, especially in the lower fat types. Textural
defects described as ‘chalkiness’ or ‘grainy’ are objec-
tionable, as consumers usually expect a smooth,
fine-bodied product (Bodyfelt et al., 1988). Exces-
sive aggregation of protein has been associated with
this kind of chalky or gritty defect. Hot-pack cheese
has a more brittle texture than cold-pack product
due to the additional heating and shearing treat-
ments. Cream cheese should have a spreadable con-
sistency as it is commonly used on bagels and in
cheesecakes. Quarg from skim milk is smooth and
white with a mild clean, acid flavour. Addition of fat
improves smoothness. In contrast to most other fresh
acid cheeses, Cottage cheese has a granular, curdy
texture instead of being a viscous, smooth or pasty
product.
The sensory or flavour attributes of acid milk prod-
ucts are very important. For many markets, fruits,
sweeteners, spices and condiments are added, which
can, to a large extent, determine the sensory properties
of these products.
Microstructure
Electron microscopy (EM) and confocal scanning laser
microscopy (CSLM) studies on acid milk gels have
shown that these gels consist of a coarse particulate
network of casein particles linked together in clusters,
chains and strands (Kalab et al., 1983; Lucey and
Singh, 1997). The network has pores or void spaces in
which the aqueous phase is confined; in fat-containing
products, the presence of (large) fat globules obscures
the finer details of pores and strands. The diameter of
these pores varies considerably, with larger pores in
gels made at a high gelation temperature (usually
30 m) or from milk with a low protein content.
There have been several EM studies on the microstruc-
ture of gels formed by acidification of heated milk
(Davies et al., 1978; Parnell-Clunies et al., 1987; Mottar
et al., 1989). Harwalkar and Kalab (1980) proposed,
based on the examination of electron micrographs,
that acid milk gels made from unheated milk had
larger protein clusters (coarse structure) than gels
made from heated milk, which they described as
highly branched (fine structure). Similar trends have
been reported for GDL-induced gels (Lucey et al.,
1998e).
Confocal laser scanning microscopy (CSLM) is a rela-
tively new (but expensive) technique which enables
samples to be observed with minimal preparation pro-
cedures due to its unique optical sectioning capabilities
and high spatial resolution (Brooker, 1995) and is very
suitable for observing the overall microstructure of
milk gels (Hassan et al., 1995; Lucey et al., 1997c,
1998b,e, 2001). A confocal micrograph of an acid-
induced gel made from heated milk is shown in Fig. 6;
its structure appears more interconnected than unheated
milk gels, especially if a small concentration of rennet
is added (Fig. 7) (Lucey et al., 2001). Confocal images
are very amenable to image analysis since the images
are already in a digital form.
Permeability
Permeability measurements provide information about
inhomogenities at the level of the gel network (i.e.,
largest pores). A simple tube method was developed by
the Wageningen group (van Dijk and Walstra, 1986) to
determine the permeability coefficient of milk gels. Gels
are made in open-ended glass tubes. After gelation the
gel tubes are placed in a measuring container full of
whey, where the level of whey is above the height of the
gel in the tubes. The pressure gradient resulting from the
difference in height of the (top of the) whey container
(actually an empty reference tube) and the gel tube is
enough to cause the flow of serum through the gel. The
permeability coefficient can be calculated as follows:
 ht2)
冤ln (h(h
  ht1)
冥
H
B (4)
[!g(t2 t1)]
where B is the permeability coefficient (m2), h is the
height of the whey in the reference tube (m), ht1 is
the height of the whey in the gel tube at t1 (m), ht2 is
the height of the whey in the gel tube at t2,  is the vis-
cosity of the whey, H is the height of the gel (m), ! is the
density of the whey and g is acceleration due to gravity.
For most acid milk gels formed at 30 °C, the value of B
is in the range ⬃1 2  10 13 m2 (Roefs et al., 1990a;
van Marle and Zoon, 1995; Lucey et al., 1998e). In gen-
eral, a very high incubation temperature, use of rennet
and conditions of rapid acidification, e.g., GDL-induced
gels, can all result in gels with high permeability.
The permeability of rennet-induced gels is in the
same range as acid-induced gels but for rennet-
induced milk gels, B increases with time, which has
been taken as evidence of ‘microsyneresis’ or breakage
of strands in the network, resulting in the formation of
larger pores (Walstra, 1993). Studies on the permeabil-
ity of acid-induced gels have shown that the value of B
 Formation, Structural Properties and Rheology of Acid-coagulated Milk Gels 113

Figure 6 Confocal laser scanning micrographs of Quarg cheese gel made from skim milk fermented with 1% (w/w) of a mesophilic
starter culture at 22 °C. Milk was pre-heated at 90 °C for 5 min. The pH of the gels was ⬃4.7. Scale bar  20 m (unpublished data
of Lucey et al., 2003).

does not change with time (Roefs et al., 1990a; Lucey
et al., 1997c); however in these studies, B was deter-
mined in aged gels. It is possible that the value of B for
acid milk gels could change with time, at least for a
short period during after gel formation. Gels with
larger pores (higher permeability) are generally less
stable and are more susceptible to whey separation
(syneresis) (Lucey et al., 1997c).
Appearance
Most acid milk gels should have a smooth, semi-
solid consistency, with no surface whey even if they
are subjected to further processing such as the addi-
tion of stabilizers. The appearance of a set gel should
be smooth with no cracks or ‘blemishes’. Defects that
are apparent at the initial gelation stage would prob-
ably require the addition of more stabilizers to pre-
vent whey separation during storage. Acid gels made
from severely heated milks with GDL had a ‘rough’
surface, with visible cracks and some whey separa-
tion (Lucey et al., 1998a,e). It was suggested that
rearrangement of the network during after gel forma-
tion might be responsible for these defects. Gels made
from severely heated milk have a lower strain at frac-
ture than gels made from unheated milk and this may
make heated gels more susceptible to localized frac-
turing of strands in the network (Lucey et al., 1997a).
The ‘transition’ in the rheological properties (as indi-
cated by the maximum in loss tangent) may increase
the susceptibility of protein–protein bonds to relax
and if these bonds have a relatively short lifetime,
114 Formation, Structural Properties and Rheology of Acid-coagulated Milk Gels

Figure 7 Confocal laser scanning micrographs of Cottage cheese gel made from skim milk fermented with 5% of a mesophilic
starter culture at 32 °C. Approximately 1 ml of standard strength rennet was added per 450 kg milk just after culture addition. The pH
of the gel was ⬃4.7. Scale bar  20 m (unpublished data of Lucey et al., 2003).

this may lead to yielding or breaking of strands (van
Vliet et al., 1991).
Whey separation and syneresis
Whey separation (wheying-off) refers to the appear-
ance of liquid (whey) on the surface of a milk gel and
is a common defect in yogurt. However, in fresh acid
cheeses, such as Cream cheese, several techniques are
used to remove some of the whey from the original
gel. Whey removal increases the total solids content
of the product, which results in increased firmness
and viscosity. Syneresis is defined as shrinkage of a gel
and this occurs concomitantly with the expulsion of
whey. It is useful to define spontaneous syneresis as
the contraction of a gel without the application of an
external force (e.g., centrifugation), and this is related
to instability of the gel network (i.e., due to large-
scale rearrangements) (see review by Walstra, 1993).
In practice, fresh acid cheese manufacturers often try
to prevent whey separation in the retail product by
adding stabilizers (e.g., xanthan gum, locust bean
gum, carrageenan) or whey protein concentrate
(WPC) prior to packaging. The causes of wheying-off
during retail storage include post-acidification, tempera-
ture fluctuations, proteolysis by the starter culture,
and physical abuse (e.g., vibration, shaking, improper
stacking).
The amount of spontaneous whey separation in acid
milk gels can be quantified using simple approaches
such as determining the amount of surface whey that is
expelled during gelation (Lucey et al., 1998a). The
quantity of whey expelled from acid milk gels as a result
of high speed centrifugation may be a useful indicator
 Formation, Structural Properties and Rheology of Acid-coagulated Milk Gels 115
of the amount of whey that can be removed during the
mechanical separation process used for Quarg or Cream
cheese products. Quantifying the amount of whey
drainage from a broken gel distributed over a screen
gives a measure of water-holding capacity, and is
more relevant to products such as Cottage cheese
where screens are used to separate the curds/whey
mixture (Lucey et al., 1998a). Some acid milk gels
may be produced by concentrating the milk (e.g., by
UF) to the desired total solids of the final product, thus
eliminating the need for a whey removal step.
It has been shown (van Dijk and Walstra, 1986)
that the one-dimensional syneresis of milk gels is
related to the flow of liquid (whey) through the net-
work and is governed by the equation of Darcy:
B p
v (5)
 x
where v is the superficial flow velocity of the syneresing
liquid through the gel (i.e., volume flow rate over the
cross-sectional area through which the liquid flows), B
is the permeability coefficient,  is the viscosity of the
liquid, p is the pressure acting on the liquid and x the
distance over which the liquid must flow.
In milk gels, a key factor in controlling syneresis is
the degree of rearrangement that occurs in the casein
network (van Vliet and Walstra, 1994). The relations
between rheological parameters (e.g., G , tan , yield
strain and stress, and the frequency dependency of
tan ) and syneresis of acid milk gels have been
discussed by Lucey (2001). Parameters that affect the
time-scale for rearrangements of bonds in a gel
include the dynamic moduli, which indicates the
strength and number of bonds in the network, the
yield stress and shear deformation at yielding, which
determine the susceptibility of the strands to break-
age, and tan , with higher values favouring the relax-
ation of bonds (van Vliet et al., 1991; Lucey et al.,
1997a,c). In freshly made gels, the number of bonds
between each junction is not yet very high, as indi-
cated by the low dynamic moduli, and tan  is higher
than in aged gels; these factors might explain why
wheying-off occurs sometimes in young but less often
in aged gels. Syneresis of acid milk gels made with
GDL increases at high gelation temperatures, high pH
values and in the presence of even low rennet levels
(van Vliet et al., 1997; Lucey, 2002a). It has been
shown recently that endogenous syneresis pressure is
generally small in acid gels made from sodium
caseinate and this results in a lesser tendency of these
gels to shrink compared to rennet-induced gels
(Lucey et al., 1997c). Acid-induced milk gels formed
by slow acidification of milk at a low temperature and
under quiescent heating exhibit little wheying-off or
spontaneous syneresis (Roefs, 1986). Surface whey
expelled during gelation is sometimes reabsorbed by
the product on cooling and storage at a low tempera-
ture (Lucey et al., 1997c). Post-acidification, product
mishandling and temperature abuse are common
causes of wheying-off in acidified milk products.
It should be noted that acid milk gels undergo
much less syneresis than rennet-induced gels even
when they are subjected to centrifugation. For this
reason, acid-coagulated cheeses have a very high mois-
ture content (e.g., 50%).
Effects of Compositional and Processing
Parameters on the Textural Properties
of Acid Milk Gels
The effects of each processing step on the textural
properties of acid milk gels are considered in the fol-
lowing section. A summary of the effects of some of
the main processing factors is given in Table 3.
Inoculation and gelation temperature
Acidification of fresh acid products by cultures is gen-
erally performed by either of two methods: slow,
12–16 h at 20–23 °C (long set) or 4–6 h at 30–32 °C
(short set). Cultures of mesophilic lactic acid bacteria
(i.e., mainly Lactococcus spp. and Leuconostoc spp.) and
sometimes probiotic species are used as cultures for
most acid-coagulated cheeses. Sometimes, fresh cheeses
are made by the addition of acid, e.g., phosphoric or
lactic acid (direct-acid-set or direct acidification)
and/or GDL.
Compared with gels made at 20 °C, acid casein gels
made at 40 °C are coarser, show more rearrangements,
are weaker and less stable (Lucey et al., 1997b,c). In
practice, other process variables (e.g., fat content, stabil-
izers, heat treatment) can help to stabilize this type of
gel. In general, an excessive rate of acid development
(e.g., use of GDL) at a high incubation temperature
(e.g., 45 °C) contributes to the ‘wheying-off’ defect and
poor gel formation. In various types of acid milk gels
formed with GDL, a lower gelation temperature (e.g.,
30 °C) results in a longer gelation time but these gels
can have higher G values than gels made at a much
higher gelation temperature (e.g., 40 °C) (Cobos et al.,
1995; Lucey et al., 1998d). This is due to a coarser gel
structure (greater rearrangements) in GDL gels formed
at high temperatures (Lucey et al., 1997c). In cultured
products, these gelation temperature-related trends
may be less obvious due to the large differences in the
rate of pH decline between cultured and GDL-induced
116 Formation, Structural Properties and Rheology of Acid-coagulated Milk Gels

Table 3 Summary of the effects of some processing conditions on the acid coagulation of milk and properties of the resulting gel

Condition Impact on acid coagulation and gel properties

Incubation Faster acid production at higher temperatures leads to shorter gelation times. At a very high temperature
temperature (e.g., 35 °C) there are more rearrangements of casein particles in the network leading to lower plateau
values for gel stiffness and an increased likelihood of whey separation than gels made at a lower tempera-
ture (e.g., 26 °C). At very high temperatures, the gelation pH may increase. At very low temperatures
(e.g., 4 °C), no coagulation of casein occurs even at pH 4.6.
Heat treatment Heat treatment of milk at a temperature 78 °C for 15 min causes enough whey protein denaturation to
greatly increase gelation pH, decrease gelation time and increase viscosity/firmness. The high isoelectric
point (5.3) of the main whey protein, -lactoglobulin, is responsible for this effect. Disulfide cross-linking of
casein strands increases gel stiffness but solubilization of CCP occurs in casein particles that are already
participating in the gel matrix, which triggers greater rearrangements and is responsible for the increase in
loss tangent observed in rheological tests.
pH Aggregation occurs as the isoelectric point of casein (#4.9) is approached. Maximum gel firmness occurs
around pH 4.6. In general, a slower rate of acidification results in slightly higher gel firmness (may also
provide more time at a low pH which should favour additional bond formation).
Ionic strength At very high ionic strength (e.g., 0.1 M NaCl), no aggregation of casein particles occurs at pH 4.6 due to
screening of electrostatic charges. A minimum concentration of Ca2 is required for acid coagulation.
Casein content Gel stiffness is proportional to casein concentration
Use of rennet The use of a very small amount of rennet in some fresh-type cheeses results in gelation occurring earlier
(i.e., at a higher pH), and greater syneresis during processing (e.g., cooking).

gels. In cultured products, gels that are made at very
low temperatures (e.g., 21 °C) are weaker than gels
made at slightly higher temperatures (eg., 26 °C). The
dynamic moduli of acid gels increase with decreasing
measuring temperature (Lucey et al., 1997a,b). Whey
separation also decreases in GDL gels made at a lower
gelation temperature (Lucey et al., 1997c, 1998a).
Acid-induced milk gels can be formed by slow acid-
ification of milk with acid (e.g., HCl) at a low tempera-
ture (e.g., 5 °C) followed by quiescent heating
(Roefs, 1986). The casein particles at pH values close
to 4.6 are very different from those at the normal,
physiological pH (Walstra, 1993). In this type of gel, it
has been proposed that the decrease in the voluminos-
ity of the casein particles after a gel has formed during
the heating step results in a ‘straightening’ of the nor-
mally tortuous strands in the network (Walstra, 1993).
Hammelehle et al. (1997, 1998) used citric acid to
form milk gels by this cold acidification procedure.
They found that close to the isoelectric point it was
harder to get homogeneous gels when the samples
were subsequently warmed. Gels were formed at a
lower heating temperature when the acidification pH
was lower. The use of a higher setting temperature
(e.g., 40 °C compared with 30 °C) resulted in firmer
gels, which is the opposite trend compared with GDL-
acidified gels. It is likely that the structure of GDL- and
directly-acidified acid milk gels is different. The
method of acidification and gel formation (e.g., GDL,
cold acidification or bacterial fermentation) has a
major impact on the structure and physical properties
of acid milk gels (Roefs, 1986; Lucey et al., 1998d).
Rapid heating of cold-acidified gels to a high tempera-
ture (e.g., 50 °C) resulted in firm gels but considerable
syneresis.
Heat treatment
Heat treatment of milk is one of the most important
process parameters affecting the texture of acid milk
gels (Mulvihill and Grufferty, 1995). Milk used for
some fresh acid cheeses, such as Quarg, is subjected to
an extensive heat treatment. Incorporation of whey
protein into fresh cheese is an important aspect of
fresh cheese manufacture because of an increased
yield. Acid whey is also considered less valuable than
rennet whey in terms of its use for the manufacture of
high value-added whey products. High heat treatment
of milk is not usually practised for Cottage cheese
since heating reduces whey syneresis, which causes
textural defects, including excessive softness and brit-
tleness. Forming a gel that is suitable for cutting is a
step unique to Cottage cheese and is not used in the
manufacture of most other fresh cheeses. Cream
cheese is manufactured from pasteurized milk (72–75 °C
for 30–90 s) (Guinee et al., 1993) as a higher heat
treatment causes difficulties due to not being able to
remove sufficient whey during the centrifugal separa-
tion process.
When milk is pre-heat treated, denatured whey pro-
teins associate with casein micelles and they cross-link
the gel network when aggregation occurs during sub-
sequent acidification of milk. The firmness and viscos-
ity of acid gels has been related to the extent of
 Formation, Structural Properties and Rheology of Acid-coagulated Milk Gels 117
denaturation of whey proteins during heat treatment
(e.g., Dannenberg and Kessler, 1988). Heat treatment
also results in a reduction in the gelation time. In gels
that are formed from pre-heated milk, gelation occurs
at a higher pH (e.g., 5.2–5.4) than from unheated milk
(pH ⬃5.0); these pH values depend on the gelation
temperature. The higher gelation pH can be attributed
to the higher isoelectric pH (⬃5.2) of the main whey
protein, -lactoglobulin, which initiates isoelectric
precipitation/aggregation at a higher pH than for
caseins which have an isoelectric point of ⬃4.6 (Lucey
et al., 1998c). In gels produced from heated milk, the
solubilization of CCP in casein particles that are
already part of the gel network can loosen the gel net-
work, which assists in curd syneresis. At lower pH val-
ues, electrostatic repulsion is weaker which facilitates
greater hydrophobic interactions and as a result, the
gel becomes firmer again and exhibits less syneresis.
In acid milk gels, syneresis is virtually absent at pH
4.6, although this pH is the point of maximum gel
firmness (Roefs, 1986). Moderate heat treatment prior
to acidification had little effect on the extent of solubil-
ization of CCP from the micelles (Law, 1996; Singh
et al., 1996). High heat treatments also increase the
dynamic moduli of acid milk gels (van Vliet and Keetels,
1995; Lucey et al., 1997a, 1998b,c) although the frac-
ture strain decreases with increasing heat treatment,
making these gels more brittle (Lucey et al., 1997a).
Heat treatment may increase the susceptibility of GDL
gels to wheying-off as the gel may undergo greater
rearrangement (Lucey et al., 1998a).
There have been a number of reports on the effects
of heat treatment on the rheological properties of acid
milk gels determined by dynamic low amplitude
(strain) oscillation (van Vliet and Keetels, 1995;
Lucey et al., 1997a, 1998b,c). van Vliet and Keetels
(1995) reported that acid skim milk gels made from
reconstituted low-heat skim milk powder (SMP) had
much lower dynamic moduli than gels made from
high-heat SMP. Lucey et al. (1997a) reported that
heating milk at a temperature 78 °C greatly
increased G compared to unheated milk (⬃15 Pa)
and produced gels with G in the range 350–450 Pa.
Increased cross-linking or bridging, by denatured
whey proteins, within gels made from heated milk
may be responsible for the increased rigidity of the
network (Lucey et al., 1997a, 1998c).
Rennet addition
Rennet is sometimes added in the manufacture of
some acid cheeses (e.g., Cottage and Quarg). The con-
centration of rennet added is very low (e.g., 0.2–10 ml
of standard strength rennet per 1000 l of milk) and it
is often an optional additive in fresh cheeses. Rennet
may be added shortly after the point when the starter
culture is added or more commonly during the fer-
mentation process as long as the pH is not too low
(typical range: pH 6.0–6.3). Some rennet (pre-diluted
rennet and other ingredients are added in a product
called ‘coagulator’) is often added when making large-
curd Cottage cheese but is rarely used when making
small curd–style, as the curd may be more easily shat-
tered during cutting. When rennet is added, the Cot-
tage cheese gel is ready to be cut at a higher pH (e.g.,
4.8) than in its absence (e.g., 4.6) or there would be
excessive loss of fines. Increasing the amount of rennet
added increases the pH of the curd at the point of
coagulation (Emmons et al., 1959); if sufficient rennet
is added, milk will coagulate close to the starting pH
value, as for rennet-coagulated cheeses. Rennet
hydrolyses some -CN and the resulting CMP dif-
fuses away from the micelles, leading to a decrease in
the zeta potential, by ⬃5–7 mV (⬃50%), which
reduces electrostatic repulsion between rennet-altered
micelles. Removal of the ‘hairs’ also results in a
decrease in the hydrodynamic diameter by ⬃5 nm,
and a loss of steric stabilization. In acid cheeses,
the rennet coagulation process proceeds slowly due to
the very low rennet level and low temperature that are
commonly used. There have been a number of recent
reports on model acid milk gels made with combined
rennet addition and concomitant acid production
(Roefs et al., 1990b; Lucey et al., 2000, 2001; Tranchant
et al., 2001). The rheological profiles are often com-
plex due to the effects of solubilization of CCP from
micelles that are already part of a gel network, and
changes in casein–casein interactions as the pH
decreases.
Solids non-fat (SNF) content
It is well known that increasing the solids non-fat
(SNF) content of milk increases the firmness and vis-
cosity of acid milk gels. The protein or SNF content
of milk can be increased by concentrating milk, e.g.,
by reverse osmosis, UF or thermal evaporation or by
dry-matter enrichment. The sources of dry-matter are
usually SMP and WPC. At similar protein levels, acid
milk gels enriched with Na caseinate have a higher
viscosity or firmness than gels enriched with SMP or
WPC. The addition of 1% WPC to milk followed by
heat treatment, resulted in an increase in G and a
reduction in the gelation time for acid milk gels
(Lucey et al., 1999). It was suggested that during heat
treatment, the added whey proteins, as well as the
original whey proteins in milk, were denatured and
associate with the casein micelles to provide additional
118 Formation, Structural Properties and Rheology of Acid-coagulated Milk Gels
cross-linkages within acid-induced gels (Lucey et al.,
1999). Substitution of up to 10–15% of the casein
by WPC has little effect on the final viscosity or sen-
sory attributes of acid milk gels but at higher levels of
substitution, flocculation and off-flavors can occur in
the product. The firmness of acid gels made from
milk with various casein to whey protein ratios was
similar ( Jelen et al., 1987).
Milk with a very high (11%) solids content is not
commonly used for Cottage cheese manufacture due
to problems with cutting the coagulum if it becomes
too firm, texture defects, reduced syneresis, increased
buffering capacity causing a reduced rate of pH
decline, and ‘stratification’ or layering of the solids can
occur in the vat during the slow coagulation process.
Both the total solids and the fat content of milk used
to make Cream cheese influence the ease of whey sep-
aration, e.g., at a low fat content there may be a high
concentration of cheese in the separator whey as the
density of the cheese (which is greatly influenced by
the fat content) becomes similar to the whey.
Fat content and homogenization
Fat provides a perception of creaminess and im-
proves the mouth-feel of acid dairy products. Homo-
genization of milk for fresh cheese manufacture helps
to prevent fat separation during storage, improves
consistency, increases whiteness and reduces whey
separation. Milk is usually homogenized at a pres-
sure in the range 10–20 MPa, at a temperature in the
range 55–65 °C prior to heat treatment of the mix.
It is considered that homogenized full-fat milk
produces a firmer gel than those made from skim
milk (Becker and Puhan, 1989). An increase in homo-
genization pressure has been reported to increase the
viscosity of full-fat acid milk gels (Puhan, 1988). In
the manufacture of high-fat acid milk gels, the use of
a higher homogenization pressure or multiple passes
to produce smaller milk fat globules in the milk
results in an increased G value and a higher yield
stress value calculated from flow curves (Sanchez
et al., 1995).
The nature of the fat globule membrane determines
the types of interaction that can occur between fat
globules and the protein matrix. Fat globules act as an
inert filler if the native fat globule membrane is intact
since this membrane does not interact with casein par-
ticles (van Vliet and Dentener-Kikkert, 1982; van Vliet,
1988). The G value of acid milk gels decreases with an
increasing volume fraction of fat, which has an intact
native fat globule membrane (van Vliet and Dentener-
Kikkert, 1982; van Vliet, 1988). In homogenized or
recombined milk, the native membrane is replaced
largely by casein and some whey proteins so that the
surface of fat particles can interact with the protein
matrix (largely casein but some denatured whey pro-
teins when the gel is made from heated milks) of acid
milk gels (van Vliet and Dentener-Kikkert, 1982; van
Vliet, 1988). In acid milk gels made from recombined
milk, G increases with an increasing volume fraction
of fat (van Vliet and Dentener-Kikkert, 1982; van Vliet,
1988; Lucey et al., 1998b). Cho et al. (1999) showed
that the G of acid milk gels made from either heated
or unheated milk is influenced by the nature of the fat
globule membrane; gels containing fat globules stabil-
ized by sodium caseinate or denatured whey proteins
had very high G values compared with those stabilized
with SMP or native whey proteins.
Homogenization or shearing of high-fat acid gels,
such as is practised in the manufacture of Cream
cheese, results in increased firmness, and brittleness
becomes more evident if shearing of the hot cheese is
excessive (Guinee et al., 1993). There is a general trend
for lower fat products to be more susceptible to a
‘chalky’ or ‘grainy’ defect (Muir, 2000). This may be due
partly to the creamy mouth-feel imparted by fat to dairy
products.
pH and calcium content
It is usually considered that the optimum pH for the
firmness/viscosity of acid milk gels is ⬃4.6. If the pH
falls below about 4.2, the gel may become weaker and
more susceptible to syneresis. A pH of 4.6–4.75 at cut-
ting is often recommended for Cottage cheese; a higher
pH gives a firmer coagulum while a lower pH gives a
softer curd (Emmons and Tuckey, 1967; Emmons and
Beckett, 1984). A higher pH at cutting probably results
in a greater retention of Ca as CCP within casein parti-
cles. Electrostatic repulsion between casein molecules
is increased by dissolving the CCP (Horne, 1998). Acid
casein gels with a very high pH value (e.g., 4.8) have
a much greater tendency to synerese than gels with a
low pH value (#4.6) (van Vliet et al., 1997).
The total calcium content of most fresh cheeses is
low due to the low pH achieved during fermentation
which solubilizes a high proportion of the CCP, which
is lost in the whey during drainage. Calcium chloride is
often added to reconstituted milk used for Cottage
cheese manufacture (e.g., White and Ryan, 1983),
although it probably has only minor effects of gel firm-
ness. Calcium fortification of fresh cheese is attractive
from a nutritional perspective. The addition of CaCl2
(even up to 0.1% which is well above the legal permit-
ted level) did not increase the Ca content of Cottage
cheese curd (Wong et al., 1976). Presumably, most of
the added CaCl2 was soluble at the low pH of acid milk
gels. Acidification of milk to pH ⬃4.9 solubilizes all
the CCP (Pyne and McGann, 1960). In acid casein
 Formation, Structural Properties and Rheology of Acid-coagulated Milk Gels 119
manufacture, Jablonka and Munro (1986) considered
that residual Ca2 on casein particles forms bridges
between negatively charged groups of the caseins (e.g.,
phosphoserine), resulting in tighter, more compact
curds and larger curd particles. Increasing the CaCl2
concentration from 0 to 50 mM reduced the rate of
casein aggregation (Bringe and Kinsella, 1993), pre-
sumably via charge neutralization/screening effects
as well as ‘salting-in’ of the proteins. The addition of
Ca-chelating agents, e.g., citrate, oxalate or ethylenedi-
aminetetraacetic acid, to milk resulted in increased
firmness of GDL-induced gels (Johnston and Murphy,
1992). The structure of casein micelles is disrupted
when CCP is chelated and this open structure presum-
ably provides additional possibilities for casein–casein
interactions in acidified milk products. Excessively
high calcium levels have been associated with a ‘bitter’
taste in fresh products, such as Quarg, although no
clear mechanism has been proposed for how this ‘bit-
terness’ develops.
Acknowledgements
The author would like to thank Tao Wang, Wonjae
Lee and Chanokphat Phadungath for preparing vari-
ous milk gel samples. The author is grateful for the
financial support for this research by the Wisconsin
Center for Dairy Research, Wisconsin Milk Marketing
Board, and the USDA Cooperative State Research,
Education and Extension Service (CSREES) project
WISO4363.
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 Starter Cultures: General Aspects
E. Parente, Dipartimento di Biologia DBAF, Università della Basilicata, Potenza, Italy
T.M. Cogan, Dairy Products Research Centre, Teagasc, Fermoy, Co. Cork, Ireland
Cheese cannot be made without the use of certain
species of lactic acid bacteria (LAB), the major func-
tions of which are to produce lactic acid from lactose
during manufacture and cause biochemical changes
during ripening, which help to develop the charac-
teristic flavour of the cheese being made. The LAB
involved are called Primary Cultures. These organisms
are also called starter bacteria because they ‘start’ (initi-
ate) the production of lactic acid. Generally, the starter
bacteria are carefully selected and deliberately added to
the milk before cheesemaking but, for some cheeses,
particularly Spanish and Italian varieties, no starter is
added. Instead, the cheesemaker relies on adventitious
contaminants present in the milk used to make the
cheese. The main species involved include Lactococcus
lactis, Leuconostoc sp., Streptococcus thermophilus,
Lactobacillus delbrueckii subsp. lactis, Lb. delbrueckii
subsp. bulgaricus and Lb. helveticus but not all of them
are used in every cheese variety. The first two organisms
are used in most cheese varieties while the latter are
used in cheeses like Emmental and Parmigiano Reg-
giano and Pizza/Mozzarella cheese, which are heated to
a high temperature during manufacture. In many arti-
sanal cheeses, especially those produced in Mediter-
ranean countries, other LAB, including Lb. casei, Lb.
plantarum, Ec. faecalis, Ec faecium, Lb. salivarius, and
Staphylococcus species are also found.
Other microorganisms are also used in cheesemak-
ing, e.g., Propionibacterium freudenreichii, Brevibac-
terium linens, Debaryomyces hansenii, Geotrichum
candidum, Penicillium roqueforti and P. camemberti.
These organisms have no function in acid production
and are called Secondary Cultures. Their major role is
to produce organoleptic and biochemical changes in or
on the cheese. These include the production of CO2 by
P. freudenreichii in Emmental cheese (‘Cheese With
Propionic Acid Fermentation’, Volume 2), the blue
veins in Blue cheese, caused by growth of P. roqueforti
(‘Blue Cheese’, Volume 2) or the velvet-like coat of
P. camemberti (mainly) which develops on Camembert
cheese during ripening (‘Surface Mould-Ripened
Cheese’, Volume 2).
Since the last edition of this book (Fox, 1993) there
has been an explosion in scientific information on
starter LAB. This has necessitated the division of the
Cheese: Chemistry, Physics and Microbiology, Third edition – Volume 1: General Aspects
ISBN: 0-1226-3652-X
Set ISBN: 0-1226-3651-1
review of starter cultures into four chapters. This chap-
ter is devoted to general aspects of starter cultures but
it cannot be considered to be exhaustive because of the
time required to digest and assimilate the considerable
literature currently available on these important bac-
teria. The approach taken is to highlight recent studies
on important aspects of starter cultures. Further infor-
mation on many aspects of starter cultures can be
obtained in the symposia on LAB held every three years
in The Netherlands (Venema et al. 1996; Konings et al.,
1999; Siezen et al., 2002), the colloquia held every few
years in France (Anonymous 1996, 1998, 2000, 2001),
Cogan and Accolas (1996) and Salminen and von
Wright (1998).
Taxonomy and Strain Identification
Except for Sc. thermophilus, there have been no
changes in the taxonomy of the starter bacteria since
the previous review by Cogan and Hill (1993). In
1984, Sc. thermophilus was classified as a sub-species
of Sc. salivarius (Farrow and Collins, 1984) but exten-
sive DNA:DNA hybridization studies under stringent
conditions and physiological data have provided evi-
dence to re-confer species rank on the organism
(Schleifer et al., 1991). A new species, Streptococcus
madedonicus, has been recently isolated from Kasseri
cheese (Tsakalidou et al., 1998). It does not hybridize
with Sc. thermophilus and differs from it in not pro-
ducing -galactosidase and in producing acid from
cellobiose, maltose and N-acetyl glucosamine; Sc.
macedonicus shows 96% similarity in 16S and 23S
rDNA sequences with those of Sc. thermophilus.
Another new species, “Sc. waius”, isolated from biofilms
formed on exposure of stainless steel surfaces to
pasteurized milk, is identical to Sc. macedonicus
(Manachini et al., 2002).
Until recently, it was difficult to distinguish between
strains of the same species but the advent of modern
molecular techniques, particularly sodium dodecyl sul-
phate polyacrylamide gel electrophoresis (SDS-PAGE),
randomly amplified polymorphic DNA (RAPD) and
pulse field gel electrophoresis (PFGE) has changed this
significantly. Many isolates from natural cheese
cultures show considerable heterogeneity and these
Copyright © 2004 Elsevier Ltd
All rights reserved
124 Starter Cultures: General Aspects
techniques have proved to be very useful in determin-
ing how many strains are present (Giraffa et al., 1998).
Separation of whole-cell proteins by SDS-PAGE or
DNA fragments by agarose gel electrophoresis results
in characteristic patterns, which can be scanned, nor-
malized and compared. RAPD is a polymerase chain
reaction (PCR) technique in which a random primer of
10 nucleotides is used as a template to generate DNA
fragments which are then separated by gel elec-
trophoresis. This is a rapid procedure. PFGE is a proced-
ure where the total DNA is extracted from the cells
and hydrolysed with rare-cutting restriction enzymes
into large fragments, which are then separated by gel
electrophoresis. This is a slow and labour-intensive
technique but the band patterns are very reproducible
and allow one to discriminate objectively between dif-
ferent strains and decide unequivocally if strains are
the same or not. PFGE was used by Boutrou et al.
(1995) to classify 18 strains of Sc. thermophilus into
two groups, and by O’Sullivan and Fitzgerald (1998) to
separate 16 strains of the same species into three
groups, which corresponded broadly with their prote-
olytic and acidifying properties. Moschetti et al. (1998)
showed that in 51 strains of Sc. thermophilus, the
16S–23S rDNA intergenic spacer region gave a single
amplification product of 350 bp; cleavage of the prod-
uct with HaeIII gave two different restriction patterns.
Considerable heterogeneity was found among 40
strains of Sc. thermophilus using RAPD-PCR and the
M13 primer; three broad clusters were found, which
were partly correlated with the source of the isolates
(Giraffa et al., 2001).
Various molecular techniques, including RAPD,
PFGE and SDS-PAGE, have also been used to character-
ize different Lb. helveticus isolates. Lombardi et al.
(2002) showed that 67 strains of Lb. helveticus isolated
from whey starters and cheese could be grouped using a
combination of genotypic (RAPD) and phenotypic
methods. The grouping corresponded with the cheese
from which the strains were isolated in the case of
Monte Veronese and Provolone cheeses but not Grana
cheese. In contrast, RAPD provided clear differentiation
between 23 strains of Lb. helveticus isolated from Grana
and Provolone cheeses (Giraffa et al., 1998). The num-
ber of isolates (23) in the latter study was small and a
greater number may have allowed less clear conclusions.
SDS-PAGE of cell-wall proteins clearly separated isolates
of Lb. heleveticus from Grana and Provolone cheese
(Gatti et al., 1999). PFGE was used to show that at least
15 different strains of Lb. helveticus are in use in the US
as starter cultures, including mixed-cultures containing
one to four strains ( Jenkins et al., 2002).
The RAPD technique has also been shown to be use-
ful for discriminating between a large group of strains of
lactococci (Tailliez et al., 1998). The analysis resulted in
three major groups, two of which, G1 and G3, contained
Lc. lactis subsp. lactis and the other Lc. lactis subsp. cre-
moris. PFGE has also been used to characterize lacto-
cocci (Tanskanen et al., 1990), and unrelated strains
showed quite different patterns. Phage-resistant deriva-
tives yielded identical or almost identical patterns to that
of the parent strain, indicating the usefulness of PFGE to
discriminate between closely related strains. PFGE has
also been used to follow the diversity of Lc. lactis in
Pecorino Sardo cheese (Mannu et al., 2000).
The divergence in the DNA sequences of Lc. lactis
subsp. lactis and Lc. lactis subsp. cremoris is estimated to
be between 25 and 30% (Godon et al., 1992). Lc. lactis
subsp. lactis differs from Lc. lactis subsp. cremoris in
9–10 bp in the sequence of the V1 region of the 16S
rRNA gene and this has allowed specific DNA probes
for the different species of lactococci and leuconostocs
to be designed (Klijn et al., 1991). A novel method for
distinguishing between Lc. lactis subsp. lactis and Lc.
lactis subsp. cremoris was proposed by Nomura et al.
(1999), who showed that Lc. lactis subsp. lactis pro-
duced -aminobutyric acid by decarboxylation of glu-
tamate while Lc. lactis subsp. cremoris did not. A
recent study (Kelly and Ward, 2002) has shown that
strains of Lc. lactis subsp. cremoris having a lactis phe-
notype can be isolated in low numbers from dairy and
plant environments; the opposite, i.e., Lc lactis subsp.
lactis with a cremoris phenotype can also be found but
is rare.
Types of Cultures
Starter cultures may be classified on the basis of their
function, their temperature of growth or their compos-
ition. Some examples are presented in Table 1.
Primary starters are involved mainly in the
production of lactic acid from lactose, which occurs
early in cheese production. Therefore, high numbers
of active cells are added to the cheese milk. However,
many of them also produce volatile compounds, e.g.,
diacetyl from citrate, which is an important flavour
component of fresh cheese, and CO2 from lactose
(heterofermentative species) and citrate (homofermen-
tative and heterofermentative species) which con-
tribute to the open texture of some cheeses. Their
proteolytic systems are also involved in flavour and
aroma development in ripening cheeses. Moreover, by
lowering the pH and Eh, by competing with spoilage
and pathogenic microorganisms and by producing
antimicrobial compounds, they also contribute to the
microbial safety of cheese.
The secondary microflora is more varied, both from a
taxonomical and a functional point of view: non-starter
 Table 1 Examples of cheese types, starter cultures and their function. Cheeses are ordered from soft to hard

Starter microflora

Primary c See
Starter Starter Volume 2,
Cheese function a type b Ln Lc Lc Cit Ec Sc Lb Ll Lh Other Chapter

Cottage, Quarg, Cream LA, D DSS,    13

MSS
Camembert, Brie LA, P, L MSS,   Geotrichum candidum, 7
DSS Penicillium camemberti
Mozzarella (Italian) LA, AR NS,       11
MSS, 
DSS 
Pizza cheese LA, P DSS    11
Roquefort, Stilton LA, C, P, DSS,    Penicillium roqueforti 8
L MSS staphylococci, yeasts
Gorgonzola LA, P, L DSS   Penicillium roqueforti 8
Tilsit LA, P, NS   Brevibacterium linens, 9
SM DSS   other coryneforms,
staphylococci, yeasts
Cheddar LA, P DSS  4
Gouda, Edam LA, C, D, P MSS    5
Emmentaler, Sbrinz, LA, C, MSS    Propionibacterium 6
Gruyere PG, P DSS   shermanii
Parmigiano-Reggiano LA, P NS    3

a LA, lactic acid; D, diacetyl; AR, aroma, other than diacetyl; C, CO2; P, proteolysis; L, lipolysis; PG, propionic acid and gas; SM, surface smear.
b NS natural starter, MSS mixed strain starter, DSS defined strain starter.
c Ln, Leuconostoc mesenteroides subsp. cremoris; Lc, Lactococcus lactis subsp. lactis (Cit ) and/or cremoris; Lc, Cit citrate-utilising Lc. lactis subsp. lactis; Ec, Enterococcus faecium
and/or faecalis; Sc, Streptococcus thermophilus; Lb, Lactobacillus delbrueckii subsp. bulgaricus; Ll, Lb. delbrueckii subsp. lactis; Lh, Lb. helveticus, , dominating species;  species
occurring in lower numbers or occasionally.
125
126 Starter Cultures: General Aspects
lactic acid bacteria (NSLAB), propionibacteria, coryne-
forms, staphylococci, yeasts and moulds may all con-
tribute to the organoleptic properties of cheeses. Because
these microorganisms play their role during ripening,
high initial numbers are not needed and natural contam-
ination, from milk and the cheese environment, is still
relied upon in many cheese varieties. However, improve-
ment in the hygiene of milk and the need for standard-
ization and acceleration of ripening have resulted in
blander-flavoured cheese. This, in turn, has prompted
the use of many secondary starters or adjuncts to
improve the sensory properties of cheese, or its health
benefits (probiotics) (see ‘Secondary and Adjunct Cul-
tures’, Volume 1).
Primary starters are usually classified as mesophilic
or thermophilic. The latter are characteristics of Italian
(Grana, Pecorino, Mozzarella) and Swiss (Emmentaler,
Sbrinz, Gruyère) cheese varieties, where a high tempera-
ture (37 °C but generally 48–52 °C) prevails during
the early phases of cheesemaking. Mesophilic starters
are used in all cheese varieties in which the temperature
of the curd during the early stage of acid production
does not exceed ⬃40 °C (Cheddar, Gouda, Edam,
Camembert, etc.). However, this distinction is losing
some of its meaning, since mesophilic and thermophilic
species are often found (or used) together in both
mixed and defined starters for the manufacture of
cheeses like Mozzarella (Limsowtin et al., 1996; Parente
et al., 1997) and Cheddar (Beresford et al., 2001).
Probably, the most common classification of starter
cultures is based on the complexity of the culture and
the way it is reproduced (Limsowtin et al., 1996). All
starter cultures available today are derived in one way
or another from natural (or artisanal) starters of
undefined composition (i.e., containing an undefined
mixture of different strains and/or species), repro-
duced daily in cheese factories by some form of back-
slopping. Reports on adding sour cream or buttermilk
to cream to improve the quality of butter in Denmark
date back to the 1860s and the use of natural whey
cultures (i.e., the addition of whey from a previous
cheesemaking batch to cheesemilk) for Grana produc-
tion dates from 1890 (Bottazzi, 1993). Natural starters
are still used widely in Europe (Limsowtin et al., 1996;
Beresford et al., 2001) and in Argentina (Reinheimer
et al., 1996). However, for many cheeses they have
been replaced by commercial mixed-strain starters
(MSS), derived from the ‘best’ natural starters and
reproduced under controlled conditions by specialized
institutions (Dairy Research Centres or commercial
starter companies) and distributed to cheese plants
which use them to build up bulk starter or for direct-
vat inoculation (see below). While the composition of
MSS is undefined, their reproduction under more con-
trolled conditions reduces the intrinsic variability
associated to the use of artisanal starters.
Natural starter cultures and commercial MSS,
because of their long history, are called traditional
starters (Limsowtin et al., 1996) as opposed to defined-
strain starters (DSS). These are composed of one or
more strains (cultures with up to 13 strains are used in
Switzerland) which were first used in New Zealand for
Cheddar cheesemaking in the 1930s. Like MSS, DSS are
selected, maintained, produced and distributed by spe-
cialized institutions. Because of their optimized, highly
reproducible, performance, and their high phage resist-
ance, DSS have replaced traditional starters in the pro-
duction of many cheese varieties, including some PDO
European varieties. While the development of DSS is
still based largely on the isolation and selection of
strains from raw milk, cheese or traditional starters
(Limsowtin et al., 1996; Wouters et al., 2002), the need
to improve the control of phage under the high selective
pressure imposed by production schedules in large-scale
cheesemaking and the availability of food-grade cloning
and gene transfer systems have led to the development
and use of genetically enhanced strains in DSS by the
introduction of natural phage resistance mechanisms
into industrial strains (Coffey and Ross, 2002; ‘Starter
Cultures: Bacteriophage’, Volume 1). These strains
are not classified as GMOs according to current Euro-
pean and US definitions (Kondo and Johansen, 2002)
and their use is not restricted. Issues related to con-
sumer acceptability have limited the commercial use of
engineered strains of starters, developed to show
improved autolysis, improved aroma-producing proper-
ties, over-expression of peptidases, novel phage resist-
ance mechanisms, etc. (Kondo and Johansen, 2002).
Natural starter cultures
Natural starter cultures are reproduced daily at the
cheese plant by some form of backslopping (i.e., the use
of an old batch of a fermented product to inoculate a
new one) and/or by application of selective pressure
(heat treatment, incubation temperature, low pH). No
special precautions are used to prevent contamination
from raw milk or from the cheesemaking environment
and control of media and culture conditions during
starter reproduction is very limited. As a result, even in
any given cheese plant, natural starters are continuously
evolving, undefined mixtures composed of several
strains and/or species of LAB. The composition and
techniques for the production of artisanal starters have
been reviewed by Limsowtin et al. (1996). Two sub-
types are recognized, whey- and milk-starters, depend-
ing on the medium and techniques used for their
reproduction.

Natural whey cultures are prepared by incubating
some of the whey drained from the cheese vat
overnight under more or less selective conditions. The
composition and the biological diversity of the culture
are strictly dependent on the selectivity of the incuba-
tion conditions. In the manufacture of Parmigiano Reg-
giano and Grana Padano cheese (see ‘Extra Hard
Varieties’, Volume 2), whey is removed for the cheese vat
at the end of cheesemaking at 48–52 °C and is incu-
bated overnight at a controlled temperature (45 °C), or
in large containers in which the temperature decreases
to 37–40 °C, to a final pH as low as 3.3 (Limsowtin
et al., 1996). The resulting whey culture (siero-fer-
mento, siero-innesto) is dominated by aciduric and/or
thermophilic strains; Lb. helveticus usually dominates
(85%), but other species (Lb. delbrueckii subsp. lactis,
Lb. fermentum, Sc. thermophilus) may be present. Sea-
sonal and geographical variations in the composition
and performance of the culture have been observed. In
a recent molecular ecology study (Cattivelli et al.,
2002) it was shown that a limited number of strains
(maximum 6) dominates the cultures, that a ‘house’-
specific flora can be identified in different plants, and
that Sc. thermophilus is found only in wheys incubated
at a low temperature. Similar whey cultures are used in
the production of pasta-filata cheese varieties in Italy
(Limsowtin et al., 1996; Parente et al., 1997), hard
cheese varieties in Argentina (Reinheimer et al., 1996),
and Comté cheese in France (Bouton et al., 2002).
Other types of whey cultures include deproteinized
whey starters (scotta-innesto) used for the manufacture
of Pecorino cheese (Limsowtin et al., 1996; Mannu
et al., 2002; see ‘Cheeses Made from Ewes’ and Goats’
Milk’, Volume 2), and deproteinized whey starters
with rennet (Fettsirtenmagenlab, Présure à la ‘recruite’)
which are used for the manufacture of Swiss-type
cheeses (Emmental, Sbrinz, Gruyère; see ‘Cheese With
Propionic Acid Fermentation’, Volume 2) in small
cheese factories in the Alps. Invariably, thermophilic
lactobacilli (Lb. helveticus, Lb. delbrueckii subsp. lactis)
dominate cultures produced under selective condi-
tions (high temperature) while streptococci (Sc. ther-
mophilus, but also lactococci and enterococci) often
dominate cultures incubated at a relatively low tem-
perature (42 °C), which usually show higher micro-
bial diversity (Parente et al., 1997).
Natural milk cultures (colture naturali in latte, lat-
toinnesti, lattofermento) are still used in small cheese-
making plants in both Southern and Northern Italy
for the production of traditional cheeses. The selective
pressure used for the development of the desired
microflora includes thermization/pasteurization of raw
milk (62–65 °C for 10–15 min) followed by incubation
at a high temperature (37–45 °C) until the desired titrat-
Starter Cultures: General Aspects 127
able acidity is reached. These cultures are usually
dominated by Sc. thermophilus but other species may be
present (Sc. macedonicus, enterococci, mesophilic lacto-
bacilli; Limsowtin et al., 1996; Andrighetto et al., 2002).
The use of natural starter cultures has both advan-
tages and disadvantages. They are an extremely valu-
able source of strains with desirable technological
properties (phage resistance, production of antimicro-
bials, aroma production), although many strains show
limited acid production ability when cultivated as pure
cultures (Cogan et al., 1997).
Fluctuations in composition result in variable per-
formance and this may not be acceptable in modern
cheesemaking practice. Natural starters are considered
to be highly tolerant to phage infection. Like the
‘practice’ MSS used in Dutch cheese manufacture
(Stadhouders and Leenders, 1984), natural cultures are
reproduced in the presence of phage, which exert select-
ive pressure, which ultimately leads to the dominance of
resistant or tolerant strains.
Occasionally, the development of highly virulent
phage attacking the dominant strains may severely
reduce culture activity, and time will be needed for the
establishment of a new equilibrium. The presence of
bacteria, like coliforms and enterococci (Coppola et al.,
1988; Parente et al., 1997), in some natural starter
cultures may also raise some concern. In Europe, the
standards of identity of many PDO cheeses require the
use of natural starter cultures, because a strict relation-
ship is believed to exist between the use of given nat-
ural starter cultures and cheese properties. Molecular
and technological characterization of Lb. helveticus
strains isolated from natural starters used for Pro-
volone and Grana cheese in Italy (Gatti et al., 1999;
Giraffa et al., 2000) has indeed shown that strains
from the cultures used for the two cheeses are differ-
ent. On the other hand, Sc. thermophilus strains isol-
ated from natural milk cultures used as starters for
PDO cheeses produced under very similar conditions
(Asiago d’Allevo, Montasio, Monte Veronese) cannot
be distinguished by RAPD-PCR (Andrighetto et al.,
2002).
Mixed-strain starters
When undefined cultures are propagated under con-
trolled conditions with a minimum of subcultures, the
stability of their composition and performance is
greatly improved, without losing the advantage of tol-
erance to phage infection (Stadhouders and Leenders,
1984). Mixed-strain starters, obtained by careful selec-
tion of natural starters, are maintained, propagated
and distributed by starter companies and research
institutions, and are widely used for the production of
128 Starter Cultures: General Aspects
cheese in Europe (Table 1). The traditional method
for the reproduction of MSS, which required several
transfers in the cheesemaking plant to build up the bulk
starter, starting from small amounts of stock cultures,
has been replaced by the use of concentrated cultures
for the inoculation of the bulk starter tank or for direct
inoculation of the cheese milk, thus minimizing the
need for transfers within the factory and the risk of fluc-
tuations in starter composition and activity.
Mixed-strain starters are usually classified as meso-
philic or thermophilic, with an optimum growth tempera-
ture of 28–30 °C and 42 °C, respectively (Limsowtin
et al., 1996). Mesophilic MSS can be further classified
on the basis of citrate fermentation and composition,
as citrate-negative ‘O’ starters (which contain acid-
producing Cit Lc. lactis subsp. lactis and cremoris) or
citrate-positive L, D and DL starters (containing Leuc.
mesenteroides subsp. cremoris, Cit Lc. lactis subsp.
lactis, or both, respectively, in addition to acid-producing
strains). Thermophilic MSS are used for the production
of Italian and Swiss cheese varieties, and usually contain
Sc. thermophilus alone or in mixtures with thermophilic
lactobacilli (Lb. delbrueckii subsp. lactis, Lb. helveticus)
(Glättli, 1990).
Like artisanal starters, MSS contain undefined mix-
ture of strains, which differ in their physiological and
technological properties (including phage resistance).
Plasmid profiles and phage sensitivity have been used
to estimate the diversity of strains in MSS, although
other molecular methods (PFGE, RAPD-PCR, etc.)
may provide a better estimate of strain diversity. In a
recent study, Bissonnette et al. (2000) evaluated the
diversity of Lc. lactis subsp. cremoris in seven MSS used
for the manufacture of Cheddar cheese in Canada by
isolating and typing a relatively large number of
strains (30) from each culture. Two MSS were domi-
nated by 2–3 strains, three by 7–9 strains but two had
a high diversity, with 18–24 distinct strains; 32 differ-
ent strains have been claimed to be present in an MSS
used for Cheddar cheesemaking in Denmark (Josephsen
et al., 1999).
Because they are derived from cultures which were
reproduced in cheese plants without protection from
disturbing phages, MSS contain many phage-resistant
strains but also harbour their own phages (Stadhouders
and Leenders, 1984; Limsowtin et al., 1996; Josephsen
et al., 1999; Bissonnette et al., 2000).
The development of MSS for the production of
Dutch cheeses at NIZO (Stadhouders and Leenders,
1984) and thermophilic MSS (Rohmischkulturen) for
the manufacture of Swiss cheese varieties by the Swiss
Federal Dairy Research Station (Glättli, 1990) are two
examples of the successful development and long-term
use of MSS (Limsowtin et al., 1996; ‘Gouda and Related
Cheeses’ and ‘Cheese With Propionic Acid Fermenta-
tion’, Volume 2).
Even if MSS have a long history of successful use
without severe inhibition by phage, one should not
be overly confident that phage infection will never be
experienced. Published studies on long-term monitor-
ing of phage/starter interaction in cheese plants using
MSS are rare. Josephsen et al. (1999) have documented
the development of virulent phages in a factory which
had been using the same MSS almost continuously
before occasional slow acidification problems were
experienced. The isolates from the MSS for which
homologous phages were detected in cheese whey
increased from 16 to 97% over 11 years, and their viru-
lence increased greatly. In fact, while phages isolated
when no acidification problem was experienced had
restricted host range, long latent times (38–52 min)
and relatively low burst sizes (35–84), phages isolated
in recent year had broader host ranges (and were able
to multiply on strains which were highly phage resist-
ant), reduced latent times (35 min) and greatly increased
burst sizes (120–200).
Defined-strain starters
Mesophilic DSS originated in New Zealand in the
1930s, as a response to the occurrence of open texture
defects in Cheddar cheese produced with MSS con-
taining Cit strains. The history of mesophilic DSS
systems in New Zealand, Australia, USA and Ireland
has been reviewed by Limsowtin et al. (1996). Since
the strain and/or species ratio in DSS is defined, their
technological performance is extremely reproducible.
This is obviously a highly desirable property in mod-
ern cheese plants with large throughputs of milk and
tight production schedules. Since only a limited num-
ber of strains are used (commonly 2–6), phage infec-
tion may have destructive consequences on starter
activity.
In fact, the history of DSS is a continuous fight to
devise measures to control phage infections. Single-
strain starters were used initially in New Zealand, but
rapid onset of destructive phage infections, with com-
plete loss of activity, occurred. These were then replaced
by pairs of phage-unrelated strains, which were rotated
daily, and measures to ensure aseptic reproduction of
the starters were implemented (Whitehead and Cox,
1936). Rotations were cumbersome to maintain and
they were replaced by an approach based on the selec-
tion of bacteriophage insensitive mutants (BIM; Heap
and Lawrence, 1976). This approach allowed develop-
ment of 3-day rotations with highly phage-resistant
strains, which were subsequently used together in a
single multiple-strain starter containing six strains

(Limsowtin et al., 1977). The difficulty of replacing
strains led to the reduction of the number of compon-
ents from six to five and finally to three. Such DSS are
in use in Australia, New Zealand, USA and Ireland. In
general, highly phage-resistant DSS are available either
through research institutions (e.g., the Australian
Starter Cultures Research Centre, or Fonterra Research
in New Zealand) or from commercial suppliers. The
strategy used for the management of DSS in Australia
has been documented thoroughly (Limsowtin et al.,
1997). Today, the selection of BIM has been largely
replaced by strategies based on the introduction of nat-
ural phage resistance mechanisms into industrial strains
(Coffey and Ross, 2002; see also ‘Starter Cultures:
Bacteriophage’, Volume 1).
Thermophilic DSS are also commercially available
for the production of a variety of Italian- and Swiss-
type cheeses. Starters composed of single or multiple
strains of Sc. thermophilus are still preferred in Italy for
the production of high-moisture Mozzarella cheese,
but associations of Sc. thermophilus and Lb. delbrueckii
subsp. bulgaricus (rod:coccus starter cultures) are used
for the manufacture of low-moisture Mozzarella cheese
(Kinstedt, 1993; Oberg and Broadbent, 1993). The use
of Lb. helveticus in place of Lb. delbrueckii subsp. bul-
garicus has been claimed to present several advantages
(Oberg et al., 1991) such as reduced make time and
improved functional properties. Phage-resistance
mechanisms are apparently less widespread among
thermophilic starter cultures than in lactococci (Coffey
and Ross, 2002). Due to the relatively narrow host
range of Sc. thermophilus phage, use of rotations and
BIMs is still relied upon to control phage infection in
thermophilic starter cultures (Moineau, 1999).
New sources of starters
Most, if not all, of the LAB found in starter cultures
can be isolated from cheese made without the deliber-
ate addition of a starter culture. Such strains are nat-
ural contaminants of milk which grow and produce
acid during cheesemaking. The ultimate source of
these bacteria remains to be determined. However, it is
generally thought that plants and plant material are
the natural habitat of Lc. lactis subsp. lactis. The habi-
tat of Lc. lactis subsp. cremoris has not been determined
but it can be isolated from dairy products.
Many of the pure cultures of starter bacteria used in
defined cultures are phage-related, implying that the
number of different strains of starter bacteria is gener-
ally limited. Therefore, efforts have been made to isol-
ate ‘new’ strains from raw milk, plants and other
natural sources (Salama et al., 1995; Cogan et al.,
1997; Wouters et al., 2002). Any potential new starter
Starter Cultures: General Aspects 129
strain must produce acid rapidly, lack off-flavour
development in milk and be resistant to a mixture of
common phage. Lc. lactis subsp. lactis but not Lc. lactis
subsp. cremoris has been isolated from red nettles,
common sow thistle, Himalayan blackberries, potato,
cucumber, corn, sweet pea, beans, cantaloupe, corn
and broccoli and many of them were good acid pro-
ducers, coagulating milk in 18 h at 21 °C (Salama et al.,
1995). In contrast, very few strains of Lc. lactis (the
sub-species was not determined) isolated from arti-
sanal dairy products were good acid producers (Cogan
et al., 1997). Some of them produce unusual flavours
in milk. For example, the combination of a ‘wild’
starter, which had low protolytic activity and high
amino acid decarboxylase activity, with a commercial
strain, which had high proteolytic activity and low
decarboxylase activity, resulted in the production of
chocolate flavour in milk, due to several branched
chain aldehydes and acids (Wouters et al., 2002).
Genome Sequence
Arguably, the most significant advance in starter cul-
tures in the past 30 years has been the determination
of the complete genome sequence of the chromosome
of Lc. lactis IL 1403 (Bolotin et al., 1999). Almost
1500 genes were located and their functions classified
on the basis of homology to human proteins. Five
potential or rudimentary prophages were identified in
the genome, implying that the ultimate source of
phage is probably the starter cell itself. The analysis
also showed that Lc. lactis has the potential to synthe-
size 20 amino acids and 4 co-factors. However, the
presence of these genes does not mean that Lc. lactis
will not require these compounds for growth. Since
then, the genomes of three other LAB, Lb. plantarum,
Lb. johnsonii and Lb. acidophilus, have also been
sequenced, and 24 other LAB including other strains
of Lb. lactis subsp. cremoris, Lb. debreuckii subsp. bul-
garicus, Lb. casei, Lb. helveticus, Sc. thermophilus are
on-going. Genome sequence projects for other non-
LAB which are important in cheese ripening have
either been completed (P. freudenreichii) or are on-
going (B. linens). The information that these data will
generate will be of considerable benefit in understand-
ing the fundamental metabolism of these bacteria,
including the production of lactic acid, proteolytic
systems, tolerances to heat, acid and salt stresses, pro-
duction of bacteriocins and other anti-microbials. As
many of them also have widely different ecological
niches, the data should also be very useful in deter-
mining why particular species occupy a particular
niche. Such data will also help in the development of
130 Starter Cultures: General Aspects
new strains or modification of common strains used as
starter cultures (Klaenhammer et al., 2002).
Metabolism of Starter Cultures
Sugar metabolism
Lactose is the major sugar in milk and its transport,
metabolism and regulation in several different starter
cultures have been reviewed (Poolman, 1993, 2002;
Cocaign-Bousquet et al., 1996) and will not be
reviewed further here. The salient features of the path-
ways used by different starter cultures are summarized
in Table 2. Application of NMR has been very useful in
understanding the flux through different pathways dur-
ing growth and in understanding regulation of different
aspects of metabolism in LAB and the literature
has been reviewed by Ramos et al. (2002). NMR has
also been useful in understanding exopolysaccharide
(EPS) production. In the case of glucose metabolism,
the results have shown that the rate of fructose-
1,6-bisphosphate consumption and the magnitude of the
PEP potential (PGA  PEP) are considerably higher
when Lc. lactis is grown under aerobic than under
anaerobic conditions, implying that NADH oxidase
activity is important.
Citrate metabolism
Citrate is present at a low concentration in milk and is
metabolized by Leuconostoc subsp. and some strains of
Lc. lactis subsp. lactis to CO2, which is responsible for
eye formation in some cheeses, and diacetyl and
acetate, which are important flavour components in
fermented milks. The latter organism was called
Sc. diacetylactis in the old literature and more recently
Lc. lactis subsp. lactis biovar diacetylactis. This name
has no taxonomic status and the correct way to refer to
it is citrate-utilizing (Cit) Lc. lactis subsp. lactis. Cit
Table 2 Salient features of lactose metabolism by starter organisms
Organism Transport a Pathway b
Lactococcus lactis PEP-PTS GLY
Leuconostoc spp. Permease PK
Sc. thermophilus Permease GLY
Lb. delbrueckii Permease GLY
Lb. helveticus Permease GLY
a PEP PTS, phosphotransferase system.
b GLY, glycolysis; PK, phosphoketolase.
c gal, phospho--galactosidase; gal, -galactosidase.
d These species metabolize only the glucose moiety of lactose.
strains of Lc. lactis differ from the more normal non-
citrate-utilizing (Cit ) strains in containing a plasmid
which encodes the transport of citrate. Citrate metabol-
ism in LAB has been reviewed by Hugenholtz (1993).
In recent years, considerable effort has been devoted to
understanding the energetics of citrate transport in
Leuc. mesenteroides and Lc. lactis (Garcia-Quintans
et al., 1989; Marty-Teyssett et al., 1996; Magni et al.,
1999). In the absence of any other carbon source, Leuc.
mesenteroides and Lc. lactis transport citrate in symport
with a proton, which leads to the generation of a pH
or proton motive force. In the presence of D-lactate and
glucose, citrate is transported by an antiport system
with lactate being extruded; in this case, citrate metabol-
ism is also more rapid. This is due to the fact that
the exchange between citrate and lactate is much
faster than the citrate/H symport system. Since
D-lactate is a product of sugar metabolism, the trans-
porter operating under physiological conditions
is likely to be that for citrate/lactate. An electrogenic
citrate/D-lactate exchange occurs, generating a proton
electro-chemical gradient across the membrane. This
may contribute significantly to the enhanced growth of
Leuc. mesenteroides during co-metabolism of glucose
and citrate.
Co-metabolism of glucose and citrate by Leuconos-
toc subsp. results in a faster growth rate. This has been
attributed to a metabolic shift in the glucose pathway,
leading to increased ATP production (Cogan, 1987).
The results of Marty-Teysset et al. (1966) suggest that
the citrate/D-lactate exchange is also involved in pro-
ducing energy. In Lc. lactis, co-metabolism of citrate
and sugar does not result in a major effect on growth
rate at neutral pH. However, at acid pH values (4.5),
the citrate transport system is induced. Metabolism of
citrate results in an increase in pH to a value at which
the consumption of glucose begins (Garcia-Quintans
et al., 1989). More recently, it has been suggested
Products
Cleavage (mol/mol Isomer of
enzyme c lactose) lactate
pgal 4 Lactate L
gal 2 Lactate  ethanol D
 2 CO2
gal 2 Lactated L
gal 2 Lactated D
gal 4 Lactate DL
 Starter Cultures: General Aspects 131

(Magni et al., 1999) that the induction of the citrate Therefore, further growth requires the hydrolysis of
metabolic pathway under acidic conditions makes milk proteins. In fact, the growth of many LAB is
the cells more resistant to the inhibitory effects of diauxic in milk; an initial fast growth rate, during
lactate. which free amino acids and peptides are used up, is
followed by a slightly slower rate during which further
peptides and amino acids are obtained by hydrolysis of
Nitrogen metabolism
casein.
Nitrogen metabolism by starters has an enormous Proteolysis is a major event in cheese ripening; the
impact on their activity and on cheese quality. To per- proteolytic system of the primary starter and of the
form their main function of acid production in milk secondary microflora contributes the production of
and curd, LAB must grow to high numbers, from hundreds of flavour compounds through the produc-
⬃1  106 cfu/ml in the inoculated milk to ⬃1  109 tion of low-molecular weight peptides and amino
cfu/g in the cheese curd; syneresis of the curd due to acids and their subsequent catabolism. The role of
expulsion of whey also contributes to the increase in proteolysis and amino acid catabolism in cheese has
cell numbers. Lactic acid bacteria are fastidious been addressed by several recent reviews (Sousa et al.,
microorganisms and are unable to synthesize many 2001; Yvon and Rijnen, 2001) and is described in
amino acids, vitamins and nucleic acid bases. Depend- detail in ‘Proteolysis in Cheese during Ripening’ and
ing on the species and the strain, LAB require from 6 ‘Catabolism of Amino Acids in Cheese During Ripening’,
to 14 different amino acids (Chopin 1993; Kunji et al., Volume 1.
1996). Although milk is rich in nitrogen, it is present The proteolytic system of LAB is composed of a
mainly as protein. It has been calculated that the cell-wall bound proteinase, transport systems for
amount of free amino acids and low-molecular weight amino acids, di- and tripeptides and oligopeptides, a
peptides present in milk can support only limited number of intracellular peptidases and some intracel-
growth (10–20% of the final biomass of a fully grown lular proteinases (Fig. 1). Several excellent reviews
culture of lactococci; Thomas and Pritchard, 1987). have been published on this topic (Kunji et al., 1996;

OUT
cell wall
membrane

IN
peptidase amino acids
Pro-specific peptidases transport systems
s
• XDAP (PepX ) aa
• proline iminopeptidase
PepI)
peptides (2–18 aa)

m
• prolidase (PepQ )
amino acid catabolism
peptide transport

m
• prolinase (PepP )
Opp DtpT DtpP

arginine deiminase pathway

systems

aldolases
general peptidases aa aminotransferases
• aminopeptidases decarboxylases
m c
(PepN , PepC )
m dehydrogenases
• dipeptidases (PepV )
m
• tripeptidases (PepT )
casein • endopeptidases
m m
(PepO , PepF )

aa biosynthetic pathways
Glu-specific peptidases protein synthesis
m
• aminopeptidases (PepA )
PrtP
aroma compounds
lysis

Figure 1 Schematic representation of nitrogen metabolism in lactic acid bacteria. The abbreviations refer to enzymes of Lactococcus
lactis. The superscripts refer to classes of peptidases (c, cysteine peptidases; m, metallo peptidases; s, serine peptidases) (adapted
from Kunji et al. (1996) and Sousa et al. (2001), Christensen et al. (1999) and Yvon and Rijnen (2001)).
132 Starter Cultures: General Aspects
Christensen et al., 1999; Siezen, 1999) and only a gen-
eral overview will be presented here.
Proteinase
Lactic starters degrade casein and large casein-derived
peptides produced by milk and coagulant enzymes by a
cell-envelope proteinase (CEP, lactocepin, EC 3.4.21.96,
also called the cell wall-bound proteinase; Kunji et al.,
1996; Siezen, 1999). All CEPs from LAB described to
date are serine-proteinases related to subtilisins.
The CEP of Lc. lactis (PrtP) is the most extensively
characterized. The proteinase gene (prtP), which may
be located on plasmids or on the chromosome, encodes
a protein of 1902 (Lc. lactis WG2 and NCDO763) or
1962 (Lc. lactis SK11) amino acids; the larger size is
due to a duplication near the C-terminus. To date, sev-
eral domains have been identified in PrtP (Fig. 2). A
pre–pro domain (PP) is needed for secretion and pro-
cessing. A signal sequence of 31–39 residues at the
N-terminus is responsible for Sec-dependent (general)
translocation of the pro-proteinase across the cell
membrane, and a proteinase maturase (PrtM), encoded
by a gene immediately upstream of prtP, cleaves the
pro-region between Thr187 and Asp188, producing a
catalytically active CEP. The catalytically active domain
(PR, residues 188–699 of PrtP) is responsible for activ-
ity and substrate specificity of CEPs and is highly con-
served. The three-dimensional structure of the PR
domain of all subtilases has been predicted (Fig. 3;
Siezen and Leunissen, 1997) and this has allowed sci-
entists to engineer the stability, catalytic activity and
specificity of the lactococcal proteinase (Siezen, 1999).
Amino acid substitutions in positions 96–107 and
I
PrtP PP PR A B H W AN
I
PrtH PP PR A B H W
I
PrtB PP PR A B W
membrane
cell wall
Figure 2 Representation of the predicted domain structure of
the cell-envelope proteinase (CEP) of Lc. lactis (PrtP), Lb. helveti-
cus (PrtH) and Lb. delbrueckii subsp. bulgaricus (PrtB). PP, pre-
pro domain; PR, protease domain; I, insert domain; A, A-domain;
B, B-domain; H, helical domain; W, cell-wall domain; AN, anchor
domain (redrawn from Siezen, 1999).
125–130 of PrtP, corresponding to the substrate bind-
ing cleft, result in a variety of specificities towards
degradation of s1-, - and -casein. All CEPs have
broad substrate specificity, and no consensus sequence
for cleavage sites has been identified.
Three further domains (I, A and B) may be important
for the stability, specificity and regulation of the activity
of the PR-domain, while a helix (H) domain positions
PR, A and B domains away from the cell surface. The
C-terminus of the proteinase is involved in binding to
the cell wall; deletion analysis show that truncated forms
lacking 130 or more residues are released into the
medium. Incubation of the cells in a Ca-free buffer
results in auto-proteolysis and release of a fragment of
135–145 kDa, which is still catalytically active. Two fur-
ther domains, the W-domain, which is a cell-wall spacer
and spans the peptidoglycan layer and a cell-wall
anchor, the AN-domain, part of which is cleaved during
translocation, anchor PrtP to the cell wall.
Further CEPs have been characterized in thermo-
philic (Lb. helveticus, PrtH; Lb. delbrueckii subsp. bulgar-
icus, PrtB) and mesophilic lactobacilli (Lb. paracasei, Lb.
rhamnosus). They all belong to the subtilase family and
share many properties with the lactococcal PrtP,
although specificities and domain structure may be dif-
ferent (Fig. 2). The catalytic domains of PrtP, PrtB and
PrtH show higher degrees of homology than the other
domains. The release of the CEP of thermophilic lacto-
bacilli into the medium requires drastic treatments
(lysozyme, osmotic shock, membrane solubilization);
although they lack the AN domain, the W-domain is
very basic and may bind to the cell wall by electrostatic
interaction.
Transport systems and peptidases
The activity of CEPs on caseins releases a large variety
of oligopeptides; although most are in the range of
4–10 residues, peptides of up to 30 residues can be
produced from -casein. No appreciable amounts of
free amino acids, di- or tripeptides are produced by the
action of CEP. It is now well recognized that all pep-
tidases of LAB are located intracellularly and therefore
peptides can be hydrolysed only if they are transported
into the cell. Although lactococci (and other LAB)
have many amino acid, and di- and tripeptide trans-
port systems, the oligopeptide transport system (Opp)
is essential for growth in milk. Opp can transport
oligopeptides containing from 4 to 18 amino acids
without any significant specificity for their compos-
ition (Detmers et al., 1998).
Once peptides reach the cytoplasm, they are
sequentially degraded by a large variety of peptidases
(Kunji et al., 1996; Christensen et al., 1999). Due to
the presence of high numbers of Pro and Glu residues
 Starter Cultures: General Aspects 133

Figure 3 Three-dimensional model of the -carbon backbone structure of the protease (PR) domain of the CEP of Lactococcus
lactis (PrtP). The conserved core of subtilases (in grey), the position where residues are inserted or deleted (in white), the substrate
binding cleft, the N- and C-termini of the PR-domain and the predicted position of bound calcium ions are shown (from Siezen,
1999).

in the caseins, general, Pro-specific and Glu-specific
peptidases are needed to liberate essential amino acids
for growth. Figure 1 shows the most important pepti-
dases in Lactococcus lactis, some of which (PepN,
PepC, PepX, PepV) have also been found in other
dairy LAB. Many other peptidases have been charac-
terized in other LAB (Christensen et al., 1999). Studies
with single or multiple peptidase-deficient mutants
have shown that, although lack of any single peptidase
does not result in complete inhibition, the growth
rates of peptidase-deficient mutants are usually lower
than the wild type (7–120% increase in generation
time; Christensen et al., 1999), with severe inhibition
for multiple mutants.
The contribution of the peptidases of lactic starters
to the release of free amino acids in cheese is now well
recognized. Although these enzymes are intracellular,
they are liberated in cheese following autolysis of the
cells. Acceleration of autolysis and over-expression of
peptidases have been used to accelerate cheese ripening
(see ‘Proteolysis in Cheese during Ripening’ and
‘Catabolism of Amino Acids in Cheese During Ripen-
ing’, Volume 1).
Amino acid degradation
Degradation of amino acids has important implica-
tions for the metabolism of starter cultures (e.g., by
providing energy in the sugar-depleted environment
of cheese), for the safety of cheese (e.g., by produc-
tion of biogenic amines by decarboxylation of Tyr,
His, Trp), and for the production of flavour and
aroma compounds. The breakdown of para-casein to
amino acids and peptides by a combination of chy-
mosin and proteinases and/or peptidases of the
starter bacteria is generally considered to be the most
important aspect of cheese ripening. However, amino
134 Starter Cultures: General Aspects
acids and peptides, of themselves, are not responsible
for flavour development in cheese. The products of
the catabolism of amino acids include alcohols, alde-
hydes, amines and organic acids and are considered
to be of major significance in flavour foundation (see
‘Proteolysis in Cheese during Ripening’ and ‘Catab-
olism of Amino Acids in Cheese During Ripening’,
Volume 1).
Amino acid catabolism in LAB and in other dairy
organisms and its relationship to cheese flavour have
been reviewed recently (Christensen et al., 1999;
Weimar et al., 1999; Yvon and Rijnen, 2001; ‘Catab-
olism of Amino Acids in Cheese During Ripening’,
Volume 1).
The arginine deiminase pathway yields energy
directly through substrate-level phosphorylation. In
addition, decarboxylation of Asp, Glu, His, Tyr and
Trp to the corresponding amines yields energy through
amine extrusion and the consequent generation of
electrochemical gradients. Histamine, tyramine and
tryptamine are biogenic amines, which have been
involved in monoamine intoxication (Christensen
et al., 1999).
While some non-lactic microorganisms (Brevibac-
terium linens, yeasts, micrococci) initiate amino acid
catabolism by elimination reactions, and threonine is
catabolized by a threonine aldolase (which produces
Gly and acetaldehyde), the first step in amino acid
catabolism in LAB is usually a transamination reac-
tion. Aminotransferases (AT) of LAB have broad and
often overlapping specificities. Aromatic amino acid
ATs (AraT) and branched chain amino acids AT (BcaT)
catalyse the first step in the catabolism of aromatic and
branched chain amino acids, respectively, resulting in
the production of -ketoacids which are potent aroma
compounds, e.g., -keto isovaleric acid, which is pro-
duced from Thr, or are converted further to aroma
compounds (alcohols, aldehydes, esters, etc.) by a
variety of enzymes. Transamination reactions generally
require -ketoglutaric acid as a substrate, and the con-
centration of this ketoacid is limiting in cheese. In
fact, addition of -ketoglutaric acid ( KG) to cheese
has been shown to greatly enhance flavour formation.
Alternatively, -ketoglutaric acid can be produced
from glutamate by strains with glutamate dehydrogen-
ase activity (Yvon and Rijnen, 2001; Tanous et al.,
2002).
Of necessity, these reactions must be capable of pro-
gressing at relatively low pH (⬃5.0) and the relatively
high salt concentrations (⬃6%), which occur in most
cheeses during ripening. Such conditions limit enzyme
activity significantly but small activities acting over
the protracted period of cheese ripening are of consid-
erable importance in generating flavour.
Lipases and esterases
Except for Parmigiano Reggiano, Pecorino and related
Italian cheeses, and Blue cheeses, limited lipolysis
occurs in cheese during ripening. Nevertheless, the
limited level, which does occur, is considered to be
important for flavour and taste perception. Esterases
have been purified from several starter and LAB,
including Lc. lactis (Holland and Coolbear, 1996;
Chich et al., 1997), Sc. thermophilus (Liu et al., 2001)
and Lb. plantarum (Gobbetti et al., 1997). All of them
are serine enzymes that preferentially hydrolyse
butyrate esters and are optimally active at pH 7.
Some of them have no activity at pH 5.0; nevertheless,
a very small amount of activity over a long time could
result in significant hydrolysis of fat during cheese ripen-
ing. The major tributyrin esterase of Lc. lactis has been
cloned, over-expressed and characterized (Fernandez
et al., 2000). The purified enzyme showed a preference
for short-chain acyl esters and also phospholipids,
suggesting that it may be involved in phospholipid
metabolism in vivo.
Growth
Chemically defined media (CDM) for the growth of
Lc. lactis, Leuc. mesenteroides and Sc. thermophilus have
been developed ( Jensen and Hammer, 1993; Cocaign-
Bousquet et al., 1995; Foucaud et al., 1997; Letort and
Juillard, 2001). Maximum specific growth rate ranges
from 0.4 to 1.0 h 1. The medium for Sc. thermophilus
contains only 20 components, including six amino acids
(glutamine, cysteine, methionine, leucine, isoleucine
and valine). Addition of pyridoxamine eliminated the
need for nucleic acid bases in the case of Sc. ther-
mophilus and Lc. lactis. Growth of Lc. lactis NCDO 2118,
which was isolated from a vegetable source, and Lc. lac-
tis IL 1403, which was isolated from a dairy source, in a
CDM were compared by Cocaign-Bousquet et al. (1995).
NCDO 2118 required no amino acid (prototrophic),
while IL 1403 required several amino acids, including
glutamate, arginine, methionine, valine, leucine or
isoleucine (auxotrophic), when the single omission
technique was used to identify a requirement. However,
in a simplified CDM, NCDO 2118 required glutamate,
methionine, isoleucine, leucine, valine and serine, indi-
cating that proto/auxotrophy is partly dependent on the
composition of the medium. The dairy strain showed
an additional requirement for arginine, histidine and
threonine.
Lactic acid bacteria do not have a functional TCA
cycle and hence most of them require the glutamate
but, surprisingly, not the aspartate, family of amino
acids. A gene cluster coding for citrate synthase, aconi-
tase and isocitrate dehydrogenase has been identified

in Lc. lactis C2 (Wang et al., 2000). Lc. lactis NCDO
2118 was able to grow in a CDM containing KG but
no glutamate, but the lag phase depended on the con-
centration of KG added (Lapujade et al., 1998). No
glutamate dehydrogenase activity, the enzyme which is
used in many bacteria to produce glutamate directly
from KG, was detected but transaminase activities,
with several amino acids as amino group donors
and KG as acceptor, were detected. Indeed, addition
of KG to milk before cheese manufacture also
increases flavour development during ripening (Yvon
and Rijnen, 2001), indicating the importance of amino
acid transferase activity in the maturation of cheese
(see later).
Niven et al. (1998) found biphasic growth of
Lc. lactis MG 4685 in milk, with a faster initial rate
(0–4 h) followed by a slower (4–8 h) one. There was
little change in the concentration of amino acids dur-
ing the first phase while the second phase correlated
with increased production of amino acids; however,
significant decreases in glycine and alanine occurred
during both growth phases.
The growth rates of Lc. lactis ML3 and Wg2
decreased rapidly above pH 7 when grown on a syn-
thetic medium containing glutamate but not glutam-
ine (Poolman and Konings, 1988). If glutamate was
replaced by glutamine, the pH of growth was extended
to 8.0, indicating that the unionized form of glutamic
acid, rather than the ionized form, is transported by
the glutamic acid/glutamine transporter in these
organisms. At alkaline pH, the rate of growth in the
absence of glutamine is limited because less unionized
glutamic acid is available at the higher pH values.
The effect of different environmental conditions
on the rate of growth of starter bacteria, particularly
Lc. lactis, has been investigated in several recent stud-
ies. Generally, lactococci produce other products of
sugar metabolism besides lactate when grown on
galactose or a low level of glucose (Thomas et al.,
1979). In contrast, Even et al. (2001) showed that Lc.
lactis subsp. lactis IL1403 retained its homolactic
behaviour on glucose and galactose in two different
minimal media of different nutritional complexities,
despite significant variations in both growth rate and
sugar consumption.
Lactic acid bacteria are essentially fermentative
organisms but they are also capable of consuming oxy-
gen with the formation of H2O2. Under these condi-
tions, various NADH oxidases and peroxidases are
produced to reduce the toxic H2O2 (Duwat et al., 2001;
van Niel et al., 2002). LAB are therefore considered to
be aerotolerant organisms. An NADH oxidase has been
purified from Lc. lactis MG1363 (Lopez de Felipe and
Hugenholtz, 2001). Oxygen can be beneficial to Lc. lac-
Starter Cultures: General Aspects 135
tis during aerobic growth if heme is also present.
Lc. lactis MG1363 grown in M17 (glucose) in the pres-
ence of 10 g haemin/ml produced increased biomass
and retained almost 100% viability over 70 days at 4 °C
(Duwat et al., 2001). Growth also resulted in changes
to a heterolactic fermentation. The results were correl-
ated with the production of cytochrome oxidase, which
is required for respiration, late in growth. Another
recent study (van Niel et al., 2002) has shown that the
intracellular concentration of pyruvate in Lc. lactis
subsp. lactis ATCC 19435 can reach 93 mM, which
rapidly destroys H2O2 non-enzymatically.
A plasmid-free strain of Cit Lc. lactis DRC1 grew
at a significantly (5%) faster rate in complex broth
than the parent strain. The slower growth rate of the
parent was due to the presence of a small (7.4 kb)
plasmid (Kobayashi et al., 2002).
Metabolic engineering
Lactic acid bacteria have a relatively simple sugar
metabolism and homofermentative strains convert
90% of metabolized sugar to lactic acid. Other prod-
ucts, some of which are commercially important, e.g.,
diacetyl, are also produced but in much smaller
amounts. Because LAB are GRAS organisms, with a
relatively simple metabolism, efforts have been made
to get them to over-produce these minor products. The
metabolic engineering of LAB to produce these prod-
ucts has been reviewed (Hoefnagel et al., 2002;
Hugenholtz et al., 2002).
Diacetyl is produced chemically from -acetolactate
(AL) which is produced from pyruvate which, in turn,
is produced from citrate. -Acetolactate is highly
unstable and breaks down to diacetyl when O2 is pre-
sent and to acetoin when O2 is absent. Acetoin pro-
duction from AL is also catalysed by AL decarboxylase
but there is no enzyme which produces diacetyl from
AL. Platteeuw et al. (1995) cloned the AL synthase
gene from Lc. lactis MG 1363 into Lc. lactis MG 5267
and obtained a 100-fold increase in AL production.
Only lactic acid was produced by the strain under
anaerobic conditions but 26 and 42% of the pyruvate
was converted to acetoin under aerobic conditions at
pH 6.8 and 6.0, respectively. An LDH-deficient strain
of Lc. lactis MG 5267, grown anaerobically, produced
significant amounts of fumarate, ethanol, acetoin and
butanediol. Under aerobic conditions, approximately
half of the pyruvate was converted to acetoin and one-
third to butanediol. To produce diacetyl in such sys-
tems, the acetolactate decarboxylase gene, as well as
the ldh gene must be inactivated. This was partially
accomplished by random mutagenesis of three strains
of a Cit strain of Lc. lactis by Monnet et al. (2000).
136 Starter Cultures: General Aspects
The strains were deficient in ALD and had much lower
LDH activity than the parent strains. The ‘double’
mutants produced up to four times more AL and ace-
toin and two times more diacetyl than the parent
under partially anaerobic (not defined) conditions and
grew very poorly in milk under aerobic conditions.
Addition of yeast extract (0.2 g/L) or catalase (70 U/ml)
increased the level of AL and resulted in the produc-
tion of 5 and 6 mM diacetyl, respectively; however,
acetoin was still produced. Recently, the ALD in Cit
Lc. lactis subsp. lactis has been shown to be a key regu-
lator of valine and leucine biosynthesis as well as
in the production of acetoin by controlling the flux
of acetolactate (Goupil-Feuillerat et al., 1997). Over-
production of NADH oxidase and inactivation of ALD
have also been shown to increase diacetyl production
in aerated cultures of Lc. lactis (Hugenholtz et al.,
2000).
Lb. helveticus has two different LDHs which pro-
duce both L and D lactate. Inactivation of the D LDH
in Lb. helveticus CNRZ 32 resulted in a strain which
produced the same amount of lactic acid as the parent
strain but all of it was in the L form (Bhowmik and
Steele, 1994). Lb. plantarum also produces both D and
L lactate. Inactivation of both enzymes resulted in a
strain which produced acetoin (mainly) and small
amounts of ethanol and mannitol from glucose
(Ferain et al., 1996).
Mannitol has a sweetness value about half that of
sucrose and, since it cannot be metabolized by
humans, it is considered a low-calorie sweetener.
Therefore, mannitol over-producing strains may have
applications in the production of functional foods.
Lc. lactis can metabolize mannitol (Neves et al., 2002)
but leuconostocs will produce mannitol during growth
on fructose (Grobben et al., 2001).
Autolysis
Cell lysis, and the consequent release of intracellular
enzymes, particularly peptidases and amino acid-
degrading enzymes, is receiving considerable attention
as an important aspect of flavour development in
cheese since Feirtag and McKay (1987) discovered that
temperatures close to the cooking temperature of
Cheddar cheese cause the lysis of many starter strains,
including Lc. lactis subsp. cremoris SK11, AM1, AM2,
US3 but not E8 or KH. This is due to induction of
temperate phage. Interestingly, the thermo-inducible
strains do not produce bitterness in cheese while
the non-inducible strains do. Since then, several
groups have identified other thermo-inducible strains
(Langsrud et al., 1987; Chapot-Chartier et al., 1994)
and the ability to lyse has become an important factor
in selecting starters for cheesemaking because of the
increased release of intracellular enzymes. Crow et al.
(1995) and Pillidge et al. (2002) reviewed autolysis in
LAB, mostly lactococci, and the consequences for
cheese ripening, particularly proteolysis. Prophage-
induced lysis should be distinguished from true
autolysins, which are also found in lactococci (Buist
et al., 1998). These findings have stimulated the study
of the effect of strains with different lytic and proteo-
lytic properties to make different cheeses, including
Cheddar (Wilkinson et al., 1994), St Paulin (Boutrou
et al., 1998; Lepeuple et al., 1998) and Gouda (Meijer
et al., 1998).
Lysis is normally measured by the release of intra-
cellular marker enzymes and concomitant increases in
soluble N and free amino acids. In Cheddar cheese
ripened at 4 or 10 °C, flavour formation was best in
the cheese made with Lc. lactis AM2, the strain show-
ing greatest lysis (Wilkinson et al., 1994). NSLAB
numbers reached 106 cfu/g within 1–2 months and
flavour was evaluated at 4 months but the contribu-
tion of NSLAB to the overall flavour of the cheese
does not appear to have been considered. Strain AM2
and a prophage-cured derivative have also been evalu-
ated in St Paulin cheese (Boutrou et al., 1998). The
parent strain underwent greater lysis and produced
greater amount of amino N; NSLAB counts were 105
cfu/g and the prophage-cured derivative produced a
bitter cheese compared with the parent strain. In
another study on St Paulin (Lepeuple et al., 1998),
five starters with different lytic and proteolytic prop-
erties were evaluated for their effect on the flavour of
the cheese. Lysis positively influenced the ripening
and flavour of the cheese but strains with low pepti-
dase activities and low lytic properties produced bitter
cheese. These studies confirm that cell lysis is import-
ant in the development of flavour in cheese during
ripening.
Autolysis appears to be a general property of LAB
as Sc. thermophilus, Lb. helveticus and several strains
of leuconostocs have also been shown to be lytic
(Sandholm and Sarimo, 1981; Lortal et al., 1997; Cibik
and Chapot-Chartier, 2000; Husson-Kao et al., 2000).
It is not clear if lysis also occurs in NSLAB. Temperate
phage is involved in Sc. thermophilus (Husson-Kao
et al., 2000) but whether phage are involved with the
other organisms was not reported. In the case of
Sc. thermophilus, autolysis occurred in response to the
depletion of lactose in the medium.
Bacteriocins
Bacteriocins are proteins produced by various bacteria,
which inhibit the growth of other bacteria. The
inhibitory host-range and the molecular mass can be
either large or small. Bacteriocins produced by LAB are

divided into three classes: lantibiotics, small heat-
stable non-lantibiotics and large heat-stable bacteri-
ocins (Nes et al., 1996). Nisin, the best known
bacteriocin, is a lantibiotic which is produced by some
strains of Lc. lactis, and is used commercially in more
than 50 countries as a food preservative to control the
growth of spoilage and pathogenic bacteria. Lantibiotics
are distinguished by the presence of unusual amino
acids, e.g., didehyroalanine and didehydrobutyrine,
which are produced by post-translational modificiation
of serine and threonine, respectively. Generally, bacteri-
ocins are single compounds but some bacteriocins com-
prise two components. For more information, see the
recent reviews of McAuliffe et al. (2001) and Twomey
et al. (2002).
Bacteriocins are receiving considerable attention
because many of them inhibit a wide range of Gram-
positive spoilage and pathogenic bacteria, especially
Listeria monocytogenes. The latter is a particular prob-
lem in cheese because it can grow at high salt concen-
trations, low temperatures and low pH, all of which
typically occur in cheese. The surface of smear-ripened
cheeses is a particular problem because of the increase
in the pH of the surface during ripening (see ‘Surface
Mould-ripened Cheeses’ and ‘Bacterial Surface-ripened
Cheeses’, Volume 2). Bacteriocins have also been used
to increase lysis of starter cells during cheese ripening
resulting in better flavour. The number of bacteriocin-
producing cells must be carefully controlled in the
latter application so that only limited reduction in the
ability of the starter culture to produce lactic acid
occurs during cheese manufacture (Ross et al., 1999).
They also have potential in cheese ripening to control
the growth of NSLAB.
Stress responses
Lactic acid bacteria are characterized by their ability to
produce large amounts of lactic and sometimes also
acetic acid during growth, which cause a significant
reduction in pH. Some starter bacteria are also sub-
jected to a considerable range of temperature during
cheese manufacture. For example, thermophilic
starters are usually grown at 37–42 °C but must with-
stand temperatures of up to 54 °C in the manufacture
of some cheeses, e.g., Emmental and Parmigiano-
Reggiano. Thus, tolerance to acid and temperature and
indeed other stresses is being actively studied. Acid
tolerances can be of two types, a general stress
response, which occurs during the stationary phase of
growth and an adaptive response, which develops dur-
ing the logarithmic phase of growth, called the loga-
rithmic acid tolerance response (LATR) (van de
Guchte et al., 2002). Induction of the LATR can also
protect LAB against temperature, salt and H2O2
Starter Cultures: General Aspects 137
stresses. Acid-adapted cells maintain a slightly higher
intracellular pH than non-adapted cells (O’Sullivan
and Condon, 1997) so that the maintenance of a trans-
membrane pH gradient (pH) via the F0F1 ATPase is
an important aspect of the ATR (van de Guchte et al.,
2002). The ability to produce NH3 from arginine, via
the deiminase pathway in many LAB, or from urea as
occurs in Sc. thermophilus, may also be important in
maintaining the pH gradient. These responses require
protein synthesis. Numerous proteins are induced by
heat in lactococci and enterococci and similar proteins
are also produced during osmotic shock but very few
of them have been characterized.
Betaine and proline are often called ‘compatible
solutes’ because they can be accumulated to high con-
centrations inside cells without affecting their physi-
ology or metabolism. Lc. lactis grown under high
osmotic strength contains high pools of proline or
betaine without any apparent effect on the physiology
of the cell (Molenaar et al., 1993). Betaine is taken up
by a high affinity transport system while proline has a
low affinity system which is repressed in complex
media.
Storage of cells is also receiving attention. Cells of
Lc. lactis MG 1363 stored at 10 °C for 4 h showed a
100-fold increase in survival to freezing at 20 °C
(Wouters et al., 1999). This was correlated with the
synthesis of several cold-induced small (7 kDa) pro-
teins. In another study, the flux of guanine and phos-
phate was implicated in the stress response of
lactococci (Duwat et al., 1999). Cells of Lc. lactis
transferred into a medium containing no glucose or
limiting glucose remained viable (106 cfu/ml) for at
least 1 year at 30 °C. However, considerable rearrange-
ment of plasmids occurred during storage (Rallu et al.,
2000). These findings may have potential for the stor-
age of cultures for cheesemaking.
Exopolysaccharide production
Smooth and creamy products, which are also low in fat
and sugar, have considerable appeal for consumers
who are conscious of their health. One way of improv-
ing the smooth texture of a product is to add suitable
polysaccharides to the product during processing. Most
of the polysaccharides used in food as thickeners and
stabilizers are obtained from plants (starch and pectin)
or different seaweeds (carrageenan, alginates). Except
for xanthan, very few are produced by microorganisms.
Exopolysaccharide production is an important charac-
teristic of many LAB involved in the production of fer-
mented milks. Producing cultures are generally
considered to be ‘ropy’ and result in the thickening of
the fermented milk. Such cultures are particularly
important in Scandinavian countries, e.g., Langfi in
138 Starter Cultures: General Aspects

Sweden and Viili in Finland. The industrial application ition (a 4:1 ratio of galactose and glucose) but different
of EPS produced by LAB is hampered by low yields, molecular masses. The levels of phosphoglucomutase,
typically 50–500 mg per litre, and efforts to improve UDP-galactose 4-epimerase and UDP-glucose pyrophos-
yields by genetic engineering and by manipulation of phorylase correlated highly with EPS production
culture conditions have been reviewed (Kleerebezem (Degeest and De Vuyst, 2000).
et al., 1999; Jolly et al., 2002). The production of EPS confers no intrinsic resist-
Lactic acid bacteria produce either homopolysac- ance to phage (Deveau et al., 2002).
charides, comprised only of fructose or glucose
residues or heteropolysaccharides, which are com-
prised of repeating units of several different sugars Preparation of Starters
including two or more of the following glucose, galact-
Lactic starters must perform one of their technological
ose, fructose and rhamnose (De Vuyst et al., 2001).
functions (acid production) early in cheesemaking and
They may be involved in a wide variety of biological
a sufficient amount of a metabolically active culture
functions, including prevention of desiccation, protec-
must be used to inoculate cheese milk. Usually, the
tion from environmental stresses, adherence to differ-
initial population of starters in cheese milk is around
ent surfaces, pathogenesis and symbioses ( Jolly et al.,
1–5  106 cfu/ml at inoculation and reaches 1–10 
2002). EPS-producing cultures have also been used to
108 cfu/ml when the curd is transferred to the moulds,
increase the moisture and improve the yield of low-fat
typically 5–6 h later in the case of Cheddar cheese. In
Mozzarella cheese (Low et al., 1998; Perry et al.,
most cheeses, during this time, the pH must decrease
1998).
from ⬃6.6 to 5.5; cells which are not fully active or
It is possible to increase EPS production by Sc. thermo-
are sub-lethally stressed at inoculation will show slower
philus by altering the levels of enzymes in carbohy-
growth and consequently slower acid production, thus
drate metabolism, particularly phosphoglucomutase
increasing the cheesemaking time. Except for natural
and UDP-glucose pyrophosphorylase (Levander et al.,
starters, most cheese plants use cultures provided in
2002). The biosynthesis of EPS has been studied in an
one of several forms (liquid, frozen, freeze-dried) by
EPS strain of Lc. lactis and a derivative carrying a
specialized industries or institutions (see above). An
plasmid encoding the EPS gene cluster (EPS strain)
outline of current systems for the preparation of starters
(Ramos et al., 2001). The concentration of UDP-glucose
is presented in Fig. 4. The traditional approach for the
and UDP-galactose, the precursors of EPS, were signifi-
build-up of a starter culture for inoculation of cheese
cantly lower in the EPS strain than in the EPS strain,
milk, requiring a number of steps from a small volume
while the concentration of the UDP-N-acetylmuramoyl
(1 ml or g) of stock culture to a large volume
pentapeptide, which is part of the cell wall, was
(100–1000 l) of bulk starter, is still in use. However, it is
greater in the EPS strain, indicating that there is
being replaced by the use of frozen or freeze-dried cul-
competition between EPS synthesis and cell growth.
tures for direct inoculation of bulk starter milk or of
These data suggest that the production of EPS could
cheese milk directly, especially in small cheesemaking
be enhanced by increasing both the flux of the EPS
plants. While the traditional system based on multiple
precursors and the lipid carrier precursors.
transfers is cheaper than the direct-to-vat system, it
Response surface analysis of different fermentation
requires skilled personnel and facilities and increases
conditions showed that the optimum temperature, pH
the risk of contamination with phage.
and casitone concentration for EPS production by
Aspects of the commercial production of starter cul-
Lb. delbrueckii subsp. bulgaricus RR were 38 °C, 5 and
tures have been thoroughly reviewed by Whitehead
30 g/L, respectively. The actual yield, 354 mg EPS/L,
et al. (1993) and Sandine (1996). Here, an overview of
was within the 95% confidence limit of the predicted
the issues related to the production, preservation and
yield (Kimmel et al., 1998). Oxygen, orotic acid and
distribution of starter cultures by companies and of the
carbon source were also important for EPS production
preparation of starters at the cheese plant is presented.
which was greatest during the stationary phase of
growth in a chemically defined medium (Petry et al.,
Propagation of starter cultures
2000). Maximum production of EPS by Lb. helveticus
occurred at pH 6.2 (Torino et al., 2001). The production of starter cultures requires careful selec-
A model describing the growth and EPS production tion of media and operating conditions to obtain opti-
by Sc. thermophilus LY03 has been developed and some mum results in terms of final cell numbers, activity
evidence for EPS degradation has been found (Degeest (prompt growth, reduced lag phase, suitable acid pro-
and De Vuyst, 1999). This strain produces two het- duction, aroma production, proteolytic ability), stability
eropolysaccharides of the same monosaccharide compos- upon storage and, in mixed cultures, composition of the
 Starter Cultures: General Aspects 139

Frozen or
freeze dried
concentrated Cheese vat
cultures
0.1–2% v/v
Frozen or A
freeze-dried
stock culures Mother culture
(1 g, 1 ml) (RSM) C B

1% v/v 1% v/v 1% v/v

Intermediate or
feeder culture
(RSM) E

Bulk starter (RSM,

starter media)

Frozen stocks

Figure 4 Examples of the production of lactic starter cultures in a cheese plant. A number of steps are necessary to build up the
bulk starter from frozen or freeze-dried stock culture. Duration and inoculum size for each step are variable, depending on the type of
culture (mesophilic, thermophilic, mixed, defined) and the temperature of incubation. To reduce time for build-up of bulk starter and
the risk of contamination, frozen or freeze-dried starter concentrates can be used to inoculate the bulk starter tank or the cheese vat.
The elements of a typical bulk starter tank are shown: A, agitator; B, sterile air inlet/outlet with HEPA filters to prevent the access of
phages during cooling and operation; C, ports for inoculation and addition of alkali for pH control; D, pH and temperature probes;
E, jacket for circulation of water or steam. Digital or analogic controllers and printer/recorders for temperature and pH, and external
alkali tanks are not shown.

starter. These, in turn, are influenced by several factors,
including the presence of disturbing phage, medium
composition and fermentation conditions (heat treat-
ment, temperature and pH control during fermentation,
duration of incubation, storage temperature, etc.).
Although cheese milk was the traditional medium
for the growth of starters in cheese plants, it has been
replaced by pre-tested, antibiotic-free reconstituted
skim milk (RSM) and by specially designed starter
media, available from starter culture companies. The
availability of pre-tested RSM allows better control of
growth prior to inoculation of milk in the cheese vat. It
can be reconstituted to a higher solid level than that of
fresh milk, thus improving the buffering capacity and
therefore the growth and activity of the culture. Doubl-
ing the concentration of solids in RSM from 8 to 16%
usually results in a doubling of the number of viable
cells (from 5–7  108 to 10–14  108 cfu/ml) with a
higher final pH (from 4.5 to 4.7). A similar result can
be obtained by increasing milk solids by ultrafiltration.
The need to increase cell numbers and improve the
activity and stability of the cultures has prompted the
development of specially designed starter media.
Although most starter media are milk- or whey-based
(whey permeate can also be used), they may contain a
range of ingredients to improve starter growth, to con-
trol pH and to inhibit phage adsorption (Table 3)
(Whitehead et al., 1993). Two of the most important
issues in the design of starter media are phage and pH
control. Measures for phage control are described in
‘Starter Cultures: Bacteriophage’, Volume 1.
pH control is important for building up starter bio-
mass, preventing acid stress and loss of activity, and con-
trolling the ratio of species and strains in mixed cultures
(Oberg and Broadbent, 1993; Whitehead et al., 1993;
Sandine, 1996). While lactobacilli and leuconostocs are
relatively acid tolerant, mesophilic and thermophilic
cocci are rapidly inhibited when the pH falls below 5.5.
Therefore, the rod:coccus ratio of thermophilic starters
may be affected significantly by the pH and pH-course
during incubation. pH control also allows complete con-
sumption of the carbohydrate source and retention of
viability during prolonged refrigerated storage of fully
grown cultures (Sandine, 1996).
Both internal and external pH control are used.
Internal pH control is achieved by the use of soluble or
insoluble buffering agents. Soluble buffers (phos-
phates) perform the dual role of pH control and phage
140 Starter Cultures: General Aspects
Table 3 Ingredients in starter media and their functions; the most commonly used ingredients are in boldface (adapted from
Whitehead et al., 1993)
Category Ingredients
Carbohydrates Lactose, glucose, maltose, sucrose
Nitrogen sources Milk proteins, whey proteins,
casein hydrolysates, peptones
Growth factors Yeast extract, Corn Steep Liquor
Chelating agents Phosphates, citrates
Antioxidants Ascorbic acid, ferrous sulphate
Buffers and neutralizers Insoluble, for internal buffering:
Trimagnesium phosphate, calcium
carbonate, encapsulated sodium carbonate.
Soluble, for internal buffering: phosphates.
Soluble, for external pH control: ammonia,
potassium or sodium hydroxide
inhibition but the concentration needed to control pH
effectively can be inhibitory to same species and may
even reduce cheese yield by chelating Ca2 in the
cheese milk. Insoluble (calcium carbonate, trimagne-
sium phosphate) or encapsulated (sodium carbonate
encapsulated in ethyl and methyl cellulose; Whitehead
et al., 1993) buffers result in a better performance, with
high cell numbers (up to 1010 cfu/ml) and prolonged
stability (up to 10 days) on refrigerated storage.
However, internal pH control is unsuitable if the pH
of the culture must be maintained at a fixed value. pH-
controlled bulk starter tanks, fitted with sterilizable
electrodes for pH measurement and computer control
with automatic addition of alkali to control the pH at
the desired set point are now readily available. The
most common neutralizers used in external pH control
are KOH, NH4OH and gaseous ammonia. NaOH and
Na2CO3 are cheaper but some starters may be
inhibited by high concentrations Na.
Other process factors that affect starter growth and
performance are the heat treatment of the growth
medium, and the temperature and the duration of
incubation. Time/temperature combinations using
during heat treatment are much higher (80–90 °C for
10–30 min is typical; higher temperatures can be used
in commercial production of thermophilic starters)
than commercial pasteurization (72 °C, 16 s). Such
conditions drastically reduce the microflora in the
medium, ensure the destruction of phage, which are
resistant to pasteurization, and reduce the redox
potential, driving off oxygen and denaturing proteins,
thus improving starter growth.
The temperature and duration of incubation are
highly dependent on the composition of the starter cul-
Function and typical concentration range
Energy and carbon source, 10–40 g/L. Glucose 1 g/L
may be used to facilitate recovery of stressed cells
Sources of amino acids, from 1 g/L for hydrolysates
to 20–30 g/L for proteins and peptones
Sources of amino acids, vitamins, nucleotides and
minerals, 2.5–5 g/L
Inhibition of phage adsorption by chelation of avail-
able Ca2, 7.5–20 g/L (under external pH control)
Prevention of oxidative stress due to H2O2
production, 1 g/L
Control of pH during fermentation to 5.5–6.5. Amount
added is highly variable depending on carbo-
hydrate concentration and target pH at the end of
fermentation
ture and on other practical considerations. The tempera-
ture of incubation may greatly affect starter composition
in mixed cultures. Incubation at 18–21 °C is usually pre-
ferred for cultures of lactococci and leuconostocs,
because both organisms have approximately the same
growth rate in this temperature range, while lactococci
grow faster at 30 °C. For thermophilic rod:coccus
cultures, a compromise (42 °C) must be found between
the optimum temperature of the moderately thermophilic
Sc. thermophilus (37–39 °C) and that of the thermophilic
Lb. delbrueckii subsp. bulgaricus and Lb. helveticus (45 °C),
although symbiosis between culture components may
overcome the problems caused by growth at sub-optimal
temperatures (Oberg and Broadbent, 1993).
In cultures without pH control, the temperature and
duration of incubation may be highly related. In gen-
eral, cultures should be refrigerated shortly after the
beginning of the stationary phase of growth; this
requirement is less critical when the carbohydrate
source is exhausted and the pH is controlled. An active
mesophilic culture reaches the stationary phase in milk
media in 6–8 h at 30 °C and in 16–18 h at 18–21 °C;
the latter combination is obviously more suitable for
overnight incubation. Thermophilic cultures may reach
the stationary phase in 6–8 h at 37 °C.
Preservation and distribution of starter cultures
While stock cultures are usually stored at the cheese
plant for only a limited time, the production and dis-
tribution of starter cultures on a commercial basis
requires suitable means for the preservation and distri-
bution of cultures in a highly active state. The cultures
may be preserved by a variety of means (chilling of

liquid cultures, drying, freezing, freeze-drying) which
expose the culture to a variety of sub-lethal and lethal
stresses (van de Guchte et al., 2002) which negatively
affect the vitality and activity (by sub-lethally damag-
ing the cells, by selectively killing some components
of the culture thus changing culture composition).
Sub-lethally stressed cells need a longer lag phase to
recover, which translates into the need for longer
resuscitation. Historically, cultures have been pro-
duced and distributed in liquid form, in air-dried form
(spray dried), as frozen cultures and freeze-dried cul-
tures. The two latter means of preservation are used
most widely in the starter industry today.
Liquid and air-dried cultures
Chilling of liquid cultures is the oldest method of pre-
servation and distribution of cultures. CaCO3 (6 g/L) is
usually added to milk to maintain a high pH and the cul-
tures are stored at a low temperature (2–5 °C). Stability
does not exceed 1 or 2 weeks and several transfers are
needed to obtain an active culture. Although chilling is
still used for daily distribution of some mixed cultures
for the production of PDO cheese in Italy, it has been
superseded by freezing and freeze-drying.
Air- or vacuum-drying of cultures was used in the
past to produce cultures in powdered forms (Sandine,
1996) but because of poor vitality and activity this means
of preservation is not used any more. Spray-drying is a
fast and economic method for removing water but cul-
tures are exposed to a variety of stresses (heat, dessic-
cation, oxidation), and survival and activity are usually
low (Teixeira et al., 1995; To and Etzel, 1997).
Frozen cultures
Freezing at a very low temperature ( 80 °C, 196 °C)
in the presence of cryoprotective agents is the best way
for preserving the vitality and activity of bacteria, and
freezing is a preliminary step in the production of freeze-
dried cultures. Several factors affect the survival of LAB
during freezing and their activity after thawing, e.g.,
species, strain, growth medium composition, culture con-
ditions, growth phase, composition of the medium used
for suspending the cells during freezing, type and
concentration of the cryoprotective agent, temperature
and rate of freezing, storage temperature, temperature
and rate of thawing (Sandine, 1996). To obtain high cell
densities prior to freezing, cells are grown under pH-
control. Stationary cells are more resistant to freezing
than exponentially growing cells. Mesophilic and ther-
mophilic cocci are more resistant than thermophilic
lactobacilli and leuconostocs; therefore, care should be
exercised in the freezing of mixed cultures to maintain
the correct strain balance. Removal of the cells from
the growth medium and their suspension in a suitable
Starter Cultures: General Aspects 141
cryoprotective medium is usually practised. RSM
(12–14% with added lactose) may be suitable as a cryo-
protectant, but other agents can also be used, e.g.,
5–15% glycerol, 5% sodium glutamate, 7% sucrose. Cul-
tures should be cooled as rapidly as possible to below
60 °C. Storage temperature should be between 20 °C
and 40 °C. Frozen cultures should be thawed as rap-
idly as possible to maximize survival.
The need to maintain the culture frozen at all times
makes frozen cultures less practical than freeze-dried
cultures for dispatch to cheese plants. However,
because of their high activity, frozen concentrated cul-
tures (see below) are still preferred as the means for
distributing starter cultures in some countries.
Freeze-dried cultures
While removing water at ambient temperature is detri-
mental to the survival and activity of starter cultures,
freeze-drying, i.e., removing water from a frozen culture
by sublimation under high vacuum, results in high levels
of survival. Freeze-drying has been used for the prepar-
ation of dairy starters for about a century (Sandine,
1996). Freeze-dried stocks containing 108–109 cfu/g are
dispatched to cheese factories in vials, serum bottles or
pouches containing a few grams of powder and need
several transfers for full reactivation.
The procedure for the preparation of freeze-dried
cultures is similar to that used for the preparation of
frozen cultures up to the freezing step, although add-
ition of antioxidants like ascorbic acid, together with
cryoprotective agents, is common. Cultures are frozen
rapidly in vials connected to manifolds or in trays and
dessiccated under high vacuum (10 Pa) for 12–24 h,
to a final aW of 0.1. Vials can be closed under vacuum,
while powders lyophilized in trays are aseptically pack-
aged in a variety of containers under an inert atmos-
phere, since dried cells are highly sensitive to oxidative
stress. Freeze-dried cultures can be dispatched and
stored at ambient temperature, although survival and
activity are improved by storage at 4 °C or 20 °C.
Concentrated starter cultures
Conventional frozen and freeze-dried cultures do not
contain enough cells for inoculation of the bulk starter
tank or the cheese milk and therefore several transfers
are needed to build up sufficient inoculum for the bulk
starter. Frozen and freeze-dried concentrated starters,
typically containing 1010–1011 cfu/g and 1011–1012 cfu/g,
respectively, for inoculation of the bulk starter (also
known as bulk sets) or the cheese milk (direct-to-vat
cultures, direct vat set cultures) are now readily avail-
able from starter companies, and are widely used in
both small and large plants. Although concentrated
starters are more expensive than starter build-up from
142 Starter Cultures: General Aspects
a stock culture, the use of concentrated starters
improves plant flexibility (because of reduction or elim-
ination of the time needed for building up the bulk
starter), reduces or eliminates the need for skilled per-
sonnel and equipment for the production of starters and
reduces the risk of phage contamination in the factory.
Starters are grown in pH-controlled milk- or whey-
based media and concentrated by bactofugation or by
microfiltration. When milk media are used, citrate (1%)
is used to solubilize the milk proteins, even when pH
control is used. Cells are then resuspended in a suitable
medium, containing cryoprotectants and antioxidants,
rapidly frozen or frozen and freeze-dried. Frozen con-
centrated cultures may be packaged directly or frozen in
pellet form and then packaged. Freeze-dried concen-
trated cultures are lyophylized in large tray freeze-dryers
and then packaged under vacuum or a nitrogen atmo-
pherein amounts suitable for inoculation of 500–1000 l
of bulk starter medium or 1000–5000 l of cheese milk.
Frozen concentrates are partially thawed by putting
the container in chlorinated (25–50 mg/kg) water at
room temperature for 20 min before adding it to milk,
where thawing is completed in 15–30 min. Freeze-
dried starters can be added directly to the bulk starter
tank or cheese vat, although rehydratation in a small
volume of milk is advisable to improve distribution.
Because of their high activity, frozen concentrated
cultures for direct-vat inoculation do not significantly
increase cheesemaking time, and are widely used in
the USA and Australia. On the other hand, sub-lethal
damage caused by freeze-drying may increase cheese-
making time by 0.5–1 h (Sandine, 1996); however,
this disadvantage may be offset by the fact that dis-
patching and handling of freeze-dried starters is
greatly simplified, and freeze-dried concentrates are
more widely used in Europe.
The choice between concentrated cultures for bulk
starter inoculation and those for direct vat inoculation
(DVI) may depend ultimately on considerations
related to costs and ease and flexibility of use. A com-
parison of costs for a cheese plant processing about
10 000 tonne/year of cheese is presented in the web
site of Australian Starter Cultures Research Centre
(http://www.ascrc.com.au/strategy.html); the cost of
concentrates for bulk starter inoculation is estimated
at US$20/tonne of cheese, compared to US$47/tonne of
cheese for cultures for DVI. However, recently aggre-
sive marketing by the culture suppliers has reduced the
price of DVI culture to US$24 per tonne of cheese
though the cost is higher for smaller cheese plants.
Concentrated starter cultures are now the preferred
way for the distribution and use of starter cultures and
a wide selection of species, strains and combinations is
available. A list of websites of some companies and
institutions providing on-line catalogs of starter cul-
tures includes:
Chr.Hansen http://www.chr-hansen.com/
Danlac http://www.danlac.com/starter-cultures.shtml
Rhodia Dairy http://www.rhodiadairy.com/products/
Swiss Federal Dairy Research Station http://www.sar.
admin.ch/
Development of concentrated starters implies higher
R&D and production costs, which are reflected in the
price of the culture; moreover, not all species and
strains are suitable for the production of concentrated
starters for DVI, and the wide diffusion of cultures for
direct vat has also reduced the diversity of cultures
available for cheesemaking.
Acknowledgement
We are grateful to Ian Powell for the information he
provided and for useful discussion.
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This Page Intentionally Left Blank
 Starter Cultures: Genetics
M.J. Callanan and R.P. Ross, Teagasc, Dairy Products Research Centre, Moorepark,
Fermoy, Co. Cork, Ireland
Introduction
Within the last three decades there has been intensive
study of the genetics of starter bacteria, ranging from
plasmid biology to genetic tool development, and lead-
ing ultimately to elucidation of the complete genome.
This has facilitated such advances as the metabolic
engineering of these commercially important bacteria
and to the improvement of particularly significant indus-
trial traits, such as increased bacteriophage resistance.
The purpose of this review is to discuss some of the
major advances which have occurred during this time,
culminating in the elucidation of the genomes of sev-
eral strains. It is worth emphasising that while the
chromosomes of a number of strains have been char-
acterised in detail, many commercially significant
traits are encoded on mobilisable elements such as
plasmids and transposons. In fact, many industrially
important strains have rich plasmid complements, and
it could be argued that much strain ‘individuality’, in
terms of industrial performance, could be attributed to
their plasmid genomes. The development of genetic
tools for strain improvement, in combination with
genomic and metabolomic technologies have opened
new possibilities for the routing or re-routing of
metabolism towards desirable metabolites such as
flavour compounds, e.g., diacetyl and vitamins, e.g.,
folate. This, together with the huge array of as yet
un-mined sequence information should lead to the
development of new and improved starter strains for
food production.
Genetics of Mesophilic Starters
Starter cultures used by the dairy industry can be
broadly divided into two types, mesophilic and thermo-
philic, based on their optimum growth temperature
(see ‘Starter Cultures: General Aspects’, Volume 1).
Mesophilic starters have a growth optimum of ⬃30 °C
and are used in the production of Cheddar, Gouda,
Edam, Blue and Camembert cheeses. Only three
species of the lactic acid bacteria (LAB) group
employed as commercial starters are considered
mesophilic, Lactococcus lactis subsp. lactis, Lc. lactis
subsp. cremoris and Leuconostoc mesenteroides subsp.
Cheese: Chemistry, Physics and Microbiology, Third edition – Volume 1: General Aspects
ISBN: 0-1226-3652-X
Set ISBN: 0-1226-3651-1
cremoris. Of all starter bacteria, Lc. lactis subsp. lactis
IL1403 and Lc. lactis subsp. cremoris MG1363 are the
most thoroughly investigated at the genetic level and
have been the workhorses for studying starter cultures.
The construction of these two plasmid-free strains from
cheese starter parents in the early 1980s was instru-
mental in their selection for further study.
Chromosome
The sequencing of the Lc. lactis subsp. lactis IL1403
genome has provided the first comprehensive insight
into the genetics of lactococcal starters. Strain IL1403
is a derivative of the Streptococcus (now Lactococcus)
lactis strain IL594, isolated from a cheese starter cul-
ture (Chopin et al., 1984). The 2365-kb circular
chromosome has a G  C content of 35.4% (Fig. 1)
and encodes 2310 open reading frames (ORFs)
(Bolotin et al., 2001). This genome is relatively small
when compared to other bacterial genomes, such as
Bacillus subtilis (4214 kb, 4099 ORFs), and most likely
reflects the specialised adaptation of Lactococcus to
growth in the nutrient-rich milk environment. As
expected, many genes required for de novo synthesis of
essential nutrients and the degradation of complex
molecules are absent from the IL1403 genome. Simi-
larly, the machinery that controls gene expression dif-
fers from the environmentally responsive Bacillus. The
proportion of transcriptional regulators is similar in
the two bacteria but IL1403 encodes much fewer
genes that respond to changing environmental condi-
tions. B. subtilis encodes 18 sigma factors and 34 two-
component systems compared with 3 sigma factors and
8 two-component systems in the lactococcal chromo-
some. There were also a few unexpected revelations
from the annotated genome sequence. For example,
strain IL1403 requires a number of amino acids
(isoleucine, valine, leucine, histidine, methionine and
glutamic acid) and vitamins (folic acid, menaquinone,
riboflavin and thioredoxin) for growth in defined
media despite having the genetic potential to synthe-
sise all these necessary growth factors (Bolotin et al.,
2001). The genes in the amino acid biosynthetic
operons appear to have mutations that may be specific
to laboratory strains. In addition, the ability of
Copyright © 2004 Elsevier Ltd
All rights reserved
150 Starter Cultures: Genetics

Figure 1 Schematic representation of the L. lactis IL1403 genome. The figure highlights the nucleotide composition of the chromo-
some and demonstrates the gross organisation of the entire genome into two divergent blocks arranged around the replication initia-
tion (0 kb) and termination (c. 1250 kb) sites. The figure (available at www.cbs.dtu.dk) was generated using Genome Atlas (Jensen
et al., 1999; Pedersen et al., 2000).

Lactococcus to respire aerobically has remained contro-
versial but the presence in the genome of the genes for
the biogenesis of menaquinone and cytochrome d
agrees with data indicating the respiratory capacity of
an organism considered to be exclusively fermentative
(Gaudu et al., 2002).
A recent examination of the genetic organisation of
the two reference lactococci, the partially sequenced
MG1363 and the completely sequenced IL1403,
revealed an average of only 85% identity at the DNA
level (Campo et al., 2002). There is, however, a high
degree of conserved gene organisation, or synteny. In
particular, the order is conserved in the oriC region of
the two genomes but not in the vicinity of the mobile
element-rich replication terminus. Overall, about 9.2%
of the Lc. lactis genome is formed by insertion ele-
ments and prophages, and this percentage reflects the
contribution of horizontal gene transfer to the
composition of the genome. For example, there is a
large chromosomal inversion between MG1363 and
IL1403 that covers nearly 50% of the genome and con-
tains the six ribosomal RNA operons (Le Bourgeois
et al., 1995). This plasticity of the Lactococcus genome
may be an important factor in optimising the genome
in response to the nutrient-rich milk environment.
Moreover, as noted by Campo et al. (2002), the com-
parative genomics of the two strains ‘demonstrates
that the IL1403 genetic content is probably not repre-
sentative for the content of all lactococcal subspecies’.
This information is important in extrapolating from
the IL1403 sequence data to other starter lactococci.
Bolotin et al. (2001) commented on the high level of
apparent non-functioning genes for amino acid
biosynthesis which may be due to the treatments used
to cure the parent of the laboratory strain of its plas-
mids. The accumulation of further genomic data on
other strains will be necessary to establish the degree
of conservation between laboratory strains and current
industrial mesophilic starter cultures.
Plasmids
The genetic investigation of starter cultures was effect-
ively initiated by McKay and co-workers at the

University of Minnesota with the demonstration that the
lactococci contain plasmids that encode traits essential
in the cheesemaking process (McKay and Baldwin,
1975). In addition to the genetic information encoded
by the chromosome, Lc. lactis species harbour numerous
large cryptic plasmids. The presence of these plasmids
helped explain the well-known and problematic instabil-
ity of key industrial traits. A rapid and reliable method
was subsequently developed that allowed the plasmids to
be isolated and visualised using agarose gels (Anderson
and McKay, 1983). Plasmid profiling remains an import-
ant method of differentiating lactococcal starter strains,
although their instability precludes the plasmids from
providing a permanent fingerprint of strain identity.
Indeed, the plasmid content of strains can vary dramatic-
ally during long-term storage. It was also quickly recog-
nised that it is possible to mobilise a number of the
plasmids via conjugation. Therefore, the enzymes neces-
sary to hydrolyse casein, transport and metabolise lactose
and citrate, produce bacteriocins and combat phage
could be transferred among industrially important
strains. The commercial importance of the plasmid-
encoded traits has resulted in numerous studies, and
some of the plasmids have been partially or even
completely sequenced (Dougherty et al., 1998; van
Kranenburg et al., 2000; Boucher et al., 2001). However,
the physiological role of the majority of these extra-chro-
mosomal elements is yet to be defined fully.
In addition to encoding key industrial traits, the
native lactococcal plasmids have provided the neces-
sary material for the development of genetic tools
required to manipulate Lc. lactis. The original lacto-
coccal cloning vectors, such as pSA3 (Dao and Ferretti,
1985), were ‘shuttle’ vectors and encoded two replica-
tion origins in order to maintain the plasmid in both
lactococcal and E. coli hosts. The ability to replicate in
E. coli was necessary to expose the plasmid to the
highly advanced genetic techniques available in that
background. Subsequent vectors have been based pri-
marily on more promiscuous replication origins, such
as those from the cryptic lactococcal plasmids, pWV01
and pSH71 (de Vos and Simons, 1994). Combined
with an antibiotic selection marker, these replicons are
stable in Gram-positive and Gram-negative hosts and
continue to form the backbone of the most useful lac-
tococcal vectors.
Genetics of industrially important traits
Lactose and citrate metabolism
The primary function of Lactococcus in an industrial
dairy fermentation is to produce lactic acid. The bac-
teria synthesise this flavoursome and spoilage-preventing
acid as a product of the fermentative conversion of lac-
Starter Cultures: Genetics 151
tose. In milk, the disaccharide lactose acts as the carbo-
hydrate source. The genetics of lactose utilisation by
lactococcal starters has been well-documented (de Vos
and Vaughan, 1994). The plasmid-encoded lac operon
consists of nine genes in the order lacABCDFEGX. They
are responsible for the transport and incorporation of
the galactose and glucose constituents of lactose into
the Embden-Meyerhof-Parnas pathway (Fig. 2). Trans-
port occurs via the lactose-specific phosphotransferase
system encoded by lacEF. The operon is directly regu-
lated at the transcriptional level by the product of the
divergently transcribed lacR gene that represses the path-
way in the presence of glucose. A second, more global,
mechanism of regulation also appears to act in the
absence of lacR (de Vos and Simons, 1994).
Citrate metabolism contributes the distinctive
diacetyl/acetate flavour and aroma to products manufac-
tured with citrate utilising (Cit) lactococci. It is also
required for CO2 production resulting in eye formation
in Dutch-type cheese. The ability to metabolise the rela-
tively low concentration of citrate in milk (8 mM) is pri-
marily dependent on the presence of citrate permease
(Fig. 2). The gene for the permease, citP, is part of the
plasmid-encoded citBRP operon. The transport genes
are not induced by citrate but are induced under condi-
tions of lactic acid stress (Garcia-Quintans et al., 1998)
reflecting the role of citrate in relieving growth inhib-
ition by lactate (Magni et al., 1999). Following trans-
port, citrate is cleaved to produce oxalacetate and
release acetate. Oxalacetate is converted to diacetyl,
acetoin and CO2 through pyruvate as an intermediary.
The distinctive nature of citrate metabolism has
prompted a number of studies aimed at manipulating
the pathway in order to increase the concentration of
desirable end products, e.g., diacetyl. One approach
has been to manipulate the citrate metabolism pathway
of Cit Lc. lactis through genetic techniques such as
gene inactivation and over-expression (Fig. 2). Inacti-
vation of lactate dehydrogenase, the enzyme responsible
for the production of lactic acid from pyruvate, resulted
in increased acetoin production (Snoep et al., 1992).
However, to efficiently convert sugars to diacetyl, inacti-
vation of -acetolactate decarboxylase (ALDB) and
over-expression of NADH-oxidase are required (Hugen-
holtz et al., 2000). The NADH-oxidase overproduction
causes the rerouting of pyruvate through NADH-
independent pathways leading to diacetyl and acetoin
since the cell no longer needs to regenerate NAD
through lactate dehydrogenase. Inactivation of aldB
ensures that the rerouted pyruvate is converted to
diacetyl and not acetoin, the product of the decarboxy-
lase. These manipulations resulted in a strain with an
increased capacity for diacetyl production. These types
of metabolic engineering studies will be further enabled
152 Starter Cultures: Genetics

Lactose

Cell wall

Transporter Cell membrane

lac genes
Glucose Galactose

2,3-butanediol galKTE

Glucose-6-P

Acetoin
aldB

Diacetyl α–acetolactate
Pyruvate Oxaloacetate
Acetate
ldh
ethanol
Citrate lysase
Lactate

Permease
(citP)

Citrate

Figure 2 Schematic of lactose (closed arrows) and citrate (open arrows) metabolism in LAB. Genes inactivated as part of meta-
bolic engineering strategies for overproducing diacetyl are indicated (see text for detail).

by our increasing knowledge of lactococcal metabolic
pathways from genome sequence analysis.
Proteolysis and amino acid catabolism
The lactococci are fastidious organisms specially adapted
to grow in the nutrient-rich growth medium that is
milk. Lactose provides a readily utilisable sugar source
for energy whereas degradation of the principal milk
protein, casein, provides nitrogen sources and supplies
the amino acids for protein synthesis. Casein break-
down is initiated by a cell envelope-associated pro-
teinase (CEP). The proteinase gene is another of the
key industrial traits located on the indigenous lacto-
coccal plasmids. The multi-domain CEPs are large
proteins (more than 1800 amino acids are encoded by
the gene) and are classified based on their caseinolytic
specificity (Kunji et al., 1996). The plasmid-encoded
genes have been sequenced for a number of lactococ-
cal strains and the sequence data exploited to investi-
gate the catalytic properties of these important enzymes.
Site-directed and cassette mutagenesis techniques have
identified the substrate-binding domain and active site
residues (Siezen et al., 1993).
The peptides produced by the proteinase are trans-
ported into the cell by three systems. The Opp system
is responsible for oligopeptides of 4–18 residues, while
the DtpT and DtpP transport hydrophilic and hydropho-
bic di- and tripeptides, respectively (Kunji et al., 1996).
Both Opp and DtpP are multi-gene systems organised
in operons. In contrast, the proton motive force-driven
DtpT transporter is encoded by a single gene (Fang
et al., 2000). Once inside the cell, the peptides are
hydrolysed to their constitutive amino acids by a set
of peptidases with various specificities. The genes for
13 peptidases have been described for the IL1403
sequence, a number of which had been studied previ-
ously (Christensen et al., 1999). Of particular interest
are the aminopeptidases, PepN and PepC, and the
proline-specific peptidases. PepN and PepC display low
substrate specificity and free amino acids by cleaving the
N-terminal end of oligopeptides. Casein is a proline-rich
substrate and lactococci have multiple peptidases with

activity against various proline-containing peptides.
The genes for X-prolyl-dipeptidyl aminopeptidase
(pepX), prolidase (pepQ), proline iminopeptidase (pepI)
and aminopeptidase P (pepP) are present on the Lc.
lactis genome. In addition, a number of peptidases
shown to cleave internal peptide bonds (endopepti-
dases) and enzymes that hydrolyse di- and tripeptides
have been described. While inactivation of the individ-
ual peptidase genes described above did not eliminate
the ability of Lactococcus to grow in milk, mutants
exhibit reduced growth rates. Moreover, strains in
which multiple peptidase genes were disrupted had
significantly reduced growth rates (Mierau et al.,
1996).
Recently, a comprehensive study on the regulation
of the proteolytic system was performed in MG1363
and some interesting observations were made (Guedon
et al., 2001a). The transcription of 16 genes encoding
12 peptidases, 2 CEPs and the 3 transport systems
were analysed in response to various environmental
factors. It was found that neither the sugar source nor
temperature effected transcription with the sole excep-
tion of pepP, which is modified by sugar. Transcription
of the 2 CEP genes, 3 aminopeptidase genes (pepX,
pepN and pepC) and the Opp transport system operon
were all regulated by the peptide content of the
medium. The remaining genes, that appeared to be
unregulated, were expressed at lower levels and it was
suggested that they probably encode enzymes involved
in cellular functions other than peptide utilisation
(Guedon et al., 2001a). In a companion study, a trans-
criptional repressor with homology to the CodY regu-
lator of Bacillus subtilis was shown to regulate
expression of the genes that were sensitive to the pep-
tide content of the medium (Guedon et al., 2001b).
The catabolism of amino acids in Lactococcus has
received significantly less attention than the proteo-
lytic system (see ‘Catabolism of Amino Acids in
Cheese during Ripening’, Volume 1). The ability to
overcome amino acid auxotrophy through degradation
of casein is well documented but the ability to synthe-
sise various amino acids has not been investigated
thoroughly. In particular, very little work has focused
on the genetic components involved. The availability
of the genome sequence will redress this situation but
at present the majority of the studies have focused on
detecting specific enzyme activities associated with the
catabolic pathways in various strains (reviewed by
Christensen et al., 1999). The catabolism of amino
acids by lactococci is likely to have an essential role in
the development of cheese flavour and aroma. Inactiva-
tion of an aromatic amino acid aminotransferase gene,
araT, of Lc. lactis NCDO 763 has already confirmed the
involvement of this enzyme in the conversion of
Starter Cultures: Genetics 153
amino acids to aroma compounds (Yvon et al., 2000).
Two further aminotransferases have been cloned and
sequenced from Lc. lactis LM0230 (Atiles et al., 2000;
Dudley and Steele, 2001) but much more work is
needed.
Bacteriocins
It is well established that starter bacteria produce
a range of substances, including lactic acid and
metabolites which aid in the preservation, and con-
tribute to the safety of many fermented food products
(Klaenhammer, 1988; Holzapfel et al., 1995). In add-
ition to the production of antimicrobial metabolites
such as lactate, almost all the different representative
species have been reported to produce antimicrobial
peptides and/or proteins which are collectively
referred to as bacteriocins. Of the many bacteriocins
isolated and studied to date, nisin is the only one
whose commercial potential has been realised. Nisin
was assessed to be safe for food use by the Joint Food
and Agriculture Organisation/World Health Organisa-
tion (FAO/WHO) Expert Committee on Food Addi-
tives in 1969, and is in use in more than 48 countries.
In the dairy industry, nisin is exploited mainly for the
prevention of clostridial growth in processed cheese,
dairy desserts and cheese spreads. Nisin has been
the subject of a wide variety of fundamental studies
as to its structure and genetics (Dutton et al., 2002).
It is classified as an antibiotic peptide, a term used
to describe a heterogeneous group of lanthionine-
containing bacteriocins that undergo extensive post-
translational modification, and has recently been
shown to exhibit inhibitory activity at nanomolar con-
centrations (Wiedemann et al., 2001).
A number of genes are involved in the production
and export of, and immunity to, nisin (Rodriguez and
Dodd, 1996). These genes are tightly linked in the
nisin cluster, composing a total of 11 genes of which
nisA encodes the nisin precursor itself. Interestingly,
the genes responsible for nisin A production and
immunity are carried on a 70 kb conjugative transpo-
son called Tn5301 from Lc. lactis NCFB894 (Dodd
et al., 1990) or Tn 5276 from Lc. lactis NIZO R5
(Rauch and de Vos, 1992) while the genetic determin-
ants for nisin Z (a natural variant of nisin, in which
the histidine at position 27 is replaced by asparagine)
are on transposon Tn5278 (Immonen et al., 1995).
Nisin synthesis is regulated by a two-component regula-
tory system made up of the membrane-bound histidine
kinase sensor protein, NisK, and the regulator, NisR
(Fig. 3). This regulatory system responds to extracellu-
lar nisin, which leads to the expression of genes
involved in immunity and synthesis/post-translational
modification (Kuipers et al., 1995).
154 Starter Cultures: Genetics
nisK nisR
Regulated gene
Nisin induction
Pi
NisK NisR
Signal transduction
Nisin
Figure 3 Protein expression using the nisin-inducible expression system (NICE).
Another lactococcal bacteriocin which has received
considerable attention in recent years is lacticin 3147,
produced by Lc. lactis strain DPC3147, which was origin-
ally isolated from an Irish kefir grain (Ryan et al., 1996).
The bacteriocin is composed of two post-translationally
modified peptides, both of which are required for opti-
mal killing activity (Ryan et al., 1999). Lacticin 3147
has a very broad spectrum of action which includes all
Gram-positive bacteria tested, including food pathogens
such as Listeria monocytogenes and Staphylococcus
aureus and food spoilage micro-organisms such as
Clostridium tyrobutyricum (Ryan et al., 1996; Galvin et al.,
1999). The native lactococcal plasmid, pMRC01,
encoding lacticin 3147 production and immunity has
been sequenced completely (Dougherty et al., 1998).
Ten genes are involved in lacticin 3147 production and
immunity and are expressed from divergent promoters
which control two clusters, namely ltnA1A2M1TM2D
and ltnRIFE. The putative functions of some of these
genes have been confirmed using a series of knock-out
deletions in single or multiple genes. Investigation of
the regulation of the lacticin 3147 gene revealed that
the promoter controlling biosynthesis (Pbac) appears to
be constitutive (McAuliffe et al., 2001). Characterisa-
tion of a downstream region revealed a stem-loop
structure within the ltnM1 gene which may act as a
rho-independent terminator, functioning as a signal for
processing of the ltnA1A2M1TM2D transcript. Further-
more, the promoter of the ltnRIFE operon (Pimm) was
shown to be regulated by the repressor LtnR.
A very useful general feature of the bacteriocins of
starter cultures is that they are frequently encoded on
mobilisable genetic elements. This has been advanta-
geous for the transfer of nisin to different starters.
Protein X
expression
NisR
gene X
However, a general feature of Nis strains is that they
are not rapid acidifiers of milk, a quality necessary for
successful cheese manufacture. The genetic determin-
ants for lactacin 3147 can also be readily transferred
between strains. In this case, the 60 kb self-transmissible
pMRC01 plasmid-encoding lacticin 3147 has been
transferred to more than 25 different lactococcal hosts,
many of which are commonly used lactococcal starters
in the cheese industry (Coakley et al., 1997; O’Sullivan
et al., 1998; Fenelon et al., 1999). The transconjugants
are lacticin 3147 producers and can be substituted for
the parent strains in commercial applications.
The genetics of bacteriocins, such as lacticin 3147
and nisin, has also become important tools for the
manipulation of starter bacteria. The best example of
this is the generation of the nisin-controlled expres-
sion (NICE) system (de Ruyter et al., 1996). It is based
on the promoter for the structural gene for the antimicro-
bial peptide, nisin (Fig. 3). The nisA promoter is
autoregulated in response to nisin through the nisPR
gene products. The system is very sensitive to nisin
concentration and by varying the nisin level in the
medium, very subtle control of genes cloned down-
stream of the nisA promoter can be achieved.
Bacteriophage
The interaction of starters and bacteriophage is cov-
ered elsewhere (see ‘Starter Cultures: Bacteriophage’,
Volume 1) but one particular aspect relates specifically
to the genetics of starter cultures, namely the effects of
prophage and other phage sequences in the bacterial
chromosome. Phage-related starter failure remains a
problem in the dairy industry, and recent studies indi-
cate that the chromosomal phage sequences contribute

to the evolution of new phage (Bouchard and
Moineau, 2000; Durmaz and Klaenhammer, 2000).
The rapid appearance of new recombinant phage is a
constant problem in combating phage infections. The
new phage can bypass the natural phage defence
mechanisms employed to protect the starter cultures.
Durmaz and Klaenhammer (2000) demonstrated that
the appearance of recombinant phages is dependent on
chromosomally encoded phage sequences. Therefore,
sequences encoded by the lactococcal chromosome
may have an injurious effect on the strain in the
cheesemaking environment. However, from an evolu-
tionary point, chromosomal phage DNA must confer
some advantage or the lysogenic cells would not be
maintained in the population. An obvious advantage is
the exclusion from super-infection. Another is the
potential of the mobile phage to exchange beneficial
genes between strains. It is tempting to assume that
lactococcal strains without prophage would be more
suitable for dairy fermentation processes but this may
not be the case.
Genetic manipulation
The genetic tools now available allow very sophisti-
cated manipulation of Lactococcus strains. Reliable
gene inactivation and expression systems are available.
The tools have evolved over three decades of research
and reflect the investment in Lactococcus genetics. One
of the key factors in the development of the genetic
tools was the discovery of efficient transformation proto-
cols for lactococci (Holo and Nes, 1995). The potential
of electroporation to introduce DNA into eukaryotic
cells was quickly adopted for bacteria. High-voltage
electric field pulses result in the permeabilisation of
the cell membrane allowing the transient passage of
macromolecules into the cell. Other methods for mobil-
ising DNA, such as conjugation and transduction,
were crucial in the early development of lactococcal
genetics (reviewed by Gasson and Fitzgerald, 1994)
but the relative simplicity and reproducibility of elec-
troporation has made it the mechanism of choice for
genetic studies. In the case of the indigenous large
plasmids, conjugation remains the most efficient
mechanism of transfer. The procedure requires that
the naturally occurring plasmid possesses a suitable
selectable marker for the transconjugant. Plasmid-
linked phage resistance has been used in numerous
conjugations since the first successful report of
improved resistance to a homologous phage as the
selection basis (Klaenhammer and Sanozky, 1985). The
presence of lactose-fermenting determinants offers an
alternative naturally occurring selectable marker. How-
ever, interference with the natural lactose-fermenting
Starter Cultures: Genetics 155
capability of the starter in order to select for the intro-
duced plasmid has diminished the usefulness of this
approach for strain improvement. As noted above with
lacticin 3147, bacteriocin production and immunity
have proven to be other suitable targets for selection in
natural conjugations.
In order to genetically investigate any organism, the
tools for cloning and manipulating the genes of the
organism must be available. The plasmid vectors cur-
rently used to clone lactococcal DNA have evolved from
shuttle vectors incorporating non-lactococcal origins to
plasmids derived from indigenous lactococcal plasmids.
These plasmids have been adapted further as integra-
tion vectors encoding expression systems. The isolation
of a temperature-sensitive (Ts) pWV01 origin of replica-
tion was instrumental in the development of an efficient
gene inactivation system for lactococci (Law et al.,
1995). An elegant two-plasmid system was developed
whereby a fragment of the gene of interest is cloned on
a vector encoding the wild-type pWV01 origin of repli-
cation, an antibiotic selection marker but no replication
protein. The replication protein is supplied by an addi-
tion plasmid encoding the Ts version of the pWV01 ori-
gin. Once the strain is shifted to a non-permissive
temperature, the plasmid encoding the replication pro-
tein is lost. The plasmid encoding the homologous
DNA is forced to integrate when antibiotic selective
pressure is maintained. The system has been employed
successfully to generate not only chromosomal muta-
tions but has recently been adapted to study genes
encoded by the native plasmids (Cotter et al., 2003).
With the availability of sophisticated genetic tools,
complex metabolic engineering of Lactococcus is feasible
and attractive due to the largely independent catabolic
and anabolic pathways in Lactococcus (Hols et al.,
1999). A number of innovative studies have demon-
strated the potential to manipulate the metabolic path-
ways of Lactococcus and exploit these bacteria as cell
factories (Hugenholtz and Smid, 2002). However, with
respect to lactococcal starter cultures, their direct
incorporation into food products means that the
recombinant DNA technologies required for metabolic
engineering cannot be used in strain development.
New food-grade technologies are being developed but
for the foreseeable future traditional mutagenesis and
natural selection of mutants with high throughput
screening facilities hold more potential for the devel-
opment of improved starter bacteria.
Leuconostoc
Leuconostocs are heterofermentative LAB that func-
tion as starters for fermented dairy products only in
association with lactococci. Their major role is to
156 Starter Cultures: Genetics
metabolise citrate to CO2 (eye formation) and diacetyl,
an important flavour component of cultured butter-
milk, cottage cheese, sour cream and ripened cream
butter. As yet, the genetics of Leuconostoc is at an early
stage and the importance of citrate metabolism has
provided the focus for most of the genetic studies of
Leuconostoc metabolism. Like their mesophilic part-
ners, the lactococci, the technologically important
genes encoding citrate and lactose utilisation are plas-
mid-encoded. The gene encoding the citrate permease,
citP, shares almost complete identity with the gene
from Lactococcus (Vaughan et al., 1995). This level of
homology suggests that the gene may have been
acquired by recent horizontal transfer between the two
LAB species. The regulation of the citrate transport
genes encoded by the citMCDEFGRP multienzymatic
complex has also been investigated in L. paramesen-
teroides (Martin et al., 2000). The results demonstrated
that the operon was induced by citrate independently
of the pH of the growth medium and that a divergently
transcribed gene, citI, upstream of the operon is
involved in regulation.
Genetics of Thermophilic Starters
The cultures that are regarded as thermophilic starters
consist of bacteria with an optimum growth tempera-
ture of ⬃45 °C. The ability to tolerate higher tempera-
tures is related to their use in the manufacture of Swiss
and Italian cheeses, that are cooked to a much higher
temperature (50–55 °C), and yoghurt. There has been
a rapid growth in recent years of genetic information
regarding the bacteria that constitute the thermophilic
starter cultures, namely Streptococcus thermophilus, Lb.
delbrueckii subsp. bulgaricus, Lb. delbrueckii subsp. lac-
tis and Lb. helveticus.
Lactobacillus
Many Lactobacillus species are used in the dairy indus-
try and some have been subject to detailed investiga-
tion. In fact, the quantity of genetic information
regarding the various lactobacilli is superseded only by
Lactococcus. In addition, the probiotic potential of
many Lactobacillus species has accelerated the accu-
mulation of genetic data. However, most of the lacto-
bacilli are used as adjuncts and for the purposes of this
review only the Lb. delbrueckii subspecies and Lb. hel-
veticus will be discussed in detail.
Chromosome
The genomes of many lactobacilli are currently being
sequenced including that of Lb. delbrueckii and Lb.
helveticus strains. The analysis of one Lactobacillus
genome, Lb. plantarum WCSF1, has been completed
and published (Kleerebezem et al., 2003). Lb. plantarum
differs from the thermophilic starters in its flexible and
adaptive behaviour and is encountered in many differ-
ent environmental niches, ranging from some dairy fer-
mentations to the human gastrointestinal tract. The
genes encoding the genetic machinery for growth in
milk, including sugar transporters, EMP and phospho-
ketolase pathways and peptidase were identified although
no proteinase homologue was found. A relatively large
number of pyruvate-dissipating enzymes with a remark-
able degree of redundancy were observed. Lc. lactis
also displays some redundancy especially in its lactate
dehydrogenases but the pyruvate-dissipating potential in
Lb. plantarum is much greater. However, by far the largest
class of proteins in the large (3.3 Mb) genome is repre-
sented by transport proteins, including many PTSs for
sugar uptake. A large number of PTS systems have
already been reported for Lb. casei (Klaenhammer et al.,
2002) and it will be interesting to determine the range
of pyruvate-dissipating enzymes and transport mechan-
isms encoded by the smaller (1.8–2.4 Mb) genomes of
the strains that remain primarily associated with milk
fermentation.
Important traits
The genes for lactose utilisation in Lb. delbrueckii and
Lb. helveticus comprise a lactose antiport permease
(lacS), a regulatory gene (lacR) and a -galactosidase
for hydrolysis of lactose to glucose and galactose. In
Lb. helveticus, the lacLM genes encoding -galactosidase
are divergently transcribed from lacR and lacS, which
are separated by 2 kb of DNA (Fortina et al., 2003).
Transcription studies confirmed the regulatory role of
LacR. In Lb. delbrueckii subsp. lactis and Lb. del-
brueckii subsp. bulgaricus, the -galactosidase (lacZ)
and permease occur in the order lacSZ. Recent investi-
gation of the regulation of the lactose operon in Lb.
delbrueckii has resulted in some interesting observa-
tions (Lapierre et al., 2002). The lacSZ genes in Lb.
delbrueckii subsp. lactis are regulated by lacR, whereas
the L. delbrueckii subsp. bulgaricus, genes were known
to be constitutive and unstable due to the presence of
insertion elements. Comparison of the lactose
metabolism systems of both species revealed that the
presence of the insertion elements alone was not suffi-
cient to deregulate the operon, and mutation in the
lacR gene must have occurred to make expression con-
stitutive in Lb. delbrueckii subsp. bulgaricus.
The proteolytic system of the thermophilic lacto-
bacilli has been investigated extensively. Cell envelope-
associated proteinases have been identified in both Lb.
delbrueckii and Lb. helveticus, and Lb. helveticus CNRZ32
may encode more than one proteinase (Pederson et al.,
1999). In addition, 18 peptidases from thermophilic

lactobacilli have been described (Christensen et al.,
1999). The mechanism of regulation has not been deter-
mined for the peptidase genes with the exception of
pepQ. A homologue of the CcpA (Central regulator of C
metabolism) catabolite regulator has been found
upstream of all LAB pepQ genes investigated and shown
to regulate the expression of pepQ in Lb. delbrueckii
subsp. lactis.
Genetic manipulation
The genetic interrogation of Lb. helveticus strains is far
advanced than Lb. delbrueckii. A major obstacle with
Lb. delbrueckii strains was inadequate transformation
protocols, a problem that has only recently been
resolved (Serror et al., 2002). In contrast, the early reso-
lution of an efficient electrotransformation protocol
for laboratory strains of Lb. helveticus allowed the
development of gene replacement technologies
(Bhowmik et al., 1993). Lactobacillus species harbour
many native plasmids (Wang and Lee, 1997) but the
tools for manipulating the strains are derived from
lactococcal studies. The ability to generate mutants
was instrumental in characterising the proteolytic sys-
tem of Lb. helveticus. In addition, it has facilitated the
first metabolic engineering studies. Inactivation of the
ldhD gene, which encodes D-lactate dehydrogenase
responsible for the production of the D-lactate isomer,
resulted in strains that produced the more desirable
L-lactate isomer only (Kyla-Nikkila et al., 2000). Similar
studies on Lb. delbrueckii await the development of the
necessary genetic tools. Moreover, both species lack
proper gene expression systems.
Streptococcus thermophilus
Streptococcus thermophilus is used in combination with
other starter bacteria for the manufacture of Swiss and
Italian cheese varieties (with Lb. helveticus or Lb. del-
brueckii subsp. lactis) and yoghurt (with Lb. del-
brueckii subsp. bulgaricus). The use of Sc. thermophilus
has increased significantly during the past two decades
because of the increase in the consumption of yoghurt
and Mozzarella cheese. Sc. thermophilus, like other
LAB, is responsible for producing lactic acid but it can
also synthesise exopolysaccharides (EPSs) that typic-
ally impart a desirable ‘ropy’ or viscous texture and
viscosity to fermented milk products. EPS-producing
cultures are particularly important in yoghurt manu-
facture and have recently been shown to improve the
functional properties of low-fat or part-skim Mozarella
cheese (Broadbent et al., 2003).
Chromosome
The sequence of two strains of Sc. thermophilus,
LMG18311 and CNRZ1066, has been completed, while
Starter Cultures: Genetics 157
a third strain, LMD-9, is near completion (Klaenham-
mer et al., 2002). Both completed genomes contain
⬃1.8 mb of sequence encoding about 1800 open read-
ing frames. They are organised as a single circular
chromosome and show 95% identity at the nucleotide
level. Analysis of the sequence also revealed several meta-
bolic features found in common with pathogenic strepto-
cocci even though Sc. thermophilus is characterised as a
GRAS (Generally Regarded As Safe) organism.
Important traits
Genes coding for metabolic pathways involved in lac-
tose metabolism, protein and peptide utilisation, and
polysaccharide production have been sequenced and
characterised. Sc. thermophilus appears to be especially
well-adapted for growth in milk with its preference for
lactose as a sugar source for glycolysis (van den
Bogaard et al., 2000). This contrasts with other LAB
that show a preference for glucose. Lactose metabol-
ism is initiated by the uptake of the sugar across the
cell membrane via a permease belonging to the glyco-
side-pentoside-hexuronide-cation symporter family
(Poolman et al., 1996). The gene for the transporter
(lacS) and -galactosidase (lacZ), required to hydro-
lyse lactose to glucose and galactose, are organised in
an operon with the gene order lacSZ. The glucose is
metabolised to lactic acid whereas in most strains
galactose cannot be metabolised and is expelled into
the external medium (Fig. 2; Gunnewijk and Poolman,
2000). However, Sc. thermophilus does encode the genes
(galKTEM) for galactose metabolism upstream of the
lacSZ. The inability to metabolise the galactose appears
to be related to poor expression of galK (Vaillancourt
et al., 2002). Transcription of the lac and the gal
genes is governed by the sugar present in the medium
and a homologue of the global regulator, CcpA. This
regulator acts to repress the expression of lacSZ, prob-
ably to match an overcapacity for lactose uptake with
rate-limiting glycolytic flux (van den Bogaard et al.,
2000).
The genetic components of the Sc. thermophilus
proteolytic system have not been investigated as
extensively as those of Lactococcus or some Lactobacil-
lus species. As a rule, the thermophilic lactobacilli
have greater proteolytic activity than Sc. thermophilus,
and this is one of the facets of their symbiotic rela-
tionship. Although most Sc. thermophilus strains
either do not express or express very low levels of
CEP activity, a proteinase gene (prtS) has been cloned
and sequenced (Fernandez-Espla et al., 2000). The
product bears similarities to the CEPs from other LAB
being a multi-domain protein belonging to the sub-
tilase family. Sc. thermophilus has also been shown to
contain at least 14 different peptidases, two of which
158 Starter Cultures: Genetics
possess biochemical activities not observed in Lacto-
coccus (Rul and Monnet, 1997). The genes for a
limited number of these peptidases have been cloned
and characterised, including two aminopeptidases
(pepN and pepC), an endopeptdiase (pepO), a X-prolyl-
dipeptidyl (pepX) gene and a unique aminopeptidase
(pepS) (Anastasiou et al., 2002). Analysis of the
genome sequence data should identify the remaining
genes corresponding to the activities detected bio-
chemically for Sc. thermophilus.
The important contribution of EPSs to the texture
and rheological properties of fermented milk products,
especially yoghurt, has prompted genetic characterisa-
tion of the large eps gene clusters. Four distinct clusters
consisting of at least nine genes have been sequenced
to date although the function of the majority of gene
products can only be inferred from sequence or struc-
tural homologies (Broadbent et al., 2003). In general,
the genes in the 5 region of the clusters appear to
encode regulators of EPS synthesis, chain length deter-
mination and membrane translocation. These open
reading frames are followed by genes, most likely
encoding the glycosyl-1-phosphate transferase glyco-
syltransferase for assembly of the polysaccharide
repeating unit and enzymes involved in repeat unit
polymerisation. The remaining genes are probably
required for membrane translocation of the polymer
subunits and production of sugar nucleotide precur-
sors. More definitive data exists for only five of the
thirteen-gene Sc. thermophilus strain Sfi6 eps cluster
(Stingele et al., 1999). The epsE, F, G, H and I genes
were cloned and over-expressed in E. coli and the data
demonstrated that EpsE catalyses the first step in the
biosynthesis of the EPS-repeating unit. It exhibits
phosphogalactosyltransferase activity and transfers
galactose to the lipophilic carrier. The second step is
performed by EpsG, which transfers an -N-acetyl-
galactosamine to the first -galactoside. The activity of
EpsF was investigated by characterising the EPS pro-
duced by an Sc. thermophilus epsF deletion mutant,
which suggested that epsF codes for the branching
galactosyltransferase. The epsI gene probably codes for
the -1,3-glucosyltransferase, since it is the only glyco-
syltransferase for which no gene has been assigned and
it exhibits similarity to other -glycosyltransferases.
These studies improve our understanding of EPS
biosynthesis and will be important for potentially novel
applications likely to emerge inside and outside the
dairy industry for polysaccharides and EPS cultures.
Genetic manipulation
Sc. thermophilus has very few native plasmids but this
has not greatly hampered the development of genetic
tools for the manipulation of these bacteria. Transform-
ation protocols have been established, and tools
employed in studying Lactococcus have been readily
adapted to Sc. thermophilus. For example, the two-
plasmid integration system described above, based on
a temperature sensitive pWV01 origin of replication,
has been successfully adapted for Sc. thermophilus
(Labarre et al., 2001). There is a requirement for a
controlled expression system analogous to the NICE
system of Lactococcus but the genomics approach is
likely to provide the basis for improved molecular
tools for the genetic investigation of Sc. thermophilus.
Genomics of Starter Bacteria
The genetics of starter bacteria and indeed biology as a
discipline are undergoing a revolution. Developments
in high throughput sequencing technologies have
facilitated the progression to genome-scale sequencing
projects. According to the Genomes Online Database
(http://wit.integratedgenomics.com/GOLD/), there are
717 prokaryotic and eukaryotic genome-sequencing
projects ongoing or completed at the time of writing
( June 2003). These include three Lc. lactis strains, ten
different Lactobacillus species and strains, four Sc.
thermophilus strains, two Oenococcus oeni strains and
one strain each of Leuconostoc mesenteroides and Pedio-
coccus pentosaceus. The availability of this information
will radically alter our understanding of starter bac-
teria. In addition to delineating the genetic comple-
ment of each of the species, comparative genomics will
allow the identification of the unique genetic traits
encoded by each of the bacterial strains. Moreover, the
genomic data opens the door for microarray and pro-
teomic technologies. Microarrays, or DNA chips, are
essentially glass slides with a representative sample of
every gene in a genome spotted onto the surface. They
can be used to detect which genes in the genome are
expressed under a specific set of conditions. Proteomics
refers to new, rapid protein identification systems
coupled with improved separation techniques that can
individually identify and quantify the proteins present
in a cell. Both microarray and proteomic technologies
rely on sequence data to produce genome-wide tran-
scription and protein expression profiles. They provide
real-time data on RNA expression, protein expression
and protein interactions. The genomics approach ultim-
ately provides a comprehensive global prospective on
the bacterium, its metabolism and response to the envir-
onment. High throughput screening technologies based
on these techniques probably represent the future of
starter genetics research. These technologies have the
potential to produce cultures that can be used readily
in industrial fermentations and avoid the need for
recombinant DNA technologies which are unlikely to

become acceptable for food production processes in
the near future.
Conclusion
As we enter the post-genomics age of starter research,
it is important to appreciate the landmark discoveries
that have enabled our detailed understanding of these
industrially relevant bacteria. These have included the
elucidation of important traits such as lactose utilisa-
tion and casein breakdown and also the development
of sophisticated genetic tools. From the genetic point
of view, given the level of complex manipulation
routinely used with Lc. lactis, it can be considered the
E. coli of the LAB, i.e., the genetic workhorse. How-
ever, tools are rapidly being developed or adapted and
applied to the other LAB. This, allied to the exponen-
tial increase in sequence data among the group, will
undoubtedly lead to the development of more reliable
and valuable strains. These enhanced strains will
likely include those engineered to produce nutritional
compounds such as folate for improved human health.
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This Page Intentionally Left Blank
 Starter Cultures: Bacteriophage
S. McGrath, National Food Biotechnology Centre, Department of Microbiology,
University College, Cork, Ireland
G.F. Fitzgerald, National Food Biotechnology Centre, Departments of Microbiology
and Food and Nutritional Sciences, University College, Cork, Ireland
D. van Sinderen, Department of Microbiology, University College, Cork, Ireland
Bacteriophage
Bacteriophages (or phage) are viruses that attack bac-
teria. Unlike prokaryotic and eukaryotic cells, viruses
are acellular and are composed of a nucleic acid core
surrounded by a proteinaceous coat, and in some cases,
a lipid-containing envelope. Viruses are obligate para-
sites and outside the host cells they are essentially non-
living organic molecules, whereas within host cells,
they exhibit various functions that are characteristic of
living systems.
Viruses are not included in the recognised kingdoms
of living organisms and they have been divided into
three large groups depending on the host utilised –
animal viruses (viruses that replicate in animals), plant
viruses (viruses that replicate within plants) and bac-
teriophage (viruses that replicate within bacterial
cells). Twort (1915) and d’Hérelle (1917) were the first
to recognise viruses that infect bacteria, and d’Herelle
coined the term ‘bacteriophages’, literally meaning
‘eaters of bacteria’.
Bacteriophage Multiplication
Depending on the type of life cycle employed, bac-
teriophage may be differentiated into two groups: lytic
or temperate. Infection of a bacterial cell by a lytic (or
virulent) bacteriophage ultimately leads to the death
and lysis of that cell. In addition to eliciting a lytic
cycle, temperate (or lysogenic) bacteriophages are also
capable of entering into a life cycle that does not result
in the death of the host cell. This non-lethal life cycle
is accomplished through the ability of the bacterio-
phage to integrate its genome in a stable manner into
that of the host cell chromosome. This integrated
prophage DNA is faithfully replicated in situ by the
host cell’s DNA replication apparatus during chromo-
somal replication, and all progeny cells will therefore
receive a copy of it. Integrated prophages may, in
response to specific stressful environmental conditions,
excise from the host genome and enter into the lytic
Cheese: Chemistry, Physics and Microbiology, Third edition – Volume 1: General Aspects
ISBN: 0-1226-3652-X
Set ISBN: 0-1226-3651-1
cycle (Fig. 1). The specific stages of the phage life
cycle will be discussed later.
Bacteriophage of Lactic Acid Bacteria
Members of the lactic acid bacteria (LAB) such as Lacto-
coccus lactis, Streptococcus thermophilus, and Lactobacil-
lus, Leuconostoc and Pediococcus spp. are commonly
used on an industrial scale in the dairy industry for the
production of fermented milk products such as cheese,
buttermilk and yoghurt.
Bacteriophages are associated with most bacterial
species and are therefore ubiquitous in environments
where their bacterial hosts are encountered. Bacterio-
phages infecting Lactococcus were first identified by
Whitehead and Cox (1935) and have since been
recognised as the major cause of disruption in dairy
fermentations. In the modern dairy industry, the dis-
ruption of lactic acid fermentations by bacteriophages
can lead to serious economic losses. Although tech-
nological advances in fermentation processes in con-
junction with stringent sanitisation regimes may have
reduced the incidence of bacteriophage infection, it
has certainly not eliminated it.
The persistent problem of bacteriophage infection
has focused research on developing phage-resistant
starter strains. These studies have involved analysis of
phage–host interactions and the characterisation of the
genetic processes essential for the phage life cycle.
Research initially concentrated on phages that infect
lactococcal spp., but more recently interest has
expanded to other LAB phages, such as those that infect
Lactobacillus spp. and Sc. thermophilus. The advent of
molecular biology research tools, such as automated
DNA sequencers and bioinformatics, has enabled the
complete sequence determination of a still growing
number of LAB bacteriophage genomes. This research
has led not only to the improvement of the bacterial
strains used in the dairy industry and the development
of phage-resistance systems, but at a more fundamental
level, has provided a detailed understanding of the
Copyright © 2004 Elsevier Ltd
All rights reserved
164 Starter Cultures: Bacteriophage
A1 A2 A3
C1 C2 C3
Figure 1 Morphological classification of bacteriophages, based on the classification schemes of Ackermann and DuBow (1987).
A, Myoviridae; B, Siphoviridae; C, Podoviridae; 1, small isometric head; 2, prolate head; 3, elongated head.
phage life cycle and an insight into the evolution of
these organisms.
Control of Bacteriophage in the Dairy Plant
Bacteriophage infection remains the largest cause of fer-
mentation disruption in the dairy industry. The continual
use of a single starter strain (or the same multiple-strain
culture) can allow phage numbers in a cheese factory to
rise to detrimental levels. This can result in the reduction
in starter viability with a subsequent reduction in the
overall rate of acid production, longer manufacturing
times and poor flavour-development. The commercial
consequences of phage infection include disruption of
production schedules, reduction in product quality (and
reduction in commercial value) and, in the most severe
cases, abandonment of production.
Since lactococcal phages were first identified, a
number of strategies for reducing their impact in dairy
fermentations have been developed. The modern dairy
plant is specifically designed to reduce the incidence
of phage infection. The area for starter culture prepar-
ation is generally physically separated from the pro-
duction area, with restricted access in order to avoid
cross-contamination by personnel. The maintenance
of a slight positive pressure in the starter room also
reduces the risk of contamination with phage from the
factory environment. Further measures include heating
of the bulk starter medium (90 °C for at least 20 min)
and the filtration of cooling air using high efficiency
particulate air (HEPA) filters. Closed fermentation vats
have been introduced and equipment coming into con-
tact with the milk is sanitised either by steaming or by
B1 B2 B3
cold disinfection with chlorine and peracetic acid (Cogan
and Hill, 1994; Limsowtin et al., 1996; Stanley, 1998).
Traditionally, bulk starter preparation involved sev-
eral scale-up steps from the mother culture through
intermediate cultures to the final bulk starter. This
process can be time-consuming and may offer the prob-
lematic phage a chance to multiply. The finding that
the majority of lactococcal phages have an absolute
dependence on calcium ions for successful infection
(Reiter, 1956) has facilitated the development of
phage-inhibitory media, incorporating Ca2 chelating
agents, such as phosphate or citrate. Various other
steps may be taken to minimise the risk of phage
infection and have been reviewed elsewhere (Cogan
and Hill, 1994; Limsowtin et al., 1996; Stanley, 1998).
The process of bulk starter preparation may be cir-
cumvented through the use of commercially available
frozen or freeze-dried concentrated cultures. Various
types are available and may be used either to inocul-
ate the bulk starter or the milk in the cheese vat
directly (direct-set) (Limsowtin et al., 1996).
Starter cultures used in the production of Cheddar
cheese may be divided into two main groups: mixed-
strain cultures and defined-strain cultures. Mixed-strain
cultures contain not only different species of bacteria
but also different strains of the same species. Approxi-
mately 90% of the bacteria in the culture contribute to
acid production whereas the other 10% are involved in
the production of flavour compounds (Cogan and Hill,
1994). The so-called P cultures (P for practice) that are
widely used in the Netherlands are an example of the
use of mixed-strain starters in the modern dairy
industry. These cultures, which are permanently

contaminated with non-disturbing phage, are used con-
tinuously without the need for rotation with phage-
unrelated cultures (Neve, 1996).
The use of defined-strain starter cultures with differ-
ent phage–host spectra within a carefully designed rota-
tion scheme has proved to be an effective means of
controlling phage proliferation. Defined-strain starters
(DSS) are usually blends of two or more phage-unrelated
strains, which may be used on a rotating basis for
cheesemaking (Cogan and Hill, 1994; Neve, 1996). This
minimises their exposure to environmental phage in the
plant and consequently curtails the accumulation of a
specific problematic phage. Heap and Lawrence (1976)
developed an elegant protocol for the identification of
phage-resistant starter strains. This method involves the
sequential culturing of strains in the presence of a cock-
tail of bacteriophage under conditions similar to those
used for cheese manufacture. Cultures identified in this
manner have been used extensively in the production of
Cheddar cheese.
The discovery that lactococci contain plasmids
(Cords et al., 1974) enabled researchers to begin unrav-
elling the genetic basis for phage resistance in these bac-
teria, in earnest. For the dairy industry, a significant
outcome of this research has been the development of
new and improved phage-resistant strains with desir-
able fermentative traits. This research has been the
focus of several reviews (Klaenhammer and Fitzgerald,
1994; Dinsmore and Klaenhammer, 1995; Garvey et al.,
1995a; Allison and Klaenhammer, 1998; Forde and
Fitzgerald, 1999; Coffey and Ross, 2002). The continu-
ous development of novel products coupled with
increasing production in the dairy industry will
undoubtedly pose new challenges for plant engineers,
food technologists and microbiologists in the preven-
tion of phage infection.
Classification of LAB Bacteriophage
A great deal of research on LAB bacteriophage has
focussed on the development of a coherent classifica-
tion scheme that would accurately reflect the evolution-
ary relationships between phages. These classification
schemes originally relied on the morphological and
serological properties of phage, phage–host interactions
and protein content. In more recent times, DNA:DNA
hybridisation and DNA sequence information have
been used.
Morphology
This is the classical method for viral classification
and relies on direct microscopic examination to charac-
terise the physical shape of the virus particle (Fig. 1).
Starter Cultures: Bacteriophage 165
The original classification was developed by Bradley
(1967) and current classification is based on the work
of Ackermann and DuBow (1987). Three distinct mor-
photypes are defined – Myoviridae (Bradley group A)
which exhibit contractile tails, Siphoviridae (B) which
have long non-contractile tails and Podoviridae (C)
which have short non-contractile tails (Ackermann and
DuBow, 1987). Almost all phages that infect LAB iden-
tified to date belong to the Siphoviridae family, although
some lactococcal phage belong to the Podoviridae
family (Jarvis et al., 1991). Members of the Siphoviridae
have been further divided into three subgroups on the
basis of head morphology (Bradley, 1967; Ackermann
and DuBow, 1987). Group B1 members have a small
isometric head, group B2 members have a prolate head,
and group B3 have an elongated head. Podoviridae
bacteriophages are similarly subdivided into three
groups (C1, C2 and C3) on the basis of head mor-
phology (Fig. 1).
The vast majority of phages that infect lactococcal
and Lactobacillus species belong to the Siphoviridae
morphotype B1 or B2. However, some exceptions
have been noted, i.e., a number of Podoviridae phages
that infect Lactococcus have been identified (Saxelin
et al., 1979, 1986; Braun et al., 1989). Sechaud et al.
(1992) identified and classified a number of morpho-
logically distinct phages that infect Lb. helveticus,
which have an isometric head and a contractile tail of
variable length. All Sc. thermophilus phages reported
to date are of the B1 morphotype of the Siphoviridae
family.
Host range
Classification schemes for LAB phage based on host
range are particularly relevant to the dairy fermenta-
tion industry. However, these schemes rarely agree
with those based on other criteria. For example, in
one study it was found that Sc. thermophilus phage
with similar host ranges exhibited limited DNA
homology, whereas phage with similar DNA restric-
tion profiles exhibited a completely different lytic
spectrum (Mata and Ritzenthaler, 1988). Similar obser-
vations were made for phages of Lactococcus (Relano
et al., 1987). These observations indicate that what-
ever the advantages these classification schemes may
have from an applied viewpoint, they are of little
taxonomic value.
Serology
Several attempts have been made to classify Sc.
thermophilus and lactococcal bacteriophage on the
basis of their serological properties (Kivi et al., 1987;
166 Starter Cultures: Bacteriophage
Mata and Ritzenthaler, 1988; Jarvis, 1989; Brüssow
et al., 1994a). However, a serious drawback of this
method is that it is based on differences in antigenic
properties exposed on the external structure of the
virion and is therefore indicative of the expression of
only a small part of the genome.
Structural protein profiles
Protein profile analysis has been commonly used to clas-
sify Sc. thermophilus bacteriophage and has been found
to generally agree with other schemes. To date, three dif-
ferent types of protein profiles have been described for
phage that infects Sc. thermophilus. Kivi et al. (1987)
described phage containing four major proteins, while
several reports have described Sc. thermophilus phage
with either two or three major structural proteins (Neve
et al., 1989; Prévots et al., 1989; Benbadis et al., 1990;
Fayard et al., 1993; Le Marrec et al., 1997; Stanley et al.,
1997). There is a correlation between the method of
DNA packaging and the number and type of structural
proteins, for both Sc. thermophilus and Lb. delbrueckii
phages (Forsman and Alatossava, 1991; Le Marrec et al.,
1997). Lactococcal phages are generally found to have
between one and three major structural proteins
together with a varying number of minor structural pro-
teins (Arendt et al., 1994; Johnsen et al., 1996; Van Sin-
deren et al., 1996). Neve (1996) reported that lactococcal
phages of different morphotypes tend to have different
protein profiles.
DNA homology
Classification of LAB phage based on DNA homology
will evaluate the entire phage genome as opposed to a
specific portion, which may encode, for example, the
structural genes. On the basis of DNA:DNA hybridisa-
tion studies, twelve genetically distinct lactococcal
Table 1 Lactococcal phage species, type phages and members (adapted from Jarvis et al., 1991)
Family Morphotype species Phage type
Siphoviridae B1 936
B1 P335
B1 P107
B1 1483
B1 P087
B1 1358
B1 BK5-T
B1 949
B2 c2
Podoviridae C2 P034
C3 KSY1
a It has been proposed that BK5-T should be assigned to the P335 species (Labrie and Moineau, 2002).
phage species have been defined (Jarvis et al., 1991)
(Table 1).
Phages that infect Sc. thermophilus appear to be
more closely related to one another than those that
infect Lc. lactis and they probably belong to a single
homology group (Neve et al., 1989; Benbadis et al.,
1990; Fayard et al., 1993; Brüssow et al., 1994a,b; Le
Marrec et al., 1997). However, the degree of homol-
ogy does vary and a number of subgroups have been
proposed (Neve et al., 1989; Prévots et al., 1989; Ben-
badis et al., 1990; Fayard et al., 1993). Four different
homology groups have been defined for phage of Lb.
delbruekii, with the majority of phages belonging to
one specific group, designated ‘A’ (Mata et al., 1986;
Lahbib-Mansais et al., 1988; Sechaud et al., 1988;
Forsman and Alatossava, 1991; Forsman, 1993). How-
ever, Lahbib-Mansais et al. (1988) have also described
a second homology group, distinct from the Lb. del-
brueckii phage group. This is comprised of five mem-
bers, all of which infect Lb. delbrueckii.
LAB Bacteriophage Epidemiology
Of the twelve lactococcal phage species described by
Jarvis et al. (1991), phage of three species, c2, 936 and
P335, represent the majority of industrial isolates. Most
of LAB phages are classified as Siphoviridae, with a non-
contractile tail and a small isometric head, such as
members of the 936 and P335 species (morphotype
B1), whereas c2 type phages have a non-contractile tail
with a prolate head and are classified as B2 morpho-
types (Ackermann and DuBow, 1987). In a survey of
Canadian dairy plants, Moineau et al. (1992) found that
members of the c2 species were isolated with the high-
est frequency whereas, in a later study conducted in
the United States, 80% of the phages identified were
Phage Members
P008 P008, F4-1, sk1, bIL41, bIL66, US3
P335 P335, LC3, r1t, Tuc2009, TP901-1,
31, 50, Q30, Q33, ul36
P107 P107
1483 1483
P087 P087
1358 1358
BK5-T BK5-Ta
949 949
c6A c2, bIL67, vML3, 197, P001
P034 P034
KSY1 KSY1
 Starter Cultures: Bacteriophage 167

classified as representatives of the 936 species (Moineau
et al., 1996). Similarly, Jarvis et al. (1991) reported that
the majority of phages identified in New Zealand, the
United States and Ireland were of the 936 species. In the
last 10 years or so, P335 type phage has been encoun-
tered with increasing frequency and it has been pro-
posed that members of this species represent an
emerging dominant phage type in industrial environ-
ments (Alatossava and Klaenhammer, 1991; Moineau
et al., 1992, 1996; Durmaz and Klaenhammer, 2000).
Prolate-headed phage
The two sequenced Lc. lactis prolate-headed phage, bIL67
(Schouler et al., 1994) and c2 (Lubbers et al., 1995), dis-
play a very similar genetic organisation (Fig. 2). The
genomes of both phages are divided into two divergently
oriented clusters consisting of the ‘early’ and ‘late’ tran-
scribed regions. The divergent clusters are separated by
the assumed origin of replication and both package their
DNA utilising a cos site. Each has a relatively small
genome, with the complete sequence of bIL67 being
22 195 bp, compared to 22 163 bp for c2.
c2 and bIL67 share about 80% of the overall
nucleotide sequence identity (Lubbers et al., 1995).
However, this is not evenly distributed along the entire
genome, with some regions sharing more than 90%
identity and others less than 40% (Lubbers et al., 1995).
The early region of c2 encompasses approximately 7 kb
of DNA, which harbours 22 putative ORFs. Similarity-
derived functions have been assigned to some of these
and they include a DNA polymerase, a recombination
protein, a sigma factor and a transcriptional regulator
( Jarvis et al., 1995; Lubbers et al., 1995). The late
region spans around 16 kb, and 17 ORFs have been
identified in this section of the genome. N-terminal
sequence analysis identified three major and eight

(A)
c2 (c2)

Early Late

cos ori cos

Replication Lysis Morphogenesis Packaging Morphogenesis Lysis

(B)
936 (sk1)

Early Middle Late

ori cos

Replication Packaging Morphogenesis Lysis

(C)
P335 (TP901-1)

Lysogenic Lytic early Lytic middle Lytic late

attP genetic switch pac/cos

Integration Replication Packaging Morphogenesis Lysis

Figure 2 Schematic representation of the genomic arrangements of the three main lactococcal phage groups: (A) c2, prolate
headed; (B) 936, small isometric headed; (c) P335, small isometric headed. Blocks represent genomic regions containing genes
involved in the bacteriophage life cycle.
168 Starter Cultures: Bacteriophage

minor structural proteins (many of which appear to be
post-translationally processed) (Lubbers et al., 1995). In
addition, two putative holins and a lysin were identified
( Jarvis et al., 1995; Lubbers et al., 1995).
Similarly, the early region of bIL67 harbours
21 ORFs spanning approximately 7 kb while the late
region consists of about 15 kb of DNA, containing 16
putative ORFs. A holin, a terminase subunit, a minor
tail subunit, lysin, DNA polymerase and a protein
involved in recombination were tentatively identified
on the genome (Schouler et al., 1994).
Small isometric-headed phage
All remaining LAB phage for which the entire genome
sequence has been determined have a small iso-
metric head. They include six Sc. thermophilus, three
Lactobacillus and thirteen lactococcal phages (Table 2).
Of the thirteen completely sequenced small isometric-
headed lactococcal phages, bIL170 and sk1 are mem-
bers of the 936 spp. Six of the remaining eleven
phages in this group were identified as prophages on
the Lc. lactis IL1403 genome, and bioinformatic analy-
sis revealed that three belong to the P335 group of
temperate phage, whereas the remaining three are
most probably satellites relying on helper phage(s) for
multiplication (Chopin et al., 2001). The remaining
five members are P335-type phages (Table 2).
Three consecutive phases of transcription (early, mid-
dle and late) were apparent for the 936-type phage, sk1
(Chandry et al., 1997) (Fig. 2). The remaining sequenced
small isometric LAB phages, include lactococcal P335
spp., and Sc. thermophilus and Lactobacillus phages, all of
which share a very similar genetic organisation. The
genomes of the lysogenic phages are arranged in two
divergent clusters separated on one side by the attach-
ment site and on the other by an intergenic region
involved in the genetic switch (Fig. 2). The genome of
the lytic Sc. thermophilus, Lactobacillus and lactococcal
P335 phage is, for the most part, transcribed in one

Table 2 List of bacteriophages infecting LAB for which the genome has been completely sequenced

Small isometric/ Number of

Cos/pac prolate headed, putative
Phage site Size/bp lytic/temperature ORFs Reference

Lactococcus
bIL67 cos 22 195 Prolate, lytic 37 Schouler et al. (1994)
c2 cos 22 163 Prolate, lytic 39 Lubbers et al. (1995)
sk1 cos 28 451 S.Ia, lytic (936) 54 Chandry et al. (1997)
biL170 cos 31 754 S.I, lytic (936) 64 Crutz-Le Coq et al. (2002)
r1-t cos 33 350 S.I, temperate (P335) 50 Van Sinderen et al. (1996)
Tuc2009 pac 38 347 S.I, temperate (P335) 56 Proux et al. (2002)
ul36 pac 36 798 S.I, temperate (P335) 59 Labrie and Moineau (2002)
BK5-T cos 40 003 S.I, temperate (BK5-T) 63 Mahanivong et al. (2001)
TP901-1 pac 36 667 S.I, temperate (P335) 56 Brondsted et al. (2001)
bIL285b 35 538 temperate (P335) 62 Chopin et al. (2001)
bIL286b 41 834 temperate (P335) 61 Chopin et al. (2001)
bIL309b 36 949 temperate (P335) 56 Chopin et al. (2001)
bIL310b 14 957 temperate 28 Chopin et al. (2001)
bIL311b 14 510 temperate 21 Chopin et al. (2001)
bIL312b 15 179 temperate 27 Chopin et al. (2001)
Lactobacillus
LL-H (Lb. delbrueckii ) pac 34 657 S.I, lytic, 52 Mikkonen et al. (1996)
g1e (Lb. plantarum) pac 42 259 S.I, temperate 62 Kodaira et al. (1997)
adh (Lb. gasseri ) cos 43 785 S.I, temperate 62 Altermann et al. (1999)
Sc. thermophilus
O1205 pac 43 075 S.I, temperate 57 Stanley et al. (1997)
7201 cos 35 466 S.I, lytic 44 Proux et al. (2002)
DT1 cos 34 820 S.I, lytic 46 Tremblay and Moineau (1999)
Sfi19 cos 37 392 S.I, lytic 44 Desiere et al. (1998)
Sfi21 cos 40 739 S.I, temperate 53 Desiere et al. (1998)
Sfi11 pac 39 807 S.I, lytic 53 Lucchini et al. (1998)

a S.I, small isometric.

b Prophage identified on the chromosome of Lc. lactis IL1403.

direction. However, transcription of these lytic genes
may be controlled in a temporal manner. For example,
transcriptional analysis of the P335-type phage, TP901-1,
revealed that genes involved in the lytic cycle are tran-
scribed in three distinct temporal phases, early, middle
and late, with maximal transcript concentrations pres-
ent after 10, 30 and 40 min post-infection, respectively
(Fig. 2).
936 phage
The first complete 936-type phage genome sequence
available was that of sk1 (Chandry et al., 1997). Sev-
eral of its 54 putative ORFs show sequence similarity to
both the small isometric- and prolate-headed morpho-
types. From a transcriptional point of view, the genome
is organised into three segments, represented by the early
(30 ORFs), the middle (4 ORFs) and the late (20 ORFs)
transcribed regions. These regions are separated from
each other by intergenic regions containing the cos site
(middle and late), a transcription terminator (late and
early) and divergent promoters (early and middle).
The early region consists of ORFs thought to specify
replication functions (the DNA polymerase subunits).
The middle region is composed of four small ORFs
just upstream of the cos site. No function has been
assigned to these ORFs. The late region of the genome
putatively encodes various structural proteins, pro-
teins involved in the DNA packaging and the lysis
functions of the phage (Chandry et al., 1997).
The complete genome sequence of bIL170 has
been published recently (Crutz-Le Coq et al., 2002).
Sixty-four ORFs were identified and the function of
16 of them was assigned by significant homology to
proteins in databases. Comparison of the bIL170
genome to that of sk1 showed that insertion/deletion
events involving one or two ORFs were the main
source of divergence in the early gene clusters. It was
proposed that such events resulted in the replacement
of a direct repeat-containing genomic fragment in
bIL170, resulting in the acquisition of a new func-
tional origin of replication by this phage (Crutz-Le Coq
et al., 2002).
Partial sequence information is available for other
936-type phages, including F4-1 (Chung et al., 1991;
Kim and Batt, 1991a), bIL41 (Parreira et al., 1996) and
bIL66 (Bidnenko et al., 1995). Sequence comparisons
indicate that these 936-type phages are very closely
related. For example, over 2 kb of the genome of sk1
(including most of the middle region) shows 84.9%
identity to the expressed middle region of bIL66
(Chandry et al., 1997). Also, a 10.2-kb segment from
the late region of bIL41 displays between 69 and 98%
identity to an homologous segment from sk1 (Parreira
et al., 1996; Chandry et al., 1997).
Starter Cultures: Bacteriophage 169
P335 phage
Members of the P335 phage species are small, isomet-
ric-headed with a genome ranging from 30 to 42 kb.
Bacteriophages included in the P335 species are het-
erogeneous, with phages utilising both cos and pac
sites for DNA packaging, and this is the only lactococ-
cal phage species that includes both virulent and tem-
perate members. The first complete P335 phage
genome sequence published was that of r1t (Van
Sinderen et al., 1996). This is a temperate phage and
its genome is arranged in two divergent clusters of 3
and 47 ORFs. This appears to be a life-cycle-specific
orientation, i.e., the orientation of the ORFs believed
to be involved in the lysogenic life cycle is opposite to
the remaining ORFs, which are associated with the
lytic life cycle. It appears, therefore, that the r1t
genome is arranged such that all of the ORFs involved
in the lytic life cycle are grouped in one large contigu-
ous gene cluster. Such a lytic cluster appears to be
composed of a number of functional modules which
are transcribed and organised in a way that reflects the
chronological order of the life cycle itself, i.e., those
ORFs required for replication are transcribed first and
are located at the proximal end of the gene cluster, fol-
lowed by modules involved in DNA packaging, mor-
phogenesis and, finally, cell lysis (Fig. 3). All other
temperate P335-type phages, the genome of which
have been sequenced, appear to exhibit this type of
gene organisation. The most recent complete P335
phage genome available, that of ul36, also represents
the first virulent P335 member to be completely
sequenced (Labrie and Moineau, 2002). Interestingly,
the genome of this lytic phage appears to be arranged
in two divergent clusters of 6 and 53 ORFs. The for-
mer represents a cryptic lysogeny module containing
genes, the deduced protein products of which display
similarities to phage integrases, repressors and a Cro
protein. An incomplete lysogeny module was identi-
fied in another lytic P335 phage, 31 (Madsen et al.,
2001). These observations indicate that some virulent
P335-type phages are derived from the temperate
phage.
Sc. thermophilus phage
The five completely sequenced Sc. thermophilus phages
appear to have a genetic arrangement very similar to
that of the P335-type species of lactococcal phage, i.e.,
they have a modular arrangement made up of replica-
tion, packaging, morphogenesis and cell lysis compon-
ents. It has been speculated that O1205 is closely
related to the P335 group of lactococcal phage (Stanley
et al., 1997). Sequence comparisons of Sc. thermophilus
phage revealed a substantial amount of homology
between them (Bruttin et al., 1997; Desiere et al., 1998,
170 Starter Cultures: Bacteriophage

Phage Phage DNA replication DNA packaging Cell lysis and

attachment to DNA and synthesis of and morphogenesis release of progeny
host cell injection phage proteins phage

Early Late
Adsorption DNA R/M Abortive
interference injection systems infection
blocking

Insertional Triggered
mutagenesis Recombinant Antisense Per
superinfection
exclusion

Figure 3 Schematic representation of LAB bacteriophage lytic life cycle showing main steps in phage development. (A) Stages at
which naturally occurring phage resistance mechanisms arrest phage development; (B) Stages at which engineered phage resist-
ance mechanisms arrest phage development.

1999, 2002; Lucchini et al., 1998, 1999a; Neve et al.,
1998; Proux et al., 2002).
Lactobacillus phage
The genomes of the Lactobacillus phages, gle (Kodaira
et al., 1997), adh (Altermann et al., 1999) and LL-H
(Mikkonen et al., 1996) have been completely sequenced
and display distinct similarities to the genomes of the
P335 group of lactococcal bacteriophage, as well as
those that infect Sc. thermophilus. The putative ORFs on
the genome of these phages are clustered into a number
of functional modules. Interestingly, although LL-H is a
lytic phage, remnants of an integrase and an attachment
site (similar to that of mv4 (Auvray et al., 1997)) can be
discerned, indicative of a direct evolutionary relation-
ship between these two Lb. delbrueckii phages (Mikko-
nen et al., 1996). Furthermore, the genes encoding
some of the structural proteins of these two phages
were shown to be highly conserved (Vasala et al., 1993).
Genome Organisation and Evolution of
LAB Bacteriophage
All genomes of phages that infect LAB analysed to date
consist of a double-stranded, linear DNA molecule
with a G  C content consistent with that of the host
chromosome (37% for lactococcal phages to ⬃48% for
phages of Lb. casei) (Forde and Fitzgerald, 1999). The
majority of chromosomes analysed are 18–40 kb in
length, although larger sizes, up to 134 kb, have been
reported (Prévots et al., 1990; Moineau et al., 1992).
Two distinct genome types have been identified, based
on the means by which the phage packages its DNA.
Some phage genomes have cohesive ends consisting of
single-stranded 3 overhangs of variable length, while
others are said to be ‘circularly permuted’ and have
‘terminal redundancy’ (Black, 1989).
Botstein (1980) has put forward ‘a theory of modular
evolution for bacteriophage’ in which he proposed that
the product of evolution is not a given virus but a fam-
ily of interchangeable genetic elements (modules), each
of which carries out a particular biological function.
Furthermore, he proposed that evolution does not act
primarily at the level of an intact virus, but at the level
of individual functional units (modules). This theory is
supported by analysis of available bacteriophage DNA
sequences, where the genomes of phage are found to be
organised in a life-cycle-specific manner, with modules
containing genes coding for integration/excision, repli-
cation, structural proteins, assembly, DNA packaging
and host cell lysis (Fig. 2). Homologous functions may
be fulfilled by a number of distinct DNA segments that
lack any sequence similarity. Particular modules may be
exchanged through recombination among phages belong-
ing to an interbreeding phage population. Hendrix et al.
(1999) have further developed this theory and proposed

a model for the genetic structure dynamics of the global
phage population in which all double-stranded (ds)
DNA phage genomes are mosaics with access, by hori-
zontal exchange, to a large common genetic pool but in
which access to the gene pool is not uniform for all
phages. Brüssow and Desiere (2001) have discussed the
evolution of phages of the Siphoviridae family using
LAB phages as a model, and have proposed the estab-
lishment of a  super-group of Siphoviridae based on
structural gene synteny. They also discuss the role of
both vertical and horizontal evolutions in relation to
these phages. The evolution of new lytic LAB bacterio-
phage due to the acquisition of genes and/or entire
DNA modules, both of which are most probably derived
from prophage sequences located on host cell chromo-
somes, has been reported in phages that infect Lactococcus
(O’Sullivan et al., 1993; Moineau et al., 1994; Bouchard
and Moineau, 2000; Durmaz and Klaenhammer, 2000).
This finding is increasingly common, is likely to be a
response to the selective pressure applied by phage
resistance systems, and demonstrates the evolutionary
flexibility of phage.
Life Cycle of LAB Bacteriophage
The individual steps that make up the bacteriophage
life cycle will be discussed, with specific reference to
phages of LAB (Fig. 4).
Lytic life cycle
Phage adsorption/ DNA injection
The exact molecular mechanisms by which LAB phage
attach to cells and inject their DNA are still not under-
stood completely. However, studies undertaken so far
indicate that the processes involved are consistent with
those of the better-characterised Gram-negative phage,
such as the T-even phage (Dreiseikelmann, 1994).
Lactococcal phages appear to attach to the host cell
at specific receptor sites which may or may not be
evenly distributed over the cell surface (Budde-Niekiel
and Teuber, 1987). The majority of phages attach in a
tail-first orientation to a carbohydrate moiety of the
cell wall. The sugars, galactose and/or rhamnose, have
been implicated in most cases (Keogh and Pettinghill,
1983; Valyasevi et al., 1990; Monteville et al., 1994);
however, more complex polysaccharide components
and cell membrane lipoproteins have also been indi-
cated (Oram, 1971; Schafer et al., 1991). This initial
phage ‘docking’ is usually reversible and phages can
detach following addition of sugars such as those men-
tioned above.
This initial reversible phase of phage adsorption is
followed by an irreversible phase. A chromosomally
Starter Cultures: Bacteriophage 171
encoded membrane-associated protein, called Phage
Infection Protein (PIP), from Lc. lactis subsp. lactis c2
has been identified as being necessary for the adsorp-
tion and subsequent DNA injection of a number of
phages that infect this strain (Valyasevi et al., 1991,
1994; Geller et al., 1993; Monteville et al., 1994).
Further studies demonstrated that although the PIP
protein is essential for infection by a number of prolate-
headed phages, PIP-mutants were still susceptible to
infection by phages of the 936 and P335 species (Kraus
and Geller, 1998). Genes homologous to pip have been
identified in all strains of Lc. lactis tested (Garbutt et al.,
1997). Analysis of the PIP protein reveals that it
possesses a putative N-terminal signal peptide and six
putative transmembrane-spanning domains (Geller et al.,
1993). Other studies have indicated that another 32-kDa
membrane-associated protein is also necessary for
phage infection of Lc. lactis subsp. lactis c2 (Valyasevi
et al., 1991). Lucchini et al. (2000) have recently identi-
fied a chromosomally encoded protein analogous to
PIP in Sc. thermophilus. Insertional inactivation of this
gene conferred a phage resistance phenotype on
Sc. thermophilus Sfi11 against all fifteen phages used in
the study (Lucchini et al., 2000).
Lysogenic/lytic switch
Regulatory regions involved in the control of the
lysis–lysogeny decision of temperate lactococcal bac-
teriophage (Boyce et al., 1995; Nauta et al., 1997; Madsen
et al., 1999), temperate Lactobacillus phage (Kodaira
et al., 1997; Ladero et al., 1998, 1999) and temperate Sc.
thermophilus phage (Stanley et al., 1997; Neve et al.,
1998; Lucchini et al., 1999b) have been described. It
has become apparent that the general mechanism for
life cycle decision is similar to that of bacteriophage ,
where CI- and Cro-like repressors play opposing roles
in a genetic switch mechanism (Ptashne, 1986). CI pre-
vents transcription of the lytic genes (and positively
regulates its own expression) by binding to specific
DNA sequences (operators) located within the immun-
ity region, whilst Cro prevents transcription of the
genes involved in the establishment of lysogenic growth
by binding the same operator sites with different affin-
ities (Ptashne, 1986).
 Cro is a small protein consisting of 66 amino acids
within which a helix-turn-helix motif can be discerned.
Cro-like proteins in LAB phages do not exhibit a great
deal of similarity, but many have been putatively identi-
fied based on their relative genome position and the
criteria outlined above (Lucchini et al., 1999b).
DNA replication
For initiation of DNA replication to occur, a specific
starting point of replication must be identified where
172 Starter Cultures: Bacteriophage

Bacterial cell

Host chromosome

Phage
particle Attachment to host cell and
injection of DNA

DNA replication
Integration of DNA into the and synthesis of
host chromosome phage proteins

Induction DNA packaging and

event phage morphogenesis
Cell division

Cell lysis and

release of
progeny phage

Integrated phage DNA replicates along

with the host chromosome

Lysogenic life cycle Lytic life cycle

Figure 4 Lytic and lysogenic life cycles of bacteriophage.

opening of the double-stranded DNA double helix
takes place prior to the recruitment of the replication
machinery. The DNA region representing the initiation
point for (phage) DNA replication, also referred to as
the origin or replication (ori) is characterised by the
presence of two or more direct repeats, which facilitate
binding of a sequence-specific duplex DNA-binding
protein. Such a nucleoprotein-binding complex gener-
ally consists of 150–250 bp of DNA and multiple
copies of a replication-specific DNA-binding protein.

Formation of the nucleoprotein complex invokes
denaturation of an A  T-rich region of DNA directly
adjacent to the ori, the single-stranded status of which
is further promoted by single-stranded binding protein.
By so marking the origin and providing a single-stranded
DNA region, the replication fork proteins can be
recruited to the correct initiation point, and nascent
strand synthesis can ensue. Examples of this type of
DNA replication initiation include the well-characterised
chromosomal replication systems employed by  phage
and E. coli (Marians, 1992).
Analysis of replication modules of LAB phage has
so far been largely at the level of database searches,
with putative functions being assigned to individual
genes on the basis of similarities to genes of known
function. However, the study of phage oris and the
exploitation of the interactions between phage/host-
encoded replication proteins and their cognate phage
oris has been quite rewarding.
The first LAB phage ori to be described was that of
the lactococcal phage, 50, followed by that of
another lactococcal phage, 31 (Hill et al., 1990a;
O’Sullivan et al., 1993). In both cases, it was shown
that the copy number of an ori-containing plasmid
drastically increases following infection by a phage
utilising the same ori for replication. Furthermore,
these plasmids also conferred a phage-resistance phe-
notype on the lactococcal host. This phenotype was
termed per for phage-encoded resistance. These observa-
tions led to the conclusion that the phage ori sequences
on the plasmid vectors were titrating essential repli-
cation functions away from the phage DNA which
were in turn driving plasmid amplification. A putative
origin of replication for the lactococcal phage,
Tuc2009, designated ori2009, has also been identified
(McGrath et al., 1999). The ori2009 sequence is located
within the gene coding for the putative replisome
organiser protein (rep2009), and its encoded protein
specifically interacts with the ori2009 DNA (McGrath
et al., 1999). In a further study, genes highly homo-
logous to rep2009, that contained sequences identical
to ori2009, were identified in two other lactococcal
phages, Q30 and Q33 (McGrath et al., 2001), while a
third rep2009 homologue was identified in another
closely related phage, ul36 (Bouchard and Moineau,
2000). The ability of plasmids harbouring ori
sequences to confer a phage-resistance phenotype
was also used to identify oris in the Sc. thermophilus
phages Sfi21, 1205 and 7201, and the Lb. casei phage
A2 (Foley et al., 1998; Moscoso and Suarez, 2000;
Stanley et al., 2000). Interestingly, it was found that
7201 appears to contain two oris, each of which is
capable of independently mediating a per phenotype
(Stanley et al., 2000). Phages infecting Sc. thermo-
Starter Cultures: Bacteriophage 173
philus have been classified into two groups on the
basis of their replication module. Members of repli-
cation group I employ an ori similar to that of 1205,
while members of replication group II use an ori
similar to one or both oris of 7201 (Stanley et al.,
2000).
Another method used to identify phage oris is based
on their ability to act as bona fide origins of replication
for plasmids. A 611-bp intergenic region located between
the early and the late gene regions of the lactococcal
phage c2 was identified as an ori in this manner
(Waterfield et al., 1996). The presence of this DNA frag-
ment was sufficient to drive plasmid replication in Lac-
tococcus strains but not in E. coli. The absence of any
ORFs within the 611-bp fragment suggests that replica-
tion of this plasmid requires only host-encoded factors.
The c2 ori contains an A/T-rich region (78% A/T),
which has several small perfect and imperfect inverted
and direct repeats, a phenomenon characteristic of ori-
gins of replication. Highly similar sequences were also
identified in two other lactococcal phages, bIL67 and
197 (Schouler et al., 1994).
Phage replication module genes likely to code for
topoisomerases, single-stranded DNA-binding proteins,
replisome organisers, DNA helicase/primases and heli-
case loader proteins have been putatively identified on
the basis of similarity to sequences in the databases.
Replication functions for phages infecting Lactococcus,
Streptococcus and Lactobacillus have been identified in
this manner (Forde and Fitzgerald, 1999).
DNA packaging
For many phages, intracellular DNA replication results
in the formation of large concatameric DNA molecules
consisting of several phage genome complements (Black,
1989). Bacteriophages may employ one of the two mech-
anisms of packaging their genome into the (pro)-capsid
prior to assembly and release of mature phage particles
from the cell. Phages whose genome contains a pac site
employ a so-called headful mechanism of DNA packag-
ing. Here, the phage DNA is initially cut at the pac site,
with each subsequent cut occurring when the prohead
has been filled with DNA. This mechanism results in
phage containing DNA molecules that are circularly per-
muted and terminally redundant, i.e., coding for more
than one unit length of genome (Streisinger et al., 1967;
Tye et al., 1974). Alternatively, phage genomes may
contain a cos site. Cutting of the concatameric DNA
molecules at these specific cos sites results in single
genomic units with 3 overhangs on the DNA which are
self-complementary (cohesive ends) (Murialdo, 1991). It
has been demonstrated that the actual DNA transloca-
tion into the prohead requires the action of several
proteins – the terminase complex, portal protein and the
174 Starter Cultures: Bacteriophage
expanded major head protein (Black, 1989). Packaging
is initiated when the terminase binds to the specific pac
or cos site. The terminase is composed of two subunits.
The small subunit which binds to, and hydrolyses ATP,
is also thought to interact with the phage DNA, while
the large subunit appears to bind to the prohead, and
may be involved in cutting the concatameric DNA mol-
ecules. One of the structural elements of the phage, the
portal protein, plays a role in forming the entrance to the
phage head. The portal protein has also been implicated
in conjunction with the terminase in initiating DNA
packaging, DNA translocation and in determining the
amount of DNA to be packaged in phages utilising the
headful mechanism. The major capsid protein also
hydrolyses ATP during the translocation of DNA (Black,
1989).
DNA sequence analysis of the regions surround-
ing cos sites has revealed the presence of several con-
served regions, which have been determined as being
essential for binding of phage terminases (Chandry et al.,
1994; Herrero et al., 1994; Nakashima et al., 1994;
Schouler et al., 1994; García et al., 1997). It has also
been reported that cos regions have a high G  C con-
tent which is thought to be necessary for stable base-
pairing of the cos region once the phage genome
has entered the cell. The majority of lactococcal
phage genomes analysed to date possess cos sites
(Klaenhammer and Fitzgerald, 1994); however, pac sites
have been identified in the lactococcal phage, Tuc2009
and TP901-1 (Arendt et al., 1994; Christiansen et al.,
1994) and the Lactobacillus phages LL-H and Mv4
(Vasala et al., 1993). Le Marrec et al. (1997) have clas-
sified a number of Sc. thermophilus phages into two
groups, depending on the DNA packaging mechanism
employed. Using Southern hybridisation, they demon-
strated that all pac-containing phages tested contained
homologs of the genes encoding the three major struc-
tural proteins of the pac-containing phage O1205,
whereas all cos-containing phages tested exhibited
homology to the gene specifying one of the structural
components of the cos-containing phage 7201.
Structural proteins
Structural protein synthesis begins immediately follow-
ing phage DNA replication. One of the most comprehen-
sive studies of the structural proteins of an LAB phage is
that of c2 (Lubbers et al., 1995). Three major struc-
tural proteins of 175, 90 and 29 kDa and eight minor
proteins of 143, 82, 66, 60, 44, 42, 32 and 28 kDa were
identified by SDS polyacrylamide gel electrophoresis
(PAGE). The genes coding for these proteins were also
identified. Several of the proteins were thought to have
undergone post-translational modification by proteolytic
cleavage. It was determined that 175, 143, 90, 82 and
66 kDa proteins had the same N-terminal amino acid
sequence, which matched the gene product specified by
the l5 gene. Similarly, two structural proteins of 29 and
28 kDa, although containing different N-terminal amino
acid sequences, were shown to be encoded by the l7
gene. Using immunogold electron microscopy, it was
shown that the structural proteins of 175 and 90 kDa
represented major head proteins, while the 29- and
60-kDa proteins were the building blocks of the major tail
and tail adsorption structures, respectively. Furthermore,
the products of the head protein gene, l5, were suggested
to be involved in forming covalently linked multimers,
including trimers, hexamers and small amounts of pen-
tamers. This type of multimerisation has been proposed
to be involved in the formation of the -icosohedral
phage head.
The techniques mentioned above, i.e., SDS-PAGE,
N-terminal amino acid sequencing, immunological
analysis, as well as homology searches of sequence data-
bases, have been used to identify structural proteins of
many other LAB phages (Hill, 1993; Klaenhammer and
Fitzgerald, 1994; Garvey et al., 1995b; Davidson et al.,
1996; Forde and Fitzgerald, 1999).
Bacteriophage lysis
Lysis of the host cell by infecting bacteriophages
results in the release of progeny phage and requires a
cell wall-degrading enzyme (lysin). Three classes of
lysin have been described to date, and they are differ-
entiated on the basis of the peptidoglycan bond hydro-
lysed (Rodgers et al., 1980). However, only lysins of
the first two classes described have been identified for
phages that infect LAB. The first class, termed glyco-
sidases, hydrolyses the glycosidic linkage between the
amino sugars of the peptidoglycan and includes endo
N-acetylglucosaminidases (or glucosaminidases) and
endo N-acetylmuramidases (muramidases or lysozymes).
The second class, N-acetyl muramoyl-L-alanine ami-
dases (amidase), hydrolyses the N-acetylmuramoyl-
L-amide linkage between the glycan strand and the
cross-linking peptide. The third class, endopepti-
dases, break the peptide chain of the peptidoglycan.
It has been proposed that lysin proteins consist of
two separate modules, with the N-terminus deter-
mining the lytic activity and the C-terminal domain
specifying cell wall-binding (García et al., 1990). In
support of this theory, a chimeric lysin protein
has been constructed by fusing the N-terminal half of
the lactococcal phage Tuc2009 lysin to the C-terminal
domain of the major pneumococcal autolysin
(Sheehan et al., 1996). This novel enzyme exhibited
a glycosidase activity capable of hydrolysing choline-
containing pneumococcal cell walls. It is noteworthy
that some lysin-encoding genes employ atypical
 Starter Cultures: Bacteriophage 175

start codons, and it has been suggested that these act
as a control mechanism to prevent premature lysis of
the host (Shearman et al., 1994).
The second part of the LAB lysis cassette is the
holin. Holins are small membrane-associated proteins,
which cause non-specific lesions in the cytoplasmic
membrane, thus allowing the lysin access to the cell
wall (Young and Bläsi, 1995). Holins have several well-
defined characteristics, although they exhibit little
similarity in either amino acid or DNA sequences.
They generally contain a hydrophilic and charge-rich
C-terminus, 2–3 hydrophobic, possible membrane-
spanning regions separated by a -turn linker region
and a hydrophilic N-terminus (Young and Bläsi, 1995).
It has been suggested that the expression of active
holin is controlled at the level of transcription, with a
methionine dual start motif identified in many holin-
encoding genes. This facilitates the synthesis of two
gene products of slightly different size, one of which
acts as an inhibitor of the other (Bläsi and Young,
1996). Three distinct holin groups have been
described (Young and Bläsi, 1995). Type I holins are at
least 87 amino acids in length and contain three pos-
sible membrane-spanning regions. Type II holins are
less than 78 residues in length and contain two pos-
sible membrane-spanning regions. Finally, a unique
holin has been identified for the E. coli phage, T4,
which was assigned to a separate group, III.
Several genes encoding phage lysins and their
deduced protein products for LAB phage have been
characterised at the molecular and biochemical levels.
These include the lysins of the lactococcal phages
P001, us3, c2, vML3, LC3, Tuc2009 and r1t, as well as
that of the Lactobacillus phage LL-H (Table 3). Sheehan
et al. (1999) have described a lysis module contained
on the genome of the temperate Sc. thermophilus phage
O1205, which contains two putative holin genes and
one lysin. Southern blot analysis revealed that at least
one or more of these genes were present in 30 other
Sc. thermophilus phages examined.
Lysogenic life cycle
The phenomenon of lysogeny was first reported in
Lactococcus by Reiter (1949). Lysogeny is widespread
in LAB, particularly in Lactococcus (Huggins and
Sandine, 1977; Jarvis, 1989; Davidson et al., 1990) and
Lactobacillus (Sechaud et al., 1988), but much less so
in Sc. thermophilus (Fayard et al., 1993; Brüssow et al.,
1994b, 1998; Le Marrec et al., 1997). It appears that the
mechanisms involved in the maintenance of lysogeny in
LAB are similar to that of phage  (see above).

Table 3 Identified genes encoding restriction/modification systems in Lc. lactis and Sc. thermophilus

R/M system or
identified subunits Type Location Reference

Lc. lactis
Lla1403I I pIL2614 Schouler et al. (1998a)
Lld I I pND861 Deng et al. (2000)
Unnamed (HsdR, HsdM, HsdS) I pAH82 O’Sullivan et al. (2000)
O’Sullivan et al. (2001)
L0308 (HsdR) I Chromosome of Lc. lactis IL1403 Bolotin et al. (2001)
L0309 (HsdM)
L0310 (HsdS)
Unnamed (HsdS) I pCIS3 Seegers et al. (2000)
Unnamed (HsdS) I pIL7 Schouler et al. (1998b)
Unnamed (HsdS) I pIL103 Schouler et al. (1998b)
Unnamed (HsdR, HsdM, HsdS) I Chromosome of Lc. lactis IL1403 Schouler et al. (1998b)
LlaI II pTR2030 Hill et al. (1989)
LlaDII II pHW393 Madsen and Josephsen (1998a)
LlaCI II pAW153 Madsen and Josephsen (1998b)
LlaBI II pJW563 Nyengaard et al. (1996)
LlaKR21 II pKR223 Twomey et al. (1998)
ScrFI II Chromosome of Lc. lactis UC503 Twomey et al. (1997)
LlaDCHI II Chromosome of Lc. lactis DCH-4 Moineau et al. (1995)
LlaFI III pND801 Su et al. (1999)
Sc. thermophilus
Unnamed (HsdS) I pCI65st O’Sullivan et al. (1999)
Unnamed (HsdR, HsdM, HsdS) I pER35 Solow and Somkuti (2001)
Unnamed (HsdS) I pER16 Solow and Somkuti (2001)
Unnamed (HsdR, HsdM, HsdS) I Chromosome Lucchini et al. (2000)
Sth368I II Chromosome Burrus et al. (2001)
176 Starter Cultures: Bacteriophage
Site-specific recombination
The integration of a prophage genome into the host
chromosome is a site-specific integration event between
the phage attachment site (attP) and the bacterial attach-
ment site (attB), which is facilitated by a phage-encoded
integrase. The first such site-specific integration system
described for LAB was that of the lactococcal phage, LC3
(Lillehaug and Birkeland, 1993), and since then, near-
identical systems have been described for three other
lactococcal phage, BK5-T, Tuc2009 and r1t, all of which
possess integrases belonging to the type I-Int family of
site-specific recombinases (Van de Guchte et al., 1994b;
Boyce et al., 1995; van Sinderen et al., 1996). The
lactococcal phage, TP901-1, has been shown to utilise
an integrative system that is significantly different from
that of other temperate LAB phage (Christiansen et al.,
1994). In this system, the integrase is replaced with a
larger resolvase-like protein, whilst the attP and attB
sequences are different from those used by other phages
(Christiansen et al., 1996).
Maintenance of lysogeny
Maintenance of the integrated prophage in the host
chromosome requires the repression of transcription of
the genes of the lytic life cycle. In , this repression is
facilitated through the use of a repressor protein
(Ptashne, 1986). Two classes of LAB phage repressors
have been identified. Class I repressors consist of
polypeptides containing 200–300 amino acids, within
which two distinct functional domains can be discerned.
The N-terminal domain contains a helix-turn-helix
motif, which is assumed to be involved in the binding
of the repressor to specific recognition sites on the
phage genome. The C-terminal domain is thought to be
involved in oligomerisation (co-operative binding of
repressor proteins) and has a conserved Ala/Gly motif
required for RecA-mediated cleavage of the hinge
region between the N- and the C-terminal domains
(Little, 1993).
A second class of LAB phage repressors has also
been identified. This group consists of proteins that are
considerably smaller than their Class I counterparts. A
helix-turn-helix motif has been identified in most
cases, and the absence of the consensus RecA-mediated
cleavage site. However, it is possible to induce phages
containing class II repressors into the lytic cycle fol-
lowing the SOS response (following treatment with
mitomycin C or UV treatment) indicating recA-mediated
cleavage of these repressors also (Madsen et al., 1999).
The majority of class I phage-repressors belong to lacto-
coccal phage (Van de Guchte et al., 1994a; Boyce et al.,
1995; Nauta et al., 1996), but these have also been
found in phages that infect Lactobacillus (García et al.,
1999); Class II repressors appear to be more common
in Sc. thermophilus phage (Stanley et al., 1997; Neve
et al., 1998), although they have been identified in
phages that infect Lactobacillus (Kakikawa et al., 2000)
and Lactococcus (Madsen and Hammer, 1998).
Superinfection exclusion
Expression of the repressor protein from an integrated
prophage may also prevent the propagation of super-
infecting phage and is said to confer ‘immunity’ on the
lysogenised bacterial host. Temperate bacteriophage
may also express so-called ‘superinfection exclusion’
activities. The latter differ from phage immunity/
repression systems in that they do not play a role in
maintaining the lysogenic state and are not specific
for homoimmune phage. Superinfection exclusion sys-
tems are well-characterised in temperate phages that
infect Gram-negative bacteria such as E. coli and
S. typhimurium (Susskind and Botstein, 1978; David
et al., 1982; Matz et al., 1982; Yu and Snyder, 1994) and
had, until recently, not been identified in LAB phage.
Bruttin et al. (1997) characterised the lysogeny
module of the Sc. thermophilus phage Sfi21. ORF203
is positioned between the genes encoding the integrase
and the repressor, and it was demonstrated that when
ORF203 was supplied on a plasmid vector it confers a
phage-resistant phenotype against 12 Sc. thermophilus
bacteriophages.
A superinfection exclusion protein (Sie2009) for the
temperate lactococcal bacteriophage Tuc2009 has recently
been described (McGrath et al., 2002a). Expression of the
Sie2009 protein from a plasmid vector confers a complete
phage resistance phenotype on Lc. lactis MG1363 against
a number of phages of the 936 species. This phage-
resistant phenotype was shown to be due to an injection-
blocking mechanism, mediated by the Sie2009 protein.
Natural Bacteriophage Resistance Systems
in LAB
Since bacteriophages were first identified as a major
cause of dairy fermentation failure, much research
effort has been directed at the development of phage-
resistance systems for use in the dairy industry. The
majority of this research to date has focussed on lacto-
coccal strains, although recently, efforts have also
been made with Sc. thermophilus (Moineau, 1999;
Coffey and Ross, 2002). Naturally occurring phage-
resistance systems have been identified in wild-type
lactococcal strains. These systems are often encoded on
native conjugative plasmids, which has facilitated the
generation of novel resistant starter strains through
food-grade, gene transfer techniques. These resistance
systems have been divided into four main groups on
the basis of their mode of action: (1) inhibition

of phage adsorption, (2) blockage of phage DNA
injection, (3) restriction/modification and (4) abortive
infection (Fig. 3). These systems have been recently
reviewed (Dinsmore and Klaenhammer, 1995; Garvey
et al., 1995a; Allison and Klaenhammer., 1998; Forde
and Fitzgerald, 1999; Coffey and Ross, 2002) and will
only be briefly discussed here.
Adsorption inhibition
Spontaneous bacteriophage-resistance mutants can be
isolated following infection of a bacterial population
with a specific phage at a high titre. Analysis of these
phage-resistant strains revealed changes in a variety of
host-encoded biochemical traits such as carbohydrate
composition, masking of cell surface characteristics or
assumed changes in specific phage protein receptors that
in many instances rendered phage unable to adsorb to
the cells (Klaenhammer and Fitzgerald, 1994; Daly et al.,
1996). However, these mutants are of only limited value
as starter cultures, since their spectrum of resistance
tends to be narrow, while their growth characteristics
also frequently undergo undesirable alterations.
Native plasmid-encoded adsorption inhibition sys-
tems have been identified in lactococci and it has been
found that these systems can be separated on the basis
of the molecular mechanism employed. These plasmids
generally direct the synthesis of cell surface antigens or
mediate the production of extracellular polysaccharides
which shield the host’s phage receptors against phage
attachment (Valyasevi et al., 1990, 1994; Schafer et al.,
1991; Forde et al., 1999). The genetic basis for adsorp-
tion inhibition remains poorly understood and it has
been proposed that plasmid-mediated adsorption block-
ing may not be a true phage defence mechanism but
rather a secondary effect of some other cellular function
(Forde and Fitzgerald, 1999). Furthermore, because of
the instability of plasmids mediating these adsorption-
inhibition phenotypes, it is possible that mixed cultures
consisting of both phage-sensitive and -resistant cells
can develop which may limit the significance of adsorp-
tion inhibition as a potent defence mechanism.
Injection blocking
Following successful adsorption to the cell wall recep-
tors, an energy-requiring, calcium-dependent, irreversible
interaction between the phage and the cytoplasmic
membrane occurs, allowing DNA translocation into the
cytoplasm (Monteville et al., 1994). As is the case for
adsorption inhibition, relatively little is known about
injection-blocking mechanisms in LAB. Early reports of
such systems in Lc. lactis and Lb. casei did little to elu-
cidate the underlying genetic mechanisms (Marshall
and Berridge, 1976; Watanabe et al., 1984). However,
Starter Cultures: Bacteriophage 177
more recently, researchers are beginning to unravel
these processes. Garvey et al. (1996) were the first to
report the identification of a plasmid-encoded injec-
tion-blocking mechanism. They demonstrated that the
naturally occurring lactococcal plasmid pNP40 confers
an early-acting resistance mechanism against c2. Fol-
lowing infection with c2, no difference in phage
adsorption was noted; however, 90% of cells harbouring
pNP40 remained viable whereas control strains without
pNP40 exhibited essentially no survival. Furthermore,
this resistance mechanism could be circumvented by
electroporation of phage DNA into resistant host cells,
whereby such transfected cells released progeny
phages. The authors concluded that the resistance was
due to an alteration in a plasma membrane component
or components required for c2 infection, but to date
the gene or genes responsible for this alteration have
not been identified (Garvey et al., 1996).
As mentioned earlier, a phage-encoded DNA injection-
blocking mechanism acting against a number of 936-
type phages has recently been reported in Lc. lactis
(McGrath et al., 2002a). The sie2009 gene (superinfec-
tion exclusion) of the temperate lactococcal phage
Tuc2009 is located on the lysogeny module. Data were
presented showing that the Sie2009 protein was associ-
ated with the cell membrane and its expression left
phage adsorption, transfection and plasmid transfor-
mation unaffected but prevented plasmid transduction
as well as phage DNA replication. The authors also
showed that similar prophage genes are widespread
not only in lactococcal genomes but also in the genomes
of many Gram-positive and -negative bacteria (McGrath
et al., 2002a).
Restriction/modification
Following successful adsorption and DNA injection,
the next obstacle in the LAB phage life cycle is pre-
sented by restriction/modification (R/M) systems. First
identified in Lc. lactis by Collins (1956), they are
found in many bacteria where they act to protect the
cell from invading foreign DNA. A R/M system has to
exhibit two enzymatic activities, i.e., restriction
endonuclease and methylase, and must also be capable
of finding its DNA recognition sequence. The methy-
lase modifies the recognition sites on the host’s DNA,
thus protecting it from restriction by the endonucle-
ase, whereas unmodified recognition sequences on for-
eign or invading DNA molecules are specifically
digested (Wilson and Murray, 1991). The severity of
restriction is dependent on the system and the phage.
In general, the efficiency of plaquing (EOP) of the
phage decreases logarithmically as the number of sites
on the phage DNA molecule increases. To date, four
178 Starter Cultures: Bacteriophage
distinct types of R/M system have been identified
(Wilson and Murray, 1991). The majority of R/M sys-
tems described in LAB are type II. These systems usu-
ally have simple co-factor requirements and molecular
organisations, cleaving at or near the recognition site.
Furthermore, type II R/M systems are generally com-
posed of two structural genes, the endonuclease
(Enase) and the methyltransferase (MTase).
The majority of R/M systems that have been char-
acterised in Lactococcus are plasmid-encoded, with
ScrFI being a notable exception (Twomey et al., 1997).
Another notable exception to the norm is that several
lactococcal type II systems contain two methylase
enzymes (ScrFI, LlaAI and LlaDCHI) (Moineau et al.,
1995; O’Sullivan et al., 1995; Twomey et al., 1997).
Little is known about the role of the dual methylases;
however, it has been shown that ScrFIBM and Scr-
FIAM independently confer protection against ScrFI
restriction (Twomey et al., 1997).
In recent years, several type I R/M systems have been
described in LAB. These systems consist of large multi-
meric proteins consisting of three subunits, HsdR which
is responsible for restriction, HsdM which mediates
host DNA methylation and HsdS which determines tar-
get recognition specificity (Hsd denotes host specificity
determinant; Bickle and Kruger, 1993). These types of
systems have been identified with increasing frequency
in LAB (Table 3) and it has been proposed that more
than 50% of lactococcal R/M systems may belong to this
category (Schouler et al., 1998b). Schouler et al.
(1998a) were the first to describe a type I system in Lac-
tococcus. This system, named Lla14031, was found to be
encoded on a native plasmid harboured by Lc. lactis
Il1403. Furthermore, introduction of plasmids encoding
single HsdS subunits elicited new R/M phenotypes,
indicating that these plasmid-encoded HsdS subunits
are able to interact with the chromosomally encoded
HsdR and HsdM subunits in trans to determine novel
R/M specificities. It was proposed that this ‘combin-
ational variation’ may represent a general strategy in
which lactococci can acquire R/M systems with novel
specificities (Schouler et al., 1998b).
O’Sullivan et al. (1999) identified a Type I HsdS sub-
unit-encoding gene on a native plasmid of Sc. thermo-
philus, NDI-6. It was demonstrated that plasmid-free
derivatives of this strain were sensitive to a bacterio-
phage which displayed no lytic growth on the parent
strain, indicating the functional role of this gene in
phage resistance. Five chromosomally encoded R/M sys-
tems have been identified in Sc. thermophilus, but little
is known about their molecular biology (Moineau,
1999). Moineau et al. (1995) have reported that the
lactococcal LlaDCHI system, when introduced into
Sc. thermophilus, conferred a strong resistance pheno-
type against a number of Sc. thermophilus phages, indi-
cating the possibility of using well-characterised lacto-
coccal systems to protect Sc. thermophilus from phage
attack. Chromosomal- and plasmid-encoded R/M sys-
tems have also been reported in Lactobacillus strains
(Auad et al., 1998; Bourniquel et al., 2002).
It is clear that host-encoded R/M systems are an
essential component of the cell’s defence systems, thus
representing an effective means of protection against
phage attack. However, it is possible for phages to
breech these defences. In a phage infection of a cell
population, a small number of phage genomes may
escape restriction and subsequently be modified by the
MTase. Such modified phage genomes will be able to
propagate to produce a phage population that will be
insensitive to the particular R/M system. Furthermore,
it has been shown that phages that infect various bac-
terial species have evolved a number of strategies such
as elimination of certain restriction sites from their
genomes, modification of bases, production of pro-
teins that inhibit host endonucleases and even the
acquisition of methylase genes (Wilson and Murray,
1991), and it is highly likely that LAB bacteriophages
have evolved similar tactics. Indeed, Hill et al. (1991)
have demonstrated that the lactococcal phage, 50,
acquired a functional methylase gene through an in vivo
genetic exchange between its genome and the phage
resistance-conferring plasmid, pTR2030. This recom-
binogenic event thus rendered 50 insensitive to
pTR2030. Higher levels of phage resistance can be
achieved through the combination of two or more R/M
systems, or by the combination of an R/M system with
other resistance systems such as abortive infection.
Abortive infection
Abortive infection (Abi) is a term used to broadly
describe any phage resistance mechanism which inter-
feres with intracellular phage development after the
phage DNA has entered the cell. Therefore, by definition,
Abi systems can interfere with such processes as genome
replication, transcription/translation, phage DNA packag-
ing and assembly, and cell lysis. Abis are generally char-
acterised by an attenuated infection due to lower numbers
of productive infections and a reduction in the numbers
of phage progeny produced (Allison and Klaenhammer,
1998). Abi-mediated resistance typically culminates in
the death of the infected cell due to corruption of host
functions, as a result of instigation of the defence activity.
Many Abi systems have been identified in Lactococcus
(Table 4), but the molecular mechanisms underlying
many of these systems remain poorly understood. How-
ever, reports of studies using phage mutants capable of
overcoming Abi systems have provided some valuable
 Starter Cultures: Bacteriophage 179

Table 4 Lactococcal abi genes for which DNA sequence data are available

Phage species
Abi Location %GC affected Mechanism Reference

AbiA pTR2030; pCI829 27 936, c2, P335 EarlyP335 Hill et al. (1990b)
AbiB Unspecified; pCI642 27 936 Late936 Cluzel et al. (1991)
AbiC pTN20 27 936, P335 Late936 Durmaz et al. (1992)
AbiD* pBF61 29 936 Late936 McLandsborough et al. (1995)
AbiD1* pIL105 26 936, c2 Latec2 Anba et al. (1995)
AbiE pNP40 29, 29 936 Late936 Garvey et al. (1995b)
AbiF* pNP40, pAJ2074 26 936 Early936 Garvey et al. (1997)
AbiG pCI750 29, 27 936, c2, P335 Late936/c2/EarlyP335 O’ Connor et al. (1996)
AbiH Chromosome 26 936, c2 – Prévots et al. (1996)
AbiI pND852 29 936, c2 Late936 Su et al. (1997)
AbiJ pND859 30 936 – Deng et al. (1997)
AbiK pSRQ800 24 936, c2 Late936/EarlyP335 Emond et al. (1997)
AbiL pND861 28, 29 936, c2 Latec2 Deng et al. (1999)
AbiN Chromosome 31 936, c2 – Prévots et al. (1998)
AbiO pPF144 26 936, c2 – Prévots and Ritzenthaler (1998)
AbiP pIL2614 27.5 936 – Schouler et al. (1998a)
AbiQ pSRQ900 28 936, c2 Late936/c2 Emond et al. (1998)
AbiR pKR223 29.8–31.6 c2 Earlyc2 Twomey et al. (2000)
AbiT pED1 33.3, 33.3 936, P335 Late936/P335 Bouchard et al. (2002)
AbiU pND001 26, 25 936, c2, P335 – Dai et al. (2001)

* AbiD, AbiD1 and AbiF are similar (26–47% identity).

insights into their mode of action (Bidnenko et al., 1995;
Dinsmore and Klaenhammer, 1997). It has been pro-
posed that Abi systems may be categorised depending on
whether they act prior to or at the level of DNA replica-
tion (early) or after replication has occurred (late)
(Garvey et al., 1995a). In lactococci, each Abi system
appears to be unique in terms of regulation, size and
nature of the Abi proteins, number of proteins required
for activity and phage affected. Nevertheless, a number of
interesting similarities have been noted. Garvey et al.
(1995a) reported that all Abi genes tested displayed an
atypical G  C content of 26–29%, compared to 37% for
lactococcal DNA. Furthermore, the proteins encoded by
Abi genes have characteristics of cytoplasmic proteins,
including the lack of an obvious secretion signal and the
presence of hydrophilic, charged residues. To our knowl-
edge, Abi systems in LAB other than lactococci have not
been studied, besides a single report of a possible Abi
mechanism in Sc. thermophilus (Larbi et al., 1992).
Tangney and Fitzgerald (2002) have reported on the
introduction of the lactococcal Abi system, AbiA, into Sc.
thermophilus. Data were presented which showed that
AbiA was effective against six Sc. thermophilus phages at
30 °C and that intracellular phage DNA replication was
affected as for phages infecting Lactococcus. However, at
37 or 42 °C, AbiA failed to have any effect on phage
propagation, indicating that this system is unsuitable
for application in standard fermentations involving
Sc. thermophilus.
Engineered Phage Resistance Systems
The extensive wealth of knowledge that has been accu-
mulated regarding LAB phage biology has enabled
researchers to develop a number of artificial or so-called
‘intelligent’ phage-resistance systems. These utilise spe-
cific genes and/or phage or host DNA sequences which
are introduced into the cell either on a plasmid vector
or by chromosomal integration. The presence of these
heterologous DNA sequences or the expression of spe-
cific genes may interfere with the phage life cycle, thus
providing a level of protection to the host strain (Fig. 3).
This topic has recently been extensively reviewed by
McGrath et al. (2002b) and will be considered only
briefly here.
Phage encoded resistance (Per)
Hill et al. (1990a) noted that supplying a specific 50
genomic DNA fragment on a plasmid vector in trans
conferred a phage-resistance phenotype on the lacto-
coccal host against 50, and that intracellular phage
DNA replication was impeded in strains harbouring
this plasmid. DNA sequence analysis revealed that
this so-called per-conferring DNA fragment contained
a number of direct and inverted repeated sequences,
a characteristic of origins of DNA replication. The
authors proposed that the per50 fragment was in fact
the origin of replication of 50 and that the resist-
ance phenotype conferred was due to the titration of
180 Starter Cultures: Bacteriophage
essential phage DNA replication factors by the plasmid-
borne oris.
The putative ori for the P335-type lactococcal bac-
teriophage, Tuc2009 (designated ori2009), has also
been used in the construction of a per system. The
ori2009 sequence is located within a gene coding for a
putative replisome organiser protein (rep2009), and a
specific protein–DNA interaction between Rep2009
and ori2009 has been demonstrated (McGrath et al.,
1999). In a further study, it was shown that cloning
multiple copies of the ori2009 sequence on a single
plasmid vector increased the level of phage resistance
conferred. Furthermore, ori2009-containing plasmids
were found to be effective against three other P335-type
phages, and DNA sequence analysis confirmed that
these three phages utilised oris identical to that of
Tuc2009. Other per systems have been constructed
for use in lactococci, Sc. thermophilus and Lb. casei
(O’Sullivan et al., 1993; Foley et al., 1998; Moscoso
and Suarez, 2000).
Per systems generally do not confer a complete
resistance phenotype and do not represent an insur-
mountable obstacle to bacteriophage proliferation. The
level of resistance conferred has been found to be
directly dependent on the copy number of the Per-
conferring fragments supplied in trans (O’Sullivan
et al., 1993; McGrath et al., 2001). Furthermore, the
incidence of per-insensitive mutant phage, capable of
replicating in Per host strains has been noted by sev-
eral authors and data have been presented supporting
the hypothesis that these mutant phages have, through
a recombinant process, acquired new DNA from their
host strains (O’Sullivan et al., 1993; Bouchard and
Moineau, 2000; McGrath et al., 2001).
Antisense mRNA
The utilisation of an antisense mRNA strategy
involves cloning of a target gene in the reverse orien-
tation relative to an active promoter. The resulting
antisense mRNA produced is assumed to form stable
hybrids with the target mRNA, thus inhibiting trans-
lation through ineffective ribosome loading, and/or
increased sensitivity to RNA-degrading enzymes (Inouye,
1988).
Recombinant antisense strategies have been used to
successfully control gene expression in animals (Izant
and Weintraub, 1984), plants (Ecker and Davis, 1986)
and bacteria (Coleman et al., 1984). Kim and Batt
(1991b) were the first to describe the use of antisense
technology for the control of bacteriophage prolifer-
ation in LAB. They cloned a gene of unknown func-
tion (gp51C) from 7–9 in the antisense orientation
under the control of a constitutive lactococcal pro-
moter. It was found that this plasmid conferred a
resistance phenotype on the host lactococcal strain
against 7–9 and a number of related phages. Further
studies on the application of this technology in lacto-
cocci targeted other genes of unknown function, a
gene encoding a major coat protein and a transcrip-
tional activator (Chung et al., 1992; Kim et al.,
1992a,b; Walker and Klaenhammer, 2000). However,
these systems were found to confer only a very modest
phage-resistance phenotype.
In an attempt to amplify the amount of anti-
sense mRNA generated within the cell, Walker and
Klaenhammer (2000) developed the so-called ‘explosive
antisense RNA strategy’. In this system, different 31
genes (two middle-expressed and four late-expressed)
were cloned between the strong Lactobacillus P6 pro-
moter and the T7 terminator (TT7) in a low-copy
number plasmid, containing the putative 31 origin of
replication (ori31). Following 31 infection of a cell
harbouring this plasmid, ori31 allows for ‘explosive’
plasmid amplification, thereby increasing the levels of
antisense transcripts late in the lytic cycle. However,
while this strategy significantly increased the concen-
tration of antisense mRNA produced, it had only a
minor impact on bacteriophage proliferation, suggest-
ing that the genes targeted were not essential or suffi-
ciently limiting for the 31 life cycle. In a report by
McGrath et al. (2001), the effectiveness of targeting a
number of different replication module genes was
studied. These included genes with putative functions
such as a topoisomerase, a single-stranded DNA-binding
protein, a replisome organiser protein, a helicase
loader, a type II methyltransferase and a Holiday junc-
tion resolvase. All constructs tested (except the con-
struct directed at the Holiday junction resolvase)
conferred a phage-resistant phenotype on the lactococ-
cal host against Tuc2009. Similar replication module
genes were identified in three other phages, Q30, Q33
and ul36, and it was demonstrated that constructs tar-
geting the putative replisome organiser protein and the
putative helicase loader provided significant protec-
tion against these phages also.
Sturino and Klaenhammer (2002) recently devel-
oped an antisense system for use in Sc. thermophilus.
This system targets a putative helicase gene which is
found on the replication module of many Sfi21-type
phages and was found to be effective against a number
of phages that infect Sc. thermophilus.
Gene replacement/insertional mutagenesis
The role of the chromosomally encoded host gene, pip
(phage infection protein), the expression of which is
required for infection of Lc. lactis subsp. lactis by a

number of phages, has been discussed earlier. A lacto-
coccal strain that is insensitive to attack by c2-type
phage has been engineered by replacing the chromoso-
mal pip gene by an allele that had been mutated in vitro
(Garbutt et al., 1997). This resulted in the production
of a food-grade lactococcal strain that contained no
recombinant DNA sequences. This type of engineered
phage resistance is advantageous because of its stable,
chromosomal location, which obviates the selective
pressure required for many plasmid-borne systems.
Lucchini et al. (2000) have described a chromo-
somal gene (orf394) of Sc. thermophilus Sfi11, the
expression of which is necessary for infection by all
tested phages. A second type of phage-resistant mutant
was also isolated which had the phenotypic character-
istics of an abortive infection system. In this instance,
it was determined that the insertional mutagenesis
event occurred adjacent to a hsdR gene encoding the R
subunit of a type I R/M system. The authors proposed
that this led to the upregulation of the hsdR gene
resulting in a more active phage-resistance phenotype
(Lucchini et al., 2000).
The development of phage-resistant LAB strains by
means of chromosomal engineering may represent one of
the most promising strategies for the generation of stable,
food-grade, strains for industrial use. This approach has
advantages over plasmid-borne systems which can be
intrinsically unstable and/or may represent a high meta-
bolic load to the cell, leading to deletions within the plas-
mid or even plasmid loss during non-selective growth.
Recombinant superinfection exclusion/immunity
systems
The recently described superinfection exclusion gene,
sie2009 (McGrath et al., 2001) has been discussed earl-
ier. When cloned under the control of a constitutive
promoter on a high-copy number plasmid, sie2009
mediates a phage-resistance phenotype in Lc. lactis
against bacteriophages of the 936-type species. Adsorp-
tion and electron microscopic analyses demonstrated
that bacteriophages adsorbed to cells expressing Sie2009
as readily as they did to a control strain, whilst intracel-
lular phage DNA replication was demonstrated not to
occur in Sie2009-expressing strains. Analysis of the
deduced Sie2009 amino acid sequence revealed that the
protein contains a putative transmembrane-spanning
domain while cell fractionation and SDS-PAGE demon-
strated that the Sie2009 protein is in fact associated with
the cell membrane. Furthermore, plasmid transduction
experiments demonstrated that the Sie2009-mediated
phage resistance phenotype is due to an injection-
blocking mechanism (McGrath et al., 2001).
Bruttin et al. (1997) described a gene from the
lysogeny module of the Sc. thermophilus phage Sfi21,
Starter Cultures: Bacteriophage 181
orf203, which when supplied in trans on a multicopy
vector provided resistance to Sc. thermophilus Sfi11
against 12 out of 25 phages tested. The orf203 gene is
located between the integrase and the repressor-
encoding genes on the lysogeny cassette of Sfi21, and
while providing protection against heteroimmune
phage it does not protect against Sfi21. The deduced
protein product of orf203 contains a hydrophobic
N-terminus, indicating that this protein may be asso-
ciated with the host cell membrane; however, the
exact mechanism by which this orf mediates a phage-
resistance phenotype remains to be elucidated.
Cruz Martin et al. (2000) recently described the
construction of a phage-resistant food-grade strain of
Lb. casei. A single copy of the A2 repressor gene
was integrated into the Lb. casei chromosome using a
site-specific integration vector, with subsequent ‘clear-
ing’ of all non-food-grade DNA by in-trans expression
of a -recombinase gene. The resulting strain was
completely immune to A2 infection during milk
fermentation.
It is likely that the naturally occurring phage-resist-
ance phenotypes attributed to some LAB strains are
due, at least in part, to the expression of superinfec-
tion exclusion and immunity factors by (defective)
prophages. The identification and characterisation of
such genes may facilitate the development of new
broad-range phage-resistance systems for many LAB.
Bacteriophage-triggered defence
A genetically engineered form of abortive infection
has been described in Lactococcus (Djordjevic et al.,
1997). This system used a phage-inducible promoter
in combination with the LlaI restriction/modification
system from a lactococcal plasmid. The middle phage-
inducible promoter (31p) was cloned upstream of
the lethal LlaIR restriction cassette so that infection
of a cell harbouring this plasmid with 31, causes the
lethal gene product of LlaIR to be produced, result-
ing in death of the host cell before the infecting phage
has a chance to reproduce itself. However, as was
found for per systems, 31 mutants considerably less-
sensitive to the 31p–LlaIR system were isolated when
phages were propagated on these strains (Djordjevic
and Klaenhammer, 1997). DNA sequence analysis
revealed that a mutation had occurred, resulting in a
single amino acid transversion in a transcriptional
activator of 31p (ORF2). Furthermore, the ability of
these mutant phages to induce the native 31p pro-
moter was demonstrated to be reduced, compared to
that of the parent 31. Pairing the 31p–LlaIR
system with other abortive infection systems, Per31
and AbiA, resulted in a reduction in numbers of 31
182 Starter Cultures: Bacteriophage
below detectable limits (Djordjevic and Klaenhammer,
1997).
The ongoing research into the natural phage defence
mechanisms as well as phages infecting other genera of
LAB will undoubtedly pave the way for the develop-
ment of similar resistance systems.
The development of engineered phage-resistance
systems in LAB has been the focus of intensive
research since the early 1990s. Besides the obvious
benefits, such as the development of phage-resistance
systems with potential industrial applications, it has
also led to an increased understanding of bacterio-
phage–host relationships, and in turn has stimulated
research in other areas. It would appear that the use of
single, strong phage-resistance systems is unsuitable
for industry, due to the selective pressure applied for
the emergence of insensitive phages. More industrially
robust strains could be developed by the stacking of
two or more such systems in a single strain or by the
introduction of engineered-resistance systems into nat-
urally phage-resistant strains.
At present, the use of the majority of these engin-
eered-resistance systems is restricted in the dairy
industry due to their recombinant origins. However,
they will be readily available if and when regulations
allow their applied use. Furthermore, some of the sys-
tems outlined above may be adapted using food-grade
methods for immediate use under current legislative
guidelines in certain jurisdictions.
Current status and future perspectives
The extensive knowledge that has been accumulated
about the physiology and genetics of LAB and their
phages has led to a detailed understanding of many
aspects of the phage–host relationship. In the past
10 years or so, the advent of modern molecular genetic
techniques, such as automated DNA sequencing and
the use of bioinformatics has resulted in a wealth of
biological information pertaining to these organisms
and their infectious parasites. This knowledge has
been utilised not only to generate phage-derived sys-
tems with the potential to prevent phage infections in
dairy fermentations (discussed above), but it was also
applied to develop sophisticated genetic tools (Raya and
Klaenhammer, 1992; Raya et al., 1992; Nauta et al.,
1997; Stoll et al., 2002). Additional biotechnological
applications may be envisaged in the light of the recent
renaissance of phage therapy, in which complete phage
or phage-encoded lytic enzymes may be used to treat
certain bacterial infections (Biswas et al., 2002; Schuch
et al., 2002; Stone, 2002).
It is clear that gene expression from lysogenic
prophages on bacterial chromosomes significantly
contributes to the host cell phenotype, from immun-
ity/exclusion systems and lysogenic conversion to
virulence (Susskind et al., 1971; Waldor, 1998;
Desiere et al., 2002). Indeed, Desiere et al. (2001)
published a report on the genome of a highly patho-
genic strain of Sc. pyogenes which contains eight
prophage elements, two of which harbour genes cod-
ing for likely virulence factors as well as sharing
extensive DNA sequence homology to two LAB
bacteriophages. These findings indicate that the sub-
stantial amount of knowledge amassed on phages
infecting dairy bacteria may be useful in gaining
insights into the molecular mechanisms underlying
virulence in certain infectious bacteria. Conversely,
it is known that commensal and probiotic bacteria
also carry prophages on their genomes, which intro-
duces the intriguing possibility that these prophages
may contain functional genes that confer an advan-
tage on these hosts and possibly play a role in
probiotic functionality.
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This Page Intentionally Left Blank
 Secondary and Adjunct Cultures
J.-F. Chamba, Institut Technique Francais de Fromages, La Roche sur Foron, France
F. Irlinger, INRA, Thiverval-Grignon, France
Introduction
Two types of cultures are used in cheesemaking: pri-
mary and secondary. The primary cultures include all
the starter lactic acid bacteria and are involved in
acid production during cheese manufacture and in
cheese ripening. The secondary and adjunct cultures
involved include yeasts, e.g., Geotrichum candidum,
Debaryomyces hansenii, moulds, e.g., Penicillum camem-
berti, P. roqueforti, and bacteria, e.g., Corynebacterium,
Staphylococcus, Micrococcus, Propionibacterium sp. and
heterofermentative lactobacilli and are involved only
in cheese ripening. Except for Propionibacterium and
the heterofermentative lactobacilli, the secondary cul-
tures grow mainly on the cheese surface (see ‘Bacterial
Surface-ripened Cheeses’, Volume 2). They are called
secondary cultures to distinguish them from the pri-
mary acid-producing starters and are as important as
the primary ones in those cheeses in which they are
found.
In the past, only a small number of secondary cul-
tures or adjuncts was used, mainly in traditional cheeses
made from raw milk. For example, in blue-veined
cheeses, P. roqueforti was added to the curd before
moulding in the form of grated, mouldy bread. In fact,
the use of moulds as adjuncts in cheesemaking pre-dates
the commercial use of lactic acid starters. Tradition-
ally, the secondary flora originated in either the milk,
the cheesemaking utensils and/or the cheese factory
environment. Like the production of traditional smear-
ripened cheese, mature cheeses were smeared, i.e.,
washed with dilute solutions of NaCl, which may also
contain some of the surface microflora (see ‘Bacterial
Surface-ripened Cheeses’, Volume 2), before young
ones. Therefore, the cheese surface microorganisms were
transferred from the old to the young cheeses. Since
then, improvement in the microbiological quality of raw
milk, the use of thermisation and bactofugation of milk,
the high level of hygiene and practice modifications in
cheese factories have reduced the sources of the indigen-
ous secondary flora. Cheese has become more bland in
taste and therefore cheese factory personnel became
aware of the decisive role played by the secondary flora
in producing good quality cheese and this, in turn, has
increased the demand for secondary starters. Today, this
need is true of most cheese varieties. In the present
Cheese: Chemistry, Physics and Microbiology, Third edition – Volume 1: General Aspects
ISBN: 0-1226-3652-X
Set ISBN: 0-1226-3651-1
chapter, the most important groups of secondary flora,
the species found in cheeses, the properties used in their
selection, and the form and use of these cultures as
adjuncts will be described. Information on how these
cultures are produced is very difficult to obtain as it is
mainly propriety to the institution producing the cul-
ture. Consequently, it cannot be reviewed in any detail.
Yeast
Yeasts are encountered and used as culture adjuncts in
many cheeses. They are used mainly in mould and bac-
terial surface-ripened cheeses because they promote the
growth of other microorganisms. For example, yeasts
are used as adjuncts in the French cheeses, Brie,
Camembert, Pont l’Evêque, Maroilles and Reblochon,
in the Belgian cheeses, Herve and Limburger, and in
the Italian cheese, Tallegio. Yeasts are also used in blue-
veined cheeses such as Danablu (Denmark), Cabrales
(Spain), Fourme d’Ambert (France), Gorgonzola (Italy)
and Stilton (UK).
Species found in cheeses
The species of yeasts isolated most frequently from
cheeses are listed in Table 1. Geotrichum sp. are often
described as intermediate between mould and yeast
and is now recognised as a yeast (Barnett et al., 1990).
Yeasts colonise numerous cheeses, particularly their
surfaces. They can grow during the early stages of
cheesemaking, e.g., during whey draining after mould-
ing and before salting. Commonly, their population
reaches 106–108 cfu cm 2 of cheese surface during the
first 5 days and remains at this level throughout ripen-
ing. Generally, their number in the interior of the
cheese is 100 or 1000 times lower.
In traditional cheeses, the source of the yeasts is
raw milk, utensils, cheese factory environment, brine
and/or use of natural whey starters used in produc-
tion (Zambonelli et al., 1996). However, Baroiller and
Schmidt (1990) have shown that the great diversity of
yeast species in milk for Camembert cheese was dras-
tically reduced by the selective action of processing.
Today, the use of yeasts as adjuncts is a common prac-
tice in modern cheese factories; they are added to the
cheese milk and/or are used in the smear preparation.
Copyright © 2004 Elsevier Ltd
All rights reserved
192 Secondary and Adjunct Cultures

Table 1 Main yeast species encountered in/on the surface
of cheese
Perfect form
Galactomyces geotrichum
Debaryomyces hansenii
Kluyveromyces marxianus var. lactis
Kluyveromyces marxianus
var. marxianus
Pichia membranifaciens
Pichia fermentans
Sacchoromyces cerevisiae
Sacchoromyces dairensis
Torulospora delbrueckii
Yarrowia lipolytica
Zygosaccharomyces rouxii
activities (Schmidt and Lenoir, 1980; Schmidt et al.,
1993). It is generally recognised that Yarrowia lipolyt-
ica, Saccharomyces cerevisiae and K. marxianus subsp.
Imperfect form
marxianus are more proteolytic than D. hansenii (Vannini
Geotrichum candidum et al., 2001). In addition, G. candidum has higher
Candida famata aminopeptidase activity than P. camemberti (Molimard
Candida sphaerica et al., 1994). Naturally, proteolytic activity is also used in
Candida kefyr
the selection of strains by suppliers of adjunct cultures.
Candida valida The high tyrosinase activity of some strains of
Candida lambica Y. lipolytica is thought to be responsible for the pro-
Candida robusta duction of brown pigments below the cheese surface
Candida dairensis (Van den Tempel and Jakobsen, 2000; Carreira et al.,
Candida colliculosa
Candida lipolytica
2001) and consequently this activity is also assessed in
Candida mogii commercial strains.

Other minor species: Candida catenulata, Candida intermedia, Lipolytic activity

Candida rugosa, Candida sake, Candida vini, Candida
zeylanoides.
From Nunez et al., 1981; Baroiller and Schmidt, 1990; Nahabieh
and Schmidt, 1990; Bârtschi et al., 1994; Eliskases-Lechner and
Ginzinger, 1995b.
Useful properties in selecting yeast adjuncts
Effect on appearance of cheese surface
The yeast flora contribute directly or indirectly to the
appearance of cheese. For example, G. candidum varies
considerably from slimy cream to velvet mould-like
depending on the strain. Consequently, the growth
behaviour of G. candidum is of great importance in
choosing the correct strain for the type of cheese being
produced.
Utilisation of residual sugars and lactate
de-acidification activity
The yeasts encountered on the surface of cheese show
varied abilities to metabolise sugars, lactate and citrate
(Eliskases-Lechner and Ginzinger, 1995b). Because
Kluyveromyces marxianus and Debaryomyces hansenii
are able to ferment lactose, their use as adjuncts is
very common. G. candidum assimilates galactose and
lactate which is of paramount importance in the ripen-
ing of mould and bacterial smear-ripened cheese. The
degradation of lactate results in de-acidification at the
cheese surface and the increased pH, in turn, stimu-
lates the growth of moulds and corynebacteria. Conse-
quently, the de-acidification activity is always taken
into account in selecting yeast strains as culture
adjuncts for soft cheese.
Proteolytic activity
Yeasts show a large diversity in proteolytic activity
between species and strains of the same species. They
have caseinolytic, aminopeptidase and carboxypeptidase
Yeasts contribute to lipolysis in cheeses, and Y. lipolytica
has the highest lipase activity of all yeast found in
cheese (Schmidt et al., 1993). In particular, Y. lipolytica
is much more lipolytic than D. hansenii and S. cerevisiae
(Van den Tempel and Jakobsen, 2000). However, it is
not used commercially. G. candidum lipase preferen-
tially releases oleic acid from milk fat (Gripon, 1993).
Although the contribution of Penicillium, Staphylococcus
and Corynebacterium spp. to lipolysis in mould, smeared
and blue-veined cheese is greater than that of yeasts,
this activity is generally used as a criterion in the selec-
tion of yeast by culture suppliers.
Production of aroma
Yeasts produce aroma compounds. They can produce
ethanol, aldehydes and esters and they degrade amino
acids to ammonia and the corresponding keto acid.
Further metabolism provides numerous compounds
such as alcohols, esters, methyl ketones and carbonyl
compounds. G. candidum produces much more aro-
matic compounds from methionine than other yeasts
tested, including D. hansenii and K. lactis (Demarigny
et al., 2000; Spinnler et al., 2001). Nevertheless, the
relationship between compounds identified by GC–MS
and the sensorial characteristics of cheese curd inocul-
ated with selected yeast were not very consistent (Mar-
tin et al., 2001). Thus, ‘the lack of progress in
describing cheese flavour in precise chemical terms’,
emphasised by Fox et al. (1993), remains topical in
this area. Starter suppliers frequently use sensorial
analysis of model cheeses instead of chemical analysis
to select strains of the surface flora. However, the
Degussa Company use both methods to characterise
their strains of surface adjuncts.
Interactions with other microorganisms
Generally, yeasts promote the growth of G. candidum,
which, in turn, reduces the occurrence of undesirable

moulds, e.g., Aspergillus, Mucor and Penicillium spp.
Therefore, the ability to inhibit Mucor spp., which pro-
duces thin strands of mycelial growth, the so-called ‘cat
hair’ defect on cheese (in French ‘poil de chat’), is one
of the criteria used in the selection of Geotrichum
strains (Gueguen and Schmidt, 1994). Hansen and
Jakobsen (1998) have shown that the growth of
P. roqueforti is not affected by D. hansenii, that its
growth and sporulation are inhibited by D. marxianus
subsp. marxianus and that its growth rate and colour
formation are increased by S. cerevisiae. The interaction
between yeast and other surface microorganisms, such
as moulds and bacteria, is taken in to consideration by
suppliers in the selection of yeast strains.
Moreover, G. candidum can inhibit Listeria monocyto-
genes by the production of two components: D-3-
phenyllactic acid and D-3-indolelactic acid (Dieuleveux
et al., 1998). This property offers an interesting criter-
ion for the selection of cheese adjuncts to increase the
safety of mould and smeared cheeses.
Forms and use of adjunct culture
For G. candidum, the diversity in the forms of adjunct
produced by culture suppliers is generally large. Besides
appearance (slimy to mould-like, and colour), several
other activities are taken into account, including
de-acidification, proteolysis, lipolysis, aroma production
(analytical and/or sensorial), sensitivity to NaCl and
ability to inhibit Mucor spp. G. candidum cultures are sold
by several companies such as Clerici-Sacco (freeze-
dried), Degussa and Chr. Hansen (liquid), Rhodia Food
(liquid, freeze-dried), Standa Industries (liquid) and
Wiesby (liquid, freeze-dried). Moreover, some Dairy
Research or Technical Centres provide Geotrichum cul-
tures for use in their respective countries, e.g., Switzer-
land. Some major cheese companies also produce
‘in-house’ cultures. These cultures can be added directly
in the cheese milk or sprayed on the cheeses, generally
after salting. The manufacturer’s recommendations are
about 105 cfu ml 1 of cheese milk or 106–107 cfu ml 1
of suspended cells for spraying.
Only a few species of yeasts are available commer-
cially and this does not reflect the diversity of yeast
found on the surface of cheese. In fact, three yeast
species, D. hansenii, K. marxianus and S. cerevisiae, are
much more frequently sold than others. Torulospora
delbrueckii (Candida valida) is used occasionally and
each yeast is sold by a different company. Sometimes,
yeasts are available as mixed cultures of yeasts and as
other surface microorganisms such as G. candidum or
Brevibacterium linens.
S. cerevisiae is recommended for blue-veined cheese
because high CO2 production by it from lactose pro-
Secondary and Adjunct Cultures 193
duces a more open textured cheese. These cultures are
added to the cheese milk and the ‘smear’ solutions at
levels of about 105–106 cfu ml 1
Moulds
Moulds are used mainly as adjuncts in two types of
cheese, viz., mould surface-ripened soft cheese, e.g.,
Brie, Camembert or goats’ milk cheeses (France), and
blue-veined cheeses, e.g., Bavarian Blue (Germany),
Bleu d’Auvergne, Bleu des Causses and Roquefort
(France), Cabrales (Spain), Gorgonzola (Italy), Danablu
(Denmark) and Stilton (UK). Moreover, a few var-
ieties of semi-hard cheeses, e.g., Tomme (France and
Switzerland) and Toma (Italy) are also surface-ripened
with moulds.
Species found in cheeses
The white mould, P. camemberti, and the blue-green
mould, P. roqueforti, are the two main species of mould
used as adjuncts. Previously, P. camemberti was called
P. caseicolum Bainier or P. candidum, for strains which
remain white during growth and P. album for strains
which develop a grey-green colour. Today, these two
phenotypic forms have been amalgamated into one
species, P. camemberti Thom (Pitt, 1979). Other Penicil-
lium spp. growing on cheese also have a white
mycelium; P. thonii, P. nalglovensis and P. verrucosum.
P. roqueforti strains can exhibit variations in colour
from yellowish-green (called viride in Italy) to dark
green. Sometimes, it can be confused with a similarly
coloured mould, P. verrucosum var. cyclopium; however,
the latter species produces a strong musty odour. This
spoilage mould can contaminate P. roqueforti cultures.
Without the use of a selected mould adjunct, many
other Penicillium spp. may be found in hard, semi-hard
and semi-soft cheeses such as Cheddar, Danbo, Port
Salut or Bel Paese. P. commune and P. nalgiovensis are the
most common (Lund et al., 1995). The appearance and
properties of P. caseifulvum allow its use as a cheese
adjunct instead of P. album. Other moulds can grow on
cheeses but they are generally undesirable. Nevertheless,
some species occur spontaneously and are desirable on
the surface of certain cheeses, e.g., Chrysosporum sul-
fureum is responsible for yellow spot formation on
St. Nectaire cheese. In some cases, these moulds are
produced as adjuncts, e.g., P. nalgiovensis, P. commune,
Trichothecium domesticum (Cylindrocarpon sp.) and
Verticillium lecanii. C. sulfureum and Sporendonema casei
(red-orange spot) encountered on some semi-hard
cheeses are no longer produced by starter companies
(Ratomahenina et al., 1995). Rhizomucor spp. are gener-
ally considered to be spoilage moulds, producing the ‘cat
194 Secondary and Adjunct Cultures
hair defect’ in most cheeses; however, it is a desirable
mould on Tomme de Savoie and St Nectaire cheese.
Consequently, three species R. fuscus, R. plumbeus and
R. sinensis are produced by ITFF, a cheese technical
centre in France.
Useful properties for which to select moulds
as adjuncts
Appearance of mould on/in cheeses
Moulds contribute directly to the appearance of the
cheese surface or, in the case of blue-veined cheeses, to
the appearance of the cheese body. The growth behav-
iour of P. camemberti varies according to the strain. The
colour and length, and density of the mycelium are very
important criteria in choosing a strain to be used as an
adjunct. The colour of P. roqueforti is also of paramount
importance in selecting strains. Generally, strains show-
ing light blue or yellowish colours are used for Gor-
gonzola cheese whereas dark green strains are used in
Danablu, Bleu des Causses, Roquefort and Stilton cheese.
Naturally, this diversity in the appearance of Penicillium
strains is indicated in the catalogues of mould suppliers.
De-acidification activity
P. camemberti and P. roqueforti are able to utilise lactic
acid as a carbon source. Therefore, their growth leads to
an increase in pH and proteolysis of cheese and conse-
quently causes the cheese to soften. This property is also
indicated by culture suppliers in their product sheets.
Proteolytic activity
Both P. camemberti and P. roqueforti have endopeptidase
and exopeptidase activities which make a major contri-
bution to proteolysis in cheese. Consequently, the ripen-
ing process begins on the surface of the mould-ripened
cheese (Gripon, 1993). The extracellular proteolytic
systems of these two moulds are somewhat similar and
they hydrolyse s1-, - and -caseins. Moreover, their
peptidases release free amino acids and have debittering
activity. Amino acids are catabolised with the production
of ammonia and other volatile compounds (Cerning
et al., 1987). Of course, these proteolytic activities are
considered by mould culture suppliers but, generally,
methods used in this characterisation are only indica-
tive. Azocasein has been suggested as a substrate for
determining proteolytic activity (Larsen et al., 1998).
Lipolytic activity
Lipolysis is much more extensive in mould-ripened
cheeses than in other varieties, especially blue-veined
cheeses and the main agents are Penicillium spp. Their
lipolytic activity varies greatly according to the strain.
Methyl ketones and their corresponding secondary
alcohols are produced by -oxidation of free fatty
acids, produced by lipolysis (see ‘Lipolysis and Catab-
olism of Fatty Acids in Cheese’, Volume 1). These
compounds contribute to the typical flavour of mould-
ripened cheeses (Cerning et al., 1987). Consequently,
the lipolytic activity of P. camemberti and P. roqueforti is
an important criterion in their selection, and this
activity is always indicated in the product specification
of the supplying companies.
Production of aroma
Besides methyl ketones and secondary alcohols, many
esters, aldehydes, volatile amines and ammonia also
contribute to the aroma of mould-ripened cheeses.
The typical mushroom note of Brie and Camembert
flavour produced by P. camemberti is mainly due to
1-octen-3-ol (Gripon, 1993). Like yeasts, mould sup-
pliers frequently use sensorial analysis of cheese mod-
els to characterise their strains and the aromatic notes
or profiles produced are given in the product sheets.
Interactions with other microorganisms
Besides the interaction with yeast described above,
Hansen and Jakobsen (1997) have shown positive and
negative interactions between 20 strains of P. roquefortii
and 15 strains of Leuconostoc, Lactococcus, Lactobacillus
and Streptococcus spp., many of which were strain-
specific. The stimulation of P. roqueforti is mainly due
to the release of amino acids, like arginine and leucine,
by the lactic acid bacteria.
Fast growth of P. camemberti prevents the establish-
ment of Rhizomucor spp. which produce a ‘cat hair’
defect on the cheese surface. This property is also used
by mould suppliers in the selection of suitable strains.
The elevated pH of the cheese surface induced by the
growth of P. camemberti, in turn, promotes the growth
of coryneform bacteria. Moulds produce tyramine, his-
tamine and tryptamine by decarboxylation of the corres-
ponding amino acid but these biogenic amines are
metabolised by coryneform bacteria such as B. linens
which possess deaminase activity (Leuschner and
Hammes, 1998).
Production of mycotoxins
P. roquefort produces several mycotoxins whereas P.
camemberti produces only one, chloplazonic acid, but
there is little risk to human health because the toxins
are present in mould-ripened cheeses at very low levels
(Gripon, 1993; ‘Toxins in Cheese’, Volume 1). Since
this production is strain-specific it must be taken into
account in the selection of moulds for use as cheese
adjuncts.
Form and use of mould adjuncts
The number of strains of P. camemberti available from
suppliers varies from 3 to 16, with the largest number

being available from Rhodia Foods. Other P. camemberti
suppliers are Clerici-Sacco, Chr. Hansen and Degussa.
The colour and the length and density of the mycelium
are always shown on product specification sheets.
Growth rates and de-acidifying, proteolytic, lipolytic
and anti-Rhizomucor activities are also given. These are
sold as liquid or freeze-dried cultures.
For P. roqueforti, 2–7 strains are available, depending
on suppliers, which differ in colour, growth rate, NaCl
and temperature sensitivities, proteolytic and lipolytic
activities and their effects on the texture and aroma of
cheese. Companies producing P. roqueforti cultures are
Clericci-Sacco, CSL, Chr Hansen, Rhodia Food and
Wiesby. The latter company also has a white mutant of
P. roqueforti, which produces the typical flavour of Blue
cheese without the blue colour. These are sold as liquid,
dried or freeze-dried cultures. Moreover, some technical
centres such as LIP, Aurillac, France, produce mould
cultures, especially P. roqueforti for private users. Other
moulds, e.g., P. album, P. nalgiovensis, Trichothecium
domestimum (cylindrocarpon) and Verticillium lecanii are
produced by Rhodia Foods. The oldest method in
which grated mouldy bread is used to inoculate the
curd of blue-veined cheese remains topical in few cases.
Traditional methods using small units on agar surface
or other solid medium to produce Penicillium conidia-
phores are still used. Nevertheless, spore production by
submerged cultivation with sufficient oxygen supply
has been developed for P. camemberti (Bockelmann
et al., 1999). Submerged batch fermentation provides
high spore yields, short fermentation intervals and
automation. P. camemberti cultures can be added
directly to the cheese milk and/or sprayed on the
cheese, generally after salting. The general recommen-
dation is ⬃106–107 spores L 1. P. roqueforti cultures are
generally inoculated directly into the cheese milk at a
level of ⬃107 spores L 1.
Coryneform Bacteria and Staphylococci
These bacteria are present on the surface of many
cheeses. They are used as adjuncts mainly on smeared
soft- and semi-hard cheeses, e.g., Epoisse, Livarot, Morbier
and Munster in France, Limburger in Belgium, Bel Paese
and Tallegio in Italy, Romadour and Tilsit in Germany,
Raclette and Appenzeller in Switzerland, Brick and Mon-
terey in the USA (Table 2). Some cultures, especially
B. linens or Staphylococcus spp. are used as an ‘enzyme
bag’ in ripened cheese without surface microorganisms.
Generally, these bacteria are isolated from soft or
semi-soft cheeses such as Camembert, Munster, Livarot
or Gubbeen, from blue-veined cheeses but also from
hard cheeses such as Gruyère, Beaufort and Comté
(Piton-Malleret and Gorrieri, 1992). Generally, counts
Secondary and Adjunct Cultures 195
of 107–109 cfu cm 2 are reached on soft smear cheeses
within the first 2 weeks of ripening, and 1013 cfu g 1
of rind in Gruyère cheese within the first 3 weeks of
ripening. The bacterial populations remain constant
thereafter until the cheese is consumed (Reps, 1993;
Eliskases-Lechner and Ginzinger, 1995a). The flora is
composed of two principal Gram-positive groups:
coryneform bacteria (irregularly shaped, catalase-posi-
tive rods) and staphylococci (catalase-positive cocci).
These two groups have certain physiological properties
which permit their growth on the cheese surface; they
are aerobic, alkalophilic, mesophilic and salt-tolerant
and cannot grow under acid conditions (see ‘Bacterial
Surface-ripened Cheeses’, Volume 2).
It has been observed that the cultivation methods
used in the laboratory are always selective and the clas-
sification of coryneform and staphylococci groups
is equivocal and confusing, because it is based on
phenotypic characteristics (Seiler, 1986; Kämpfer et al.,
1993; Irlinger et al., 1997). In recent years, molecular
approaches, such as ribotyping, amplified fragment
polymorphism and randomly amplified polymorphic
DNA, have been developed and could give significant
insight into specific isolates and dominant microbial
populations during cheese manufacture. However, these
techniques are laborious and time-consuming for moni-
toring population dynamics and have not been used to
any great extent in classifying the microorganisms.
Coryneform bacteria
Coryneform bacteria include organisms from the genera
Arthrobacter, Brevibacterium, Corynebacterium, Microbac-
terium. The term has no taxonomic significance except
that bacteria in these genera are generally small irregu-
larly shaped rods. For a long time, B. linens was consi-
dered to be the typical, orange-red pigmented, red
smear-cheese bacterium due to its role in colouring the
surface of the cheese and its ability to produce typical
flavours. For that reason, B. linens is the main adjunct
culture available and used for smear-cheeses. Neverthe-
less, some authors have cast doubt on the exclusive
importance of B. linens in the cheese smear. The max-
imum proportion of B. linens found in Tilsit cheese
ranges from 0 to 15% (Eliskases-Lechner and Ginzinger,
1995a). This species was not isolated from the surface of
Gubbeen, an Irish smear cheese, even though the cheese
surface was deliberately smeared with this species at the
beginning of ripening (Brennan et al., 2002); however,
9.3% of isolates were B. linens-like but had different
Pulse-Field Gel Electrophoresis patterns than the deliber-
ately inoculated strain. Moreover, none of the other bre-
vibacteria isolated from cheese, e.g., B. casei, have been
found on Tilsit, Brick and other German cheeses or on
196

Table 2 Diversity and identification of the smear bacteria flora from different cheeses

Number of Characterisation Identification of

Cheese isolates methods Gram-positive bacteria Authors

Limburger, 372 Phenotypic from Seiler (1986) 61% Yellow and white colonies of which: Seiler (1986)
Romadour, – 29.5% Corynebacterium: mainly C. ammoniagenes, C. variabilis
Weinkäse, and unidentified
Handkäse, Munster, – 33% Arthrobacter: A. nicotianae and unidentified
Camembert, – 1% Rhodococcus sp.
Appenzeller 31% Brevibacterium: B. linens
8% Yellow and white non-clustered isolates unidentified
Tilsit 386 Phenotypic from Seiler (1986) 45% Arthrobacter: mainly A. globiformis, A. citreus, A. nicotianae Eliskases-Lechner
and some unidentified and Ginzinger
24.5% Corynebacterium: mainly C. ammoniagenes, C. variabilis (1995a)
and unidentified
21% Brevibacterium: mainly B. linens
5% Curtobacterium: mainly Cu. poinsettiae, Cu. betae, Cu. oxydans
and Cu. helvolum
2% Microbacterium: mainly Mb. imperiale
2% Clavibacter: mainly Cl. insidiosum
0.5% unidentified
Brick cheeses: 195 Phenotypic from Seiler 41% Corynebacterium: mainly C. ammoniagenes, C. variabilis Valdès-Stauber et al.
Limburger, (1986) and unidentified (1997a)
Romadur, 24%: Arthrobacter: mainly A. nicotianae and unidentified
Weinkäse, Munster, 17% Brevibacterium: mainly B. linens
Harzer, Tilsit 10% Rhodococcus: mainly R. fascians
5% Microbacterium: mainly Mb. imperiale, Mb. oxydans and
unidentified
2% Cellulomonas: C. cellulans
Tilsit unpublished Phenotypic from Bergey 5–15% Staphylococcus: mainly S. equorum, small numbers of Bockelmann et al.
(1986) S. saprophyticus and S. sciuri (1997a)
16s rRNA sequencing 75–95% Coryneform bacteria: mainly unidentified, B. linens
(some isolates) and Arthrobacter sp.
Gubbeen 400 Molecular identification 2.5% Staphylococcus sp. Brennan et al. (2002)
methods: 49% Corynebacterium casei
RAPD-PCR, 25.5% Corynebacterium mooreparkense
PFGE 12.5% Microbacterium gubbeenense
1.25% Corynebacterium flavescens
9.25% unidentified coryneforms

Gubbeen (Seiler, 1986; Eliskases-Lechner and Ginzinger,
1995a; Bockelmann et al., 1997a; Brennan et al., 2002).
The occurrence of yellow-pigmented Arthrobacter
strains, especially A. nicotianae, on surface-ripened
cheeses has been reported (Marcellino and Benson,
1992; Valdès-Stauber et al., 1997). Yellow coryneform
isolates from several varieties of Austrian cheese
were classified as A. globiformis (Eliskases-Lechner
and Ginzinger, 1995a). This was confirmed by Bock-
elmann et al. (1997a), who identified several yellow-
pigmented strains as A. globiformis.
Some corynebacteria are major components of the
microflora of surface-ripened cheese. The dominant
genus differs depending on the type of cheese studied.
In Comté, only 12% of the surface flora was assigned to
the genus Corynebacterium (Piton, 1988; Piton and
Fontanier, 1990) while in Brick cheeses it was the dom-
inant genus (nearly 50% of isolates) (Valdès-Stauber
et al., 1997). In cheese smears, C. ammoniagenes (pre-
viously named Brevibacterium ammoniagenes) and
C. variabilis (previously named Caseobacter polymorphus)
were reported when the key of Seiler (1986) was used
to identify the isolates. Two newly described species,
C. mooreparkense and C. casei, were isolated from the
surface of an Irish smear-ripened cheese. C. ammonia-
genes and C. variabile were their nearest known phylo-
genetic neighbours (Brennan et al., 2001a, 2002).
The genus, Brachybacterium, including three new
species isolated from milk and Beaufort, Gruyère and
Camembert cheeses (Brachybacterium nesternkovii,
B. alimentarium and B. tyrofermentans; Gvozdiak et al.,
1992; Schubert et al., 1996; Lefresne, 2000), may
be present on other smear cheeses. Brachybacteria are
highly salt-tolerant and yellow in colour. However, the
incidence of Brachybacteium on cheeses has not been
studied systematically.
Microbacterium sp. are widely distributed in various
environments and three species have been isolated
from milk products or cheese, M. lacticium (Collins
et al., 1986), M. liquefaciens (Collins et al., 1983) and
M. gubbeenense (Brennan et al., 2001b). Moreover,
Eliskases-Lechner and Ginzinger (1995a), Valdès-
Stauber et al. (1997) and Brennan et al. (2002) isolated
8, 7 and 50 Microbacterium strains from Tilsit, Brick
and Gubbeen cheeses, respectively. Brennan et al.
(2002) showed that, generally, M. gubbeenense was isol-
ated more frequently late in ripening.
Until the mid-1970s, micrococci were erroneously
considered to constitute a major portion of the second-
ary flora of cheeses and to be important for flavour
development. The Micrococcus strains isolated from
various cheeses were most probably misclassified
staphylococci. However, some reports confirm that
micrococci, especially Kocuria varians and Micrococcus
Secondary and Adjunct Cultures 197
luteus, play a role in goats’ milk cheese, but compared
to staphylococci their numbers are not very high and
they decrease rapidly during ripening (Michaux, 1983;
Massa and Turtura, 1989; Vernozy-Rozand et al., 1996;
Caceres et al., 1997).
Staphylococcus
Many investigators have noted the predominance of
novobiocin-resistant and coagulase-negative staphylo-
cocci in cheeses, particularly in hard varieties, made
from ewes’ or goats’ milk (Delarras and Laban, 1981;
Garcia et al., 1988; Massa and Turtura, 1989). Coagulase-
negative staphylococci are found mainly in high cell
numbers early in ripening and they make up 5–25% of
total surface cell counts (Bockelmann et al., 1997a).
Staphylococcus spp. are replaced by coryneform bacteria
after about 15 days ripening (Brennan et al., 2002). The
prevailing species in soft, smeared cheeses are Staph.
equorum, Staph. vitulinus and Staph. xylosus (Irlinger
et al., 1997). This last species is commercially available
as an adjunct and, is also used as a starter in fermented
sausage. A new coagulase-negative and novobiocin-
resistant Staphylococcus, S. fleuretti, has been isolated
from raw goats’ milk cheese (Vernozy-Rozand et al.,
2000). In addition, a new subspecies, Staph. succinus
subsp. casei, isolated from a Swiss surface-ripened
cheese, has been described (Place et al., 2002). This
species has been used as a starter component in typical
Swiss cheeses. Naturally, it is necessary to characterise
these strains carefully and to confirm that they are food
grade.
Useful properties for selecting surface bacteria as
adjuncts
Growth
All species encountered on the cheese surface are salt
tolerant, e.g., B. linens tolerates up to 15% of NaCl
(Ferchichi et al., 1985; Collin and Law, 1989). Their
minimal pH for growth and their sensitivity to the ripen-
ing temperature are the main determinants in colonising
the cheese surface. The growth rate at pH 5.8 differs sig-
nificantly between strains of B. linens. Staphylococci pro-
mote the growth of other smear bacteria at the
beginning of ripening because they grow rapidly at pH
5.5 and below (Bockelmann et al., 1997a). Conse-
quently, sensitivity to pH and temperature are criteria
used in the selection of surface bacteria strains.
Effect on the colour of cheese surface
The colour of the cheese surface is an important char-
acteristic of smear-ripened cheeses. The contribution of
the surface bacteria varies according to genus, species
and strain. Most strains of B. linens produce distinctive
red-orange carotenoid-type pigments but numerous
198 Secondary and Adjunct Cultures
strains are non-pigmented. Corynebacterium spp. are
similar. Arthrobacter are generally yellow-pigmented
and produce the typical red-brown colour of smear-
cheeses by conversion of yellow pigments (Bockel-
mann et al., 1997a). Brachybacterium spp. found in
cheeses are yellow-pigmented and staphylococci pro-
duce orange pigments.
Of course, the colour exhibited by surface bacteria
is a major criterion used in the screening and in the
selection of strains as adjuncts. Several tests are used,
the most relevant being the development of colour on
cheese models which takes into account the inter-
actions between surface microorganisms.
Proteolysis, peptidolysis and amino acid catabolism
Although the caseinolytic activity varies greatly
between species and strains, this property of surface
bacteria has only a small additional effect on cheese
proteolysis. However, their peptidase activities and
amino acid catabolism are much more important for
the production of aroma compounds (Gobbetti et al.,
2001; Curtin et al., 2002). Their demethiolase activity
produces sulphur compounds, particularly methanthiol
from methionine (Brennan et al., 2002). Their deamin-
ase activity produces ammonia and degrades biogenic
amines (Leuschner and Hammes, 1998). Moreover,
proteolysis plays a role in the production of the typical
colour of the cheese surface (Bockelmann et al.,
1997b). Naturally, these properties are taken into con-
sideration by culture suppliers in the process of strains
selection.
Lipolysis
Staphylococci have higher lipolytic activity than other
surface bacteria (Bergère and Tourneur, 1992; Curtin
et al., 2002). However, this property is sometimes
shown in the product sheets of suppliers, but the
methods used to characterise it are not indicated.
Antimicrobial activities
B. linens produces antimicrobial substances which
inhibit the growth of many Gram-positive food-poi-
soning bacteria as well as several yeasts and moulds
(Maisnier-Patinaud and Richard, 1995; Motta and
Brandelli, 2002). Some isolates of M. lacticum show
anti-listerial activity (Carminati et al., 1999). Kocuria
varians NCC 1482 produces variacin, an antibiotic of
the same class of antimicrobial peptides as nisin. It
inhibits food-borne pathogens such as species of Ente-
rococcus sp., Staphylococcus aureus, Bacillus cereus and
Clostridium botulinum (Pridmore et al., 1996;
O’Mahony et al., 2001). Staphylococci produce many
anti-bacterial substances such as antibiotics (Brennan
et al., 2002). A strain of Staph. equorum was found to
produce a macrocyclic peptide antibiotic, micrococcin
P1, on soft cheese and to inhibit Listeria monocyto-
genes (Carnio et al., 2000). Naturally, these antimicro-
bial activities provide interesting criteria for the
selection of strains in order to control the safety of
smear-ripened cheeses using specific adjuncts.
Form and use of adjunct culture
Like yeasts, only a few species and strains of these bac-
teria are commercially available and this does not
reflect the complexity of the cheese surface bacteria
described previously. Of course, several strains of
B. linens are often marketed by the main suppliers
(Crerici-Sacco, Degussa, Chr Hansen, Rhodia Food
and Wiesby). In contrast, A. nicotianae, A. globiformis
and C. flavescens are available from only two com-
panies (Degussa and Rhodia Food). Some Dairy
Research or Technical Centres may provide coryne-
form cultures for their domestic cheese producers. For
staphylococci, S. xyloasus or S. carnosus is recom-
mended by Chr. Hansen and Rhodia Food, whereas
other companies (Bioprox, Degussa and Wiesby) do
not indicate the identity of the staphylococcal adjuncts
in their product sheets. These surface bacterial cul-
tures are always available in freeze-dried form but
Wiesby market their cultures in three forms, liquid,
frozen and freeze-dried. Several mixed-cultures are
supplied by Degussa, Rhodia Food and Wiesby, which
contain mixed bacterial species or mixtures of yeasts
and bacteria. In using adjunct cultures, the manufac-
turers recommend direct inoculation of the cheese
milk to obtain 5.104–105 cfu ml 1 or spraying a smear
solution on the cheese surface.
Propionic Acid Bacteria
Propionic acid bacteria (PAB) are used mainly as
adjuncts in cheeses with eyes which are also called
Swiss-type cheeses, particularly Emmental, Jarlsberg
and Maasdam (see ‘Cheese With Propionic Acid Fer-
mentation’, Volume 2). They could also be used in
other hard- or semi-hard cheeses for their protective
effects and their contribution to taste and aroma.
Species found in cheeses and characteristics
The bacteria that produce propionic acid were named
Propionibacterium by Orla-Jensen in 1898 and their
fermentation was studied by Pasteur and Fitz. The PAB
were seriously classified only in 1928 by Van Niel but
most of the current classification keys emerged from
the work of Cummins and Johnson (1981). The ‘clas-
sical’ or ‘dairy’ PAB should be differentiated from the

cutaneous ones which occur on the human skin. Four
species are currently classified as ‘dairy’ PAB: P. acido-
propionici, P. freudenreichii, P. jensenii and P. thoenii. The
other two ‘classical’ species, P. cyclohexanicum and
P. microaerophilus, were not isolated from dairy prod-
ucts (Kusano et al., 1997; Koussemon et al., 2001).
P. freudenreichii is the most common species found in
cheese and, consequently, is the one used as an
adjunct. Of course, the number of PAB is high around
109 cfu g l in hard cooked cheeses such as Emmental
which are ripened in a warm room. However, signifi-
cant numbers of PAB, between 107 and 109 cfu g l,
are also found in semi-hard cheeses, e.g., Abondance,
Appenzell, Gouda, Maasdam, Morbier, Tomme de
Savoie and some ewes’ milk cheeses. This is not sur-
prising because PAB are part of the natural flora of raw
milk and they can grow at a low temperature.
Propionic acid bacteria are Gram positive, non-
motile, non-sporing, small rods (0.5–0.8 m  1 to
5 m). PAB are pleomorphic, paired small rods or
coccoid-shaped cells, often in clumps with Chinese
characters; sometimes filamentous shapes are seen.
Their genome size is between 1.6 and 3.1 kb, their
G  C content ranges from 65 to 67% and plasmids
occur frequently (1–3). PAB are anaerobic to aerotol-
erant mesophiles and many strains are able to grow
slowly at a temperature as low as 3 °C. They are able
to metabolise many different carbon sources: sugars
(lactose, glucose, galactose, fructose), alcohols (gly-
cerols, erythritol, adonitol), organic acids (lactate,
citrate, aspartate). Biotin and pantothenic acid are
essential factors for their growth (Cummins and
Johnson, 1981). Optimum growth occurs in the pH
range 6.5–7, and pH 5.0–5.2 is the lowest limit for
the growth of most strains. Low temperatures and
high sodium chloride concentrations (up to 3%)
enhance the inhibitory effect of low pH on the
growth of PAB (Hettinga and Reinbold, 1972a,b,c).
Useful properties for selecting PAB as adjuncts
Lactate metabolism
Orla-Jensen was the pioneer researcher on the prop-
ionic acid fermentation in Emmental cheese. He was
the first to show the relationship between this
fermentation and eye formation (von Freudenreich
and Orla-Jensen, 1906). Already in 1878, Fitz had
established the well-known stoichiometric equation:
3 lactate : 2 propionate  1 acetate  1 CO2  H2O.
Nevertheless, this fermentation balance is often dif-
ferent in cheese where strong propionic acid fermenta-
tion occurs, such as Emmental. This suggests that PAB
Secondary and Adjunct Cultures 199
have a more complex lactate metabolism. Recently, the
use of in vivo 13C-NMR was used to follow more pre-
cisely carbon metabolism in PAB and indicated the
presence of six other minor pathways (Deborde et al.,
1999; Deborde and Boyaval, 2000).
In hard cheese, the rate of the propionic acid fer-
mentation and CO2 production is decisive for the suc-
cessful formation of the desirable round, shiny eyes. A
fast lactate fermentation is needed to accumulate CO2
in the cheese (there must be a balance between CO2
production in the cheese and CO2 diffusion from the
cheese). Consequently, the growth rates of PAB under
conditions encountered in cheese during the ripening
(pH 5.2, 1–2% NaCl, 18–22 °C) is the first criterion in
the selection of PAB. For this purpose, the use of mini-
cheesemaking as described by Richoux and Kerjean
(1995) is better than in vitro studies.
Acetic and propionic acids also contribute to the
preservation and the taste of cheese.
Proteolytic activities and amino acid catabolism
The caseinolytic potential of PAB is estimated to be
5–15 times less than that of lactococci (El Soda et al.,
1992; Dupuis et al., 1995). Propionic acid bacteria con-
tain numerous peptidases, including a wide spectrum
of general aminopeptidase activities and many activ-
ities towards proline-containing peptides, e.g., proline
aminopeptidase, X-prolyl-dipeptidyl aminopeptidase,
prolinase and prolidase (Lemée et al., 1998; Gagnaire
et al., 1999; Stepaniak, 2000). They are mainly intracel-
lular but, unfortunately, autolysis of PAB in cheese is
limited and slow. It is less important than autolysis of
lactic acid bacteria (Sahlstrom et al., 1989; Lemée et al.,
1995; Valence et al., 1998; see Ostlie et al., 1999). Con-
sequently, these proteolytic activities may be omitted in
the selection of PAB as secondary cultures.
Propionibacteria are able to catabolise amino acids,
especially aspartic acid, asparagine, alanine, valine,
serine, tyrosine, glutamic acid, arginine, cysteine and
methionine, to different flavour compounds (Keenan
and Bills, 1968; Brendenhaug and Langsrud, 1985).
But today, the production of aroma compounds by PAB
remains measurable only in real cheeses. However,
their ability to metabolise aspartate is easily checked
in the laboratory. This aspartate deamination pathway
leads to high CO2 production which can provoke a
late blowing defect in aged Emmental cheese
(Fröhlich-Wyder et al., 2002).
Lipolysis
Propionic acid bacteria are well known for their
high lipolytic activity and have 10–100 times more
activity than lactic acid bacteria (Oterholm et al., 1970;
Dupuis et al., 1993). In vitro studies, as well as data
200 Secondary and Adjunct Cultures
from experimental cheeses, have shown that PAB
release FFAs in cheese (Chamba and Perreard, 2002).
P. freudenreichii exhibits the highest lipolytic activity
but it is strain-dependant. This activity on lipids in
cheese produce aroma compounds through the release
of free fatty acids and their subsequent catabolism.
This is an important property in choosing strains as
adjuncts.
Unfortunately, the available laboratory methods to
characterise the lipolytic activity of PAB do not correl-
ate well with their lipolysis in cheese (Kerjean et al.,
2000). In order to use the lipolytic activity as a screen-
ing criterion for PAB, improvements in analytical
methods are needed.
Probiotic properties
Propionic acid bacteria can survive in the digestive
tract and reach and maintain high populations in the
human intestine (Bouglé et al., 1999; Jan et al., 2002).
They are able to inhibit undesirable intestinal bacteria,
and have a growth-promoting effect on bifidobacteria
(Kaneko et al., 1994). Some dairy strains produce
nitric oxide with a positive effect on intestinal peristal-
sis. To date, it seems that probiotic properties have not
been taken into consideration in selecting strains to be
used as cheese adjuncts.
Form and use of adjunct cultures
Swiss cheesemakers were the pioneers in the use of
PAB as adjuncts; their Dairy Research Station at
Liebefeld-Berne began to produce selected propioni-
bacteria cultures as early as 1926. The use of PAB cul-
ture is justified for three main reasons:
• to replace the low level of PAB in the cheese milk
(natural or after treatment) which often is unable to
reach the necessary level to obtain a satisfactory
propionic acid fermentation;
• to increase the rate of propionic acid fermentation in
order to obtain the desirable openings or eyes (the
balance between CO2 production and CO2 diffusion);
• to better control propionic acid fermentation and
cheese quality.
Generally, PAB are not cultivated in the cheese fac-
tories because their cultivation is laborious, time-
consuming and sensitive to microbial contamination. In
the past, liquid cultures were the normal form; today,
concentrated frozen or freeze-dried cultures are com-
monly supplied by specialised companies or Dairy
Research Centres. These cultures are added directly to
the milk in the cheese vat. Production of PAB is done
in a sterile fermenter using complex media containing
lactate. After the desired number of bacteria has been
attained (⬃109 cfu ml 1), the culture is concentrated
by centrifugation or microfiltration. Then, the biomass
is frozen or freeze-dried after addition of a cryopro-
tectant(s). The bacterial concentration of commer-
cially freeze-dried cultures is about 1010 cfu g 1. The
main commercial suppliers are Clerici Sacco, Centro
Sperimentale del Latte (CSL), CSK, Chr-Hansen,
Rhodhia Foods, Standa Industrie and Wiesby. Most
commercial strains are P. freudenreichii and, generally,
the number available is limited (1–3). In contrast, the
portfolio of Standa Industrie contains a large choice of
cultures (⬃15) with various technological abilities. In
addition, the frequent and strong interaction between
PAB and lactic acid bacteria must be taken into consid-
eration in the choice of the starters associations
(Chamba, 1994; Kerjean et al., 2000).
The manufacturer’s recommendations for propioni-
bacteria are to use ⬃103 cfu ml 1 of milk for Emmen-
tal-type cheese and between 105 and 106 cfu/ml for
other cheeses. Below this amount, the risks of a slow,
propionic acid fermentation and several defects, e.g.,
brown spots, and blind cheese, are high.
Heterofermentative Lactobacilli
In spite of not being added deliberately to the milk,
heterofermentative lactobacilli grow to high numbers
(⬃108 cfu g 1) in many hard and semi-hard cheeses,
especially in the major ripened cheeses produced such
as Cheddar and Emmental. Their use as adjuncts is in
its infancy. Very few culture producers produce hetero-
fermentative lactobacilli as adjuncts. However, their
use could increase in the future.
Species found in cheeses
It is generally recognised that Lactobacillus paracasei
subsp. paracasei, Lb. rhamnosus, Lb. plantarum and
Lb. curvatus are the main species of facultative heterofer-
mentative lactobacilli (FHL) in cheese (Jordan and
Cogan, 1993; Lindberg et al., 1996; Coppola et al., 1997;
Bouton et al., 1998; Crow et al., 2001). They are Gram
positive, non-motile and catalase negative, their G  C
content ranges from 44 to 47%. Under the microscope,
FHL appear as short rods. They are aerotolerant and
mesophilic and are able to grow at 15 °C; Lb. paracasei
subsp. paracasei and Lb. rhamnosus can grow at 45 °C
but Lb. plantarum cannot. Many sugars, such as lactose,
glucose, galactose, fructose and especially ribose, are fer-
mented with production of L- or DL-lactate, but without
CO2 production, and some strains metabolise lactate,
citrate, amino acids and glycolipids (Williams et al.,
2000). The fermentation of pentoses results in produc-
tion of lactic and acetic acids. The heat resistance of

heterofermentative lactobacilli varies according to the
species and strain. Lb. paracasei and Lb. rhamnosus are
more resistant than Lb. plantarum. They can survive pas-
teurisation at 72–75 °C for 15 s (Jordan and Cogan,
1999). Likewise, the two former species resist the cook-
ing temperature, 50–55 °C for at least 1 h, used in hard
cheeses such as Emmental and Grana.
Lb. brevis, Lb. buchneri and Lb. fermentum are the
main obligatory heterofermentative lactobacilli encoun-
tered in cheese. The C  G content of Lb. fermentum, at
52–54%, differs from other lactobacilli. These three
species produce DL lactate, ethanol and CO2 from glu-
cose. Gluconate is also fermented and arginine is
metabolised with the production of NH3. Lb. brevis and
Lb. buchnerii can grow at 15 °C but Lb. fermentum can-
not; Lb. brevis does not grow at 45 °C, but Lb. buchneri
and Lb. fermentum can. The obligatory heterofermenta-
tive lactobacilli have been considered as a spoilage bac-
teria in cheese for a long time, but this opinion should
be revised. Generally, FHL are present in cheese in
much higher numbers than obligate heterofermenting
lactobacilli.
Heterofermentative lactobacilli are regarded as an
adventitious flora in cheese and they originate in the
raw milk and factory environment. This flora can reach
108 cfu g 1 in most, if not all, ripened cheeses. In spite
of this observation, which was made over 30 years ago,
the role of heterofermenting lactobacilli in flavour for-
mation in cheese is still unclear compared to the
homofermentative starter lactobacilli. The use of het-
erofermentative lactobacilli as cheese adjunct culture is
still at an early stage.
Useful properties to select heterofermentative
lactobacilli as adjuncts
Heterofermentative lactobacilli exhibit a large diversity
of properties and their effects on cheese characteristics
vary from negative to no effect to positive effects.
These properties are strongly strain-dependant and
offer several ways for their selection as adjuncts.
Proteolysis and amino acids catabolism
The proteinase activity of heterofermentative lacto-
bacilli seems to be lower than that of homofermenta-
tive lactobacilli and their contribution to casein
hydrolysis during ripening of Cheddar cheese appears
to be relatively small (Lynch et al., 1997). In contrast,
the peptidase activities, at least in certain strains, con-
tribute to the hydrolysis of bitter peptides to non-bitter
peptides with the release of free amino acids (Gagnaire
et al., 2001). This is the case for Lb. casei subsp. casei
LLG which has an active aminopeptidase and a pro-
line-specific peptidase with debittering activity (Park
et al., 1995). Likewise, Lb. curvatus DPC 2024 has a
Secondary and Adjunct Cultures 201
PepN-like aminopeptidase, which shows broad sub-
strate specificity (Magboul and McSweeney, 1999).
Amino acid catabolism and the production of
aroma compounds by heterofermentative lactobacilli,
especially their glutamate dehydrogenase activity,
appear to be one of the determinant properties for
their use as cheese adjuncts. This activity also provides
-ketoglutarate for transamination of other amino
acids to produce aroma compounds (Rijnen et al.,
2000). This activity is strain-dependant; about half of
Lb. plantarum and Lb. paracasei strains have glutamate
dehydrogenase activity (Tanous et al., 2002). More-
over, Lb. fermentum and Lb. reuteuri produce aroma
compounds from sulphur amino acids but Lb. brevis,
Lb. paracasei and Lb. curvatus do not (de Angelis et al.,
2002). Consequently, measurement of aminopeptidase
activity and the ability to catabolise amino acids
should be taken into account in their selection.
Formation of biogenic amines
Amino acid decarboxylase activity of obligately hetero-
fermentative lactobacilli, particularly Lb. buchneri, has
been implicated in biogenic amine production in Swiss
cheeses and outbreaks of food poisoning (Sumner et al.,
1985; Joosten and Northolt, 1987). It is certainly an
exceptional case since the use of Lb. fermentum and Lb.
buchnerii as adjuncts in Emmental cheese produced less
than 10 mg kg 1 of histamine and 50 mg kg 1 of tyra-
mine (Chamba, 2000). However, Crow et al. (2001) take
the ability to form biogenic amines into consideration in
screening heterofermentative lactobacilli as cheese
adjuncts.
Lipolytic activities
Like other lactic acid bacteria, heterofermentative
lactobacilli are generally considered to be weakly
lipolytic in comparison with other microorganisms,
such as PAB, corynebacteria, yeast and moulds in
cheese (Knaut and Mazurek, 1974; Khalid and Marth,
1990; Fox et al., 1993; Gobbetti et al., 1997). There-
fore, lipolytic activity can be omitted in the selection
of heterofermentative lactobacilli as adjuncts.
Antagonistic activities
The production of inhibitory metabolites and bacterio-
cins by heterofermentative lactobacilli is also problem-
atic in cheese. The first one was observed against PAB in
Swiss cheese. Lb. casei and Lb. rhamnosus produce
acetate, formate and small amounts of diacetyl from cit-
rate in cheese and interfere negatively with the growth of
P. freudenreichii (Jimeno et al., 1995). Swiss researchers
use this ability to prevent secondary fermentation,
which causes cracks and splits in Emmental cheese.
Antimicrobial activities of lactobacilli have been
known and recognised for more than 14 years. Many
202 Secondary and Adjunct Cultures
heterofermentative lactobacilli such as Lb. brevis, Lb. cur-
vatus, Lb. fermentum, Lb. plantarum and Lb. rhamnosus
produce bacteriocins. Generally, these inhibit several
Gram-positive bacteria, including enterococci, clostridia,
S. aureus and Listeria spp., but, unfortunately, lactic acid
bacteria are also frequently inhibited (Klaenhammer
et al., 1994; Malik et al., 1994). It is possible that this
property will become important in the future use of het-
erofermentative lactobacilli as cheese adjuncts.
An interesting inhibitory activity against the spoilage
bacterium, Clostridium tyrobutyricum, is produced by
Lb. rhamnosus LC705 (DSM7051). This was patented
and is commercially available (Maÿra-Mäkinen and
Suomalainen, 1996). This adjunct was tested success-
fully in Emmental and Gouda cheeses. In Gouda cheese,
Lb. rhamnosus LC705 provides an efficient substitute for
nitrate.
Probiotic properties
Some strains of heterofermentative lactobacilli, Lb. casei
Shirota, Lb. plantarum DSM9843, Lb. rhamnosus GG,
have shown probiotic capabilities such as the prevention
and treatment of diarrhoeal disease, intestinal inflamma-
tion or permeability disorders, immunomodulation and
tumour prevention (Huis In’t Veld and Marteau, 1997).
Their ability to survive in the gastro-intestinal tract and
colonise the intestine, especially exopolysaccharide-
producing strains, could be useful in the development of
probiotic dairy products (Chabot et al., 2001). In this
way, Gardiner et al. (1998) have shown that cheese is a
better vector than fermented milk for increasing the
numbers of lactobacilli in the intestines of piglets. The
use of heterofermentative lactobacilli selected for their
probiotic properties as cheese adjuncts could be a
possibility in the future.
Form and use of adjunct cultures
The statement of Fox et al. (1993) ‘The contribution of
NSLAB to cheese ripening and quality is a vexed ques-
tion’ remains topical. The main reason for this statement
is certainly the high versatility of heterofermentative
lactobacilli. Their effects are strain-specific, strongly
affected by the technological context and probably by
the interactions with other microorganism in cheese.
This is probably why the supply of heterofermentative
lactobacilli as adjuncts is still limited.
Nowadays, in modern cheese factories, cheesemak-
ing conditions are better and more clearly understood
and good hygiene is practised. The bulk milk is of good
quality; the cheese milk is standardised (for fat and pro-
tein), clarified and thermised. Technological and envir-
onmental parameters of cheesemaking and ripening are
under control. Moreover, efficient starters are used. Nev-
ertheless, NSLAB, especially heterofermentative lacto-
bacilli, remain an uncontrolled part of the cheese micro-
bial ecosystem. For that reason, their use as adjuncts
should increase in the next years in order to:
• Overcome the probable negative effect encountered by
the growth of the indigenous flora. For this purpose,
useful strains of heterofermentative lactobacilli must
be able to grow faster than the indigenous NSLAB
without affecting the characteristics of the cheese.
• Improve cheese quality by using adjuncts with
desirable properties.
In Switzerland, the use of FHL as adjuncts is com-
mon for Emmental cheese to prevent late blowing.
This adjunct is supplied by the Federal Dairy Research
Institute, Liebefeld-Berne, and the recommended level
of inoculation is around 1  104 cfu ml 1 of cheese
milk.
For Cheddar cheese, Crow et al. (2001) provide a
very good description of the screening process to select
NSLAB as starter adjuncts which are used at levels of
300–1000 cfu ml 1 of vat milk. They maintain that
‘NSLAB adjuncts are required for improved flavour
control in aged cheeses such as mature Cheddar as
uncontrolled adventitious strains of NSLAB can cause
defects’. From this sentence, can we understand that
the use of NSLAB as cheese adjuncts is now a common
practice in New Zealand?
Today, the portfolios of heterofermentative lacto-
bacilli usable as cheese adjunct from starter suppliers
are small. Heterofermentative lactobacilli are frequently
marketed as probiotics, for example by Clericii-Sacco,
CSL, DSM Food Specialities, Chr. Hansen or Rhodia
Foods. Only a few cultures are available and often
these are sold as mixed cultures with other lactic acid
bacteria or with surface-ripening microorganisms.
Wiesby market Lb. rhamnosus strain LC 705 which
inhibits Cl. tyrobutyricum. Generally, these cultures are
in freeze-dried form for direct inoculation of the vat
milk or sometimes for bulk starter preparation.
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bacterium freudeinreichii: first evidence by using species-
specific lysis-markers. J. Dairy Res. 65, 609–620.
Van den Tempel, T. and Jakobsen, M. (2000). The technolog-
ical characteristics of Debaryomyces hansenii and Yrrowia
lipolytica and their potential as starter cultures for pro-
duction of Danablu. Int. Dairy J. 10, 263–270.
Vannini, L., Baldi, D. and Lanciotti, R. (2001). Use of
Fourier transform infrared spectroscopy to evaluate the
proteolytic activity of Yarrowia lipolytica and its contribu-
tion to cheese ripening. Int. J. Food Microbiol. 69,
113–123.
Vernozy-Rozand, C., Mazuy, C., Perrin, G., Hahond, F.,
Bes, M., Brun, Y. and Fleurette, J. (1996). Identification of
Micrococcaceae isolated from goat milk and cheese in the
Poitou-Charentes region. Int. J. Food Microbiol. 30,
373–378.
Vernozy-Rozand, C., Mazuy, C., Meugnier, H., Bes, M.,
Lasne, Y., Etienne, J. and Freney, J. (2000). Staphylococcus
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Syst. Evol. Microbiol. 50, 1521–1527.
von Freudenreich, E. and Orla-Jensen, S. (1906). Uber die in
EmmentalkerKäse statfindene Propionsaüregärung. Zent.
Bakteriol. Usw. Abt. 17, 529–543.
Williams, A.G., Withers, S.E. and Banks, J.M. (2000). Energy
sources of non-starter lactic acid bacteria isolated from
Cheddar cheese. Int. Dairy J. 10, 17–23.
Zambonelli, C., Montanari, G., Passarelli, P. and Rainieri, S.
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261–270.
 Salt in Cheese: Physical, Chemical
and Biological Aspects
T.P. Guinee and P.F. Fox, Dairy Products Research Centre, Moorepark, Fermoy,
Ireland. Department of Food and Nutritional Sciences, University College, Cork, Ireland
Introduction
The use of salt (NaCl) as a food preservative dates from
pre-historic times and, together with fermentation and
dehydration (air/sun), is one of the classical methods of
food preservation. So useful and widespread was the use
of salt as a food preservative in Classical and Medieval
times that it was a major item of trade and was used as a
form of currency in exchange for goods and labour. It is
perhaps a little surprising that Man discovered the
application of salt in food preservation so early in civil-
ization since, in contrast to fermentation and dehydra-
tion, salting is not a ‘natural event’ in foods but requires
a conscious act. It is interesting that the three classical
methods of food preservation, i.e., fermentation, dehy-
dration and salting, are exploited in cheese manufacture
and, in fact, are interdependent. The fourth common
method for food preservation, i.e, use of high and/or
low temperatures, was less widespread than the others
because the exploitation of low temperatures was con-
fined to relatively few areas until the development of
mechanical refrigeration about 1870 and, although
heating was probably used to extend the shelf-life of
foods throughout civilization, its controlled use dates
from the work of Nicolas Appert (1809) and Louis
Pasteur (1860–1864). In modern cheese technology,
temperature control complements the other three meth-
ods of food preservation.
The level (%, w/w) of salt in cheese ranges from ⬃0.7
in Swiss to ⬃6 for Domiati (see Table 1). Salt, together
with the desired pH, water activity and redox potential,
contributes to minimization of spoilage and prevention
of the growth of pathogens in cheese (see Naguib et al.,
1979; Russell and Gould, 1991; Eckner et al., 1994;
Guraya et al., 1998; Bolton and Frank, 1999; Erkmen,
2001). In addition to its preservative effect, NaCl plays
two other important roles in foods. Man requires ⬃2.4 g Na,
i.e., ⬃6 g NaCl, per day (Kaplan, 2000) and although
this requirement can be met through the indigenous
Na content of foods, added NaCl is a major source in
modern western diets. In fact, western diets contain
approximately two to three times more Na than is neces-
sary and excessive intakes of Na have toxic, or at least
undesirable, physiological effects, the most significant of
Cheese: Chemistry, Physics and Microbiology, Third edition – Volume 1: General Aspects
ISBN: 0-1226-3652-X
Set ISBN: 0-1226-3651-1
which are hypertension and increased calcium excretion
which may lead to osteoporosis (see Abernethy, 1979;
Anonymous, 1980, 1983; Moses, 1980; Schroeder et al.,
1988; Midgley et al., 1996; Beard et al., 1997; McCarron,
1997; Beilin, 1999; Cutler, 1999; Feldman and Schmidt,
1999; Korhonen et al., 1999, 2000; Cappuccio et al., 2000;
Kaplan, 2000; McCarron and Reusser, 2000).
Cheese, even when consumed in large amounts (see
‘Cheese: An Overview’, Volume 1), as in France and
Switzerland, makes a relatively small contribution to
dietary Na intake, although it may be a major contrib-
utor in individual cases where large amounts of high-
salt cheese, e.g., Blue, Feta or Domiati, are consumed.
Nevertheless, there is interest in many western coun-
tries in the production of low-Na cheese, for at least
certain sectors in the population, but, as discussed in
‘Reduced Sodium Cheese’, this has significant reper-
cussions in cheese manufacture. The most common
approach at present is to replace some or all of the
NaCl by KCl, but apart from cost, this practice
adversely affects the taste of cheese since the taste of
KCl is distinctly different from that of NaCl and a bit-
ter flavour (not due to abnormal proteolysis) is
detectable in cheese containing 1%, w/w, KCl (see
‘Reduced Sodium Cheese’ for discussion on low-
sodium cheese).
The third major feature of the use of NaCl in foods
is its direct contribution to flavour. The taste of salt is
highly appreciated by many and saltiness is regarded
as one of the four basic flavours. Presumably, the char-
acteristic taste of NaCl resides in the Na moiety since
KCl has a distinctly different flavour sensation. At
least part of the desirability of salt flavour is acquired
but while one can easily adjust to the flavour of foods
without added salt, the flavour of salt-free cheese is
insipid and ‘watery’, even to somebody not ‘addicted’
to salt; the use of 0.8%, w/w, NaCl is probably suffi-
cient to overcome the insipid taste (Schroeder et al.,
1988).
In this chapter, we will concentrate on the signifi-
cance of NaCl in cheese ripening rather than on its
dietary and direct flavour effects. NaCl influences
cheese ripening principally through its effects on
Copyright © 2004 Elsevier Ltd
All rights reserved
208 Salt in Cheese: Physical, Chemical and Biological Aspects

Table 1 Typical composition of major cheeses (from various sources)

Cheese Moisture (%, w/w) Salt (%, w/w) S/M a (%, w/w) pH

Blue 42 4.5 10.5 6.5

Brick 40 1.9 4.8 6.4
Bulgarian White 32 3.5 10.9 5.0
Camembert 52 2.5 4.8 6.9
Cheddar 37 1.5 4.1 5.5
Edam 43 2.0 4.7 5.7
Emmental 35 0.7 2.0 5.6
Gouda 41 2.0 4.9 5.8
Grana (Parmesan) 31 2.6 8.4 5.4
Gruyère 33 1.1 3.3 5.7
Limburger 45 2.0 4.4 6.8
Muenster 43 1.8 4.2 6.2
Provolone 42 3.0 7.1 5.4
Pecorino Romano 23 5.5 23.9 5.4
Roquefort 40 3.5 8.8 6.4
Domiati 55 6.0 10.9 4.6
Feta 53 3.0 5.7 4.5

a S/M  salt-in-moisture.

water activity but it probably has some more specific
effects also. Among the principal effects of salt are:
• control of microbial growth and activity;
• control of the various enzyme activities in cheese;
• syneresis of the curd and thus in a reduction in
cheese moisture, which also influences the above;
• physical changes in cheese proteins which influence
cheese texture, protein solubility and probably pro-
tein conformation.
Control of Microbial Growth
Probably the most extreme example of the use of NaCl
for this purpose in cheese is in the manufacture of
Domiati cheese from milk to which 12–15%, w/w,
NaCl is added to inhibit bacterial growth and thus
maintain milk quality (Naguib et al., 1979; Sußmuth,
1998; ‘Cheese Varieties Ripened in Brine’, Volume 2).
For all other major varieties, NaCl is added after curd
formation but nevertheless it plays a major role in reg-
ulating and controlling cheese microflora.
The simplest example of this is the contribution of
NaCl to the regulation of cheese pH, which in turn
influences cheese ripening and texture.
The pH of cheese may be regulated by:
• reducing the amount of residual lactose in the curds by
washing the curds with water, as practised in the manu-
facture of Dutch-type, Tallegio and Cottage cheeses;
• the natural buffering capacity of the cheese and the
toxic effect of the lactate anion which establishes a nat-
ural lower limit to pH (⬃4.5), e.g., Blue, Camembert,
hard Italian varieties;
• addition of salt.
The use of salt, together with buffering capacity, to
regulate the final pH appears to be confined almost
exclusively to British-type cheeses, i.e., dry-salted var-
ieties such as Cheddar, Cheshire and Stilton. The curds
for most, if not all, non-British cheeses are placed
in moulds while the pH is still high (6.0) and acid
development continues during pressing. Since a level of
NaCl 1.5%, w/w, inhibits starter activity, such cheeses
are salted by immersion in brine or by surface applica-
tion of dry salt. In British cheeses, the pH has almost
reached its ultimate value at hooping and salt is added
to maintain the pH at that desired value. One could
probably argue that the method of salting cheese that
predominates in a certain region reflects the form of salt
available locally; in regions where salt deposits occur,
dry salt was readily available and thus permitted the
manufacture of cheese in which dry salt was added to
the curd or to the surface of the cheese; in regions
where salt was prepared by evaporation of sea water, it
would have been more convenient to salt the cheese by
immersion in concentrated brine rather than wait for
crystallization.
Curd for Cheddar and similar varieties contains
⬃0.6–1.0%, w/w, lactose at hooping (Turner and Thomas,
1980); this is fermented during the early stages of ripen-
ing by continued starter activity but this depends
strongly on the salt-in-moisture (S/M) level in the curd
and the salt tolerance of the starter. Irvine and Price
(1961) showed that acid development by six commer-
cial lactic acid cultures in reconstituted 10%, w/v, skim
milk powder (RSM) was either stimulated or not
affected by a low level (1%, w/w) of NaCl but was
strongly inhibited by 2.5%, w/w, NaCl. However, even
at 5%, w/w, NaCl, acid was produced by all starters to
 Salt in Cheese: Physical, Chemical and Biological Aspects 209
a level ⬃45–55% of the maximum. In the same study,
portions (454 g) of curd at pH ⬃6.05 were taken after
whey drainage and placed in brine containing 0–5%,
w/w, NaCl at ⬃38 °C for 2 h; a sample of the curd held
in the cheese whey was used as a control sample (it is
assumed that the S/M equilibrium between the brine
and curd moisture was rapid because of the high tem-
perature and the open structure of the mass of curd par-
ticles). The pH decreased to a minimum of 5.53 at 2%,
w/w, NaCl brine but did not fall below 5.9 at 5%, w/w,
NaCl brine; the pH of curd held in 4%, w/w, NaCl brine
was similar to that of curd held in water (0%, w/w NaCl
brine) (Fig. 1). The pH decreased to 5.65, 5.53, 5.62
and 5.90 at 0, 2, 4 and 5%, w/w, NaCl brine, respect-
ively (Fig. 1). These results suggest that starter activity
is stimulated by 2%, w/w, NaCl. Overall, the experi-
ments of Irvine and Price (1961) suggest that the
growth of lactococci in Cheddar curd is generally not
inhibited by #4%, w/w, S/M and that the inhibitory
effect of NaCl is less in curd than in RSM. This conclu-
sion is supported by the results of Schroeder et al.
(1988) who found that varying S/M level from 0.18 to
4.1%, w/w, had little effect on the starter population in
1-day-old Cheddar cheese made with a six-strain cul-
ture of Lactococcus lactis supsp. cremoris.
The pH at which salt was added to RSM and curd
(6.7 and 6.05, respectively) in the study of Irvine and
Price (1961) was much higher than that (⬃5.25–5.35)
at which full-fat Cheddar is salted in practice and, there-
fore, may not reflect the full combined inhibitory effect
of salt and low pH. However, the pH of half-fat Cheddar
which was salted at pH ⬃5.75, rather than at 5.3, to
increase the moisture level (Guinee et al., 1998; Fenelon
5.9
5.85
5.8
5.75
Cheese pH
5.7
5.65
5.6
5.55
5.5
0 1
Concentration of brine, %, w/w, NaCl
Figure 1 Influence of NaCl concentration on the pH of Cheddar cheese curds after holding in the brine for 2 h at 37.7 °C; the pH of
the mass of curd particles at the time of placing in the brine was 6.05 (redrawn from Irvine and Price, 1961).
et al., 1999), decreased to 5.2 at 1 day, i.e., similar to that
of half-fat Cheddar salted at pH 5.3. This observation
confirms the findings of Irvine and Price (1961), i.e.,
that S/M #4%, w/w, has little inhibitory effect on starter
lactococci in Cheddar curd, and suggests that the pH of
Cheddar is controlled by a combination of salting and
buffering capacity. Cheshire cheese has a considerably
lower pH than Cheddar (e.g., typically 4.7–4.9 com-
pared to 5.1–5.3). This low pH is probably primarily due
to a high level of starter (⬃4%) and, consequently, a very
rapid rate of acidification, which causes extensive solu-
bilization of colloidal calcium phosphate and a reduction
in buffering capacity (Lucey and Fox, 1993). As a result,
although Cheshire is salted at a level and at a curd acid-
ity similar to that for Cheddar (Robinson and Wilbey,
1998), the pH of the former is lower, because of
the lower buffering capacity, a higher moisture level, a
higher lactate content and, hence, a higher lactate-
to-buffering ratio.
This importance of S/M in controlling the pH
of Cheddar curd is also evident from the data of
O’Connor (1974). Curd (presumably at ⬃pH 5.3) was
salted at a varying level in the range 0.5–6%, w/w
(Fig. 2). The pH decreased after salting, presumably
due to the action of starter, at S/M levels 5%, w/w,
but starter activity decreased abruptly at higher values
of S/M, and the pH remained high or increased. The
grade assigned to the cheese also decreased sharply at
S/M levels 5%, w/w. The control of pH and lactose
metabolism by S/M concentration in commercial
Cheddar cheese, produced with a linear S/M gradient
of 4–6%, w/w, within a single cheese, was clearly
demonstrated (Fig. 3) by Thomas and Pearce (1981).
2 3 4 5
210 Salt in Cheese: Physical, Chemical and Biological Aspects

28 The above studies show that inhibition of starter

(maximum 30 points) occurs within quite a narrow S/M range (Fig. 2),
emphasizing the importance of precise control of S/M
Total grade score

26
level. However, since the sensitivity of starter cultures
to salt varies, the influence of NaCl concentration on
24 post-salting acid production in cheese obviously
depends on the starter used and a general value for
22 S/M cannot be definitely stated. At pH 5.3, Lactococ-
cus lactis subsp. lactis strains are generally more salt-
tolerant than strains of Lc. lactis subsp. cremoris but
20
there is also considerable variation in salt sensitivity
5.5 between strains of Lc. lactis subsp. cremoris (Martley
and Lawrence, 1972; Turner and Thomas, 1980). If
5.4 starter activity is inhibited after manufacture, residual
lactose will be metabolized by non-starter lactic acid
bacteria (NSLAB). However, the number of NSLAB
5.3
present, which is influenced by the level of contamin-
pH

ation at salting, level of S/M, NSLAB strain, rapidity

5.2
with which pressed curd is cooled and ripening
temperature (Fryer, 1982; Jordan and Cogan, 1993;
5.1 Bechaz et al., 1998), is usually insufficient (e.g.,
#1000 cfu/g) to cause significant lactose metabolism
5.0 for several days and, consequently, the pH falls slowly.
0 1 2 3 4 5 6 7 8 9
In the study by Turner and Thomas (1980), NSLAB,
Salt-in-moisture, %, w/w
mainly Pediococcus, were more salt-tolerant than starter
Figure 2 Relationship between the salt-in-moisture (S/M) level bacteria and metabolized the lactose with the produc-
and the pH () at eight weeks, and between the S/M and the tion of DL-lactate and the racemization of L-lactate.
total grade score (maximum 30) (●) of cheeses made from the Non-starter lactic acid bacteria grew in all cheeses but
same vat but salted at different levels (drawn from data of
O’Connor, 1974, from Lawrence and Gilles, 1982).
their growth was markedly dependent on temperature
and they had little influence on lactose or lactate
concentration until numbers exceeded 106–107 cfu/ml.
The high salt tolerance of NSLAB was confirmed by
1 5.4
Jordan and Cogan (1993) who found that ⬃90% of
NSLAB strains (Lactobacillus casei, Lb. plantarum and
Lb. curvatus) isolated from commercial Cheddar grew
0.8 in the presence of 6%, w/w, NaCl while 58% grew in
5.3
Lactose, g/100 g cheese

the presence of 8%, w/w, NaCl. Similarly, Lane et al.

(1997) reported that ⬃6%, w/w, S/M was required to
0.6 retard the growth of NSLAB in Cheddar cheese and
NSLAB numbers after ripening for 6 months were
pH

5.2
approximately equal at all S/M levels (2.8–6.1%,
0.4 w/w). The greater salt tolerance of NSLAB was clearly
apparent from the study of Thomas and Pearce
5.1
(1981), which showed that the fermentation of lac-
0.2 tose to D-lactate and the racemization of L-lactate in
cheeses with 6%, w/w, S/M occurred relatively late
(90–180 days) during ripening. However, the results
0 5.0 of Bechaz et al. (1998), which showed significantly
4.0 4.5 5.0 5.5 6.0 higher populations of NSLAB in reduced-salt Cheddar
Salt-in-moisture, g/100 g (1.0%, w/w) than in the control (1.8%, w/w), suggest
that salt level has a major effect on the growth of
Figure 3 Effect of salt-in-moisture concentration on lactose
concentration () and pH () within a single block of Cheddar NSLAB. The salt resistance of lactococci and other
analysed at 14 days after manufacture (redrawn from Thomas bacterial species isolated from African cheeses was
and Pearce, 1981). studied in detail by Sußmuth (1998).
 Salt in Cheese: Physical, Chemical and Biological Aspects 211
Although acid production can be uncoupled from cell
growth, it is likely that acid production at low salt levels
will be accompanied by high cell numbers which tend to
lead to bitterness (Lowrie and Lawrence, 1972). Not sur-
prisingly, bitterness in Cheddar cheese is markedly influ-
enced by S/M level over a very narrow range; Lc. lactis
subsp. cremoris HP generally yielded bitter cheese at S/M
levels 4.3%, w/w, but rarely at 4.9%, w/w (Lawrence
and Gilles, 1969).
In the foregoing discussion on the influence of NaCl
on the fermentation of residual lactose in cheese curd
by starter microorganisms, it has been assumed that the
NaCl is distributed throughout the cheese within a
very short period after salting. However, this is not so.
Cheddar cheese curd is usually milled into quite large
particles (chips) of cross-section 2 cm  2 cm or larger.
Obviously, dry salt applied to the surface of such chips
requires a considerable period of time to diffuse to the
centre of the chips and to attain an inhibitory level
throughout (see ‘Factors that influence salt diffusion in
cheese during salting’). Consequently, starter bacteria
will continue to grow and produce acid at the centre of
a chip for a considerable period after growth at the sur-
face has ceased.
Experimental support for this is provided by the
experiments of Hoecker and Hammer (1944) who
measured the levels of salt and moisture and pH at
the surface and centre of individual chips, prised
from a block of Cheddar cheese, over a 72 h period
after salting and pressing. Their data showed that the
pH fell faster and to a lower value at the centre,
where NaCl concentration was lower, than at the sur-
face. In one experiment, the difference in pH per-
sisted for 72 h but in a duplicate experiment the
difference in pH had essentially disappeared after 48 h.
Thomas and Pearce (1981) showed that a higher
level of added salt is required to inhibit lactose
metabolism when the curd is milled into large chips
than smaller ones.
In surface-salted Meshanger cheese, Noomen (1977)
showed considerable zonal variations in lactose and
pH throughout the cheese in response to variations in
S/M concentration. Similarly, Pavia et al. (1999) showed
that a decreasing S/M gradient from the surface (⬃9%,
w/w) to the centre (⬃0.2%, w/w) of freshly brine-
salted Manchego cheese was paralleled by a pH gradi-
ent in the same direction and a lactate gradient in the
opposite direction, indicating inhibition of the starter
culture. Streptococcus salivarius subsp. thermophilus is
considerably less salt-tolerant than Lc. lactis subsp. lac-
tis (Rüegg and Blanc, 1981); its critical NaCl concen-
tration is 0.4 M (2.34%, w/w), corresponding to an aw
of 0.984, compared with 1.1 M NaCl (aw  0.965) for
Lc. lactis subsp. lactis; Lb. delbrueckii subsp. helveticus
and Lb. lactis subsp. lactis were also less salt tolerant,
being inhibited by 0.95 M and 0.90 M NaCl, respectively.
Data on the sensitivity of propionibacteria to NaCl
appear to be variable: Orla-Jensen (1931) reported that
concentrations of NaCl as low as 0.5%, w/w, are suffi-
cient to reduce the growth of Propionibacterium in a
medium containing calcium lactate. However, Antila
(1954) reported that 3%, w/w, NaCl is necessary to
reduce growth. In fact, salt tolerance appears to be
strain- and pH-dependent (Rollman and Sjostrom,
1946); in a lactate medium, 6%, w/w, NaCl was req-
uired to inhibit the growth of a fast-growing strain of
Propionibacterium at pH 7.0 and 3%, w/w, at pH 5.2,
whereas a slow-growing strain was more salt-tolerant at
pH 5.2 than at pH 7.0. The data of Rüegg and Blanc
(1981) show that P. shermanii was the most salt tolerant
of the starter species investigated; its critical NaCl con-
centration was 1.15 M (⬃6.7%, w/w; aw  0.955).
Boyaval et al. (1999) studied the effect of varying NaCl
concentration (0–0.8 M; ⬃0.0–4.8%, w/w, S/M) on the
growth of P. freudenreichii subsp. shermanii CIP 103027
in a chemically-defined medium (modified M63), Yeast
Extract-Lactate medium (YEL), or 10% (w/v) reconsti-
tuted skim milk at pH 7.0 and at 30 °C under quiescent
conditions. Increasing NaCl concentration in modified
M63 resulted in a progressive decrease in cell growth,
with a 50% reduction at 0.3 M and almost complete
inhibition at 0.7 M. Growth inhibition was due to the
osmotic effect rather than NaCl per se, as reflected by the
similar inhibition of growth on substitution of NaCl by
KCl or sucrose. In YEL or milk, 0.79 and 0.25 M NaCl,
respectively, were needed to double the generation time,
and concentrations of 1.5 M and 1.0 M to effectively
inhibit growth completely. Differences in the effect of
salt on growth rate in the three media were attributed to
the presence of different types and levels of osmoprotect-
ive compounds, such as choline and glycine-betaine in
YEL and various choline derivatives and carnitine in
milk. While some inhibition of P. shermanii is expected
in Emmental cheese, the aqueous phase of which has an
osmolarity ⬃0.7 M NaCl (Salvat-Brunaud et al., 1997),
the presence of osmoprotective compounds in milk
assists growth (Boyaval et al., 1999). Interestingly,
Emmental cheese, which contains ⬃0.7%, w/w, is the
least heavily salted among major cheese varieties.
Blue cheeses are among the most heavily salted var-
ieties, with 3–5%, w/w, NaCl (Stilton 3%, w/w).
Ripening in these varieties is dominated by Penicillium
roqueforti and consequently good growth of this mould
is paramount. Germination of P. roqueforti spores is
stimulated by 1%, w/w, NaCl but inhibited by 3–6%,
w/w, NaCl, depending on strain. However, the growth of
germinated spores on malt extract agar or in cheese
curd is less dependent on NaCl concentration than is
212 Salt in Cheese: Physical, Chemical and Biological Aspects
germination, and some strains grow in cheese curd
containing 10%, w/w, NaCl, although growth is retarded
compared to that in curd containing less NaCl
(Godinho and Fox, 1981a,b). Morris (1981) reported
that it is fairly common commercial practice to add 1%,
w/w, NaCl to Blue cheese curd before hooping, possibly
to stimulate spore germination, although it also serves
to give the cheese a more open structure which facili-
tates mould growth. Since most Blue cheeses are surface-
salted, a salt gradient from the surface to the centre
exists for a considerable period after manufacture; a
high initial level of salt in the outside zone of the cheese
may inhibit spore germination at a critical time and a
mould-free zone at the outside is a common defect in
Blue cheeses (Godinho and Fox, 1981b).
Growth of P. camemberti is also stimulated by low levels
of NaCl; 0.8%, w/w, NaCl, mould growth on Camembert
cheese is poor and patchy (O’Nulain, 1986).
Influence of NaCl on Enzyme Activity
in Cheese
Coagulant
With the exception of Emmental and similar high-
cooked cheeses, the initial proteolysis in cheese is
catalysed by residual coagulant. Polyacrylamide gel
electrophoresis of cheese during ripening has shown
that in hard and semi-hard, bacterially-ripened cheeses,
s1-casein undergoes considerable proteolysis but
-casein remains unchanged until an advanced stage of
ripening (Ledford et al., 1966; Phelan et al., 1973;
Creamer, 1975; Visser and de Groot-Mostert, 1977; Yun
et al., 1995; Kristiansen et al., 1999; Fenelon and
Guinee, 2000; Feeney et al., 2001; ‘Proteolysis in Cheese
during Ripening’, Volume 1). A similar pattern is evident
during the early phases of mould-ripened cheeses, when
the coagulant is the principal ripening agent (Godinho
and Fox, 1982; Hewedi and Fox, 1984) but fungal pro-
teinases dominate in these cheeses during the later
phases of ripening (see ‘Surface Mould-ripened Cheeses’
and ‘Blue Cheese’, Volume 2).
The hydrolysis of s1-casein by milk clotting enzymes
is greatly influenced by the concentration of NaCl. The
proteolytic activity of chymosin, pepsins, Rhizomucor
miehei and Cryphonectria parasitica rennets on dilute
casein fractions is stimulated by increasing NaCl concen-
tration to an optimum at ⬃6%, w/w (Fox and Walley,
1971; Gouda, 1987). Activity is inhibited at higher NaCl
levels, but limited proteolysis of s1-casein occurs up to
20%, w/w, NaCl (Fox and Walley, 1971; Gouda, 1987).
However, degradation of s1-casein is retarded by
very low levels of salt in Cheddar (Phelan et al., 1973;
Thomas and Pearce, 1981; Kelly et al., 1996; Mistry and
Kasperson, 1998) and on salting at 1.36%, w/w (S/M 
2.55%, w/w) in Mozzarella (Guo et al., 1997). The
inverse relationship between casein degradation and salt
concentration in cheese is mirrored by the reduction in
the level of pH 4.6-soluble N and/or water-soluble N (as %,
w/w, total N), and/or NPN in Blue (Godinho and Fox,
1982), Camembert (O’Nulain, 1986; Table 2), Cheddar
(Thakur et al., 1975; Kelly et al., 1996), Danbo (Kris-
tiansen et al., 1999), Ragusano (Licitra et al., 2000),
Romano (Guinee and Fox, 1984; Fox and Guinee,
1987), Feta (Pappas et al., 1996) and other cheeses (Wis-
niewska et al., 1990). In contrast to the above trends, the
level of water-soluble N in serum expressed on centrifu-
gation of unsalted low-moisture Mozzarella cheese at its
unadjusted pH is significantly lower than that from
salted cheese (Guo et al., 1997). However, the higher
water-soluble N in the former is due to an increase
in casein hydration as a result of a salting-in-effect at a
S/M level of ⬃2.6%, w/w (see ‘Effect of NaCl on casein
hydration in model systems and in cheese’) rather than
to proteolysis which is very low in low-moisture Moz-
zarella. The latter effect is somewhat similar to the large
increase in soluble N obtained on adding sodium citrate
or sodium phosphate emulsifying salts during the manu-
facture of processed cheese, even though the level of
pH 4.6-soluble N remains essentially constant (see
‘Pasta-Filata Cheeses’, ‘Pasteurized Processed Cheese and
Substitute/Imitation Cheese Products’ and ‘Cheese as an
Ingredient’, Volume 2).
In contrast to the trends noted for s1-casein, proteo-
lysis of -casein in dilute solution by chymosin or
pepsins is strongly inhibited by 5%, w/w, and com-
pletely inhibited by 10%, w/w, NaCl (Fox and Walley,
1971; Lane and Fox, 1999). Sucrose and glycerol selec-
tively inhibit proteolysis of -casein by chymosin and
pepsins (Creamer, 1971; Al-Mzaien, 1985). KCl, LiCl,
NH4Cl and CaCl2 are as effective as NaCl in inhibit-
ing the proteolysis of -casein (O’Nulain, 1986). Since
the inhibitory effect of solutes is substrate- rather
than enzyme-specific, it appears that NaCl and similar
solutes cause some conformational changes in -casein
(Barford et al., 1988) which render its chymosin (pepsin)-
susceptible bonds less accessible to the enzyme. The
nature of these conformational changes does not appear
to have been investigated but may arise from the
strongly hydrophobic nature of -casein.
-Casein undergoes significantly less breakdown
than s1-casein in most cheese varieties. The resist-
ance of -casein in cheese to proteolysis is not depend-
ent solely on the salt concentration since it is also quite
resistant to proteolysis in salt-free, and low S/M (e.g.,
2.7%, w/w) cheese (Phelan et al., 1973; Kelly et al.,
1996), suggesting that a high protein concentration is
sufficient to induce the necessary conformational
 Salt in Cheese: Physical, Chemical and Biological Aspects 213

Table 2 Influence of NaCl on pH and proteolysis in Camembert cheese (4-weeks old)a

Water-soluble N pH 4.6-soluble N 70% ethanol-soluble 5% PTA-soluble N

NaCl (%, w/w) Zoneb pH (% total N) (% total N) N (% total N) (% of total N) c

0.20 I 5.5 36.0 43.3 23.7 16.8

O 6.4 100.0 54.4 35.7 18.9
0.70 I 5.3 28.7 29.1 15.8 10.4
O 6.1 100.0 39.2 28.7 15.5
0.93 I 5.2 17.9 17.3 13.3 12.1
O 6.0 100.0 49.5 32.8 15.2
1.14 I 5.2 22.5 23.8 15.8 8.1
O 6.2 93.7 43.4 28.4 10.4
1.73 I 5.1 26.6 28.3 15.8 8.8
O 6.4 85.3 37.1 22.7 10.1
5.0
2.4 I 5.1 22.2 23.1 18.0 8.3
O 5.0 63.2 29.8 26.1 9.3
6.3

a Compiled from data of O’Nulain (1986).

b I and O correspond to the inner and outer portions of the cheese.
c PTA  phosphotungstic acid.

change(s). A level of S/M 4.9%, w/w, is necessary
to prevent the development of bitterness in cheese
(Lawrence and Gilles, 1969).
The inhibitory effect of NaCl on proteolysis of
sodium caseinate, s1-casein and -casein is pH-
dependent, with the extent of inhibition generally
decreasing with pH in the range 6.6–5.4 (Fox and
Walley, 1971; Mulvihill and Fox, 1980; Lane and Fox,
1999). At low pH, NaCl also alters the proteolytic
specificity of chymosin and pepsins: NaCl (2.5%, w/w)
inhibits the formation of -III but promotes the for-
mation of -IV and -V (Mulvihill and Fox, 1978).
Formation of the s1-casein peptides, s1-VII and
s1-VIII, in solution is stimulated by NaCl (5%, w/w)
and these peptides are also formed in cheese (Mulvihill
and Fox, 1980). The proteolytic activity of R. miehei and
C. parasitica rennets on -casein is less strongly
inhibited by NaCl than that of chymosin or pepsins
(Phelan, 1985; Gouda, 1987).
Milk proteinase
Milk contains several indigenous proteinases, the most
significant, alkaline milk proteinase (plasmin), is almost
exclusively associated with the casein micelles at the
normal pH of milk, but dissociates from the micelles as
the pH is reduced (Humbert and Alais, 1979; Fox,
1981; Visser, 1981; Reimerdes, 1982; Grufferty and Fox,
1988a; Sousa et al., 2001; Nielsen, 2002; Visser and van
den Berg, 2002). Richardson and Elston (1984) reported
that the dissociation of plasmin from the casein micelles
is pH- and time-dependent and that it occurs at pH 5.7
and possibly higher, but Grufferty and Fox (1988a)
found no dissociation on holding at a pH 4.9 for 4 h.
This implies that all the plasmin in milk should be
present in the curd for most rennet-coagulated cheese
varieties. However, the concentration of plasmin in
Swiss-type cheese is two to three times that in Cheddar
(Richardson and Pearce, 1981; Lawrence et al., 1983)
while the activity in Cheshire cheese is very low
(Lawrence et al., 1983), suggesting that the plasmin
content of cheese may be influenced by the pH at
hooping (Lawrence et al., 1983). The difference in
plasmin level between Cheddar and Swiss cheeses is
considered unlikely to be due to pH dependent dissoci-
ation of the enzyme as the pH of both cheeses is 6.1
and 6.4 at whey drainage (Grufferty and Fox, 1988b).
The differences may to be due to different rates of plas-
minogen activation in the two cheeses due to different
processing conditions, especially cooking temperature
(Ollikainen and Nyberg, 1988; Farkye and Fox, 1990),
and possibly the higher pH in Swiss cheese during
ripening (Grufferty and Fox, 1988b). The increase in
pH in Swiss-type cheese during ripening is paralleled
by a large increase in plasmin activity (Ollikainen and
Nyberg, 1988). Owing to the relatively high buffering
capacity of Swiss-type cheese (as affected by the reten-
tion of colloidal calcium phosphate due to the rela-
tively high pitching pH (i.e., ⬃6.4 compared to ⬃6.1
for Cheddar)), its relatively high protein level (i.e.,
⬃29 compared to 24% for Cheddar) and the propionic
acid fermentation, during which lactic acid is con-
verted to the weaker propionic and acetic acids, the pH
214 Salt in Cheese: Physical, Chemical and Biological Aspects
of Swiss does not fall as low as, and rises more rapidly
than, that of Cheddar.
The role of plasmin in cheese ripening has not been
studied extensively but the presence of -caseins in most
cheese suggests at least some activity (see Farkye and
Fox, 1992). Plasmin appears to make a significant con-
tribution to the maturation of Gouda (Creamer, 1976;
Visser and de Groot-Mostert, 1977), possibly because of
the removal of proteinase inhibitors by washing during
curd manufacture, and in Romano-type cheese (Guinee
and Fox, 1984) and in Swiss (Richardson and Pearce,
1981; Sweeney, 1984; Ollikainen and Nyberg, 1988;
Ollikainen and Kivelä, 1989) in which the coagulant is
extensively denatured by the high cooking temperature
(Matheson, 1981). However, it has only a limited role in
the ripening of Cheddar (Green and Foster, 1974;
Creamer, 1976; Fenelon and Guinee, 2000; Kubis et al.,
2001) and soft Meshanger-type cheese (Noomen, 1978).
The coagulant is also extensively denatured in low-
moisture Mozzarella because of plasticization of the curd
at ⬃58–60 °C (Feeney et al., 2001; see ‘Pasta-Filata
Cheeses’, Volume 2), but plasmin makes little contribu-
tion to proteolysis, as reflected by the low levels of
-casein (Yun et al., 1993a; Feeney et al., 2001) except
where C. parasitica, a rennet substitute with a high pro-
teolytic activity on -casein, is used (Yun et al., 1993b).
However, degradation of -casein is very substantial in
Mozzarella made from milk pre-acidified to ⬃5.6 prior
to rennet addition (Feeney et al., 2002; Guinee et al.,
2002). This effect concurs with the findings of Grufferty
and Fox (1988b) who reported no dissociation of plas-
min from the casein micelles in milk at pH 4.9.
Noomen (1978) suggested that plasmin may make a sig-
nificant contribution to proteolysis in soft cheeses with a
surface flora, in which the pH rises markedly during
ripening to a value more favourable for plasmin activity.
The presence of -caseins in Camembert-type cheese,
Cooleeney (Sousa and McSweeney, 2001) and blue-
veined cheeses, including Stilton, Danablu, Cashel,
Chetwynd and Gorgonzola (Zarmpoutis et al., 1998),
suggests a high level of plasmin activity, which is
expected in view of the high pH of these cheeses. The
addition of plasmin to milk, at levels which increased
the activity by 3- to 4-fold the indigenous level normally
found in the cheese, resulted in increased degradation of
-casein and level of pH 4.6-soluble N (Farkye and Fox,
1992); the organoleptic quality of the plasmin-enriched
cheese was superior to that of the control and the ripen-
ing rate was accelerated considerably.
Noomen (1978) showed that the activity of alkaline
milk proteinase in simulated cheese was stimulated by
concentrations of NaCl up to a maximum at 2%, w/w,
but was inhibited by higher concentrations of NaCl,
although some activity remained at 8%, w/w, NaCl.
Milk also contains an acid proteinase, cathepsin D,
which apparently has a specificity similar to chymosin
(Kaminogawa and Yamauchi, 1972; Kaminogawa et al.,
1980; Larsen and Petersen, 1995; Hurley et al., 2000a).
About 80% of cathepsin D is in the serum and although
it partially survives pasteurization (Larsen et al., 2000),
its contribution to proteolysis in most cheeses is prob-
ably low. However, it has been claimed to make a con-
tribution to proteolysis in Feta coagulated with GDL
(Wium et al., 1998) or in Quarg (Hurley et al., 2000b).
To our knowledge, the influence of NaCl on the activ-
ity of acid milk proteinases has not been investigated.
Microbial enzymes
There appears to be relatively little information on the
influence of NaCl on microbial enzymes in cheese.
Indirect evidence, e.g., in relation to bitterness in
cheese (Lawrence and Gilles, 1969; Sullivan and
Jago, 1972; Stadhouders and Hup, 1975; Thomas and
Pearce, 1981) suggests that the activity of starter pro-
teinase is inhibited by a moderately high level of NaCl.
P. roqueforti lipases (Morris and Jezeski, 1953) and
proteinases (Madkor, 1985) are inhibited by NaCl
concentrations 6%, w/w. Vafopoulou-Matrojiannaki
(1999) found that an increase in S/M from 3 to 6%,
w/w, reduced the activity of intracellular aminopep-
tidase, dipeptidylaminopeptidase and carboxypepti-
dase, but had little effect on the intracellular esterase
activity of Leuconostoc mesenteroides subsp. mesen-
teroides strain K1G8.
Gobbetti et al. (1999a) studied the interactive effects
of pH (5.5–7.0), S/M (0.0–7.5%, w/w) and temperature
(4–16 °C), under conditions designed to simulate the
cheese environment, on the peptidase activities (amino-
peptidases N and A, and proline iminopeptidase) of 11
strains of NSLAB bacteria isolated from cheese: Lb. casei
subsp. casei 2107, 2756, 2788; Lb. plantarum 2788,
2789, 2741; Lb. casei subsp. pseudoplantarum 2745 and
2742; and Lb. curvatus 2771 and 2770. A low pH and a
high S/M level markedly inhibited the peptidases of
Lb. casei subsp. pseudoplantarum and Lb. curvatus. In
contrast, the peptidases of Lb. casei subsp. casei and
Lb. plantarum were quite insensitive to pH and not very
sensitive to NaCl. The aminopeptidase activities (espe-
cially A) of the latter strains were less sensitive than the
proline iminopeptidases to the combined effects of salt,
temperature and pH. In a subsequent study, Gobbetti
et al. (1999b) investigated the effects of S/M (2.5 to
7.5%, w/w), pH (5.0–5.7) and aw on the proteolytic and
lipolytic activities of starter and NSLAB, including
Lb. delbrueckii subsp. bulgaricus, Lc. lactis subsp. lactis
T12 and Lb. plantarum 2739. The effect of S/M was both
enzyme- and species-specific. These authors concluded

that interactions between these three variables were
mainly responsible for changes in enzyme activity
under conditions simulating cheesemaking. The com-
bined effects of NaCl and pH did not significantly
influence the lipase/esterase activity of Lb. plantarum
2739 and it was suggested that strains like 2739 might
be responsible for a moderate level of lipolysis during
long-term ripening of cheese.
The cell envelope-associated proteinase (lactocepin)
of Lc. lactis subsp. lactis BN1 and Lc. lactis subsp. cre-
moris SK11 was stabilized by NaCl (5%, w/v), espe-
cially at pH 5.2 (i.e., cheese-like conditions) and by the
humectants, polyethylene glycol and sorbitol (Reid and
Coolbear, 1998, 1999). The specificity of both proteinases
on s1-, - and -caseins was changed considerably by
NaCl level and water activity.
Influence of NaCl on the Water Activity (aw)
of Cheese
The preservative action of NaCl is due to its effect on
the water activity (aw) of the medium:
p NaCl per litre of H2O and [NaCl] is the concentration
aw 
po
where p and po are the vapour pressure of the water in
a system and of pure water, respectively. If the system
is at equilibrium with its gaseous atmosphere, then
aw  ERH/100, where ERH is the equilibrium relative
humidity.
Due to the presence of various solutes in foods, the
vapour pressure of water in a food system is always
less than that of pure water, i.e., aw 1.0. The rela-
tionship between aw and the moisture content of food
is shown in Fig. 4. Three zones are usually evident:
• Zone I represents monolayer water that is tightly
bound to polar groups in the food, e.g., the 9OH
I II
g water/g dry matter
0 0.25 0.5
Water activity, aw
Figure 4 Idealized relationship between the water activity (aw)
of food and its water content.
Salt in Cheese: Physical, Chemical and Biological Aspects 215
group of carbohydrates, or the 9NH 3 and 9COO
groups of proteins;
• Zone II consists of multilayer water in addition to
the monolayer water;
• Zone III contains bulk phase water in addition to
monolayer and multilayer water.
Comprehensive discussions on the general concept
of water activity in relation to foods are provided by
Duckworth (1975), Rockland and Stewart (1981),
Simatos and Multon (1985), Rockland and Beuchat
(1987) and Fennema (1996). More specific aspects in
relation to dairy products are discussed by Kinsella
and Fox (1986) and Roos (1997).
The aw of food depends on its moisture content
and the concentration of low molecular mass solutes
(Russell and Gould, 1991). The aw of young cheese is
determined almost entirely by the concentration of
NaCl in the aqueous phase:
aw  1 0.033 [NaClm]  1 0.00565 [NaCl]
where [NaClm] is the molality of NaCl, i.e., moles
of NaCl as g/100 g cheese moisture (Marcos, 1993).
This equation was used to construct the nomograph
shown in Fig. 5, which facilitates the calculation of aw.
The salt content of cheese varies from ⬃0.7%, w/w,
for Emmental to ⬃5%, w/w, for Domiati (Table 1).
Other compounds, including lactic and other acids,
amino acids, very small peptides and calcium phos-
phate, in addition to NaCl, contribute to the depression
60
1.00 0
55 1
0.99
50 0.98
III
2
45 0.97
40 0.96
3
% H2O aw % NaCl
0.75 1
Figure 5 Nomograph for estimation of water activity (aw) of fresh
cheese from the percentages of moisture and salt. Examples: for
respective salt and moisture levels (%, w/w) of 57 and 1.5, or 44.5
and 2.0, respectively, then aw  0.95 or 0.974, respectively (from
Marcos and Esteban, 1982).
216 Salt in Cheese: Physical, Chemical and Biological Aspects

of aw, especially in extra-mature cheeses. Salt increases
the osmotic pressure of the aqueous phase of foods,
causing dehydration of bacterial cells, killing them or,
at least, preventing their growth. The minimum water
activity for the growth of various microorganisms in
foods is shown in Table 3. Typical values for the aw of
some cheese varieties are shown in Table 4. It will be
apparent from Table 4 that the aw of most cheese vari-
eties is not low enough to prevent the growth of yeasts
and moulds and many bacteria but in combination
with a low pH and low temperature, is quite effective in
controlling microbial growth.
Measurement of the salt content of cheese is an
important quality control step in cheese production. As
Table 3 Water activity (aw) of some cheese varieties*
described above, the aw of cheese can be calculated
from its composition but can also be determined exper-
imentally (see Marcos, 1993).
The concentration and distribution of salt in cheese
have a major influence on various aspects of cheese
quality, as discussed in ‘Introduction’, ‘Control of
Microbial Growth’, ‘Influence of NaCl on Enzyme
Activity in Cheese’, ‘Influence of NaCl on the Water
Activity (aw) of Cheese’ and ‘Overall Influence of NaCl
on Cheese Ripening and Quality’.
The aw of cheese, factors that affect it and related
aspects have been reviewed by Acker (1969), Rüegg
and Blanc (1977, 1981), Streit et al. (1979), Rockland
and Nishi (1980), Marcos et al. (1981), Rüegg (1985),
Fernandez-Salguero et al. (1986), Larsen and Anon
(1989a,b, 1990), Marcos (1993) and Hardy (2000).

aw Cheese Overall Influence of NaCl on Cheese

1.00 Fresh cheese curd, Ricotta
Ripening and Quality
0.99 Beaumont, Cottage, Fresh, Quarg Cheddar cheese
0.98 Belle des Champs, Münster, Pyrénées,
Processed, Taleggio The influence of salt-in-cheese moisture (S/M) on lac-
0.97 Brie, Camembert, Emmental, Fontina, Limburger, tose metabolism in young Cheddar cheese has already
Saint Paulin, Serra da Estrêla
been discussed. There appears to be little information
0.96 Appenzeller, Chaumes, Edam, Fontal, Havarti,
Mimolette, Norvegia, Samsø, Tilsit available on the influence of %, w/w, S/M on lipolysis
0.95 Bleu de Bresse, Cheddar, Gorgonzola, Gouda, in Cheddar and other cheeses. However, Thakur et al.
Gruyère, Manchego (1975) compared lipolysis in salted (1.48–1.79%, w/w,
0.94 Idiazábal, Majorero, Mozzarella, Norzola, NaCl) and unsalted Cheddar; the concentration of
Raclette, Romano, Sbrinz, Stilton
volatile acids was significantly higher in the unsalted
0.93 Danablu, Edelpilzkäse, Normanna, Torta del
Casar than in the salted cheese mainly due to acetic acid,
0.92 Castellano, Parmesan, Roncal, Zamorano which is presumably a product of lactose metabolism.
0.91 Provolone, Roquefort The concentrations of all individual fatty acids, except
0.90 Cabrales, Gamalost, Gudbrandsdalsost, Primost linoleic and linolenic (at certain ages), were also
higher in the unsalted cheese than in the control; the
* Compiled from various sources.
authors did not comment on the markedly lower lev-
els of linoleic acid in the unsalted cheese. However,
Lindsay et al. (1982) found little difference between
Table 4 Minimum water activity (aw) for microbial growth in the level of free fatty acids in cheeses with low (3.5%,
foods* w/w) or intermediate (4–2%, w/w) S/M levels except
for myristic and palmitic acids which were consider-
Pathogen Minimum aw
ably higher in the higher-salt cheese. Reduced-sodium
Shigella spp. 0.96 cheeses will be discussed in more detail in ‘Reduced
Yersinia enterocolitica 0.96 Sodium Cheese’.
Vibrio parahaemolyticus 0.94 Proteolysis is considerably more extensive in unsalted
Pseudomonas spp. 0.95
than in salted Cheddar cheese and consequently
E. coli 0.95
Clostridium botulinum 0.94 the body of the former is less firm (Thakur et al.,
Salmonella spp. 0.94 1975; Thomas and Pearce, 1981; Schroeder et al., 1988;
Listeria monocytogenes 0.92 Kelly et al., 1996). Wisniewska et al. (1990) reported
Micrococcus spp. 0.87 that the salt content Cheddar, Gouda, Tilsit, Roquefort
Staphylococcus aureus (aerobic) 0.86
and Camembert was inversely related to the levels of
Most yeasts and moulds 0.80
Osmophilic yeasts and moulds 0.55 primary and secondary proteolysis and directly to the
time required to attain proper ‘organoleptic’ character-
* Compiled from various sources. istics; the authors suggested that reducing the salt
 Salt in Cheese: Physical, Chemical and Biological Aspects 217
content may offer a possible means of accelerating
cheese ripening. However, as discussed below, a low
level of NaCl has been found to adversely affect the
quality of Cheddar cheese and a relatively narrow
desirable range has been prescribed for premium qual-
ity (see also ‘Cheddar Cheese and Related Dry-salted
Cheese Varieties’, Volume 2). A linear relationship
between the extent of degradation of both s1- and
-caseins in young (1 month) cheese and %, w/w, S/M
is apparent from the data of Thomas and Pearce
(1981) and Kelly et al. (1996). During the normal
ripening of Cheddar cheese, sl-casein is the principal
substrate for proteolysis with little degradation of
-casein (see ‘Coagulant’); proteolysis of -casein is
more extensive at low salt levels (Phelan et al., 1973;
Kelly et al., 1996). However, Thomas and Pearce
(1981) noted that while the normal products of
-casein degradation (-CNf1-192, -CNf1-189 and
-CNf1-165 produced by rennets, and -caseins by
milk proteinase) were not apparent in their studies,
the concentration of unhydrolysed -casein decreased,
suggesting that proteolysis of -casein in low-salt
cheese may be due to bacterial proteinases. Kelly et al.
(1996) noted that cleavage of Leu192–193 in -casein
and Leu101–Lys102 in s1-casein was particularly sensi-
tive to the salt concentration in Cheddar cheese. In
contrast to primary proteolysis, the level of secondary
proteolysis, as measured by the level of 5% (w/v)
phosphotungstic acid-soluble N, tended to be higher
in salted (2.7–5.7%, w/w, S/M) than in unsalted Ched-
dar at 12 and 24 weeks; no effect of S/M was apparent
at 5 weeks.
At least five studies (O’Connor, 1971; Gilles and
Lawrence, 1973; Fox, 1975; Pearce and Gilles, 1979;
Lelievre and Gilles, 1982) have attempted to relate the
quality of Cheddar cheese to its composition. While
these authors agree that the moisture content, %, w/w,
S/M and pH are the key determinants of cheese quality,
they disagree as to the relative importance of these
three parameters.
In a study of 300 Scottish Cheddar cheeses, O’Connor
(1971) found that flavour and aroma, texture and total
score were not correlated with moisture content but
were significantly correlated with %, w/w, NaCl and
particularly with pH. Salt content and pH were them-
selves strongly correlated, as were salt and moisture; a
very wide variation in composition was noted. Based
on analysis of cheese made at the New Zealand Dairy
Research Institute over many years and also by com-
mercial cheese factories in New Zealand, Gilles and
Lawrence (1973) proposed a grading scheme for
young (14-day-old) Cheddar cheese. The influence of
cheese composition on quality and compositional
grading of Cheddar cheese is discussed in ‘Factors that
Affect the Quality of Cheese’, Volume 1 and ‘Cheddar
Cheese and Related Dry-salted Cheese Varieties’, Volume
2; suffice it to record here that the S/M specified for
premium and First Grade Cheddar in New Zealand are
4.0–6.0 and 4.7–5.7, respectively (Lawrence et al.,
1993).
Fox (1975) assessed the influence of moisture, salt
and pH on the grade of 123, 10-week-old Irish Ched-
dar cheeses (70 high quality and 53 ‘rejects’) from six
factories and 27 extra-mature, high-quality Cheddars.
The composition of the cheeses varied widely and
while the correlations between grade and any of the
compositional factors were poor, a high percentage of
cheeses with compositional extremes was down-
graded, especially those with low salt (1.4%, w/w),
high moisture (39%, w/w) or high pH (pH 5.4). In
the samples studied, salt concentration seemed to
exercise the strongest influence on cheese quality and
the lowest percentage of downgraded cheeses can be
expected in the salt range 1.6–1.8%, w/w (S/M range,
4.0–4.9%, w/w). The composition of high quality extra-
mature cheeses also varied widely but less than that of
the young cheeses. Although the mean salt level was
identical for both groups of cheeses, the spread was
much narrower for the mature cheeses and only three
had 1.7%, w/w, NaCl. The mean moisture content of
the mature cheeses was 1%, w/w, lower than that of
the regular cheeses.
The grading ratio (ratio of high to low grading
cheeses) for 486 14-day-old cheeses produced at the
New Zealand Dairy Research Institute was most highly
correlated with the percentage of moisture in non-
fat-substances (MNFS) and second best with the
percentage of salt (Pearce and Gilles, 1979). The opti-
mum compositional ranges were: MNFS 52–54%, w/w;
S/M 4.2–5.2%, w/w; pH 4.95–5.15. Cheese with an
S/M of 3.1%, w/w, received the highest grade in a study
by Knox (1978) although there was little difference in
grade in the S/M range 3.1–5.2%; quality declined
markedly at S/M 6.4%, w/w.
A very extensive study of the relationship of the
grade and composition of nearly 10 000 cheeses pro-
duced in five commercial New Zealand factories was
undertaken by Lelievre and Gilles (1982). As in previ-
ous studies, considerable compositional variation was
evident but the variation was considerably less for
some factories than others. While the precise relation-
ship between grade and composition varied from plant
to plant, certain generalizations emerged:
• within the compositional ranges suggested by Gilles
and Lawrence (1973) for ‘premium’ quality cheese,
composition does not have a decisive influence on
grade, which falls off outside this range;
218 Salt in Cheese: Physical, Chemical and Biological Aspects
• composition alone does not provide a basis for grad-
ing as currently acceptable to the dairy industry
(New Zealand);
• MNFS was again found to be the dominant factor
influencing quality;
• within the recommended compositional bands,
grades declined marginally as MNFS increased from
51 to 55%, w/w, increased slightly as S/M decreased
from 6 to 4%, w/w, while pH had no consistent effect
within the range 4.9–5.2 and FDM had no influence
in the range 50–57%, w/w. The authors stress that
since specific inter-plant relationships exist between
grade and composition, each plant should determine
the optimum compositional parameters pertinent to
that plant.
Apart from the acid flavour associated with low-salt
cheese, bitterness has been reported consistently as a
flavour defect in such cheeses. A complex correlation
exists between the propensity of a cheese to develop
bitterness and starter culture, pH, rate of acid develop-
ment and %, w/w, S/M. There is still some controversy
on the development of bitterness (see Lowrie and
Lawrence, 1972; Mills and Thomas, 1980; Stadhouders
et al., 1983; Gomez et al., 1997; McSweeney, 1997;
Smit et al., 1998, 2002; Kirin, 2001; Morales et al.,
2001; Broadbent et al., 2002), but the subject will not
be reviewed here.
From the compositional viewpoint, S/M, %, w/w,
appears to be the most important factor influencing bit-
terness (Lawrence and Gilles, 1969). The probability of
bitterness developing is greatly increased at S/M 4.9%,
w/w; pH, in the normal range encountered for Ched-
dar, i.e., 4.9–5.3, where para-casein is most soluble
(Creamer, 1985) and therefore most susceptible to pro-
teolysis, has little effect except at low S/M values, i.e.,
4.9%, w/w. Rennet has maximum activity on para-
casein in salt solutions between 2.5 and 4%, w/w
(Stadhouders, 1962). The bitterness of peptides is
strongly correlated with hydrophobicity (Guigoz and
Solms, 1976; Bumberger and Bleitz, 1993). The bitter
peptides in cheese appear to arise primarily from
-casein (see Hill et al., 1974; Visser et al., 1983a,b;
Dinakar et al., 1989; Vandeweghe, 1994; Casal and
Gomez, 1999; Frister et al., 2000) which might be
expected since -casein is the most hydrophobic
casein (Swaisgood, 2003); however, peptides from
s1- and s2-caseins, especially those containing pro-
line, probably also contribute to bitterness in cheese
(Lee and Warthesen, 1995; Kai Ping Lee, 1996; Frister
et al., 2000). The effectiveness of NaCl in preventing
bitterness is very likely due to the strong inhibition of
-casein hydrolysis by NaCl (Fox and Walley, 1971;
Phelan et al., 1973; Mulvihill and Fox, 1978; Pearce,
1982; Stadhouders et al., 1983; Gouda, 1987; Banks
et al., 1993; Kelly et al., 1996; Mistry and Kasperson,
1998). However, Laan et al. (1998) found that the
addition of salt (4%, w/w) and Ca (120 mM), at levels
to simulate those in cheese, increased the aminopep-
tidase activity of starter lactococci and non-starter
lactobacilli isolated from Cheddar cheese. Such
aminopeptidase activities, which debitter -casein
hydrolysates (Kai Ping Lee, 1996; Parra et al., 1999;
Barry et al., 2000; Bouchier et al., 2001), are considered
important in reducing the risk of bitterness in cheese.
The protein matrix in young cheese appears to con-
sist of s1-casein molecules linked through hydropho-
bic interactions between their amino-terminal regions;
the primary site for rennet action on s1-casein is
Phe239Phe24 (Hill et al., 1974) or Phe249Val25
(Creamer and Richardson, 1974), hydrolysis of which
weakens the matrix. This specific cleavage is consid-
ered to be primarily responsible for the loss of firm-
ness during the early stages of ripening (de Jong,
1976; Creamer and Olson, 1982; Fenelon and Guinee,
2000; ‘Cheese as an Ingredient’, Volume 2). Hard and
semi-hard cheese, such as Cheddar, becomes shorter
also during maturation (Visser, 1991; Fenelon and
Guinee, 2000). Luyten (1988) found that increased
s1-casein breakdown in Gouda cheese had little effect
on shortness (which may be best described as the
inverse of fracture strain). Indeed, the increase in
shortness of Gouda cheese on ripening was attributed
more to in-depth proteolysis (e.g., NPN formation)
than to gross proteolysis. The increase in shortness
with maturation may arise as a result of an upward
shift in pH away from pH 5.2–5.35 where casein
hydration as a function of pH in the range 4.6–6.0
(Creamer, 1985) and fracture strain (Luyten et al.,
1987; Visser, 1991) are maximal (see ‘Cheese as an
Ingredient’, Volume 2). Indeed, this seems highly
probable when one considers the production of pasta-
filata-type cheeses such as Mozzarella and Kashkaval;
the cheeses flow and stretch over a narrow pH range,
5.2–5.35, outside which flow is very restricted unless
some processing changes, such as reduction of calcium
level or plasticizing in hot dilute brine (so as to par-
tially solubilize the casein), are implemented (see
‘Pasta-Filata Cheeses’ and ‘Cheese as an Ingredient’,
Volume 2). During the time required for the pH to fall
from ⬃6.1 at pitching to ⬃5.2 at stretching, little or
no degradation of s1-casein occurs. It is probable that
both mechanisms (i.e., NPN formation with conse-
quent movement of pH from the point of maximum
fracture strain, and hydrolysis of s1-casein) con-
tribute to the age-related rheological/textural changes
to different extents depending on the variety and the
ratio of primary-to-secondary proteolysis. Owing to its
 Salt in Cheese: Physical, Chemical and Biological Aspects 219
effects on primary and secondary proteolysis, the salt
content of cheese has a major influence on its rheo-
logical properties, as discussed in ‘Effect of NaCl on
cheese rheology’.
Blue cheese
The influence of NaCl concentration on the principal
ripening events in Blue cheese was studied by
Godinho and Fox (1981a,b,c, 1982). Proteolysis, as
measured by polyacrylamide gel electrophoresis and
the formation of 12% TCA-soluble N, was invariably
lower in the outer (high salt) region than in the mid-
dle or centre (lower salt) zones; the differences were
apparent both before visible mould growth (during the
first two weeks when the coagulant is the principal
proteolytic agent) and during the mould phase (after
two weeks) (Godinho and Fox, 1982; Hewedi and
Fox, 1984). There was a strong negative correlation
between salt concentration and TCA-soluble N. Unfor-
tunately, the formation of amino acid N (e.g., PTA-
soluble N) or other more detailed characterizations of
proteolysis were not investigated. With a few excep-
tions, the pH increased faster at the centre than in the
outer region of the cheese, indicating that the catabol-
ism of amino acids or lactic acid is also influenced by
NaCl concentration.
Lipolysis in Blue cheese is also influenced by salt
concentration, with maximum activity occurring at
4–6%, w/w, NaCl (Godinho and Fox, 1981c). How-
ever, the concentration of methyl ketones was rela-
tively independent of salt concentration.
Camembert cheese
The ripening of the surface mould-ripened cheeses,
Camembert and Brie, is characterized by a very
marked softening, almost liquefaction, of the body
from the surface to the centre. This ripening pattern is
mainly due to the combination of s1-casein hydroly-
sis and the decreasing pH gradient from the surface to
the centre, due to the production of ammonia by the
surface mould, P. camemberti, and its inward diffusion,
and the catabolism of lactic acid, and outward diffu-
sion of calcium (Le Graet et al., 1983; Noomen, 1983;
Karahadian and Lindsay, 1987). Proteolysis by the
coagulant and starter proteinases is also important
and although the proteinases excreted by P. camem-
berti undergo only very limited diffusion in the cheese
(Noomen, 1983), peptides produced by them do,
apparently, diffuse into the cheese (see ‘Surface
Mould-ripened Cheeses’, Volume 2 and ‘Biochemistry
of Cheese Ripening: Introduction and Overview’,
‘Metabolism of Residual Lactose and of Lactate and
Citrate’, ‘Lipolysis and Catabolism of Fatty Acids in
Cheese’, ‘Proteolysis in Cheese during Ripening’,
‘Catabolism of Amino Acids in Cheese During Ripening’,
Volume 1).
In this variety also, NaCl concentration has a major
influence on proteolysis and pH changes, as well as on
surface mould growth (Table 2).
Other cheeses
Feta and Domiati are special in the sense that they
are stored in brine, containing typically 6–8%, w/w,
NaCl, after manufacture. The high level of salt strongly
affects the microflora, enzymology and ripening of
these cheeses (see ‘Cheese Varieties Ripened in Brine’,
Volume 2). In addition to the inward migration of salt,
outward diffusion of low molecular mass water-soluble
compounds (e.g., small peptides, amino acids, lactate,
volatile water-soluble acids, and minerals) occurs and
these accumulate in the brine. Studies on the diffusion
of these molecules are lacking. Pappas et al. (1996)
studied the effects of S/M level (4.3–5.8%, w/w, S/M)
in Feta cheese, by altering the duration of dry-salting
prior to storage in 7–8%, w/w, brine. Increasing S/M
level reduced the moisture content and the levels of
pH 4.6-soluble N and lipolysis (as measured by acid
degree value), or organoleptic characteristics.
El-Sissi and Neamat-Allah (1996) studied the effect
of different salt levels in cheesemilk (5, 7, 9 and 12%,
w/w, added NaCl) on the ripening and quality of
Domiati; the corresponding S/M levels in the cheese
were ⬃6.0. 8.3, 9.5 and 12.7%, w/w. Increasing the salt
level increased yield (2.9–3.5 kg/100 kg), moisture
(⬃61–69%, w/w), pH (⬃5.3–6.5) and reduced the
level of pH 4.6-soluble N (% total N) and of total
volatile fatty acids; these effects were most dramatic
as the S/M level was increased from 9.5 to 12.8%,
w/w. The development of the desired flavour and
texture/body characteristics was prevented at 12.8%,
w/w, S/M (even after 4 weeks storage in pasteurized
whey at 14 °C) and delayed at 9.5%, w/w, S/M, com-
pared to cheese with a lower S/M level. The authors
recommended a S/M level of 5–9%, w/w, for accelerat-
ing the ripening of Domiati.
Nájera et al. (1994) reported that the concentra-
tions of individual (C4–C18 and C18:1) and total
FFAs in Idiazabal cheese increased on increasing the
brining time from 12 to 24 or 36 h; however, no
details on cheese composition were given. Kaya et al.
(1999) studied the effect of storage time in brines of
different NaCl concentration on the ripening and qual-
ity of a Turkish white pickled cheese, Gaziantep, the
manufacture of which does not involve the addition
of a starter culture. Increasing the S/M level in the
experimental Gaziantep cheese from 8.7 to 24%, w/w,
220 Salt in Cheese: Physical, Chemical and Biological Aspects
resulted in significant reductions in moisture content
and the level of FFAs, and increases the firmness and
peroxide value. Sensory analysis showed that increas-
ing the S/M level to 13.2%, w/w, increases the inten-
sity of off-flavours, metallic, oxidized, rancid and
bitterness.
Kristiansen et al. (1999) investigated the effects of
varying S/M on proteolysis in Danbo-type cheese
brine-salted for different times. Increasing S/M, in the
range 0.1–6.4%, w/w, significantly reduced the level of
MNFS, the degradation of -casein and the levels of
pH 4.6 and 5% (w/v) phosphotungstic acid-soluble N
over the 10-week ripening period. In contrast, higher
salt levels had little effect on the primary breakdown
of s1-casein. Capillary gel electrophoresis showed
that the hydrolysis of -casein in dilute solution
(0.5%, w/v, in sodium phosphate buffer, pH 7.0) by
chymosin or bovine pepsin was strongly inhibited by
5%, w/w, S/M, while that by plasmin was not
(Kristiansen et al., 1999); the opposite trend was
noted for s1-casein. These results concur with those
of Lane and Fox (1999) who studied the effects of
salt (0–10%, w/w, S/M) and pH on the proteolysis of
-casein and sodium caseinate in dilute (0.5%, w/v)
solutions. The negative effect of salt on proteolysis is
associated with its effect on ionic strength, which
affects casein hydration and conformation (see ‘Effect
of NaCl on Casein Hydration and the Physical Proper-
ties of Cheese’), and the concomitant reductions in the
level of MNFS and aw (Creamer, 1971; Lawrence and
Gilles, 1980; Rüegg and Blanc, 1981; van den Berg and
Bruin, 1981).
A higher S/M level (i.e., 3.0%, w/w) in accoustically
brined Mahon cheese, compared to the conventionally
brined control cheese (2.8%, w/w) gave higher levels of
all individual FFAs analysed (apart from caprylic and
stearic acids) and an increase (9%) in the concentra-
tion of total FFAs (Sánchez et al., 2001). This trend is
similar to that noted by Nájera et al. (1994) for Idiaza-
bal cheeses but disagrees with that reported by Thakur
et al. (1975) and Lindsay et al. (1982) for Cheddar
(who reported that increasing the salt level from 0.03
to 1.78, and from 1.25 to 1.5%, w/w, respectively, led to
reductions in the levels of individual and/or total FFAs)
and to that of Freitas and Malcata (1996) for Picante, a
hard Portuguese cheese.
Effect of NaCl on Casein Hydration and the
Physical Properties of Cheese
The extent of the hydration and aggregation of casein
has a major impact on the formation and textural/func-
tional characteristics of dairy products, including
cheeses (see Fox and McSweeney, 2003). Indeed, the
manufacture of many protein-based products and
ingredients, such as cheese, yoghurt and casein, is
based on a limited destabilization and aggregation of
the casein micelles. The extent of casein aggregation,
or hydration, affects the microstructure and nature of
attractions between protein molecules within the pro-
tein phase of dairy products containing protein. Con-
sequently, it has a major influence on several aspects
of product quality: rheology, texture and cooking char-
acteristics of cheese (see ‘Rheology and Texture of
Cheese’, Volume 1 and ‘Pasta-Filata Cheeses’ and ‘Cheese
as an Ingredient’, Volume 2); texture and mouth-feel
of yoghurt; and re-hydration characteristics of casein
in food formulation (see Fox and McSweeney, 2003).
Apart from Domiati, all cheeses are salted after ren-
net coagulation and curd formation, at a level ranging
from ⬃2.0%, w/w, in Emmental to ⬃12%, w/w, in
Feta. The practice of adding salt to the curd, rather
than to the milk, has been deliberate as the early
cheesemakers would have soon discovered that its
addition prior to renneting severely impaired or pre-
vented the coagulation of milk (Fig. 6) and curd
syneresis (Cheeseman, 1962; Grufferty and Fox, 1985;
Walstra et al., 1985; Pearse and Mackinlay, 1989;
Abou-El-Nour, 1998). The adverse effects of salt at the
concentrations used in cheese on curd formation are
probably a consequence of the solubilization of col-
loidal calcium phosphate as a result of a sodium–calcium
interchange, and the positive effect of salt on casein
hydration, which impairs casein aggregation. In
Domiati cheese, where 5–15%, w/w, NaCl is added to
the milk (Abou-El-Nour, 1998), the effects of NaCl in
curd formation are off-set by the use of water buffalo
milk, which has a higher casein content than bovine
milk (see Kosikowski and Mistry, 1997), or by the forti-
fication of milk with skim milk powder, and/or the add-
ition of CaCl2 (M.M. Hewedi, personal communication).
Owing to the importance of casein hydration on
cheese quality, the effects of NaCl on hydration are
discussed below.
Effect of NaCl on casein hydration in model
systems and in cheese
The significance of casein hydration, as affected by
NaCl, on the physical properties of cheese has been
demonstrated using dilute model systems. Creamer
(1985) studied the effect of NaCl on casein hydration
in rennet-treated skim milk at pH values in the range
4.6–6.6, by measuring the levels of moisture and
protein in the para-casein pellet obtained on ultracen-
trifugation at 81 000 g for 2 h. The addition of 5%,
w/w, NaCl to the milk increased the levels of serum Ca
and casein hydration at all pH values, with a maxi-
mum in the pH range 5.2–5.3 (Fig. 7). The increase in
 Salt in Cheese: Physical, Chemical and Biological Aspects 221

80

70

60
Elastic shear modulus, G′, Pa

50

40

30

20

10

0
0 1000 2000 3000 4000 5000 6000
Time from renent addition, s

Figure 6 Influence of added salt on the firmness, as measured by the elastic shear modulus (G ), of reduced-fat milk (2.0%, w/w,
fat; 3.3%, w/w, protein; 4.7%, w/w, lactose). Salt was added at levels of 0 (), 1 (), 2 (), 4, 6, 8 or 12%, w/w, and stirred into the
milk for 30 min prior to measurement of G using dynamic low amplitude oscillation strain rheometry; no coagulation was detected at
salt levels 4%, w/w. The pH of the milks at the different salt levels was 6.66, 6.48, 6.42, 6.38, 6.35, 6.25 and 6.27, respectively
(T.P. Guinee, unpublished results).

casein hydration with NaCl may be attributed to the

Supernatent para-casein, % of total para-casein

80
binding of Na by the casein (Gál and Bánky, 1971;
70 Hardy and Steinberg, 1984) and the displacement of
calcium or calcium phosphate from the para-casein by
60 the Na. In effect, the addition of NaCl appears to
50 create a sodium–calcium ion exchange effect with
the para-casein, somewhat similar to that observed
40 between emulsifying salts (sodium phosphates and
sodium citrates) and the casein matrix during the
30
manufacture of processed cheese products (see ‘Pas-
20 teurized Processed Cheese and Substitute/Imitation
Cheese Products’, Volume 2). Indeed, an inverse rela-
10
tionship between casein hydration and casein-bound
0 calcium in model systems has been reported by many
4.7 5.0 5.3 5.6 5.9 6.2 6.5 6.8 7.1 7.4 investigators. In these studies, casein hydration was
pH measured directly or indirectly by determining the
Figure 7 Effect of salt, added at 5 %, w/w, on the solubility of a levels of water and protein in pellets obtained on
5%, w/w, dispersion of rennet casein (80%, w/w, protein; 7.1%, ultracentrifugation of milk (Sood et al., 1979, 1980;
w/w, ash). The rennet casein was thoroughly mixed with water Guillaume et al., 2002) or the uptake of water by
() or 5%, w/w, NaCl () and the pH adjusted to different values casein(ate)s with different levels of added calcium
using 20%, w/v, lactic acid. The dispersions were held at 15 °C
on exposure to environments with different aw values
for 22 h and then centrifuged at 3000 g for 30 min at 15 °C. The
protein content of the supernatant was measured and in the range 0.58–0.95 (Rüegg and Moor, 1986). Inter-
expressed as a percentage of the total protein (T.P. Guinee and estingly, a study by Pastorino et al. (2003b) showed
B.T. O’Kennedy unpublished results). that the addition of calcium, at a level of 0–1.4%, w/w,
222 Salt in Cheese: Physical, Chemical and Biological Aspects
to Mozzarella cheese by high pressure injection of a
40% (w/w) CaCl2 solution resulted in marked ‘weep-
ing’ and water loss, a reduction in pH and a more
aggregated para-casein matrix interspersed with large
voids containing free water; the reduction in mois-
ture was ⬃12%, w/w, at 1.4%, w/w, Ca. These findings
indicate that the addition of calcium increases casein–
casein interactions and thereby reduces casein hydration.
Conversely, the levels of moisture and non-expressible
serum, which is an index of protein hydration, in
Mozzarella cheese increase as the calcium content is
reduced (Guinee et al., 2002).
In Mozzarella cheese, the effect of salt on casein
hydration has been measured by determining the level
of expressible serum (ES), i.e., the serum released on
centrifugation of the cheese at ⬃12 500 g at 25 °C, and
the level of serum protein (Guo et al., 1997). The level
of ES in brine-salted Mozzarella (49.5%, w/w, moisture;
1.4%, w/w, NaCl; 2.8%, w/w, salt-in-moisture) decreased
from ⬃16 to 1 g/100 g cheese over the first 10 days of
ripening, indicating an increase in the water-binding
capacity of the protein (Guo et al., 1997); the level of
water-soluble protein (WSP) in the serum increased
from ⬃5 to 10%, w/w, over the same time period. In
contrast, the levels of ES and WSP for the unsalted
cheese (0.13%, w/w, NaCl) changed from ⬃19 to
14 g/100 g, and from ⬃3 to 5%, w/w, respectively, dur-
ing storage. However, the level of pH 4.6-soluble pro-
tein in the sera from both the salted and unsalted
cheeses was similar, indicating that the differences in
WSP were due to the solubilization of intact casein by
the added NaCl rather than to differences in proteolysis
of the casein. Thus, electrophoretic analysis of the sera
showed that the levels of intact s1-, s2-, - and para-
-casein in the ES of brine-salted Mozzarella were
higher than those for the unsalted cheese at all times
over a 10-day storage period. A similar trend was
reported for fat-free Mozzarella for which the curd was
dry-salted to different levels (0, 0.5 or 1.0%, w/w) prior
to stretching in hot water containing 0, 5 or 10%, w/w,
NaCl (Paulson et al., 1998). At all times throughout the
24-day storage period, the level of ES in cheeses from
curds stretched in hot water decreased as the level of
dry-salting prior to stretching was increased to give a
salt level of 0.14–0.68%, w/w (0.2–1.1%, w/w, S/M) in
the cheese. No ES was obtained at any stage from
cheeses made from curds stretched in hot solutions
containing 5 or 10%, w/w, NaCl and with salt levels
of 0.85–2.18%, w/w (i.e., 1.4–3.5%, w/w, S/M). These
results indicate a higher water-binding capacity of the
protein matrix of cheeses as the S/M level increased to
0.85%, w/w.
Dry-salting of cheese (by mixing milled curd pieces
and salt prior to moulding or further curd treatments)
offers several advantages over brine-salting, including
savings in plant space (occupied by brine tanks) and
labour, more uniform salt-in-moisture distribution (at
least initially), less zonal variations in texture, melting
properties and quality (see ‘Effect of salt content on
cooking properties’ and ‘Attainment of salt and mois-
ture equilibria after salting’). Consequently, the effect of
dry-salting Mozzarella cheese prior to plasticization in
hot water as an alternative to brining the plasticized
curd in cold brine has been investigated (Fernandez and
Kosikowski, 1986; Paulson et al., 1998; Guinee et al.,
2000). Plasticization of curd in hot salt solutions con-
taining 5 or 10 (Paulson et al., 1998) or 18 (Piacquadio
et al., 2001) %, w/w, NaCl has also been investigated;
this form of salting is similar to plasticizing dry-salted
curd in hot-water. A combination of dry-salting (e.g.,
1%, w/w) and plasticizing in hot dilute brine (e.g., 5%,
w/w, NaCl) has also been examined (Paulson et al.,
1998; Guinee et al., 2000). The use of dry salting at
4.6%, w/w, or a combination of dry salting (to 1%, w/w,
NaCl) and plasticizing in hot dilute brine (i.e., 5%, w/w,
NaCl) gave the desired S/M level (e.g., 2.3%, w/w) and a
moisture content which was higher (by ⬃2–3%, w/w)
than that of conventionally brine-salted Mozzarella
(Paulson et al., 1998; Guinee et al., 2000). The higher
moisture content suggests a salting-in of the casein and
a concomitant increase in the degree of para-casein
hydration in the cheeses which were dry-salted or dry-
salted and platicized in hot dilute brine. In contrast to
the above, Fernandez and Kosikowski (1986) reported
that stretching in hot brine (10%, w/w, NaCl) resulted
in a marked decrease in moisture content (3–4.5%,
w/w) in low-moisture Mozzarella.
The above findings for Mozzarella are consistent with
those from the model systems described in this section
which showed that a low concentration of NaCl
(⬃5–6%, w/w, S/M) enhances the solubilization of casein
or para-casein (Hardy and Steinberg, 1984; Creamer,
1985). Such a trend is expected as the protein matrix of
Mozzarella, and all rennet-curd cheeses, may be consid-
ered as highly concentrated hydrated para-casein.
The influence of low salt concentrations on casein
hydration in cheese is also apparent from:
• the higher level of 5% salt-soluble N than water-
soluble N in Cheddar and other cheese varieties,
e.g., 93 and 26% total N for 6-month-old Cheddar
(Reville and Fox, 1978);
• the uptake of water by cheese in dilute brines, espe-
cially if calcium is absent (Geurts et al., 1972;
Guinee and Fox, 1986a, 1993).
On salting in brines of typical composition (e.g.,
18–22%, w/w, NaCl and 0.5%, w/w, CaCl2), cheese
looses water during brining, resulting in a net weight

loss, as discussed in ‘Moisture level’. In contrast, salt-
ing in freshly prepared dilute brine (e.g., #12%, w/w,
NaCl) without added calcium has been found to
increase the moisture content in the rind region of
Gouda (Fig. 8) and in Romano cheese slices (Fig. 9;
Guinee and Fox, 1986a). This phenomenon can lead
to a defect known as soft-rind or rind rot (soft, slimy,
sticky surface) in Gouda and other cheeses which are
salted in freshly prepared brines without calcium
(Geurts et al., 1972), unless casein hydration is
reduced by adding CaCl2 and reducing the pH to
⬃4.8–5.0. The problem rarely occurs in mature (stable
composition and pH) well-used brine because of the
accumulation of soluble calcium which migrates from
the cheese with the moisture.
Effect of NaCl on cheese microstructure
Owing to its effect on protein hydration, salt has a
major influence on the microstructure of cheese. Scan-
ning electron microscopy has shown that the protein
matrix of salted non-fat Mozzarella (⬃0.25 or 3.5%,
w/w, S/M) or Muenster (⬃1.2, 3.6, or 6.7%, w/w, S/M)
cheeses is more swollen, homogeneous and contin-
uous, and has a higher specific surface area than their
unsalted counterparts (Paulson et al., 1998; Pastorino
et al., 2003a). Moreover, the unsalted cheeses had large
24
20
Salt-in-cheese moisture, %, w/w
16
12
8 firmness, max, f, and sensory hardness for Camembert
4 Schroeder et al., 1988), reduced-fat Cheddar
0 28
0 1 2 3
Distance from cheese surface, cm
Figure 8 Moisture content (open symbols) and salt-in-moisture
concentration (closed symbols) in Gouda cheese as a function
of distance from the salting surface after unidimensional brine-
salting for 4 days at 20 °C in 5 ( , ), 12 (,), 20 (, ) or
24.8 (,●) %, w/w, NaCl solution (without calcium) (redrawn
from Guinee, 1985)
Salt in Cheese: Physical, Chemical and Biological Aspects 223
open channels with free serum (whey pockets) distrib-
uted throughout the matrix, which acted as light
scattering surfaces and gave the cheese an opaque
white colour, compared to a translucent, waxy appear-
ance in the salted cheeses in which such fissures were
fewer and smaller. These observations suggest that
salt increases casein hydration and are consistent with
the positive relationships between the NaCl content
(at low levels, i.e., ⬃6%, w/w, S/M) and casein hydra-
tion in dilute casein systems and in cheese (see ‘Effect
of casein hydration in model systems and in cheese’).
Effect of NaCl on cheese rheology
The influence of NaCl on cheese texture is most obvi-
ous on comparing salt-free cheeses with their salted
counterparts; the former is generally weak, soft, pasty
and/or adhesive depending on age. In contrast, high
salt concentrations lead to shortness, crumbliness,
dryness and hardness, as observed for curds held in
brine for an excessively long time. The relationship
between cheese rheology/texture and salt level is also
evident on visual examination of brine-salted cheese
on completion of salting; the outside rind region is
hard, brittle, dry and white while the inside is more
pliable, waxy, softer and more translucent. The influ-
ence of NaCl on cheese texture is probably due to
its effects on composition (moisture content, MNFS),
para-casein hydration/solubility and conformation, age-
44
related effects on pH (see Table 2; Furtado et al., 1982)
and proteolysis. The effects of salt are discussed in more
42 detail below.
Numerous investigators have studied the effects of
40 salt concentration, or salt-in-moisture (S/M), on rheo-
logical properties such as firmness (max, e.g., force
required to attain a given compression, or to push a
Moisture, %, w/w
38
probe to a given depth into cheese), fracture stress
36 (f), fracture strain ( f) and/or sensory hardness.
These studies have shown that increases in S/M
34 within the range 0.4–12%, w/w, result in increased
(Pagana and Hardy, 1986; Lesage et al., 1992), Ched-
32
dar (⬃0.2–6%, w/w, S/M; Thakur et al., 1975;
30
(2.7–4.5%, w/w, S/M; Mistry and Kasperson, 1998),
Feta (6.7–10.5%, w/w, S/M; Prasad and Alvarez, 1999;
2.0–5.5%, w/w, S/M; Pappas et al., 1996), Gaziantep
4 5 6
cheese (Kaya, 2002), Mozzarella (0.5 and 5.14%, w/w,
S/M; Cervantes et al., 1983), Muenster (0.3–7.45%,
S/M; Pastorino et al., 2003a), and low pH model
cheese made from ultrafiltered skim milk retentate
(2.0 and 4.7%, w/w, S/M; Euston et al., 2002). The
increase in max and f may be attributed in part to the
concomitant changes in composition, e.g., reduction
224 Salt in Cheese: Physical, Chemical and Biological Aspects

14

12

g water lost per 100 g cheese

g NaCl gained per 100 g cheese
10

8 8

6 6

4 4

2 2

0 0

–2
0 100 240 0 100 240 0 100 200 0 100 200 0 100 200
Brining time, min

Figure 9 Moisture loss (open symbols) and salt uptake (closed symbols) by Romano-type cheese slices (0.5 cm thick; 7 cm diam-
eter) brine-salted in 6.5 ( , ), 10.7 (,), 14.8 (,), 18.9 (, ) or 24.9 (,), %, w/w, NaCl solution (without calcium) at 20 °C,
as a function of time in the brine (redrawn from Guinee and Fox, 1986a).

in moisture level and increase in protein, and the
effects of salt on proteolysis (see ‘Cheese as an Ingred-
ient’, Volume 2). The effect of salt on composition is
very evident in brine-salted cheeses, in which there is
generally an increasing moisture gradient from the
surface to the centre, and a salt gradient in the oppo-
site direction (see ‘Salt Absorption and Diffusion into
Cheese’) at the end of salting. However, even in
cheeses where the other compositional parameters
were relatively constant (Visser, 1991; Euston et al.,
2002), salt had a major effect on cheese rheology. This
suggests that in addition to its indirect effects on
rheology via its influence on gross composition, salt
also exerts more direct effects on rheology, e.g., by
promoting changes in the degree of casein hydration
and aggregation which alters the ratio of viscous to
elastic character in the cheese. Thus, Euston et al.
(2002) noted an effect of the interaction between salt
level and pH on the rheology of model cheeses with
similar gross composition.
Pagana and Hardy (1986) reported an inverse linear
relationship between the brittleness of Camembert
cheese, as measured by fracture strain, and S/M level in
the range ⬃3–21%, w/w. Visser (1991) noted that the
fracture strain of model Gouda cheeses increased
monotonically with S/M in the range 0 to ⬃4.5%, w/w,
then decreased sharply to a value which was about half
the maximum at 5.5%, w/w, S/M and remained rela-
tively constant thereafter as the S/M was increased to
11.3%, w/w. Similar to f and max, the effect of salt on
f is probably also attributable to a salting-in effect with
a concomitant increase in para-casein hydration as S/M
increases to ⬃5%, w/w, and a salting-out effect with a
concomitant loss in casein hydration at higher S/M
levels (Geurts et al., 1972; Guinee and Fox, 1986a;
‘Cheese as an Ingredient’, Volume 2). An increase in
casein hydration would impart a more viscous (and
less elastic) character to the cheese and a transition
from elastic fracture behaviour to plastic fracture behav-
iour, which would necessitate a higher strain for frac-
ture (see ‘Rheology and Texture of Cheese’, Volume 1).
Conversely, a lower degree of casein hydration at the
higher S/M (5%, w/w) would favour a more elastic
casein matrix and an elastic fracture behaviour, i.e., a
shorter, firmer, more brittle cheese.
Effect of salt content on cooking properties
The development of acceptable cooking characteristics
in cheese, such as Mozzarella and Cheddar, generally
requires an ageing period (see ‘Pasta-Filata Cheeses’
and ‘Cheese as an Ingredient’, Volume 2), the duration
of which depends, inter alia, on the cheese type, man-
ufacturing conditions, ripening conditions and the
specifications set by the customer. Age-related bio-
chemical and microstructural changes which con-
tribute to the development of the desired cooking
characteristics include: increases in proteolysis, casein
hydration and non-globular fat; a concomitant swelling
of the protein matrix; and decreases in the levels of
aggregation and continuity of the para-casein matrix.
The increase in casein hydration leads to an enhanced
water-binding capacity of the protein matrix, improved
moisture retention during cooking, easier displace-
ment of neighbouring planes of the protein matrix
in the heated cheese mass, and improved (higher)
 Salt in Cheese: Physical, Chemical and Biological Aspects 225
flowability and stretchability of the heated cheese.
Moreover, the heated aged cheese is generally more
moist and less prone to blistering and charring than
fresh cheese.
Salt level has a marked influence on the cooking
properties of low-moisture part-skim Mozzarella (Apos-
tolopoulos et al., 1994) and non-fat Mozzarella cheese
(Paulson et al., 1998), a trend expected because of its
influence on casein hydration, as discussed in ‘Effect of
NaCl on casein hydration in model systems and in
cheese’. In these studies, salt level was varied by brin-
ing in salt solutions of different concentration, or by
dry salting at 0.0, 0.5 or 1.0%, w/w, NaCl prior to
stretching the curd in hot water containing 0, 5 or 10%,
w/w, NaCl (Paulson et al., 1998). The flowability of
melted cheeses increased fairly linearly with salt level
in the range 0.1–0.5%, w/w (S/M ⬇ 0.2–1.2%, w/w)
and changed only slightly as the salt level was further
increased to ⬃2.2%, w/w (S/M ⬇ 0.2 to 3.3%, w/w).
The increase in flowability coincided with an increase
in free oil and water-binding capacity of the para-casein
matrix, as reflected by a decrease in the level of ES.
Moreover, the protein matrix of the salted cheese was
more swollen and uniform than that of unsalted cheese
which had fissures thought to be pockets of free
unbound water (Paulson et al., 1998). Thus, Kindstedt
and co-workers (Kindstedt, 1990, 1995; Kindstedt
et al., 1992) found that zonal variations in salt level,
which occur in brine-salted Mozzarella, lead to varia-
tions in cooking properties at different locations in the
cheese throughout a 16-day ageing period. Melted
cheese from the high-salt (⬃3.04%, w/w, at day 2, and
1.7%, w/w, at day 16) surface region had a higher
apparent viscosity and a low level of free oil, and was
tough and chewy. In contrast, cheese from the low-salt
interior (⬃0.38%, w/w, at day 2, and 0.9%, w/w, at day
16) had a markedly lower apparent viscosity and was
smooth, soft, fluid and gelatinous. After 16 days, the
melted interior sample had become excessively soft
while the exterior sample still remained unacceptably
tough and chewy. The above trends suggest a salting-in
effect of the para-casein matrix with a concomitant
increase in hydration at low S/M levels (e.g., 1.5%,
w/w) and a salting-out effect and decrease in para-
casein hydration at the high salt levels (especially
at 6.3%, w/w, S/M). Consequently, Kindstedt and Guo
(1997) concluded that, in addition to proteolysis, the
increase in casein hydration, at 3–4%, w/w, S/M is a
major factor contributing to the development of the
desirable cooking properties in low-moisture part-skim
Mozzarella during storage. In contrast to the above
studies, Pastorino et al. (2003a) reported no effect of
salt in the range 0.14–2.8%, w/w (0.34–7.4%, w/w,
S/M) on the degrees of melt and flowability of 40-day-old
Muenster cheese in which salt level was varied by the
number of injections of brine.
Reduced Sodium Cheese
While the physiological requirement of Na as a dietary con-
stituent is universally accepted, there is growing concern
that an excess (⬃2.4 g/day for healthy adults) induces
physiological defects, including hypertension (see Beard
et al., 1997; Beilin, 1999; Kaplan, 2000). Such concern has
led to a recommendation for a reduced dietary intake of Na,
classification of foods (high, medium, low) according to
sodium level, declaration of sodium level on the food labels
and an increased demand for reduced-sodium foods,
including cheese (see Demott, 1985; Petik, 1987; Schroeder
et al., 1988; Morris and Dillon, 1992; Narhinen et al.,
1998).
In addition to its preservative effect, salt in cheese
exerts a major influence on cheese composition, micro-
flora, ripening rate, texture, flavour and quality (see
‘Control of Microbial Growth’, ‘Influence of NaCl on
Enzyme Activity in Cheese’, ‘Influence of NaCl on the
Water Activity (aw) of Cheese’, ‘Overall Influence of
NaCl on Cheese Ripening and Quality’ and ‘Effect of
Salt on Cheese Composition’). The sodium level (%,
w/w) in cheese ranges from ⬃0.26, 0.62 and 2.6,
respectively, for Swiss, Cheddar and Domiati (United
States Department of Agriculture, 1976; Volume 2
Kindstedt and Kosikowski, 1988). Approaches to reduce
the Na level in cheese include:
• reducing the level of added salt per se (Kosikowski,
1983; Wyatt, 1983; Lindsay et al., 1985);
• partial or complete substitution of NaCl by other
salts such as KCl, MgCl2 or CaCl2 (Fitzgerald and
Buckley, 1985; Demott et al., 1986; Lefier et al.,
1987; Anonymous, 1993; Iwanczak et al., 1996;
Katsiari et al., 1997; Salem and Abeid, 1997; Abou-El-
Nour, 1998);
• a reduced salt level in combination with flavour-
enhancing substances such as autolysed yeast extract
(Karahadian and Lindsay, 1984; Demott et al., 1986);
• the use of ultrafiltration and reverse osmosis reten-
tate supplemented milks to alter the mineral level in
the cheese (Kosikowski, 1983, 1985; Kindstedt and
Kosikowski, 1984b; Lindsay et al., 1985);
• alterations of cheesemaking procedure, e.g., wash-
ing curd at a low temperature (⬃20 °C; to remove
lactose) and heating moulded curd to a core tempera-
ture curd of 85 °C (Drews, 1991); or high intensity
centrifugation of milk and re-incorporation of the
bacteria-rich portion after sterilization followed by
normal cheese manufacture apart from a shorter
brining time (Wessanen, 1983).
226 Salt in Cheese: Physical, Chemical and Biological Aspects
Attempts to produce low-sodium processed cheese
products include the partial substitution of sodium
phosphates with the corresponding potassium phos-
phates, the use of flavour enhancers (e.g., monosodium
glutamate, glucono-delta-lactone, enzyme-modified cheese
and cheese pastes) (Karahadian and Lindsay, 1984) and
the use of selected cheese blends and dairy ingredients
in the production of emulsifying salt-free processed
cheese foods and spreads.
Cheddar cheese
Schroeder et al. (1988) showed that it was possible
to reduce the salt content of Cheddar to 1.12%, w/w,
without ill-effects on quality; cheese with 0.73%, w/w,
NaCl was also acceptable. Fitzgerald and Buckley
(1985) studied the influence of KCl, MgCl2, CaCl2
and 1:1 mixtures of these salts with NaCl on the qual-
ity of Cheddar cheese salted to give an ionic strength
similar to the control (i.e., 1.44%, w/w, NaCl in the
cheese) ripened at 4 $C over a 4-month period. The
use of KCl, MgCl2 or CaCl2 alone resulted in oversoft
cheeses with very bitter and unacceptable flavours.
These defects may be attributed to the higher mois-
ture level and greater proteolysis and lipolysis in the
case of MgCl2 and CaCl2 but not in the case of KCl,
where the moisture and water-soluble N levels were
similar to, or slightly higher than, respectively, the
controls. Similarly, the cheeses with salt mixtures gen-
erally had higher levels of proteolysis and lipolysis
than the control but the blends had little effect on
the hardness or firmness, except in the case of the
NaCl/MgCl2 blend which gave softer cheese than
the control. Both flavour and texture scores for the
CaCl2/NaCl- and MgCl2/NaCl-salted cheeses were sig-
nificantly lower than the controls. A KCl/NaCl (1:1)
mixture gave cheese at 16 weeks which was not
significantly different from the control in terms of
proteolysis, texture flavour and acceptability.
Similar results with NaCl/KCl (1:1) mixtures were
observed for Cheddar (Lindsay et al., 1982), and
Camembert, Gouda and Camping type cheeses
(Iwanczak et al., 1996). Moreover, both of these stud-
ies reported that reduction of salt (i.e., NaCl or 1:1
NaCl/KCl mixtures) by ⬃30% in these cheeses resulted
in no major differences in flavour, texture or accept-
ability scores (Lindsay et al., 1982) or improved them
(Iwanczak et al., 1996). In Cheddar cheese (⬃34%,
w/w, moisture) salted with NaCl/KCl mixtures, free
fatty acid levels were higher and grading scores were
somewhat lower due to a slight bitterness (Lindsay
et al., 1982). Reddy and Marth (1995) studied the
starter and NSLAB populations in Cheddar cheeses
made with different starters and salted at a level of
1.5–1.75%, w/w, with NaCl, KCl or mixtures of
NaCl/KCl (2:1, 1:1, 1:2 and 3:4). For a given starter
system, the type of salt did not effect the predominant
bacterial species in the cheese. Kosikowski (1983,
1985) found that in Cheddar cheeses with a reduced
level of NaCl (i.e., 1.05%; ⬃3.0%, w/w, S/M), increas-
ing the protein content of the milk, from ⬃3.36 to
6.26%, w/w, prior to renneting, by supplementing the
cheese milk with increasing amounts of ultrafiltered
milk (4.5:1 retentate), was paralleled by a decrease
in moisture and increases in the Ca (from ⬃590
to 730 mg/100 g) and P (from ⬃470 to 556 mg/100 g)
levels and in the scores for flavour, body and texture
during ripening at 10 °C over 4 months. Grading
scores for flavour and texture increased to an optimum
at a milk protein concentration of 4.97–6.26% in the
supplemented milks; at the lower milk protein level,
the cheeses become progressively more acidic, bitter,
pasty and devoid of cheese flavour. The enhancing
effect of increased milk protein level on grading score
was attributed to the increased buffering capacity
which prevented a rapid decline in pH (in the absence
of a normal salt level) during moulding and pressing
and hence excessive loss of calcium and phosphorus
which influence cheese structure and rate of proteoly-
sis (Kosikowski, 1983; Kindstedt and Kosikowski,
1984a,b, 1986). However, the results of Kosikowski
(1983) could not be confirmed under practically iden-
tical conditions by Lindsay et al. (1985). Contrary to
the results of Kosikowski (1983), the latter group
found that:
• the calcium level in low-sodium (1%, w/w, NaCl)
Cheddar made from control milk was not signifi-
cantly lower than that made from milk supple-
mented with retentate;
• the grading scores of cheeses made from supple-
mented milk were of the same magnitude as, or
slightly lower than, those of the control ‘non-
supplemented’ low-salt Cheddar. Moreover, the for-
mer cheeses were generally softer and had a less
intense cheese flavour.
The inclusion of reverse osmosis (RO) retentate in
UF retentate-supplemented milk gave low-salt Cheddar
(1%, w/w NaCl) with grading scores similar to the con-
trol (Lindsay et al., 1985); such cheeses had a unique
sharpness which could be used to enhance the flavour
of other cheese products, such as processed cheese.
Undoubtedly, the quality of commercial reduced-
sodium cheese depends on many factors, including
pitching pH, the type and amount of residual coagu-
lant in the cheese, types and counts of starter
and non-starter bacteria, composition and ripening
temperature. Ranges of compositional parameters for
 Salt in Cheese: Physical, Chemical and Biological Aspects 227
good quality (New Zealand) Cheddar as proposed by
Lelievre and Gilles (1982) are: 4.0–6.0%, w/w, S/M;
50–57%, w/w, FDM; 50–56%, w/w, MNFS and pH
5.0–5.4; outside these ranges quality deteriorates
sharply. With the modern continuous production
methods for Cheddar, in combination with rapid
cooling of blocks, it may be possible (though some-
what more expensive) to produce consistently high-
quality Cheddar by reducing the MNFS, keeping the
pH close to 5.1, avoiding the use of bitter starters
and microbial rennets, and ripening at a low tempera-
ture (i.e., 5 °C).
Cottage cheese
Because of its relatively large serving size (⬃112 g
compared to ⬃66 g for other cheeses), Cottage cheese
has been viewed as a potentially high source of
dietary sodium (Marsh et al., 1980). Hence, much
interest has focused on various ways of reducing the
salt level of Cottage cheese. Wyatt (1983) evaluated
preference scores for Cottage cheeses in which the
NaCl content was reduced stepwise from 1%, w/w,
(control commercial cheese) to 0.25%, w/w, NaCl. It
was concluded that a 35% reduction in salt did not
influence consumer response to the cheese compared
to the control; however, reduction by 50% or greater
resulted in significantly lower scores. Demott et al.
(1986) also evaluated consumer reactions to low-
sodium Cottage cheese salted with various mixtures
of KCl and NaCl and found that the sodium level
could be reduced by 50% (by adding 1.26%, w/w, of
an NaCl/KCl mixture instead of 1.26%, w/w, NaCl)
without affecting grading scores. Reducing the
sodium level by more than 50% resulted in a signifi-
cant reduction in score (Demott et al., 1984, 1986).
Lindsay et al. (1985) also found that a 50% reduction
caused no significant changes in consumer acceptabil-
ity. However, the use of substitutes, i.e., KCl or
KCl/NaCl, to reduce the level of sodium by 50% gave a
significant reduction in quality.
Other cheeses
Martens et al. (1976) reported the successful manufac-
ture of low-sodium Gouda cheese using mixtures of
NaCl and KCl in curd manufacture and brining. While
the Na and K levels (mg %) in the control cheese were
⬃830–650 and 120, those of the reduced-sodium
cheese were 200 and 200, respectively. However,
reduction of salt in dry matter (SDM) in Gouda cheese
by ⬃20% is claimed to increase significantly the sus-
ceptibility to butyric acid fermentation (van den Berg
et al., 1986). To prevent such undesirable fermentation
in cheeses at salt levels 3.8%, w/w, SDM requires
process modifications such as bactofugation of the
milk and reduction of the cheese moisture level. Lefier
et al. (1987) reported the production of low-sodium
Gruyère (⬃45 mg Na/100 g compared to 272 mg/100 g
in the control) by replacing NaCl by MgCl2. While the
degree of proteolysis and the concentrations of free
fatty acids were similar in both cheeses, the cheese
containing MgCl2 had a more bitter taste and a softer
body than the control, but was organoleptically
acceptable. Partial replacement (50%) of NaCl in
brines with CaCl2, MgCl2, KCl, or a mixture of CaCl2,
MgCl2 and KCl did not significantly affect the level of
proteolysis, as measured by pH 4.6-soluble N (per-
centage total N) and NPN in Turkish White
cheese (Güven and Karaca, 2001). However, the pH 4.6-
soluble N (percentage total N) was numerically high-
est for the cheeses salted in the NaCl brine and lowest
in those salted in NaCl:CaCl2  KCl  MgCl2 brines
during most of the ripening period. Katsiari et al.
(1997) reported that the sodium content of Feta
cheeses was successfully reduced by 50%, by partial
replacement of NaCl with KCl, without affecting gross
composition, water activity, lipolysis (Katsiari et al.,
2000b), proteolyis (Katsiari et al., 2000a), or sensory
or textural properties. Similarly, a 50% reduction in
the sodium content of Kefalograviera cheese, by sub-
stituting KCl for NaCl, had no effect on lipolysis or
proteolysis (Katsiari et al., 2001a,b).
Processed cheese products contain a relatively high
level of Na (1.0–1.5%, w/w; USDA, 1976) because of
the inclusion of sodium phosphate emulsifying salts
in their formulation. Karahadian and Lindsay (1984)
produced acceptable low-sodium processed cheese
(75% reduction, 0.34%, w/w, Na in product) by
using reduced-sodium Cheddar cheese and/or various
combinations of potassium-based emulsifying salts
(citrates, phosphates). A similar process for reduced-
sodium processed cheese, based on the selective use
of sodium-, potassium- and calcium-based phosphates,
was patented by Henson (1999). The most efficient
means for reducing Na in processed cheese products is
by eliminating the emulsifying salts, i.e., as in emulsify-
ing salt-free processed cheese foods and spreads which
have been commercially available since 1988. The pro-
duction of such products requires careful blending of
cheeses (i.e., high and low calcium cheeses, cheeses
with varying pH and degree of proteolysis) and alter-
ation of processing conditions so as to obtain a stable
emulsion (McAuliffe and O’Mullane, 1989; Guinee,
1991). In the latter products, lack of saltiness is easily
overcome by adding ingredients such as monosodium
glutamate, autolysed yeast extract, ‘high-cured cheese’,
cheese powders, enzyme-modified cheese, cheese pastes
and/or acidulants.
228 Salt in Cheese: Physical, Chemical and Biological Aspects
Salt Absorption and Diffusion into Cheese
Methods of salting
There are three principal methods of salting cheese curd:
• direct addition and mixing of dry salt crystals to
broken or milled curd pieces at the end of manufac-
ture, e.g., Cheddar and Cottage;
• rubbing of dry salt or a salt slurry to the surface of
the moulded curds, e.g., blue-veined cheeses;
• immersion of moulded cheese in a brine solution,
e.g., Edam, Gouda, Saint Paulin, Provolone, etc.
Sometimes, a combination of the above methods is
used, e.g., Emmental, Parmesan, Romano and Brick.
Other methods which have been used less frequently,
mainly in experimental studies, include: brine injection
under pressure, e.g., 17 MPa (Lee et al., 1978; Pastorino
et al., 2003a); high pressure brining, e.g., at isostatic
pressures up to 500 MPa (Messens et al., 1999), acoustic
brining in an experimental scale (28 l) brine vat with
an acoustic system generating high intensity (300 W)
ultrasonic (30 kHz) waves (Sánchez et al., 2000); vac-
uum impregnation brining at a vacuum of 3.7 kPa,
absolute (Pavia et al., 1999). In high pressure brining,
cheese portions were placed in high-density polythene
containers, which were filled with brine, closed, placed
in an isostatic press and pressurized.
Mechanism of salt absorption and diffusion
in cheese
Brine-salted cheese
When cheese is placed in brine there is a net move-
ment of NaCl molecules, as Na and Cl , from the
brine into the cheese as a consequence of the osmotic
pressure difference between the cheese moisture and
the brine. Consequently, the water in the cheese dif-
fuses out through the cheese matrix so as to restore
osmotic pressure equilibrium. Gels, including cheese,
consist of a three-dimensional network of strands of
fused para-casein micelles in clusters, which gives the
mass its structure and a certain degree of rigidity and
elasticity; the properties of the inter-penetrating fluid
are generally not appreciably different from those of
corresponding solutions. It would appear, therefore,
that NaCl molecules diffusing in cheese moisture,
while having a longer distance to travel than in solu-
tion (diffusing ions must travel a circuitous route to
by-pass obstructing protein strands and fat globules
through which they cannot penetrate), would not be
appreciably affected otherwise. However, based on the
mobilities of NaCl and H2O in Gouda-type cheeses
brine-salted under model conditions to obey Fick’s law
for unidimensional brine flow, Geurts et al. (1974b)
concluded that the penetration of salt into cheese and
the concomitant outward migration of water could be
described as an impeded diffusion process, i.e., NaCl
and H2O molecules move in response to their respective
concentration gradients but their diffusion rates are
much lower than those in pure solution (Georgakis,
1973; Resmini et al., 1974) due to a variety of imped-
ing factors. The diffusion coefficient (D) for NaCl in
cheese moisture is typically ⬃0.2 cm2/d, though it
varies from ⬃0.1 to 0.45 cm2/d with cheese compos-
ition and brining conditions (Geurts et al., 1974b;
Guinee and Fox, 1983a; Guinee, 1985; Turhan and
Kaletunç, 1992; Turhan and Gunasekaran, 1999; Simal
et al., 2001), compared to 1.0 cm2/d for NaCl in pure
H2O at 12.5 °C.
Geurts et al. (1974b) used the term ‘pseudo-diffusion
coefficient’ in relation to the movement of NaCl in
cheese moisture since the value of the observed coeffi-
cient depended on the net effect of many interfering
factors on true diffusion. The discrepancies between
the true- and pseudo-diffusion coefficients, i.e., D and
D*, respectively, were explained by a simplified model
of cheese structure consisting of moisture and discrete
spherical fat globules dispersed in a protein matrix
comprised of discrete spherical protein particles and
15% (w/v) bound water. Based on theoretical consider-
ations, the impedance of various compositional and
structural features intrinsic to the model structure
was formulated and their effects on D quantified.
The principal factors responsible for impedance of
NaCl diffusion in cheese, as postulated by Geurts et al.
(1974b), are:
1. The larger outward migration of water, com-
pared to inward flux of NaCl, during brining. The
pores (estimated to be ⬃2.5 nm wide) of the protein
matrix exert a sieving effect on both the inward-
diffusing NaCl molecules and outward-moving H2O
molecules but the effect is more pronounced on the
former because of their greater effective diffusion
radius, which is approximately twice that of the H2O
molecules. Hence, during brining, the H2O flux is
approximately twice the NaCl flux. The net outflow
of H2O during brining causes the plane of zero mass
transfer (a plane where the average flux of all diffus-
ing species is zero) to recede from the cheese/brine
interface into the brine and hence reduces the appar-
ent rate of NaCl diffusion due to the additional path
length through which the NaCl molecules must
migrate. The interference, which is most pronounced
when moisture loss is high, e.g., when using con-
centrated brines or high brining temperature, was
estimated to reduce the diffusion coefficient by 20%;
hence D* ⬇0.8D.
 Salt in Cheese: Physical, Chemical and Biological Aspects 229
2. The high relative viscosity of the aqueous
phase of cheese. The viscosity of cheese moisture
(juice) is about 1.27 times that of pure water at
12.5 °C due to the presence of dissolved materials,
e.g., acids, salts and nitrogenous compounds. When
the NaCl molecules enter the cheese, they encounter
collisions/obstructions with the dissolved substances,
an occurrence which retards movement. Moreover,
the charge fields of these substances also affect the
movement of the diffusing ions. Both of these factors
reduce NaCl mobility and contribute to the reduction
of the pseudo-diffusion coefficient by a factor of
1/rel.
3. Obstructions of fat globules and globular protein
particles. On proceeding from one parallel plane to
another within the cheese, the diffusing molecules
must travel by a circuitous route to bypass obstructing
particles. Hence, the real distance travelled by diffus-
ing molecule proceeding from the rind (cheese–brine
interface) to a point x cm inside the rind is x cm
rather than x cm (apparent distance), where  is the
tortuosity factor. The ratio of the real to the apparent
distance travelled is a measure of the obstructions
caused by fat globules (f) and protein particles (p).
These reduce the diffusion coefficient by a factor of
1/(fp). Theoretically, f can vary from /2 for a
close-pack arrangement to 1 for a very low-fat system,
e.g., skim milk cheese. Typical values of f and p are
1.32 and 1.35, respectively, for a Gouda cheese con-
taining 29% fat and 43% moisture (Geurts et al.,
1974b). However, the values of f and p in experi-
mental Gouda cheese vary markedly with composition
(Tables 5 and 6).
4. The mechanical sieving effect of the pores of the
protein matrix on migrating ions. The relatively nar-
row pore width of the protein matrix exerts a fric-
tional effect on the diffusing NaCl and H2O,
analogous to the restriction of movement of a sphere
through a pipe with a diameter which varies some-
what and which at its smallest is comparable to that
the sphere. In cheese, the pore width, estimated to be
typically ⬃2.5 nm, is not much larger than the com-
bined diameters of the Na and Cl , estimated to be
⬃0.6 nm (see Geurts et al., 1974b); moreover, consider-
ation of cheese microstructure suggests that pores are
not uniform in diameter or in orientation (aspect).
The sieving effect (S) reduces the relative diffusion
rates of the diffusing NaCl and water from 1 in pure
solution to ⬃0.5 and 0.75, respectively, in cheese
moisture. As the effective pore restriction on the dif-
fusion of NaCl in cheese moisture is determined by its
effect on the larger molecule, i.e., NaCl, the pseudo-
diffusion coefficient was estimated to be reduced by a
factor of S  0.5.
5. Frictional effects of protein-bound water. Water
binding in cheese (0.1–0.15 g H2O/g para-casein;
Geurts et al., 1974a) accounts for 10–15% of the total
cheese moisture. The bound water increases the
effective diameter of protein particles of which the
matrix is composed, and, thereby, reduces the relative
pore width of the protein matrix. This in turn
increases the sieving effect on the migrating ions, as
discussed in 4, and the tortuosity factor (p) as dis-
cussed in 3.
Beginning from a simplified model of cheese struc-
ture and considering the relative effects of the interfer-
ing factors discussed, Geurts et al. (1974b) postulated
a theoretical ‘pseudo-diffusion’ coefficient,
0.8  DS
D* 
r!rel
While the model cheese structure adopted by
Geurts et al. (1974b) is simplified in view of the
results of electronmicroscopic examinations of cheese
structure (Kimber et al., 1974; de Jong, 1978; Kaláb,
1995; Guinee et al., 1998; Auty et al., 2001), the
calculated impedance derived from it was sufficient
to explain the very low diffusion coefficient of NaCl
in cheese moisture and the variations of D* with vari-
ations in cheese composition and brining conditions.
A number of later studies (Minarik, 1985; Luna and
Bressan, 1986, 1987; Luna and Chavez, 1992; Payne
and Morison, 1999) on the modelling of salt and
water diffusion in semi-hard cheeses have verified the
low diffusivity of salt in cheese moisture. Using devel-
oped prediction models, these studies found a close
correlation between the experimental data from earl-
ier studies (Geurts et al., 1974b; Guinee and Fox,
1983a) and predicted salt and moisture levels in the
cheese.
Direct mixing of salt with milled curd
When dry salt is distributed over the surface of milled
curd or curd granules, some NaCl dissolves in the sur-
face moisture and diffuses slowly inwards a short dis-
tance (Breene et al., 1965; Sutherland, 1974). This
causes a counterflow of whey from the curd to the sur-
face which dissolves the remaining salt crystals and, in
effect, creates a supersaturated brine solution around
each particle, provided mixing of curd and salt is ade-
quate. However, because of the relatively large surface
area to volume ratio of the curd as a whole, salt uptake
occurs from many surfaces simultaneously and less
time is required for uptake of an adequate amount of
salt in dry-salting milled curd (10–20 min) than in
brining whole cheeses (0.5–5 days depending on the
230

Table 5 Influence of cheese composition on salt diffusion in cheese moisture

Properties of unsalted cheese Obstructing factors Diffusion coefficients

Volume Volume Relative

Moisture fraction of fraction of pore width Fat Protein
Cheese Fat Moisture Solids Fat-in-dry in-fat-free fat phase protein of protein tortuosity tortuosity D* D* f
code (%) (%) not-fat (%) matter (%) cheese (%) (f) matrix (p) matrix (y/dp) (f) (p) (cm2/day) (cm2/day)

1 0.00 53.00 47.00 0.00 53.00 0.000 0.466 0.132 1.000 1.425 0.136 0.136
2 10.88 49.00 40.12 21.33 54.98 0.127 0.442 0.152 1.117 1.409 0.153 0.171
3 19.88 45.93 34.19 36.77 57.33 0.227 0.413 0.178 1.229 1.389 0.203 0.249
4 26.25 43.48 30.27 46.44 58.96 0.297 0.394 0.198 1.296 1.378 0.227 0.294
5 0.00 52.90 47.10 0.00 52.90 0.000 0.467 0.132 1.000 1.425 0.140 0.140
6 10.00 48.93 41.07 19.58 54.37 0.117 0.449 0.146 1.105 1.414 0.202 0.223
7 18.25 44.90 36.85 33.12 54.92 0.207 0.442 0.152 1.208 1.409 0.205 0.248
8 30.28 40.50 29.22 50.89 58.09 0.340 0.404 0.187 1.333 1.383 0.236 0.315
9 18.18 49.20 32.62 37.59 60.13 0.207 0.380 0.213 1.208 1.365 0.295 0.356
10 20.00 47.94 32.06 38.42 59.93 0.227 0.382 0.211 1.229 1.366 0.263 0.323
11 21.00 45.02 37.98 38.20 56.99 0.240 0.417 0.174 1.242 1.392 0.207 0.257
12 21.00 44.44 34.60 37.77 56.25 0.240 0.427 0.165 1.242 1.400 0.176 0.218
13 29.00 41.11 28.89 49.24 57.90 0.326 0.406 0.185 1.322 1.384 0.216 0.285
14 27.66 44.02 28.32 49.41 60.85 0.310 0.371 0.223 1.308 1.358 0.247 0.324

For calculation of f and p it was assumed that: (a) cheese moisture contained 5% dissolved solids, density  1 g/ml; (b) the protein matrix consisted of protein  15% water bound
and had a specific gravity of 1.25; (c) the specific gravity of fat  0.93. Cheeses were from four trials, i.e cheeses 1–4, from trial 1; 5–8, from trial 2; 9–12, from trial 3; and 13, 14 from
trial 4. All cheeses were salted in 18.5% NaCl at 20 °C for 3–4 days (from Guinee, 1985).
 Table 6 Experimentally determined diffusion coefficient (D*) of salt in moisture in cheese varying in properties and brined under different conditions. Calculation of diffusion
coefficient in moisture (D*v) and of relative pore width of the protein matrix (y/d)e in fat-free cheese

Diffusion coefficient
Properties of non-salted cheese Calculation of factors (cm2/day)

Brine Fat Moisture

(g NaCl/ g fat in content content
100 g H2O) 100 g DM pH (%) (%) v v e (y/d)e D* D* v

19.7 12 5.00 5.3 53.0 0.06 1.04 0.42 0.171 0.164 0.170
19.0 12 5.01 4.9 54.0 0.06 1.04 0.41 0.181 0.185 0.192
19.5 12 4.99 5.0 55.0 0.06 1.04 0.40 0.191 0.162 0.168
20.2 22 5.00 10.9 50.2 0.13 1.12 0.42 0.171 0.152 0.171
19.8 50 5.10 33.0 36.2 0.37 1.36 0.44 0.153 0.100 0.136
20.5 50 4.79 31.2 41.1 0.35 1.34 0.37 0.225 0.160 0.215
20.0 50 5.42 30.9 41.5 0.34 1.33 0.37 0.225 0.160 0.213
19.6 50 5.09 30.4 42.2 0.34 1.33 0.37 0.225 0.172 0.229
20.4 50 5.02 29.9 42.5 0.33 1.32 0.36 0.238 0.185 0.245
34.8 50 5.09 29.9 42.5 0.33 1.32 0.36 0.238 0.148 0.196
14.0 50 5.10 30.2 42.5 0.34 1.33 0.36 0.238 0.194 0.258
19.7 50 5.07 29.2 42.9 0.33 1.32 0.37 0.225 0.187 0.248
13.8 50 5.18 29.5 43.0 0.33 1.32 0.36 0.238 0.177 0.234
20.0 50 5.64 28.9 43.4 0.32 1.32 0.36 0.238 0.168 0.221
13.8 50 4.98 26.9 48.0 0.30 1.30 0.31 0.312 0.235 0.305
20.1 50 4.92 26.6 49.0 0.29 1.29 0.30 0.330 0.258 0.333
19.4 50 5.09 25.8 50.1 0.29 1.29 0.29 0.349 0.239 0.309
20.0 62 5.00 39.1 38.5 0.43 1.40 0.34 0.265 0.179 0.251
20.1 62 4.98 37.1 40.8 0.41 1.39 0.32 0.295 0.224 0.311

v  volume fraction of fat in cheese, calculated from fat content.

v  tortuosity factor of fat, a function of v.
e  volume fraction of protein matrix in fat-free cheese.
(y/d)e  relative pore width of this matrix.
D*  pseudo-diffusion coefficient in moisture in cheese.
In calculating v and e it is assumed that cheese moisture contains 5% dissolved substances (density of solution  1), that the protein matrix consists of protein  15% water
(density 1.25), and that the density of the fat is 0.93. All experiments were carried out at about 12.5 °C (from Geurts et al., 1974b).
231
232 Salt in Cheese: Physical, Chemical and Biological Aspects

dimensions). Some of the ‘brine’ on the surface of curd 0.21

particles drains away through the curd mass while

Diffusion coefficeint, D*, cm2/d

more is physically expelled from the curd particles
during pressing and is lost in the ‘press whey’. As the
salt/surface area ratio is usually low, and the period
of contact of the curd surface with the concentrated
brine layer is relatively short (i.e., 20 min mellow-
ing period), little localized surface protein contraction
occurs compared to that in dry-salted, moulded curds
(Sutherland, 1974).
Dry surface-salting of moulded pressed cheese curd
A block of curd can be regarded as a very large particle
and solution of dry salt in the surface moisture layer is
a pre-requisite for salt absorption in this method also.
The counter flow of moisture from the cheese creates a
supersaturated brine layer on the cheese surface and
salt uptake then occurs by an impeded diffusion
process. Because the surface is in contact with a con-
centrated brine for a long time (several days), there is
considerable contraction of the curd surface (salting
out of protein) and this probably leads to relatively
high moisture losses from the surface region and
hence a reduction in the inward mobility of NaCl
which accounts for the lower rate of salt uptake in this
method than in brining (Godinho and Fox, 1981b;
Melilli et al., 2003).
Factors influencing salt absorption by cheese
The only pre-requisite for salt absorption by cheese is
the existence of a salt-in-moisture gradient between the
cheese and the salting medium. However, the quantity
of salt absorbed depends on the intrinsic properties of
the cheese, the conditions of salting and the duration of
salting. As the different procedures of salting all involve
salt absorption via an impeded diffusion process, the
general factors affecting salt uptake by cheese apply
0.2
0.19
0.18
0.17
0.16
0 5 10 15 20 25
Brine concentration, %, w/w, NaCl
Figure 10 The effect of brine concentration on the diffusion
coefficient, D*, of salt in the moisture phase of experimental
Gouda cheese (43.6%, w/w, moisture; 49.1%, w/w, fat-in-dry
matter) salted for 4 days at 20 °C (redrawn from Guinee, 1985).
concentration in the range 5–25%, w/w (Fig. 11;
Breene et al., 1965; Sutherland, 1974; Guinee, 1985;
Guinee and Fox, 1986a; Chamba, 1988; Kaya et al.,
1999; Prasad and Alvarez, 1999; Melilli et al., 2003).
This is due to the reduction in the concentration gra-
dient of S/M between the cheese moisture and the
brine. Hence, in model brining experiments, in which
cheese slices of different thickness were completely
submerged in brine, there was a sharp decrease in the
rate of salt absorption (per unit weight) as the differ-
ence between the NaCl concentration in the cheese
moisture and the brine decreased, especially when the
7
6
Salt in cheese, %, w/w

equally to granules or milled curd pieces on mixing

5
with dry salt and to moulded cheeses which are brined
and/or dry salted. Certain peculiarities of the salting of
milled curd pieces, as in Cheddar, which affect salt 4
absorption will be discussed separately.
3
Brine concentration and concentration gradient
It is generally accepted that an increase in brine con-
centration results in greater salt absorption and an 2
increased salt-in-moisture level in the cheese (Figs 8, 9);
Breene et al., 1965; Geurts et al., 1980; Godinho and 1
5 10 15 20 25
Fox, 1981b; Guinee and Fox, 1986a; Apostolopoulos
et al., 1994; Pappas et al., 1996; Kaya et al., 1999). Brine concentration, %, w/w, NaCl
While the rate of NaCl diffusion is scarcely affected by
Figure 11 Salt level in Romano-type cheese slices (0.5 cm
brine concentration in the range 5–20% (Guinee, thick; 7 cm diameter) as a function of brine concentration after
1985; Geurts et al., 1974b; Fig. 10), the rate of uptake salting for 50 (), 100 () or 200 (●) min in 20%, w/w, NaCl
increases at a diminishing rate with increasing brine brine containing 0.5%, w/w, Ca (redrawn from Guinee, 1985).

initial difference was large (Fig. 12; Guinee and Fox,
1986a). A somewhat similar situation applies to dry-
salted cheese: the increase in salt-in-moisture level in
Cheddar curd is not proportional to the increase in the
level of dry salt added to the milled curd (O’Connor,
1974; Gilles, 1976). This is attributed to increased salt
losses with increased salting level, which reflects the
decreasing effect of the driving force (concentration
gradient) in raising the quantity of salt absorbed as the
salt-in-moisture level in the cheese approaches that of
the brine.
While increasing the NaCl concentration in the
brine from ⬃19%, w/w, to 25% or 31%, w/w, at 20 °C,
results in an increase in the level of salt absorbed by
brine-salted Gouda cheeses (Geurts et al., 1980;
Guinee, 1985), Romano cheese slices (Guinee and Fox,
1986a), brine-salted Cheddar cheese cubes (Breene
et al., 1965), the diffusion coefficient, D*, and hence,
the depth of penetration of salt into the cheese,
decreases sharply (Fig. 10).
In contrast, Melilli et al. (2003) reported a markedly
lower mean salt content (⬃35%) in Ragusano cheese,
at day 8, on raising the NaCl concentration in the brine
from 18 to 30%, w/w. The discrepancy between the
results of Melilli et al. (2003) and previous studies
(Breene et al., 1965; Geurts et al., 1980; Guinee, 1985;
Guinee and Fox, 1986a) may be related to differences
in the lactate content of the cheese, which is likely to
be lower in Ragusano than in Gouda, Cheddar or
Romano, because of the loss of lactate during plasti-
cization of the Ragusano curd in hot water at a curd-
0.35
g NaCl absorbed per 100 g cheese per min
0.30
0.25
0.20
0.15
0.10
0.05
0.00
0 5 10 15
Δ NaCl level (%, w/w) between
cheese moisture and brine
Figure 12 Influence of NaCl concentration gradient between
the cheese moisture and the brine on salt uptake by Romano-
type cheese slices (0.5 cm thick; 7 cm diameter) salted in 20%,
w/w, NaCl brine containing 0.5%, w/w, Ca (redrawn from Guinee
and Fox, 1986a).
Salt in Cheese: Physical, Chemical and Biological Aspects 233
to-water ratio of ⬃1:3. Owing to the higher concentra-
tion of lactate in the moisture phase of cheeses than in
the brine, an outward migration of lactate form the
cheese to the brine and a counter-flow of water from
the brine to cheese would be expected in all cheeses
during brine-salting. We are not aware of studies on
this but the data of Pavia et al. (1999) show lower con-
centrations of lactate in the surface than in the centre
of Manchego cheese. The quantity of lactate lost and
water gained as a result of this mutual lactate-water dif-
fusion process depends on the lactate concentration in
the cheese moisture prior to salting, with a higher lac-
tate concentration leading to a higher water uptake
by the cheese. Hence, all other factors being equal, a
low level of lactate-in-moisture before brine-salting is
expected to result in a greater net loss of water from
the cheese that occurs as a result of the mutual salt-
water diffusion process during brining. Consequently,
salting of Ragusano cheese, for which the lactate level
is expected to be lower than that of Gouda and other
non-plasticized cheeses, in high-NaCl brine (e.g.,
25–30%, w/w) probably leads to a relatively large water
loss, especially from the surface layer of the cheese. A
high level of dehydration in the surface layer would
impede salt absorption because of the concomitant
increases in protein content and the consequent reduc-
tion in pore width of the protein matrix (see ‘Moisture
content of cheese’).
Method of brine-salting
High pressure (HP) treatment of cheese during brining
at 100–500 MPa, at 20 °C for 15–30 min, did not signifi-
cantly affect salt uptake in Gouda cheese but in some
cases reduced moisture loss (Messens et al., 1999).
Similarly, vacuum impregnation brining at 3.7 kPa,
absolute, did not effect the mean S/M level in
Manchego-type cheese (Pavia et al., 1999). However,
in contrast to observations with HP-brining (Messens
et al., 1999), vacuum impregnation brining gave a more
uniform S/M distribution immediately after brining
than conventional brine-salting.
The quantity of salt taken up by Mozzarella on
brine injection under pressure was directly propor-
tional to the number of injections for salt levels of
0–3%, w/w (Pastorino et al., 2003a); the results of
Lee et al. (1978) suggest that salt retention on pres-
sure injection is proportional to the square root of
20
pressure and decreases with the diameter of the jet
orifice.
Cheese geometry
It is generally agreed that the rate of salt absorption
increases with increasing surface area to volume ratio
of the cheese (Breene et al., 1965; Gilles, 1976; Guinee
234 Salt in Cheese: Physical, Chemical and Biological Aspects
and Fox, 1986a). This is most readily observed on
comparing the rate of salt uptake by milled curd (e.g.,
Cheddar) and whole moulded cheeses (Brick, Emmental,
Romano or Blue-type cheeses) in brine; in the former,
salt absorption occurs from many surfaces simultan-
eously, and the time required to attain a fixed level of
salt is very much less than for brine-salted moulded
cheeses. While at first sight it may appear that smaller
cheeses would have a higher mean salt content than
larger ones after brining for equal intervals, this applies
only to cheeses of the same shape and relative dimen-
sions since salt uptake is linearly related to the surface
area to volume ratio of the cheese (Geurts et al., 1980;
Guinee and Fox, 1986a,b).
In addition to its influence on the surface area to
volume ratio, cheese shape also affects the rate of salt
absorption via its effect on: (i) the number of direc-
tions of salt penetration from the salting medium into
the cheese and (ii) the ratio of planar to curved surface
area of the cheese (Geurts et al., 1980; Guinee and
Fox, 1983b, 1986a,b). Geurts et al. (1980) found that
on brining Edam-type cheese, the quantity of NaCl
absorbed per cm2 cheese surface was greater for an
infinite slab than for a sphere, and the relative reduc-
tion in salt uptake through curved surfaces increased
with brining time and with the degree of curvature. In
Romano-type cheeses with approximately equal sur-
face area to volume ratios, the rate of salt absorption
by rectangular-shaped cheeses (volume: 4000 cm3; three
effective directions of salt penetration) was higher
than that by cylindrical cheese (volume: 3400 cm3;
two effective directions of salt penetration at any time
during a 9-day brining period (Fig. 13; Guinee and
Fox, 1986b). For cylindrical, spherical or rectangular
cheeses with a volume 1000 cm3, the surface area is
0.12
Salt uptake, g/cm2 cheese surface
0.1
0.08
0.06
0.04
0.02
0 0
0 3
Brining time, days
Figure 13 Influence of cheese shape on salt uptake (, ) and moisture loss (,) by Romano-type cheese during salting in
19.5%, w/w, NaCl brine at 23 °C. Rectangular cheeses (,), cylindrical cheeses ( ,) (redrawn from Guinee and Fox, 1986c).
607, 483 and 600 cm2, respectively, i.e., surface area to
volume ratios of 0.607, 0.483 and 0.600, respectively.
As discussed below, the rate of salt uptake is affected
by whether the surface is flat or curved; for the above
cheeses, the area of the curved surface is 275, 483 and
0 cm2, respectively.
Salting time
It is well established that the quantity of salt absorbed
increases with salting time (Byers and Price, 1937;
Hoecker and Hammer, 1944; Breene et al., 1965;
Sutherland, 1974; Geurts et al., 1980; Godinho and
Fox, 1981b; Luna and Bressan, 1986; Guinee and Fox,
1986a,b; Turhan and Kaletunç, 1992; Nájera et al.,
1994; Kristiansen et al., 1999; Messens et al., 1999;
Melilli et al., 2003). However, the rate of salt absorp-
tion decreases with time due to a decrease in the NaCl
concentration gradient between the cheese moisture
and the brine (Geurts et al., 1980; Guinee and Fox,
1986a,b; Melilli et al., 2003). Indeed, the quantity of
salt taken up by a cheese is proportional to the square
root of brining time, √t (Geurts et al., 1980; Guinee
and Fox, 1986a; Messens et al., 1999). However, as the
curvature of the cheese surface increases, the propor-
tionality of salt uptake with √t is lost and the relative
reduction of salt uptake per unit area of cheese surface
increases with increasing degree of curvature, and with
time (Geurts et al., 1980). This implies that for cheeses
with an equal volume and composition, brined under
the same conditions, the rate of salt absorption per unit
surface area (and hence the cheese as a whole) would
be in the order: rectangular  cylindrical  spherical
(Guinee and Fox, 1986b); however, aspects of cheese
other than geometry affect the mean salt level, as dis-
cussed above. Geurts et al. (1980) derived a theoretical
(a) 0.18 (b)
Water loss, g/cm2 cheese surface
0.16
0.14
0.12
0.1
0.08
0.06
0.04
0.02
6 0 3 6 9
Brining time, days
 Salt in Cheese: Physical, Chemical and Biological Aspects 235
relationship for the quantity of salt absorbed through a
flat surface as a function of brining time:
Mt  2(C Co) (D*t/ )1/2 Vw (1)
where Mt  quantity of salt absorbed over time, g NaCl/
cm2, C  salt content of brine, g NaCl/ml, Co  ori-
ginal salt content of the cheese, g/ml, t  duration of the
salting period, days, D*  pseudo-diffusion coefficient,
cm2/day, Vw  average water content throughout the
cheese at time t, g/g.
Applying this theoretical relationship to their model
brining experiments on cylindrical Gouda cheeses brine-
salted by unidimensional diffusion through one of the
planar surfaces in contact with the brine (Geurts et al.,
1974b), Geurts et al. (1980) found that the predicted
values for the quantity of salt absorbed per cm2 planar
surface (Mp) were in close agreement with the experi-
mental values (Mt) over a three-day brining period: Mt
 0.98Mp. The strong relationship between Mt and Mp
confirms the accuracy of the experimentally deter-
mined D* values.
Temperature of curd and brine
In model brining experiments, Breene et al. (1965)
studied the effect of curd temperature (27, 32, 38, 43 °C)
on salt uptake by Cheddar curd cubes (1 cm3) in
brines (20 or 25%, w/w, NaCl) at the same tem-
perature as the curd. Salt uptake was lowest at 32 °C,
similar at 27 and 38 °C, and highest at 43 °C. The
reduction in salt content on changing the brining tem-
perature from either 27 or 38 °C to 32 °C was ⬃6.5%.
The low salt uptake at 32 °C was attributed to a layer
of exuded fat on the surface of the curd particles
which impeded salt uptake; less fat was exuded at
lower temperatures while at temperatures 32 °C,
exuded fat was liquid and dispersed in the brine.
Increasing brine temperature increases the mobility of
NaCl and salt absorption in Gouda (Geurts et al.,
1974b; Guinee, 1985), Emmental (Chamba, 1988),
Romano-type (Guinee, 1985; Guinee and Fox, 1986a)
and Turkish White (Turhan and Kaletunç, 1992)
cheeses partly due to an increase in true diffusion and
partly to an increase in the effective pore width of the
protein matrix as non-solvent water decreases with
increasing temperature (Geurts et al., 1974b). The
increase in D* for Romano and Gouda cheeses in the
range 5–25 °C in 19.2% NaCl brine was similar
at ⬃0.0083 cm2/day (Guinee, 1985). Geurts et al.
(1974b) reported that D* for Gouda cheeses salted in
⬃20% NaCl increased by ⬃40–50% on increasing the
temperature from 12.5 to 25 °C, compared to an
increase in D in pure water of ⬃20% over the same
temperature range.
Initial salt-in-moisture level of curd and pre-salting
Brine-salting of cheese can be an expensive process in
terms of space, maintenance cost and corrosiveness of
the brine. Consequently, pre-salting of cheese as a
means of reducing the brining time of Gouda (Guinee,
1985) and Ragusano (Melilli et al., 2003) cheeses has
been investigated.
Guinee (1985) mixed Gouda curds, immediately
after whey drainage, with varying quantities of dry
salt to give an S/M level ranging from ⬃0.25 (control,
unsalted curd) to ⬃14%, w/w, in the (pre-salted)
curd. The salted curds were left undisturbed for ⬃5
min and then moulded and pressed in the usual man-
ner; the moulded cheeses were stored at the brine
temperature (15 °C) for 1 day, covered with wax on
three sides and the unwaxed side placed in contact
with 19.2%, w/w, NaCl for 3 days. On completion of
brine-salting, the S/M levels decreased with distance
from the cheese–brine interface until it approached
that of the pre-salted curd while moisture showed the
opposite trend (Fig. 14a). Increasing the level of pre-
salting and, hence, the S/M level in the curd prior to
brine-salting, increased the levels of salt and S/M and
reduced the level of moisture in the final cheese. A
similar trend was noted for Ragusano cheese which
was pre-salted by adding dry salt at a level of 4%, w/w,
prior to plasticization in dilute brine (4.5%, w/w),
and brine-salted for 1–24 days in 18%, w/w, NaCl at
18 °C (Melilli et al., 2003). In agreement with earlier
studies, which showed that the difference in salt-in-
moisture gradient between the cheese moisture and
the brine was a major determinant of the quantity
of salt absorbed (see ‘Brine concentration and con-
centration gradient’; Equation 1; ‘Salting time’), the
magnitude of the increase in S/M decreased with the
level of pre-salting (Fig. 14b) indicating a decrease in
the quantity of salt absorbed per unit surface area
of cheese (Guinee, 1985). However, the S/M level
increased in all cheeses and approached closer to that
of the brine with the level of pre-salting prior to brin-
ing. A similar trend was noted for Feta cheese stored
in brines of different salt concentration (Prasad and
Alvarez, 1999). Indeed, the S/M level in the outer
(rind; 5 mm thick) layer of cheeses pre-salted to
6%, w/w, S/M exceeded the NaCl concentration in
the brine at the end of the 3-day-brining period, to a
degree which increased with the level of pre-salting
(Fig. 14a). This occurrence was attributed to an
intense ‘salting-out’ and shrinkage of the protein
matrix in the rind layer of these cheeses because of
the very high level of S/M (⬃19–22%, w/w) (Guinee,
1985). Hence, the moisture level in rind of the brine-
salted cheese decreased markedly on pre-salting to
6%, w/w, S/M (Fig. 14b).
236 Salt in Cheese: Physical, Chemical and Biological Aspects
(a)
22.00
20.00
18.00
Salt-in-moisture, %, w/w
16.00
14.00
12.00
10.00
8.00
6.00
4.00
2.00
0.00
0 1 2 3 4 5
Distance from cheese surface, cm
Figure 14 Salt-in-moisture concentration (a) and moisture (b) as a function of distance from cheese/brine interface in experimental
Gouda-type cheeses which were pre-salted to 0.5 ( ), 4.8 (), 6.0 ( ), 8.4 (), 9.5 (), 10.5 () or 12.5 (●) %, w/w, NaCl before
brine-salting at 15 °C in 19.2%, w/w, NaCl brine with ⬃0.5% %, w/w, Ca. The drained curds were thoroughly mixed with dry salt,
added at the desired quantity; the salted curds were held at room temperature for 5 min, then pressed, and brine-salted; initial NaCl
concentration in brine (redrawn from Guinee, 1985).
Initial moisture content of the curd
Geurts et al. (1974b) showed that the quantity of salt
absorbed by experimental Edam and Gouda-type
cheeses during brine-salting generally increased as the
initial moisture content of the curd increased, with
the effect becoming more pronounced with the dur-
ation of brining. Similar results were obtained by Byers
and Price (1937) for brine-salted Brick cheese. This
increased salt uptake by the experimental cheeses con-
curs with the linear increase in predicted salt uptake
through a flat cheese surface as the water content
increases (see ‘Cheese geometry’). The increased salt
uptake undoubtedly reflects the concomitant increase
in D* as moisture level increases. A high moisture
content leads to a lower protein level, a lower volume
fraction of the protein matrix occluding the moisture
through which NaCl diffuse, an increase in the rela-
tive pore width of the protein matrix and hence
a reduced frictional effect on the inward-diffusing
Na and Cl .
On dry-salting milled Cheddar curd, the reverse situ-
ation occurs: as the initial moisture level increases, the
rate of salt absorption decreases giving lower salt and
S/M values in the cheese for a fixed salting level
(Sutherland, 1974; Gilles, 1976). Such decreases were
attributed to greater whey and salt losses from the
high-moisture curds; an increase in curd moisture con-
tent from 39.1 to 43.4%, w/w, caused a 30% increase in
the amount of whey drained off and a decrease in salt
(b)
44
40
Moisture, %, w/w
36
32
28
6 0 1 2 3 4 5 6 7
Distance from cheese surface, cm
retention from 59 to 43%, w/w, of the amount applied
(Sutherland, 1974). Thus, while the extent of salt pene-
tration within each granule increases, there is less salt
available for uptake as the initial curd moisture
increases (salt causes loss of moisture from the curd
and at the same time is itself removed).
The apparent discrepancy between the effects of
moisture content on salt uptake in brined-salted and
dry-salted cheeses may be due to differences in the
degree of contact between the salting medium and the
curd, and in the length of contact time between curd
and salting medium. On increasing the moisture con-
tent of dry-salted cheeses, the higher outflow of mois-
ture may result in ‘excess brine’ which percolates
through the spaces between the chips, drains away,
loses contact with the chips and thereby lowers the
effective amount of salt available for uptake. This does
not happen when cheese is submerged in brine, as in
brine-salting. Moreover, there is sufficient time for a
lactate/water (brine) mutual diffusion process during
brine-salting whereas this is not expected during dry-
salting as the water ‘drawn out’ of the cheese by the
applied dry salt quickly drains away through the curd
bed and loses contact with most of the curd. As the
lactate level in cheese increases with moisture content,
a greater influx of water as a result of the lactate/water
diffusion process is expected. Consequently, an increase
in the moisture level in cheese prior to brine-salting is
expected to reduce the net water loss per given weight
 Salt in Cheese: Physical, Chemical and Biological Aspects 237
of cheese and increase salt penetration and uptake
during subsequent brine-salting. This hypothesis con-
curs with the increase in D* (see ‘Factors that influ-
ence salt diffusion in cheese during salting’), and the
tendency of the flux ratio (i.e., the ratio of water lost
to salt absorbed during brining) to decrease (Geurts
et al., 1980) as the moisture level of Gouda cheese pre-
brining is increased.
pH of curd and brine
While D* for Gouda cheese was not influenced by
cheese pH in the range 4.7–5.7 (Geurts et al., 1974b),
the rate of salt uptake was higher at pH 4.7 than at
5.7 even though the initial moisture content of the
cheeses was similar (Geurts et al., 1980). This finding
was consistent with that of previous studies, which
showed higher NaCl uptake at low, than at high,
cheese pH (Geurts et al., 1980). The higher salt uptake
at lower cheese pH coincides with a lower water
loss during brining (per unit weight of salt gained),
which may be attributed to a higher lactate level in
the low-pH cheese (Geurts et al., 1980). A higher lac-
tate level pre-brining would reduce the net loss of
water during brining (see ‘Brine concentration and
concentration gradient’ and ‘Initial moisture content
of the curd’).
A number of investigators have examined the effect
of titratable acidity at salting on salt retention by Ched-
dar cheese curd. However, Cheddar curd dry-salted at
low acidity retained more salt than more acidic cheeses
(Lawrence and Gilles, 1969, 1982; Gilles, 1976). Since
low-acid curd normally contains more moisture than
high-acid curd, one might expect more syneresis and
higher salt losses, less available salt for absorption, and
therefore less salt uptake in the low-acid curd. How-
ever, for a given salt availability, the rate of salt diffu-
sion and salt uptake would be expected to increase as
the level of moisture in the curd increases, as discussed
in ‘Initial moisture content of the curd’). Lawrence and
Gilles (1969) suggested that the observed difference in
salt retention may be due to the higher degree of curd
hydration at the higher pH values (i.e., ⬃5.3), which
may effect a higher retention of salt by the curd struc-
ture per se (see Dolby, 1941; Creamer, 1985; ‘Effect of
NaCl on casein hydration in model systems and in
cheese’).
In practice, the pH of brine is adjusted to ⬃5.0–5.3,
which is close to that of most brine-salted cheeses.
Acidification of the brine has a preservative effect and
also minimizes the risk of surface defects (e.g., velvety,
non-drying rind) associated with an imbalance in
[H] which affects the level of casein hydration (see
‘Effect of NaCl on casein hydration in model systems
and in cheese’). While little information is available on
the effect of brine pH on salt uptake (Geurts et al.,
1980), it is conceivable that excessive lowering of the
pH (e.g., 4.6) would lead to protein precipitation and a
high loss of water at the cheese surface, which in turn
would reduce salt uptake.
Factors that affect salt uptake in Cheddar curd
Method of salting
Breene et al. (1965) showed that salting of milled
Cheddar curd by brining gives a higher rate of salt
absorption and a higher level of salt-in-moisture in the
pressed curd than dry salting. Differences in absorp-
tion rate were explained on the basis of availability of
salt at the surfaces of the curd. When dry salt is placed
on freshly milled curd, a portion dissolves in the sur-
face moisture, creating a very thin layer of super-
saturated brine. The salt-in-moisture gradient between
the brine and the cheese moisture results in mutual
movements of salt and water in opposite directions
in response to their respective concentration gradients.
Some water is also ‘squeezed out’ of the curd due to
localized surface contraction (salting-out of the protein
matrix) as a result of contact with the super-saturated
brine. The level of moisture in the curd, which influ-
ences whey release, affects the rate of solution of sur-
face salt. When curd is placed in brine, salt absorption
begins immediately through all surfaces. Release of whey
occurs, as in dry salting, but its extent is not a limiting
factor (Sutherland, 1974).
Level of salting
As expected, an increase in salting level (especially
when the level is low) increases the rate of salt
absorption by, and whey drainage from, cheese, thus
giving higher levels of salt and salt-in-moisture and a
lower level of moisture in the cheese after salting for
a fixed time (Breene et al., 1965; O’Connor, 1970,
1971, 1973b, 1974; Gilles, 1976; Guinee, 1985; Kelly
et al., 1996). However, the relationship is curvilinear
(Fig. 15; O’Connor, 1973b), i.e., the increase in the
salt and salt-moisture levels in the cheese is not pro-
portional to the level of salt added, especially at the
higher salting levels, because of higher salt losses at
increased salting levels. Although these principles are
probably generally applicable, the precise relation-
ship between salt loss and retention depends on the
pH and moisture content of the curd and the period
of time allowed for salt diffusion into the curd. These
inter-relationships have been studied by Sutherland
(1974) and Gilles (1976). Sutherland (1974) showed
that the volume of whey released from the curd and
the percentage of added salt lost increased linearly
with the level of salt added (over a narrower range
than that used by O’Connor) while the percentage
238 Salt in Cheese: Physical, Chemical and Biological Aspects
Salt in cheese, %, w/w
3.0
1.5
0.0
0 2 4 6
Salting level, g/100 g
Figure 15 Relationship between salting level of curd and the
concentration of salt (●) and salt-in-moisture () in Cheddar
cheeses, prepared from batches of curd from the same vat
(redrawn from Lawrence and Gilles (1982) using the data of
O’Connor (1974)).
of moisture decreased and the percentage of salt, salt-
in-moisture and pH of the cheeses increased in a
curvilinear fashion as the level of added salt was
increased. The level of salt added had no significant
effect on the loss of fat (⬃0.25 kg/100 kg curd). Kelly
et al. (1996) reported a linear increase in salt and S/M
and a linear decrease in moisture with salting level in
the range 0–3.0%, w/w.
Curd temperature
Increasing the temperature of Cheddar curd chips
from 24 to 41 °C resulted in a marked increase in the
percentage of added salt lost during holding (mellow-
ing), a decrease in the level lost during pressing, and a
slight increase in the percentage lost overall during
holding and pressing (Sutherland, 1974). Conse-
quently, the levels of salt and S/M decreased by ⬃11%
on raising the curd temperature from 24 to 41 °C and
the percentage of fat lost increased markedly, e.g., by
⬃0.6 kg fat/tonne curd per 1 °C rise in temperature in
the normal working temperature range of 29–35 °C.
The pH and moisture level in the finished cheese were
essentially unaffected by salting temperature.
Degree of mixing of salt and curd
Extending the stirring time of salted Cheddar curd
from 20 s to 6 min caused a significant increase in
salt and S/M levels, i.e., from 1.53 to 1.97%, and 4.41
9.0 to 5.71%, respectively (Sutherland, 1974). Undoubt-
edly, better mixing leads to salt absorption from more
faces and hence there is less ‘free’ salt to be lost
7.5 in the press whey. Hence, salt losses decreased with
the duration of stirring time even though there was
little effect on the volume of whey released (Sutherland,
Salt-in-moisture, %, w/w
6.0 1974). For a given mixing period, increasing the
surface area of the curd by reducing the size of the
curd chips results in a significant increase in salt
4.5
level (Gilles, 1976). Increased mixing time resulted
in a higher loss of fat, probably as a consequence of
3.0
shrinkage at the surface of the curd chips and a con-
comitant increase in the loss of fat in the salt/water
(Sutherland, 1974).
1.5 Mechanical salting procedures give more uniform
distribution of salt in Cheddar cheese than hand or
semi-automated salting systems (O’Connor, 1968,
0.0 1970, 1973b; Fox, 1974; Knox, 1978). Because of the
significance of salt level and distribution in relation to
cheese quality, salting of Cheddar curd at factory level
is a carefully controlled operation. It is performed on
enclosed inclined, perforated (to allow whey drainage)
belts where a single, or twin, oscillating boom distrib-
utes the salt, delivered from an overhead metering
device according to a sensor which measures the bed
depth, onto the moving curd bed and overhead stir-
rers continuously mix the curds. Improved means of
salting cheese curds, e.g., trommel salt mixers, are
being developed (Zahlus, 1986; Cosentino et al., 1987;
Ryskowski et al., 1989; ‘General Aspects of Cheese
Technology’, Volume 2).
Holding time between salting and pressing
(mellowing period)
Extending the holding time between salting and press-
ing increases the salt and S/M levels in the pressed
Cheddar cheese (Breene et al., 1965; Sutherland, 1974;
Gilles, 1976), e.g., by ⬃0.3%, w/w, on increasing time
from 15 to 30 min. The increase is attributed to a
higher total absorption and hence a reduction in the
physical loss of salt.
Curd depth during holding
When the depth of salted Cheddar curd during hold-
ing was increased from 12.7 to 68.0 cm, the moisture,
salt and S/M levels decreased from 35.1 to 34.9%, 1.81
to 1.68%, and 5.1 to 4.8%, respectively (Sutherland,
1974).
Moisture content of the curd
Sutherland (1974) and Gilles (1976) studied the
effect of moisture content, which was varied by
altering agitation speed, degree of whey removal at
half-whey-off stage, and/or the level of dry-stirring,
on salt uptake in Cheddar. Increasing the moisture
 Salt in Cheese: Physical, Chemical and Biological Aspects 239
content of the curd before salting from 37 to 45%,
w/w, resulted in reductions (⬃42%) in the levels of
salt and S/M, and in pH, and an increase in moisture
content. These changes coincided with an increase
in the level of whey released and a reduction in
salt retention, e.g., from ⬃59 to 39% of total salt on
increasing the moisture level from 39 to 43.5%, w/w
(Sutherland, 1974).
Other factors
As well as confirming the work of Sutherland (1974),
Gilles (1976) showed that salt particle size has little
effect on salt retention, milling the curd to smaller
particles increases salt retention, and extensive flow
and development of a fibrous structure during ched-
daring leads to increased variation in S/M levels.
Indeed, Gilles (1976) maintained that the best way to
regulate the salt content of cheese is to control its
moisture content (which can be best done by dry
stirring). Regulating moisture content by altering
the level of salt added was considered undesirable
because of the influence of several factors on salt
retention and the effects of salt concentration on
cheese quality.
The interaction of some factors influencing salt
uptake in Cheddar-type curd and brine- or dry-salted
cheeses is summarized in Figs 16 and 17, respectively.
Factors that influence salt diffusion in cheese during
salting
While it is well established that the diffusion coeffi-
cient of salt in cheese moisture is much lower than
that in pure water (see ‘Mechanism of salt absorption
and diffusion in cheese’), there is relatively little inform-
ation on the factors which influence the movement of
NaCl in cheese during salting. The first such study
was made by Georgakis (1973), who related the diffu-
sion of NaCl in Greek Feta to cheese surface area,
Manufacturing conditions
Curd acidity Curd particle
at salting size at milling
Moisture Salt uptake
Salting
content rate
of curd
Extent of
mixing
Dry salting
Salt-in-moisture Method of
salting
Brine salting
Figure 16 Principal factors that affect the uptake of salt by
Cheddar curd (from Fox, P.F., ed., Cheese: Chemistry, Physics
and Microbiology, 2nd edn, Chapman & Hall, p. 281).
Manufacturing conditions
Cheese dimensions
at salting
Curd acidity Shape Surface area Size
at salting to volume
ratio
Moisture
content
of curd
Salt uptake Brining
conditions
(e.g. temperature,
% NaCl)
Salt-in-moisture
Figure 17 Principal factors that affect the uptake of salt by
brine-salted cheeses (from Fox, P.F., ed., Cheese: Chemistry,
Physics and Microbiology, 2nd edn, Chapman & Hall, p. 281).
duration of salting, brine concentration and the fat
and moisture contents of the cheese.
In model brining experiments, Geurts et al. (1974b)
quantified the influence of variations in cheese com-
position and brining conditions on the pseudo-diffu-
sion coefficient (D*) of NaCl in the moisture phase
of Gouda cheese. The factors which affect the move-
ment of salt in cheese during brining presumably also
apply to the cheese after brining and hence have a
decisive effect on the rate of attainment of equilib-
rium of S/M and of moisture; under normal brining con-
ditions (e.g., 18–20%, w/w, NaCl; 0.2% Ca; 10–20 °C),
moisture and salt move in opposite directions as
a consequence of diffusion (Geurts et al., 1974b;
Guinee and Fox, 1983b, 1986a,b). Although continu-
ing physico-chemical and structural changes during
ripening may alter the situation somewhat, it is
worth noting that the diffusion coefficient for NaCl
in the moisture phase of a dry-salted, 12-week-old
Cheddar (50% FDM, 37.9% H2O) (Sutherland, 1974)
corresponded well with that found by Geurts et al.
(1974b) for brine-salted Gouda cheese of similar
composition.
The influence of the various factors on NaCl diffu-
sion in Gouda cheese has been studied by Geurts et al.
(1974b), Guinee (1985), and Turhan and Kaletunç
(1992).
Concentration gradient
The concentration gradient between cheese and brine
is determined by the difference in the level of salt in
the brine and the S/M of the cheese, while the concen-
tration gradient between neighbouring regions within
240 Salt in Cheese: Physical, Chemical and Biological Aspects
a cheese loaf is determined by the difference in S/M
level between the two regions. The gradient changes
with time either for cheeses which are matured after
removal from the brine (as for most cheeses) or
matured in the brine (e.g., Feta, Gaziantep) until S/M
equilibrium is attained (Godinho and Fox, 1981b;
Guinee and Fox, 1986a,b,c; Pappas et al., 1996; Kaya
et al., 1999; Messens et al., 1999; Pavia et al., 1999;
Prasad and Alvarez, 1999; Melilli et al., 2003).
While the concentration gradient is a major determin-
ant of the rate of salt absorption by cheese during salt-
ing, it scarcely affects the mobility of the diffusing
species at concentrations in the range 5 to 20%, w/w,
NaCl (Geurts et al., 1974b; Guinee, 1985). However,
a sharp drop (⬃18%) in D* for Gouda-type cheese
occurred when the salt content of the brine was increased
from 20 to 24.8%, w/w, at 20 °C, i.e., from ⬃0.205
to 0.17 cm2/day (Guinee, 1985). Similarly, increasing
the brine concentration from 15 to 20%, w/w, NaCl
reduced the moisture diffusivity in Turkish white
cheese, with the reduction at 20 °C (⬃17%) being more
pronounced than that at 4 or 12.5 °C (⬃8%) (Turhan
and Gunasekaran, 1999). While the apparent D*
decreases on increasing NaCl level in the brine to 25%,
w/w, especially at high temperatures, the true value
would be somewhat higher if allowance was made for
relatively high water loss which in effect causes the
plane of zero mass transfer of all diffusing species to
recede further from the cheese/brine interface into the
brine. However, since S/M seldom reaches 20%, w/w,
in cheese, except in the rind layer, the large inter-zonal
variations in S/M level at the end of brining should not
significantly alter the rate of attainment of S/M equilib-
rium within a given cheese loaf or between loaves of the
same variety.
Temperature of brine and cheese
Increasing brine (and curd) temperature is paralleled by
increasing diffusion mobilities of NaCl and H2O in
cheese (Geurts et al., 1974b; Turhan and Kaletunç,
1992); an increase of ⬃0.008 cm2/day/°C was found for
Gouda-type cheeses for brine temperature in the range
5–25 °C (Guinee, 1985). This increase was attributed
(Geurts et al., 1974b) to an increase in true diffusion and
to some effect on diffusion-interfering factors, i.e., possi-
ble decreases in the relative viscosity of cheese moisture
and the amount of protein-bound water which effects an
increase in the relative pore width of the protein matrix
(in cheese, water non-solvent for sugars decreases with
increasing temperature; Geurts et al., 1974a).
Because of the large effect of temperature on D*,
the higher the storage temperature, the shorter should
be the time required for the equilibration of salt and
moisture levels within the cheese mass after salting.
Concentration of calcium in the brine
Preparation of brine requires the addition of calcium
(normally added as a CaCl2 solution) so as to mini-
mize moisture uptake by the exterior of the cheese
(Figs 8, 9) and the associated risk of defects such as
rind rot and soft rind, especially where the NaCl con-
centration in the brine is relatively low, e.g., 15%,
w/w (Geurts et al., 1972; Guinee and Fox, 1986a). The
latter defects are due to diffusion of calcium from the
cheese moisture to the brine which ultimately results
in the solubilization, and loss, of protein-bound cal-
cium to an extent depending, inter alia, on the level of
calcium in the cheese and brining conditions (pH,
temperature, NaCl level, duration, size of cheese rela-
tive to brine volume). The depletion of cheese calcium
leads to hydration of the para-casein and associated
moisture uptake and softening (see ‘Effect of NaCl on
casein hydration in model systems and in cheese’).
Typical levels of CaCl2 added give a calcium level in
the brine (e.g., 0.2–0.3%, w/w) which is probably
somewhat lower than that of some semi-hard brine-
salted cheeses, such as Gouda; however, these levels
are adequate to prevent the occurrence of the above
defects with typical NaCl level (19–21%, w/w) and
brine temperature (⬃12 °C). For hard and semi-hard
cheeses, the percentage of total cheese calcium which
is soluble increases with decreasing pH, e.g., from ⬃20%
for commercial low-moisture part-skim Mozzarella
(mean pH ⬃5.5) to ⬃37% in Cheddar at pH 5.2
(Guinee et al., 2000). Hence, the concentration of cal-
cium in the serum phase is ⬃0.72 and 0.34%, w/w, for
Cheddar and Mozzarella, respectively. The level of
serum calcium for other hard and semi-hard cheeses
such as Gouda, Masdammer and Emmental, with a pH
of ⬃5.3 to 5.4 before brining is probably similar to
that for Mozzarella; the level for low-calcium, soft
cheeses with a low pH (e.g., 4.6–5.0) before salting,
e.g., Camembert, Cheshire, Brie and Blue-types, are
undoubtedly much lower than 0.34%, w/w. Typical
levels of total calcium in Emmental, Mozzarella,
Camembert, Blue and Stilton are 1000, 710, 530, 475
and 400 mg/100g, respectively. Hence, the minimum
calcium level required in the brine depends on the
type of cheese being salted.
Increasing the concentration of Ca in freshly pre-
pared brine (19%, w/w, NaCl; 15 °C) from 0 to 1.8%,
w/w, reduced the moisture level (⬃3%, w/w) and
increased the salt content (⬃1.5%, w/w) of the outer
0.5 cm layer of Gouda cheese (Guinee, 1985). How-
ever, the increase in calcium had little effect otherwise
on cheese composition or on D* (⬃4% reduction).
Consequently, the levels of total calcium and serum
calcium probably have little influence on the rate at
which equilibrium of S/M is attained post-brining.
 Salt in Cheese: Physical, Chemical and Biological Aspects 241

Moisture content of cheese

It is generally accepted that the moisture content of
cheese affects the rates of salt absorption and/or diffu-
sion (McDowall and Whelan, 1933; Byers and Price,
1937; Georgakis, 1973). However, from calculations of
diffusion coefficients it has been shown (Geurts et al.,
1974b; Guinee, 1985) that for two cheeses of the same
variety, the rate of diffusion is not necessarily higher in
the higher moisture cheese; the diffusion coefficient
depends on the ratios of fat to solids-not-fat (SNF) and
moisture to SNF. These factors affect the volume frac-
tions of the fat and protein phases, which in turn deter-
mine the impedance to the diffusing molecules (see
‘Brine-salted cheese’). The results in Table 5 indicate
the importance of cheese composition, and hence struc-
ture, in salt diffusion. The diffusion coefficient for NaCl
in the cheese moisture (D*) increased with increasing
FDM when the percentage of SNF (and hence protein
tortuosity, p) decreased and the relative pore width of
the protein matrix (/dp) increased (e.g., cheeses 1–8,
Table 5) but decreased when both the FDM and SNF
levels increased (cheeses 9 and 10). In some instances
(e.g., cheeses 2, 6 and 9 with ⬃49%, w/w, H2O or
cheeses 12 and 14 which had ⬃44%, w/w, H2O) the
moisture contents were approximately equal but the
D*-values differed considerably due to differences in fat
(and hence fat tortuosity, f) and protein (SNF). In con-
trast, D* was almost equal for other cheeses (e.g., 11
and 13) which differed appreciably in moisture level.
Similar results, shown in Table 6, were obtained by
Geurts et al. (1974b). Therefore, while it is difficult to
elucidate the effect of moisture, or indeed of any one
compositional parameter separately, on salt flux, the
effect of moisture is discussed separately below.
Within a series of cheeses of the same variety with
equal FDM, D* increased curvilinearly with moisture
content (Fig. 18; Geurts et al., 1974b). Considering
cheeses 9–12, Table 5, it is apparent that the contribu-
tion of the decreasing fat tortuosity (f) (with increasing
moisture content) to the increase in D* was small (D*
and D* f increased by a factor of 1.7 and 1.6, respect-
ively, when the moisture content increased from 44.5
to 49.2%). The principal factor affecting the increase
in salt flux was the reduced frictional effect on the dif-
fusing molecules as the volume fraction of the protein
matrix (p) decreased; hence the relative pore width
(y/dp) increased concomitantly with increasing mois-
ture content (see Tables 5 and 6).

0.4

0.3
D* (cm2/day)

60 50 40 30 20 10

0.2

0.1

30 40 50 60
Moisture content of cheese (%)

Figure 18 Diffusion coefficient of NaCl in cheese moisture (D*) as a function of the initial moisture content of the cheese. Param-
eter is g fat/100 g DM in unsalted cheese. The solid lines are experimental values, broken lines are extrapolations (from Geurts et al.,
1974b).
242 Salt in Cheese: Physical, Chemical and Biological Aspects

The relationship between the diffusion coefficient in
the fat-free cheese, D* f, and the relative pore width of
the protein matrix is seen in Fig. 19 (Geurts et al.,
1974b); D* f can be considered as the ‘theoretical’
value of D* for a system with the same structural fea-
tures as cheese but from which the impedance to salt
diffusion, due to the physical presence of fat globules,
has been eliminated. While the decrease in the protein
tortuosity (p) contributes to the increase in D* associ-
ated with increasing moisture content (e.g., cheeses
9–12, Table 5), its effect is small as it varies little
within the range of p values encountered. The role of
moisture as the main compositional factor affecting salt
flux has been confirmed by Morris et al. (1985) who
found that the D* values for different commercial
cheese varieties (⬃37.3–49% H2O; ⬃23.5–27.5% fat;
⬃40.5–49.5% FDM; ⬃28–35% SNF) were directly
related to the moisture content of the unsalted cheeses
(Fig. 20). Of the variation in D* which could be
attributed to compositional factors (⬃70% of total vari-
ation) in the latter cheeses, ⬃49, 29 and 22% could be
attributed to variations in the relative pore width of the
protein matrix, the protein tortuosity and fat tortuosity,
respectively (Guinee, 1985). The strong positive correl-
ation between moisture content of the cheese and D*
suggests that S/M equilibrium in brine-salted cheese is
attained more rapidly with increasing moisture con-
tent. This is indeed the case as reflected by the much
shorter time required for attainment of equilibrium in
soft cheeses compared to hard cheeses, e.g., 3 days
in Camembert (Hardy, 2000), ⬃45 days in a 2.2 kg
Blue cheese (mean moisture, ⬃40%, w/w; Godinho and
Fox, 1981b), and ⬃60 days for a 3 kg cylindrical
Romano-type cheese (mean moisture, ⬃40%, w/w;
Guinee and Fox, 1986c). However, the rate of attain-
ment of equilibrium, as discussed later, is very depend-
ent on cheese dimensions and weight.
While D* is strongly dependent on the composition
and structure of the unsalted cheese, especially the
moisture content, it is, surprisingly, scarcely affected by
variations in composition along the different planes of
a cheese resulting from salt uptake and moisture loss
during brine salting. This is reflected by: (i) the consist-
ency of D* over the region of salt and water movement

0.3
D*λ f (cm2/day)

0.2

0.1

0.1 0.2 0.3

Relative pore width of protein matrix (y/dp)

Figure 19 Diffusion coefficient of NaCl in the moisture in fat-free cheese (D * f), as a function of the relative pore width of the
protein matrix (y/dp). Brine concentration, 19–20 g NaCl/100 g H2O; temperature, 12.5 °C; 50%, w/w, fat in DM; pH 5, unless stated
otherwise. g fat/100 g DM: ●, 12; , 22; , 62; , pH 4.79; , pH 5.50;  brine concentration, 14 g NaCl/100 g H2O (from Geurts
et al., 1974b).
 Salt in Cheese: Physical, Chemical and Biological Aspects 243
0.23
B
0.21 C
D As discussed in ‘Mechanism of salt absorption and dif-
D ∗, cm2/day
0.19
E
0.17
F
G of protein aggregates and occludes fat globules, both
0.15
H
I
0.13
35 39 43 47
Moisture, g/100 g
Figure 20 Dependence of the pseudo-diffusion coefficient of
salt in cheese moisture (D*) on the initial moisture content of
cheese salted in ⬃20%, w/w, NaCl brine at 15–16 °C. Blue
cheese (A, B), Gouda (C, D), Romano-type cheese (E), Jarlsberg
(F), Emmental (G, I), unsalted milled Cheddar (H) (from Morris
et al., 1985).
and with time (Geurts et al., 1974b; Guinee and Fox,
1983a) and (ii) the almost-constant D* value for brine
concentrations in the range of 5–20% NaCl (Geurts
et al., 1974b; Guinee, 1985). Thus, while pre-salting
the curd to different S/M levels prior to brine-salting
(as described in ‘Initial salt-in-moisture level of curd
and pre-salting’) reduced the level of salt absorbed, it
had little effect on D*, which increased from 0.18 to
0.19 cm2/day on increasing the S/M level from 0.3 to
4.7%, w/w, or the penetration depth of the salt during
subsequent brine-salting (Fig. 14a) (Guinee, 1985).
These results confirm that compositional changes
accompanying salt uptake have little, or no, effect on
D*. Consideration of the physico-chemical changes in
cheese associated with the physical presence of salt per se
and ageing may provide a tentative explanation (Geurts
et al., 1974a). Salting and ageing of cheese are paral-
leled by a reduction in the amount of protein-bound
water (i.e., water unavailable as a solvent) and a
decrease in the mean diameter of the protein particles
(Geurts et al., 1974a,b) and hence an increase in the
effective moisture concentration and relative pore
width of the protein network. Such changes possibly
offset the impeding effects of moisture loss during brin-
ing on salt flux, and hence D* remains essentially con-
stant during brining. Moreover, as concentration
gradient in the range 0–20% S/M has no effect on
D* (see ‘Concentration gradient’), the variations in salt
gradient within the cheese during salting or post-
A salting should have little effect on salt diffusion in
cheese moisture.
Fat content of cheese
fusion in cheese’, the diffusion coefficient of NaCl in
cheese moisture is much lower than that in pure solu-
tion, i.e., ⬃0.2 cm2/day compared to ⬃1.0 cm2/day at
12.5 °C. This is because salt diffusion in cheese takes
place in moisture held in a matrix which is comprised
of which obstruct the movement of diffusing mol-
ecules and increase the real distance travelled by a
salt molecule on proceeding from one parallel plane
to another (see ‘Brine-salted cheese’). Therefore, the
51 physical presence of fat per se reduces the apparent
D-value due to the inverse of its tortuosity, i.e., 1/f.
However, D* increases with fat content in cheeses
with equal moisture content (Fig. 18). In unidimen-
sional brine salting experiments, Geurts et al. (1974b)
observed that for Gouda cheese with 50% moisture,
but with 11 or 26% fat, the D* values were 0.15 and
0.25 cm2/day, respectively. While the fat tortuosity fac-
tor increased with fat level, i.e., 1.12 and 1.29 at 11
and 26% fat, respectively, the relative pore width of the
protein matrix also increased (i.e., 0.17 and 0.35 at 11
and 26% fat, respectively). Hence, the increase in D*
with fat content is not due to fat per se (which actu-
ally reduces D* by a factor of 1/f) but rather to the
concomitant decrease in the protein volume fraction
and, hence, the increase in the relative pore width of
the protein matrix. The reduction in the sieve-effect of
the protein matrix on the salt molecules overrides the
increased obstruction caused by increasing fat levels
and, hence, D* increases. Indeed, for cheeses of equal
moisture content in fat-free cheese (i.e., cheeses with
equal protein volume fractions), D* is always higher in
cheese with the lower fat content (Geurts et al.,
1974b). However, in practice, reducing the fat level
of cheese, while increasing the moisture percentage
per se, results in a reduction in the level of moisture in
non-fat substances and an increase in protein level
(see Fenelon and Guinee, 1999). Hence, the concomit-
ant increases in p and p and reduction in relative
pore width would be expected to reduce D* signifi-
cantly, unless the reduction in fat content is small
(e.g., 1–3%, w/w) and the cheesemaking process modi-
fied to prevent a reduction in the MNFS.
From the foregoing, it is apparent that the effect of
varying any cheese compositional parameter on salt
mobility depends on the concomitant changes it
causes in the cheese structure (i.e., the ratios of fat to
solids-not-fat, and solids-to-moisture). Since increasing
244 Salt in Cheese: Physical, Chemical and Biological Aspects
the fat level in cheese reduces syneresis (Whitehead,
1948; Marshall, 1982; Walstra et al., 1985; van Dijk
and Walstra, 1986; Pearse and Mackinlay, 1989), D*
should generally increase with increasing fat content
due to the concomitant decrease in p.
Cheese geometry
Cheese geometry influences the rate of attainment of
salt-in-moisture equilibrium via its effect on the rela-
tive dimensions of the cheese. Guinee and Fox (1986b)
working with commercial Romano-type cheeses of dif-
ferent shapes, showed that at any time during storage,
the net difference in S/M concentration along layers of
the cheeses increased with layer length. This observa-
tion is consistent with the fact that the depth of salt
penetration during brining is proportional to the
square root of brining time (Geurts et al., 1974b;
Guinee and Fox, 1986a). Using differently shaped
cheeses, it was found that the rate of attainment of S/M
equilibrium is not necessarily directly proportional to
the volume when comparing cheeses of the same var-
iety (Guinee and Fox, 1986c).
Attainment of salt and moisture equilibria
after salting
Brine- and surface dry-salted cheeses
In cheeses which are salted by immersion in brine
and/or by surface application of dry salt there is a large
decreasing salt gradient from the surface to the centre
and a decreasing moisture gradient in the opposite
direction at the completion of salting (see Fig. 21;
Guinee, 1985; Guinee and Fox, 1986a, 1993; Turhan
and Kaletunç, 1992; Messens et al., 1999; Pavia et al.,
1999; Turhan and Gunasekaran, 1999; Melilli et al.,
2003). Due to the slow diffusion of salt from the rind
inwards, these gradients disappear slowly and equilib-
rium of S/M is practically reached at some stage of
ripening, depending on cheese type, size of cheese and
curing conditions (Fig. 22; Guinee and Fox, 1986b;
Messens et al., 1999; Pavia et al., 1999).
Salt absorption is a relatively rapid event, varying
from 15–30 min for salt uptake by Cheddar-type
curd chips (Sutherland, 1977, 2002) and ⬃7.5 h
(Camembert) to ⬃15 days (e.g., Parmesan). In con-
trast, diffusion of salt and moisture post-salting, and
hence the rate of attainment of S/M equilibrium
throughout the cheese mass, is a slow process, e.g.,
10–12 days for Limburger (McDowall and Whelan,
1933; Kelly and Marqurdt, 1939), 8–12 weeks for
Gouda (Morris, 1961), Brick (Beyers and Price,
1937), Blue (Godinho and Fox, 1981b) and
Romano-type cheeses (Guinee and Fox, 1983b,
1986b), ⬃40 days for Feta (Georgakis, 1973) and
⬃10 months for Parmesan (Resmini et al., 1974).
44
20
42
16
40
Moisture, %, w/w
Salt-in-moisture, %, w/w
12
38
8
36
4
34
32
0 1 2 3 4 5 6 7
Distance from cheese surface, cm
Figure 21 Moisture content (open symbols) and salt-in-moisture
(g NaCl/100 g H2O) (closed symbols) in Gouda cheese (initial
fat-in-dry matter 49.1%, w/w; moisture, 43.64%, w/w; pH, 5.26)
as a function of distance from the salting surface after unidi-
mensional brine salting (20.3%, w/w, NaCl) for 1 (, ), 2 (,●),
3 (,) and 4 ( , ) days at 15 °C (redrawn from Guinee,
1985).
Sutherland (2002) reported 7–10 days for Camembert
(0.25 kg, flat disc; 55%, w/w, moisture), 4–6 weeks
for Edam (2.5 kg sphere; 46%, w/w, moisture), 7–9
weeks for Gouda (10 kg wheel; 42%, w/w, moisture)
and 4 months for Emmental (60–130 kg wheel;
36%, w/w, moisture). However, on consideration of these
data and times reported elsewhere (see ‘Moisture con-
tent of cheese’), it is clear that intra- and inter-variety
differences occur as a result of variations in cheese
dimensions, surface area-to-volume ratio, composition
and brining conditions (which may affect cheese com-
position and distribution of salt and moisture at the
end of brining).
Though the importance of the mean, and the uniform-
ity of, S/M levels in cheese in relation to quality have
received much attention (see ‘Overall Influences of NaCl
on Cheese Ripening and Quality’ and ‘Effect of NaCl
on Casein Hydration and the Physical Properties of
Cheese’), the factors that affect the diffusion of NaCl and
moisture in cheese after salting and hence the rate of
attainment of equilibrium have received little study.
However, as for diffusion during the brine/dry-salting

22
Top surface layer, A1
Centre layer, I1, I2
20
Bottom surface layer, A2
18
16
14
Salt-in-moisture, g/100 g
12
10
8
6 chips (3/8 in  3/8 in  2 in) in 25% brine for 5 min
4
2 potentially useful and appears to warrant further
0 O’Connor (1968) assessed the uniformity of salt
–5 –4 –3 –2 –1 0 1
Distance from cheese surface, cm
Figure 22 The mean salt-in-moisture levels in discs A1/A2,
B1/B2, C1/C2 etc. (as indicated) of cylindrical Romano-type
cheese salted in 19.5% NaCl brine at 23 °C for 1 (●), 3 (), or
5 (■) days or salted for 5 days and stored wrapped at 10 °C for
30 () or 83 () days (redrawn from Guinee and Fox, 1986b).
process per se, the salt diffusion during ripening/storage,
and thus the rate of attainment of S/M equilibrium
within a cheese loaf, is undoubtedly similarly affected by:
• cheese dimensions and shape, which determine the
distance between the high salt exterior zones and
low salt interior zones at the end of salting (Guinee
and Fox, 1986c);
• temperature;
• concentration gradient between different zones;
• cheese composition (see ‘Factors that influence salt
diffusion in cheese during salting’).
Though not investigated to date, conditions of rela-
tive humidity, rate of air circulation, and frequency of
turning the cheese during storage possibly alter the
rate of attainment of salt and moisture equilibria as a
result of alterations in cheese moisture.
Salt in Cheese: Physical, Chemical and Biological Aspects 245
Cheddar and dry-salted varieties
Salt is fairly uniformly distributed in Cheddar-type
cheese initially, as salt is mixed with the milled curd.
However, in contrast to brine-salting or surface
dry-salting, complete equilibrium is slow and rarely, if
ever, reached (McDowall and Whelan, 1933; Morris,
1961; O’Connor, 1968, 1971, 1973a; Sutherland, 1977;
Thomas and Pearce, 1981; Morris et al., 1985; Wiles
and Baldwin, 1996). Thus, significant intra- and inter-
block variations in salt concentration occur in mature
commercial Cheddar cheese, giving rise to considerable
variations in the rate of ripening and grading quality
(O’Connor, 1971, 1973b, 1974; Fox, 1975; Thakur
et al., 1975; Sutherland, 1977; Thomas and Pearce,
1981; Lawrence et al., 1984; Kelly et al., 1996; ‘Ched-
dar Cheese and Related Dry-salted Cheese Varieties’,
Volume 2).
A preliminary investigation of brine-salting Ched-
dar curd chips (Breene et al., 1965) showed that ade-
quate uptake of salt could be accomplished by placing
and holding for 15 min after removal from the brine
before pressing. Considering the problems encoun-
tered in controlling salt uniformity using the dry-
salting procedures, brine-salting of Cheddar could be
investigation.
2 3 4 5 distribution in cheeses salted by hand, semi-automatic
or fully automatic systems; one plug from every 10th
cheese per vat was analysed. While the range of mean
salt content was relatively narrow, there was very con-
siderable intra-vat and inter-vat variation in salt con-
tent, with greatest variation in the hand-salted cheese
and least with the fully automated system; an inverse
correlation between salt and moisture contents was
apparent. Further evidence of high within-vat varia-
tion in salt distribution is provided by O’Connor
(1973b).
The findings of O’Connor (1968, 1973b) were con-
firmed and extended by Fox (1974) who showed that, in
general, mechanical salting systems gave more uniform
salt distribution than hand-salting systems or a semi-
automatic system. Considerable within-block variability
(12 samples per 20 kg block) in salt concentration was
also demonstrated. Morris (1961) also found very large
differences in the salt content of blocks from the same
vat (spread of 0.6%, w/w, on a mean of 1.38%, w/w).
All the foregoing investigators stress the impor-
tance of inter- and intra-block variations in salt con-
tent, which is inversely related to moisture content.
Since it is generally agreed that the quality of cheese is
strongly dependent on moisture, S/M and pH (which
was not reported in any of the above studies) (see
246 Salt in Cheese: Physical, Chemical and Biological Aspects
‘Cheddar Cheese and Related Dry-salted Cheese Vari-
eties’, Volume 2), it might reasonably be expected that
the quality of cheese also varies between blocks from
the same vat and even within the same block. It is nor-
mal cheese-grading practice to grade a vat of cheese on
the basis of 1–3 plugs taken from a single cheese per
vat, at any one time; obviously, the quality of this sam-
ple may not be representative of the vat. For similar
reasons, calculation of mass balances in cheese factor-
ies on the basis of a few plug samples per vat may be
very inaccurate.
In practice, salting of Cheddar is performed
mechanically on salting belts where the salt is deliv-
ered by oscillating booms in proportion to curd depth
and the salted curd is mixed by rotating peg mixers
mounted over the belt. The method of salt application
used in Cheddar cheese manufacture would appear to
be particularly amenable to ensuring accurate control
of salt concentration with respect to both level and
uniformity. However, in commercial practice, it has
been difficult to achieve uniform distribution (Morris,
1961; Fox, 1974), possibly because of the many fac-
tors which influence salt uptake by curd (Fig. 16) and
the design of salting equipment capable of giving ade-
quate mixing in the time allowed. Undoubtedly, a
more consistent salt level and distribution could be
obtained by:
• the production of curd with more consistent
composition,
– e.g., via standardization of milk protein level (i.e.,
by ultrafiltration or addition of milk ingredients)
and protein-to-fat ratio;
– a more defined cheesemaking process where
stages/operations are regulated (e.g., levels of rennet
and starter in proportion to casein level; pH at set,
drain and milling; firmness at cut; faster curd/whey
emptying, especially from larger vats; washing to
constant lactose level in the curd);
• more thorough mixing of salt and curd, e.g., via the
use of curd/salt mixers or tumblers (Sutherland, 2002;
‘General Aspects of Cheese Technology’, Volume 2);
• better control over the temperature/humidity condi-
tions of salt storage and salting room to ensure uni-
form delivery to salt application device;
• optimum performance of the curd mill by regular
maintenance to ensure uniform chip size.
Salt and moisture equilibration during storage
Although salt is fairly well distributed in Cheddar
cheese during the initial salting, in contrast to brine
and/or dry salted cheese, full equilibrium is approached
slowly. Sutherland (1977), who prepared Cheddar
cheeses (9.5 kg) with regions of high and low salt,
found that equilibria of salt, moisture, and hence, S/M,
were not established after 25 weeks at 13 °C. Samples
situated 7.6 cm apart, which showed an initial differ-
ence in S/M concentration of 4.27%, still showed a dif-
ference of 1.56% at the end of the 25-week period. As
zones of high and low salt within commercial cheese
blocks (⬃20 kg, ripened at 4–7 °C) are likely to be
more widely separated, it was concluded that equil-
ibrium of S/M within such cheeses is unlikely. A simi-
lar study by Thomas and Pearce (1981) showed that
there was only a very slight shift towards equilibrium
of S/M during a 6-month ripening period in Cheddar
cheeses prepared with an approximately linear S/M
gradient diagonally across the blocks. Equilibration of
NaCl in Cheddar cheese intentionally prepared with
poor salt distribution was studied by Morris et al.
(1985). Salt and moisture analyses were performed on
samples taken from 32 selected locations in 20 kg
blocks (stored at 10 °C) over a 24-week ripening
period (a similar sampling pattern was used on each of
six occasions); the results indicated that there was
only a slight equilibration of salt over the 24-week
period.
Hoecker and Hammer (1944), who measured the
salt and moisture levels at the surface and centre of
individual chips, prized from a block of Cheddar, over
a 72 h period after pressing, found that salt and mois-
ture equilibria were established within individual
chips 48 and 24 h after hooping, respectively (a com-
parable study by Morris et al. (1985) gave almost iden-
tical results). However, analysis of two 4-month-old
cheeses showed significant intra- and inter-block vari-
ation in both variables. Hence, while salt and moisture
equilibria are attained relatively rapidly within chips
because of the short distance over which NaCl mol-
ecules have to diffuse from the surface to the centre,
the variations throughout the block, as a result of the
different quantities of salt absorbed by individual
chips, do not disappear during normal ripening. The
foregoing observations suggest that movement of salt
and water across the chip boundaries, and hence the
cheese mass as a whole, even where a concentration
gradient exists, is inhibited because:
• the contracted protein layers (salting-out of protein
at chip surfaces possibly occurs because of the high
initial S/M concentration before equilibrium is
established) at the surface of individual chips;
• and/or microspaces between milled curd (chips)
junctions which break the continuity of the inter-
penetrating gel fluid/moisture (in which salt is
dissolved);
• lack of a continuous S/M gradient in combination
with an impeded diffusion process.
 Salt in Cheese: Physical, Chemical and Biological Aspects 247

Indeed, milling results in the development of a 2.5

‘skin’ which has fewer fat globules and hence a denser (a) (b)
protein matrix than the interior of the chip (Brooker,
1979). Moreover, light microscopy studies (Rammell,

NaCl, %(w/w)
1960; Kaláb et al., 1982; Lowrie et al., 1982) show that
the ‘skin’ at milled curd junctions appears much 2.0
thicker than that of the curd granules. Observations by
Morris (1961) on salt diffusion in Cheddar cheese
lend support to the view that the milled curd pieces
‘trap’ absorbed salt; the spread in salt level within indi-
vidual cheeses at 3 weeks was the same as that
1.5
observed immediately after hooping. Days
Morris et al. (1985), who also studied salt diffusion 1
in model Cheddar cheese systems, found that equilib- 3
rium was established rapidly in cheeses prepared from 14
alternate discs (2 cm thick) of salted and unsalted, 28
56
unmilled curd, but not in model cheeses prepared
from alternate layers (2 cm thick) of salted and
unsalted chips. In agreement with the results of a simi-
lar experiment by McDowall and Whelan (1933),
NaCl diffusion across the interface formed between
the salted and unsalted layers of milled curd was very 0.5
slow (Fig. 23). Morris et al. (1985) suggested that the
fragmented structure of Cheddar cheese (due to its
construction from chips) may retard salt diffusion but
a further experiment, the results of which showed that
4 3 2 1 1 2 3 4
the diffusion coefficient for NaCl in the moisture
phase of a brine-salted block of Cheddar prepared Distance from interface between
(a) unsalted and (b) salted curds, cm
from unsalted chips at 0.15 cm2/day was as expected
from its moisture content (see Fig. 20), could not ver- Figure 23 Distribution of salt (NaCl) throughout a 7.5-cm Ched-
ify this. Thus, it appears that the slow diffusion of dar ‘cheese’ prepared from half-salted curd and half-unsalted curd
at 1, 3, 14, 28 and 56 days after manufacture. The preparation of
NaCl in the moisture phase of Cheddar is due to con-
the ‘cheese’ involved half-filling aluminium cans with unsalted
tracted protein layers between salted chips, which pos- chips (2  2  2 cm) of Cheddar curd and pressing lightly; filling
sibly offer a very tight screening effect on the diffusing the remainder of the can with salted (4%, w/w, NaCl) chips of
molecules, and thereby override the effect of low dis- Cheddar curd; pressing the cheese in the can overnight; storing
continuous gradients in various directions in commer- cheeses in cans at 5 °C (from Morris et al., 1985).
cial Cheddar or even at interfaces between salted and
unsalted regions where the concentration gradient is
high (see ‘Concentration gradient’). The absence of a etrating the surface of curd granules (no light
continuous salt-in-moisture gradient and the fact that microscopy studies have been reported on unsalted,
salt diffusion in moisture is impeded, as discussed in milled Cheddar curd).
‘Mechanism of salt absorption and diffusion in cheese’
and ‘Factors that influence salt diffusion in cheese Effect of Salt on Cheese Composition
during salting’, also reduces the tendency for salt and
moisture equilibrium, especially between locations in In the light of studies (O’Connor, 1968, 1971, 1974;
a block which are far apart. Sutherland, 1974; Gilles, 1976; Morris et al., 1985; Kelly
The surface of chips in unsalted milled Cheddar et al., 1996) showing that varying salting levels in Ched-
would not be as dense as those in dry-salted milled dar cheese manufacture are associated with large compo-
Cheddar due to their higher moisture and fat contents. sitional variations in the cheese, the effect of salt on the
Hence, the sieve-effect of the matrix on the diffusing gross composition of cheese merits brief discussion.
molecules would be much lower than in the latter.
Moisture level
Indeed, the impedance on the salt molecules penetrat-
ing the surface layers of milled Cheddar chips during The moisture content of cheese curd is influenced primar-
brining is possibly similar to that encountered on pen- ily by syneresis of the cheese curd during manufacture
248 Salt in Cheese: Physical, Chemical and Biological Aspects
which is, in turn, influenced by the composition of the
cheese milk, i.e., fat, protein and calcium levels, the
level of rennet used, firmness of the gel at cutting, and
curd treatments during manufacture, i.e., size of curd
cut, degree of curd agitation, cooking rate, cooking
temperature, rate of acid development, extent of dry-
stirring of curd and depth of curd during cheddaring
and size of pressed cheese (Whitehead, 1948; Emmons
et al., 1959; Lawrence, 1959a,b; Aiyar and Wallace, 1970;
Lelievre, 1977; Geurts, 1978; Marshall, 1982; Walstra
et al., 1985; van Dijk and Walstra, 1986; Fenelon and
Guinee, 1999; ‘Cheddar Cheese and Related Dry-
salted Cheese Varieties’, Volume 2). Further syneresis
occurs on addition of salt after milling (e.g., for Ched-
dar and Cheshire), during pressing and brine and/or
dry salting.
It is generally accepted that there is an inverse rela-
tionship between the levels of salt and moisture in
cheese. This is most readily observed in brine and/or
dry salted moulded cheeses during, or immediately
after, salting, where a decreasing salt gradient from
surface to the centre is accompanied by a decreasing
moisture gradient in the opposite direction (see Fig. 21;
McDowall and Whelan, 1933; Beyers and Price,
1937; Geurts et al., 1972, 1974b; Godinho and Fox,
1981b; Guinee and Fox, 1983a,b, 1986a,b; Turhan and
Kaletunç, 1992; Messens et al., 1999; Licitra et al., 2000;
Melilli et al., 2003). The inverse relationship between
salt and moisture levels is also observed in cheeses
where the mean salt level is varied by brine- or surface
dry-salting for different times (Guinee and Fox, 1986b;
Freitas and Malcata, 1996; Kristiansen et al., 1999) or
for similar times in brines of different NaCl levels
(Guinee and Fox, 1986a; Pappas et al., 1996; Kaya
et al., 1999). A similar trend was observed in brine-
injected Muenster cheese for which the salt content
increased linearly with the number of injections,
applied 24 h apart (Pastorino et al., 2003a).
O’Connor (1971) found that there is a negative cor-
relation between the salt and moisture concentrations
in commercial Scottish Cheddar cheeses. Although
there was considerable scatter, the data of Fox (1975)
show an inverse correlation between the percentage of
moisture and percentage of NaCl in 123 commercial
Irish Cheddar cheeses. Direct evidence of this relation-
ship is also apparent from the work of O’Connor (1970,
1973a,b) and Kelly et al. (1996) for Cheddar cheeses
from the same batch of curd salted at different levels.
An inverse correlation between %, w/w, moisture
and %, w/w, NaCl in Cheddar cheese is not surprising
since a considerable volume of whey is released from
Cheddar curd following salting and during pressing
(Sutherland, 1974). The amount of whey released is
directly related to the amount of salt added to the curd;
roughly half of the whey is released during mellowing
and the remainder during pressing. Although other fac-
tors, e.g., curd temperature, stirring time after salting,
depth of curd, degree of mixing of salt and curd, and
duration of mellowing period influence the ratio of
whey released during mellowing to that released on
pressing, the overall level of whey released was not sig-
nificantly influenced by these factors (Sutherland,
1974). The moisture content of the cheese was
inversely related to salting level (Sutherland, 1974).
Geurts et al. (1974b) expressed the relative fluxes of
NaCl and H2O during the unidimensional brine-salting
of Gouda-type cheese in terms of the flux ratio, p:
Wx ⬇ pSx
where W and S are the changes (from the unsalted
cheese) in the g H2O and g NaCl, respectively, per 100 g
cheese solids-not-salt in planes of cheese x cm from
the cheese/brine interface; the minus sign indicates the
loss of water from the cheese to the brine. Experimen-
tal values for W and S are shown in Fig. 24, together
with theoretical curves calculated for various values of p.
The experimental curve for W approximated the theor-
etical curve for p  2 (i.e., when the amount of H2O
leaving the cheese is twice that of the NaCl entering)
but varied from 1.5 at the salt front to 2.34 at the
20
44
4
Salt in moisture (g NaCl/ 100 g H2O)
3
16
Moisture content of cheese (%)
2 40
–1
12
36
8
32
4
28
0
0 10 20 30 40 50 60 70
Distance from cheese surface (mm)
Figure 24 Moisture (●) and salt () content of a full-cream
Gouda cheese after 8 days of brining, as a function of penetra-
tion depth; pH 5.64, brine concentration 20.5 g NaCl/100 g H2O,
temperature 12.6 °C. Experimental moisture values (●); (1) mois-
ture content calculated from salt content and a flux ratio
(g water:g salt) p  2.5; (2) the same, but p varies from 1.7 at
the cheese–brine interface to 2.9 in the cheese surface; (3) the
same, but p  1; (4) the same, but p  0 (from Geurts et al.,
1974b).
 Salt in Cheese: Physical, Chemical and Biological Aspects 249
brine/cheese interface and was always 1. While a
similar trend in p values was observed by Guinee and
Fox (1983a) for commercial Romano-type cheese
(salted for 9 days in 19.3% NaCl brine), the value of p
varied more, i.e., from 3.75 at the rind to 1 in a
region between the rind and the salt front. Guinee
(1985) concluded that the value of p at a particular
location within the region of salt and water movement
depends on the concentrations of NaCl and H2O at the
location; indeed, this is possibly the reason why p
decreases from the rind inwards (Geurts et al., 1974b;
Guinee and Fox, 1983a), along which significant vari-
ations of salt and moisture occur as a result of salt
uptake. Indeed, changes in cheese texture and appear-
ance corresponding to the changes in p which occur
in the region of high salt and moisture movement are
visible when a brined cheese is cut parallel to the
direction of brine movement, during or shortly after
brining (Geurts et al., 1974b; Guinee and Fox, 1983a;
Bochtler, 1987). In the outermost region (0.3–1.3 cm
depending on the duration of brining) bordering the
brine, where the S/M level is high, e.g., 12%, w/w,
the cheese is hard, brittle, dry and white (indicative of
salting-out), whereas further removed from the inter-
face, where percentage of S/M 3% and 10%, the
cheese is soft, yellowish and somewhat waxy translu-
cent (indicative of protein hydration and swelling);
between the ‘waxy’ layer and the salt front, the cheese
had a uniform appearance and resembled the unsalted
cheese (Guinee, 1985). It is noteworthy that pockets
of free serum in unsalted non-fat Mozzarella provide
light scattering surfaces and thereby contribute to
opaqueness of the cheese (Paulson et al., 1998); in
contrast, the increase in protein hydration in salted
Mozzarella led to a significant reduction in the level of
free serum and a more translucent appearance. The
extent of the outer dry white layer in brine-salted
cheeses is augmented by a low cheese moisture and a
low brine pH, i.e., 4.6 (Bochtler, 1987); such factors
contribute further to protein insolubility.
Since the average flux ratio over the region of salt and
water movement is 1, there is a net outflow of water
which accounts for the commonly observed volume
reduction in cheese during brining and/or dry salting.
Perhaps unexpectedly, salt uptake during brining is
sometimes accompanied by an increase in moisture con-
tent in the vicinity of the cheese–brine interface (see
‘Effect of NaCl on casein hydration in model systems
and in cheese’ and ‘Concentration of calcium in the
brine’), especially in weak brines (10%, w/v, NaCl)
without calcium (see Figs 8, 9). Such an effect is associ-
ated with the ‘soft rind’ defect and swelling in cheese
and is attributed to a salting-in of the protein matrix in
low percentage NaCl brines which results in increased
protein solubility. There are several reports on the com-
position and maintenance of brines and brining times in
relation to cheese quality (Jakubowski, 1968; Geurts
et al., 1972; de Vries, 1979; Brazhnikov et al., 1986;
Blanchard, 1987; Cohen-Maurel, 1987; Chamba, 1988;
Schaegis, 1988).
Salt content
Higher salt levels in Cheddar cheese usually coincide
with increased fat content (O’Connor, 1971, 1974;
Thakur et al., 1975), probably due to the greater loss
of water than salt uptake during salting. Thus, it
is noteworthy that the moisture content of Cheddar is
inversely related to the salt content (O’Connor, 1971;
Sutherland, 1974; Jordan and Cogan, 1993; Kelly
et al., 1996). However, on considering the findings of
Breene et al. (1965), the fat content may decrease,
especially at high salting levels if the curd temperature
at salting exceeds 32 °C.
Lactose content and pH
As discussed in ‘Control of Microbial Growth’, the lac-
tose content and pH of cheese are strongly influenced by
the level and time of salt application (Fox et al., 1990).
Conclusion
Clearly, salt plays a multi-faceted role in cheese ripening
with an influence on the physical, chemical and micro-
biological attributes of the mature cheese. While a con-
siderable amount of information is currently available
on many aspects of the significance of salt in cheese and
on salt diffusion in cheese curd, many gaps persist, e.g.,
its effects on individual enzymes, protein–protein inter-
actions (and its consequences in hydrolysis, rheology
and functionality), effect on the growth of pathogens,
interaction with pH and other hurdles in controlling
cheese microbiology.
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This Page Intentionally Left Blank
 Application of Membrane Separation
Technology to Cheese Production
V.V. Mistry, Dairy Science Department, South Dakota State University, Brookings, USA
J.-L. Maubois, Laboratoire de Recherches Laitières, Institut National de la Recherche
Agronomique, Rennes Cedex, France

Introduction buffaloes, production of milk powders with good

cheesemaking properties (Maubois et al., 1973), restor-
Membrane processing has revolutionized the dairy
ation of the rennet coagulation properties of ultrahigh
industry in many interesting ways and has led to signifi-
temperature (UHT)-treated milk (Maubois et al., 1972;
cant new process and product development. This revo-
Ferron-Baumy et al., 1991), on-farm concentration of milk
lution has been in the making for the past 34 years and
(Maubois, 1979), removal of somatic cells (Le Squeren
has encompassed not only ultrafiltration and nanofiltra-
and Canteri, 1995; Maubois, 2002) and bacteria from
tion but, more recently, microfiltration as well. In 2001,
cheese milk by microfiltration (Trouvé et al., 1991;
more than 450 000 tonnes of cheese were made using
Saboya and Maubois, 2000) and casein enrichment of
ultrafiltration technology. The history of cheesemaking
cheese milk by microfiltration (Fauquant et al., 1988;
using membranes commenced in the late 1960s with
Maubois et al., 2001). These developments were cata-
the invention of the MMV process (Maubois et al., 1969;
lysed by improvements in membrane components
Maubois and Mocquot, 1971, 1975). This process, named
such as the development of mineral and ceramic mem-
after its inventors (Maubois, Mocquot and Vassal), opened
branes, studies on physico-chemical equilibria of UF
up new avenues for significant advances in cheesemaking,
retentates, characterization of the rheological behav-
including improvements in plant efficiency, increases in
iour of protein-enriched milks, studies on the growth
cheese yield, development of a continuous process and
and activity of starter bacteria in liquid pre-cheeses
possibilities for creating new cheese varieties. As a result,
and in the resulting cheeses, and more importantly by
numerous plants all over the world, but mainly Europe,
the generation of new ideas and the acceptance of new
now use this process to manufacture a wide range of
cheesemaking concepts in laboratories and in cheese
cheeses (Korolczuk et al., 1986; Maubois, 2002).
plants around the world.
Since the introduction of the MMV process, com-
In this chapter, cheesemaking using UF, reverse
mercial applications of membranes in the cheese indus-
osmosis and microfiltration will be discussed as well as
try, as well as research efforts aimed at developing new
other cheese-related applications using these processes.
applications and understanding and improving current
Initially, some membrane terms, membrane design and
applications, have expanded all over the world. For
configuration will be discussed briefly.
instance, it was reported by Kosikowski (1986a) that
during the period 1979–1983, a total of 213 scientific
papers were published dealing with membrane separa-
Membrane Design and Configuration
tions in food processing. Of these, publications dealing
with cheese formed the largest category at 25%. Publi- Membrane technology is a broad term that encompasses
cations dealing with cheese and whey, combined, repre- several molecular separation processes. Each process
sented 50% of the total. In a more recent literature requires its own specialized equipment and has its own
search, it was found that more than 1200 publications characteristics that make it suitable for some applica-
on the application of membranes in food processing tions but not for others. Reverse osmosis and ultrafiltra-
appeared between 1995 and 2002. These data clearly tion are two of the most commonly used membrane
illustrate the magnitude of effort that has been invested processes in the dairy industry (Glover, 1985; Van der
in developing and understanding applications of mem- Horst and Hanemaaijer, 1990) but nanofiltration and
branes in the food industry. microfiltration have emerged and have demonstrated
Since 1969, cheese-related applications of mem- tremendous potential for dairy applications for the
branes have expanded into numerous areas, including future (Gregory, 1987; Eriksson, 1988; Fauquant et al.,
the manufacture of fresh, soft, semi-hard and hard 1988; O’Shea, 1990; Kelly et al., 1992; Saboya and
cheeses from the milk of cows, goats, ewes or water Maubois, 2000; Maubois, 2002).
Cheese: Chemistry, Physics and Microbiology, Third edition – Volume 1: General Aspects Copyright © 2004 Elsevier Ltd
ISBN: 0-1226-3652-X All rights reserved
Set ISBN: 0-1226-3651-1
262 Application of Membrane Separation Technology to Cheese Production
Definitions
Ultrafiltration (UF)
Ultrafiltration is a process which selectively separ-
ates macromolecules having a molecular weight of
1000–200 000 Da from solvent and dissolved solutes.
With cross-flow over a membrane surface at a rela-
tively low pressure (less than 1000 kPa), UF produces
from milk a permeate (also called ultrafiltrate) con-
taining water, lactose, soluble minerals, non-protein
nitrogen and water-soluble vitamins, and a retentate in
which the proteins, fat and colloidal salts are increased
in proportion to the amount of permeate removed
(Maubois et al., 1969; Maubois and Mocquot, 1971;
Glover, 1985).
Reverse osmosis (RO)
Reverse osmosis (sometimes known as hyperfiltration)
is a dewatering process which operates at pressures at
least five to ten times higher than those used for UF
(Glover et al., 1978; Glover, 1985; Dziezak, 1990).
Reverse osmosis membranes separate solutes with a
molecular weight of approximately 150 Da or less.
Hence, fat, proteins, lactose and all undissociated miner-
als are retained and concentrated by the membrane, and
only water and some ionized minerals pass through.
Microfiltration (MF)
Microfiltration is a process which selectively separates
particles with a molecular weight greater than 200 000
Da. According to the membrane pore size, milk mater-
ials removed by MF include somatic cells, fat globules,
bacteria, casein micelles (Saboya and Maubois, 2000),
aggregated whey components (Maubois et al., 2001),
-casein (Terré et al., 1987) and -lactoglobulin
(Maubois, 1988; Léonil et al., 1997).
Nanofiltration (NF)
Nanofiltration, also known as loose-RO (Horton,
1986), falls between RO and UF. It separates low
molecular weight compounds (200 and 1000 Da) from
larger molecules. Small ionized molecules, such as dis-
solved mineral salts, are removed, at a rate inversely
proportional to their valency, along with water,
whereas other materials such as lactose, proteins and
fat are retained, making it suitable for desalting cheese
whey (Daufin et al., 1998a), for recovering of water
generated during thermal concentration of milk or
whey or for recycling cleaning solutions (Daufin et al.,
1998b).
Membrane configuration
Four basic configurations are currently available for
UF, RO, MF and NF applications: (1) tubular, (2) hol-
low fibre, (3) plate and frame and (4) spiral-wound
(Maubois and Brulé, 1982; Cheryan, 1998). The char-
acteristics of each of these configurations are described
below.
Tubular
In this configuration, feed flows through a tube,
85–600 cm long and 3–25 mm inside-diameter. The
inside wall of the tube is lined with the membrane and
the outside consists of support material. Several tubes
may be connected in series or in parallel as a bundle
and are housed in a stainless steel cartridge. A multi-
channel tubular geometry was developed in France
(Gillot and Garcera, 1986) and in the USA (Renner
and Abd El-Salam, 1991) for MF and UF mineral
membranes (Fig. 1).
Tubular membranes are easy to clean and allow
recirculation of liquids with a high level of solids and
viscosity (Mahaut et al., 1982; Maubois and Brulé,
1982; Cheryan, 1998). However, they have the lowest
surface area-to-volume ratio and therefore require a
high feed flow rate and consequently, a high running
energy (0.6–1.0 kW/m2) (Maubois and Brulé, 1982).
Reverse osmosis operations are conducted at high
pressures; hence tubular membranes for RO require add-
itional support material to withstand the high pressures.
Hollow fibre
Hollow fibre membranes can be thought of as the
tubular-type except that they are self-supporting. Hol-
low fibres also have a much smaller diameter than the
tubular-type membranes. The diameter of each fibre
ranges from 0.19 to 1.25 mm (Cheryan, 1998). In a
see-through casing, 50–3000 such fibres may be bun-
dled together in parallel. Each such unit is referred to
as a cartridge. Hollow fibre membranes have a very
high surface area-to-volume ratio, providing for very
low floor space requirements.
As in the tubular design, feed flows through the
inside of the fibres, and permeate is collected outside in
the casing. A disadvantage with this system is that even if
only one fibre fails, the entire cartridge must be replaced.
Replacement costs of membranes are, therefore, high. On
the other hand, since hollow fibres are self-supporting,
operating pressures are low (Maubois and Brulé, 1982).
Transmembrane pressure is limited to 170–270 kPa.
While this is an advantage in terms of energy consump-
tion (0.2 kW/m2), this configuration may not be suitable
for applications requiring high pressures. One of the
greatest advantages of hollow fibre membranes, as bun-
dled tubular membranes, is the ability to backflush. This
aids in cleaning the membrane as well as in preventing a
build-up of fouling material on the surface.
Hollow fibres such as those described above are
used for UF and MF applications. For RO applications,
 Application of Membrane Separation Technology to Cheese Production 263

Figure 1 An arrangement of multi-channel geometry ceramic membranes (courtesy of GEA filtration, Hudson, WI, USA). (See
Colour plate 1.)

even smaller fibres, known as hollow fine fibres, are
used (Cheryan, 1998). In these fibres, feed flows from
the outside of the fibre to the inside.
Plate and frame
This configuration consists of a stack of plates and flat
sheet membranes, much like a filter press arrangement
(Fig. 2). The flat sheet membrane and its support are
sandwiched together in large numbers to form a mod-
ule. Feed flows parallel to the membrane surface, and
permeate is chanelled out of the module. Plate and
frame configurations are available in horizontal as well
as vertical designs. The surface area-to-volume ratio is
between hollow fibre and tubular designs. The required
pumping energy is around 0.5–0.7 kW/m2 (Maubois
and Brulé, 1982).
Spiral-wound
This configuration is the most widely used in the dairy
and food industries and is also the most inexpensive
(Fig. 3). Spiral-wound membranes consist of two flat
sheet membranes along with spacers wrapped around

Figure 2 Plate and frame UF system (courtesy of GEA filtration, Hudson, WI, USA). (See Colour plate 2.)
264 Application of Membrane Separation Technology to Cheese Production

Figure 3 Spiral-wound UF membranes (courtesy of GEA filtration, Hudson, WI, USA). (See Colour plate 3.)

a perforated permeate-collecting tube (Cheryan, 1998).

As feed passes over the membrane surface, permeate
spirals its way to the centre of the tube. Spacers are
included in the assembly to promote turbulence,
thereby minimizing fouling, but sometimes cleaning
and sanitizing difficulties are experienced, especially
when highly viscous retentates are recirculated. Spiral-
wound membranes are available for UF, RO, MF and
NF applications. The nature of the membrane support
and the general design permit operation at high trans-
membrane pressures without damaging the membrane.

Vibrating membrane system

Developed by the Pall Corporation (Boston, MA), this
is a relatively new concept in which vibration energy is
used to reduce the thickness of the fouling layer and
consequently to improve the flux rate. This is accom-
plished by vibrating a disc filter stack at 50–60 Hz on
the vertical axis to generate shear rates of 100 000–
150 000 s 1 on the membrane surface. Membranes are
bonded to both sides of stainless steel discs, which are
then arranged in a stack to obtain a total membrane
surface area of up to 40 m2 (Fig. 4). With this system,
it has been possible to separate milk proteins effect- Figure 4 Vibrating membrane system (courtesy of Pall Corpor-
ively. While commercial applications have not been ation, Portsmouth, UK). (See Colour plate 4.)
 Application of Membrane Separation Technology to Cheese Production 265
developed yet, there is potential for the future (Hurwitz
and Brantley, 2000).
Good quality membrane material is critical for the
proper operation of UF, RO, MF or NF plants. Cellu-
lose acetate was the most common material for UF and
RO membranes but these have now been almost com-
pletely replaced by polysulphone membranes, especially
for UF applications. Numerous other materials have
been assessed, e.g., polyamide, polyimide, polyvinyl-
idene fluoride, etc. Mineral membranes, specifically
zirconium oxide, or titanium oxide supported by car-
bon or by alumina and ceramic membranes are now
being used increasingly for UF, MF and even NF. These
materials have high mechanical strength and tolerate
wider pH and temperature ranges than polymeric
membranes. They are more expensive but have a sub-
stantially longer life than organic membranes (at least
5 years compared to 18 months).
The above overview is concerned only with mem-
brane processes used in cheese applications. Other
membrane processes, such as dialysis and electro-
dialysis and details of UF, RO, MF and NF, such as
flux rates, thermodynamics of operation, fouling and
concentration polarization have been discussed in depth
elsewhere (Cheryan, 1998; Meireles-Masbernat et al.,
1998) and will not be addressed here.
Membrane Applications in Cheesemaking
Ultrafiltration is the most widely used membrane
process for cheesemaking and is fairly well-advanced.
Microfiltration techniques for the removal of bacteria,
separation of milk fat globules and enrichment of
micellar casein have been developed and have already
entered industrial operations.
To successfully make cheese by UF or MF, specific
properties of the protein-enriched products must be
well understood because they strongly determine the
quality of the end products, as well as economy of the
use of the membrane technology.
Properties of UF retentates
Buffering capacity
If milk is ultrafiltrated at its normal pH (6.6–6.8),
mineral salts (Ca, Mg, P) bound to casein micelles are
concentrated in the same proportion as proteins. This
results in an increase in the buffering capacity of UF
retentates which will consequently modify the basic
parameters of the cheesemaking process – acidification
kinetics by lactic acid bacteria, ultimate pH value, ren-
net coagulation kinetics and rheological characteristics
of the curd, activity of ripening enzymes, lysis of
mesophilic lactic acid bacteria during ripening (Saboya
et al., 2001), growth and rate of survival of spoilage
flora (Rash and Kosikowski, 1982) and water-holding
capacity of the cheese mass during ripening. A similar
increase in buffering capacity is also observed when
milk is specifically enriched in micellar casein by the
use of 0.1 m MF membranes.
According to the volumetric concentration factor
(F) (ratio of the volumes of milk and retentate),
higher production of lactic acid by lactic starter bac-
teria is required to obtain optimum pH in cheese, usu-
ally 5.2 in hard cheeses and 4.6 in soft and fresh
cheeses. For the latter category, the increase in
required lactic acid production was quantified by Brulé
et al. (1974) and was expressed as:
QL  4.4 F  1.5
where QL is g of lactic acid per kg of pH 6.7 UF retentate.
Consequently, for most cheese varieties, use of pH
6.7 retentates results in acid-tasting products (Maubois,
1979). On the other hand, a large quantity of calcium
salts is released into the aqueous phase of cheese curd
during acidification. Ionic strength is strongly increased
and casein micelle aggregation is modified. Cheese
texture is crumbly or sandy (Brulé et al., 1975), and
spreadability and stretching properties are poor (Green
and Grandison, 1987). The buffering effect of pH 6.7
UF retentates leads to higher numbers of lactic starter
bacteria in curd and resulting cheese than in non-UF
curd and cheese (Mistry and Kosikowski, 1985;
Goudédranche et al., 1986). While this may result in
bitterness in some cheeses, such as Quarg (Mortensen,
1985), it has been used to advantage in the development
of a new bulk starter (Mistry and Kosikowski, 1986a).
This starter is manufactured by fermenting a whole or
skim milk retentate containing 12% protein with a
mesophilic lactic culture at 22 °C for 12–15 h (Fig. 5).
It has a built-in internal pH control mechanism due to
the buffering capacity of the UF retentate, which
maintains the pH of the starter steady at 5–5.2. It,
therefore, has a greater activity than traditional bulk
starters made from pasteurized milk and maintains its
activity for 10–12 h at room temperature. The UF
retentate starter also has a high protein content which
contributes to increased cheese yield (Mistry and
Kosikowski, 1986b; Mistry, 1990), making it suitable
for either traditional or UF cheesemaking.
The increased buffering capacity of pH 6.7 UF milk
could also offer a favourable environment for the
growth and survival of certain bacteria such as
enteropathogenic E. coli (Rash and Kosikowski, 1982).
This underscores the importance of adjusting the min-
eral content of UF retentates to avoid the unfavourable
consequences in cheesemaking due to the increase in
buffering capacity of pH 6.7 UF milk. This aspect was
266 Application of Membrane Separation Technology to Cheese Production
Figure 5 Flow diagram for the production of retentate starter
(derived from Mistry and Kosikowski, 1986a).
emphasized by Brulé et al. (1974), who suggested sev-
eral suitable ways for adjusting the mineral salts con-
tent of UF retentates, which is specific for each cheese
variety. The first method involves a reduction in milk
pH before or during ultrafiltration by the growth of a
lactic starter or by any approved acidifying agent
(glucono--lacatone or organic acids in some coun-
tries). Acidification leads to solubilization of colloidal
calcium and magnesium phosphate salts, which pass
into the permeate. Reduction of milk pH from 6.6 to
6.0 and 5.6 increases the Ca content of UF permeate
from 0.38 to 0.50 and 0.80 g per kg, respectively. Con-
sequently, a 5X UF retentate obtained at pH 5.6 has a
Ca content 2.6 times that in milk instead of 3.8 times
for the 5X UF retentate prepared at pH 6.6 (Brulé
et al., 1974). The second method, which eventually
can be combined with the first one, is the addition of
NaCl (0.5–0.9%, w/w) to UF retentate during or after
ultrafiltration. The increase of ionic strength resulting
from NaCl addition reduces the ionization of casein
phosphoseryl groups and consequently leads to solubil-
ization of colloidal calcium in the permeate or in the
aqueous phase of UF retentate (up to 15–18%, w/w,
depending on the pH and amount of NaCl added)
(Brulé et al., 1974). An increase of ionic strength also
lowers the isoelectric pH of casein, which may offer an
increased security margin to the cheesemaker for
handling acidified UF retentate.
Addition of NaCl to milk or reduction of the pH by
acidification reduces UF flux because of increased
membrane fouling but it is obvious that any cheese-
maker will prefer to have satisfactory cheeses even at
the expense of reduced performance of UF equipment
rather than defective cheeses resulting from a process
involving the highest UF flux. It must also be remem-
bered that milk having a pH lower than 5.0, when
ultrafiltered, leads to a higher UF flux than that
observed at pH 6.7 because of the weak texture of the
polarization layer at the isoelectric pH of casein (Mahaut
et al., 1982).
Rheological behaviour of UF retentates
Milk can be considered as a Newtonian liquid, while
UF retentates behave differently. The higher the pro-
tein content and/or the lower the temperature, the
more pseudoplastic is their behaviour (Culioli et al.,
1974). Such a rheological behaviour must be taken
into consideration in the design and in the operating
parameters of UF equipment (for example, in the
restarting procedure after an electrical failure to avoid
a hydraulic ram).
The viscosity of UF retentates increases markedly
with an increase in their protein content. At 30 °C, at a
shear rate of 437.4 s 1, the observed viscosity is 45 cP
at a protein content of 19.6%, w/w, and 370 cP at a
protein content of 20.6%, w/w (Culioli et al., 1974;
Goudédranche et al., 1980). The manufacture of semi-
hard or related cheese from these highly viscous UF
retentates requires removal of all dissolved gases
which are entrapped in the liquid and the use of special
mixing devices (static and dynamic) to enable thorough
blending of rennet and lactic starters (Maubois, 1987).
If dissolved gases are not removed by application of a
vacuum, a spongy curd is obtained and the appearance
and the taste of the cheese are poor. If rennet is not
mixed satisfactorily, the resulting curd will be flaky
due to localized coagulation.
Rennet coagulation
If the same amount of rennet is added to equal vol-
umes of milk or UF retentate, the rennet-clotting time
is not affected by the increase in protein content (% P)
but the time from clotting to cutting is reduced
(Maubois and Mocquot, 1971; Garnot et al., 1982;
Lucisano et al., 1985; Garnot, 1988; Maubois, 1989).
This is the net result of numerous phenomena – there
is an increase in the velocity of the enzyme reaction as
the protein content is increased (Garnot, 1988) but
 Application of Membrane Separation Technology to Cheese Production 267
the degree of proteolysis at gelation decreases as % P
increases. At pH 6.6 and at the normal casein content
of milk, coagulation occurs when 80–90% of the
-casein has been hydrolysed. However, in a 4X UF
retentate, hydrolysis of only 50% is necessary for curd
formation (Dalgleish, 1980). Because the secondary
phase of rennet action is a diffusion-controlled process,
an increase in protein content leads to a sharp increase
in the rate of aggregation (Garnot, 1988). The final firm-
ness of rennet-induced coagulum is generally directly
related to the casein content (Maubois and Mocquot,
1971, 1975; Maubois et al., 1972, 1973; Korolczuk et al.,
1986; Kosikowski, 1986b; Ferron-Baumy et al., 1991).
This is of particular importance when low-concentrated
retentates (LCR) are used to make cheese because tradi-
tional equipment is employed. This would require
stronger knives and agitators to handle the firmer,
stronger coagulum (Kosikowski et al., 1985b).
If the primary phase of -casein hydrolysis by ren-
net is slightly affected in UHT milk (Ferron-Baumy et al.,
1991), coagulation does not occur owing to the
increased electronegativity of the casein micelles
resulting from the covalent binding of -lactoglobulin
with -casein (Dalgleish, 1990). Erdem (2000) has
suggested that a decrease in the surface hydrophobic-
ity of proteins due to UF is the cause of the unique
renneting properties of UF milk. Increasing the pro-
tein content by UF before or after UHT treatment
restores curdforming ability (Maubois et al., 1972).
According to Ferron-Baumy et al. (1991), such a phe-
nomenon would result from lowering the zeta poten-
tial of casein micelles on UF. This hypothesis, which
must be confirmed by direct observations, agrees with
the fact that UF retentates coagulate at a lower degree
of -casein hydrolysis than normal milk.
Applications of UF in cheesemaking
Cheesemaking using UF can be divided into three
main categories:
1. protein-standardized milk;
2. intermediate or medium concentrated retentates;
3. liquid pre-cheeses, i.e., UF retentates having the
composition of the cheese variety to be made.
Protein-standardized milk
The protein content of milk collected by dairy plants
varies according to season due to multiple factors –
stage of lactation, weather, feeding and breed of lactat-
ing cows. Such a variation in the composition of the
incoming milk requires adjustment of processing
parameters by cheesemakers. Moreover, at a low pro-
tein content the rennet-induced coagulum is weak and
leads to relatively high losses of caseins as fines in
whey. A slight increase in the protein content by UF
eliminates these difficulties. In many cheese plants,
generally those using highly mechanized equipment,
the protein content of cheese milk is increased to
3.7–4.5% throughout the year (Korolczuk et al., 1986;
Mietton, 1990). Protein-standardized milk is used in
Europe for the manufacture of Camembert cheese
(Korolczuk et al., 1986) using an Alpma coagulator or
similar equipment. It is also used for semi-hard and hard,
cooked cheese. In the USA, the acronym, LCR (low-
concentrated retentates), was proposed (Kosikowski,
1986b) to characterize this use of UF in cheesemaking.
Several pilot plant and industrial studies have reported
on the use of the LCR concept for Cheddar and other
hard cheeses using either direct concentration or sup-
plementation (Chapman et al., 1974; Kealey and
Kosikowski, 1985; Kosikowski et al., 1985b; Sharma
et al., 1989). These studies concluded that the opti-
mum degree of concentration for making these cheese
varieties is between 1.7:1 and 1.8:1. In the LCR or pro-
tein standardization process, cheese is made using con-
ventional equipment and a cheese plant can easily
adapt this application of UF. Manufacturers of UF
equipment have now proposed specially designed
ultrafiltration systems that are equipped with in-line
protein and fat sensors (Friis, 1985). This will make it
possible to determine the fat and protein content of
the incoming milk and to standardize the cheese milk
for fat and protein simultaneously.
The cost of UF for this application is balanced by
a slight increase in manufacturing efficiency due to
increased production of cheese per vat per day, reduced
rennet requirements, improved quality of cheese
(Kosikowski, 1986b) and a slight increase in yield
(generally less than 1% for most varieties). This
increase in yield results from reduced losses of fat and
casein particles in whey and better retention of whey
proteins in the aqueous phase of cheese. The increase
in retention of whey proteins with LCR is relatively
small compared to the MMV process. The effect of this
on cheese yield can be estimated according to the for-
mula proposed by Vandeweghe (2000). Another
advantage is the possible added value of the resulting
whey, which has an increased content of protein/total
solids. However, it must be said that in industrial situ-
ations these advantages are minor. Therefore, it is
somewhat surprising that a large number of UF plants
have been installed for protein standardization in
Europe. An indirect but important advantage of the
LCR/protein standardization concept is the utilization
of permeate to reduce the protein content of fluid
UHT milk to the minimum required by law – 2.8%,
w/w, in most EC countries but 3.15%, w/w, in France.
Such a practice, which is forbidden in the EU since
268 Application of Membrane Separation Technology to Cheese Production
1997, had led to considerable profit for many UHT
milk processors. The payback of UF investment was
less than six months. While it is possible to detect
dilution of milk with water, it is impossible to detect
dilution with permeate.
A number of cheese varieties have been made using
the LCR concept. These include Cottage (Mattews
et al., 1976; Athar et al., 1983; Kosikowski et al., 1985a;
Kealey and Kosikowski, 1986b; Zall and Chen, 1986),
Mozzarella (Fernandez and Kosikowski, 1986a,b) and
Saint Paulin (Abrahamsen, 1986). Industrial and pilot-
plant trials with Cottage cheese indicate that a concen-
tration ratio in the range of 1.2:1.7 is optimum for
yield, flavour and body characteristics. Above these
levels, the texture becomes firm and the cheese has a
flat flavour. Thermization of milk (74 °C for 10 s)
prior to UF gives the highest increase in Cottage
cheese yield compared with thermization after UF or
no thermization (Zall and Chen, 1986).
Good-quality low-moisture Mozzarella cheese with
excellent stretching and melting properties can be pro-
duced from low-concentration retentates at 1.75:1.0
ratios (Fernandez and Kosikowski, 1986a,b). Cheese
from higher concentrates was firmer and had greater fat
losses in the brine. Using LCR, it is possible to produce
both starter-acidified and directly acidified Mozzarella.
The LCR concept has also been applied to Brick
and Colby cheese (Bush et al., 1983). LCR Brick
cheese had a lower pH and higher fat losses in whey
than in controls. The cheese was firmer and more
mealy and scored lower in overall preference than con-
trol cheese. For Colby, the use of UF made it possible
to eliminate the curdwashing step. Sensory scores
were similar to those of controls. Reduction in cook-
ing time and rennet usage was reported. In studies on
Edam cheese by Pahkala et al. (1985), 2:1, 4:1 and 6:1
retentates were used. LCR (2:1 concentration) pro-
duced the best cheese with the fewest defects. In this
cheese, the rate of proteolysis of s1-casein was similar
to that in control cheese but that of -casein was
slower. With UF Danbo cheese, made from 2:1 diafil-
tered UF milk, a slight increase in yield, a 50% reduc-
tion in rennet requirements, and a 40% increase in
the cheesemaking capacity of vats was possible (Qvist
et al., 1985).
The LCR concept for cheese appears to have been
well-accepted commercially for cheeses such as Ched-
dar, mainly because of increased yield without the
need for additional equipment. Its application in cor-
recting the effects of seasonal variation in milk
composition on Cheddar cheesemaking has been stud-
ied (Broome et al., 1998a,b). It was suggested that
when milk was ultrafiltered up to 4.5%, w/w, protein,
the moisture content of Cheddar cheese was optimal
and the yield increased. A recent study demonstrated
the possibility to further enhance the impact of UF on
cheesemaking by homogenizing the cream (Oommen
et al., 2000). Cream containing 35%, w/w, fat was
homogenized and mixed with skim milk and UF milk
to obtain 6%, w/w, protein for Cheddar cheesemaking.
Fat recovery in the cheese was 96.8% compared with
94.7% for the control cheese. It was possible to also
improve the meltability of cheese as well as its texture.
The LCR concept has been used to improve the
quality of low-fat cheeses. Ultrafiltered sweet butter-
milk has been used to manufacture low-fat Cheddar
(Mistry et al., 1996; Turcot et al., 2002), Mozzarella
(Poduval and Mistry, 1999) and Processed cheese
(Raval and Mistry, 1999). The UF of buttermilk allows
for the selective concentration of its phospholipid con-
tent, which may play a role in developing the texture
of cheese.
Medium or intermediate concentrated retentates
Numerous cheese varieties, ranging from soft to hard,
have been made from medium-concentrated reten-
tates. In this approach, cheese is made by using spe-
cially designed equipment able to cut and handle the
firm gel resulting from the coagulation of 2:1–5:1 con-
centrated retentates, eventually diafiltrated with pure,
salted or acidified water. The main application, which
is in industrial operation, is the manufacture of struc-
tured Feta cheese (Hansen, 1985).
APV-sirocurd process. An Australian dairy research
team, in collaboration with the APV firm, devel-
oped this process. Commercial plants were installed in
Australia and USA but the process has been discon-
tinued for technical and economic reasons. The details
of the process have been discussed by Mistry and
Maubois (1993). This process had the potential to
increase cheese yield by 6–8% under continuous and
automated conditions.
Structured Feta-like cheese. This approach in mak-
ing Feta-like cheese was developed in Denmark in
response to consumer demand from many Mediter-
ranean countries where people desired cheeses having
an appearance and texture (presence of mechanical
holes) similar to those of traditional products, charac-
teristics they did not find in UF Feta-like cheese made
from liquid pre-cheeses (Mortensen, 1985). Pasteur-
ized, fat-standardized milk, generally homogenized at
18 MPa and 60 °C, is ultrafiltered at 50 °C. The final
concentrate contains 28.5%, w/w, TS, which corres-
ponds to a concentration factor of 3:1. Lipase, starter
culture or glucono--lactone are added to the UF
retentate, previously homogenized at 5 MPa at 65 °C,
heat-treated to 80 °C for 60 s and cooled to 34 °C.
 Application of Membrane Separation Technology to Cheese Production 269
After a short storage period in a tank, the UF retentate
is pumped to specially designed Alfa-Laval Alcurd or
Pasilac equipment. Rennet is added in-line. In both
types of equipment, rennet is mixed thoroughly and
the UF retentate coagulates in tubes. The resulting
coagulum is removed from the tubes, cut into cubes,
moulded and drained (16–24 h at 10–14 °C) until the
pH has decreased to 4.8 (Skovhauge, 1987). The prod-
uct of this process is virtually indistinguishable from
the traditional product. A yield increase of about 14%
on a solids basis is claimed over the traditional process
(Mortensen, 1985), a far smaller value than that
obtained with the process using liquid pre-cheese
(30%). Such a difference explains why only a few
plants produce structured Feta-like cheese.
Other cheeses produced from medium concentration
retentates. Several experiments on the use of UF
retentates of up to 5:1 concentration have been reported
for making Havarti, a semi-soft cheese of Danish origin,
containing approximately 26%, w/w, fat and 56%, w/w,
solids (Bundgaard et al., 1972; Qvist et al., 1986, 1987;
Qvist, 1987). Cheese milk used in these experiments
was not pre-acidified, and diafiltration was not per-
formed with acidified water. Consequently, the buffer-
ing capacity of the UF retentates was high and it was
reported that more starter was required than with the
traditional process or the use of specially selected cul-
tures (Skovhauge, 1987). However, the taste and the
flavour of UF cheeses were similar to the traditional
product. The texture was, nevertheless, softer and the
melting properties poorer. A 10% saving in the cost of
skim milk cheese manufacture was claimed, resulting in
a net profit of US$42 000 per year for a production of
600 tonnes of cheese (Skovhauge, 1987).
In experiments with Gouda cheese (Spangler et al.,
1989, 1990), whole milk was first ultrafiltrated to
3.3:1 and then diafiltered to 3.6:1–5:1 concentration.
Gouda cheese produced from 5:1 retentate was similar
in moisture, hardness and proteolysis to controls pro-
duced from non-UF milk. A savings of 33% in the cost
of rennet was reported. Flavour development in UF
Gouda cheese could be accelerated by using a combin-
ation of liposome-entrapped enzyme and freeze-
shocked Lactobacillus helveticus cells.
Attempts have also been made to manufacture Blue
cheese from UF milk (Mahaut and Maubois, 1978;
Maubois, 1979; Abd El-Salam et al., 1988). According
to French studies (Mahaut and Maubois, 1978), the
organoleptic qualities of cheese made from UF reten-
tates with a protein content ranging from 3.2 to 10%
and treated in traditional cheese vats were similar to
reference cheese. Above 12%, w/w, protein, modifica-
tions of cheesemaking parameters and new cutting
and handling equipment were required to produce sat-
isfactory Blue cheese. That was successfully accom-
plished in 1996 by the French Guilloteau Society
which produced the award-winning ‘La Roche’ variety.
Egyptian workers describe the use of recombined
ultrafiltered milk for making Blue cheese (Abd El-Salam
et al., 1988; Abdou et al., 1988; Dawood et al., 1988).
General considerations on the use of intermediate
UF retentates. Benefits accruing from the use of
intermediate UF retentates for making any cheese var-
iety must be substantial enough to justify substitution
of traditional cheesemaking technology. Moreover, the
organoleptic quality must be acceptable to the con-
sumer. Investments involve not only UF equipment, as
in the LCR concept, but also additional equipment,
such as curd makers. Increased cheese yield is strongly
related to the volume concentration factor (F) and to
the difference between the composition of the UF
retentate and the final cheese (Jacobsen, 1985). The
saving of skim milk increases logarithmically as the dif-
ference becomes smaller but UF-operating costs also
increase logarithmically with F. The economic study to
be made by cheese plants to assess investment must
also take into consideration the potential value of the
two by-products obtained – drained whey, which con-
tains more protein and fat than normal whey, and per-
meate; each is of interest to a different downstream
industrial network. Minor benefits also accrue from
reduced rennet consumption and reduced requirement
in volume and floor space.
Liquid pre-cheeses (LPC concept)
In this approach, cheese milk is concentrated by UF
to the composition of the drained curd being made
before addition of rennet. There is very little whey
drainage, and there is no need for cheese vats
(Maubois et al., 1969; Maubois and Mocquot, 1971).
This principle was first applied to Camembert cheese
(Maubois et al., 1969; Maubois, 1979) but many
applications have been developed successfully for the
manufacture of other cheese varieties, ranging from
‘fromages frais’ or Quarg to semi-hard cheeses, such as
Saint Paulin.
Fresh unripened cheeses. In early attempts to apply
UF for the manufacture of cheese varieties belonging
to this category, milk was preconcentrated prior to
starter and rennet addition. The cheese produced had
a highly acid and metallic taste, frequently associated
with bitterness. These defects were attributable to the
high mineral content of the curd and consequently its
high buffering capacity (Brulé et al., 1975; Mahaut
et al., 1982; Lawrence, 1987). Some reduction of acid
flavour was observed when pre-acidified (pH 6.0) milk
270 Application of Membrane Separation Technology to Cheese Production
was ultrafiltered or when milk was concentrated to a
higher degree than necessary and subsequently diluted
with water (Brulé et al., 1975). Introduction of new
membranes, such as mineral membranes and specially
designed membrane supports that permit the UF of
high-viscosity products (Maubois, 1979; Herbertz,
1984a), has made it possible to solve this organoleptic
defect completely by using the process initially pro-
posed by Stenne (1973), i.e., first fermenting the milk
to pH 4.6 with a conventional mesophilic culture,
adding rennet and then ultrafiltering to remove lactose
and mineral salts but retaining whey proteins. A high
initial flux rate and a decrease in flux rate with con-
centration were observed when pH 4.6 milk was ultra-
filtered (Mahaut et al., 1982), both phenomena being
attributable to the highly porous structure of the
polarization layer (Maubois, 1979). Because of the rela-
tively high protein content (12%), this use of UF was
successfully developed for the manufacture of Quarg, a
German cheese variety (Baurle et al., 1984; Anon,
1984b,c; Herbertz, 1984b, 1985; Patel et al., 1986;
Koch International GmbH, 1987). For application to
similar French cheeses, which contain much less pro-
tein, it was necessary to develop specially designed UF
equipment, which minimized the mechanical shear
stress applied to the retentate (Fig. 6). The viscosity of
pH 4.6-acidified curds decreases markedly with the
increase of mechanical treatment imposed during cen-
trifugal drainage or UF concentration (Mahaut, 1990).
Thanks to UF technology, it was possible to make
fresh unripened cheeses from buttermilk. Interest in
using this new starting material, which has poor ren-
Figure 6 Commercial UF system for the production of fresh cheese from pH 4.6 milk (courtesy of TIA, Bollene, France). (See Colour
plate 5.)
neting characteristics, because of the high heat treat-
ment generally applied to cream before churning, is
due to its high content in phospholipids, which give a
very unctuous texture.
Many other fresh unripened cheese varieties are
now made according to the LPC approach or the MMV
process. Some examples include Ricotta (Maubois and
Kosikowski, 1978; Skovhauge, 1988), Cream cheese
(Covacevich and Kosikowski, 1977; Resmini et al.,
1984; Dos Santos Neves and Ducruet, 1988) and
Mascarpone (Resmini et al., 1984; Sordi, 1984).
The manufacture of Ricotta presents special prob-
lems because of the complexity of precipitation and
requirements for suitable texture and flavour (Maubois
and Kosikowski, 1978; Kosikowski and Mistry, 1997).
In one UF process (Maubois and Kosikowski, 1978),
whole milk is acidified to pH 5.9 with lactic starter,
acid whey powder or food-grade acid and ultrafiltered
at 55–60 °C to 12%, w/w, protein. The acidified liquid
pre-cheese is heated in a scraped-surface heat exchanger
at 80 °C and filled directly into packages. In another
process (Skovhauge, 1988), milk or whey is pasteur-
ized, acidified to pH 6.3 and ultrafiltered to 30%, w/w,
solids at 50 °C. The retentate is heated to 90 °C at a
pressure of 1–1.5 bar, following which the pressure is
reduced to atmospheric to aid curd formation. The
product is cooled to 70 °C, packaged and chilled to
10 °C. No whey drainage occurs.
Another interesting application of UF for the manufac-
ture of cheese varieties belonging to this high-moisture
category is the procedure developed for the produc-
tion of ‘Faisselles’ or ‘country cheese’. Traditionally,
 Application of Membrane Separation Technology to Cheese Production 271
this cheese is made from whole milk inoculated at
18–22 °C with a mesophilic starter and rennet. After
overnight cooling to 12–16 °C, pieces of coagulum are
scooped by hand into moulds for slight whey
drainage. The drained curd is then removed, always
by hand, from the moulds and gently laid down in
the retail cups. This production was disappearing
because of the increasing cost of labour. Use of UF
has allowed the draining step to be eliminated and
consequently labour requirements are reduced sub-
stantially. The production of this cheese variety has
now reached its former maximum level (Maubois,
1985).
Soft cheese. Camembert, a French surface-moulded
cheese variety, was the first cheese to be made accord-
ing to the MMV principle (Maubois et al., 1969;
Maubois and Mocquot, 1971). Several recipes were
proposed (Maubois and Mocquot, 1975; Maubois,
1979, 1989) to optimize the use of UF with industrial
cheesemaking constraints (24-h production of UF
retentate with moulding for 16 h) and to obtain the
very delicate equilibrium in calcium salts in the curd
required to obtain texture and flavour similar to trad-
itional Camembert cheese. The procedure used with
the continuous moulding and demoulding equipment,
‘Camatic’, developed by Alfa-Laval (Hansen, 1981;
Gutter, 1984; Korolczuk et al., 1986), is the following –
HTST-pasteurized milk is ultrafiltered at 50 °C to a
pre-cheese concentration of 5:1 and a total solids con-
tent of 35%, w/w. The resulting pre-cheese is cooled to
30 °C, and 2%, w/w, mesophilic lactic starter and
0.75%, w/w, NaCl are added. Then, the mixture is
allowed to acidify to pH 5.5 and is automatically filled
into forms with online inoculation with rennet. Curd
wheels develop rapidly and are continuously and gen-
tly moved in the Camatic equipment for 45 min. After
being inverted once, a continuous electric current is
applied to each cheese between the air-exposed surface
in contact with a carbon electrode and the stainless
steel cup holding the cheese. Limited electrolysis of
whey occurs and the use of an air injector allows per-
fect demoulding of the wheels onto cheese trays.
Then, the fresh Camembert cheeses are brined for
about 30 min, removed, sprayed with Penicillium
camemberti spores and held for 12 days at 11–12 °C to
permit development of the white mould covering. A
yield increase between 12 and 15% is obtained. Several
units of Camatic equipment have been sold, mainly in
Germany. In France, UF Camembert cheeses have
encountered ‘psycho-commercial’ difficulties. The
organoleptic qualities of UF Camembert were indistin-
guishable from those of traditional cheese but the bulk
density of the UF cheese paste is much higher than
that of traditional cheese because there are no mechan-
ical openings. Since French consumers are accustomed
to buying Camembert cheese by the piece and not by
weight, they are conscious of the volume of this cheese
variety and get the impression that they receive less
cheese for their money when buying UF Camembert
(Qvist et al., 1985; Maubois, 1987).
The commercial failure of UF Camembert has led
French cheesemakers to develop new varieties, most
of which have achieved a very rapidly growing pro-
duction. For example, ‘Pavé d’Affinois’ was developed
in 1982 and reached a production of 5000 tonnes in
2000 (Fig. 7). This cheese is made from 4:1 UF whole
milk retentate fermented with a thermophilic lactic
starter. Rennet is added and the mixture is poured into
rectangular plastic trays that are 5 cm high. The trays
are set in an incubator at 43 °C for 6 h to allow acidifi-
cation and coagulation to occur. After cooling to room
temperature, the cheese slabs are removed from the
trays and cut with an automatic dividing knife to 96
pieces, each having the size and form of a small rec-
tangular paving stone (approximately 7  5  5 cm).
The resulting fresh cheeses are ripened for 10 days, as
for Camembert (Korolczuk et al., 1986). Several other
soft cheese varieties (bacterial and mould surface-
ripened, Blue) have been developed according to the
same principles and have achieved commercial success
(Maubois, 2002).
The greatest success worldwide of the MMV
process is unquestionably the manufacture of Feta-like
cheese (Fig. 8) (Hansen, 1980, 1984; Mortensen, 1985;
Lawrence, 1987). Until the EU regulations changed
market dynamics in 1997, Feta accounted for 35% of
all cheese produced in Denmark and more than 90%
of it was produced by UF.
Most of this cheese was manufactured for the Iran
market but UF Feta cheese has been produced in Iran
itself for several years (Ziabary and Hoffmann, 2001).
The LPC concept for making Feta has made feasible an
old dream of cheesemakers – to make the cheese in its
retail package. Yield increases of 30% were reported,
higher than could be expected from the retention of
whey proteins in the cheese (22% at most). The differ-
ence must be related not only to the total elimination
of curd particle and fat losses arising from the coagula-
tion and curd cutting inside the retail tins but also to
the retention in the curd of all the -caseinomacropep-
tide (4%, w/w, of the casein content of the LPC).
A similar concept was applied to the manufacture of
Domiati, an Egyptian cheese variety – 5:1 UF whole
milk retentate was homogenized, 5%, w/w, NaCl, 2%,
w/w, lactic starter and lipase–rennet mixture were
added prior to pouring into 18-kg tins (Al Khamy,
1988) or Tetra Pak packages.
272 Application of Membrane Separation Technology to Cheese Production

Figure 7 A selection of cheeses made by UF using the liquid pre-cheese concept. (See Colour plate 6.)

UF processes for Mozzarella have been reported
since the mid-1970s (Covacevich and Kosikowski,
1978). In one of the first attempts to use the MMV
principle for Mozzarella cheese (Covacevich and
Kosikowski, 1978), retentates were adjusted to 33.6%
solids with freeze-dried retentate, and then blended
with 69% fat cream to 45–50% solids. This mixture was
fermented, and rennet curd was prepared. It was con-
cluded that diafiltration is required to produce good
flavour, stretch and melting characteristics. In a subse-
quent continuous process for low-moisture Mozzarella
(Resmini et al., 1984; Kosikowski, 1986b), pasteurized
skim milk was pre-acidified to pH 6.0 to reduce its cal-
cium content. It was then ultrafiltered/diafiltered at
54 °C to approximately 10:1 concentration. The reten-
tate was blended with cream to obtain 20%, w/w, fat,
28%, w/w, protein, and then dosed with starter and ren-
net. Coagulation occurred continuously, followed by
conventional stretching and moulding at pH 5.2. Some
problems encountered with this process included poor

Figure 8 A UF plant for producing Feta cheese by the MMV process (courtesy of TIA, Bollene, France). (See Colour plate 7.)
 Application of Membrane Separation Technology to Cheese Production 273

stretching characteristics of the cheese. Lack of proper w/w, solids. On the other hand, cheese base made by UF
stretching of UF Mozzarella cheese may be attributed can satisfactorily replace the young cheese component
to the incorporation of large quantities of whey pro- in the manufacture of conventional processed cheese.
teins and their denaturation during cooking, an improper An Australian process (Ernstrom et al., 1980) was
calcium:protein ratio (Hansen, 1987; Lawrence, 1987), commercialized in the USA (Kosikowski, 1986b). In
inadequate removal of dissolved gases and incomplete this process, whole milk or whole milk acidified to pH
blending of rennet (Maubois, 1987). 5.7 was ultrafiltered to 40% of its original weight and
then diafiltered to 20% of its original weight. The
Semi-hard cheeses. Saint Paulin is a bacterial sur-
product was fermented with a lactic starter for 16 h at
face-ripened, semi-hard cheese of French origin and
30 °C and then vacuum-evaporated to 64%, w/w,
contains approximately 47%, w/w, moisture and 2.5%,
solids. A similar process was developed in Denmark
w/w, salt (Kosikowski and Mistry 1997). In the manu-
(Madsen and Bjerre, 1981b).
facture of this cheese by UF, it is necessary to obtain at
Spraying pre-fermented LPC onto the surface of
least 21%, w/w, protein (45%, w/w, solids) (Maubois,
fresh cheese curd instead of using hand or mechanical
1979). This is more easily attainable with mineral mem-
washing can also represent an interesting improve-
branes (Goudédranche et al., 1980) than with polymeric
ment of the process for making bacterial surface-
membranes. Procedures have been developed for both
ripened varieties because it allows for a concentrated
brine-salted and dry-salted cheese. Increases in cheese
layer of lactic acid bacteria on the surface of the cheese
yield up to 19% may be realized, with 85% savings in
and thereby avoids generalized contamination by
rennet (Goudédranche et al., 1980). Acid flavour and
undesirable bacteria such as Listeria or Pseudomonas
slow ripening of UF Saint Paulin cheese can be con-
and accelerates ripening.
trolled by reducing the lactose and ash content of the
By means of ultrafiltration and drying, a pre-cheese
retentate to less than 1.9% each (Delbeke, 1987). The
powder can be produced for subsequent reconstitution
flavour of UF Saint Paulin may be improved by adding
and conversion into cheese (Maubois et al., 1973;
lysozyme at 0.5–1.0 g/l (Goudédranche et al., 1986),
Glover, 1985). The primary use is for export to coun-
which increases the proteolytic count and reduces the
tries with low milk production or where the milk sup-
mesophilic count of the cheese or by adding broken
ply is very seasonal. In the importing country, the user
lactococci cells (Saboya et al., 2001).
needs only to add water, starter and rennet to make
A new cheese variety, with propionic bacteria fer-
cheese (Maubois and Fauconneau, 1977; Madsen and
mentation, has been studied in France (Ducruet et al.,
Bjerre, 1981a). Such powders could also be used in
1981; Maubois, 1987). The procedure includes the
dairy countries for home-cheesemaking (Maubois
preparation of a 7.5:1 retentate in two steps – first with
et al., 1973; Le Graet and Maubois, 1979). Pre-cheese
continuous diafiltration at 3.0:1 concentration regulated
powders offer many advantages to both exporting and
by a refractometric sensor inserted in the permeate
importing countries – cheesemaking characteristics are
line, followed by heat treatment at 4.0:1 concentra-
better than those of even low-heat normal milk powders
tion, and second, with continuous ultrafiltration to
(Maubois et al., 1973; Le Graet and Maubois, 1979;
7.5:1 using specially designed equipment (short car-
Lablée, 1982; Mahaut and Maubois, 1988), cheese-
tridges and positive displacement recirculation
makers in the importing country have no whey prob-
pumps) for handling highly viscous products. Original
lem, and economy of production is favourable for both
mixing devices for starters and rennet addition were
countries since both spray drying and transport costs
used. The moulding equipment includes a vacuum
are cheaper than those for normal milk powder
step for removing dissolved gases and a special injec-
(Maubois and Fauconneau, 1977). However, this
tion head for pouring renneted LPC into two-part
application of UF has found very few uses, mainly
spherical or cylindrical moulds.
because of the regulations of dairy-exporting coun-
Other applications of the LPC concept. Jolly and tries, such as the USA and the EC, which subsidize the
Kosikowski (1975) pioneered an original application export of milk solids regardless of the amount of liq-
of UF in processed cheesemaking by proposing the uid milk used to make 1 kg of these milk solids
substitution of UF skim milk retentate previously (Maubois and Fauconneau, 1977).
incubated with blue mould spores for aged cheeses.
Some interesting results were reported by Sood and Cheese quality
Kosikowski (1979) for the replacement of Cheddar Texture. Although UF cheeses offer moderate to sig-
cheese – fully acceptable processed cheese was nificant yield benefits and have been well-accepted by
obtained by substituting 40% of aged Cheddar cheese consumers, they possess some inherent characteristics
by enzyme-treated UF retentate containing up to 30%, that make them unique with respect to composition,
274 Application of Membrane Separation Technology to Cheese Production
ripening characteristics and texture qualities. It has
even been suggested (Lawrence, 1987; Lawrence et al.,
1987) that separate standards of identity for UF
cheeses would be advisable, and that a new range of
cheese varieties should be developed rather than
duplicating traditional varieties.
Most texture defects of UF cheeses, such as sandi-
ness, firmness or crumbliness, are caused by the higher
content of Ca salts if UF retentates of pH 6.7 are used
for making cheese. Mineralization of the drained curd
(total Ca and repartitioning of Ca between the casein
matrix and the soluble phase) play an essential role in
the rheology of the cheese variety (Maubois and
Kosikowski, 1978; Kindstedt and Guo, 1998). The min-
eral content may be adjusted as described above under
Properties of UF Retentates.
One of the most notable characteristics of UF
cheese is the incorporation of whey proteins in the
cheese. The quantity of whey proteins retained depends
on the variety and the degree of UF concentration. If
all the whey proteins of milk are retained, they will
represent approximately 20% of the total protein in
the cheese. Lower quantities will be retained when the
LCR method is used. Part of the casein is replaced by
whey proteins, which act as an inert filler and may
soften the cheese (De Koning et al., 1981). On the
other hand, the water-binding capacity of whey pro-
teins is much higher than that of casein, and UF
cheeses are less susceptible to drying during retailing
than traditional cheeses. UF cheeses also contain more
Ca phosphate salts and -GMP, both components with
interesting nutritional and nutraceutical properties
(Maubois et al., 2001).
Proteolysis and ripening characteristics. It has been
commonly observed that UF cheese ripens more
slowly than traditional cheese (De Koning et al., 1981;
Hickey et al., 1983; Creamer et al., 1987; Lawrence et al.,
1987, Furtado and Partridge, 1988; Harper et al.,
1989; Guinee et al., 1994, Broome et al., 1998a,b).
Generally, the larger the amount of whey proteins
incorporated, the slower the flavour development.
Large variations in the flavour quality of UF cheese
have also been observed and these have been attributed
to the varying levels of immunoglobulin and proteose-
peptones in the whey proteins (Lawrence et al., 1987).
The effect of whey proteins on flavour development is
less pronounced in LCR cheeses due to the smaller
quantities of whey proteins present but is more signifi-
cant in cheeses made from higher retentate concentra-
tions and those that are ripened for long periods.
The retarded maturation could be due to several
reasons. The high content of -lactoglobulin in UF
cheeses could inhibit to some extent the general prote-
olytic activity of rennet (Creamer et al., 1987) and
plasmin (Visser, 1981). Undenatured whey proteins
found in UF cheeses are resistant to proteolysis by
these proteases, as well as by starter-derived enzymes.
The high buffering capacity of UF cheeses prepared
from pH 6.7 ultrafiltered milk is the most probable
cause. Indeed, it retards or even completely inhibits
the rate of autolysis of mesophilic lactic starter
(Goudédranche et al., 1986; Saboya et al., 2001) and
consequently hydrolysis of the casein network. The
rate of s1-casein breakdown, as well as of -casein,
has been found to be reduced in UF cheese (Creamer
et al., 1987). The rate of flavour development of UF
cheese may be improved by adding flavour-producing
enzymes (Spangler et al., 1990) or cell extracts (Saboya
et al., 2001) which are totally retained in the retentate,
contrary to what happens in traditional cheesemaking
where 80–90% of the added enzyme is lost in the
whey. Non-starter bacteria or slow-acidifying lactic
micro-organisms may be used for their proteolytic and
flavour production potential because UF cheesemak-
ing permits separate management of acidification,
drainage and ripening flora.
Functionality. When cheeses are used as ingredients,
various characteristics become important. These
include melting behaviour, shredding ability, viscosity
and stretchability (Kosikowski and Mistry, 1997). The
ultrafiltration of milk prior to cheesemaking alters the
physico-chemical properties of cheese in a way that has a
distinct impact on some of these functional properties.
Acharya and Mistry (2002) reported that the meltabil-
ity of processed cheese manufactured from Cheddar
cheese base made from ultrafiltered milk (up to 6%
protein) is lower than that from control (regular)
Cheddar. These cheeses also had the highest calcium
content. These observations were also true for the base
Cheddar cheese; Cheddar cheese made from UF milk
containing 6% protein had a melting value of 62 mm
compared to 77 mm for the control. Likewise, the vis-
cosity of molten processed cheese at 80 °C was signifi-
cantly higher for the UF cheese (1043 cP) than for the
control (557 cP). Methods to reduce the calcium con-
tent of cheese, such as pre-acidification, should help
improve functionally.
Madsen and Qvist (1998) attributed the impaired
melting of UF Mozzarella cheese to the presence of whey
proteins and suggested the use of proteolytic enzymes to
accelerate the degradation of casein to improve melting.
The primary cause of the altered functionality
appears to be the difference in the calcium equilibrium
and its relationship with the casein structure but the
texture and the proteolysis characteristics discussed
earlier are also involved. For example, the reduction in
 Application of Membrane Separation Technology to Cheese Production 275
the rate of proteolysis and the presence of whey proteins
impact directly on the melting characteristics of cheese.
The consumer is the final judge of cheese quality and
the success of any new cheese or traditional cheese made
with new technology, such as membrane separations, will
depend to a large extent on acceptance by consumers.
Reverse osmosis in cheesemaking
Richardson (1929) proposed the use of evaporated
milk in cheesemaking. This idea was revived some 30
years later by Stenne (1964), but within the frame-
work of an original combination with the observa-
tions of Berridge (1951) on the separation of the
primary and secondary phases of rennet action at a
low temperature. The same basic idea lies behind
increasing the solids content of cheese milk by
adding dried milk or by concentration using reverse
osmosis.
For Cheddar cheesemaking, reverse osmosis was
proposed for preconcentrating whole milk to 20–25%
solids (Agbevavi et al., 1983; Barbano and Bynum,
1984; Bynum and Barbano, 1985; Mayes, 1985;
Schmidt et al., 1986). Cheesemaking is conducted in
traditional equipment, and the gross composition of
the resulting cheese is identical to that of cheese from
unconcentrated milk. The amount of starter and rennet
required are reduced by 50 and 60%, respectively
(Agbevavi et al., 1983), and with a 20% milk volume
reduction by RO, a 2–3% increase in cheese yield can
be expected (Barbano and Bynum, 1984). However, fat
losses in the whey increase with increasing solids con-
centration due to a partial homogenization effect dur-
ing processing. A sudden release of pressure during RO
can induce lipolysis in the milk and cheese. A 15% vol-
ume reduction, representing 1.8:1 concentration, has
been reported to be optimal (Barbano et al., 1983). For
Cottage cheese, a 5% increase in yield can be realized
with an 8% skim milk volume reduction by RO
(Barbano, 1986). Such yield increases result from the
entrapment of concentrated whey within the network
formed by calcium paracaseinate in the cheese.
Depending on the degree of concentration, the same
consequences for cheese quality result from a greater
retention of minerals as described for UF retentates. In
RO cheese, there is a high concentration of residual
lactose, which may lead to a resumption of lactic acid
fermentation after several days in the ripening room,
when sufficient lactic acid has been consumed by
ripening micro-organisms. This is almost always very
detrimental to organoleptic qualities (Richard et al.,
2000).
Reverse osmosis is widely used for processing whey
but it is doubtful whether it will find widespread
acceptance in cheesemaking. However, there are several
plants in the USA that already use thermal evaporators
to pre-concentrate cheese milk (Sandfort, 1983; Honer,
1984). Excess lactose and minerals in the curd are
removed by washing with water. Benefits of the overall
process must therefore be examined closely.
On-farm concentration
Interest in on-farm concentration of milk started in
1974 in France (Maubois, 1979), and in 1977, the Alfa-
Laval company developed an on-farm UF-processing
unit (Kosikowski, 1985). It was believed at that time
that on-farm concentration of milk by UF would reduce
milk transportation costs due to reduction in milk vol-
ume. An increase in cheese yield was also anticipated.
At four French farms, milk was concentrated to 2:1 and
then delivered to a cheese plant for the manufacture of
Emmental and St Paulin cheeses (Anon, 1984a). Perme-
ate produced at the farms was fed to cows, resulting in
savings in feed. The French on-farm operation ther-
mized the 2:1 UF milk prior to cooling and delivery.
The microbiology of on-farm UF milk was favourable
(Benard et al., 1981). Slack et al. (1982a,b) studied the
economics of on-farm ultrafiltration in the US by using
milk concentrated 2:1–3:1. Economic advantages were
possible when the on-farm concentration concept was
used on farms with 100–1000 cows. In a year-long
study in California, a 900-cow herd was used to study
the feasibility of on-farm UF (Zall, 1987). Retentate
from this farm was delivered to cheese plants for
cheesemaking. The regulatory aspect of this operation
was not fully resolved. The use of RO to concentrate
milk on the farm has been evaluated in Australia (Cox
and Langdon, 1985; Cox et al., 1985).
Another approach to treating milk by UF and ther-
mization at the producer level was studied in France
(Kosikowski, 1985). During two years, milk collected
from 22 farms located on an island south of Brittany
was concentrated 2:1 every second day. The retentate
was HTST pasteurized and cooled to 2 °C. Pooled
retentates were shipped to a dairy plant twice a week.
The mesophilic flora of the UF retentate was on aver-
age 7700 cfu/ml. A net benefit of 0.0482 FF/litre of
milk was achieved.
While concentration of milk on the farm showed
promise in its early years, the idea was abandoned after
various large-scale attempts because of unfavourable eco-
nomics (capital investment and membrane-replacement
costs relative to returns), safety of the process on the
farm and regulatory considerations. These problems have
apparently been overcome, and the North American Milk
Products Company has been successfully using the
process since 1997 (Fig. 9) in Texas, California and New
276 Application of Membrane Separation Technology to Cheese Production
Figure 9 Farm with a UF facility in Dexter, New Mexico, USA (courtesy of North American Milk Products, LLC, St Louis, Missouri, USA).
(See Colour plate 8.)
Mexico in the USA (Fassbender, 2001). In this process,
raw milk is ultrafiltered at 8 °C to approximately 3.5X
(28%, w/w, total solids and 10%, w/w, true protein). The
total bacterial count of the product is less than 300 000
per ml and is classified as a Grade A product according
to the US Food and Drug Administration. The concen-
trate is then shipped by truck at 4 °C to several cheese
plants in other parts of the country where it is used
to standardize milk to 13.5–15%, w/w, total solids for
making several varieties of cheeses (Cheddar, Monterey
Jack, Mozzarella). In total, an excess of 1 million kilo-
grams of raw milk is processed per day. Permeate is used
for animal feeding or spread on land; other applications
are being developed.
Applications of microfiltration in cheesemaking
Microfiltration, curiously often referred to as cross-
flow microfiltration whereas the terms ‘cross-flow UF’
and ‘cross-flow RO’ are never used, is a relatively new
processing technique in the dairy industry. Introduc-
tion really started with the development of mineral
MF membranes made from alumina (Gillot and
Garcera, 1986) or from zirconium oxide supported
on carbon (Cacciola and Leung, 1980). In 1990, the
total area installed in the world dairy industry was
less than 750 m2 (Van der Horst and Hanemaaijer,
1990), but studies have projected a potential market
six to seven times higher than that for UF. In dairy-
ing, MF applications have gained increasing attention
because of the wide range of available pore size,
which makes it possible to separate and fractionate
all milk particles.
Microbial epuration of raw milk by MF
Decontamination of raw milk is generally achieved
through heat treatment. Various combinations of time–
temperature treatments can be used, depending on the
desired bacteriocidal effect. While heat treatment is neces-
sary to ensure the safety of milk and milk products, it
almost always induces irreversible modifications of milk
components, alters physio-chemical equilibria and also
adversely affects the organoleptic quality and cheese-
making properties.
As with bactofugation, MF allows the heat treatment
for decontaminating milk to be minimized, but MF
appears to be more efficient than bactofugation. Holm
et al. (1986) and Piot et al. (1987) were the first to sug-
gest the use of MF for the removal of bacteria from
milk. Initially, permeation of milk components and
retention of bacteria were very satisfactory, but serious
fouling of the MF membrane occurred rapidly. To over-
come this, a new hydraulic concept, developed by Sand-
blom (1974), could be applied because of the
development of new MF ceramic membranes with a
highly permeable structure and a multichannel geom-
etry (Gillot and Garcera, 1986). It includes a recircula-
tion loop of micro-filtrate, which permits a constant
and low transmembrane pressure all along the MF
tubular membrane in spite of a high retentate recircula-
tion velocity (7 m/s). Commercialized equipment using
this concept for the removal of bacteria, named ‘Bacto-
catch’, is used as follows (Holm et al., 1986; Maubois,
1990); raw skim milk is microfiltered continuously using
1.4 m pore size membranes at a temperature between
35 and 50 °C. Retentate flow from the corresponding
 Application of Membrane Separation Technology to Cheese Production 277
loop generally represents 5% of the entering milk flow
but it can be reduced to 0.5% by using a second MF
equipment in cascade. This retentate, which contains
the bacteria and somatic cells in the milk, may be used
for animal feed after heat treament, or it may be
blended continuously with cream for fat standardiza-
tion. The cream–retentate mixture is subjected to a
moderate UHT treatment (115–120 °C for 3 s), cooled
and incorporated into the microfiltrate (Fig. 10).
Because of the high content of thermostable enzymes
(present in the retained bacteria and somatic cells) in
the MF retentate, such a practice could have negative
effects on cheese quality and is inadvisable. Fat stand-
ardization must be done only with heat-treated cream.
MF fluxes ranging from 500 to 700 l/h m2 are
obtained for 10 h, with an average bacterial removal of
99.6% (Malmberg and Holm, 1988; Vincens and Tabard,
1988; Meersohn, 1989; Olesen and Jensen, 1989; Trouvé
et al., 1991) regardless of the initial count in the raw
skim milk. Improvement of the degree of bacterial
removal to 99.96% was obtained by using double-layer
MF membranes (Saboya and Maubois, 2000). Morph-
ology of bacterial cells and cellular volume slightly influ-
ence membrane retention. High retention levels (greater
than 99.98%; 99.998% with the double layer MF mem-
brane) observed for spore-forming bacteria, such as
Bacillus cereus (Olesen and Jensen, 1989) or Clostridium
Figure 10 Flow diagram for the treatment of raw milk by the
Bactocatch process (Maubois, 1990).
tyrobutyricum (Trouvé et al., 1991), are likely due to
binding of bacterial spores to a part of the cell wall, con-
sequently resulting in an apparently larger cell size.
Retention of Listeria monocytogenes, Brucella abortus,
Salmonella typhimurium and Mycobacterium tuberculosis
during the Bactocatch process, using double-layer MF
membranes, shows a decimal reduction of 3.4, 4.0, 3.5
and 3.7, respectively (Madec et al., 1992; Saboya and
Maubois, 2000). Such results mean that MF cheese skim
milk will contain less than 1 cfu/l of these pathogenic
bacteria, bearing in mind the usual level of contamin-
ation at the farm. Such results have led French regula-
tory authorities to permit the provisional use of MF milk
for the making of Protected Designation of Origin
(PDO) raw milk cheeses (CNA, 2002).
While treatment of milk by the Bactocatch process
tremendously improves the hygienic quality and shelf-
life of manufactured dairy products (Malmberg and
Holm, 1988; Meersohn, 1989; Kosikowski and Mistry,
1990; Maubois, 1990; Saboya and Maubois, 2000), it
also raises the problem of how to make good quality
cheese from ultra-clean cheese milk, often described
by cheesemakers as ‘dead milk’. Extensive research
must be conducted to understand fully the growth of
lactic starters in these ultra-clean cheese milks. It
appears from French studies (Maubois, 1990) that
each cheese variety requires an independent study. For
example, satisfactory distribution of eyes in Emmental
cheese made from Bactocatch-treated milk requires
the incorporation of specific non-starter lactic acid
bacteria, such as heterolactic strains, along with
mesophilic lactic, thermophilic and propionic starters
added to this milk at the start of cheesemaking
(Maubois, 1990, 2002). The development of typical
flavour in Camembert cheese requires the addition of
Hafnia alvei, a bacterial species able to produce
volatile sulphur compounds from methionine in the
cheese (Cousin, 1994)
Casein enrichment of cheese milk by MF
Microfiltration of skim milk with 0.1 m pore size mem-
branes enables the selective separation of micellar casein,
i.e., native calcium phosphocaseinate (Fauquant et al.,
1988; Maubois et al., 2001). Depending on the amount of
microfiltrate (MMF) removed (referred to by Maubois as
‘ideal whey’ because of its sterility and composition), the
casein content of the retentate increases. Consequently,
applications of MF retentates, such as those aforemen-
tioned for UF retentates in cheesemaking, can be done
but prior removal of microfiltrate from the cheese milk
offers the cheesemaker a method for optimizing both the
cheese process and the value of derived co-products
(Maubois et al., 2001). Rennet coagulation of a 20%
casein-enriched cheesemilk is improved (Saint-Gelais
278 Application of Membrane Separation Technology to Cheese Production
et al., 1998; Maubois et al., 2001), leading to a significant
decrease in curd fines and fat in the whey and conse-
quently an increase in cheese yield of 2–4% (Daviau,
2000). MMF is easily transformed by UF and diafiltration
to a highly purified whey protein isolate (WPI)
(protein/total solids ratio0.975) with very interesting
functional properties such as gelling, foaming and solu-
bility (Bacher and Konigsfeldt, 2000). This WPI is also a
convenient starting material for the purification of major
and minor whey proteins (Maubois et al., 2001). Partial
removal of -lactoglobulin from milk through this use of
0.1 m MF membranes according to the process of
Quiblier et al. (1992) eliminates the detrimental effects
of the heat treatments caused in drying and consequently
allows the production of new powders showing very
interesting cheesemaking characteristics (Garem et al.,
2000). Microfiltration membranes of 0.1 m pore size
combined with diafiltration with water allows the prepar-
ation of a new dairy product. This product, called micel-
lar casein powder (Schuck et al., 1994), has potential
either for fortifying yoghurt and cheese milks with pro-
teins or Ca, for replacing cheese curd in processed cheese
formulations or for preparing individual caseins or
-casein glycomacropeptide (Maubois et al., 2001).
These are examples of strategies to produce nutraceutical
derivatives from milk proteins (Maubois and Ollivier,
1997).
Casein enrichment by MF has been used for Moz-
zarella and Cheddar cheeses. For Mozzarella (Brandsma
and Rizvi, 1999), concentrations of 17.9% were achieved
with 0.2 m membranes. Calcium content was reduced
by approximately 20% by lowering the pH of the reten-
tate to 6. For Cheddar cheesemaking, milk was microfil-
tered 2-fold (4.2% casein) such that casein was 87% of
true protein (Neocleous et al., 2002a,b). Fat recovery in
cheese was not affected but protein recovery increased.
Selective fractionation of globular milk fat
Separation of milk fat into small and large globules
was proposed by Goudédranche et al. (2000) through
the use of special ceramic MF membranes under
hydraulic conditions which cause no damage to the
native fat globule membrane (FGM). Using a patented
process, cheese made from milk with small globules
had a higher yield and a smoother and finer texture,
probably because of the interaction of FGM with the
cheese casein matrix, and the differences in triglycerides
content of the fat globules according to their size.
Modifications of ␣s /␤-casein ratio by MF
Hydrophobic binding entraps most of the -casein in
micelles. On cooling milk or a caseinate suspension to
a temperature lower than 5 °C, -casein is solubilized.
This soluble -casein can be separated by 0.2 m pore
size MF membranes (Terré et al., 1987) or by 100 000 Da
cut-off UF membranes (Murphy and Fox, 1991a). The
retentates have a casein content with an increased s/-
casein ratio and the microfiltrate is a solution of almost
pure -casein (Terré et al., 1987). Such a separation
process may allow in the future, if the economics are
favourable, cheesemaking from milk with a variable
s/-casein content (Terré et al., 1987; Murphy and
Fox, 1991b). Existing knowledge on cheese made from
goats’ or ewes’ milk and on the role played by the
degradation of each individual caseins in the develop-
ment of cheese flavour suggests that a large range of
new cheese varieties might be possible from -casein-
adjusted milks.
Recent developments in ceramic membrane tech-
nology allow the MF permeate recirculation loop to be
removed, and thus reduce the investment cost per m2
of installed equipment with a saving of consumed
energy. The first system, known as ‘Membralox GP®’,
was developed by Garcera and Toujas (1998). The
required counter-pressure on the permeate side is
obtained by a continuous variation of porosity of the
membrane support. In the second system, named
‘Isoflux®’, the required UTP (uniform transmembrane
pressure) is obtained by a continuous gradient of
membrane thickness. Both developments should be
used for well-defined applications, i.e., a product and a
selective separation.
Milk protein concentrates
Milk protein concentrates (MPC) have emerged over
the past decade utilizing UF, MF or a combination of
various concentration technologies and have become
important products for cheesemaking (Mistry, 2002).
They present interesting new technical possibilities
in cheesemaking and have also initiated intense dis-
cussion concerning their impact on trade and local
milk production in some countries, particularly the US.
Since there are no specific standards of identity in
any country for MPC, they, like whey protein concen-
trates (WPC), cover a wide range of compositional
parameters (in the dried product, the milk protein
content may range from 35 to over 85%) and func-
tional characteristics. Unlike WPCs, MPCs contain
both major milk protein groups in proportions similar
to milk.
Manufacturing technologies for MPC include UF,
diafiltration and spray drying, if the end product is to
be in the dry form (Mistry and Hassan, 1991). It is
essential to have raw milk of good quality (low total
and spore counts). Skim milk is ultrafiltered and diafil-
tered to approximately 21% total protein. With diafil-
tration, the lactose content is reduced such that the
 Application of Membrane Separation Technology to Cheese Production 279
final product contains less than 0.5% lactose. The
diafiltered product is then spray-dried to less than 5%
moisture. This dried or liquid product can be used to
supplement milk for cheesemaking using techniques
described earlier. For example, in experiments with
Gouda cheese, it was shown that fortification of milk
with 1% MPC increased cheese yield (Mistry and Pulgar,
1996). This was attributed to reduced losses of casein
and better retention of whey proteins. Milk protein
concentrates have introduced new possibilities in
cheesemaking, as demonstrated by recent patents
(Bhaskar et al., 2001; Blazey et al., 2001; Moran et al.,
2001a,b). Moran et al. (2001a) developed a continuous
method for manufacturing Process cheese in which
acidified milk is ultrafiltered and diafiltered to a con-
centration factor of 4–7, followed by evaporation up to
70% solids to give reduced-calcium pre-cheese. Process
cheese is made from this pre-cheese in the traditional
manner using flavouring agents and emulsifiers. No
cheese base is required, but, instead, high protein con-
centrates are used as the base material along with
flavouring agents.
Concentrates with a high micellar casein content
have been developed using microfiltration (Schuck
et al., 1994). The casein content of such powder is
approximately 90% and forms almost 96% of total pro-
tein (Saboya and Maubois, 2000). When used in
cheesemaking, increases in cheese yield have been
reported (Caron et al., 1997). Such powders also pro-
vide flexibility in usage regardless of the extent of heat
treatment because the -casein–-lactoglobulin com-
plex typically found in NDM does not exist due to the
removal of the -lactoglobulin during microfiltration.
Concluding Remarks
Membrane technologies have, during the last 20 years,
opened new avenues for improving traditional cheese-
making procedures and consequently improved not
only the overall quality of a number of cheese varieties
but also increased net profit resulting from this transfor-
mation of milk. Membrane processing has truly evolved
from processes requiring highly specialized equipment
to those that have now become an integral part of the
cheesemaking operation. They have also allowed the
survival of cheese varieties that require unacceptable
and tedious manual labour by the traditional process. In
addition, they have led to the creation of new cheeses in
response to consumer demand.
On the other hand, because of difficulties encoun-
tered in making cheeses with satisfactory organoleptic
qualities through membrane technologies, an impres-
sive amount of knowledge has been acquired in numer-
ous fields of dairy science – protein biochemistry and
physiochemistry, inter-relationships between protein
and minerals, dairy microbiology, rheology, etc.
The future of the use of these technologies in the
world dairy industry is very promising. Many new
cheese varieties might be prepared by combining the
properties of mineral-adjusted UF retentates and
enzymic abilities of lactic starters. Microfiltration has
opened new and much diversified avenues for research
and technology. Some have already quickly penetrated
the cheese industry. In the future, numerous ideas
for cheese scientists and technologists may also origin-
ate from microfiltration applications. For example,
somatic cells are the only milk components which con-
tain all the genome of the producing animal. Their
specific separation by MF and the use of molecular
genetics could be the starting point for determining
the origin (producing cows) of all dairy products
except those made from MF milk. Removal of the
entire contaminating flora by MF also offers a means to
study precisely how each type of starter bacteria added
to the cheese milk will act on ripening of different
cheese varieties.
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This Page Intentionally Left Blank
 The Microbiology of Cheese
Ripening
T. Beresford, Dairy Products Research Centre, Teagasc, Moorepark, Fermoy,
Cork, Ireland
A. Williams, CHARIS Food Research, Hannah Research Institute, Scotland, UK
Introduction
Micro-organisms, including bacteria, yeast and
moulds, are present in cheese throughout ripening
and contribute, in a positive manner, to the matur-
ation process either directly through their metabolic
activity or indirectly through the release of enzymes
into the cheese matrix through autolysis. The cheese-
maker encourages the growth of such organisms;
however, other micro-organisms, such as food-borne
pathogens, have a negative impact on cheese quality,
and thus technologies to remove or prevent their
entry to cheese are required. In this chapter we will
review the major groups of those micro-organisms
which contribute in a positive manner to cheese
ripening.
The microflora associated with cheese ripening is
extremely diverse; however, it may be conveniently
divided into two groups – the starter lactic acid bac-
teria (LAB) and the secondary microflora. Starter
bacteria are primarily responsible for acid produc-
tion during manufacture and, thus, need to be cap-
able of producing sufficient acid to reduce the pH of
milk rapidly; a useful rule of thumb is a pH5.3 in
milk in 6 h at 30–37 °C, depending on the cheese
variety. The secondary microflora do not play any
active role during cheese manufacture but are
involved with the starter bacteria in the ripening
process. Using this approach, Lactococcus, Strepto-
coccus thermophilus, Lactobacillus delbrueckii and Lb.
helveticus are regarded as starter bacteria. Enter-
ococcus has been regarded as a starter by some
researchers; however, as most strains isolated from
cheese are not significant acid-producers (Cogan
et al., 1997), they will be regarded as part of the sec-
ondary microflora within this review. The secondary
microflora may be divided into a number of primary
groups including: (i) non-starter lactic acid bacteria
(NSLAB) consisting of non-starter lactobacilli, Pedio-
coccus, Enterococcus and Leuconostoc, (ii) propionic
acid bacteria (PAB), (iii) moulds and (iv) bacteria
and yeast, which grow on the surface of smear-
ripened cheeses.
Cheese: Chemistry, Physics and Microbiology, Third edition – Volume 1: General Aspects
ISBN: 0-1226-3652-X
Set ISBN: 0-1226-3651-1
Techniques used to Study Micro-organisms
in Cheese
When studying the micro-organisms in cheese it is
important that the complete flora is monitored and that
the individual components are accurately identified and
characterised. Approaches used to achieve these object-
ives include methods that: (1) depend on cultivation
followed by phenotypic characterisation, (2) depend on
cultivation followed by molecular characterisation and
(3) are culture-independent methods. These approaches
and their associated advantages and disadvantages were
reviewed recently (Beresford et al., 2001). Useful media
are discussed in Cogan and Beresford (2002).
Source of Micro-organisms in Cheese
Micro-organisms gain entry into the cheese either by
deliberate addition as part of the starter culture or are
naturally associated with the ingredients used in cheese
production. Thus, the manufacturing technology is
central to defining the biodiversity of the cheese flora.
Milk in the udder of healthy animals is essentially
sterile; however, during milking and storage, opportun-
ities for contamination occur. Milk extracted from the
udder at farm level under hygienic milking conditions
can routinely contain 5  103 cfu ml 1 (Fox et al.,
2000). The rapidity and degree of milk cooling post-
milking has a significant impact on the microbial flora.
Milk cooled to 15–21 °C is dominated by mesophilic
micro-organisms, particularly Lactococcus and Enter-
obacter species (Bramley and McKinnon, 1990). Cool-
ing milk to 4 °C will greatly retard the growth of most
micro-organisms, but psychrotrophic bacteria, such as
Pseudomonas, Flavobacterium and Acinetobacter will
continue to grow slowly and dominate the flora. Pas-
teurisation, which is part of the manufacturing process
for most commercial cheeses kills ⬃99.9% of the bac-
teria found in raw milk. However, Bacillus and Clostrid-
ium spores and thermoduric organisms, e.g., Micrococcus,
Microbacterium and Enterococcus, will survive pasteurisa-
tion and gain entry into the cheese.
Copyright © 2004 Elsevier Ltd
All rights reserved
288 The Microbiology of Cheese Ripening
The most prevalent micro-organisms in cheese, par-
ticularly early in ripening, are the starter bacteria (see
‘Starter Cultures: General Aspects’, Volume 1). Other
ingredients used in cheese manufacture include rennet
and salt. During preparation, rennets undergo a series
of extraction and purification steps, and 15–20% NaCl
is added to them to inhibit microbial growth during
storage. Little information is available regarding the
microbial flora of commercial rennet. However, it
is generally considered that they add little to the
microbial load of cheese. Salt may be added either
(i) directly to the milled cheese curd, (ii) rubbed to the
surface of moulded curds as a dry salt or salt slurry or
(iii) by immersion of moulded cheese in a brine solu-
tion containing 15–23% NaCl. While rubbing dry salt
to the cheese surface aids transfer of micro-organisms
from the cheesemaker’s hands and the environment to
the cheese surface, it is unlikely that salt is directly
responsible for the addition of any flora. Industrial
brines are used repeatedly and are pasteurised infre-
quently. While the relatively high salt content of brine
inhibits the growth of most micro-organisms, leaching
of proteins and other nitrogenous compounds from
the cheese into the brine may enhance the survival of
micro-organisms that gain access to the brine. A num-
ber of studies have indicated that the microflora of
commercial brines include LAB, e.g., Lb. paracasei,
Lb. casei and Lb. plantarum (Bintsis et al., 2000) and
yeast, e.g., Debaromyces hansenii and Candida versatilis
(Seiler and Busse, 1990). The presence of such
microbes in the brine contributes subsequently to the
surface flora developing on the cheese; however, it is
unlikely to add to the internal flora.
Complex microbial communities composed of bac-
teria, yeast and mould develop on the surface of
smear- and mould-ripened cheeses during ripening
(‘Bacterial Surface-ripened Cheeses’, Volume 2). The
smear micro-organisms may evolve due to natural con-
tamination from the milk or the ripening room envir-
onment or result from deliberate inoculation of the
surface. Traditionally in the production of some smear-
cheeses, a process referred to as ‘old-young’ smearing
is used to promote the development of the smear. Mix-
tures of defined strains are also being developed for
direct application to the cheese surface (Bockelmann,
2002). Deliberate addition of specific strains of Penicil-
lium roqueforti or P. camemberti is now common in
large-scale production of mould-ripened cheeses.
Traditional Swiss Emmental cheese is made from raw
milk and the propionic acid fermentation depends on
the presence of ‘wild’ PAB in the milk. However, in
Emmental made from pasteurised milk, PAB are added
to the milk at the beginning of manufacture to ensure
that they are present at ⬃105 cfu g 1 of milk.
Factors Influencing Growth of
Micro-organisms in Cheese
The manufacturing process plays a major role in defin-
ing the final environmental conditions of the cheese.
This environment is highly selective and exerts a major
impact on growth and survival of micro-organisms dur-
ing processing and ripening. The manufacture of most
cheese varieties involves coagulation at temperatures of
30–37 °C, followed by cooking to 37–54 °C. The coagu-
lation temperature will facilitate the growth of most
micro-organisms; however, the temperature achieved
during cooking has the potential to inhibit the growth of
some organisms. For example, Swiss-type cheese is
cooked to 52–54 °C and is maintained above 50 °C for
up to 5 h. This heat treatment is considered to play an
important role in controlling the growth of starters and
undesirable micro-organisms (Steffen et al., 1993). The
manufacturing process also influences the gross compos-
ition of the cheese which is best defined by the four
parameters – salt-in-moisture, moisture in non-fat sub-
stance, fat in dry-matter and pH (Gilles and Lawrence,
1973). These parameters in turn influence the environ-
ment in which the micro-organisms proliferate. The pri-
mary environmental factors controlling growth of
micro-organisms in cheese include water and salt con-
tent, pH, presence of organic acids and nitrate, redox
potential and ripening temperature, and were reviewed
recently (Beresford et al., 2001).
Starter Bacteria
The primary function of starter bacteria is to produce
sufficient acid during cheese manufacture to reduce
the pH of milk to the desired level. However, they also
contribute to cheese ripening since their enzymes are
involved in proteolysis, lipolysis and conversion of
amino acids to flavour compounds (Fox and Wallace,
1997). Starter cultures are reviewed in ‘Starter Cul-
tures: General Aspects’, Volume 1; thus, discussion on
starters in this chapter will be limited to their behav-
iour in cheese during ripening.
Starters provide the most significant contribution to
the microbial biomass in young curd, typically attaining
densities of 108 cfu g 1 within one day of manufac-
ture. This biomass represents considerable biocatalytic
potential for cheese-ripening reactions. However, the
majority of the starter enzymes are intracellular and do
not have immediate access to the cheese matrix. During
cheese ripening, many starters loose viability and release
their intracellular enzymes due to autolysis. Feirtag and
McKay (1987) first reported this phenomenon for lacto-
cocci, and observed that some strains lost viability when
incubated at 40 °C due to lysis by thermo-inducible

phage. Interest was further stimulated when it was
demonstrated that cheese manufactured with autolytic
strains was more flavoursome. The pathways of autolysis
have been studied extensively and two main mech-
anisms are currently recognised involving induction of
lysogenic phage or defects in cell wall synthesis. The
relationship between lysogeny and lysis of lactococci in
Cheddar cheese has recently been demonstrated for a
large number of lactococcal strains (O’Sullivan et al.,
2000). Bacterial cell wall synthesis is a complex process.
Modifications to the specificity of the enzymes involved
or their levels of production can result in defective cell
walls. Muraminidase is the major autolytic enzyme in
lactococci (Niskasaari, 1989). Wilkinson et al. (1994)
studied starter autolysis by assaying cell viability and
release of intracellular enzymes in Cheddar cheese and
concluded that Lc. lactis subsp. cremoris strains had dif-
ferent autolytic patterns (Fig. 1). Levels of proteolysis, as
measured by free amino acids in the cheese, correlated
with the autolytic phenotype, being higher in the most
autolytic strain (AM2) and lowest in the least autolytic
strain (HP). It has been reported (Crow et al., 1995) that
intact cells ferment lactose, remove oxygen and initiate a
number of flavour reactions, while autolysed cells accel-
erate peptidolytic processes. Bacteriocin production is
common among lactococci, and some bacteriocins
induce lysis in susceptible starter strains. This phenom-
enon was used to induce lysis of starter lactococci dur-
ing cheese ripening and resulted in cheese with
improved flavour (Morgan et al., 1997).
Autolysis has also been reported for Lb. helveticus in
Grana (Botazzi et al., 1992), in Swiss-type (Gagnaire
et al., 1998; Valence et al., 1998) and Cheddar cheeses
(Kiernan et al., 2000). The extent of autolysis varied
between strains (5–7-fold) and had a direct impact on
the degree of proteolysis in cheese (Valence et al.,
2000). The mechanism of autolysis in Lb. helveticus
has not been fully elucidated; however, many strains
are lysogenic (Carminati et al., 1997). Six of eight
10
Population size (log cfu g–1)
9
8
7
6
5
4
3
2
1
0
0 10
Figure 1 Changes in the populations of three lactococcal starters during ripening of Cheddar cheese. G11/C25 (), HP ( ) and
AM2 ( ) (redrawn from Wilkinson et al. (1994)).
The Microbiology of Cheese Ripening 289
lysogenic strains grew during the manufacture of Swiss
cheese, exhausted the galactose and lysed extensively
early in ripening (Deutsch et al., 2002). Phage was
detected in four of the cheeses at day 1, strongly impli-
cating the role of phage induction in autolysis. Little
investigation has occurred regarding autolysis in Lb.
delbrueckii (Kang et al., 1998).
Autolysis of Sc. thermophilus in cheese has received
little attention. Autolysis was reported in a number
of strains at the end of growth in laboratory media
(Sandholm and Sarimo, 1981; Thomas and Crow, 1983a;
Husson-Kao et al., 1999). Prophage-induction triggered
by environmental signals, e.g., low pH, may contribute
to autolysis of Sc. thermophilus (Husson-Kao et al.,
2000).
Non-starter Bacteria
Non-starter lactic acid bacteria are a significant pro-
portion of the microbial population of, probably, all
ripened cheese varieties. Except for leuconostocs,
NSLAB are not deliberately added as part of the starter
culture or as secondary adjunct cultures but are
adventitious contaminants, which grow during ripen-
ing. They do not contribute to acid production during
cheese manufacture, but impact on flavour develop-
ment in the ripening cheese. The principal bacterial
groups involved are non-starter lactobacilli, leuconos-
tocs, pedicocci and enterococci.
Non-starter lactobacilli
Non-starter lactobacilli constitute the majority of the
NSLAB population in most cheese varieties during
ripening (Beresford et al., 2001). They grow at 2–53 °C
and are acid-tolerant with an optimal pH for growth of
5.5–6.2. They have been subdivided into three groups,
viz., obligate homofermenters, facultative heterofer-
menters or obligate heterofermenters (Kandler and
20 30 40 50 60 70
Ripening period (days)
290 The Microbiology of Cheese Ripening
Weiss, 1986). The obligate homofermenters include
the starter bacteria Lb. delbrueckii and Lb. helveticus.
The non-starter lactobacilli frequently recovered from
cheese are facultative heterofermenters and are often
referred to as the facultatively heterofermentative lacto-
bacilli (FHL). The obligate heterofermenters are detected
less frequently in cheese.
Information on the non-starter Lactobacillus popula-
tion of over 50 different cheese varieties is summarised
in Table 1. Studies on non-starter lactobacilli occurring
in Cheddar cheese produced in various countries con-
sistently report the dominance of Lb. paracasei and
Lb. plantarum. Other species that are frequently
detected as minor components of the population
include Lb. curvatus, Lb. casei, Lb. brevis and Lb. rham-
nosus. Several other species, including Lb. bifermentans,
Lb. buchneri, Lb. collinoides, Lb. farciminis, Lb. fermen-
tum, Lb. kefiri, Lb. parabuchneri and Lb. paraplantarum
are occasionally reported in commercially manufactured
Cheddar cheese (Williams and Banks, 1997; Fitzsimons
et al., 1999; Chandry et al., 2002). Lb. paracasei and Lb.
plantarum are also prevalent in many other cheese var-
ieties (Table 1). Non-starter lactobacilli were present in
19 of 35 European artisanal cheeses and were a major
component of the microflora of Kasseri, Feta, Serra da
Estrela, Gredos and Majorero cheeses (Cogan et al.,
1997). The range of species detected is almost identical
to that described for Cheddar cheese. The limited range
of species reported suggests that few species of Lacto-
bacillus are capable of surviving the environmental con-
ditions that pertain in cheese. The factors required to
facilitate proliferation in cheese have not been deter-
mined, although an ability to utilise the available
growth substrates and an inherent resistance to the
adverse pH and salinity are imperative.
Pediococci
Although pediococci have been used as adjunct cultures
to improve the flavour of Cheddar and Feta cheeses, they
also occur along with, and on occasions can predom-
inate, the non-starter population (Law et al., 1976;
Bhowmik et al., 1990; Vafopoulou-Mastrojiannaki et al.,
1990; Bhowmik and Marth, 1990a). Pediococcus acidilac-
tici and Pd. pentosaceus are isolated most frequently from
cheese. The presence of pediococci in Cheddar cheese
was first reported by Dacre (1958) who found that they
comprised ⬃25% of the bacterial population after
6 months of maturation. Pediococci have subsequently
been reported in the non-starter flora of Cheddar cheese
manufactured in the UK, Canada and the USA (Fryer and
Sharpe, 1966; Elliott and Mulligan, 1968; Litopoulou-
Tzanetaki et al., 1989), in Manchego and Serra da Estrela
cheeses (Nunez, 1976; Tavaria and Malcata, 1998), Parmi-
giano Reggiano and Sicilian artisanal cheeses (Coppola
et al., 1997; Randazzo et al., 2002), Comté (Bouton et al.,
1998) and Feta and other white-brined cheeses
(Tzanetakis and Litopoulou-Tzanetaki, 1989, 1992;
Bintsis and Papademas, 2002; Hayaloglu et al., 2002).
Leuconostoc spp.
Many leuconostocs produce diacetyl and acetoin from
citrate and are widely used in mixed-strain L and DL cul-
tures (Dellaglio et al., 1995). The CO2 produced is
responsible for eye formation in Dutch-type cheese. The
identity of strains in starters has not always been estab-
lished, although the application of molecular tech-
niques has indicated that dairy starters were principally
Leuc. lactis and the three subspecies of Leuc. mesen-
teroides (Morea et al., 1999; Server-Busson et al., 1999).
While isolation of leuconostocs is not restricted to the
cheeses produced with leuconstoc-containing starters,
their apparent infrequent occurrence may, in part, be
due to their poor growth on the selective media used
(Mathot et al., 1994). Leuconostoc spp. have been isol-
ated from artisanal cheese produced from raw milk and
white-brined cheese varieties (Aran, 1998; Bintsis and
Papademas, 2002; Hayaloglu et al., 2002) and from
French (Cibik et al., 2000), Greek (Litopoulou-Tzane-
taki, 1990) and Italian (Coppola et al., 1988, 2001;
Morea et al., 1999; Randazzo et al., 2002) cheeses. Sev-
eral cheeses produced from ovine and/or caprine milks
on the Iberian peninsula contain Leuconostoc spp.
(Poullet et al., 1993; Garcia et al., 1995; Macedo et al.,
1995; Centeno et al., 1996a; Arizcun et al., 1997a;
Estepar et al., 1999; Dahl et al., 2000; Freitas and Mal-
cata, 2000; Fontan et al., 2001; Menendez et al., 2001;
Pérez et al., 2002).
Enterococci
Enterococci occur widely in the environment but are
associated principally with the gastrointestinal tract
and, because of this, their presence in food products is
often perceived as an indicator of poor hygiene. How-
ever, enterococci have a history of safe use in dairy
products and additionally may exhibit probiotic char-
acteristics or produce bacteriocins (Franz et al., 1999).
Conversely, they are regarded as emerging nosocomial
pathogens of humans and have been implicated in the
aetiology of bacteraemia, endocarditis and in infec-
tions of the urinary tract, the central nervous system,
the pelvis, the abdomen and the neonate. Many
exhibit resistance to vancomycin and other antibiotics
and also possess recognised virulence factors (Franz
et al., 1999; Giraffa, 2002). Gene transfer mechanisms
in enterococci have been described, and the intra- and
inter-species transfer of antibiotic resistance genes has
been reported (Noble et al., 1992).
 Table 1 Occurrence of non-starter lactobacilli in different cheese varieties

Dominant non-starter lactobacilli*

Country of
Cheese variety origin ace aci ali bre cas cur cyp far fer hil pub pca ppl pen pla rha Reference

Cheddar Australia   Broome et al.

(1990a)
Cheddar  Chandry et al.
(2002)
Cheddar New Zealand   Crow et al. (2001)
Cheddar Ireland    Jordan and Cogan
(1993)
Cheddar     Fitzsimons et al.
(1999)
Cheddar  Fitzsimons et al.
(2001)
Cheddar UK    Naylor and Sharpe
(1958a,b)
Cheddar   Williams and Banks
(1997)
Cheddar     Williams et al.
(2002a)
Cheddar USA  Swearingen et al.
(2001)
Jarlberg, Norway/    Lindberg et al.
Norvegia, Sweden (1996)
Greve,
Gouda
Herrgård Sweden  Antonsson et al.
(2001)
Comté France    Bouton et al. (1998)
Comté    Grappin et al.
(1999)
Comté   Berthier et al.
(2001)
Emmental  Thierry et al. (1998)
(Swiss type)
Vacherin   Sozzi and Maret
Mont d’Or (1973)
Afuega’l Pita Spain   Cuesta et al. (1996)
Armada   Tornadijo et al.
(1995)
Artisanal    López and Mayo
(1997)
291

continued
292

Table 1 continued

Dominant non-starter lactobacilli*

Country of
Cheese variety origin ace aci ali bre cas cur cyp far fer hil pub pca ppl pen pla rha Reference

Arzua   Centeno et al.

(1996a)
Cabrales  Nunez (1978)
Casar de     Poullet et al. (1993)
Caceres
Idiazabal     Elortondo et al.
(1998)
La Serena Freitas and Malcata
(2000)
Leon  Medina et al. (1995)
Mahon  Ramos et al. (1982)
Majorero      Fontecha et al.
(1990)
Manchego Freitas and Malcata
(2000)
Penamellera    Estepar et al.
(1999)
Roncal Freitas and Malcata
(2000)
Vadeon López-Díaz et al.
(2000)
Azeitas Portugal    Freitas and Malcata
(2000)
Evora     Freitas and Malcata
(2000)
Picante da    Freitas et al. (1996)
Beira Baixa
Serra da Estrela  Macedo et al.
(1995)
Serra da Estrela   Roserio and
Barbosa (1996)
Serra da Estrela     Tavaria and
Malcata (1998)
Caciocavallo Italy   Gobbetti et al.
Pugliese (2002)
Caciocavallo    Corsetti et al.
Silano (2001b)
 Canestrato      Albenzio et al.
Pugliese (2001)
Casu Axedu   Ledda (1996)
Ewe milk      De Angelis et al.
(2001)
Fiore Sardo   Mannu et al. (2000)
Fontina   Cocconcelli et al.
(1996a)
Fossa (Pit)    Gobbetti et al.
(1999b)
Montasio  Lombardi et al.
(1995)
Mozzarella    Morea et al. (1998)
Parmigiano   Coppola et al.
Reggiano (1997)
Pecorino Sardo  Mannu et al. (2002)
Pecorino Toscano    Bizzarro et al. (2000)
Ricotta Forte     Baruzzi et al. (2000)
Scamorza  Baruzzi et al. (2002)
Altamurana
Toma   Cocconcelli et al.
(1996b)
Batzos Greece    Nikolaou et al.
(2002)
Domiati     Bintsis and
Papademas
(2002)
Feta     Bintsis and
Papademas
(2002)
Feta Teleme  Tzanetakis and
Litopoulou-
Tzanetaki (1992)
Kefalotyri   Litopoulou-
Tzanetaki (1990)
Beyaz Turkey   Durlu-Ozkaya et al.
(2001)
Beyaz Peynir Hayaloglu et al.
(2002)
Kashar Aran (1998)
Domiati Egypt    El Zayat et al.
(1995)

continued
293
294

Table 1 continued

Dominant non-starter lactobacilli*

Country of
Cheese variety origin ace aci ali bre cas cur cyp far fer hil pub pca ppl pen pla rha Reference

Halloumi Cyprus     Papademas and

Robinson (2000)
Pichtogalo Crete     Papageorgiou et al.
Chanion (1998)
Ragusano Sicily  Randazzo et al.
(2002)
Tenerife Tenerife   Zárate et al. (1997)
caprine milk

* The abbreviations used for the non-starter lactobacilli are as follows: ace, Lb. acetotolerans; aci, Lb. acidophilus; ali, Lb. alimentarius; bre, Lb. brevis; cas, Lb. casei; cur, Lb. curvatus;
cyp, Lb. cypricasei; far, Lb. farciminis; fer, Lb. fermentum; hil, Lb. hilgardii; pbu, Lb. parabuchneri; pca, Lb. paracasei; ppl, Lb. paraplantarum; pen, Lb. pentosus; pla, Lb. plantarum; rha,
Lb. rhamnosus.
 The Microbiology of Cheese Ripening 295

Enterococci are a major component of the bacterial
population of cheeses produced in Italy (Senini et al.,
1997; Suzzi et al., 2000; Andrighetto et al., 2001),
France (Bouton et al., 1998), Spain, Portugal (Freitas
and Malcata, 2000), Greece (Papageorgiou et al., 1998;
Nikolaou et al., 2002), Turkey, The Balkans (Bintsis
and Papademas, 2002; Hayaloglu et al., 2002) and
Egypt (Hemati et al., 1998). Their numbers at the end
of ripening range from 105 to 107 cfu g 1, although
numbers vary with cheese type (Fig. 2A). The species
isolated most frequently are Ec. faecalis, Ec. faecium
and Ec. durans.
Source of NSLAB
Non-starter lactic acid bacteria are present in cheeses
made from both raw and heat-treated milk. Hygienically
produced raw milk may contain ⬃102 lactobacilli ml 1
and it is probable that the milk is the principal source
of organisms in cheeses made from raw milk.

8
7
6
5
4
3
2
1
A
0
0 20 40 60 80 100 120 140 160 180 200

10
9
Population size (log cfu g–1 )

8
7
6
5
4
3
2
1
B
0
0 20 40 60 80 100 120 140 160 180 200
10
9
8
7
6
5
4
3
2
1
C
0
0 20 40 60 80 100 120 140 160 180 200

Ripening period (days)

Figure 2 Changes in the populations of (A) enterococci, (B) non-starter lactobacilli and (C) Leuconostoc spp. during ripening of
Afuega’l Pitu (; Cuesta et al., 1996), Armada ( ; Tornadijo et al., 1995), Cabrales (; Nunez, 1978), Canestrato Pugliese
( ; Albenzio et al., 2001), Cheddar (×; Dasen et al., 2003), Fossa Pit ( ; Avellini et al., 1999), La Serena (; Fernandez del Pozo
et al., 1988), Penamellera (; Estepar et al., 1999), Serra da Estrela ( ; Dahl et al., 2000), Swiss-type (; Beuvier et al., 1997 and
Demarigny et al., 1996) and Tenerife caprine (; Zárate et al., 1997) cheeses.
296 The Microbiology of Cheese Ripening
The diversity of the non-starter population is greater in
cheeses made from raw than from pasteurised milk,
and Berthier et al. (2001) were able to demonstrate,
using molecular techniques, that most of the non-
starter lactobacilli in Comté originated from the milk.
The presence of high numbers of enterococci in arti-
sanal cheeses is usually associated with poor hygienic
practices (Franz et al., 1999), although bovine faeces
was not considered to be the source of enterococci in
the farm-house raw milk Cheddar type cheese studied
by Gelsomino et al. (2002).
Although some lactobacilli are inactivated by pas-
teurisation (Turner et al., 1986), other strains may sur-
vive the heat treatment and proliferate in the cheese
during ripening (McSweeney et al., 1994; Jordan and
Cogan, 1999). Enterococci are also likely to survive
pasteurisation. The production of natural milk cultures
from pasteurised milk that is incubated at 42–44 °C
for 12–15 h inevitably promotes the selection of heat-
tolerant LAB, including enterococci (Giraffa et al., 1997).
It has been suggested (Martley and Crow, 1993) that
milk is not the principal source of NSLAB in cheeses
made from pasteurised milk and that the manufacturing
equipment was the more probable source. Serological
typing methods confirmed that air-borne lactobacilli in
the plant during cheesemaking are recovered from the
cheese (Naylor and Sharpe, 1958b). Non-starter lactic
acid bacteria have been isolated from the floor and drains
in the dairy environment and from the surfaces of equip-
ment used in cheese manufacture and vacuum packaging
(Somers et al., 2001). Lactobacilli are able to form and
persist in biofilms on cheesemaking equipment and
could be re-isolated from batches of cheese produced
after the plant had been cleaned, implying that they sur-
vive cleaning and sanitising treatments (Somers et al.,
2001). The source of enterococci in cheese milk has been
identified as the milking equipment (Gelsomino et al.,
2002). The proposition that a factory-specific flora could
impart distinctive flavour characteristics to the cheese
produced in a given plant (Chapman and Sharpe, 1981)
implies that contamination during manufacture is a crit-
ical component of the process. However, studies of the
non-starter Lactobacillus populations of cheeses made in
a single plant over an extended time period failed to
detect the recurrence of specific strains (Fitzsimons et al.,
2001; Williams et al., 2002a), indicating that, if the
contamination was from within the plant, the source was
either intermittent or at a low level.
Factors affecting growth and survival of NSLAB
Environmental conditions
Non-starter lactic acid bacteria, in particular non-
starter lactobacilli and enterococci, are not adversely
affected by environmental conditions in the cheese
curd and are able to proliferate during maturation.
Non-starter lactobacilli have a generation time of
approximately 8.5 days in cheese ripened at 6 °C
(Jordan and Cogan, 1993) and viable cells can be
recovered from cheese stored at 10 °C for 3 years. The
growth rate and final population density of non-starter
lactobacilli and enterococci are not affected signifi-
cantly over the pH range, salt and moisture levels that
normally occur in the curd during Cheddar cheese
manufacture (Lane et al., 1997). Their growth rate is
temperature-dependent but ripening temperature had
little influence on the final numbers of lactobacilli in
the cheese. Rapid block cooling and ripening at low
temperatures reduces their growth rates (Folkertsma
et al., 1996). In cheese ripened at 1 °C, the non-starter
lactobacillus population was 3 log cycles lower than in
a cheese ripened at 8 °C (Shakeel-Ur-Rehman et al.,
2000).
Nutrient availability
Non-starter lactic acid bacteria require an energy
source for growth. The level of residual lactose in fresh
curd is usually low but nevertheless some is likely to
be present when the non-starter Lactobacillus popula-
tion is becoming established in the cheese. However,
the subsequent increase in the non-starter Lactobacil-
lus population is likely to occur after the lactose has
been utilised, indicating that it is not the sole energy
source (Turner and Thomas, 1980). Waldron (1997)
showed that growth of mesophilic lactobacilli was
independent of the lactose content of the cheese.
Mesophilic lactobacilli possess glycoside hydrolases
(Williams and Banks, 1997) and can utilise sugars
derived from the glycomacropeptide of casein and the
glycoproteins of the milk-fat globule membrane (Fox
et al., 1998; Diggin et al., 1999; Williams et al., 2000).
In addition, starter culture autolysis during ripening
releases sugars, e.g., ribose (Thomas, 1987; Rapposch
et al., 1999). However, studies by Lane et al. (1997) on
the growth rate of non-starter lactobacilli in cheeses
made with fast- and slow-lysing starter cultures indi-
cated that cell lysate was not a major source of growth
substrates.
Citrate is present in small amounts (⬃8 mmol kg 1)
in unripened Cheddar cheese but is not used as an
energy source by non-starter lactobacilli (Palles et al.,
1998; Williams et al., 2000); high numbers of lacto-
bacilli also develop in cheese in which there has been
no significant citrate utilisation (Jordan and Cogan,
1993). Lipids and bacterial catabolites are not effective
substrates but peptides and amino acids are
catabolised by lactobacilli, provided that a keto acid
acceptor is present to facilitate the aminotransferase
involved (Tammam et al., 2000; Williams et al., 2000,

2001). Proteolytic products formed during ripening
stimulate the growth of Lb. casei (Nath and Ledford,
1972), whilst Laht et al. (2002) concluded that argin-
ine was one of the main energy sources for non-starter
lactobacilli in Swiss-type cheese. The ATP available
from arginine metabolism is theoretically sufficient to
support growth to 108 cfu g 1.
Interactions
The microbial flora of cheese are complex and it is
inevitable that interactions between members of the
population will occur. The complex nature of cheese
ecosystems complicates the interpretation of these inter-
actions; however, Martley and Crow (1993) were able to
demonstrate interactions between NSLAB during ripen-
ing. It has also been reported that Lb. casei, Lb. rhamno-
sus and Lb. plantarum inhibit PAB and enterococci in
cheese as a result of competition for limiting nutrients
(Jimeno et al., 1995; Lynch et al., 1996).
In addition to the inter-species competition for
nutrients, some of the metabolic products formed by
NSLAB, e.g., lactate, acetate and formate, may interfere
with the growth of other species (Lindgren and Dobro-
gosz, 1990; Vandenbergh, 1993; Ouwehand, 1998). In
addition, many NSLAB are able to produce bacter-
iocins with broad or narrow spectra of activity (Stiles,
1994; Ouwehand, 1998; Franz et al., 1999). The inclu-
sion of bacteriocin-producing starter LAB offers a
means of suppressing the growth of undesirable bac-
teria. This approach has been used to control the growth
of both food-borne pathogens and NSLAB in cheese
(Ryan et al., 1996; Buyong et al., 1998; Benkerroum
et al., 2000; Benech et al., 2002), and as a means of
manipulating the ripening process (Fenelon et al., 1999;
Oumer et al., 2001; Garde et al., 2002).
Non-starter lactic acid bacteria may undergo autoly-
sis during ripening. However, Kiernan et al. (2000)
were unable to find evidence for the autolysis of
mesophilic lactobacilli during the ripening of Cheddar
cheese. The ability of Leuconostoc spp. to autolyse is
strain-dependent (Cibik and Chapot-Chartier, 2000).
Population dynamics
Although the population size remains relatively sta-
ble from 3 months to the end of maturation (Fig. 2),
the population is not static but is in a dynamic state as
the balance of the species and the strains change.
Cheddar cheese
The initial non-starter Lactobacillus level in commer-
cially produced Cheddar curd is ⬃102 cfu g 1 which
increases to ⬃107 cfu g 1 within 3 months of manu-
facture and remains at this level throughout the
remainder of maturation (Peterson and Marshall,
The Microbiology of Cheese Ripening 297
1990; Fox et al., 1998). However, the heterogeneity of
the population of non-starter lactobacilli in Cheddar
cheese decreases during ripening. While several
species are detected in young cheeses, they are
replaced, as the cheese ages, by strains of Lb. paracasei
which dominate throughout the remainder of the
ripening period (Fitzsimons et al., 2001; Williams
et al., 2002a). Similar dynamics was observed in New
Zealand Cheddar except that the dominant species
were Lb. paracasei and Lb. rhamnosus (Crow et al.,
2001). Population shifts at strain level also occur dur-
ing the ripening of Cheddar cheese. Although some
strains may persist throughout ripening while others
may recur in the latter stages of ripening, having been
undetected in the intervening period, the overall trend
is for the number of strains present to decline during
maturation. Most mature cheeses are dominated by no
more than six strains (Crow et al., 2001; Fitzsimons
et al., 2001; Williams et al., 2002a; Dasen et al., 2003).
Swiss-type cheeses
The number of non-starter lactobacilli and enterococci
are higher in Swiss-type cheese made from raw milk
than in cheese made from pasteurised milk but the
diversity of non-starter lactobacilli declines during
ripening (Beuvier et al., 1997). The population of
young cheese was comprised of Lb. paracasei, Lb. plan-
tarum and Lb. brevis but as the cheese matured Lb.
paracasei dominated (Demarigny et al., 1996). Similar
results were obtained in Emmental cheese made from
thermised milk (Thierry et al., 1998). The non-starter
Lactobacillus population in Comté cheese was domin-
ated by Lb. paracasei, although Lb. plantarum and
Lb. rhamnosus were also present (Bouton et al., 1998;
Grappin et al., 1999). Enterococci remained at low
levels throughout ripening (Fig. 2A). Berthier et al.
(2001), using PCR methods, detected fewer strains in
the mature cheese than at other stages throughout
ripening.
Spanish artisanal cheeses
There is very little information available on changes in
the species profiles in Spanish cheeses during ripen-
ing. Most data are restricted to counts of the dominant
genera at various times during maturation, and signifi-
cant variation occurs between the different groups of
bacteria in different cheeses (Fig. 2A–C). This vari-
ation may be due to the artisinal nature of many of
these cheeses. Lb. plantarum and Lb. brevis dominated,
and the population remained constant at ⬃108 cfu g 1
throughout the 60-day ripening of Afuega’l Pitu cheese
but the numbers of leuconostoc and enterococci
decreased during ripening (Cuesta et al., 1996). In San
Simon cheese, enterococcal numbers were maximal
298 The Microbiology of Cheese Ripening
during the first week of ripening, and stayed constant
throughout the remainder of the 6-week maturation
period (Fontan et al., 2001). The numbers of leu-
conostoc, lactobacilli and enterococci varied little dur-
ing the ripening of Penamellera cheese (Estepar et al.,
1999), but leuconostocs were one of the major groups
at the drying room stage in the maturation of Cabrales
cheese (Nunez, 1978). Lb. plantarum was dominant in
the cheese’s interior during the cave-ripening stage.
Non-starter lactobacilli and leuconostocs varied little
during the ripening of La Serena and ovine milk
cheeses, coagulated with Cyanara extracts (Fernandez
del Pozo et al., 1988; Vioque et al., 2000), and the
enterococcal population declined; in contrast, the pro-
portion of enterococci in Manchego cheese increased
throughout ripening (Ordoñez et al., 1978).
Variations in the individual populations during
the ripening of caprine milk cheeses were cheese-
specific. The development of the LAB population
of Valdeón, a hand-made blue cheese, exhibited an
initial dominance of lactococci and enterococci.
However, from the drying stage, lactobacilli and leu-
conostoc replaced the lactococci, and the mature
cheese was dominated by enterococci (López-Díaz
et al., 2000). The population of 1-week old Armada
cheese was dominated by lactococci whereas Lb. casei
and Lb. plantarum were the most abundant during
the latter stages of ripening (Tornadijo et al., 1995).
The numbers of enterococci, leuconostocs and lacto-
bacilli declined during ripening in summer-made
cheese, but the latter two microbial groups remained
at ⬃108 cfu g 1 throughout the 16-week period of
ripening in autumn-made cheese. Leuconostoc and
enterococcal numbers remained at ⬃107 cfu g 1 dur-
ing the 60-day ripening period of Tenerife cheese
whereas the numbers of lactobacilli increased from
105 cfu g 1 after 2 days to 107 cfu g 1 at the end
of ripening. Lb. plantarum was dominant in the
young cheese whereas in the mature cheese Lb. para-
casei was predominant among the lactobacilli (Zárate
et al., 1997).
Portuguese cheese varieties
The microbiology of Appélation d’Origine Protegée
(AOP) Portuguese cheeses was reviewed by Freitas
and Malcata (2000). The more dominant LAB in
9-day-old Picante da Beira Baixa cheese were Leuc.
mesenteroides (19%), Lb. plantarum (15%), Lb. paraca-
sei (15%) and Ec. faecalis (8%). Leuconostoc spp. were
not detectable after 40 days, with Lb. plantarum and
Lb. paracasei persisting throughout ripening (Freitas
et al., 1996). Lb. brevis and Lb. fermentum were also
detected in the mature cheese. The geographical loca-
tion and season of manufacture influence the micro-
bial population of Serra da Estrela (Tavaria and Malcata,
2000). The numbers of LAB are maximal after a ripen-
ing period of 7 days, and, in this population, Leuc. lac-
tis and enterococci are the most abundant (Macedo
et al., 1995; Dahl et al., 2000). The proportion of Leuc.
mesenteroides and Lb. plantarum in the population
tended to increase throughout ripening, whilst that
of Ec. faecium, Ec. faecalis and Lb. pentosus declined
(Tavaria and Malcata, 1998).
Italian cheese varieties
Ricotta forte is produced by ripening cottage ricotta
cheese for 12 months, during which time the curds are
mixed regularly to prevent mould growth. At the end
of ripening, the dominant lactobacilli are Lb. paracasei,
Lb. acetotolerans, Lb. alimentarius and Lb. brevis, and of
these only Lb. paracasei was detected early in ripening.
Lb. kefiri, Lb. gasseri, Lb. hilgardii, Lb. plantarum, Lb.
paraplantarum and Lb. zeae were detected transiently
(Baruzzi et al., 2000). Major shifts in species profile
also occurred during the ripening of the pasta-filata
cheese, Caciocavallo Pugliese. The two dominant
species at the end of the 60-day ripening period were
Lb. parabuchneri and Lb. paracasei, while Lb. fermen-
tum was the dominant species in the young cheese
(Gobbetti et al., 2002). The proportion of Ec. faecalis
and Ec. durans in the population decreased from ~6%
to 0.1% during ripening whilst that of Pd. pentosaceus
increased. The Lactobacillus community involved in
traditional Mozzarella production has been investi-
gated (Morea et al., 1998). Lb. fermentum, which was
dominant in the natural whey starter, was not detected
during manufacture or ripening. Following heat treat-
ment associated with the stretching process the com-
plexity of the population decreased from 11 to 5
strains of Lb. casei, Lb. fermentum, Lb. plantarum and
Weissella hellenica. Only Lb. plantarum was detected
during ripening.
The most numerous non-starter lactobacilli in
Scamorza Altamurana cheese ripened for 6 days were
Lb. fermentum and Lb. paracasei (Baruzzi et al., 2002).
Lb. paracasei was not detected in the whey or curd
whereas Lb. fermentum was present at all stages in the
manufacturing process. Lb. gasseri and W. viridescens
were also detected in the whey but not in the ripening
curd whereas Ec. durans was only detected in the
mature cheese. Dramatic changes in the diversity of
the microbial communities of the Sicilian artisanal pasta-
filata-type cheese, Ragusano, during the manufacturing
process were revealed by classical and culture-
independent PCR and density gradient gel electro-
phoresis techniques (Randazzo et al., 2002). Mesophilic
LAB, including Leuconostoc spp. and Lc. lactis, dominated
the raw milk population but disappeared during cooking

and fermentation of the curd. However, Lb. delbrueckii
and Lb. fermentum grew during ripening, and entero-
cocci were also present in reasonable numbers as the
microbial population stabilised in 15- and 30-day-old
cheeses.
Marked shifts in the species profiles have also been
observed in cheeses ripened for longer periods of time.
Non-starter lactobacilli and enterococci increased dur-
ing the 60-day ripening period of Pecorino Sardo
ovine milk cheese, though significant differences
occurred between batches (Mannu et al., 2002).
Lb. casei numbers were constant throughout the ripen-
ing of one batch of cheese, appeared after 60 days in a
second batch whereas, in a third batch, although they
were present in high numbers during ripening, they
only became dominant when the starter LAB popula-
tion declined. The numbers of non-starter lactobacilli
in traditional farm-house Fiore Sardo cheese increased
from 105 cfu g 1 in 1-day-old cheese to 108 cfu g 1
after ripening for 30 days; the population then
decreased slowly, and by 7 months the level was
reduced to 104 cfu g 1 (Mannu et al., 2000). Lb. plan-
tarum decreased dramatically during maturation
whereas Lb. paracasei, when present, dominated the
cheese microflora. Lb. paracasei was also dominant in
Montasio cheese, and although it was not detectable
immediately after manufacture its population
increased to 107 cfu g 1 during the first month of
ripening and remained at that level up to 120 days
(Lombardi et al., 1995).
Enterococcal numbers decreased by one to two
orders of magnitude during the 9-week ripening of
Canestrato Pugliese cheese (Albenzio et al., 2001),
whilst the non-starter Lactobacillus population
increased to 28 days and remained at that level for
the remainder of maturation (Fig. 2A,B). The popu-
lation in cheese made from raw milk (108 cfu g 1)
was 3 log cycles higher than that in cheeses made
from thermised or pasteurised milk. A similar popu-
lation developed during the 60-day maturation
period of Fossa (pit) cheese; the numbers of non-
starter lactobacilli then declined by two orders of
magnitude during the 3-month aging process (Avellini
et al., 1999).
Prolonged ripening times are a feature of Parmi-
giano Reggiano cheese. During ripening the number of
non-starter lactobacilli decreased from 108 cfu g 1
after 5 months to approximately 104 cfu g 1 at 24
months (Coppola et al., 1997). Lb. paracasei/Lb. casei
and Lb. rhamnosus, which persisted throughout the
24-month maturation, dominated the population, and
Pd. acidilactici was present for 22 months. Enterococci
disappeared during ripening of Parmesan for 14
months (Thompson and Marth, 1986).
The Microbiology of Cheese Ripening 299
Greek and eastern European cheeses
Kefalotyri is a hard, salted cheese, which traditionally
is produced without starters. Lactobacilli and entero-
cocci are present throughout the 120-day ripening
period. Lactobacilli predominate for 30 days when
enterococci became increasingly dominant (Litopoulou-
Tzanetaki, 1990). The proportion of Lb. plantarum,
Ec. faecium, Ec. durans and Pediococcus spp. recovered
increased during ripening whereas the proportion of Lb.
casei remained at 16–20% throughout the period. Lb.
brevis, Lb. buchneri and Leuconostoc spp. decreased dur-
ing ripening and were absent in 120-day-old cheese.
Ripening-related changes in the LAB population of two
other Greek ovine milk soft cheeses, Feta and Teleme,
have also been studied (Tzanetakis and Litopoulou-
Tzanetaki, 1992). Lb. plantarum was the dominant
isolate recovered from both cheeses. Phenotypic char-
acterisation of isolates indicated that different strains
dominate at different stages of ripening in Feta cheese
(Xanthopoulos et al., 2000). The proportion of entero-
cocci and pediococci in Feta after ripening for 90 days
was lower than that in the original curd. After 180
days of ripening, the proportion of enterococci in
Teleme cheese had increased from the levels in the
curd whereas that of the Leuconostoc spp. present was
similar at both stages after a transient increase
between 30 and 60 days.
In Turkish white cheeses, species of Enterococcus, Lac-
tobacillus, Leuconostoc and Pediococcus dominate during
ripening (Bintsis and Papademas, 2002; Hayaloglu et al.,
2002). Enterococci numbers remained constant during
the ripening of Turkish Kashar cheese, with Ec. faecium
and Ec. durans being the most frequently isolated, while
Lb. casei, Lb. plantarum and Lb. rhamnosus were the
dominant non-starter lactobacilli in the mature cheese
(Aran, 1998).
Significance of NSLAB in cheese manufacture
Influence of non-starter lactobacilli on cheese
quality
Non-starter lactobacilli can impact on cheese quality
in both beneficial and detrimental ways; however, an
increasing number of studies have shown that selected
adjunct strains of Lactobacillus spp. positively influ-
ence cheese quality (Fox et al., 1998; Table 2). Ched-
dar cheese produced under controlled bacteriological
conditions in aseptic vats can develop full mature
flavour in the absence of non-starter lactobacilli,
although non-starter lactobacilli are believed to add
desirable flavour notes and reduce harshness and bit-
terness associated with some starter cultures
(McSweeney et al., 1994; Shakeel-Ur-Rehman et al.,
2000). The presence of non-starter lactobacilli in com-
mercial cheese is associated with the development of
300

Table 2 Examples of the observed effects of adjunct non-starter lactobacilli on cheese characteristics

Cheese variety Adjunct cultures Flavour/texture effects Reference

Arzua-Ulloa Lb. casei, Lb. casei pseudoplantarum Improved acidification during ripening; s1-casein degradation Menendez et al. (2000)
(syn Lb. paracasei), Lb. plantarum increased; -casein degradation decreased; soluble nitrogen,
volatile fatty acid, diacetyl and acetoin levels increased;
less bitter/astringent, more acid flavour; texture improved
Cheddar Lb. casei Increased peptide degradation; enhanced flavour Broome et al. (1990b)
development and intensity
Cheddar Lb. brevis, Fruity flavours, open texture, late-gassing defects Laleye et al. (1990)
Lb. fermentum, Lb. casei, Lb. casei pseudo- Prevented late-gassing
plantarum (syn Lb. paracasei)
Cheddar Lb. casei Increased flavour intensity Tré
panier et al. (1991a,b)
Cheddar Lb. casei, Lb. casei pseudoplantarum (syn Higher free amino acid levels; increased flavour McSweeney et al. (1994)
Lb. paracasei), Lb. curvatus, Lb. plantarum intensity and acceptability
Cheddar Lb. casei, Lb. casei pseudoplantarum Higher free amino acid levels; different free Lynch et al. (1996)
(syn Lb. paracasei), Lb. curvatus, amino acid profiles; increased flavour
Lb. plantarum acceptability and cheese quality
Cheddar Lb. casei Sensory characteristics, flavour and texture influenced Muir et al. (1996)
Cheddar Lb. paracasei, Lb. plantarum Increased free amino acid levels; significant differences Lynch et al. (1999)
in some important sensory attributes; apparent
acceleration of ripening as assessed by
effects on aroma/flavour intensity, perceived
maturity and creamy/milky flavour notes
Cheddar Lb. paracasei, Lb. rhamnosus Improved flavour quality and increased maturation rates Crow et al. (2001)
Edam (reduced fat) Lb. reuteri Proteolysis increased; free amino acid levels higher; Tungjaroenchai et al. (2001)
texture quality improved; no flavour benefits detected
Emmental Lb. casei Concentrations of ethanol, propan-1-ol, 2-methylbutanol, Rychlik et al. (1997)
3-methylbutanol, 2,3-pentandione, 2-methylbutanal
and 3-methylbutanal affected. No significant
impact on flavour
Herrgå
rd-type Lb. casei pseudoplantarum Overall quality and texture improved; flavour quality and Antonsson et al. (2002)
(syn Lb. paracasei), Lb. plantarum intensity improved; off-flavour intensity reduced
Mozzarella Lb. casei Cheese stretch reduced at 1–7 days of ripening; melt not Merrill et al. (1996)
(reduced fat) affected; cook colour increased
Norvegia Lb. paracasei pH, free amino acid, -aminobutyric acid and cysteine Skeie et al. (2001)
concentrations reduced; lactate level increased;
more rapid decline in starter numbers
Spanish bovine Lb. plantarum Lower pH, reduced casein degradation and soluble nitrogen Gomez et al. (1996)
milk semi-hard during early stages. Reduced bitterness. Higher elastic
modulus, breaking force and hardness values.
Negative impact on flavour
St Paulin type Lb. plantarum Free amino acid levels increased and peptide profiles affected Hynes et al. (2001)

more intense Cheddar flavour in a shorter time (Reiter
et al., 1967). Obligate heterofermentative lactobacilli
that occur in the latter stages of ripening have been
associated with the occurrence of undesirable flavours
and textures in Cheddar (Dacre, 1953; Laleye et al.,
1987; Khalid and Marth, 1990) and flavours in Gouda
(Kleter, 1977). The racemisation of L-lactate to D-lactate
by non-starter lactobacilli can result in the occurrence
of a surface white spot defect as a consequence of cal-
cium D-lactate crystal deposition in the mature cheese
(Thomas and Crow, 1983b). However, Sherwood
(1939) observed that Lb. casei and Lb. plantarum could
improve Cheddar flavour, and although the inclusion
of non-starter lactobacilli accelerated flavour develop-
ment, Law et al. (1976) were unable to attribute the
improved flavour obtained with a curd-derived whole
reference flora to any specific non-starter group. Later
investigations by Puchades et al. (1989), Broome et al.
(1990b) and Lee et al. (1990a,b) established that
cheeses containing adjuncts of Lb. casei and Lb. plan-
tarum developed higher levels of free amino acids and
received higher flavour intensity scores than control
cheeses; Lb. brevis-containing cheese had an inferior
flavour.
Typically, the inclusion of adjunct strains of non-
starter lactobacilli results in improved flavour inten-
sity, increased aroma and accelerated ripening.
Although primary proteolysis was not affected by the
adjunct cultures, the levels of small peptides and free
amino acids were higher than in the control cheese
(Table 2). The same volatiles tend to be present in
both control and adjunct-containing cheeses but their
relative concentrations differ significantly (Dasen et al.,
1999). An alternative strategy to accelerate cheese ripen-
ing is the use of attenuated cultures (El Soda et al.,
2000). Cheddar cheese made with attenuated adjunct
strains of Lb. casei had improved sensory and textural
characteristics (Trépanier et al., 1992; Madkor et al.,
2000).
The presence of adventitious NSLAB introduces
variability into the ripening process that cannot be
easily controlled by the cheesemaker. The species and
strain composition of the non-starter Lactobacillus
population exhibits not only inter-factory differences
(Williams and Banks, 1997; Fitzsimons et al., 1999;
Antonsson et al., 2001; Berthier et al., 2001; Crow
et al., 2001; De Angelis et al., 2001), but also differ-
ences in cheeses produced at the same factory on dif-
ferent days and in cheeses from different vats on the
same day (Naylor and Sharpe, 1958b; Fitzsimons
et al., 2001; Williams et al., 2002a). The relationship
of these population differences to between-batch
variations in the quality of the cheese has not been
established.
The Microbiology of Cheese Ripening 301
Use of other NSLAB as adjunct cultures
Pediococci enhanced the flavour of Feta (Vafopoulou-
Mastrojiannaki et al., 1990) and low-fat Cheddar
cheeses (Bhowmik et al., 1990). In contrast, Law et al.
(1976) observed that pediococci alone had no impact
on flavour development in Cheddar cheese, but were
effective in combination with other starter bacteria.
There are contradictory reports on the role of
enterococci. Although unsuitable as starters due to
their low milk-acidifying ability and poor extracellular
proteolytic activity, many strains have beneficial meta-
bolic traits (Sarantinopoulos et al., 2001; Delgado et al.,
2002). Enterococci impart desirable flavours to a
number of cheeses, including Cheddar (Jensen et al.,
1975; Gardiner et al., 1999b), Manchego (Ordoñez
et al., 1978) and Cebreiro (Centeno et al., 1999). In
contrast, high numbers of enterococci resulted in the
deterioration of the sensory properties of Parmesan
and a Spanish blue cheese (Thompson and Marth,
1986; López-Díaz et al., 1995). Recent results show
that three strains of Ec. faecalis, two of Ec. faecium,
one of Ec. casseliflavus and one of Ec. durans had no
effect on the flavour of Cheddar cheese (Rea and
Cogan, unpublished). The perceived beneficial role
of enterococci on flavour has resulted in their inclu-
sion in defined-strain starter cultures for Mozzarella
(Coppola et al., 1988; Parente et al., 1989), Feta
(Litopoulou-Tzanetaki et al., 1993), Venaco (Casalta
and Zennaro, 1997) and Cebreiro (Centeno et al.,
1996b) cheeses. The use of enterococci as adjuncts
will also depend on the resolution of outstanding
safety issues.
Adjunct NSLAB as probiotics
Lactic acid bacteria have a long history of safe use in
foods and there is now considerable interest in their
probiotic potential (Mattila-Sandholm et al., 1999).
Cheese is effective as a functional food and as a con-
venient vehicle for the introduction of probiotic cul-
tures into the diet because, in comparison with yoghurt
and other fermented milk products, cheese has a solid
matrix, and a higher pH, buffering capacity and fat
content, which help protect the probiotic strain during
intestinal transit to the site of action (Ross et al., 2002).
Cheddar cheese is an effective carrier for probiotic Lb.
paracasei (Gardiner et al., 1998), Ec. faecium (Gardiner
et al., 1999a,b) and two bifidobacteria (McBrearty
et al., 2001). The probiotic Lb. paracasei adjuncts had
no adverse effects on the flavour and sensory character-
istics of the cheese (Gardiner et al., 1998), whereas
after ripening for 6 months, Cheddar containing the
Ec. faecium adjunct exhibited improved flavour over
the control (Gardiner et al., 1999b). Bulgarian yellow
cheese and Argentinian Fresco cheese have also been
302 The Microbiology of Cheese Ripening
used for the delivery of probiotic strains (Vinderola
et al., 2000; Atanassova et al., 2001).
Biochemical activities of NSLAB that are important
in cheese ripening
The range and extent of the activities of the principal
NSLAB, which establish during ripening, determine
their overall impact on cheese quality. The use of
NSLAB to manipulate or accelerate cheese flavour
development requires effective pre-screening to iden-
tify isolates that have suitable metabolic capabilities.
Historically, the choice of adjunct strains for evalu-
ation in cheesemaking trials has been random and not
based on biochemical criteria, and consequently the
effects of adjuncts were often inconclusive. The use of
strains with defined metabolic attributes as adjuncts
offers the potential to specifically manipulate cheese
flavour development (Williams et al., 2000; Tanous
et al., 2002).
Citrate utilisation
Although citrate is present at low levels in milk, it is
the precursor of diacetyl and acetate, important
flavour components of some cheese varieties; the CO2
produced is responsible for eye formation in Dutch
cheeses and can affect the texture of other varieties.
Other products of citrate metabolism, acetoin and
2,3-butanediol, do not impart flavour. It is probable
that citrate is catabolised by the adventitious lacto-
bacilli, although it is not used as an energy source
(Palles et al., 1998; Williams et al., 2000). Leuconostoc
spp. also have the ability to co-metabolise sugar(s)
and citrate but the excess pyruvate produced is
reduced to D-lactate (Hugenholtz, 1993). Enterococci
also metabolise citrate and can form acetalydehyde,
acetoin and diacetyl, metabolic capabilities that have
resulted in the inclusion of enterococci in starter cul-
tures for Cebreiro (Centeno et al., 1996b), Feta
(Litopoulou-Tzanetaki et al., 1993) and Mozzarella
cheeses (Coppola et al., 1988; Parente et al., 1989).
The production of diacetyl from glucose by pediococci
has also been reported (Ray, 1995).
Proteolysis
Lactic acid bacteria possess a complex, well-characterised,
proteolytic system, which enables them to meet their
amino acid requirements from the hydrolysis of milk
proteins (Christensen et al., 1999). The peptides and
the amino acids released also contribute to, and act as
precursors for, flavour development in cheese. In Ched-
dar cheese, primary proteolysis is effected by the added
chymosin and endogenous milk enzymes, whilst small
peptides and free amino acids are released from the pri-
mary products by the action of the LAB proteolytic
enzymes (Fox and McSweeney, 1996). Starter bacteria
make a greater contribution to protein breakdown than
NSLAB (Lane and Fox, 1996; Lynch et al., 1996, 1997),
whose principal contribution appears to be peptidolysis
and the release of free amino acids (Williams and Banks,
1997; Muehlenkamp-Ulate and Warthesen, 1999).
Peptides are hydrolysed intracellularly by a variety of
endopeptidases, aminopeptidases, dipeptidases and tripep-
tidases, some of which are proline-specific and some
of which have overlapping specificities (Christensen
et al., 1999). Wide-ranging peptidolytic activities have
been detected in non-starter lactobacilli (Khalid and
Marth, 1990; Williams and Banks, 1997; Williams et al.,
1998), pediococci (Bhowmik and Marth, 1990b;
Vafopoulou-Mastrojiannaki et al., 1994) and Leuconostoc
spp. (El Shafei et al., 1990), although activities in entero-
cocci, isolated from cheese, are generally low (Arizcun
et al., 1997b; Hemati et al., 1998; Sarantinopoulos et al.,
2001).
Amino acid catabolism
Although peptides and amino acids contribute to
cheese flavour, attempts to increase their formation by
over-expression of enzyme activity (Christensen et al.,
1995) or the addition of free amino acids to the curd
at the manufacturing stage (Wallace and Fox, 1997)
have not been successful in enhancing flavour. The
implication, therefore, is that the transformation of
amino acids rather than their release is the rate-limiting
step in flavour formation. The catabolism of amino
acids can result in the formation of many compounds
that contribute to cheese flavour (McSweeney and
Sousa, 2000). Degradative mechanisms potentially
include deamination, decarboxylation, desulphur-
ation, oxidation and reduction reactions resulting in
the formation of amines, aldehydes, alcohols, indoles,
carboxylic acids and sulphur-containing moieties
(Yvon and Rijnen, 2001; see ‘Catabolism of Amino
Acids in Cheese during Ripening’, Volume 1). The
range of amino acid converting enzymes in cheese lac-
tobacilli is restricted (Yvon and Rijnen, 2001).
Amino acid breakdown by LAB is initiated by
an -ketoglutarate-dependent transaminase. The result-
ant -keto acids are subjected to further enzymatic or
chemical reactions to hydroxyacids, aldehydes, alco-
hols and carboxylic acids (Yvon and Rijnen, 2001).
Cell-free extracts (Groot and De Bont, 1998; Klein
et al., 2001) and non-proliferating suspensions of lacto-
bacilli (Kieronczyk et al., 2001), leuconostoc and ente-
rococci (Tavaria et al., 2002) generate important cheese
flavour compounds from amino acids. Branched-chain
and aromatic amino acid aminotransferase activities have
been detected in non-starter lactobacilli (Gummalla and
Broadbent, 1999, 2001; Curtin et al., 2001; Hansen

et al., 2001; Williams et al., 2001, 2002b). The degrad-
ation of sulphur-containing amino acids proceeds via
an aminotransferase (Dias and Weimer, 1998; Amarita
et al., 2001) or cystathionine lyase-mediated path-
way (Smacchi and Gobbetti, 1998). The addition of
-ketoglutaric acid to cheese curd enhances the conver-
sion of amino acids into aroma compounds (Yvon et al.,
1998; Banks et al., 2001).
Lipolysis
Screening studies using natural substrates, trigly-
cerides and synthetic chromogenic substrates have
confirmed the presence of lipase and esterase activities
in non-starter lactobacilli (Khalid and Marth, 1990).
In general, the enzymes are intracellular and activities
are strain-specific. In the majority of strains, activities
increased as the carbon chain length of the fatty acid
decreased. A 65-kDa intracellular lipase from Lb. plan-
tarum has been purified (Gobbetti et al., 1996). Intracel-
lular esterases have also been purified and characterised
from Lb. plantarum (Andersen et al., 1995; Gobbetti
et al., 1997a), Lb. casei (Castillo et al., 1999) and Lb.
fermentum (Gobbetti et al., 1997b). They are all serine-
dependent enzymes with an estimated molecular mass
ranging from 70 to 105 kDa (subunit mass 25–40
kDa). The response of esterase activity to the effects of
salt, temperature and pH is strain-dependent (Gobbetti
et al., 1999a) but retention of this activity during
ripening is important for flavour formation both from
lipolysis and ester formation.
The beneficial effect of enterococci in cheesemaking
has been attributed to the hydrolysis of milk fat by
esterases (Tsakalidou et al., 1993). The released fatty
acids can be further converted into methyl ketones and
thioesters which have been implicated as cheese flavour
compounds. A survey confirmed that food isolates were
lipolytic and hydrolysed all triglycerides from tributyrin
to tristearin with decreasing efficiency as the carbon
chain length of the fatty acid increased (Sarantinopoulos
et al., 2001). Multiple esterase activities were present
10
Population size (log cfu g–1 )
9
8 Room, 22 °C
6
5
4
0 5
Figure 3 Changes in the population of PAB during ripening of Swiss-type cheese (redrawn from Turner et al. (1983)).
The Microbiology of Cheese Ripening 303
and whereas Ec. faecalis strains were the most lipoly-
tic, Ec. faecium strains were more esterolytic. Pedio-
cocci and some Leuconostoc spp. are also actively
esterolytic (Bhowmik and Marth, 1989; Vafopoulou-
Mastrojiannaki et al., 1994; Katz et al., 2002).
Propionic Acid Bacteria
Propionic acid bacteria are usually found in Swiss-type
cheeses where they grow during ripening and contribute
to the characteristic flavour and appearance of these
cheeses. Their primary contribution is their ability to
metabolise lactic acid present in the cheese curd:
3 Lactate : 2 Propionate  Acetate  CO2  H2O
The CO2 produced is responsible for formation of
large eyes that are a feature of these cheeses and the
acetic and propionic acids contribute to flavour
development. Propionic acid bacteria in the cheese
milk survive the relatively high cooking temperature,
⬃54 °C, used in the manufacture of these cheeses
and their growth is stimulated by increasing the
ripening temperature to 18–22 °C (Fig. 3). Propionic
acid bacteria will typically attain levels of 108–109
cfu g 1 cheese after a few weeks, at which time the
cheese is cooled to limit further growth (Steffen
et al., 1993).
Studies on autolysis of PAB are limited and while
spontaneous autolysis of P. freudenreichii occurs in syn-
thetic media (Lemée et al., 1995), no evidence of its
autolysis was detected during cheese ripening (Valence
et al., 1998). Propionic acid bacteria have been impli-
cated in late blowing of Grana cheese. Scanning elec-
tron microscopy demonstrated the presence of
damaged cells of P. freudenreichii, suggesting that
autolysis did occur in Grana cheese (Cappa et al.,
1997). Bacteriophage infection of P. freudenreichii
Transfer to Hot
10 15 20 25 30 35 40
Ripening period (days)
304 The Microbiology of Cheese Ripening
occurs in Swiss-type cheese and may contribute to
PAB lysis during cheese ripening (Gautier et al., 1995).
Interactions between PAB and other bacteria are
important during cheese ripening. Propionic acid bac-
teria do not grow well in milk-based media; however,
proteolysis of casein by rennet and starter bacteria
stimulates growth (Baer, 1995). Further, Piveteau et al.
(2000) demonstrated that growth of PAB in milk or
whey did not occur unless the initial cell density was
106 cfu ml 1. Growth inhibition appeared to be due
to a heat-stable inhibitor(s) present in the whey.
Pre-growth of some LAB, used as starter cultures in
Swiss-type cheese manufacture, in milk removed the
inhibition. Antagonistic interactions between PAB and
various LAB were reported by Alekseeva et al. (1983).
Nine of twenty-two strains of LAB tested were antagon-
istic for PAB; Lc. lactis subsp. lactis had the greatest
inhibitory effect, while Lc. lactis subsp. cremoris, Sc.
thermophilus and Lb. helveticus were compatible with
P. freudenreichii and P. shermanii. Inhibition of PAB by
Lb. rhamnosus and Lb. casei has also been reported by
Jimeno et al. (1995). Interactions between 14 LAB,
including strains of Lb. helveticus, Lb. acidophilus, Lb.
lactis, Sc. thermophilus and Lc. lactis and P. freuden-
reichii or P. acidipropionici in whey were investigated by
Piveteau et al. (1995). No inhibition was observed, and
Lb. helveticus and Sc. thermophilus stimulated the
growth of the PAB.
Micrococcus and Staphylococcus
Micrococci and staphylococci have traditionally been
placed in the family Micrococcaceae; however, phylogen-
etically they are not closely related. Micrococci have a
high GC content and are related to the actinomycetes
whereas staphylococci have a low GC content and are
found in the clostridal branch of the eubacteria. Most
micrococci and staphylococci grow in 5% NaCl and
are considered by some authors to contribute to the
ripening process. Many of the media used for the isol-
ation of micrococci and staphylococci from cheese are
not very selective and do not distinguish between
them. Confirmation of genus requires further bio-
chemical or molecular analysis. As many of the
reported studies do not include such characterisation,
care must be taken when drawing conclusions regard-
ing the type of bacteria isolated (Fig. 4).
Micrococci
Micrococci are obligate aerobes with optimum growth
temperatures of 25–37 °C. The genus Micrococcus has
been recently divided into Micrococcus, Kocuria,
Nesterenkonia, Kytococcus and Dermacoccus based on a
phylogenetic and chemotaxonomic study (Stackebrandt
et al., 1995). They have been isolated from a variety
of cheeses, including Cheddar, Iberian and white-
brined cheeses (Bhowmik and Marth, 1990a; Freitas
and Malcata, 2000; Bintsis and Papademas, 2002).
However, the ripening temperature and the absence of
oxygen internally in most cheese varieties inhibit their
growth and thus they are present at lower numbers
than the other microbial groups.
Micrococci possess a range of hydrolytic enzymes
that could contribute to cheese ripening (Bhowmik and
Marth, 1990a). The population in Tenerife cheese dur-
ing ripening ranged from 106 to 108 cfu g 1 and it was
proposed that their lipolytic activity could have con-
tributed to flavour development (Zárate et al., 1997).
Micrococci are also believed to contribute positively to
the maturation of surface-ripened Taleggio cheese
(Gobbetti et al., 1997c). However, attempts to improve
the flavour of low-fat Cheddar with a Micrococcus sp.
adjunct were not successful (Bhowmik et al., 1990).
The adjunct-containing cheese contained higher levels
of acetate and, although the sulphur volatiles were not
affected, an intense off-flavour developed.
Staphylococci
Staphylococci are facultative anaerobes, but growth is
more rapid and abundant under aerobic conditions. Most
strains grow in the presence of 15% NaCl and between 18
and 40 °C. They have been isolated from a number of
cheese varieties and form a significant portion of the sur-
face flora of some cheeses (Cuesta et al., 1996; Aran,
1998; Avellini et al., 1999; Albenzio et al., 2001; Corsetti
et al., 2001a). Batch, geographical location, year and sea-
son of manufacture affect their numbers in Serra da
Estrela and Caciocavallo Silano cheeses (Tavaria and
Malcata, 2000; Corsetti et al., 2001b). In Serra da Estrela
cheese the major staphylococci are Staph. xylosus, Staph.
aureus and Staph. epidermidis, with Staph. xylosus predom-
inating at the end of ripening (Macedo et al., 1995).
Lower numbers of Staph. simulans and Staph. hominis
were present. Similar species have been identified in other
ovine and caprine milk cheeses (Fernandez del Pozo
et al., 1988; Freitas and Malcata, 2000). Staph. aureus is a
recognised food-borne pathogen and, although present
during the initial ripening stages of Serra da Estrela
cheese, it showed a tendency to disappear during matura-
tion (Fernandez del Pozo et al., 1988; Macedo et al.,
1995). The contribution of staphylococci to flavour devel-
opment in cheese has not been clearly defined.
Moulds
Moulds contribute to ripening of many cheeses, particu-
larly surface mould-ripened cheeses like Camembert
and Brie, which depend on growth of P. camemberti
 The Microbiology of Cheese Ripening 305

8
7
6
5
4
3
2
1
A
0
0 10 20 30 40 50 60 70

10
9
Population size (log cfu g–1 )

8
7
6
5
4
3
2
1 B
0
0 20 40 60 80 100 120 140

9
8
7
6
5
4
3
2
1
C
0
0 20 40 60 80 100 120 140 160 180 200

Ripening period (days)

Figure 4 Changes in the population of micrococci and/or staphylococci during ripening of: (A) Tenerife ( ), Afuega’l Pitu ( ), La
Serena (), Penamellera (surface) (), Penamellera (interior) () and Fossa Pit (); (B) Kashar (; Aran, 1998)), Cabrales (sur-
face) (), Cabrales (interior) (), Armada ( ), Taleggio (surface) ( ; Gobbetti et al., 1997c) and Taleggio (interior) (; Gobbetti
et al., 1997c) and (C) Swiss type (a) (), Swiss type (b) ( ), Picante de Beira Baixa (; Freitas et al., 1996), Serra da Estrela ()
and Canestrato Pugliese ( ). Where not indicated data were collected from sources as outlined for Fig. 2.

on the cheese surface, and blue-veined cheeses, like
Roquefort, Gorgonzola, Stilton and Danish Blue which
depend on the growth of P. roqueforti within the cheese
matrix.
In Camembert and Brie, P. camemberti develops on
the cheese surface 6–7 days post-manufacture. Once
fully grown, the surface is covered with a white ‘mat’ of
mould hyphae. P. camemberti metabolises lactate to CO2
and H2O and contributes to proteolysis, resulting in
production of NH3. This results in deacidification of the
cheese surface within 3 weeks and the establishment of
a pH gradient from the surface (basic) to the interior
306 The Microbiology of Cheese Ripening
(acidic). The increase in pH and breakdown of
s1-casein by rennet are responsible for the softening of
the curd which gradually extends towards the centre,
and is visible in a cross-section of the cheese
(see ‘Metabolism of Residual Lactose and of Lactate and
Citrate’, Volume 1; ‘Surface Mould-ripened Cheeses’,
Volume 2).
During the production of most blue-veined cheeses,
a water suspension of P. roqueforti spores is added to
the milk prior to setting, or spores are dusted onto the
curd. Following whey drainage and salting, the cheese
is pierced, which facilitates the diffusion of oxygen
into the interior of the cheese and growth of P. roque-
forti. Gas production by heterofermentative LAB and
yeasts, results in curd-openness, which further aids
the diffusion of oxygen (Devoyod et al., 1972). The
production of methyl ketones by P. roqueforti is
inhibitory to further mould growth, and may be a fac-
tor in preventing excessive mould development in
blue-veined cheese (Girolami and Knight, 1955; see
‘Blue Cheese’, Volume 2).
Moulds are associated with a range of other cheese
varieties; however, the moulds involved and their
impact on ripening are less well understood. A complex
fungal flora comprising Penicillium, Mucor, Cladospo-
rium, Geotrichum, Epicoccum and Sporotrichum develop
on the surface of the French cheeses, St Nectaire and
Tome de Savoie, while Penicillium, and Rhizomucor,
have been reported on the surface of the Italian cheese,
Taleggio and Geotrichum on that of Robiola (Gripon,
1993). The surface of the Norwegian cheese, Gam-
melost, is sprayed with Rhizomucor (Oterholm, 1984),
while P. roqueforti is sometimes introduced to the cheese
interior after piercing (Gripon, 1993).
Yeast
Yeasts occur naturally in many cheeses, but particu-
larly in those made from raw milk. The low pH, mois-
ture content, temperature and high salinity, favour the
growth of yeast, and numbers on the surface can reach
105–108 cfu g 1 (Fleet, 1990). Their role in deacidifi-
cation and the formation of metabolites such as
ethanol, acetaldehyde and CO2 is beneficial. However,
they can also cause spoilage. Fruity and bitter off-
flavours, gassy and open texture have been attributed
to yeast activity. There is considerable diversity in the
yeast flora although Debaromyces hansenii is the domin-
ant one on smear- and surface-ripened cheeses such
as Limburger, Tilsit, St Nectaire, Roquefort, Camem-
bert and Cabrales (Fox et al., 2000), Danish Blue (van
den Tempel and Jakobsen, 1998), white-brined
cheeses (Bintsis and Papademas, 2002) and various
Spanish and Portuguese AOP cheeses (Freitas and
Malcata, 2000). Many commercial smear preparations
include Candida utilis, Geotrichum candidum and
Kluyveromyces lactis together with D. hansenii. Other
yeasts frequently found include Candida, Geotrichum,
Kluyveromyces, Pichia, Rhodotorula, Saccharomyces,
Trichosporon, Torulospora, Yarrowia and Zygosaccharo-
myces spp. Changes in the total yeast population over
the maturation period have been monitored for several
cheese varieties (Fig. 5), and there is evidence that
the yeast population of the traditional Greek cheese,
Anevato, was affected by the season of manufacture
(Hatzikamari et al., 1999). Although there is consider-
able information on the population size and species
composition there is very little information on the
changes in species and strain profiles throughout
ripening. van den Tempel and Jakobsen (1998)
reported that D. hansenii, C. rugosa, Y. lipolytica and
Zygosaccharomyces spp. were the dominant species in
Danish Blue cheese ripened for 1 or 14 days, but after
28 days only D. hansenii and C. rugosa were found.
D. hansenii was the dominant species throughout the
ripening of Danbo, whereas Trichosporon, Rhodotorula
and Candida spp. were detected in the initial stages
(Petersen et al., 2002). Restriction fragment length
polymorphism of mitochondrial DNA confirmed that
several strains of D. hansenii were present from the
beginning of ripening, and a succession of strains
occurred during maturation. A sequential appearance
of yeasts on the surface of the ripening curd of St
Nectaire cheese over a 2-month period has been
observed (Marcellino and Benson, 1992). The surface
was initially colonised by Debaromyces and Torulopsis
spp. but within 4 days rapid growth of G. candidum
and filamentous fungi occurred and rind thickening
continued up to 2 months as the fungal hyphae pene-
trated into the curd. The involvement of yeasts in the
maturation process necessitates that further insights
into their population changes during ripening be
sought.
Yeasts are located not only on the cheese surface
but are also found within the curd. Yeast levels in the
curd of Camembert are 1 log lower compared to the
surface (Schmidt and Lenoir, 1980). Most studies on
the microflora of Cheddar cheese neglect to monitor
the presence of yeasts although a high proportion of
Australian and South African cheeses sampled con-
tained yeasts (Fleet and Mian, 1987; Lues et al., 1999;
Welthagen and Vijoen, 1999). In these studies, the
population in the majority of cheeses exceeded
105 cfu g 1 at some stage during maturation, a level at
which the population can impact on flavour develop-
ment. The yeast population declined from 105 cfu g 1
to 103 cfu g 1 over a 3-month ripening period in one
trial in cheeses manufactured in open vats, whilst in a
 The Microbiology of Cheese Ripening 307

9
8
7
6
5
4
3
2
1
A
0
0 10 20 30 40 50 60 70

9
8
Population size (log cfu g–1 )

7
6
5
4
3
2
1
B
0
0 20 40 60 80 100 120 140

9
8
7
6
5
4
3
2
1
C
0
0 20 40 60 80 100 120 140 160 180 200
Ripening period (days)

Figure 5 Changes in the population of yeast during ripening of: (A) Tenerife ( ), Afuega’l Pitu ( ), La Serena (), Penamellera
(surface) () and Penamellera (interior) (); (B) Kashar (), Cabrales (surface) (), Cabrales (interior) () and Armada ( ) and (C)
Ovine (ewes’) milk cheese (; Vioque et al., 2000), Swiss type ( ), Picante Beira () and Serra da Estrela (). Where not indicated
data were collected from sources as outlined for Figs 2 and 3.

different cheese production (Welthagen and Vijoen,
1999) the number increased transiently from 102 to
106 cfu g 1 over the first 40 days of ripening before
declining. The involvement of yeast in the ripening
process of Cheddar cheese is uncertain.
Yeasts possess proteolytic and lipolytic enzymes
(van den Tempel and Jakobsen, 2000; Klein et al.,
2002), form volatile sulphur compounds (Bonnarme
et al., 2001) and are able to develop appropriate
flavour and aroma notes in cheese curd (Martin et al.,
1999; Wyder and Puhan, 1999b).
Surface Smear Micro-organisms
Many European cheeses are characterised by a com-
plex surface ‘smear’ flora that consists of yeasts
308 The Microbiology of Cheese Ripening
and Gram-positive cocci, mainly Staphylococcus, and
irregular rod-shaped coryneform bacteria that are clas-
sified within families of the Actinobacteria including,
Arthrobacter, Brevibacterium, Corynebacterium and
Microbacterium (Bockelmann and Hoppe-Seyler, 2001;
Brennan et al., 2002). The surface smear may develop
from the deliberate inoculation, after salting, with combi-
nations of specific strains or a defined starter culture
(Bockelmann, 2002), but more traditionally the growth of
the surface microflora is initiated by ‘old-young’ smearing.
The microbiology of the smear is complex and not
fully characterised. Ripening conditions (12–16 °C;
RH 90%) and repeated smearing result in rapid smear
development. It is believed that yeasts develop initially,
oxidise the lactate to CO2 and water and release ammo-
nia by deamination of amino acids. This results in the
pH on the surface increasing to a level favourable for
bacterial growth. A number of yeast genera have been
isolated from the smear population (Eliskases-Lechner
and Ginzinger, 1995; Wyder and Puhan, 1999a;
Corsetti et al., 2001a). Studies on the evolution of yeast
indicate that the highest numbers of 108–109 cfu g 1
are reached after about 7 days of ripening (Eliskases-
Lechner and Ginzinger, 1995). The population is not
static; however, and a succession of species and strains
occurs during ripening (Petersen et al., 2002). Coryne-
form bacteria are dominant in the surface flora for most
of the ripening period and although there are reports of
the isolation and identification of species from many
different genera, there is little information on changes
that occur in the population profile during ripening
(Beresford et al., 2001). In smears developed from
defined starters, the initial high numbers of A. nico-
tianae declined in aged cheese whilst the numbers of B.
linens were generally variable and low throughout
ripening (Bockelmann, 2002). A recent study (Brennan
et al., 2002) in which the bacteria in the smear popula-
tion were identified, using a polyphasic approach, at
four different stages during ripening, found little evi-
dence for microbial succession. The absence of species
progression during ripening may reflect the manufac-
turing processes used as the cheese surface was washed
frequently with the resultant disruption of micro-
colonies and widespread distribution of the released
cells over the cheese surface.
The interactions between and within the yeast and
the bacterial populations are essential for smear develop-
ment and cheese ripening. The progression of microbial
growth on the cheese surface is a consequence of these
interactions. This development, depicted schematically
by Bockelmann (2002), results in the establishment of a
complex stable smear population and the development
of the typical colour, texture and flavour of the cheese
variety. In view of the impact that different species may
exert during ripening, a systematic assessment of the
dynamics of the bacterial smear population during
maturation is warranted (see ‘Bacterial Surface-ripened
Cheeses’, Volume 2).
Summary
A major diversity of microbial flora is associated with
cheese ripening. This flora may result from deliberate
addition or through adventitious colonisation. The
application of molecular techniques to the study of
cheese microbiology is providing a valuable insight into
the behaviour of individual strains and populations dur-
ing ripening. Further characterisation of metabolic
potential of the cheese flora is required to elucidate the
methods by which these micro-organisms influence
cheese quality. The diversity of the flora at species and
strain level provides a major biotechnological resource
that offers the potential for manipulation in the devel-
opment of new and innovative cheese products.
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 Raw Milk Cheeses
E. Beuvier and S. Buchin, Station de Recherches en Technologie et Analyses
Laitières, Institut National de La Recherche Agronomique, Poligny, France
Introduction
About 700 000 tonnes of raw milk cheeses (R cheeses)
are produced annually in Europe, particularly in France,
Italy and Switzerland, and they represent a significant
proportion of the cheese produced (approximately 10%
of the total cheese production in the European Union
and Switzerland) (Grappin and Beuvier, 1997). Due to
large-scale production and widespread areas of pro-
duction, R cheeses have a true social and economic
importance in several European countries (Cogan and
Rea, 1996; Grappin, 1997). However, contrasting situ-
ations exist in Europe concerning R cheese production;
for example, 191 000 tonnes per year (20% of the total
ripened cheese production) was produced in France in
1999 (CNIEL, 2002) and close to 5 000 tonnes (1.5% of
the total cheese production) in Spain in 2001 (Valentin-
Gamazo, personal communication). These cheeses rep-
resent many years of tradition, constitute the product of
a specific territory, evolve from a rural civilisation and
could be considered a type of handcraft. Moreover, in
Europe, some traditional R cheeses have a protected des-
ignation of origin (PDO) (Bertozzi and Panari, 1994).
Cheese manufacture is constantly evolving and
there is a tendency to consolidate numerous small
units into larger ones for most varieties of cheese. This
implies changes in milk production, with consequences
for the quality of the milk. In particular, milk collec-
tion has changed; milk is collected over a wider area,
resulting in co-mingled milks, and increased transport
and storage time before processing. This induces the
development of microbial populations which are dif-
ferent from those present in milk at the farm (Mocquot,
1986). One of the consequences of this is the need for
milk that is more and more microbiologically ‘clean’
due to the improvement in hygiene on the farm,
which, in turn, is enforced particularly by European
microbial standards (Directive 92/46/EEC) (Odet, 1999).
For example, at present, it is common to find in
France, and in particular in the provinces of Franche-
Comté and Rhône-Alpes, raw milk with a total count on
the farm of less than 5 000 cfu ml 1 (Bouton; Michel,
personal communication). Another consequence is the
modification of the cheese manufacturing practices. In
order to destroy pathogens and standardise the milk
Cheese: Chemistry, Physics and Microbiology, Third edition – Volume 1: General Aspects
ISBN: 0-1226-3652-X
Set ISBN: 0-1226-3651-1
microflora, pasteurisation of milk has become wide-
spread. The use of raw milk and ‘wild’ starters requires
constant adaptation of the technological conditions to
ensure a good-quality product. In contrast, the use of
selected starters, however unspecific, is now in general
use. Combined with the standardisation of milk and
the general use of a secondary microflora, this leads
to cheeses with a more constant and uniform quality
(Mocquot, 1986).
Faced with this change in milk production and
cheese manufacture, there is a need for knowledge of
the natural biodiversity of microorganisms, their role,
and the need and the way of preserving it. This is the
reason why, over the last decade, much experimental
work has been carried out, mainly in Europe, in order
to demonstrate the specific characteristics of R cheeses.
This has led to numerous publications from different
laboratories in which R cheeses have been compared
with pasteurised (P cheese) and/or microfiltered milk
cheeses (MF cheese), in which most of the natural
microflora is removed. In contrast to pasteurisation,
microfiltration of milk eliminates a great part of the
indigenous microflora without concomitant heat-
induced changes in enzymes, except for the cream
which is heat-treated (by pasteurisation or higher heat
treatment); microfiltration is applied to skim milk.
Moreover, if heat treatment leads to a selection of the
microorganisms according to heat sensitivity, microfil-
tration reduces the level of microorganisms more or less
according to their morphology and volume (Grappin
and Beuvier, 1997; Saboya and Maubois, 2000).
Otherwise, heat treatment also acts on natural milk
enzymes; this effect is more marked in P cheese than in
MF cheese in which only cream (including sometimes
retentate) is heat-treated. In this chapter, the knowledge
of the contribution of raw milk to the development of
the biochemical and sensory characteristics of R cheeses
is summarised. Other studies (not direct comparisons)
dealing with R and P cheeses are also considered when
necessary. Moreover, recent work is used to give some
examples of the diversity of the microflora of R cheeses.
This chapter focuses particularly on biochemical and
sensory aspects, with data on the microbiology of
cheeses. Only a little information on pathogenic aspects
Copyright © 2004 Elsevier Ltd
All rights reserved
320 Raw Milk Cheeses
of R cheeses is given because pathogens are covered in
‘Growth and Survival of Microbial Pathogens in Cheese’,
Volume 1.
Microbiological Aspects
Levels of microorganisms
Generally, there is an opposite trend in numbers
between starter lactic acid bacteria (LAB) and non-
starter lactic acid bacteria (NSLAB) during cheese matur-
ation. Starter counts are high, usually 108–109 cfu g 1,
at the beginning of ripening, and decrease regularly
by two or more log cycles during ageing, whereas
adventitious microorganisms, which are initially at a
level often 103–104 cfu g 1, grow during ripening,
generally reaching higher counts than the initial num-
bers of starter in mature cheese. McSweeney et al.
(1993) and Beuvier et al. (1997), working on Cheddar
and Swiss-type cheeses, respectively, found that the
decline in mesophilic or thermophilic starter bacteria
was slower in R cheeses than in P or MF cheeses, with a
difference between R and P and MF cheeses at the end
of ripening of one or two log cycles for the streptococci
or lactobacilli (Beuvier et al., 1997). Adventitious bac-
teria in R, P or MF cheeses generally had similar growth
curves but in R cheeses, they reached a plateau more
rapidly, because of their high number at the beginning
of ripening. Non-starter lactic acid bacteria (NSLAB),
such as mesophilic lactobacilli, grow regularly during
ripening, reaching 107–108 cfu g 1 in R cheeses and
one or two log cycles lower in MF or P cheeses at the
end of ripening (Fig. 1) (McSweeney et al., 1993; Beu-
vier et al., 1997; Buchin et al., 1998; Eliskases-Lechner
et al., 1999; Xanthopoulos et al., 2000; Rehman et al.,
2000c; Albenzio et al., 2001; Buffa et al., 2001b).
Other groups of microorganisms, such as entero-
cocci and propionibacteria, which occur naturally in
raw milk, are found at higher numbers in R Raclette
cheese (Klantschitsch et al., 2000) and Swiss-type
cheese (Beuvier et al., 1997) than in P and MF cheeses.
At the end of ripening, enterococci counts ranged
between 106 and 107 cfu g 1 in R Raclette and 106 cfu g 1
in R Swiss-type cheeses. In corresponding cheeses
made from P or MF milk, enterococcal counts were
four or five logs lower than in Raclette, and one to
three logs lower, in Swiss-type cheeses. Likewise, pro-
pionibacteria reached 107–108 cfu g 1 in R Raclette
cheese, at ripening temperatures of 17 and 20 °C, and
10 8 cfu g 1 in R Swiss-type cheese, whilst propioni-
bacteria counts did not exceed 102 cfu g 1 in P and MF
Raclette cheeses (Klantschitsch et al., 2000), and 104
cfu g 1 in P Swiss-type cheese. On average, 107 cfu g 1
was found in MF Swiss-type cheese, probably due to
14 °C 18 °C
9
8
7
6
log cfu g–1
5 Raw
Microfiltered
4
Pasteurised
3
2
1
0
1 15 29 43 57 71
Days of ripening
Figure 1 Evolution of mesophilic lactobacilli during the ripen-
ing of Swiss-type cheese made from raw, pasteurised or micro-
filtered milk (from Beuvier et al., 1997).
good growth of residual propionibacteria during ripen-
ing (Beuvier et al., 1997). Enterococcal counts were
found to be 3.5 logs higher in R semi-hard (Morbier-
type) cheese than in P cheese (Buchin et al., 1998) and
on average 1.5 logs higher in 25 R Castellano cheeses
(a hard Spanish cheese made from ewes’ milk) than in
25 P Castellano cheeses, collected at the market
(Román-Blanco et al., 1999). Coliforms, Enterobacteri-
aceae and Gram- bacteria were generally found at
higher levels during ripening of R than in P or MF
cheeses; the differences depended on the cheese variety
(McSweeney et al., 1993; Buchin et al., 1998; Xan-
thopoulos et al., 2000; Albenzio et al., 2001). There
was no difference in coliform populations between R
and P Swiss-type cheeses, as coliforms do not survive
the manufacturing process (Beuvier et al., 1997;
Eliskases-Lechner et al., 1999).
Diversity of microorganisms
Generally, in studies comparing R and P cheeses, R
cheeses are characterised by a natural highly variable,
‘rich’ microflora; this microbial diversity is not found in
P cheeses (Grappin and Beuvier, 1997). Indeed,
McSweeney et al. (1993) showed that strains of
Lactobacillus spp., which dominated the non-starter
microflora in Cheddar cheese, were much more hetero-
geneous in the R than in the P cheese. Compared to raw
milk, combined pasteurisation and microfiltration of
milk resulted in a significant alteration of the Lacto-
bacillus species and strain profile in the cheese (elimin-
ation of most of the Lb. paracasei strains and all the Lb.
plantarum strains (Dasen et al., 2003). In Canestrato
Pugliese cheese (a hard Italian cheese), Polymerase
chain reaction-Randomly Amplified Polymorphic DNA

(RAPD) revealed a greater diversity analysis in Lb. plan-
tarum strains in R cheese than in P cheese (Albenzio
et al., 2001). De Angelis et al. (2001) found that, in gen-
eral, commercial Italian cheeses produced from raw
ewes’ milk contained a larger number of more diverse
strains of NSLAB, especially mesophilic lactobacilli,
than commercial Italian cheeses produced from pas-
teurised milk. The LAB populations found in farm-
house-produced Pecorino Sardo (an Italian semi-cooked
ewes’ milk cheese), made with raw milk and without
the addition of a starter culture, were different from
those found in the industrially manufactured cheese
made from thermised or pasteurised milk inoculated
with autochthonous thermophilic whey starters. More-
over, the intra-species and intra-genus genetic diver-
sity in the LAB population is higher in the former
(58–80%) than in the latter (20–28%) (Mannu et al.,
2002). These authors concluded that the use of raw
milk in the farmhouse cheese leads to the preservation
of its wild microbiota and, consequently, the microbial
diversity of natural LAB in cheese.
New molecular techniques, such as denaturing gel
gradient electrophoresis (DGGE) and temporal tem-
perature gel electrophoresis (TTGE), based on the
analysis of nucleic acids, without cultivation of
microorganisms, were carried out to study the com-
plexity of the microbial communities in cheese. Cop-
pola et al. (2001) observed that artisanal Mozzarella
cheeses made from raw milk were distinguished from
industrial ones, made with pasteurised milk, by the
appearance of a greater number of bands in the DNA
profiles, reflecting a higher species diversity, in the for-
mer than in the latter cheese. Similar results were
obtained by Ogier et al. (2002) in Camembert cheese.
R Camembert cheese showed the most complex pro-
files; eight bands were detected in it, compared to five
for P Camembert cheese. The results obtained with
this approach showed that it could be possible to dis-
criminate traditional products from industrial ones
using culture-independent methods.
In order to prevent the loss of microbial biodiver-
sity in traditional R cheeses, several studies have been
carried out in the past few years to increase informa-
tion on the natural microflora of R cheeses. Raw milk
cheeses have a very diverse microflora at the genus
level and in species within the same genus (Estepar
et al., 1999; Hatzikamari et al., 1999; Mannu et al.,
2000; Callon et al., 2001; Prodromou et al., 2001; Garcia
et al., 2002; Fortina et al., 2003). For example, in Ave-
nato cheese, a traditional spreadable Greek cheese
made from raw goats’ or ewes’ milk, 12 different
species of LAB were isolated and identified throughout
manufacture and storage: three species of Lactococ-
cus (Lc. lactis, Lc. garviae and Lc. raffinolactis), three
Raw Milk Cheeses 321
species of Leuconostoc (Ln. mesenteroides, with the two
subspecies mesenteroides and dextranicum, Ln. para-
mesenteroides and Ln. lactis) and six species of Lactobacil-
lus (Lb. plantarum, Lb. coryneformis, Lb. paracasei, Lb.
brevis, Lb. bifermentans and Lb. viridescens) (Hatzika-
mari et al., 1999). Likewise, among cocci isolated from
Piemontese Toma cheese, an Italian semi-cooked
cheese, two species of Lactococcus, six species of Strepto-
coccus and three species of Enterococcus were identi-
fied throughout manufacture and ripening (Fortina
et al., 2003).
Raw milk cheeses also have a large diversity in terms
of strains within the same species (Berthier et al., 2001;
Andrighetto et al., 2002; Bouton et al., 2002). For
example, in two individual Comté cheeses, made in two
factories called ‘fruitières’, known to produce cheeses
with different sensory properties, 13 and 15 different
strains of Lb. paracasei and Lb. rhamnosus species were
detected throughout cheesemaking and ripening
(Berthier et al., 2001). Those strains isolated during
ripening originated, for the most part, from the raw
milk. A recent study of Marcellino et al. (2001) reported
great genetic diversity in the PCR–RAPD patterns of
Geotrichum candidum strains isolated from traditional R
cheeses, from seven regions of France; 48 different
strains among the 64 isolates tested were found. All this
microbial diversity contributes to the diversity in the
flavour of R cheeses.
Hygienic aspects
Table 1 indicates the microbiological standards set by
EEC directive 92/46 for the four pathogens, Listeria
monocytogenes, Staphylococcus aureus, Salmonella spp.,
and pathogenic E. coli, that constitute a major threat
to the safety of cheese.
Overall, dairy products and cheese, in particular,
have a remarkably good safety record (Alterkruse et al.,
1998; Neaves, 2000). According to the data available,
milk and milk products were implicated in 5% of the
total 3839 bacterial outbreaks reported in France from
1988 to 1997 and in 1–5% of the total bacterial out-
breaks in six other countries: USA, Finland, The
Netherlands, England and Wales, Germany and Poland
(De Buyser et al., 2001). In all those countries, R
cheeses were as frequently involved as P cheeses (30%
each) in outbreaks reported between 1983 and 1997
(surveillance period varied according to the country).
For the other 40%, milk treatment was unspecified (De
Buyser et al., 2001). Analysis of unpublished data on
food-borne disease outbreaks, listeriosis excluded, col-
lected by the coordinator of the French surveillance
system from 1992 to 1997, revealed 69 documented
outbreaks involving milk and milk products as the
322 Raw Milk Cheeses
Table 1 Microbiological criteria for European raw and heat-
treated milk cheeses (Directive 92/46 EEC)
Microorganism ma Mb nc
Listeria No L. monocyto- 5 0
monocytogenes genes in 25 g
Salmonella No Salmonella 5 0
in 25 g
Staphylococcus 103 cfu g 1 104 cfu g 1 5
aureus
Escherichia coli 104 cfu g 1 105 cfu g 1 5
a m: threshold value for the number of bacteria; the result is
considered satisfactory if the number of bacteria in all sample
units does not exceed ‘m ’.
b M: maximum value for the number of bacteria, the result is
considered unsatisfactory if one or more sample units con-
tains ‘M ’ or more.
c n: number of sample units comprising the sample.
d c: number of sample units where the bacterial count may be
between ‘m ’ and ‘M ’; the sample is still considered acceptable
if the bacterial count of the other sample units is ‘m ’ or less.
vehicle confirmed by the isolation of the etiological
agent. Thirty R cheeses and thirty unspecified milk
cheeses were involved. In these cheeses, the etiologic
agents were S. aureus (88%), Salmonella (8%) and
E. coli (3%) (De Buyser et al., 2001).
Finally, according to the results mentioned above
and the recent review by Donnelly (2001), it has never
been clearly demonstrated that R cheeses are less safe
than those made from pasteurised milk.
As noted by Grappin and Beuvier (1997), the
growth of milk pathogens in R cheese is highly
dependent on the variety of cheese and on the technol-
ogy involved. It is well documented that pathogens will
grow more easily in cheese with high moisture, high
pH and low salt content, than in cooked, long-ripened
cheeses.
The occurrence of pathogenic bacteria has been
reported in certain soft cheeses (high moisture and high
pH) made from raw milk (Ryser and Marth, 1987;
Centeno et al., 1994b; Eppert et al., 1995; Freitas and
Malcata, 2000; De Buyser et al., 2001; Menendez et al.,
2001). Generally, surface-ripened cheeses represent a
greater risk for the transmission of pathogens than other
cheeses (Johnson et al., 1990a). Indeed, Maher and
Murphy (2000) demonstrated that the rind of two Irish
smear-ripened, farmhouse cheeses produced from raw
milk may provide favourable conditions for the growth
of many undesirable pathogenic microorganisms such as
E. coli and S. aureus. However, the occurrence of
pathogens such as L. monocytogenes is not solely a prob-
lem for R cheeses. Rudolf and Scherer (2001) have
reported higher incidences of L. monocytogenes in red
smear-ripened cheeses made from pasteurised milk
(8.0%) than in cheeses made from raw milk (4.8%). In
another study (Loncarevic et al., 1995), however,
L. monocytogenes was found less frequently in soft and
cd
semi-soft cheeses made from heat-treated milk than in R
cheeses. In the case of cheeses made from pasteurised
milk, post-contamination is involved, as demonstrated,
for example, by Canillac and Mourey (1993). They
2
found that contamination by L. monocytogenes was prin-
cipally due to the washing with brushing machines.
2 Although S. aureus can be found in R cheeses, and,
in particular, in semi-hard cheeses, only a small per-
centage (7%) of S. aureus strains isolated from different
varieties of French R cheeses were able to produce
enterotoxin(s) at a detectable level in vitro (Meyrand
and Vernozy-Rozand, 1999; Lamprell et al., 2003). If
one considers that the conditions in vitro optimise
enterotoxin synthesis, one can reasonably conclude
that the risk of enterotoxin production in cheese is low.
Studies on cheeses made from artificially inoculated
milk have been carried out to show the behaviour
(growth, survival or inhibition) of pathogens in these
products. Bachmann and Spahr (1995) studied the
behaviour of E. coli, S. typhimurium, S. aureus and
L. monocytogenes, during the manufacture of an
Emmental-type, hard cooked cheese, made from raw
milk. They inoculated milk with pathogens at levels
between 104 and 106 cfu ml 1. One week after manu-
facture, the pathogens were not detectable in the cheese.
Panari et al. (2001) did a similar study on Parmigiano-
Reggiano, an Italian hard cooked cheese made from raw
milk. None of the inoculated pathogens were detected
24 h after manufacture. The rapid disappearance of
pathogens depends on the specific technology of these
varieties of cheese: a high cooking temperature (53–55 °C)
for a long time period (45–60 min) associated with the
fast growth of thermophilic LAB which lowers the
pH to 5.2–5.0 within a few hours and prevents
the development of the other bacteria. Yousef and
Marth (1990) also demonstrated that Parmesan cheese
was not a favourable medium for the survival of
L. monocytogenes. These results show that hard cooked
cheeses are hygienically safe. This is not necessarily valid
for the surface of these cheeses, which is considered to
be part of the packing (Bachmann and Spahr, 1995).
Neaves (2000) indicated that E. coli and Salmon-
ella die slowly during the maturation of hard
cheeses and, for these products, the use of a min-
imum maturation time forms a requirement of hazard
analysis critical control point (HACCP) plans. It also
forms the basis of US legislation that requires that all
imported cheeses made from unpasteurised milk be
matured for at least 60 days, so that Salmonella and
E. coli will be presumed to have died before the
cheese is consumed.
 Raw Milk Cheeses 323

Bachmann and Spahr (1995) demonstrated that Proteolysis

Swiss semi-hard R cheese (Tilsiter-type) inoculated
with 104 and 106 cfu ml 1 of E. coli, S. typhimurium,
S. aureus and L. monocytogenes, was except for L.
monocytogenes, free of these pathogens at the end of
ripening (90 days). These authors concluded that
potentially pathogenic bacteria, except L. monocyto-
genes, do not survive the manufacture of this cheese
produced with good manufacturing practices, because
of the synergistic effect of active antimicrobial enzyme
systems in the fresh milk, the antagonistic starter cul-
ture flora, fast acidification and antimicrobial activities
of LAB, combined with the specific brining and ripen-
ing conditions.
Biochemical Aspects
Cheese is a biochemically dynamic product and under-
goes significant changes during ripening (McSweeney
and Sousa, 2000). Proteins, carbohydrates and fat are
metabolised by both microbial activities (starter and
non-starter microorganisms), and by the action of
indigenous milk enzymes and residual coagulant. Pro-
teolysis (degradation of the casein matrix to a range of
peptides and free amino acids (FAAs)) has a direct and
indirect role in the formation of texture and flavour of
cheeses. Other biochemical reactions such as lipolysis
(liberation of free fatty acids (FFAs)), metabolism of
residual lactose, lactate and citrate, and the formation
of volatile compounds are also extremely important in
the development of flavour compounds (Grappin and
Beuvier, 1997; McSweeney and Sousa, 2000).
According to Grappin et al. (1985) and Rank et al.
(1985), proteolysis can be considered as two phenom-
ena. Primary proteolysis represents the extent of break-
down of the native casein and is estimated by
electrophoresis. Secondary proteolysis is the further
degradation that leads to the formation of peptides and
FAAs; the global amount of these compounds is meas-
ured by nitrogen fractionation and their proportions by
high performance liquid chromatography (HPLC).
Primary proteolysis
In most cheese varieties, primary hydrolysis of caseins
is caused by the residual coagulant, plasmin, and per-
haps by cathepsin D (acid protease) and other somatic
cell proteinases. Although LAB are weakly proteolytic,
they possess proteinases that also degrade casein.
Traditional secondary starters, e.g., Penicillium roque-
forti, P. camemberti and Brevibacterium linens, have
extracellular proteinases which act on s1- and
-caseins (Grappin and Beuvier, 1997; McSweeney
and Sousa, 2000). Likewise, yeasts might also exhibit
some endopeptidase activity on both s- and -caseins
(Xanthopoulos et al., 2000).
Table 2 summarises the data from different studies
on the degradation of s1- and -caseins in R, P and MF
cheeses.
The more pronounced degradation of s1-casein in R
Swiss-type, including Bergkäse, Raclette and Anevato
cheeses, may be attributed to enzymes of the raw milk
microflora, which is almost totally eliminated by pas-
teurisation. Mesophilic lactobacilli, which are part of the

Table 2 Primary proteolysis: levels of native casein in raw (R), pasteurised (P) and microfiltered (MF) milk
cheeses

Cheese s1-Casein -Casein Reference

Swiss-type R  MF  P R  P  MF Beuvier et al. (1997)

Swiss-type (Bergkäse) RP RP Ginzinger et al. (1999b)
Canestrato Pugliese RP RP Albenzio et al. (2001)
Cheddar RP RP Lau et al. (1991)
Cheddar R  P  MF R  P  MF McSweeney et al. (1993)
Cheddar RP RP Rehman et al. (2000c)
Raclette RP RP Gallmann and Puhan (1982)
St Paulin RP RP Beuvier (1990)
Morbier-type RP RP Buchin et al. (1998)
Manchego RP RP Gaya et al. (1990)
Manchego RP RP Gomez et al. (1999)
Idiazabal R  P ( s-casein) 90 da R  P 180 d Mendia et al. (2000)
Arzua (soft cheese) RP RP Centeno et al. (1994)
Avenato RP RP Xanthopoulos et al. (2000)
Travnicki (Feta type) RP RP Saric et al. (2002)

a 90d: 90 days of ripening.

324 Raw Milk Cheeses
indigenous milk microflora in Swiss-type, and other
cheeses (Demarigny et al., 1996; Eliskases-Lechner et al.,
1999), and yeasts in Avenato cheese, are able to hydro-
lyse s1-casein significantly (Khalid and Marth, 1990;
Xanthopoulos et al., 2000). The possible inactivation of
cathepsin D by pasteurisation may also be involved,
explaining the similar degradation of s1-casein in R and
MF Swiss-type cheeses, which was higher than in the
corresponding P cheese (Beuvier et al., 1997). On the
other hand, the lower level of s1-casein found in P
Manchego, St Paulin, Arzua and Travnicki cheeses can
be explained by greater retention of rennet in the curd
(Grappin and Beuvier, 1997).
The higher level of -casein observed in R Swiss-
type cheese, including Bergkäse, and in Manchego, Idi-
azabal and Canestrato Pugliese cheeses, than in P
cheeses, is explained by a lower plasmin activity in the
former cheeses, since pasteurisation of milk increases
plasmin activity due to inactivation of plasmin
inhibitors and/or increases in the activity of plasmino-
gen activators (Fox and Stepaniak, 1993). This activa-
tion is confirmed by the higher levels of -caseins found
in P cheeses (Beuvier et al., 1997; Ginzinger et al.,
1999b). On the other hand, the lower levels of -casein
in R Raclette, St Paulin, Arzua and Avenato cheeses may
be explained by the higher proteolytic activity of the
indigenous microflora. Gallmann and Puhan (1982),
Centeno et al. (1994a, 1996, 1999) and Sarantinopoulos
et al. (2002) clearly demonstrated that raw milk
microorganisms, such as Pseudomonas fluorescens,
micrococci and enterococci, can hydrolyse -casein.
Lau et al. (1991) suggested that interactions between
denatured whey proteins and caseins, which reduced
the accessibility of the caseins to proteases, affected the
degradation of -casein in P Cheddar cheese.
These results suggest that the breakdown of s1-
and -caseins is highly dependent on the cheese var-
iety and reflects different manufacturing parameters
(cooking temperature, pH at drainage) and different
physico-chemical characteristics (pH, salt-in-moisture,
moisture), and their effect on the activity of enzymes.
Secondary proteolysis
Nitrogen (N) fractionation is commonly used to quan-
tify the extent of proteolysis in cheese. Table 3 sum-
marises the results of different investigations on
nitrogen fractions from different cheese varieties.
Generally, the proportion of total N soluble in 12%
TCA or in PTA, and the amount of free amino acids
(FAAs) is higher in R cheeses than in P cheeses, reflect-
ing more extensive proteolysis due to aminopeptidases
of indigenous milk microorganisms. However, some-
times a discrepancy occurs and the R cheese shows less
secondary proteolysis, for reason(s) which are not clear.
In most of the studies where the RP–HPLC peptide
profiles were analysed, P and MF cheeses were close,
but differed significantly from R cheeses. In R cheeses,
some peaks present in the hydrophobic zone were
smaller in Cheddar (Lau et al., 1991; McSweeney et al.,
1993) and in Swiss-type cheeses (Ginzinger et al.,
1999b) than in P and MF cheeses; larger peaks corres-
ponding to hydrophilic peptides (Lau et al., 1991;
McSweeney et al., 1993) or to peptides containing
phenylalanine or tryptophan residues (Ginzinger et al.,
1999b) have been observed in cheeses made from P or
MF milk. In contrast, some peaks containing hydropho-
bic peptides were larger in P than in R Canestrato
Pugliese cheese (Albenzio et al., 2001). In the study by
Beuvier et al. (1997) on Swiss-type cheeses, some peaks
present in the medium hydrophobicity zone were
smaller in the R cheeses.
The composition of the FAAs also varied signifi-
cantly with the milk treatment in all varieties of cheese
and differed from one study to another. Nevertheless,
some amino acids appeared to be characteristic of the
treatment, maybe because of some typical metabolic
pathways due to the activity of specific enzymes.
Hard Spanish cheeses, such as Idiazabal or Mahon,
asparagine and glutamine, as well as taurine were
characteristic of P cheeses (Frau et al., 1997; Ordonez
et al., 1999; Mendia et al., 2000). According to Frau
et al. (1997), asparaginase and glutaminase would be
denaturated by pasteurisation. Aspartic acid was
higher in R than in P cheeses (Frau et al., 1997;
Ordonez et al., 1999; Albenzio et al., 2001). Serine
and tyrosine were also found preferentially in P milk
cheeses of these varieties, as well as in P or MF
Swiss-type cheeses (Skie and Ardö, 2000), whereas
-aminobutyric acid seems to be characteristic of R
cheeses (Ordonez et al., 1999; Mendia et al., 2000;
Skie and Ardö, 2000). According to the latter authors,
serine would be degraded to pyruvate by lactobacilli
and decarboxylation of glutamine, tyrosine and histi-
dine would result in -aminobutyric acid, tyramine
and histamine, respectively.
The influence of the indigenous microflora on the
decarboxylation of amino acids was confirmed in some
studies by the measurement of biogenic amines in
ripened cheeses. In general, R cheeses contained more
biogenic amines than P cheeses, e.g., in semi-soft
(Schneller et al., 1997) Manchego (Inigo et al., 1986)
and Swiss-type cheeses (Ginzinger et al., 1999b). Inigo
et al. (1986) related this to the staphylococci and micro-
cocci, whereas Schneller et al. (1997) concluded that
enterococci, and Enterobacteriaceae are able to produce
biogenic amines in cheese.
Overall, the differences between R and P or MF
cheeses concern mainly secondary proteolysis.
 Raw Milk Cheeses 325

Table 3 Secondary proteolysis: levels of N fractions of raw (R), pasteurised (P) and microfiltered (MF) milk cheeses (soluble N as
a % total N)

high molecular weight low

Cheese Water b pH 4.6 c 12% TCAd PTAe FAAfs Reference

Swiss-type R  MF R  MF Bouton and Grappin

(1995)
Swiss-type R  MF R  MF Demarigny et al. (1997)
Swiss-type R  P  MF R  P  MF Beuvier et al. (1997)
Bergkäse RP RP RP RP Ginzinger et al. (1999b)
(Swiss-type)
Canestrato R  P R  P R  P Albenzio et al. (2001)
Pugliese
Cheddar R  MF  P R  MF  P Mc Sweeney et al.
(1993)
Cheddar RP RP Lau et al. (1991)
Cheddar RP RP R  P 240 da Rosenberg et al.
R  P 470 d (1995)
Cheddar R P Rehman et al. (2000c)
Raclette R  P  MF R  P  MF Klantschitsch et al.
60 d (2000)
R  P  MF
90 d
Morbier-type RP RP Buchin et al. (1998)
Manchego RP RP RP Gaya et al. (1990)
Idiazabal (ovine) RP R  P or R  P or Ordonez et al. (1999)
R#P RP
depending depending
on starter on starter
Idiazabal (ovine) RP R  P 180 d RP Mendia et al. (2000)
90–180 d 90–180 d

a 240 d: 240 days of ripening.

b Water: water-soluble nitrogen.
c pH 4.6, pH 4.6 soluble nitrogen.
d 12% TCA: 12% trichloroacetic acid-soluble nitrogen.
e PTA: nitrogen soluble in 5% phosphotungstic acid.
f FAAs: free amino acids.

Lipolysis
The level of lipolysis and its impact on cheese quality
depend on the cheese variety. It is limited in Swiss-type
and semi-hard cheeses, where a level higher than
0.25–1.5% (Cheddar, depending on the commercial
quality), 1.5% (Gouda), 1% (Emmental) or 2% (Comté),
is considered to induce flavour defects such as rancid
(Choisy et al., 1997). In contrast, lipolysis is much
more extensive in some hard Italian, mould-ripened
(5–20%), blue-veined (18–25%) and goat-milk cheeses,
where lipolysis is essential for typical flavour formation.
Lipolysis in cheese is due to the activity of indigenous
milk lipoprotein lipase (LPL), to lipases or esterases of
starter bacteria or the native microflora, or to added
pregastric esterases (Deeth and Fitz-Gerald, 1983;
Gripon, 1993).
Table 4 summarises the differences between FFAs
obtained in R, P or MF cheeses.
In semi-hard and hard, uncooked cheeses, the total
amount of FFAs appears to be lower in P than in R
cheeses (Table 4); more than 50% lower in Cheddar
(McSweeney et al., 1993; Shakeel-Ur-Rehman et al.,
2000b,c) and 38% lower in Manchego (Gaya et al.,
1990). This was attributed to the inactivation of LPL by
pasteurisation. This inactivation varies according to the
severity of the heat treatment since heating for 15 s at 70
or 75 °C results in a residual activity of 2% and 0%,
respectively (Andrews et al., 1987). In the study of
McSweeney et al. (1993), lipolysis in MF Cheddar
cheeses was comparable to that in P cheeses; so it is
likely that lipases of the native microflora (NSLAB in this
case) could also play a role. According to Choisy et al.
(1997), LPL would have a lesser impact on the lipolysis
of R cheeses than microbial or technological enzymes.
Some fatty acids do not originate from lipolysis,
e.g., only fatty acids with six or more carbon atoms
326 Table 4 Total amounts and composition of free fatty acids in raw (R), pasteurised (P) and microfiltered (MF) milk cheeses

Total free fatty acids

Cheese Age Method (units) R P MF Individual free fatty acids Reference

Cheddar 120 days Global: CSM1 (g 13 7 C49C59C69C79C8: R  P, Shakeel-Ur-

palmitate/g C99C109C10:19C119C129C149C14:19C169C16:1: R  P Rehman
cheese) et al. (2000c)
Cheddar 180 days Global: CSM (g 216 142 Shakeel-Ur-
8 °C palmitate/g Rehman
cheese) et al. (2000b)
180 days 1 °C 135 103
Cheddar 180 days C49C59C69C79C89C99C109C10:19C119C129C149 Shakeel-Ur-
C14:19C159C16: R  P Rehman
C16:1: R  P et al. (2000a)
Cheddar 27 weeks Global: CSM (meq 4.7 2.2 2.1 C69C89C109C129C149C169C18: R  P/MF McSweeney
palmitate/kg et al. (1993)
cheese)
Morbier-type 5 weeks C5: R  P Buchin et al.
semi-hard C49C69C79C89C9: R  P (1998)
cheese
Manchego 24 h Global: titration 12.86 5.86 Gaya et al.
(meq/100 g fat) (1990)
4 months 8 °C 11.09 6.95
4 months 16 °C 16.21 9.87
Idiazabal 180 days C29C18:3: GC 15.5/8.3 10.8/6.5 C49C69C89C109C16:19C189C18:1: R  P, Chavarri
winter/ (mmol/kg C129C149C169C18:29C18:3: R  P et al. (2000)
spring cheese)
180 days 11.4 13.8
summer
Canestrato 63 days C4:09C18:3: HPLC 1673 1397 C69C109C18:2: R  P, Albenzio
Pugliese (mg/kg cheese) C49C89C129C149C169C189C18:1—C18:3: R  P et al. (2001)
Goat pressed 60 days C4:09C18:2: GC 8 6 C49C69C109C129C149C169C189C18:1—C18:2: R  P, Buffa et al.
cheese (mg/g fat) C8: R  P (2001a)
Serra Ovine Fresh C4:09C18:3: HPLC 3538 3002 Sousa and
soft cheese (mg/kgcheese) Malcata (1997)
68 days 6517 8115 C49C69C89C10: P  R
C129C149C169C189C18:1- C18:29C18:3: R  P
Goat soft Fresh (2 weeks C4:0—C20:4: GC 1.5 1.4 Morgan
cheese 4 °C) (g/100 g fat) et al. (2001)
10 days 12 °C 9.2 7.2
 4 weeks 4 °C
10 days 12 °C 12.9 10.3
 8 weeks 4 °C

1 CSM: copper soap method.


come exclusively from lipolysis. The proportion of
short-chain fatty acids in total FFAs varies according
to the extent of lipolysis of the cheeses; it can reach
50–80% (mol/mol) in hard-cooked cheeses. Thus, the
validity of global measurement to estimate lipolysis in
such cheeses is questionable, and it can be concluded
that the examination of individual FFAs is more
informative.
The higher level of lipolysis in R compared with P
cheeses affects different fatty acids: all FFAs from C6
to C16 in Cheddar cheeses (Shakeel-Ur-Rehman et al.,
2000a), all the major fatty acids in a goats’ milk hard
cheese (Buffa et al., 2001a), only short-chain (C4–C8)
fatty acids in Cheddar (Shakeel-Ur-Rehman et al.,
2000c) and only C6, C8 and C18:1 in Canestrato
Pugliese cheese (Albenzio et al., 2001). All the major
fatty acids in Cheddar were affected to the same extent
by pasteurisation or MF (McSweeney et al., 1993). In
the study by Chavarri et al. (2000), the amounts of
short-chain FFAs (C4 to C10) and of C16:1, C18:0,
C18:1 in Idiazabal were affected by pasteurisation.
But, the effect of pasteurisation varied according to the
season; amounts of FFA were higher in R cheeses
made in winter and spring, but lower in those made in
summer. In the study by Buchin et al. (1998) on R and
P Morbier-type, semi-hard cheeses, no differences were
observed in the levels of C6 to C9 fatty acids.
The attribution of the observed differences in lipoly-
sis, either to LPL or to microbial lipases, is difficult.
Inactivation of LPL was suggested by Gaya et al.
(1990). Chavarri et al. (1998) found that depending on
the period of the trial, LPL was inactivated by 73–95%,
by pasteurisation of milk for the manufacture of Idiaza-
bal. Short-chain fatty acids were released preferentially
during ripening, showing microbial participation in
lipolysis, but the balance between the different FFAs
was not affected by pasteurisation, indicating that
microbial lipolysis was not significantly affected by
pasteurisation (Chavarri et al., 2000).
Albenzio et al. (2001) attributed the slight differ-
ences in lipolysis between R and P cheeses to differ-
ences in lipase and esterase activities of the milk
microflora (NSLAB). Shakeel-Ur-Rehman et al. (2000a)
also attributed the differences observed in Cheddar to
the activity of NSLAB, because NSLAB were highest in
the most highly lipolysed cheeses. However, agreement
between NSLAB counts and FFA levels did not occur in
all cheeses, so the authors supposed that not only num-
ber but also species or strains play a role. In hard
cheeses, such as Cheddar, where moulds are not pre-
sent, LAB could be the main cause of lipolysis (Choisy
et al., 1997). In particular, starter LAB may contribute
to the release of fatty acids, because they have lipase
and esterase activities (Deeth and Fitz-Gerald, 1983).
Raw Milk Cheeses 327
The role of starter LAB in the observed differences is also
questionable; modification of their activity due to inter-
action with the natural microflora cannot be excluded.
Such differences could be expected in soft cheeses, but
the studies on this type of cheese show contradictory
results. In goats’ milk mould-ripened cheeses, Morgan
et al. (2001) found higher lipolysis, determined by a
global method, in R cheeses than in P cheeses. The
extent of lipolysis in mature cheeses was related to that
of the milk. Pasteurisation was supposed to inactivate
LPL. But, in this study, pasteurisation also induced
lower moisture cheeses, which could reduce the activity
of the lipolytic surface microflora. In contrast, Sousa
and Malcata (1997) found higher lipolysis in P cheeses
made from ovine milk (Serra) than in R cheeses, despite
higher levels in the fresh cheese. However comparison
between the two types of cheese was made difficult by
a higher fat retention in P than in R cheeses, and the
amounts of FFAs were expressed per weight of cheese.
Thus, in both studies, interpretation of the results is
difficult because of the difficulties in controlling the
cheese composition.
In Swiss-type cheeses, the level of C6 could be a
good indicator of the extent of lipolysis, as suggested by
Kuszdal-Savoie (Choisy et al., 1997). In the studies of
Bouton and Grappin (1995), Beuvier et al. (1997) and
Demarigny et al. (1997), the variations in C4 followed
those of C6, which indicates that, in these cases, C4 was
mainly from lipolysis, and not from the growth of
clostridia on lactate. Bouton and Grappin (1995) and
Beuvier et al. (1997) found no differences in these two
acids in cheeses (R, P, MF or P with the addition of the
natural microflora), whereas Demarigny et al. (1997)
found that R cheeses tended to be more lipolysed than
MF cheeses, depending on the season of the year and
the age of the cheese. In the latter cheeses, lipolysis may
have been of microbial origin. On the one hand, the
heating of the milk in the vat, performed in this type of
technology, would inactivate the LPL (Chamba and
Perreard, 2002), which would explain why no differ-
ences were observed between P and MF cheeses (Beuvier
et al., 1997). On the other hand, thermophilic starter
LAB and propionibacteria have lipolytic activity in Swiss-
type cheeses (Deeth and Fitz-Gerald, 1983). According
to Chamba and Perreard (2002), the lipolytic activity of
propionibacteria is far greater than that of starter bac-
teria. Moreover, it could be strain-dependant, which
could also explain the variability in the above results.
Volatile compounds
Comparisons of the volatile compounds in R and P or
MF cheeses, have been undertaken only recently and
are not very numerous. The same volatile compounds
328 Raw Milk Cheeses
were present in each type of cheese within the same
study, whether the microflora was present or removed
from the milk. Differences were observed in the levels
of many volatile compounds, R cheeses generally hav-
ing higher levels than P or MF cheeses, with excep-
tions in certain chemical families.
Volatile fatty acids (VFAs)
Volatile fatty acids in cheese are the products of various
metabolic pathways, mostly microbial. Acetic acid may
have different origins, e.g., from the catabolism of lac-
tose, citrate, amino acids, or the propionic fermentation
(PF). Propionic acid is one of the end products of lactose
or lactate fermentation by propionibacteria. Butyric acid
can originate from the catabolism of triglycerides, but
also from lactate fermentation by clostridia. Hexanoic
acid (C6) is found only in triglycerides and is liberated
by lipolysis (its behaviour is described under Lipolysis
and catabolism of fatty acids in cheese. Branched-chain
VFAs result from catabolism of the amino acids, e.g.,
2-methyl propanoic (isobutyric), 2-methyl butanoic,
3-methyl butanoic (isovaleric) acids from valine,
isoleucine and leucine, respectively.
These compounds are major volatile compounds
in cheese, and participate significantly in the flavour
of many cheese varieties, as suggested by results from
olfactometry: acetic acid has a characteristic vinegar
flavour, propionic acid a pungent, fruity one, butyric,
isobutyric and isovaleric acids have similar flavours,
associated with cheesy, sweaty, rancid and sour notes
(Yvon and Rijnen, 2001; Curioni and Bosset, 2002).
Table 5 summarises the differences between VFAs
obtained in R, P or MF cheeses.
The PF is one of the major fermentations that occur
in Swiss-type cheeses (Curioni and Bosset, 2002). In the
studies of Bouton and Grappin (1995), Beuvier et al.
(1997), Demarigny et al. (1997) and Eliskases-Lechner
et al. (1999), the presence of the raw milk microflora
was associated with higher levels of acetic and propionic
acids and lower levels of lactic acid in the ripened
cheeses. These results showed a greater PF in R cheeses,
due to the presence of the native propionibacteria,
which are eliminated by pasteurisation or microfiltra-
tion. It should be noted that no propionibacteria were
added to the cheeses studied, as required in the manu-
facture of Bergkäse or Comté. In contrast, in a hard
cheese such as Emmental, in which propionibacteria are
used as starters to ensure a high level of PF, the effect
of native populations of propionibacteria seems to
disappear. Buchin et al. (2002) compared R and MF
Emmental-type cheeses: the total population of pro-
pionibacteria tended to be lower in R cheeses from mid-
ripening, with a subsequent lower level of propionic acid
at the end of ripening.
Generally, acetic acid is more important in Swiss-
type cheeses made from R milk than in those from P or
MF milk (Bouton and Grappin, 1995; Demarigny et al.,
1997; Eliskases-Lechner et al., 1999). This was related
to the presence of propionibacteria and facultatively
heterofermentative lactobacilli (FHL) in the R milk.
In Swiss-type cheeses, the level of butyric acid tended
to be higher in R than in MF cheeses (Demarigny et al.,
1997). Because no butyric acid bacteria were found in
these cheeses, the origin of this compound could not be
butyric fermentation. It is likely that it was liberated from
triglycerides by the lipolytic activity of the micro-
organisms present in the cheese, particularly propioni-
bacteria (Chamba and Perreard, 2002). Caproic acid (C6)
followed the same trend as butyric acid, but the differ-
ences between R and MF cheeses were less marked (see
‘Lipolysis’). In contrast, Bouton and Grappin (1995) and
Beuvier et al. (1997) observed no differences between R,
P, MF or P cheeses with added native microflora.
The level of isovaleric acid was higher in R cheeses
than in P or MF cheeses (Beuvier et al., 1997; Demarigny
et al., 1997). The occurrence of this compound was cor-
related with FHL, propionibacteria or enterococci popu-
lations, and at least the former two populations may be
involved (Langsrud and Reinbold, 1973; Paulsen et al.,
1980; Thierry and Maillard, 2002). However, Buchin
et al. (2002) found that the production of isovaleric acid
was positively correlated with the level of propionibac-
teria, but negatively with the levels of starter LAB, native
mesophilic lactobacilli or enterococci.
The influence of the native microflora on VFA pro-
duction was also observed in other cheese varieties, e.g.,
Cheddar (Shakeel-Ur-Rehman et al., 2000a,c), Raclette
(Klantschitsch et al., 2000) and Morbier-type cheese
(Buchin et al., 1998). In Cheddar, several VFAs, e.g.,
acetic, propionic, butyric, valeric and caproic acids, were
found at higher levels in R than in P cheeses. Moreover,
the levels of all these compounds increased at a ripening
temperature of 8 °C, compared with 1 °C, and the levels
in cheeses of the different acids, except caproic,
increased with the proportion of raw milk in several
blends of R and P milk. The authors attributed all these
differences to the presence of NSLAB in the raw milk,
because their presence and growth in cheese were
closely related to the production of fatty acids. NSLAB
were mostly eliminated by pasteurisation, and the higher
the R:P milk ratio or the higher the ripening tempera-
ture, the higher was the number of these bacteria in
cheese, at all times during ripening. The influence of the
presence of the native microflora on the production of
acetic and propionic acids in a semi-hard, Morbier-type
cheese was confirmed by Buchin et al. (1998) (compari-
son of R and P cheeses), by Klantschitsch et al. (2000) in
Raclette (comparison of R, P and MF cheeses) and by
 Table 5 Volatile fatty acids in raw (R), pasteurised (P) and microfiltered (MF) milk cheeses

2-Methyl 3-Methyl 4-Methyl

propanoic Butanoic 2-Methyl butanoic Pentanoic pentanoic Hexanoic Heptanoic Octanoic Nonanoic
Cheese Formic Acetic Propanoic (isobutyric) (C4) butanoic (isovaleric) (C5) (isocaproic) (C6) (C7) (C8) (C9) Reference

Swiss-type RP RP RP Eliskases-

Lechner et al.
(1999)
Swiss-type R  MF R  MF n.d.a n.d. Bouton and
Grappin
(1995)
Swiss-type R  MF varb var R  MF var Demarigny
RPM RPM et al. (1997)
Swiss-type F F n.d. R  P  MF n.d. Beuvier et al.
(1997)
Emmental R  MF R  MF R  MF R  MF Buchin et al.
(unpublished)
Cheddar n.d. RP n.d. RP RP RP RP RP RP RP n.d. Shakeel-Ur-
Rehman
et al. (2000c)
Cheddar RP R (8 °C) R (1 °C)  RP n.d. R (1 °C)  RP RP RP RP RP Shakeel-Ur-
 R (1 °C) R (8 °C)  P R (8°C) P Rehman
P et al. (2000a)
Morbier-type RP RP n.d. n.d. n.d. n.d. RP n.d. n.d. n.d. n.d. n.d. Buchin
(semi-hard et al. (1998)
cheese)
Raclette R  P, MF RP Klantschitsch
MF et al. (2000)
(90 d,
17 °C)
Roncal RP RP n.d. n.d. n.d. Ortigosa
(240 d) (240 d) et al. (2001)

a n.d.: no difference.
b var: variable.
329
330 Raw Milk Cheeses
Ortigosa et al. (2001) in ovine Roncal cheese (compari-
son of R, P and P  Lb. casei). In Cheddar cheese, the
variations in VFAs originating from amino acid catabol-
ism were not the same as those of other VFAs. In a study
by Shakeel-Ur-Rehman et al. (2000c), 2- and 3-methyl
butanoic acids were found at higher levels in P cheeses
than in R cheeses, and in another of their studies
(Shakeel-Ur-Rehman et al., 2000a), 2-methyl propanoic
and 3-methyl butanoic acids were found at higher levels
in cheeses ripened at low temperature (1 °C compared
to 8 °C). These compounds correlated negatively with
the number of NSLAB in the cheeses, so the authors
hypothesised that NSLAB have a minor role in the for-
mation of these compounds or that NSLAB broke them
down further.
Carbonyl compounds
Tables 6 and 7 summarise the differences between
ketones and aldehydes found in R, P or MF cheeses.
Diacetyl (2,3-butanedione) and acetoin (3-hydroxy-
2-butanone) are products of the metabolism of citrate by
Lc. lactis spp. lactis bv. diacetylactis, Leuconostoc spp. or
some Lactobacillus species (McSweeney and Sousa,
2000). They are important aroma compounds in numer-
ous varieties of cheese (Curioni and Bosset, 2002). Nev-
ertheless, they tend to decrease during ripening. In
6-month-old Cheddar, diacetyl is present in quantities
too low to participate in flavour (Urbach, 1997). In
cheese, diacetyl can be converted into acetoin, then 2,3-
butanediol and 2-butanone and finally 2-butanol
(Urbach, 1993). It is notable that 2-butanone and
2-butanol, in contrast to diacetyl and acetoin, generally
increase during ripening, like other methyl ketones and
secondary alcohols (Bosset and Liardon, 1985; Barlow
et al., 1989; Urbach, 1993; Carbonell et al., 2002).
Therefore, it is likely that the transformation of diacetyl
into the more reduced compounds progresses through-
out ripening due to the enzymatic activities of micro-
organisms, so the raw milk microflora can be expected
to influence these compounds. Indeed, diacetyl and ace-
toin were found in lower quantities in R cheeses than
in P or MF cheeses in semi-hard (Buchin et al., 1998;
Shakeel-Ur-Rehman et al., 2000a,c; Ortigosa et al., 2001)
or Swiss-type cheeses (Buchin et al., unpublished). The
contrasting result was observed in only one study on
Morbier-type cheese (Buchin et al., unpublished). The
quantities of 2-butanone in ripened cheese were affected
differently by the presence of the raw milk microflora.
Ortigosa et al. (2001) found less 2-butanone in Roncal
cheese made from raw ewes’ milk than in cheese made
from P milk, but the opposite was true for the Morbier-
type cheeses studied by Buchin et al. (1998). These
contradictory results could be expected because the
amount of 2-butanone in a ripened cheese arises from
the balance between its formation and its reduction to
2-butanol. It is difficult to establish a correlation between
the presence of microbial populations and the level of
2-butanone at a given time of ripening, because of the
continuous production and degradation of this com-
pound. To follow the evolution of microbial growth and
the level of 2-butanone throughout the ripening period
may provide valuable information. In contrast, as
expected, 2-butanol was more abundant in R cheeses
than in P cheeses in all studies (Buchin et al., 1998;
Shakeel-Ur-Rehman et al., 2000c; Ortigosa et al., 2001).
The other carbonyl compounds found in cheeses are
methyl ketones and aldehydes. Their characteristics
have been summarised in a recent review by Curioni
and Bosset (2002). Methyl ketones originate from the
-oxidation of fatty acids by microorganisms. Their
aromatic impact is of primary importance in blue and
surface mould-ripened cheeses, but they are also likely
to have an influence on the flavour of other varieties,
e.g., 2-heptanone in Emmental, Gruyère and Grana
Padano. This ketone is typical of blue cheese flavour,
whereas the others have fruity, floral or musty notes.
Straight-chain aldehydes originate from the oxidation
of unsaturated fatty acids. They are characterised by
green, fatty odours. The branched-chain aldehydes
with four or five carbon atoms originate from the
catabolism of the amino acids, valine (2-methyl
propanal), leucine (2-methyl butanal) and isoleucine
(3-methyl butanal). In cheese, they have green or malty
notes.
As previously noted, the variations of these com-
pounds in the presence of the raw milk microflora
differed according to the study. In the studies of Shakeel-
Ur-Rehman et al. (2000c) and Buchin et al. (1998)
on Cheddar and Morbier-type cheese, respectively, R
cheeses contained less of the different methyl ketones
and aldehydes than P cheeses. This is in contrast with
the results of the studies of Shakeel-Ur-Rehman et al.
(2000a) comparing R and P Cheddar, and Buchin et al.
(2002) comparing R and MF Morbier-type cheeses. The
results of Buchin et al. (2002) showed less ketones but
more aldehydes in R Swiss-type cheeses compared to MF
ones.
The explanation for these contrasting results may be
the same as that for the products of pyruvate metab-
olism. Methyl ketones and aldehydes are intermediate
products in the degradation of fatty acids or amino
acids. Due to the enzymatic activities of microorganisms
in cheese, methyl ketones are progressively reduced to
2-alkanols, aldehydes are oxidised to acids or reduced
to n-alkanols. Therefore, their levels depend on the bal-
ance between production and degradation, which is
linked to the degree of maturity of the cheese. The
maturity of the cheeses for each of the studies may be
 Raw Milk Cheeses 331

Table 6 Ketones in raw (R), pasteurised (P) and microfiltered (MF) milk cheeses

Cheese Emmental Cheddar Cheddar Semi-hard Semi-hard Roncal

Propanone R  MF n.d.a n.d. n.d.

3-Hydroxy-2- R  MF RP RP RP R  MF n.d.
butanone (acetoin)
2,3-Butanedione R  MF RP n.d. RP
(diacetyl) (120 d)c
2-Butanone R  MF RP R  MF RP
(120 d)
3-Methyl 2- n.d.
butanone
2-Pentanone R  MF RP n.d. RP
(240 d)d
2,3-Pentanedione R  MF RP R  MF n.d.
4-Methyl 2- n.d.
pentanone
3-Methyl 2- RP
pentanone
Cyclopentanone n.d.
2-Hexanone R  MF n.d. RP n.d. n.d.
4-Methyl n.d. n.d.
cyclohexanone
Cyclohexanone n.d. n.d.
2-Heptanone R  MF RP RP RP R  MF varb
3-Methyl 2- n.d.
heptanone
5-Methyl 2- R  MF
heptanone
6-Methyl 2- n.d.
heptanone
6-Methyl-5-hepten- n.d. n.d. R  MF
2-one
2-Octanone R  MF n.d. RP n.d.
2-Nonanone R  MF RP RP n.d. n.d.
8-Nonen-2-one RP PR
2-Decanone RP n.d.
2-Undecanone n.d. RP RP
2-Dodecanone RP RP
2-Tridecanone RP RP
2-Pentadecanone n.d. RP
Acetophenone n.d. R  MF
Reference Buchin et al. Shakeel-Ur- Shakeel-Ur- Buchin et al. Buchin et al. Ortigosa
(unpublished) Rehman et al. Rehman et al. (1998) (unpublished) et al.
(2000c) (2000a) (2001)

a n.d.: no difference.
b var: variable.
c 120 d: 120 days of ripening.
d 240 d: 240 days of ripening.

different, which could explain the different results from
one study to another, even using the same technology.
Alcohols
Table 8 summarises the differences between alcohols
obtained in R, P or MF cheeses.
The observation of the levels of alcohols in the
cheeses confirms the previous hypothesis. The presence
of the native microflora in milk has a major influence on
the production of alcohols in cheeses. All studies agreed
that R cheeses contained greater amounts of the different
alcohols, compared to P or MF cheeses (Buchin et al.,
1998; Shakeel-Ur-Rehman et al., 2000a,c; Ortigosa et al.,
2001; Buchin et al., 2002). This was valid for n-alkanols
as well as 2-alkanols. Because alcohols result from the
reduction of previously formed compounds, it can be
concluded that the raw milk microflora induced faster
cheese ripening.
332 Raw Milk Cheeses

Table 7 Aldehydes in raw (R), pasteurised (P) and microfiltered (MF) milk cheeses

Cheese Emmental Cheddar Cheddar Semi-hard Semi-hard Roncal

Acetaldehyde R  MF n.d.
Propanal n.d.
2-Methyl propanal R  MF R  MF var
Butanal P  R (240 d)c
3-Methyl butanal R  MF n.d. R  MF n.d.
2-Methyl butanal R  MF RP R  P (120 d)d
2-Methyl butenal n.d. n.d.
Pentanal n.d. n.d. n.d.
Hexanal n.d. n.d. n.d.
Heptanal PR n.d. n.d. R  MF
2,4-Heptadienal PR
Octanal n.d. n.d.
Nonanal R  MF RP n.d. n.d. R  MF R  P (240 d)
Decanal R  MF n.d. R  MF R  P (240 d)
Undecanal n.d.
Dodecanal n.d.
Tetradecanal n.d. RP
Hexadecanal varb RP
Furancarboxaldehyde RP var
Benzaldehyde n.d. RP RP n.d.
Phenylacetaldehyde var RP
Reference Buchin et al. Shakeel-Ur- Shakeel-Ur- Buchin et al. Buchin et al. Ortigosa
(unpublished) Rehman et al. Rehman et al. (1998) (unpublished) et al. (2001)
(2000c) (2000a)

a n.d.: no difference.
b var: variable.
c 240 d: 240 days of ripening.
d 120 d: 120 days of ripening.

Esters
Esters are formed by the condensation of an acid and
an alcohol. In cheese, this reaction may be spontan-
eous, or may be mediated by microbial esterases.
According to Urbach (1995), esters are not likely to
be formed by the starter culture, although Yvon and
Rijnen (2001) reported that esterification reactions
can be mediated by various LAB, including lactococci,
lactobacilli, Sc. thermophilus, leuconostocs and pedio-
cocci. This ability is highly strain-dependent. Esters
generally have fruity odours and may also influ-
ence cheese flavour. They are particularly numerous in
hard cheeses such as Swiss Emmental and Parmesan,
in which they play an important role in flavour
(Urbach, 1997). In contrast, their presence in Cheddar
cheese is limited; in fact, fruity flavour in this cheese is
a defect.
Table 9 summarises the differences between esters
obtained in R, P or MF cheeses.
In all studies (Buchin et al., 1998, 2002; Shakeel-Ur-
Rehman et al., 2000a,c; Ortigosa et al., 2001), whatever
the cheese variety, the presence of the raw milk micro-
flora was linked to a greater formation of esters. As
expected, ethyl esters were the most important, in rela-
tion to the levels of ethanol, and they were more diver-
sified in Swiss-type cheese (Buchin et al., unpublished).
Sulphur compounds
Sulphur compounds contribute to cheese flavour. They
are numerous in mould- or smear-surface cheeses, and
provide typical cabbage or garlic flavours (Urbach, 1997;
Yvon and Rijnen, 2001). Hydrogen sulphide, methional,
methanethiol, dimethyldisulphide and dimethyltrisul-
phide are related to Cheddar flavour (McSweeney and
Sousa, 2000); methional, methanethiol and dimethyl-
trisulphide are key flavour compounds in Emmental
cheese (Rychlik and Bosset, 2001), 3-methylthio-1-
propanol is present in premium quality Cheddar cheese,
ethyl 3-methylthiopropanoate in Parmesan cheese, while
methanethiol is related to unpleasant odours in Grana
cheese (Urbach, 1997).
The sulphur compounds in cheese derive from the
sulphur amino acids. Several mechanisms are involved
in their formation. In the reducing environment of
cheese, purely chemical decomposition of methionine or
cysteine could occur to produce compounds such
as methanethiol or H2S. A negative redox potential is
a necessary condition for the production of volatile
 Raw Milk Cheeses 333

Table 8 Alcohols in raw (R), pasteurised (P) or microfiltered (MF) milk cheeses

Cheese Emmental Cheddar Cheddar Semi-hard Semi-hard Roncal

Ethanol R  MF RP n.d. R  P (240 d)a

1-Propanol R  MF RP R  MF R  P (120 d)b
2-Methyl propanol PR10d  P R  P (240 d)
1-Butanol R  MF PR10  P RP RP R  MF n.d.
3-Methyl butanol R  MF PR10  P n.dc n.d. R  MF R  P (240 d)
1-Pentanol RP RP n.d.
1-Hexanol PR10  P n.d. RP n.d. n.d.
1-Heptanol n.d. RP
1-Octanol PR10  P RP
1-Nonanol PR10  P n.d.
1-Decanol PR10  P
2-Ethyl 1-decanol n.d.
2-Propanol R  MF RP n.d. R  P (240 d)
2-Propen-1-ol n.d. n.d.
2-Butanol R  MF PR10  P RP R  MF R  P (120 d)
3-Methyl 2-butanol RP
2-Pentanol R  MF RP RP RP n.d. R  P (120 d)
2-Hexanol PR10  P
2-Heptanol R  MF RP R  MF
2-Octanol PR10  P
2-Nonanol PR10  P
2-Decanol PR10  P
3-Methyl 3-buten-1-ol R  MF n.d. R  MF n.d.
3-Methyl 2-buten-1-ol n.d.
3-Penten-2-ol n.d.
2-Methyl 3-pentanol n.d. n.d.
2,3-Butanediol n.d.
1,3-Butanediol n.d.
Furan methanol PR10  P n.d.
Phenol n.d. n.d.
Phenethyl alcohol RP n.d.
Reference Buchin et al. Shakeel-Ur- Shakeel-Ur- Buchin et al. Buchin et al. Ortigosa et al.
(unpublished) Rehman Rehman (1998) (unpublished) (2001)
et al. et al.
(2000c) (2000a)

a 240 d: 240 days of ripening.

b 120 d: 120 days of ripening.
c n.d.: no difference.
d PR10: mix of 90% pasteurised milk with 10% of raw milk.

sulphydryl compounds in cheese, but enzymatic reac-
tions may also be involved (Urbach, 1997). On the one
hand, native milk enzymes may produce disulphide
linkages as precursors of sulphydryl groups, and heating
of milk stops the production of H2S and reduces the pro-
duction of methanethiol by inactivating these enzymes
(Urbach, 1995). On the other hand, the surface micro-
organisms of smear cheeses are high producers of sulphur
compounds, like methanethiol or methylthioesters. Lac-
tic acid bacteria may also contribute to the production of
sulphur compounds. Cheese lactobacilli can produce
H2S and Lc. lactis has the ability to cleave methionine
and produce methanethiol (Yvon and Rijnen, 2001), and
starters may contribute by providing a reducing environ-
ment. The further formation of dimethyldisulphide or
dimethyltrisulphide and of most of the methyl thioesters
from methanethiol is due to chemical rather than bio-
logical reactions.
Table 10 summarises the differences in sulphur
compounds between R, P or MF cheeses.
As expected, comparisons of R and P cheeses
showed higher levels of sulphur compounds in the R
cheeses (Buchin et al., 1998; Shakeel-Ur-Rehman et al.,
2000c; Ortigosa et al., 2001), except in the study by
334 Raw Milk Cheeses

Table 9 Esters in raw (R), pasteurised (P) and microfiltered (MF) milk cheeses

Cheese Emmental Cheddar Cheddar Semi-hard Semi-hard Roncal

Methyl acetate R  MF R  MF
Methyl propanoate n.d.a R  MF
Methyl butanoate n.d. n.d.
Methyl hexanoate n.d. n.d.
Methyl octanoate n.d.
Ethyl methanoate n.d.
Ethyl acetate R  MF R  MF R  P (120 d)b
Ethyl propanoate R  MF R  MF
Ethyl butanoate R  MF n.d.
Ethyl hexanoate R  MF RP RP RP R  MF R  P (240 d)c
Ethyl heptanoate n.d. n.d.
Ethyl octanoate R  MF RP RP R  MF
Ethyl decanoate PR10d  P RP n.d.
Ethyl dodecanoate PR10  P RP
Ethyl tetradecanoate RP RP
Propyl acetate n.d. R  MF
Propyl propanoate n.d. R  MF
Propyl butanoate n.d. R  MF
Butyl acetate R  MF n.d. n.d.
Butyl propanoate n.d.
Pentyl acetate n.d.
1-Methyl-propyl acetate R  MF
2-Methyl-propyl R  MF
propanoate
2-Methyl-propyl R  MF R  MF
butanoate
3-Methyl-butyl acetate R  MF R  MF
2-Methyl-butyl R  MF n.d.
butanoate
3-Methyl-butyl n.d. n.d.
butanoate
Reference Buchin et al. Shakeel-Ur- Shakeel-Ur- Buchin et al. Buchin et al. Ortigosa et al.
(unpublished) Rehman Rehman (1998) (unpublished) (2001)
et al. et al.
(2000c) (2000a)

a n.d.: no difference.
b 120 d: 120 days of ripening.
c 240 d: 240 days of ripening.
d PR10: mix of 90% pasteurised milk with 10% of raw milk.

Shakeel-Ur-Rehman et al. (2000a), where dimethyl-
disulphide and dimethyltrisulphide were absent from R
cheeses. In comparisons of R and MF cheeses, Buchin
et al. (2002) found no differences in Morbier-type cheeses,
and only a higher level of dimethyldisulphide in raw
milk Swiss-type cheeses. This would indicate that the
inactivation of native enzymes by heating the milk may
be a major event in the diminution of sulphur compound
formation, compared to the elimination of the native
flora. It is noteworthy that in all these studies, the diver-
sity of the sulphur compounds reported was very poor.
Lactones, hydrocarbons
Table 11 summarises the differences in lactones between
R and P cheeses.
Lactones are the result of spontaneous cyclisation of
the hydroxy-acids naturally present in milk fat. In the
studies by Shakeel-Ur-Rehman et al. (2000a,c), heat-
treatment of the milk influenced the levels of some
lactones, but the results were inconsistent. Their occur-
rence in cheese may also be linked to feeding (Urbach,
1997). Whether aliphatic or aromatic, the levels of
hydrocarbons in cheeses do not seem to be influenced
by the presence of the native microflora in milk.
In conclusion, the presence of the native microflora
in R cheeses is of primary importance for the formation
of most volatile compounds. Nevertheless, considering
the present state of knowledge, it is difficult to establish
precisely the role of this microflora. This role can be
direct, in transforming the milk constituents into volatile
 Raw Milk Cheeses 335

Table 10 Sulphur compounds in raw (R), pasteurised (P) and microfiltered (MF) milk cheeses

Cheese Emmental Cheddar Cheddar Semi-hard Semi-hard Roncal

Carbon sulphide n.d.a n.d.

Carbon disulphide n.d.
Dimethyl sulphide n.d. n.d. R  P (240 d)b
Dimethyl disulphide R  MF n.d. RP RP n.d. n.d.
Dimethyl trisulphide n.d. RP RP
Methional RP RP
Methane sulfonylbis n.d.
Reference Buchin et al. Shakeel-Ur- Shakeel-Ur- Buchin et al. Buchin et al. Ortigosa et al.
(unpublished) Rehman Rehman (1998) (unpublished) (2001)
et al. et al.
(2000c) (2000a)

a n.d: no difference.
b 240 d: 240 days of ripening.

compounds, or indirect, by modifying the composition Sensory Aspects

of the cheese, with the production of precursors of
In order to avoid any ambiguity, due to the different
volatile compounds or of molecules that influence
use of the same terms by different authors, we have
chemical reactions or the activity of other microorgan-
chosen to define sensory perceptions as follows: odour
isms. In particular, the activities of the indigenous popu-
is perceived by the nose, with no introduction of the
lations can interfere with those of the starter bacteria.
food into the mouth, while flavour is the perception of
Within the complexity of the native milk microflora, it is
the food during mastication, either retronasaly or by
difficult presently to establish the role of each popula-
the tongue (five basic tastes: sweet, acid, bitter, salty,
tion, at the species and at the strain level. It is likely that
umami).
many of the metabolic pathways producing volatile com-
pounds are strain-dependant (Yvon and Rijnen, 2001), Flavour/odour
which would make their elucidation all the more diffi-
cult. The development of molecular techniques for the Table 12 summarises the differences between the flavour
discrimination of microbial populations at the strain and odour attributes reported for R, P or MF cheeses.
level could be very beneficial to such studies. This situ- Raw milk cheeses ripen faster than cheeses made
ation underlines the importance of maintaining a high from milk, the microflora of which has been removed.
diversity of strains in the milk, to retain the diversity of As a consequence, R cheeses tend to develop a stronger
the molecules produced. odour/flavour at the same age than those made from P
or MF milk (Johnson et al., 1990b; Lau et al., 1991).
This has been observed in all types of cheese studied:
Cheddar (McSweeney et al., 1993; Shakeel-Ur-Rehman
Table 11 Lactones in raw (R) and pasteurised (P) milk cheeses et al., 2000a,b), Manchego (Gaya et al., 1990; Fernandez-
Garcia et al., 2002; Gomez-Ruiz et al., 2002), Raclette
Cheese Cheddar Cheddar (Gallmann and Puhan, 1982), other hard and semi-hard
cheeses (Lau et al., 1991; Van den Berg and Exterkate,
-Octanolactone PR10b n.d.a
-Decanolactone PR RP
1993; Buchin et al., 1998; Skie and Ardö, 2000),
-Dodecanolactone n.d. RP Bergkäse (Ginzinger et al., 1999a), Swiss-type cheeses
-Hexadecanolactone n.d. n.d. (Bouton and Grappin, 1995; Beuvier et al., 1997;
-Decanolactone n.d. n.d. Demarigny et al., 1997) and soft goats’ milk cheese
-Dodecanolactone RP RP (Morgan et al., 2001). In all cases, this phenomenon
-Dodecenolactone RP RP
seems to be directly linked to the activity of the indigen-
Reference Shakeel-Ur- Shakeel-Ur- ous microflora of the milk. In Cheddar, it has been
Rehman et al. Rehman et al.
attributed, in part, to the presence of NSLAB (com-
(2000c) (2000a)
posed mainly of lactobacilli, but also of pediococci and
a n.d.: no difference. micrococci) in the raw milk, which are the major
b PR10: mix of 90% pasteurised milk with 10% of raw milk. part of the natural microflora of this variety of cheese
336 Raw Milk Cheeses

Table 12 Characteristic flavour and odour attributes of raw (R), pasteurised (P) and microfiltered (MF) milk cheeses

Cheese variety Raw milk Pasteurised milk Microfiltered milk Reference

Bergkäse Odour: intense Ginzinger et al.

Flavour: intense Flavour: bitter (1999a)
Swiss-type Flavour: intense, Flavour: bitter Bouton and
typical, acid, pungent Grappin (1995)
Swiss-type Flavour: intense, Flavour: acid, Beuvier et al.
pungent, salty bitter, salty (1997)
Cheddar Odour: intense, Odour: musty Shakeel-Ur-
creamy/milky, Rehman et al.
fruity/sweet, (2000a)
acid/sharp, pungent
Flavour: intense, sour/
acid, sulphur/eggy,
bitter, rancid, unclean
Cheddar Only cheeses ripened Shakeel-Ur-
at 8 °C (vs 1 °C) Rehman et al.
Odour: intense, acid (2000b)
Flavour: intense, sour
Cheddar Odour: intense, fruity/ Shakeel-Ur-
sweet, pungent Rehman et al.
Flavour: sour/acid (2000c)
Semi-hard Flavour: intense, of acid Buchin et al.
cheese, milk, of rind, (1998)
Morbier-type Flavour: of fresh milk,
fruity, of garlic, spicy,
animal, chemical,
rancid, bitter, pungent
Semi-hard Flavour: intense, animal, Skie and
round-eyed spicy, sour Ardö (2000)
cheese
Roncal Odour: intense (120 d), Odour: animal (120 d) Ortigosa et al.
animal (240 d) (2001)
Flavour: characteristic, Flavour: torrefied
pungent (240 d), animal (240 d)
Aftertaste:intense
Idiazabal Odour: characteristic, Odour: sweet Mendia et al.
pungent, sour (1999)
Flavour: characteristic, Flavour: sweet, bitter,
pungent, salty sour
Aftertaste: characteristic, Aftertaste: bitter, sour
pungent
Idiazabal Flavour: characteristic, Flavour: sweet Ordonez et al.
creamy, pungent, acid (1999)
Odour: sweet

(McSweeney et al., 1993). In Swiss-type cheeses
(Beuvier et al., 1997), flavour intensity was correlated
with counts of FHL, propionibacteria and enterococci,
which occur naturally in the raw milk. In pasteurised
milk cheeses, denaturation of enzymes and whey
proteins by the heat treatment may also be involved;
the aggregation of whey proteins on the surface of
the caseins micelles also prevents proteolysis of the
caseins.
This difference in maturity is enhanced by the tem-
perature of ripening and depends on the age of the
cheese (Klantschitsch et al., 2000; Shakeel-Ur-Rehman
et al., 2000b).
Besides the intensity of flavour, differences in the
flavour profile of cheese can be observed. The flavour
of the ripened cheese is richer and more complex when
the indigenous microflora is present in the milk to be
processed. Some observations are constant from one
study to another, whereas others vary.
In almost all studies comparing cheeses made from
raw milk and raw milk after elimination of the micro-
flora, the R cheeses received a higher score for the
pungent attribute. Similarly, acid, sour or rancid charac-
teristics were also generally higher in these cheeses. It is
likely that these sensory attributes are related to the
presence of volatile and FFAs (Curioni and Bosset, 2002;

Gomez-Ruiz et al., 2002). In general, R cheeses are char-
acterised by more ‘strong’ attributes, such as animal, gar-
lic, spicy, sulphur and unclean. All these characteristics
of R cheeses are linked to the notion of higher maturity,
expressed from a sensory point of view, but also revealed
by physico-chemical patterns, i.e., a greater degree of
proteolysis, a higher content of most volatile com-
pounds, and sometimes greater lipolysis (Fig. 2).
The distribution of milder attributes, such as fruit,
milk or sweet, differs with the study; they can be char-
acteristic of cheeses made either from R or P milk. In
Idiazabal cheese, Ordonez et al. (1999) found a relation-
ship between the sweet taste and the amounts of free
proline and asparagine, which were higher in P cheeses.
Fruity notes may be linked to some methyl ketones
such as 2-nonanone (Gomez-Ruiz et al., 2002) or esters
(Ortigosa et al., 2001; Gomez-Ruiz et al., 2002). Milky
notes are characteristic of diacetyl and acetoin (Gomez-
Ruiz et al., 2002).
The relationship between bitterness and the presence
of the microflora depends on the variety of cheese.
When differences were observed in relation to the milk
treatment, R semi-hard cheeses were more bitter than
P (Buchin et al., 1998; Shakeel-Ur-Rehman et al.,
2000a) or MF cheeses (Buchin et al., 2002). In contrast,
hard cheeses made from R milk were less bitter than
those made from MF or P milk (Bouton and Grappin,
1995; Beuvier et al., 1997; Mendia et al., 1999;
Ginzinger et al., 1999a). On the one hand, it seems that
the presence of the indigenous microflora is involved,
Axis 3 14 %
Acid milk
diMe trisulphide
3Me 2butanol Garlic isopentanone
Animal
1propanol diMe disulphide Fruit
2heptanone 2,3pentanedione
2butanol Bitter C2 Intense Axis 1
2pentanol 1hexanol diacetyl 43 %
C3 2butanone Chemical acetoin
Et hexanoate Rancid 2pentanone 3me butanal
ethanol 3me 2pentanone
Pungent heptane
1butanol
C5 Fresh milk
Figure 2 Distribution of volatile compounds and flavour attrib-
utes (additional variables, italicized and boldfaced) within R ( )
and P () semi-hard Morbier-type cheese using principal
component analysis C2, C3, C5: acetic, propionic, valeric acids
(from Buchin et al., 1998).
Raw Milk Cheeses 337
because P and MF cheeses were similar and differed
from R cheeses. On the other hand, the heat treatment
is likely to play a role. In the study by Beuvier et al.
(1997), where R, P, MF and P  indigenous microflora
(PR) milks were processed, the most bitter cheeses were
P and PR. Bitterness is attributed mainly to the presence
of hydrophobic peptides, resulting from the hydrolysis
of caseins, mostly s1- and -. Bitterness in cheese
results from the balance between the production of bit-
ter peptides by the action of rennet (preferentially in
semi-hard cheeses), plasmin (preferentially in hard-
cooked cheeses), bacterial proteinases and peptidases,
and their further degradation by bacterial peptidases.
The role of the respective proteolytic systems and their
interactions are known to differ according to the cheese
variety (Bergère and Lenoir, 1997). In Bergkäse, a Swiss-
type cheese, Ginzinger et al. (1999a) found more
hydrophobic peptides in P cheeses, which were also
more bitter than R cheeses. This distribution of peptides
was confirmed in Cheddar by Lau et al. (1991). Accord-
ing to Gomez et al. (1997), bitterness of peptide origin
is more likely to be masked by other flavour compon-
ents in R than in P cheeses. Bergère and Lenoir (1997)
pointed out that other components such as indole,
amino acids, amines, amides, long-chain ketones or
monoglycerides could contribute to the bitter taste of
cheese. Thus, Ordonez et al. (1999) found a relation-
ship between bitterness and the amounts of arginine
and aromatic amino acids in Idiazabal, though no differ-
ences in bitterness were found between R and P
cheeses.
It is likely that the presence of the R milk microflora
affects the flavour characteristics in two ways: on the
one hand, acceleration of ripening by faster metabolic
pathways, and, on the other hand, the occurrence of a
greater variety of metabolic pathways, specific to par-
ticular strains of bacteria, which is also influenced by
the microbial diversity.
The acceptability of R cheeses is also dependent on
the cheese variety. Cheddar cheese made from raw milk
is, in general, of lower quality than that made from
pasteurised milk ( Johnson et al., 1990b; McSweeney
et al., 1993). In the study of Shakeel-Ur-Rehman et al.
(2000b), R Cheddar cheese received higher flavour
scores than P cheeses, but this was dependent on the
ripening temperature, as higher temperatures (8 °C
instead of 1 °C) led to defects in R cheeses after
6 months. In the study by Morgan et al. (2001), soft
goat’s milk cheeses had more flavour defects when
made from raw than from pasteurised milk, in relation-
ship to microflora and lipolysis levels. Moreover,
although the ‘goat’ flavour of these cheeses is linked to
the liberation of particular fatty acids (Le Quéré et al.,
1996), no differences were found in this attribute.
338 Raw Milk Cheeses
According to Klantschitsch et al. (2000), the quality of
Raclette cheese in relation to raw milk is related to
ripening temperature and time; to avoid flavour and
openness defects, R cheeses should be ripened for less
than 90 days at 11 °C or 60 days at 14 °C, whereas P or
MF milk cheeses can be ripened at 17 °C for 90 days.
These differences in acceptability are of course related
to the speed of ripening, since the presence of the
native microflora accelerates biochemical transforma-
tions in the cheese. The difference in maturity, and
hence in the occurrence of defects, is more perceptible
in soft or semi-hard cheeses, because of their high
moisture content; biochemical activities are favoured
by the presence of water. Thus, besides the elimination
of pathogens, pasteurisation is useful in this type of
cheese to obtain a longer shelf-life by slowing the
ripening and delaying the occurrence of flavour
defects. The consumer of these cheeses is used to the
milder flavour provided by pasteurised milk, and may
regard the stronger flavour of R cheeses as a defect. It
is likely that these varieties of cheese made from raw
milk would be appreciated mostly by ‘connoisseurs’.
Conversely, Swiss-type cheeses, hard Italian cheeses
( Johnson et al., 1990b; Bouton and Grappin, 1995), or
hard Spanish ovine cheeses, like Idiazabal (Ordonez
et al., 1999; Chavarri et al., 2000), are preferred when
made from raw milk. Because of their low moisture
content, hard cheeses ripen more slowly than soft or
semi-hard ones. The presence of the natural microflora
in the raw milk may have a lesser influence on the
speed of ripening and on the shelf-life of these cheeses.
The use of raw milk does not induce defects, and may
even reduce some, e.g., bitterness. Moreover, the more
complex flavour provided by raw milk may be appreci-
ated by the consumer of hard cheeses. The loss of
microflora and, to a lesser extent, of native milk enzyme
activities, in pasteurised milk, affects the typical flavour
of these cheeses. In Idiazabal cheese, the level of the
sensory scores was related to the level of lipolysis, the
less lipolysed cheeses being rated ‘rather mild’, suggest-
ing that this cheese requires a minimum level of lipoly-
sis to develop its characteristic flavour (Chavarri et al.,
2000). In hard Italian cheeses such as Romano, Parmesan
or Asiago, the inhibition of milk lipase (LPL) in pas-
teurised milk may be detrimental to the development of
typical flavour (Johnson et al., 1990b). In goats’ milk
cheeses, the preservation of LPL activity can be import-
ant for the development of the ‘goat’ flavour, linked to the
liberation of typical goat-flavoured fatty acids from gly-
cerides (Le Quéré et al., 1996). According to Ordonez
et al. (1999) and Chavarri et al. (2000), the characteristic
Idiazabal flavour is related to the extent of proteolysis. In
Swiss-type cheeses, Bouton and Grappin (1995) found a
relationship between the extent of primary proteolysis
and the flavour intensity, whereas the typical flavour was
related to the concentration of propionic acid.
The presence of the raw milk microflora contributes
to the sensory diversity of raw milk cheeses. This has
been supposed by Shakeel-Ur-Rehman et al. (2000b) for
Cheddar cheese, and shown in Swiss-type cheese models
by Beuvier et al. (1997) and Demarigny et al. (1997).
There is a higher heterogeneity in the sensory character-
istics of cheeses when the native microflora was retained
than when it was removed from milk (Fig. 3).
The diversity of R cheeses is likely to depend on the
level but also on the nature of the strains present in the
microflora. Whether the strains in themselves have dif-
ferent metabolic potentialities or interfere by affecting
the activity of starter bacteria has not yet been eluci-
dated. Nevertheless, Bouton and Grappin (1995) have
shown an interaction between the composition of
starter mixtures and the raw milk microflora in the bio-
chemical transformations and sensory characteristics of
Swiss-type cheeses. Thus, whatever the mechanisms
involved, the preservation of the microbial diversity in
raw milk seems to contribute to the diversity of cheeses
such as Swiss-type cheeses, particularly Comté. This
diversity in the sensory characteristics is a point of
major interest in the production of PDO cheeses.
Texture
The texture of cheeses is the macroscopic expression of
the structure of the cheese matrix, i.e., its composition
and organisation. The texture is formed during two
Axis 2
17 %
R
MF Ferm Entero
Elastic Granular MesoLb PAB
pH Axis 1
iC5 55 %
PTA
P C3
P + bact Pungent
Bitter
αs1-CN Salted Aroma
C2
Acid intensity
γ-CN
NaCl
Figure 3 Distribution of physico-chemical, microbiological and
flavour criteria (additional variables, italicized and boldfaced)
within raw (R), pasteurised (P), microfiltered (MF) and pas-
teurised  microorganisms contained in retentate (P  Bact)
milk using principal component analysis. MesoLb: mesophilic
lactobacilli; Entero:enterococci; PAB: propionibacteria; C2: acetic
acid; C3: propionic acid; iC5: isoavaleric acid; PTA: PTA-soluble
N (from Beuvier et al., 1997).

stages of cheese processing: manufacturing and ripen-
ing. The events that occur during these two steps are
different in nature.
During manufacture, the cheese matrix is formed.
It begins with coagulation of the milk, where the
caseins organise themselves into a network, entrap-
ping fat globules, water pockets and gas bubbles. The
initial structure of the network is thus determined by
the composition of the milk, and also by the techno-
logical conditions of coagulation: renneting param-
eters and work in the vat which influence the
moisture content of the curd. The network is then
modified by acidification due to the fermentation of
lactose, that begins in the vat and continues in the
mould. Acidification influences the extent of mineral-
isation of the caseins, and thus their hydration as well
as their interactions.
During ripening, changes occur in the matrix
through the influence of the loss of water and proteoly-
sis. Proteolysis begins with coagulation in the vat; this
is essentially primary proteolysis, i.e., internal hydroly-
sis of casein molecules, by the coagulant or indigenous
enzymes of milk, such as plasmin. Secondary proteoly-
sis occurs essentially during ripening, by the action of
peptidases of microorganisms. Proteolysis weakens the
structure of the casein matrix.
It can be easily supposed that removal of the native
microflora from raw milk may alter the texture of sub-
sequent cheeses by two major mechanisms. On the
one hand, the heat treatment of the milk used to
destroy the microflora may alter the structure of the
casein matrix by denaturation of whey proteins or the
loss of water, or modify the proteolysis patterns by
denaturation, activation or modified retention of
enzymes. On the other hand, the elimination of most
of the indigenous microflora, either by heating or
microfiltration, may modify the biochemical changes
in cheeses, in particular proteolysis (Grappin and
Beuvier, 1997).
Among all the articles in which R, P or MF cheeses
were compared, few deal with cheese texture. Some
work resulted in no differences related to the treatment
of milk: no clear differences between R/P/MF milks
(McSweeney et al., 1993) and R/P milks (Shakeel-Ur-
Rehman et al., 1999) in Cheddar cheese, no sensory tex-
tural differences between R/MF milk Swiss-type cheeses
(Bouton and Grappin, 1995) or rheological differences
between R/P Bergkäse cheese (Ginzinger et al., 1999a).
The comparison of R and P cheeses from a texture point
of view is difficult, because of the differences in the
behaviour of milk during the coagulation step, due to
the heat treatment. Depending on the cheesemaking
procedures, contradictory findings have been reported,
in terms of moisture, on the compositional differences of
Raw Milk Cheeses 339
cheeses (Lau et al., 1990; Buffa et al., 2001b), or pH
(Buffa et al., 2001b). Texture differences are thus diffi-
cult to interpret.
The results of Beuvier et al. (1997) seem to indicate
that in Swiss-type cheeses, sensory texture characteris-
tics of R cheeses are influenced by both the heat treat-
ment of milk and the activity of the indigenous
microflora. They showed in a comparison of R, P, MF
and P cheeses to which the indigenous microflora con-
tained in microfiltration retentate had been added, that
R cheeses had a firmer and more granular texture.
Proteolysis appears to be the main factor responsible
for differences in texture between R and P cheeses.
According to Shakeel-Ur-Rehman et al. (2000a), the
increase in chymosin retention and in plasmin activity
by heat-treatment of milk is the major cause of texture
differences between R and P Cheddar cheeses ripened
at 1 or 8 °C. While the temperature influenced all tex-
ture descriptors, milk treatment influenced only rub-
beriness (P  R) and graininess (R  P). They
attributed the texture characteristics mostly to differ-
ences in water-soluble N (WSN) (Shakeel-Ur-Rehman
et al., 2000b), in which enzymes such as chymosin or
plasmin have more influence than the activity of the
indigenous microflora.
Gaya et al. (1990) found a lower fracturability, elas-
ticity and hardness in Manchego cheese made from R
ewes’ milk than in P cheeses, whatever the ripening
time (2 or 4 months) and the ripening temperature
(between 8 and 16 °C). They attributed these differ-
ences to higher secondary proteolysis in R cheeses,
measured by pH 4.6-, TCA- and PTA- soluble N. Buffa
et al. (2001b) studied the rheological characteristics of
goats’ milk semi-hard cheeses made from R or P milk.
R cheeses were firmer, less fracturable, and more cohe-
sive than P ones. These characteristics were attributed
to the levels of moisture and WSN: the lower the mois-
ture and more intact the caseins, the less the fractur-
ability and deformability. Fracture stress was higher for
R cheeses, i.e., a lower fracturability than the P cheeses.
This parameter was correlated with the levels of mois-
ture and WSN: the less the moisture and more intact
the caseins, the less the fracturability. Fracture strain,
which describes the deformability of cheese, was
higher for R cheeses, but only at one day. It could be
due to the higher pH of these cheeses at this stage,
water being partly absorbed to hydrate the negative
charges formed in caseins with high pH values. This
parameter has the same correlation with moisture and
WSN as previously – deformability decreases when the
hydration of proteins decreases and when elastic struc-
tural elements disappear. The microstructure of R
cheeses was more regular, with a closed protein matrix,
and smaller and more uniform fat globules, whereas
340 Raw Milk Cheeses
P cheeses had an open structure with irregular cavities.
As a consequence, differences in colour were observed.
Rosenberg et al. (1995) measured the viscoelastic
characteristics, G (storage modulus) and G (loss modu-
lus), of Cheddar cheeses. These parameters were higher
in R cheeses than in P cheeses ripened for 8 months. In
P cheeses, they were found to be related to the extent
of proteolysis; a higher G signified a higher elastic
behaviour of the matrix with the accumulation of prote-
olysis products. The authors explained this observation
by the binding of water by the ionic groups liberated by
the cleavage of peptide bounds. This relation with the
extent of proteolysis was not observed in R cheeses,
maybe because of different proteolytic activities during
the ripening of these cheeses, as revealed by differences
in peptide composition.
Mendia et al. (1999) found more graininess and
firmness and less creaminess and elasticity in R ewes’
milk Idiazabal cheeses than in P cheeses. These differ-
ences were attributed to the slower maturation of P
cheeses. This was confirmed by the fact that the differ-
ences diminished with increases in ripening time, and
were thought to be linked to the moisture content.
For certain types of cheese consumed mainly in a
melted form, such as Raclette, it is more interesting to
evaluate the texture characteristics of the cheese after
melting. Melting properties were evaluated in Raclette
cheeses made from R, P or MF milk and mixtures of
the three types of milk in different proportions
(Klantschitsch et al., 2000). R cheeses had a longer
consistency than P/MF cheeses after 90 days ripening.
According to the authors, this is related to the proteoly-
sis patterns, proteolysis ‘in width’, pH 4.6 N/TN (lower
in MF) leading to longer consistency and higher vis-
cosity, proteolysis ‘in depth’ (NPN/TN) leading to
shorter consistency. The viscosity did not differ
between the cheeses. The firmness of melted cheese
was also higher in R than in P/MF cheeses after 90 days
ripening, with a score indicating insufficient melting
quality. Fat separation increased more rapidly with
ripening time in R than in P cheeses. Softening and
dropping points were in the range for good melting
quality in all cheeses ripened at 11 or 14 °C, but only
in the MF cheeses ripened at 17 °C. The effect of the
microflora on the melting quality of Raclette is depen-
dant on the ripening temperature and time; a high
temperature (17 °C) is detrimental when using raw
milk, whereas, in the case of microfiltered milk, it is
useful to accelerate ripening.
In all these studies, the lack of microbial investiga-
tions made it difficult to establish a relationship
between the microbial populations, whether of indigen-
ous or starter origin, and the characteristics of texture.
Nevertheless, when the observed differences were
attributed to the secondary proteolysis, microbial activ-
ity was involved.
Conclusion
Microbial communities play an essential role in the
control of sensory qualities of cheese. They are more
diverse and complex in R cheeses for which milk
undergoes no treatment to reduce the microflora. They
contribute to the development of a typical cheese taste
and flavour. Diversity of the sensory qualities is a spe-
cific feature of R cheese. Elimination of the raw milk
microflora by pasteurisation or microfiltration defini-
tively leads to different cheeses from a sensorial point of
view. Still, is it necessary to have raw milk that is suffi-
ciently rich in terms of quantity and diversity of
microorganisms?
As outlined at the beginning, the improvement in
hygienic practices on farms has led to a ‘clean’ raw milk,
with low microbial counts (Odet, 1999). Raw milk with
a low level of microbes could induce a reduction in sen-
sorial diversity of cheese due to a reduction of microbial
diversity. Indeed, Dasen et al. (2003) have observed that
the strain diversity of mesophilic lactobacilli in raw
milk experimental Cheddar cheese was close to that
observed in industrial Cheddar cheese manufactured
with pasteurised milk. The former was made from raw
milk with a total of around 10 000 cfu ml 1. The fact
that raw milk tends to be more and more microbiologic-
ally ‘clean’ implies that there is a risk that the sensorial
differences between R and P cheeses will be erased.
Some experiments in progress, particularly in
France, aim to evaluate dairy farming practices, includ-
ing milking practices, on the raw milk microflora in
terms of quantity and diversity. Recently, Michel et al.
(2001) observed links between milking practices and
the bacteriological quality of milk, showing that it is
possible to manage the microbial quality of milk on the
farm to promote the technologically ‘useful’ microflora,
while maintaining pathogens at a low level. This is a
good way to keep the natural microflora in R cheese
production, in terms of quantity and diversity, in order
to preserve their sensorial diversity. To add selected
microorganisms could enhance the aroma of cheese, but
the cheese would have a more uniform flavour, a char-
acteristic which is not sought by both the producers
and the consumers, because diversity of flavour is consid-
ered a special feature of traditional R cheeses (Grappin
and Beuvier, 1997).
Otherwise, according to Montel (2002), microbial
communities may play a key role in the microbiological
safety of R cheese. This potential role is supported
by several studies in which cheeses or milk, with
a more complex microflora, were less contaminated by

L. monocytogenes than those with a less diversified flora
(Brouillaud-Delattre et al., 1997; Eppert et al., 1997).
Thus, well-monitored microbial diversity, from farm to
cheese, by acting as a barrier against pathogens, may be
a trump card for cheese safety (Montel, 2002).
According to Stanton et al. (1998), cheeses, because
of their high fat content and their texture, could offer
protection to the living microorganisms contained
within them, especially at the moment of their passage
into the gastrointestinal tract of the consumer. More and
more studies demonstrate the beneficial effects on health
of strains of microorganisms and give hope for other
discoveries in R cheeses, which are rich in microorgan-
isms (Bouton, 2001; Moreau and Vuitton, 2002).
The preservation of the microbial diversity in raw
milk, essential to obtain cheeses with greater sensorial
diversity, more and more appreciated by (European)
consumers, potentially useful to fight against pathogens
and potentially useful for health, is a challenge for milk
and cheese producers, and researchers, to take up over
the next years.
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This Page Intentionally Left Blank
 Biochemistry of Cheese Ripening:
Introduction and Overview
P.L.H. McSweeney, Department of Food and Nutritional Sciences, University College,
Cork, Ireland
Introduction
As discussed in ‘Cheese: An Overview’, Volume 1, rennet-
coagulated cheeses are ripened (matured) for a period
ranging from 2 weeks (e.g., Mozzarella) to 2 or more
years (e.g., Parmigiano Reggiano or extra-mature
Cheddar) during which the flavour and texture char-
acteristic of the variety develop. Ripening usually
involves changes to the microflora of the cheese,
including death and lysis of the starter cells, develop-
ment of an adventitious non-starter microflora and, in
many cheeses, growth of a secondary microflora (e.g.,
Propionibacterium freudenreichii subsp. shermanii in
Swiss cheese, moulds in mould-ripened varieties and a
complex Gram-positive bacterial microflora on the
surface of smear-ripened cheeses). The metabolic
activity of the secondary microflora often dominates
flavour development, and in some cases, e.g., white-
mould cheeses, the texture, of varieties in which they
grow. The microbiology of cheese during ripening is
discussed in ‘The Microbiology of Cheese Ripening’,
Volume 1. As discussed in ‘Rheology and Texture of
Cheese’, Volume 1, ripening usually involves the soft-
ening of cheese texture, as a consequence of the
hydrolysis of the casein matrix, changes in the water-
binding ability of the curd and changes in pH (which
may cause other changes such as the migration and
precipitation of calcium phosphate). The flavour of
cheese curd immediately after manufacture is rather
bland and indeed it can be difficult to differentiate
the flavours of certain varieties at this stage. During
ripening, cheese flavour develops due to the produc-
tion of a wide range of sapid compounds by the bio-
chemical pathways described below. Volatile flavour
compounds are of particular importance to cheese
flavour and are discussed in ‘Sensory Character of
Cheese and its Evaluation’, Volume 1. Quantification
of the volatile flavour compounds of cheese are
described in ‘Instrumental Techniques’, Volume 1.
Biochemical reactions which occur in cheese during
ripening are usually grouped into four major cat-
egories: (1) glycolysis of residual lactose and catabol-
ism of lactate, (2) catabolism of citrate, which is very
important in certain varieties, (3) lipolysis and the
Cheese: Chemistry, Physics and Microbiology, Third edition – Volume 1: General Aspects
ISBN: 0-1226-3652-X
Set ISBN: 0-1226-3651-1
catabolism of free fatty acids and (4) proteolysis and
the catabolism of amino acids (Fig. 1). These reactions
are discussed in ‘Metabolism of Residual Lactose and
of Lactate and Citrate’, ‘Lipolysis and Catabolism of
Fatty Acids in Cheese’, ‘Proteolysis in Cheese during
Ripening’, ‘Catabolism of Amino Acids in Cheese dur-
ing Ripening’, Volume 1. Since the biochemistry of
cheese ripening is complex, the purpose of this chap-
ter is to present an overview of the principal biochem-
ical pathways which contribute to cheese ripening and
to discuss the role of the principal ripening agents in
cheese and the acceleration of cheese ripening. Aspects
of cheese ripening common to many varieties are dis-
cussed in ‘Metabolism of Residual Lactose and of Lac-
tate and Citrate’, ‘Lipolysis and Catabolism of Fatty
Acids in Cheese’, ‘Proteolysis in Cheese during Ripen-
ing’, ‘Catabolism of Amino Acids in Cheese during
Ripening’, Volume 1; ripening of specific varieties is dis-
cussed in the relevant chapters in Volume 2.
Glycolysis of Residual Lactose and
Catabolism of Lactate
Since cheeses are fermented dairy products, the metab-
olism of lactose to lactate is essential in the manufac-
ture of all varieties. Cheese curd contains a low level
of residual lactose which is metabolised rapidly early
in ripening to lactate which may be catabolised subse-
quently via a range of pathways. Catabolism of lactate
probably occurs in all cheeses and is particularly
important in surface mould-ripened varieties (e.g.,
Camembert) and in Swiss cheese. These reactions were
reviewed by Fox et al. (1990, 1993) and McSweeney
and Sousa (2000) and are discussed in detail in
‘Metabolism of Residual Lactose and of Lactate and
Citrate’, Volume 1.
The pathway through which lactose is metabolised
depends on the starter type (see ‘Starter Cultures: Gen-
eral Aspects’, Volume 1; Cogan and Hill, 1993; Fox
et al., 2000; McSweeney and Sousa, 2000; Broome
et al., 2003). The final step in the glycolysis of lactose
is the conversion of pyruvate to lactate which is cata-
lysed by lactate dehydrogenase (LDH). Depending on
Copyright © 2004 Elsevier Ltd
All rights reserved
348 Biochemistry of Cheese Ripening: Introduction and Overview

O
Triglyceride COOH
(a) (b) (c) CH2

O O HO C COOH
Caseins O
C
O H2C
O
C Lactose
COOH
O
Chymosin O
C
and plasmin Fermentation Citrate
O by starter
se
Intermediate- pa
sized peptides Li

Proteinases from
Lactococcus
Other products
H H
Short peptides
O
C C
Peptidases of C OH OH
H3C COOH H3C
Lactococcus OH COOH
and NSLAB Fatty acid
D-Lactate
L-Lactate
Racemisation
by NSLAB
Amino acids

Fatty acid catabolism

Interactions between Interactions between
products

e
Am products

at
ct
in
o

la
ac

of
id

m
ca

lis
ta
bo

bo
lis

ta
m

Ca
te
m of citra
Metabolis
VOLATILE FLAVOUR COMPOUNDS

Figure 1 General overview of the biochemical pathways which operate in cheese during ripening.

the type of LDH (D- or L-LDH) in the cell, D- (e.g.,
Lb. delbrueckii subsp. bulgaricus), L- (e.g., Lactococcus,
Sc. thermophilus) or D/L- (e.g., Lb. helveticus) lactate is
the end product of glycolysis which converts 1 mol of
lactose to 4 mol of lactate with the production of
4 mol of ATP. Unlike most lactic acid bacteria (LAB),
Leuconostoc spp. use the phosphoketolase pathway to
metabolise lactose; the end products of this pathway
are lactate, ethanol and CO2 and thus differ from that
of the glycolytic pathway. Although essential for cheese
manufacture, the metabolism of lactose to lactate is
essentially complete at the end of manufacture or dur-
ing the early stages of ripening. Most lactose in milk is
lost in the whey and that which is retained in the curd
is metabolised rapidly after drainage. However, the
activity of the starter is greatly reduced at the end of
manufacture or soon thereafter due to the combination
of low pH, high NaCl and lack of a fermentable carbo-
hydrate. The inhibition of acid production is particu-
larly abrupt in dry-salted varieties (e.g., Cheddar)
where NaCl concentration reaches equilibrium much
faster than in brine-salted cheeses. Fresh cheese curd
contains a low level of lactose which, in the case of
Cheddar cheese, is reduced to trace levels within one
month of ripening by the (albeit reduced) activity of
the starter or by the action of the non-starter lactic
acid bacteria (NSLAB). Lactate contributes to the
flavour of cheese, particularly early during maturation,
but the major effect of acidification on flavour devel-
opment is indirect since, together with the buffering
capacity of the curd, it influences pH and thus the
growth of the secondary flora and the activity of ripening
enzymes.
Lactate is an important substrate for a range of reac-
tions which contribute positively or negatively to
cheese ripening. L-Lactate, produced by Lactococcus,
can be racemised to DL-lactate by the NSLAB flora
in Cheddar and Dutch-type cheeses. DL-Lactate is less
soluble than L-lactate, resulting in the formation of
Ca-D-lactate crystals which appear as white specks on
the surface of the mature cheese. Lactate can also be
metabolised to acetate and CO2 by some members of
the NSLAB flora, although this oxidative pathway is
relatively minor in cheese due to its low oxidation–
reduction (redox) potential (c. 250 mV) and is lim-
ited by the availability of O2. Late gas blowing is a
defect in certain hard and semi-hard varieties caused by
the anaerobic catabolism of lactate to butyrate and H2
 Biochemistry of Cheese Ripening: Introduction and Overview 349
by Clostridium tyrobutyricum. This problem can be over-
come by good hygiene, addition of NaNO3 or lysozyme
or by the physical removal of the spores by bactofuga-
tion or microfiltration.
However, catabolism of lactate is particularly
important in Swiss and surface mould-ripened cheeses.
In the former, lactate is catabolised by Propionibacterium
freudenreichii subsp. shermanii to propionate, acetate,
H2O and CO2. Propionate and acetate contribute to
the flavour of Swiss cheese; CO2 migrates through the
curd to points of weakness where it collects to form
the large eyes characteristic of Swiss-type cheese. The
oxidative catabolism of lactate to H2O and CO2 by
Penicillium camemberti at the surface of Camembert
and Brie-type cheeses is of great indirect importance to
their ripening. The catabolism of lactic acid causes a
large increase in the pH of the surface of these cheeses
which leads to a pH gradient from the surface to the
core and to the migration of lactate towards the sur-
face. The high pH at the surface causes precipitation of
calcium phosphate, which, in turn, causes the migra-
tion of calcium and phosphate to the cheese surface.
These changes lead to the characteristic softening of
surface mould-ripened cheese which, when mature,
have an almost liquid-like consistency. Oxidative
metabolism of lactate is also of significance at the sur-
face of smear-ripened cheeses (e.g., Tilst or Limburger)
where, early in ripening, yeasts deacidify the surface
which encourages the growth of the Gram-positive
bacteria characteristic of the surface. Oxidative metab-
olism of lactate probably also occurs in Blue cheese
but its effect is less important than in surface mould-
ripened cheese since P. roqueforti is distributed
throughout the cheese and thus gradients do not
develop across the cheese mass.
Lipolysis and Metabolism of Fatty Acids
Studies in which milk fat was replaced with other
lipids have demonstrated that milk fat is essential for
the development of the flavour of Cheddar and prob-
ably all other ripened cheeses. As in all high-fat foods,
lipids present in cheese can undergo hydrolytic or
oxidative degradation; the latter is generally con-
sidered not to be important in cheese, primarily due to
its low redox potential. Lipolysis in cheese during
ripening is discussed in detail in ‘Lipolysis and Catab-
olism of Fatty Acids in Cheese’, Volume 1.
As discussed by McSweeney and Sousa (2000) and
Collins et al. (2003a), lipases in cheese originate from a
number of sources. Milk contains an indigenous
lipoprotein lipase (LPL), which contributes to lipolysis
in cheese during ripening. Lipoprotein lipase activity
is more important in cheese made from raw milk than
in that made from pasteurised milk since the enzyme is
extensively inactivated by pasteurisation. Rennet paste,
used as coagulant in certain Italian cheese varieties,
contains a potent lipase, pregastric esterase, which is
responsible for lipolysis in cheeses such as Provolone
and the Pecorino varieties. Lactic acid bacteria are
weakly lipolytic, but their enzymes have been shown to
contribute to the low level of lipolysis characteristic of
Cheddar cheese (Collins et al., 2003b). Likewise,
Pr. freudenreichii subsp. shermanii possesses a lipase
which, together with enzymes from the thermophilic
starter organisms, contributes to the low level of lipoly-
sis in Swiss cheese. Penicillium roqueforti produces
potent extracellular lipases which are responsible for
the extensive lipolysis characteristic of Blue cheese.
P. camemberti and the complex Gram-positive surface
microflora of smear cheeses also produce extracellular
lipases which contribute to lipolysis in surface-bacterial
or white mould-ripened varieties. The level of lipolysis
in cheese is determined using various non-specific tech-
niques (e.g., solvent extraction and titration of the fatty
acids with alcoholic KOH or by the formation of
coloured Cu soaps) or by quantitation of individual
fatty acids, usually by gas chromatography (see Collins
et al., 2003a).
Fatty acids have a direct impact on the flavour of
many cheese varieties. In particular, C4–C10 acids are
strongly flavoured. Levels of fatty acids vary considerably
between varieties. Many internal bacterially ripened
varieties (e.g., Edam, Swiss and Cheddar) contain
low levels of fatty acids (c. 200–1000 mg kg 1). Very
high levels of fatty acids are found in Blue cheese
(c. 30 000 mg kg 1). In addition to their direct role in
cheese flavour, fatty acids are important precursors for
the production of other volatile flavour compounds
during ripening (Fig. 2). Fatty acid esters are produced
by reaction of fatty acids with an alcohol; ethyl esters
are most common in cheese. Thioesters are formed by
reaction of a fatty acid with a thiol compound formed
via the catabolism of sulphur-containing amino acids.
Fatty acid lactones are cyclic compounds formed by the
intramolecular esterification of hydroxyacids; - and
-lactones contribute to the flavour of a number of
cheese varieties. The principal class of volatile flavour
compounds in Blue cheese is n-methyl ketones (alkan-
2-ones) which are produced from fatty acids by partial
-oxidation. n-Methyl ketones may be reduced to the
corresponding secondary alcohols. Fatty acid catabol-
ism is summarised in Fig. 2 and is discussed in detail
in ‘Lipolysis and Catabolism of Fatty Acids in Cheese’,
Volume 1. Volatile flavour compounds in cheese,
including those derived from fatty acids, are usually
quantified using gas chromatography–mass spectrom-
etry (GC–MS; see ‘Instrumental Techniques’, Volume 1).
350 Biochemistry of Cheese Ripening: Introduction and Overview

Triglyceride

O
C
O O
C
O
O
C
O

e
pas OH O
Li C OH

γ- or δ-hydroxy fatty acids

Partial glycerides
H 2O

C
O
O γ- or δ-lactones
C OH

Fatty acids
R-SH
Thiols
CH3CH2OH
Ethanol
Partial β-oxidation

O CO2 O

C S-R O C CH3
C OCH2CH3 Alkan-2-ones
Thioesters
Ethyl esters

OH

CH CH3
Alkan-2-ols

Figure 2 Pathways for the production of flavour compounds from fatty acids during cheese ripening.

Proteolysis and Catabolism of Amino Acids

Proteolysis is the most complex, and in most varieties,
the most important biochemical event which occurs
during cheese ripening. Proteolysis has been discussed
in reviews by Grappin et al. (1985), Rank et al. (1985),
Fox (1989), Fox et al. (1993, 1994, 1995), Fox and
McSweeney (1996), McSweeney and Sousa (2000) and
Sousa et al. (2001) and is covered in detail in ‘Proteoly-
sis in Cheese during Ripening’, Volume 1. Proteolysis is
very important for cheese texture by hydrolysing the
para-casein matrix which gives cheese its structure and
by increasing the water-binding capacity of the curd
(i.e., to the new -carboxylic and -amino groups pro-
duced on cleavage of peptide bonds). Proteolysis may
indirectly affect texture by increasing pH through the
production of NH3 following amino acid catabolism.
Peptides may have a direct impact on cheese flavour
(some are bitter) or they may provide a brothy back-
ground flavour to cheese. However, recent research has
indicated that the major role of proteolysis in cheese
flavour is in the production of amino acids which act as
precursors for a range of catabolic reactions which pro-
duce many important volatile flavour compounds (see
McSweeney and Sousa, 2000; Yvon and Rijnen, 2001).
In most cheese varieties, the initial hydrolysis of
caseins is caused by the coagulant and to a lesser
extent by plasmin and perhaps somatic cell proteinases
(e.g., cathepsin D) which result in the formation of
large (water-insoluble) and intermediate-sized (water-
soluble) peptides which are subsequently hydrolysed
by the coagulant and enzymes from the starter and
non-starter flora of the cheese. The production of
 Biochemistry of Cheese Ripening: Introduction and Overview 351
small peptides and amino acids is caused by the action
of microbial proteinases and peptidases, respectively.
Preparations of selected aspartyl proteinases are
used to coagulate milk. Chymosin (EC 3.4.23.4) is the
principal proteinase (88–94%) in traditional calf ren-
nets, the remainder being pepsin (EC 3.4.23.1) (Rothe
et al., 1977). Although, the principal role of the coagu-
lant in cheesemaking is to coagulate milk, some activ-
ity is retained in the curd, depending on factors such
as coagulant type, cooking temperature and pH at
drainage, and contributes to proteolysis in many var-
ieties (Creamer et al., 1985).
Plasmin (fibrinolysin; EC 3.4.21.7) is the dominant
indigenous proteinase in milk and is produced from its
inactive precursor, plasminogen, by a system of plas-
minogen activators (PA). Inhibitors of plasmin and of
PA are also present in milk. Plasmin, which is opti-
mally active at pH 7.5 and 37 °C, is most active in
high-cook cheeses due to denaturation of inhibitors
and increased activation of plasmin and in cheeses in
which the pH increases during ripening (e.g., Blue
cheese or the surfaces of white-mould and smear-
ripened varieties). Plasmin is most active on -casein,
hydrolysing it at three sites to produce the -caseins
and some proteose peptones. Milk contains somatic
(white blood) cells, which contain lysosomes, which
in turn, contain many proteolytic enzymes. To date,
cathepsin D (see review by Hurley et al., 2000) and
cathepsin B (Magboul et al., 2001) have been con-
firmed in milk.
Lactic acid bacteria (Lactococcus, Lactobacillus,
Streptococcus) possess very comprehensive proteolytic
systems that have been studied extensively and reviewed
(e.g., Fox and McSweeney, 1996; Kunji et al., 1996; Law
and Haandrikman, 1997; Christensen et al., 1999).
Lactic acid bacteria possess a cell envelope-associated
proteinase (PrtP or lactocepin), 3–4 intracellular pro-
teinases, intracellular oligoendopeptidases (PepO, PepF),
a number of aminopeptidases (PepN, PepC, PepG,
PepA, PepL), a pyrolidone carboxyl peptidase (PCP), a
dipeptidylaminopeptidase (PepX), a proline iminopep-
tidase (PepI), an aminopeptidase P (PepP), a prolinase
(PepR), a prolidase (PepQ), general dipeptidases
(PepV, PepD, PepDA) and a general tripeptidase
(PepT). They also possess oligopeptide, di/tripeptide
and amino acid transport systems (Fig. 3). This prote-
olytic system is necessary to enable the LAB to grow to
high numbers in milk (109–1010 cfu ml 1), which
contains only low levels of small peptides and amino
acids. PrtP contributes to the formation of small
peptides in cheese, probably by hydrolysing larger
peptides produced from s1-casein by chymosin or
from -casein by plasmin, whereas the aminopepti-
dases, dipeptidases and tripeptidases (which are intra-
Cell envelope- Intracellular proteinases
associated Endopeptidases
proteinase (PepO, PepF)
(CEP, PrtP, Aminopeptidases
Lactocepin) (PepN, PepC, PepA, PCP, PepL)
Proline-specific peptidases
(PepX, PepI, PepR, PepQ, PepP)
Caseins Dipeptidase
(PepV, PepD, PepDA)
Tripeptidase LYSIS
(PepT)
Amino acids
No carboxypeptidase
CHEESE
Transport Di, tripeptides
systems Oligopeptides
Figure 3 Summary of the proteolytic system of Lactococcus.
The proteolytic systems of other lactic acid bacteria are gener-
ally similar.
cellular) are responsible for the release of free amino
acids after the cells have lysed.
Non-starter lactic acid bacteria, although present
initially at low numbers (50 cfu g 1 in Cheddar
made from pasteurised milk, and probably in other
cheeses), grow at a rate largely governed by ripening
temperature to reach ⬃107 cfu g 1 within 4 weeks and
remain relatively constant thereafter. The activity of
the NSLAB appears to supplement the proteolytic
action of the starter. Non-starter lactic acid bacteria in
cheese are discussed in ‘The Microbiology of Cheese
Ripening’, Volume 1.
In many cheese varieties, a secondary microflora
(secondary starter) is added intentionally and/or
encouraged to grow by environmental conditions and
has a diverse range of functions, depending on the
organisms used. A number of different LAB, e.g.,
strains of Lactobacillus, have been added to Cheddar
cheese with the objective of improving flavour or
accelerating ripening. These have proteolytic systems
similar to those of other species of LAB. Brevibacterium
linens is the best-studied smear microorganism; it
secretes an extracellular proteinase and aminopepti-
dase, and possesses a number of intracellular pepti-
dases, which contribute to proteolysis at the surface of
smear-ripened cheeses (see Rattray and Fox, 1999).
Penicillium roqueforti produces potent extracellular
aspartyl and metalloproteinases and various peptidases
which are major contributors to the extensive proteoly-
sis found in Blue cheese. P. camemberti secretes active
metallo and aspartyl proteinases which contribute to
proteolysis in Camembert and Brie-type cheeses.
The final products of proteolysis are amino acids,
the concentration of which depends on the cheese var-
iety. The concentration of amino acids in cheese at a
given stage of ripening is the net result of the liberation
of amino acids from the caseins by proteolysis and
352 Biochemistry of Cheese Ripening: Introduction and Overview

their catabolism or transformation into other amino
acids by the cheese microflora. The principal amino
acids in Cheddar cheese are Glu, Leu, Arg, Lys, Phe
and Ser.
Medium and small peptides contribute to a brothy
background flavour in many cheese varieties; short,
hydrophobic peptides are bitter. Amino acids con-
tribute directly to cheese flavour as some amino acids
taste sweet (e.g., Gly, Ser, Thr, Ala, Pro), sour (e.g.,
His, Glu, Asp) or bitter (e.g., Arg, Met, Val, Leu, Phe,
Tyr, Ile, Trp). However, research in the last decade has
shown that accelerating proteolysis does not necessar-
ily accelerate flavour development, suggesting that
the production of amino acids is not the rate-limiting
step in the development of cheese flavour. It is now
generally believed that the principal role of proteoly-
sis in the production of flavour compounds is the
liberation of amino acids as precursors for a com-
plex series of catabolic reactions that produce many
important volatile flavour compounds. Amino acid
catabolism was reviewed by Yvon and Rijnen (2001),
and is discussed in detail in ‘Catabolism of Amino
Acids in Cheese During Ripening’, Volume 1 and sum-
marised in Fig. 4. Amino acid catabolism appears to
proceed via two major pathways – transaminase action
and elimination reactions. Transaminases catalyse the
transfer of the -amino group from an amino acid to
an -keto acid (usually -ketoglutarate) with the pro-
duction of the corresponding amino acid and an
-keto acid corresponding to the amino acid substrate
(cf. Fig. 5 for leucine). The second pathway, which is
initiated by elimination reactions, is particularly
important in the production of volatile sulphur com-
pounds from the side chain of methionine. In addition,
decarboxylases remove the carboxylic acid group of
amino acids to produce amines, some of which have
physiological effects (see ‘Toxins in Cheese’, Volume 1).
Decarboxylases may also act on -keto acids to
produce aldehydes, which in turn may be oxidised to
carboxylic acids or reduced to primary alcohols. The
-amino group of amino acids may be removed by the
action of deaminases, with the formation of a car-
boxylic acid and ammonia. In addition, the side
chains of amino acids may be degraded by the action
of various lysases (see ‘Catabolism of Amino Acids in
Cheese during Ripening’, Volume 1).
Ripening Agents in Cheese
Agents from five, and possibly six, sources are involved
in the ripening of cheese:
• enzymes from the coagulant;
• indigenous milk enzymes;

Coagulant
Casein Large and small polypeptides
Plasmin
Starter proteinases

Small peptides

Starter and non-starter peptidases

Small peptides Amino acids

Deaminases
NH3
Decarboxylases
α-Keto acid 1 CO2
Acids Lyases

Transaminases Amines
Carbonyls

α-Amino acid

α-Keto acid 2

Various compounds
(e.g., sulphur compounds)

Figure 4 Summary of proteolysis and amino acid catabolism in cheese during ripening.
 Biochemistry of Cheese Ripening: Introduction and Overview 353

O O

O C C OH H2 N CH C OH

α-Ketoglutarate CH2 CH2

Glutamic acid
CH2 CH2 OH

CH2
C O C O

CH2
OH OH

CH CH3
CO2
O O
CH3
O H
H2N CH C OH O C C OH C
TRANSAMINASE DECARBOXYLASE 3-Methylbutanol
CH2 CH2 CH2

CH CH3 O OH
CH CH3 CH CH3 C

CH3 CH3 CH3 CH2

Leucine 2-Keto-4-methylpentanoic acid 3-Methylbutanal CH CH3

CH3
DECARBOXYLASE

DE
AM 3-Methylbutanoic acid
IN
CO2 AS
E O

C OH
NH3
CH2

CH2
NH2
CH CH3
CH2
CH3
CH2

CH CH3
4-Methylpentanoic acid

CH3

3-Methylbutylamine

Figure 5 Catabolism of leucine initiated by transaminase, deaminase or decarboxylase action and volatile flavour compounds
which may be formed from this amino acid. Similar catabolic pathways operate for the other branched-chain aliphatic amino acids
(isoleucine and valine).

• starter bacteria and their enzymes, which are released Non-starter bacteria may be eliminated by using an
after the cells have died and lysed; aseptic bucket milking technique, developed by Perry
• enzymes from secondary starters (e.g., Propionibac- and McGillivray (1964); the teat cups and clusters were
terium freudenreichii subsp. shermanii, Gram-positive chemically sterilised and the bucket steam-sterilised.
bacteria on the surface of smear-ripened cheese, Cows were screened for the bacteriological quality of
yeasts and moulds, such as Penicillium roqueforti their milk and animals with counts 100 cfu ml%l
and P. camemberti), which are of major importance selected; prior to milking, their udders were cleaned with
in some varieties; a quaternary ammonium solution. An essentially similar
• non-starter bacteria, i.e., organisms that either sur- approach was used by O’Keeffe et al. (1976a), who
vive pasteurisation of the cheese milk or gain access obtained milk with a total bacterial count 500 cfu ml%1.
to the pasteurised milk or curd during manufacture; Kleter and de Vries (1974) included a cooling coil
and, in certain circumstances, between the cluster and the bucket and succeeded in
• exogenous enzymes added to accelerate cheese achieving counts averaging 46 cfu ml%1. This approach
ripening. was also used by Visser (1976). Reiter et al. (1969) with-
drew milk aseptically by means of a teat cannula, but the
There has been interest for about 40 years in quantities obtained (l l) were sufficient to produce
developing model systems in which to quantify the cheeses of only about 100 g. More recently, it has been
contribution of each of these agents to cheese ripen- our experience (McSweeney et al., 1994; Lynch et al.,
ing. The techniques developed eliminate one or more 1996, 1997) that special precautions for milking are
of the above agents, thereby enabling its role to be unnecessary; good quality raw milk pasteurised at 78 °C
assessed, directly or indirectly. for 15 s is suitable for aseptic cheesemaking.
354 Biochemistry of Cheese Ripening: Introduction and Overview
Having collected low-count milk, a heating step is
usually used to reduce bacterial counts further. Perry and
McGillivray (1964) used batch pasteurisation (68 °C 
5 min) in a steam-jacketed cheese vat. Chapman et al.
(1966), who did not use an aseptic milking technique,
used HTST pasteurisation (71.6 °C  17 s) to produce
low-count milk. Reiter et al. (1967), Kleter and de
Vries (1974) and Visser (1976, 1977a) also used HTST
pasteurisation. An LTLT regime (63 °C  30 min) was
used by Reiter et al. (1969) and O’Keeffe et al.
(1976a). Le Bars et al. (1975) used a UHT treatment
and offset the ill-effects of the high heat treatment on
the rennetability of milk by using a higher rennet con-
centration, a higher setting temperature and adding
CaCl2. Tyndallisation (three successive cycles of heat-
ing at 75 °C  5 min) or treatment with H2O2 fol-
lowed by catalase was used by Roberts et al. (1995) to
treat milk for the production of aseptic cheese curd.
Both treatments were successful, although H2O2
caused the development of oxidised off-flavours.
Cheese with a controlled microflora must be manu-
factured under aseptic conditions. Enclosed vats
equipped with integral rubber gauntlets were used by
Mabbitt et al. (1959) and modified by Perry and
McGillivray (1964) to include pressurised or sterile
air. Chapman et al. (1966) and Reiter et al. (1967,
1969) used a similar technique. Le Bars et al. (1975)
made cheese in an aseptic room (5  3 m) with a fil-
tered air supply and the cheesemakers were clothed in
sterile garments. O’Keeffe et al. (1975, 1976a,b),
McSweeney et al. (1994) and Lynch et al. (1996, 1997)
made cheese in 20-l vats set in thermostatically con-
trolled water baths in a laminar air-flow unit. If the
cheese curd is to be acidified chemically, antibiotics
should be added to the cheesemilk to inhibit the
growth of any surviving (or contaminating) bacteria.
Nisin, penicillin and streptomycin were used by Le
Bars et al. (1975) and O’Keeffe et al. (1976a). Addition
of antibiotics is probably necessary to achieve aseptic
starter-free cheese.
Acidification of cheese curd to ⬃pH 5, which is an
essential element of cheese manufacture, is normally
achieved by in situ production of lactic acid by a cul-
ture of LAB (starter). If the contribution of starter to
cheese ripening is to be assessed, the use of starter
must be avoided and acidification is then accom-
plished by pre-formed acid or acidogen. Early workers
used dilute acid for direct acidification but encoun-
tered difficulties in controlling the pH. Mabbitt et al.
(1955) largely overcame this problem by using an aci-
dogen, gluconic acid--lactone (GDL), which hydroly-
ses to gluconic acid at a predictable rate in aqueous
solutions. O’Keeffe et al. (1975) found that GDL, used
as recommended by Mabbitt et al. (1955), caused
excessively rapid acidification, leading to extensive
demineralisation of the casein micelles. Demineralisa-
tion was considered to be responsible for the exces-
sively rapid rate of proteolysis observed in chemically
acidified cheese but this may have been due to
increased retention or activity of rennet in over-acid
curd (Creamer et al., 1985). O’Keeffe et al. (1975) over-
came excessively rapid acidification by using incre-
mental addition of lactic acid to mimic the pH drop
during cooking, followed by the addition of GDL to
the curd after whey drainage.
Roberts et al. (1995) developed an aseptic system
for making cheese curd which was then used to pro-
duce slurries. The system consisted of a 1L cylindrical
polypropylene ‘vat’ fitted with a lid and a stainless
steel cutter/stirrer. The milk was coagulated, cut,
cooked and stirred in the sealed ‘vat’. At the end of
cooking, the lid was replaced by a steel mesh screen
through which the whey was drained off.
Role of rennet in cheese ripening
The manufacture of rennet-free cheese is necessary if
the contribution of rennet to ripening is to be asses-
sed. Since rennet must be used to form a para-casein
curd, the approach usually adopted is to inactivate the
rennet after it has completed the first stage of rennet-
induced coagulation.
Four techniques have been developed to achieve
this objective. Visser (1976) used cheesemilk which
had been depleted of Ca and Mg by treatment with an
ion-exchange resin; at the reduced Ca concentration,
the enzymatic phase of renneting could be completed
without coagulation. The rennet was subsequently inac-
tivated by heat treatment (72 °C  15 s), the milk
cooled to 5 °C and CaCl2 added. To induce coagulation,
the renneted milk was heated dielectrically to avoid agi-
tation. Cheesemaking was then completed in aseptic
vats. This technique was used by Visser (1977a,b,c) and
Visser and de Groot-Mostert (1977).
Porcine pepsin is very unstable at pH values near or
above neutrality. O’Keeffe et al. (1977) used porcine
pepsin as coagulant; after the gel had formed, it was cut
and the pH of the curds–whey mixture raised to ⬃7
using NaOH; this technique has been used subsequently
by Lane et al. (1997) with satisfactory results. Mulvihill
et al. (1979) demonstrated the potential of piglet chy-
mosin for the manufacture of rennet-free cheese; this
enzyme hydrolyses bovine -casein but appears to be
inactive on s1- or -caseins or to be inactivated rapidly
during the early stages of cheesemaking. Its use in
cheesemaking was demonstrated in small-scale experi-
ments. Meinardi et al. (1998) developed methodology
and equipment for the production of rennet-free cheese
 Biochemistry of Cheese Ripening: Introduction and Overview 355
using pH-inactivated pepsin. Pasteurised milk supple-
mented with CaCl2 was cooled to 6 °C in a cylindrical
glass vessel (15 l) equipped with a series of glass tubes
through which water could be circulated to control the
temperature and to heat the contents of the vessel with-
out stirring. Porcine pepsin was added and the first stage
of rennet action allowed to progress. The coagulant was
inactivated by titrating the milk to pH 7.8 using NaOH
and holding at this pH for 45 min. The milk was
adjusted to pH 7.0 using HCl; starter was then added
and addition of HCl continued until the milk reached
pH 6.5.
Shakeel-Ur-Rehman et al. (1999) added pepstatin A
(isovaleryl-Val-Val-statine-Ala-statine), a potent inhibitor
of aspartyl proteinases, to the curds–whey mixture dur-
ing cooking; results indicated that this compound very
effectively inhibited chymosin action in cheese during
ripening. Immobilised rennets have been suggested as
another approach to making rennet-free curd (e.g., Fox
et al., 1993) but leaching of the enzyme from the solid
support makes this technique unsuitable. Furthermore,
properly immobilised rennets are unable to coagulate
milk as the Phe9Met bond of -casein may not be able
to reach the active site of immobilised chymosin and the
rate of diffusion of large casein micelles to immobilised
rennet is very slow compared to the rate of diffusion of
the small chymosin molecule towards the casein micelle
(Beeby, 1979).
Plasmin
Eliminating the proteolytic activity of plasmin presents
more difficulties than eliminating the action of the
coagulant. The contribution of plasmin to proteolysis
in cheese has been assessed indirectly, i.e., in cheese
from which all other agents have been eliminated
(e.g., Visser and de Groot-Mostert, 1977). Plasmin is
inhibited by soybean trypsin inhibitor which should
be suitable for the inhibition of plasmin activity in
cheese but no studies to evaluate this approach have
been reported. The high heat stability of plasmin and
the finding that its activity is increased by high cook-
ing temperatures (Farkye and Fox, 1990) suggest that
a model system could be developed in which aseptic
curd is produced, the rennet denatured by a suitable
cooking temperature and the curd acidified by GDL;
such a system would allow plasmin to act in isolation.
6-Aminohexanoic acid (AHA) is an inhibitor of
plasmin and/or plasminogen activators but does not
inhibit chymosin or bacterial peptidases. It was used by
Farkye and Fox (1991) to assess the role of plasmin in
Cheddar cheese made without aseptic precautions and
with a normal lactic acid starter. -Casein bands on
electrophoretograms were less intense in cheeses con-
taining AHA than in the control, suggesting that plas-
min plays a role in Cheddar cheese ripening. It was
necessary to use a high concentration of AHA to inhibit
the plasmin in cheese curd and this appeared to cause
increased syneresis and consequently reduced the mois-
ture content of the cheese. Further, since AHA contains
N, the background level of soluble N was increased
greatly. Several specific irreversible inhibitors of serine
proteinases were described by Harper et al. (1985) who
recommended dichloroisocoumarin; as far as we are
aware, none of these inhibitors have been used in stud-
ies on cheese.
Since most of the potential plasmin activity in cheese
is in the form of its inactive precursor, plasminogen, it
is possible to increase plasmin activity in cheese by activ-
ation of plasminogen to plasmin using exogenous plas-
min inhibitors. Barrett et al. (1999) used urokinase to
activate plasminogen to plasmin, while Upadhyay et al.
(unpublished) used streptokinase, a plasminogen activa-
tor produced by the mastitis pathogen, Streptococcus
uberis. In both studies, the rate of proteolysis was accel-
erated on activation of plasminogen to plasmin.
Since plasmin associates with the casein micelles,
most exogenous plasmin added to milk is retained in
the curd, unlike many exogenous enzymes added to
cheese milk, which are lost in the whey. Studies in
which exogenous plasmin was added to milk include
Farkye and Fox (1992), Farkye and Landkammer
(1992) and O’Farrell et al. (2002). Increasing the level
of plasmin in milk increased the rate of primary
proteolysis, but did not greatly increase the production
of secondary proteolysis products.
Cathepsin D
Cathepsin D is a lysosomal proteinase found at low
levels in milk and which has a very similar specificity
on the caseins to chymosin (McSweeney et al., 1995).
Thus, it is difficult to assess the role of cathepsin D in
proteolysis in rennet-coagulated cheeses due to the
presence of the much greater amount of chymosin (or
other coagulant). The hydrolysis of s1-casein to s1-CN
(f24-199) in ‘rennet-free’ cheeses (e.g., Lane et al., 1997)
and in high-cook varieties such as Swiss (in which
much of the rennet is inactivated; see for example
Cooney et al., 2000) has been attributed to the action
of cathepsin D. However, it is also possible that the
production of s1-CN (f24-199) in these cheeses was
due to a low level of residual chymosin activity rather
than cathepsin D which is largely inactivated on pas-
teurisation (Hayes et al., 2000). However, clear evi-
dence for a minor role for cathepsin D in proteolysis in
cheese during ripening has emerged from the study of
Quarg, an acid-curd cheese (Hurley et al., 2000) and
356 Biochemistry of Cheese Ripening: Introduction and Overview
a pickled Feta-type cheese made from ultrafiltered
milk in which no rennet was used in manufacture
(Wium et al., 1998).
Other indigenous enzymes
As discussed by Fox (2003), milk contains about 60
indigenous enzymes, of which about 20 have been isol-
ated and characterised in detail. However, the contribu-
tions of only plasmin, cathepsin D and lipoprotein
lipase (see Proteolysis in Cheese during Ripening and
‘Lipolysis and Catabolism of Fatty Acids in Cheese’,
Volume 1) to cheese ripening have been investigated. It
is possible that other indigenous enzymes, e.g., xan-
thine oxidase, sulphydryl oxidase or acid phosphatase
may contribute to cheese ripening, although these
enzymes have not been studied in this context.
Starter enzymes
Advances in the genetics of LAB have permitted study
of the roles of specific bacterial enzymes in cheese
during ripening. The first enzyme to be studied in
this way was lactocepin (cell envelope-associated pro-
teinase, PrtP) of Lactococcus. The gene for this enzyme
is plasmid-encoded, and therefore it is easy to produce
PrtP mutants. The role of this enzyme has been stud-
ied by comparison of cheese made with PrtP or
PrtP strains (e.g., Farkye et al., 1990; Law et al.,
1993; Lane and Fox, 1997; Broadbent et al., 2002) or
cheeses made with control strains and starters with
enhanced PrtP activities (e.g., Law et al., 1993). The
principal role of PrtP during cheese ripening appears
to be the degradation of intermediate-sized peptides
produced from the caseins by the action of chymosin
or plasmin.
The genes for peptidases are chromosomally encoded
and therefore more sophisticated techniques are required
to prepare mutants with different peptidase genes than
those that were used to produce PrtP strains. Strains
deficient in specific peptidases (e.g., Christensen et al.,
1995; Meyer and Spahni, 1998), strains which over-
produce specific peptidases (e.g., McGarry et al., 1994;
Christensen et al., 1995) or strains which express pepti-
dase genes from other organisms (e.g., Wegmann et al.,
1999; Luoma et al., 2001; Courtin et al., 2002; Joutsjoki
et al., 2002) have been developed. Although the object-
ive of many of these studies has been to study the role of
peptidases in nitrogen metabolism in LAB in milk, some
(e.g., McGarry et al., 1994; Christensen et al., 1995;
Courtin et al., 2002) have made cheeses using these
mutant strains and thus information is available on the
roles of peptidases in cheese during ripening which
appears to degrade polypeptides to shorter peptides,
usually with the release of amino acids.
Recent research has suggested that the principal
role of proteolytic enzymes is the production of amino
acids as precursors for a range of catabolic reactions
which produce volatile flavour compounds. The genet-
ics of enzymes involved in amino acid catabolism is
now an active area of research and it is expected that
mutant strains of LAB which are deficient in, or over-
produce, specific amino acid catabolic enzymes will be
produced in the near future. Cheese made using such
strains or their wild types as starters would give very
clear insights into the role of specific amino acid cata-
bolic enzymes in the development of cheese flavour.
NSLAB and their enzymes
Non-starter lactic acid bacteria affect cheese quality
and almost certainly contribute to the intensity of
flavour, although sometimes they may cause off-
flavours in cheese. The role of NSLAB in cheese ripen-
ing has been studied actively, principally with a view
to explaining the differences observed between cheese
made from raw or pasteurised milk. Comparison of
raw and pasteurised milk cheese (e.g., Lau et al., 1990,
1991; McSweeney et al., 1993; Shakeel-Ur-Rehman et al.,
1999) generally showed that raw milk cheese ripens
more quickly and develops a stronger flavour than
pasteurised milk cheese. Descriptive sensory analysis
of the flavour profiles of raw and pasteurised milk
Cheddar cheese (Muir et al., 1997) showed that raw
milk cheese is more intensely flavoured than pas-
teurised milk cheese but has higher ratings for certain
atypical flavours and is more variable.
The role of NSLAB in cheese has been studied by
physically removing them by microfiltration (e.g.,
McSweeney et al., 1993; Bouton and Grappin, 1995;
Beuvier et al., 1997; Roy et al., 1997) or by inhibiting
their growth by the addition of antibiotics at salting
(e.g., Walsh et al., 1996; Shakeel-Ur-Rehman et al.,
1999) or by the use of bacteriocin-producing starters
(Fenelon et al., 1999). Several investigators have
added selected strains of NSLAB to pasteurised milk as
adjuncts (e.g., Broome et al., 1990; Muir et al., 1996).
Cheese has been made under controlled bacterio-
logical conditions in an attempt to prevent NSLAB
gaining access to the cheese from the environment
(e.g., McSweeney et al., 1994; Lynch et al., 1996,
1997). Raw milk (Shakeel-Ur-Rehman et al., 2000c) or
MF retentate from raw skim milk (Beauvier et al.,
1997) has been added to pasteurised cheesemilk as an
inoculum of NSLAB. Blends of as little as 1% raw milk
with 99% pasteurised milk influence the quality of
cheese (Shakeel-Ur-Rehman et al., 2000c). Since the
rate of growth of NSLAB is strongly affected by tempera-
ture (see Folkertsma et al., 1996), Shakeel-Ur-Rehman
 Biochemistry of Cheese Ripening: Introduction and Overview 357
et al. (2000a,b) successfully prevented the growth of
NSLAB in raw milk Cheddar by ripening at 1 °C.
The results of these studies suggest that the differ-
ences observed between cheese made from raw and pas-
teurised milk are due principally to heat-induced
changes to the NSLAB microflora, although pasteurisa-
tion largely inactivates the indigenous lipoprotein lipase,
which results in a reduced level of lipolysis in pas-
teurised milk cheese. Since the proteolytic systems of
NSLAB are generally similar to those of other LAB, they
appear to contribute to proteolysis in a similar way to
the starter, but to a lesser extent since maximum NSLAB
numbers in cheese (often c. 107–108 cfu g 1) are lower
than maximum numbers of starter (c. 109–1010 cfu g 1).
Acceleration of Cheese Ripening
Cheese ripening is a slow, and consequently an expen-
sive, process. The expense of cheese ripening arises
principally from the inventory cost associated with
holding a large amount of cheese in storage and the
capital cost of providing a ripening facility adequate to
hold sufficient cheese during ripening. The tempera-
ture and, in certain cases, the relative humidity of
ripening rooms must be controlled, adding to the cost
of cheese ripening. Thus, acceleration of cheese ripening
has received considerable attention in the scientific
literature. This topic has been reviewed by Fox
(1988/89), El Soda and Pandian (1991), Wilkinson
(1993), Fox et al. (1996) and Upadhyay and McSweeney
(2003).
Various approaches have been used to accelerate
the ripening of cheese, including the use of an ele-
vated ripening temperature, addition of exogenous
enzymes or attenuated starters, use of adjunct cul-
tures, use of genetically modified starter bacteria and
high-pressure treatments. As novel processing tech-
nologies become available, it is likely that they will
find an application to accelerate cheese ripening.
Certain approaches (in particular the use of attenu-
ated starters and adjunct cultures) are also used
commercially to intensify the flavour of hard cheese
and low-fat variants thereof, without necessarily
reducing ripening time. Enzyme-modified cheeses
are products in which a cheese-like flavour develops
rapidly in a base material of young cheese (or some-
times caseinates) using cocktails of exogenous
enzymes (and sometimes LAB) (see Kilcawley et al.,
1998; ‘Cheese as an Ingredient’, Volume 2).
The simplest and the most successful approach to
accelerate ripening studied to date is an elevated
ripening temperature (e.g., Folkertsma et al., 1996).
Modification of the ripening temperature is used to
control the rate of flavour development in hard cheese,
and ripening at an elevated temperature (e.g., c. 16 °C)
results in the rapid development of flavour, although
problems can occur with texture but this is not a seri-
ous drawback if the cheese is to be used in certain
ingredient applications. Recent advances in the genet-
ics of LAB and a greater understanding of the role of
specific enzymes in the generation of volatile flavour
compounds in cheese during ripening will facilitate
the development of genetically modified starter strains
to enhance flavour development.
Acknowledgements
The author wishes to express his sincere thanks
to Ms Niamh O’Sullivan, Ms. Patricia O’Connell and
Ms Anne Cahalane for their assistance in preparing
the typescripts of this and the other sub-chapters
on cheese ripening.
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 Biochemistry of Cheese Ripening: Introduction and Overview 359
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 Metabolism of Residual Lactose
and of Lactate and Citrate
P.L.H. McSweeney and P.F. Fox, Department of Food and Nutritional Sciences,
University College, Cork, Ireland.
Metabolism of Lactose in Cheese
During the manufacture of cheese curd, lactose is con-
verted to lactic acid (mainly the L-isomer) by the
starter bacteria (see ‘Starter Cultures: General Aspects’,
Volume 1). In the case of Cheddar-type cheeses, most
of the lactic acid is produced in the vat before salting
and moulding whereas for most other varieties, acidifi-
cation occurs mainly after the curds have been placed
in moulds (see Fox et al., 2000). For many common
varieties, the pH of the curd reaches ⬃5.0–5.3 within
⬃12 h from the start of cheesemaking.
The rate and extent of acidification has a major
impact on cheese texture via demineralization of the
casein micelles (see Creamer et al., 1985, 1988;
Lawrence et al., 1987; Fox et al., 1990) and on cheese
proteolysis owing to the increased susceptibility of
demineralized casein micelles to proteolysis (O’Keeffe
et al., 1975) and/or greater retention of chymosin at
low pH (Holmes et al., 1977; Stadhouders et al., 1977;
Visser, 1977; Creamer et al., 1985; Garnot et al., 1987).
However, such aspects will not be considered here.
Although ⬃98% of the lactose is removed in the
whey as lactose or lactate during the manufacture of
Cheddar (Huffman and Kristoffersen, 1984), Cheddar
cheese curd contains 0.8–1.0% lactose at milling.
Under normal circumstances, this residual lactose is
metabolized quickly, predominantly to L-lactate, mainly
through the activity of the starter bacteria (see ‘Starter
Cultures: General Aspects’, Volume 1). The complete
and rapid metabolism of the lactose and its constituent
monosaccharides in cheese curd is essential for the
production of good quality cheese since the presence of
a fermentable carbohydrate may lead to the develop-
ment of an undesirable secondary flora (see Fox et al.,
1990, 2000). In Cheddar cheese, the residual lactose is
fermented at a rate and to an extent dependent on the
salt-in-moisture (S/M) content of the curd (Turner and
Thomas, 1980). Lactococcus lactis subsp. cremoris is
more salt-sensitive than Lc. lactis subsp. lactis, which
in turn is more sensitive than non-starter lactic acid
bacteria (NSLAB; Turner and Thomas, 1980). Since
some NSLAB are facultatively heterofermentative, the
Cheese: Chemistry, Physics and Microbiology, Third edition – Volume 1: General Aspects
ISBN: 0-1226-3652-X
Set ISBN: 0-1226-3651-1
% S/M may determine the products of lactose fermenta-
tion post-manufacture. At low S/M concentrations and
low populations of NSLAB, residual lactose is con-
verted mainly to L-lactate by the starter. At high popu-
lations of NSLAB, e.g., at a high storage temperature,
considerable amounts of D-lactate are formed, partly by
fermentation of residual lactose and partly by isomer-
ization of L-lactate (Turner and Thomas, 1980). At high
S/M levels (e.g., 6%) or at low NSLAB populations the
concentration of lactose falls very slowly and changes
in levels of lactate are slight. The quality of cheese is
strongly influenced by the fermentation of residual lac-
tose, as is evident from the data of O’Connor (1974).
The pH decreases after salting, presumably due to the
continued action of the starter at S/M levels 5%, but
at higher level of S/M, starter activity decreases
abruptly, as indicated by the high level of residual lac-
tose and high pH. The quality of the cheeses also
decreases sharply at 5% S/M (O’Connor, 1974).
In Cheddar cheese made from milk supplemented
with lactose to a concentration of 8% (⬃2.5% lactose in
the cheese curd), lactose persisted and the pH contin-
ued to fall throughout a 9-month ripening period; the
cheese had a harsh, strong over-acid flavour (Waldron,
1997; Shakeel-ur-Rehman and Fox, unpublished).
In certain varieties (e.g., Edam, Gouda, Samsoe,
Havarti), some whey is removed during curd manufac-
ture and replaced with hot water. Probably, the initial
function of this step was to cook the curds (on farms
lacking steam-generating facilities and jacketed cheese
vats) but it removes some lactose and reduces the ratio
of lactose to buffering substances and hence acts to
control pH (Walstra et al., 1993). Dutch-type cheese
curd contains up to ⬃1.4% lactose at pressing but this
decreases to 0.1% after ⬃12 h and to undetectable
levels after brining. The lactose is fermented by the
starter to L-lactate.
The curd for some Cheddar-type cheeses (washed-
curd Cheddar and Monterey Jack) is washed to reduce
its lactose content. The lactose in washed-curd Ched-
dar is exhausted rapidly, the pH, which remains higher
Copyright © 2004 Elsevier Ltd
All rights reserved
362 Metabolism of Residual Lactose and of Lactate and Citrate
than normal for Cheddar, rises during ripening (in
common with most varieties) and the cheese has a
mild clean flavour (Waldron, 1997; Shakeel-ur-Rehman
and Fox, unpublished).
The fermentation of lactose in Swiss-type cheeses is
quite complicated (see Mocquot, 1979; Turner et al.,
1983; Fox et al., 1990). Typically, 30 min after moulding,
Emmental contains ⬃1.7% lactose which is metabolized
rapidly by Streptococcus thermophilus to a low level
within 12 h, with the production of up to 0.8% L-lactate
(Turner et al., 1983). Only the glucose moiety of lactose
is metabolized by Sc. thermophilus and, consequently,
galactose accumulates to a maximum of ⬃0.7% at ⬃10 h.
The lactobacilli metabolize lactose and galactose to a
mixture of D- and L-lactate, reaching ⬃0.35 and 1.2%,
respectively, at day 14, by which time all sugars are nor-
mally completely metabolized. Thereafter, the concen-
trations of L- and D-lactate change little until the cheese
is transferred to the hot room, when the propionic acid
bacteria begin to grow. Changes in the concentrations of
lactose, galactose, glucose, D- and L-lactate and their
degradation products in Swiss-type cheese were studied
by Turner et al. (1983) and are shown in Fig. 1.
Changes to Lactate During Ripening
Lactate is an important substrate for a series of reac-
tions in cheese during ripening (Fig. 2):
Figure 1 Relationship between lactose and lactate metabolism, growth of propionic acid bacteria and production of propionate and
acetate in Swiss cheese (Turner et al., 1983).
• in most cheeses, L-lactate is racemized to D-lactate
by the NSLAB flora;
• lactate is catabolized in Swiss-type cheese by Propi-
onibacterium freudenreichii subsp. shermanii which is
important for the development of characteristic eyes
and flavour;
• lactate is catabolized to CO2 and H2O by Penicillium
camemberti in surface mould-ripened cheeses, such
as Camembert and Brie, which is important for tex-
ture development;
• in the presence of O2, some members of the NSLAB
flora, particularly pediococci, can oxidize lactate to
formate and acetate;
• lactate can be metabolized anaerobically by Clostrid-
ium tyrobutyricum leading to defects known as ‘late
gas blowing’.
Racemization of lactate
The concentrations of lactate in Camembert, Swiss,
Cheddar and Dutch-type cheeses are 1.0, 1.4, 1.5 and
1.2%, respectively (Raadsveld, 1957; O’Connor, 1974;
Turner and Thomas, 1980; Thomas and Pearce, 1981;
Turner et al., 1983; Karahadian and Lindsay, 1987; Wal-
stra et al., 1993). Turner and Thomas (1980), Thomas
and Pearce (1981) and Tinson et al. (1982) showed that
experimental and commercial Cheddar cheese contains
a considerable concentration of D-lactate, which could
 Metabolism of Residual Lactose and of Lactate and Citrate 363

DL-Lactate

Non-starter lactic
acid bacteria
1

sp.
Clo rium
Butyrate, H2 strid ib acte Propionate, acetate
ium pion
sp. H Pro H2O, CO2
5 2
C
HO
COOH

co ally ia c
dio eci ter cti
H3C

pe sp ac la
(e id b rter

Pe
Lactic acid

i)

nic eas
ac -sta

cc

illi ts
y
n

um
No
4 3

sp
.
Formate, CO2, H2O
acetate, CO2

Figure 2 Pathways by which lactate is metabolized in cheese during ripening. (1) racemization, (2) metabolism by Propionibac-
terium freudenreichii subsp. shermanii in Swiss cheese, (3) oxidative metabolism of lactate, (4) conversion to formate, ethanol and
acetate and (5) anaerobic metabolism of lactate to butyrate and H2 which leads to late gas blowing.

be formed from residual lactose by lactobacilli or by dar cheese and P. pentosaceus NCDO 1220 were capable
racemization of L-lactate. Racemization of L-lactate is of converting L-lactate to D-lactate, eventually pro-
likely to occur more rapidly in cheese made from ducing a racemic mixture, while only 5 of 16 Lacto-
raw milk than in pasteurized milk cheese due to bacillus isolates were capable of racemizing L-lactate,
higher numbers of NSLAB and a more diverse non- at much slower rates and to a lesser extent than the
starter microflora in the former. Commercial Gouda pediococci. However, pediococci constitute only a
contains a relatively low proportion of D-lactate, prob- small proportion of the microflora of Cheddar cheese
ably due to the short ripening time. The level of L- or ( Jordan and Cogan, 1993; Crow et al., 2001) and thus
D-lactate in Camembert is very low (Gripon, 1993) racemization of lactate in Cheddar and similar
due to the catabolism of lactate by the mould, as dis- cheeses is presumably mainly a consequence of the
cussed below. growth of non-starter lactobacilli. Both lactobacilli
Racemization presumably involves oxidation of and pediococci possess L()-LDH and D( )-LDH,
L-lactate by L-lactate dehydrogenase (LDH) to pyru- both of which are NAD dependent. Racemization of
vate which is then reduced to D-lactate by D-LDH. L-lactate by cell suspensions of both pediococci and
Except in cases where the post-milling activity of the lactobacilli is pH dependent (optima: 4.0–5.2 and
starter is suppressed, racemization is likely to be the 4.5–6.0, respectively) and is retarded by an NaCl
principal mechanism (Thomas and Crow, 1983). concentration 5% or 2% for pediococci and lacto-
Racemization of lactate is a major change in cheese bacilli, respectively (Thomas and Crow, 1983). Racem-
during ripening, transforming up to approximately ization of lactate in a Cheddar cheese inoculated with
half the lactate or ⬃0.7% of the cheese mass (Thomas pediococci was complete after ⬃19 days, while it
and Crow, 1983). required ⬃3 months in a control cheese with a much
lower number of NSLAB, especially pediococci (Thomas
and Crow, 1983).
The racemization of L-lactate is probably not signifi-
cant from the favour viewpoint. However, Ca-lactate
H H may crystallize in cheese, causing undesirable white
specks, especially on cut surfaces (Pearce et al., 1973;
C C Severn et al., 1986; Dybing et al., 1988). Such crystals
HO OH
COOH HOOC are harmless, but they may cause consumers to reject
H3C CH3
cheese as being mouldy or containing foreign bodies
L(+)-Lactic acid D(–)-Lactic acid (Dybing et al., 1988). The solubility of Ca-DL-lactate
is lower than that of pure Ca-L-lactate (Thomas and
Crow, 1983; Dybing et al., 1988) and hence racemiza-
tion of lactate favours the development of crystals in
Thomas and Crow (1983) showed that pediococci cheese. Dybing et al. (1988) calculated that the
isolated from cheese racemize L-lactate more actively amount of available lactate in cheese can potentially
than lactobacilli; all 27 pediococci isolated from Ched- create enough Ca-lactate pentahydrate to exceed its
364 Metabolism of Residual Lactose and of Lactate and Citrate
solubility only slightly at 0 °C. Thus, crystal formation
is favoured if microbial metabolism increases the con-
centration of D- relative to L-lactate, due to the lower
solubility of Ca-DL-lactate. Crystal growth requires
nucleation centres which may be bacterial cells,
microcrystals of calcium phosphate or undissolved
CaCO3. Increased levels of residual lactose, which
favour the growth of NSLAB, can facilitate production
of Ca-lactate crystals (Pearce et al., 1973; Sutherland and
Jameson, 1981). Likewise, factors which increase the
release of casein-bound Ca (e.g., low pH or high
salt which causes the ion-exchange of Na for
Ca2; Dybing et al., 1988) or reduce the solubility of
Ca-lactate (e.g., a lower ripening temperature) favour
crystal formation.
Oxidation of lactate
Lactate can be metabolized by LAB, depending on
strain, to acetate, ethanol, formate and CO2 (see Fox
et al., 2000). Pediococci, if present in cheese together
with high concentrations of O2, produce l mol of
acetate and 1 mol of CO2 and consume 1 mol of O2
per mol of lactate utilized (Thomas et al., 1985). The
pH optimum for oxidation is 5–6 and depends on
the lactate concentration. The concentration of lac-
tate in cheese exceeds that required for optimal oxi-
dation, and lactate is not oxidized until all sugars
have been exhausted. However, the oxidation of
L-lactate to acetate occurs to a very limited extent in
cheese wrapped in film due to the low level of O2
available.
The oxidative activity of suspensions of starter
and NSLAB isolated from cheese on lactose, lactate,
citrate, amino acids and peptides was studied by
Thomas (1986). Starter bacteria were active mainly
on lactose, with low activity on enzyme-hydrolysed
casein; Lb. casei oxidized citrate, while Lb. plan-
tarum, Lb. brevis and P. pentosaceus oxidized lactose,
peptides, L- and D-lactate, but not citrate. These
results suggest that the oxidation of lactate to acetate
in cheese depends on the NSLAB population and on
the availability of O2, which is determined by the
size of the block and the oxygen permeability of the
packaging material (Thomas, 1987). Acetate, which
may also be produced by starter bacteria from lac-
tose (Thomas et al., 1979), citrate or from amino
acids by starter bacteria and lactobacilli (Nakae and
Elliott, 1965), is usually present at high concentra-
tions in most, or all, cheeses and is considered to
contribute to cheese flavour, although a high con-
centration may cause off-flavours (Aston and Dulley,
1982).
Oxidative metabolism of lactate in surface
mould-ripened varieties
The catabolism of lactate is very extensive in surface
mould-ripened varieties, e.g., Camembert and Brie.
The concentration of lactate in these cheeses at day 1
is ⬃1.0%, produced mainly or exclusively by the
mesophilic starter, and hence, presumably, is L-lactate.
Secondary organisms quickly colonize and dominate
the surface of these cheeses (Addis et al., 2001), initially
Geotrichum candidum and yeasts (e.g., Kluyveromyces
lactis, Debaryomyces hansenii and Saccharomyces cere-
visiae; Gripon, 1993), followed by a dense growth of
Penicillium camemberti (Mollimard et al., 1995) and,
particularly in traditional manufacture, by low numbers
of Gram-positive organisms similar to those found on
the surface of smear-ripened cheeses, which do not
colonize the cheese surface until the pH has increased to
5.8 (see ‘Surface Mould-ripened Cheeses’, Volume 2).
G. candidum and P. camemberti rapidly metabolize
lactate to CO2 and H2O, causing an increase in pH.
Deacidification occurs initially at the surface, resulting
in a pH gradient from the surface to the centre and
causing lactate to diffuse outwards. When the lactate
has been exhausted, P. camemberti metabolizes pro-
teins, producing NH3 which diffuses inwards, further
increasing the pH. The concentration of calcium phos-
phate at the surface exceeds its solubility at the high
pH and precipitates as a layer of Ca3(PO4)2 on the sur-
face, thereby causing a calcium phosphate gradient
within the cheese, resulting in its outwards diffusion;
reduction of the concentration of calcium phosphate
in the interior helps to soften the body of the cheese
(Fig. 3). In addition to softening the texture, changes
to the cheese matrix may influence cheese flavour by
changing the rates of migration or release of flavour
compounds (Engel et al., 2001). The elevated pH stimu-
lates the action of plasmin, which, together with
residual coagulant, is responsible for proteolysis in
this cheese rather than proteinases secreted by the sur-
face microorganisms, which, although very potent, dif-
fuse into the cheese to only a very limited extent,
although peptides or other low molecular weight com-
pounds produced by them at the surface may diffuse
into the body of the cheese (Sousa and McSweeney,
2001; Churchill et al., 2003). The combined action of
increased pH, loss of calcium (which affects to the
integrity of the protein network) and proteolysis are
necessary for the very considerable softening of the
body of Brie and Camembert (see Noomen, 1983;
Lenoir, 1984; Karahadian and Lindsay, 1987; Sousa
and McSweeney, 2001). Changes which occur in
Camembert-type cheese during ripening are indicated
in Fig. 3.
 Metabolism of Residual Lactose and of Lactate and Citrate 365

Growth of Penicillium camemberti at surface

Ammonia produced at surface

Lactate metabolized at surface by proteolysis diffuses into cheese
Ca3(PO4)2 precipitates pH High pH

Cheese
softens
Low pH from
surface

Concentration gradient
towards

Direction of migration
score
Migration of soluble Ca, PO43– and
lactate towards surface

Figure 3 Schematic representation of the changes which occur in Camembert-type cheese during ripening as a consequence
of the growth of Penicillium camemberti at the surface.

Anaerobic metabolism of lactate by Clostridium Late gas blowing and accompanying off-flavours are
tyrobutyricum defects associated with certain hard cheeses resulting
Gas (CO2 or H2) production by microorganisms may from the anaerobic metabolism of lactate (or glucose) by
occur in cheese during ripening and may be desirable Clostridium tyrobutyricum (and perhaps other clostridia;
(e.g., eye production in Swiss and Dutch-type cheeses) see Ingham et al., 1998) to butyrate and H2 (Fox et al.,
or a defect. Organisms responsible for gas production 1995; Klijn et al., 1995; Fig. 4); Cl. tyrobutyricum prefer-
in cheese are summarized in Table 1. entially utilizes D-lactate in a mixture of both isomers
(Huchet et al., 1997). Late gas blowing is a problem only
in certain varieties, principally those that are brine-
salted, owing to the time lag for NaCl to reach an
Table 1 Major microbial groups responsible for producing gas inhibitory level throughout cheese (Kleter et al., 1984).
in cheese (modified from Mullan, 2000) Cheddar cheese is not susceptible to late gas blowing
mainly because it is dry-salted. The combined effects of
Gaseous pH, lactate, glycerol and NaCl on the growth of vegeta-
Microorganism Substrate product(s)
tive cells of Cl. tyrobutyricum were studied by Huchet
Clostridium Lactate CO2, H2 et al. (1995) who found that the growth of this organism
tyrobutyricum is very sensitive to changes in these parameters within
Lactobacillus casei Citrate CO2 the ranges found during the manufacture of Emmental
Lactobacillus brevis Lactose CO2 cheese (pH, 5.3–5.9; NaCl level, 0–0.6%; lactic acid con-
Streptococcus Urea CO2
thermophilus
centrations, 0–1.6%; aw, 0.965–0.99). Late gas blowing
Coliforms Lactose CO2, H2 may be avoided by minimizing spore numbers in milk
Yeasts Lactose CO2 by good hygiene and avoiding the feeding of silage
Citrate-positive Citrate CO2 (Driehuis and Elferink, 2000). Germination of spores
lactococci and the growth of the vegetative cells may be inhibited
Leuconostoc Lactose/citrate CO2
mesenteroides
by the use of lysozyme or NO3 or perhaps by biological
Leuconostoc Lactose/citrate CO2 control (e.g., Carminati et al., 2001, who used co-inocula
dextranicum of Sc. thermophilus), or in the case of processed cheese,
Propionibacterium Lactate CO2 by long-chain polyphosphates (Loessner et al., 1997).
freudenreichii Spores may be removed from milk by bactofugation
subsp. shermanii
or microfiltration (see McSweeney and Sousa, 2000).
366 Metabolism of Residual Lactose and of Lactate and Citrate

H OH
H O
HO
HO H
H OH
O
H OH
Glucose
O
NAD 2Pi
Pi Butyrate

2ADP
CoA Acetyl-CoA
NADH2
Acetyl-P

Acetate 2ATP
ADP NADH2 NAD
O
ATP OH
CoA O
S O O
2 2 O
Butyryl-CoA O
Pyruvate Lactate
NAD
2H2
2Fd
NADH2 2 CO2
2 CoA

O 2Fd+H2
CoA
S
Crotonyl-CoA O
CoA
2 S

H2O Acetyl-CoA

CoA
OH O
O O
CoA
S CoA
S
L-β-Hydroxybutyryl-CoA
Acetoacetyl-CoA
NAD NADH2

Figure 4 Pathway for the formation of butyrate and H2 from glucose, lactose or lactate by Clostridium tyrobutyricum (Fd: ferredoxin)
(McSweeney and Sousa, 2000).

Bactofugation of milk, an increased level of NaCl in
cheese and a reduced ripening temperature are effective
measures for preventing or reducing gas production by
Clostridium spp. (Su and Ingham, 2000).
Lactate metabolism by Propionibacterium
In Swiss-type cheese, Propionibacterium freudenreichii
subsp. shermanii metabolizes lactate to propionate,
acetate, CO2 and H2O (Piveteau, 1999).
3 CH3CH(OH)COOH : 2 CH3CH2COOH  CH3COOH  CO2  H2O
Lactic acid propionic acid acetic acid
The CO2 generated migrates through the cheese
mass to points of weakness where it accumulates as
eyes, a characteristic feature of Swiss cheese. Carbohy-
drate metabolism in Swiss cheese is summarized in
Fig. 1. Since propionibacteria are very sensitive to
NaCl (Richoux et al., 1998), Swiss-type cheeses con-
tain a low level of salt. Eye development in Swiss
cheese, which is discussed in ‘Cheese with Propionic
acid Fermentation’, Volume 2 and by Steffen et al.
(1993) and Polychroniadou (2001), depends mainly
on:
• Rate and quantity of CO2 production.
• Number and size of loci suitable for future eye
development.
• CO2 pressure and diffusion rate.
• Cheese texture and temperature (Steffen et al.,
1993).
Relatively little of the total amount of CO2 produced
by the propionic acid fermentation in Swiss cheese
remains trapped in the eyes; in a cheese of c. 80 kg,
 Metabolism of Residual Lactose and of Lactate and Citrate 367
120 l CO2 are produced during ripening. Approximately
60 l remain dissolved in the cheese mass, ⬃40 l are lost
from the cheese and only c. 20 l remain in the eyes
(Steffen et al., 1993).
L-Lactate is metabolized preferentially to D-lactate
by propionic acid bacteria (Crow, 1986) to reach 0.2%
after ⬃20 days in the hot room (Turner et al., 1983).
In fact, the concentration of D-lactate continues to
increase to ⬃0.4% during the early days in the warm
room, before being metabolized by propionic acid bac-
teria. Increasing the number of starter lactobacilli
accelerates sugar metabolism and causes higher con-
centrations of both D- and L-lactate but suppresses
the growth of propionibacteria (due to a lower pH in the
cheese) and thus delays the production of propi-
onate and acetate. In the absence of lactobacilli or with
Gal lactobacilli, galactose accumulates and no D-lactate
is formed. Therefore, the proportion of lactobacilli in the
starter probably influences the production of CO2 and
volatile acids.
Citrate Metabolism
The relatively low concentration of citrate in milk
(⬃8 mmol l 1) belies the importance of its metabol-
ism in many cheeses made using a mesophilic culture
(for reviews see Cogan, 1985; Cogan and Daly, 1987;
Fox et al., 1990; Cogan and Hill, 1993; ‘Starter Cul-
tures: General Aspects’, Volume 1). Approximately
94% of the citrate in milk is soluble and most of it is
lost in the whey; however, the concentration of citrate
in the aqueous phase of cheese is ⬃3 times that in
whey (Fryer et al., 1970), presumably reflecting the
concentration of colloidal citrate; Cheddar cheese con-
tains 0.2–0.5% citrate.
Citrate is not metabolized by most strains of Lc. lac-
tis subsp. lactis or Lc. lactis subsp. cremoris, but is
metabolized, with the production of diacetyl, acetate,
acetoin and CO2, by citrate-positive (Cit) strains of
lactococci (formerly referred to as Lc. lactis subsp. lac-
tis biovar diacetylactis or Streptococcus diacetilactis) in
which the trait is plasmid-encoded, and Leuconostoc
mesenteriodes subsp. cremoris and Ln. lactis. It is not
metabolized by Sc. thermophilus or by thermophilic
lactobacilli (see Fox et al., 2000).
Citrate is not used as an energy source by Cit lacto-
cocci or Leuconostoc spp., but it is metabolized very rap-
idly in the presence of a fermentable carbohydrate by
the pathway outlined in Fig. 5. Enzymes in this pathway
have been characterized (see Cogan and Hill, 1993;
O’Sullivan et al., 2001). The CO2 produced on citrate
metabolism is responsible for the characteristic eyes of
Dutch-type cheese and for the undesirable openness and
floating curd defects in Cheddar and Cottage cheese,
respectively. Due mainly to the formation of diacetyl,
citrate metabolism is very significant in aroma/flavour
development in Cottage cheese (e.g., Antinone et al.,
1994), Quarg (Mohr et al., 1997), and many fermented
milks, particularly cultured ‘buttermilk’ (Ulberth, 1991;
Gaafar, 1992; Laye et al., 1993; Hernandez et al., 1995;
Rankin and Bodyfelt, 1995). Diacetyl also contributes
to the flavour of Dutch-type and Cheddar cheeses
(McGugan, 1975; Manning, 1979a,b; Dacremont and
Vickers, 1994; Christensen and Reineccius, 1995; Milo
and Reineccius, 1997). However, diacetyl is produced in
small amounts (0.11 mmol l 1 in milk); acetoin pro-
duction is usually 10–50 times greater. According to
the pathway shown in Fig. 5, 1 mol acetate should be
produced from 1 mol citrate. However, studies suggest
that c. 1.2 mol acetate are actually produced per mol
citrate used; the excess probably results from small
amounts of acetate produced on the metabolism of sugars.
Acetate produced from citrate may also contribute to
cheese flavour. There are few data on the production of
2,3-butanediol by starters (see Fox et al., 2000).
In cheese with a controlled microflora, Fryer et al.
(1970) showed that in cheese made using Lc. lactis
subsp. cremoris, citrate remained constant at 0.2% up
to 3 months, but decreased to 0.1% at 6 months.
Cheese made using Lc. lactis subsp. cremoris plus a
Cit strain of Lactococcus contained no citrate at
3 months. Although Lb. casei could metabolize citrate
in milk, the concentration of citrate in cheese made
using Lc. lactis subsp. cremoris and Lb. casei decreased
at about the same rate as in cheese made with Lc. lactis
subsp. cremoris alone.
Although the pathway shown in Fig. 5 is probably
the major route for the metabolism of citrate by LAB,
the possibility that lactate may be formed from the
pyruvate produced from citrate cannot be overlooked.
Three out of four strains of Cit Leuconostoc growing
on glucose plus citrate produced no diacetyl or acetoin
and more lactate than could be accounted for in terms
of the amount of glucose used, suggesting that pyru-
vate derived from citrate was being reduced to lactate
(Cogan, 1987).
Citrate may be metabolized by some strains of faculta-
tively heterofermentative lactobacilli, which are compon-
ents of the NSLAB flora, to acetoin, acetate and
probably diacetyl (Palles et al., 1998) by the same path-
way as Cit lactococci and Leuconostoc spp. (Fig. 5).
Thomas (1987) showed that citrate in Cheddar cheese
decreased slowly to almost zero at 6 months, presumably
as a result of metabolism by lactobacilli which became
the major component of the NSLAB flora. Inoculation of
cheese milk with Lb. plantarum accelerated the depletion
of citrate. Pediococci did not appear to utilize citrate.
368 Metabolism of Residual Lactose and of Lactate and Citrate

OH
O O

O O

O O

OH OH Citrate
OH H
H O H O
H HO
HO O H O
H OH H OH Citrate
H H H OH lyase O

Lactose Acetate
O O

O
O

O
Oxalacetate

Acetolactate
decarboxylase CO2

OH
NAD+ NADH O TPP CO2 O

O O
TPP
O O
Lactate Lactate dehydrogenase
Pyruvate Acetaldehyde TPP

Acetolactate
synthase

OH Acetyl-CoA
Other metabolic pathways
O
Diacetyl
O
O synthase
CoASH

α-Acetolactate
TPP
CO2

Spontaneous?
OH O
Acetoin
dehydrogenase
O O

Acetoin + Diacetyl
NAD(P) NAD(P)H
NAD(P)H
Butanediol
dehydrogenase
OH
NAD(P)+

OH

2,3-Butanediol

Figure 5 Pathways for citrate metabolism in citrate-positive strains of Lactococcus and Leuconostoc species (modified from Cogan
and Hill, 1993).
 Metabolism of Residual Lactose and of Lactate and Citrate 369
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This Page Intentionally Left Blank
 Lipolysis and Catabolism of Fatty
Acids in Cheese
Y.F. Collins, Teagasc, Dairy Products Research Centre, Ireland
P.L.H. McSweeney, Department of Food and Nutritional Sciences, University College,
Cork, Ireland
M.G. Wilkinson, Department of Life Sciences, University of Limerick, Ireland
Introduction
Lipids present in foods may undergo oxidative or
hydrolytic degradation (McSweeney and Sousa, 2000).
However, lipid oxidation does not occur to a signifi-
cant extent in cheese, probably due to its low redox
potential ( 250 mV) (Fox and Wallace, 1997; Fox
et al., 2000; McSweeney and Sousa, 2000). Enzymatic
hydrolysis (lipolysis) of triglycerides to fatty acids and
glycerol, mono- or di-glycerides is considered to be
essential for flavour development in cheese (McSweeney
and Sousa, 2000). Lipolysis is an important biochem-
ical event during cheese ripening and has been studied
extensively in varieties such as Blue and hard Italian
cheeses where extensive lipolysis occurs and is a major
pathway for flavour generation. However, in the case
of cheeses such as Cheddar and Gouda, in which the
level of lipolysis during ripening is low, the contribu-
tion of lipolysis to cheese quality and flavour has
received relatively little attention. Free fatty acids
(FFAs) are important precursors of several volatile
compounds which contribute to flavour (McSweeney
and Sousa, 2000; Collins et al., 2003a,b).
Lipolytic Agents in Cheese
Lipolytic enzymes may be classified as esterases or
lipases, which are distinguished according to three
main characteristics: length of the hydrolysed acyl
ester chain, physico-chemical nature of the substrate
(whether emulsified or not) and enzymatic kinetics
(esterases have classical Michaelis Menten-type kinet-
ics while lipases, since they are active only at an inter-
face, obey interfacial Michaelis Menten-type kinetics)
(Chich et al., 1997; Villeneuve and Foglia, 1997;
Deeth and Touch, 2000). Unfortunately, the terms
‘esterases’ and ‘lipases’ are often used interchangeably
in the scientific literature.
In general, lipolytic enzymes are specific for fatty
acids esterified at the sn-1 or sn-3 positions of trigly-
cerides (Deeth and Touch, 2000). Initially, triglycerides
are hydrolysed to 1,2- and 2,3-diglycerides and later to
Cheese: Chemistry, Physics and Microbiology, Third edition – Volume 1: General Aspects
ISBN: 0-1226-3652-X
Set ISBN: 0-1226-3651-1
2-monoglycerides; butyrate, as well as the other short-
and medium-chain acids, are located mainly at the sn-1
and sn-3 positions in milk lipids and thus are preferen-
tially released by lipolytic enzymes (Parodi, 1971;
Christie, 1995; Deeth and Touch, 2000).
Lipases in cheese originate from six possible
sources:
• milk
• rennet paste
• starter bacteria
• secondary starter microorganisms
• non-starter lactic acid bacteria (NSLAB)
• exogenous lipase preparations
(Deeth and Fitz-Gerald, 1995; Fox and Wallace, 1997;
McSweeney and Sousa, 2000).
Milk contains a very potent indigenous lipoprotein
lipase (LPL) (Olivecrona and Bengtsson-Olivecrona,
1991; Fox and Stepaniak, 1993; Fox et al., 1993;
Olivecrona et al., 2003). The enzyme consists of 450
amino acid residues, with an overall molecular mass of
55 kDa and in milk exists as a dimer of identical sub-
units. The enzyme has an isoelectric point above pH 9,
with a positive charge at physiological pH (Olivecrona
and Bengtsson-Olivecrona, 1991). The lipase is of
blood origin and is involved in the metabolism of
plasma triglycerides; its presence in milk is due to
leakage through the mammary cell membrane. Bovine
milk contains 10–20 nmol lipase/l which, under opti-
mum conditions (37 °C, pH 7, in the presence of an
apolipoprotein activator, apo-CII), could theoretically
release sufficient FFAs within 10 s to cause perceptible
hydrolytic rancidity in milk (Walstra and Jenness,
1984). This does not occur under normal circum-
stances as LPL and fat are compartmentalized, c. 90%
of the LPL in milk is associated with the casein
micelles while the triglycerides in milk occur as glob-
ules surrounded by a lipoprotein membrane, the milk
fat globule membrane (MFGM). If the MFGM is dam-
aged, e.g., due to agitation, foaming, homogenization,
inappropriate milking or milk-handling techniques,
significant lipolysis may occur, resulting in off-flavours
Copyright © 2004 Elsevier Ltd
All rights reserved
374 Lipolysis and Catabolism of Fatty Acids in Cheese
in cheese and other dairy products (Fox et al., 2000).
Lipoprotein lipase has a preference for medium-chain
triglycerides (MCT) with a 2-fold faster rate of hydroly-
sis of emulsions containing triC6:0, triC8:0, triC10:0 or
triC12:0 compared to long-chain triglyceride (LCT)
emulsions containing triC16:0, triC18:0, triC18:1, triC18:2,
triC18:3 or triC20:0 (Deckelbaum et al., 1990). The dif-
ferent hydrolysis rates were attributed to higher con-
centrations of MCT at the emulsion surface due to
higher mobility of such triglycerides compared with
LCT emulsions (Deckelbaum et al., 1990).
Lipoprotein lipase is relatively non-specific for fatty
acid type but is specific for the acids at the sn-1 and sn-3
positions of mono-, di- and tri-glycerides (Olivecrona
et al., 1992). Therefore, short- and medium-chain fatty
acids are released preferentially by LPL. In raw milk
cheeses, LPL activity is significant. According to Deeth
and Fitz-Gerald (1983), it is generally accepted that
high-temperature short-time (HTST) pasteurization
(72 °C for 15 s) very extensively inactivates the
enzyme. However, it may contribute to lipolysis in pas-
teurized milk cheese, as heating at 78 °C  10 s is
required for its complete inactivation (Driessen, 1989).
Commercial rennet extracts are free from lipolytic
activity, but rennet paste, used in the manufacture of
some hard Italian varieties (e.g., Provolone and
Pecorino cheeses), contains a lipase, pregastric esterase
(PGE) (Nelson et al., 1977; Hamosh, 1990; Fox,
2003). Although the term ‘esterase’ suggests that this
enzyme acts only on water-soluble esters, it is in fact
able to hydrolyse water-insoluble triglycerides contain-
ing long-chain fatty acids and hence ‘esterase’ is a
misnomer as this enzyme is a lipase (Hamosh, 1990).
Pregastric esterase is highly specific for short chain
acids esterified at the sn-3 position (Nelson et al.,
1977; Fox and Stepaniak, 1993; Fox, 2003). Pregastric
esterases from different species are optimally active at
32–42 °C, pH 4.8–5.5 in presence of 0.5 M NaCl. The
enzyme has an isoelectric point above pH 9 with a posi-
tive charge at physiological pH (see Fox, 2003). Suck-
ling stimulates the secretion of PGE by glands at the
base of the tongue, and it is washed into the aboma-
sum by the milk. Rennet paste is prepared from the
abomasa of calves, kids or lambs slaughtered after
suckling. The abomasum is partially dried and ground
into a paste, which is slurried in milk before being
added to cheesemilk (Fox and Stepaniak, 1993). Some
interspecies differences in specificity have been
reported for calf, kid and lamb PGEs which result in
slight differences in the flavour characteristics of
cheese, depending on the source of PGE (Nelson et al.,
1977; Fox and Stepaniak, 1993). Due to concerns
regarding the hygienic quality of rennet pastes,
research has been focused on identifiying exogenous
lipases which could be blended with rennet extracts to
produce substitutes for rennet pastes. Most lipases
investigated are unsuitable due to incorrect speci-
ficity; certain fungal lipases (e.g., a lipase secreted by
Rhizomucor miehei) may be acceptable alternatives to
rennet paste (Fox, 1988).
Lipases and esterases of LAB appear to be the prin-
cipal lipolytic agents in Cheddar and Dutch-type
cheeses made from pasteurized milk (Fox et al., 2000).
Evidence for this comes from studies on aseptic starter-
free cheeses acidified with gluconic acid--lactone,
where very low levels of FFAs are released during
ripening (Reiter et al., 1967) and from the relationship
between autolysis of starter cells and FFA levels dur-
ing ripening (Collins et al., 2003a). Lactic acid bac-
teria possess esterolytic/lipolytic enzymes capable of
hydrolysing a range of derivatives of FFAs, tri-, di-
and mono-glyceride substrates (Holland and Coolbear,
1996; Chich et al., 1997; Fox and Wallace, 1997; Liu
et al., 2001). Despite the presence of these enzymes,
LAB, especially Lactococcus and Lactobacillus spp.
are weakly lipolytic in comparison to species such
as Pseudomonas, Acinetobacter and Flavobacterium
(Stadhouders and Veringa, 1973; Fox et al., 1993;
Chich et al., 1997). However, because they are present
at high numbers over an extended ripening period,
LAB are responsible for the liberation of significant
levels of FFAs in many cheese varieties which do not
have a strongly lipolytic secondary flora.
Lipases and esterases of LAB appear to be intracel-
lular and a number have been isolated and character-
ized (Chich et al., 1997; Castillo et al., 1999; Liu et al.,
2001). The presence of lipases and esterases has been
demonstrated (Piatkiewicz, 1987) in nine strains of
lactococci, including Lc. lactis subsp. lactis, citrate-
positive lactococci and Lc. lactis subsp. cremoris.
-Naphthyl laurate (C12:0) and -naphthyl acetate
(C2:0) were the substrates used to determine lipase and
esterase activities, respectively; esterase activity was
higher than lipase activity in all strains. Kamaly et al.
(1990) reported the presence of lipases in the cell-free
extract of a number of strains of Lc. lactis subsp. lactis
and Lc. lactis subsp. cremoris; these lipases were, in
general, optimally active at 37 °C and at pH 7–8.5.
Lc. lactis subsp. cremoris showed higher lipolytic activ-
ity on tributyrin and milk fat emulsions than Lc. lactis
subsp. lactis. The activity of all lipases was stimulated
by reduced glutathione and low (c. 2%) concentra-
tions of NaCl but inhibited by high NaCl concentra-
tions (c. 20%). Chich et al. (1997) reported esterolytic
activity on -naphthyl butyrate by an intracellular
extract of Lc. lactis subsp. lactis NCDO 763; the
enzyme was active on p-NP esters from C2 to C12, with
pH and temperature optima 7.0–8.0 and 55 °C.
 Lipolysis and Catabolism of Fatty Acids in Cheese 375
El-Soda et al. (1986) found intracellular esterolytic
activities in four species of lactobacilli: Lb. helveticus,
Lb. delbrueckii subsp. bulgaricus, Lb. delbrueckii subsp.
lactis and Lb. acidophilus. All lactobacilli showed activ-
ity on p-nitrophenyl (p-NP) derivatives of fatty acids
up to C5:0; Lb. delbrueckii subsp. lactis and Lb. acido-
philus showed the highest esterolytic activity. None of
the microorganisms tested hydrolysed o- and p-NP
derivatives of C6:0 to C14:0. Khalid and Marth (1990)
quantified the lipolytic activity of Lb. casei L-7, Lb. casei
L-14, Lb. plantarum L-34 and Lb. helveticus L-53 on
milk fat, olive oil and tributyrin emulsions; the three
substrates were hydrolysed by all lactobacilli with the
exception of Lb. casei L-7, which did not hydrolyse
olive oil. According to Lee and Lee (1990), esterolytic
and lipolytic enzymes are released by lysis of Lb. casei
subsp. casei LLG cells. Maximum lipolytic activity was
observed at pH 7.2 and 37 °C and activity was in-
hibited by Ag and Hg2 and stimulated by Mg2 and
Ca2 (Lee and Lee, 1990). Lb. fermentum contains a
cell surface-associated esterase which is optimally
active on C4:0 but which can hydrolyse -naphthyl
esters of fatty acids from C2:0 to C10:0 (Gobbetti et al.,
1997).
Gobbetti et al. (1996) reported the purification of
an intracellular lipase from a strain of Lb. plantarum
isolated from Cheddar cheese. This enzyme had a
molecular mass of 65 kDa and was optimally active at
pH 7.5 and 35 °C; it was relatively heat stable at 65 °C
but was irreversibly inactivated on heating at 75 °C for
2 min. The enzyme was most active on tributyrin, with
less activity on trilaurin and tripalmitin and no activity
on triolein. When activity was determined on -naphthyl
esters from C2 to C18:1, the enzyme was most active
on -naphthyl butyrate, with significant activity on
-naphthyl esters of acetate, caproate, caprylate and
laurate, and low activity on -naphthyl caprate, myris-
tate, palmitate, stearate and oleate.
Liu et al. (2001) identified three intracellular esterases
in Streptococcus thermophilus, two of which were puri-
fied to homogeneity and designated esterase I and II,
with a molecular mass of ⬃34 and 60 kDa, respect-
ively. Differences in substrate specificity between
esterases I and II were noted; esterase I hydrolysed p-NP
esters of short chain acids from C2 to C8 while esterase
II hydrolysed C2–C6 p-NP esters only. Both enzymes
had maximum activity on p-NP butyrate. Only
esterase I was tested on a range of glyceride substrates;
it hydrolysed di- and mono-glycerides containing fatty
acids up to C14:0 but tributyrin was the only trigly-
ceride it could hydrolyse. The impact of cheese compos-
ition on esterase I activity on p-NP butyrate substrate
indicated that activity was reduced by decreasing pH
in the range 5.5–8.0, and decreasing water activity in
the range aw 0.99–0.80. Interestingly, esterase activity
increased on increasing the NaCl concentration from
3.7 to 7.5%, w/v; this stimulatory effect of NaCl on
esterase activity had not been reported previously. The
genetic characterization of lipolytic enzymes of LAB by
Fernandez et al. (2000) confirmed the intracellular
nature of a tributyrin esterase in Lc. lactis subsp. cre-
moris B1014. These workers showed that the 744 base
pair estA gene encoded for a 258 amino acid protein of
molecular mass ⬃29 kDa; however, this gene did not
encode for a signal sequence required for extracellular
secretion. Cloning of the gene and up to 170-fold over-
production of this enzyme was possible using a nisin-
controlled expression system which allowed detailed
characterization of the specificity and kinetics of the
enzyme. The esterase showed highest activity on
short-chain p-NP-esters of fatty acids, with highest
activity on p-NP hexanoate (C6:0). This enzyme was
not active on p-NP esters of fatty acids of chain length
longer than C12:0. Tributyrin was readily hydrolysed
while activity was also detected on phospholipids.
However, increasing the concentration of phospho-
lipids to levels favouring micelle formation did not
lead to an increase in enzyme activity through inter-
facial activation, confirming the esterolytic nature of
this enzyme.
Since the esterases/lipases of LAB appear to be
intracellular, they must be released into the cheese
matrix through cell autolysis for maximum efficiency.
However to date, few studies have been undertaken to
establish whether a relationship exists between the
extent of autolysis of LAB and lipolysis in various
cheese varieties. Early work by Walker and Keen
(1974) showed that Cheddar cheese made with Lc. lactis
subsp. cremoris AM2 developed higher levels of odd-
numbered C3—C15 methyl ketones than cheese made
with Lc. lactis subsp. cremoris HP which indicated that
properties of the starter strain may influence the con-
centrations of these compounds in cheese. However,
these workers did not monitor cell viability or autoly-
sis in cheese during ripening. Wilkinson et al. (1994)
demonstrated that Lc. lactis subsp. cremoris AM2 is
more autolytic than Lc. lactis subsp. cremoris HP and
that secondary proteolysis is higher in Cheddar cheese
made using the former strain. Collins et al. (2003a)
studied the influence of starter autolysis on lipolysis
during a 238-day ripening period, as measured by the
release of FFAs (C4:0–C18:3) in Cheddar cheese made
using Lc. lactis subsp. cremoris AM2 or Lc. lactis subsp.
cremoris HP as starter. These workers found that
cheese made using the highly autolytic starter, Lc. lac-
tis subsp. cremoris AM2, developed significantly higher
levels of caprylate (C8:0), myristate (C14:0), palmitate
(C16:0) and stearate (C18:0) during ripening than
376 Lipolysis and Catabolism of Fatty Acids in Cheese
cheese made with the less autolytic strain, Lc. lactis
subsp. cremoris HP. Cell-free extracts prepared from
both strains had generally similar levels of activity on
a triolein emulsion (lipase) or a p-NP butyrate
(esterase) and these workers suggested that there is a
relationship between early autolysis of these starter
bacteria and lipolysis, possibly as a result of elevated
release of intracellular lipolytic activity in cheese.
Freitas et al. (1999) assayed proteolytic and lipoly-
tic activities of Enterococcus faecium, Ec. faecalis, Lb.
plantarum and Lb. paracasei and three species of yeasts
(Debaryomyces hansenii, Yarrowia lipolytica and Crypto-
coccus laurentii) isolated from Picante cheese. High
lipolytic activity was reported for Y. lipolytica, using
tributyrin as substrate; the other species studied were
less lipolytic.
Brevibacterium linens is a constituent of the microflora
of surface smear-ripened cheeses (e.g., Limburger)
which undergo a significant level of lipolysis during
ripening. Lipolytic activity has been demonstrated
in B. linens using emulsified olive oil as substrate (San
Clemente and Vadehra, 1967). Sørhaug and Ordal
(1974) reported esterolytic and lipolytic activities in
five strains of B. linens using tributyrin and olive oil as
substrates. Welch Baillargeon et al. (1989) reported
lipase activity in three strains of Geotrichum candidum.
Emulsified esters of oleic or palmitic acid were used as
substrates; optimum pH and temperature for lipolytic
activity were pH 7 and 37 °C, respectively. Lipases of
different strains of G. candidum may be classified
either as type A strains, which are not highly specific
for unsaturated substrates with cis-9 double bonds, or
type B strains, which are specific for cis-9 18:1 ( Jacobsen
and Poulsen, 1995).
In mould-ripened cheeses, such as Brie, Camembert
and Roquefort, Penicillium spp. are significant agents
of lipolysis (Gripon, 1993; McSweeney and Sousa,
2000). P. roqueforti possesses two lipases, one with a
pH optimum of 7.5–8, the other with a more alkaline
pH optimum (9–9.5) (Morris and Jezeski, 1953; Kman
et al., 1966; Niki et al., 1966). The lipase with the
lower pH optimum is more active on tricaproin while
that with the higher pH optimum is more active on
tributyrin (Menassa and Lamberet, 1982). P. camem-
berti produces an extracellular lipase, which is opti-
mally active on tributyrin at pH 9 and 35 °C
(Lamberet and Lenoir, 1976).
Propionic acid bacteria are between 10 and 100
times more lipolytic than LAB (Knaut and Mazurek,
1974; Dupuis, 1994). Using emulsified tributyrin
as substrate, Oterholm et al. (1970) showed that P.
freudenreichii subsp. shermanii possesses an intracellu-
lar lipase with pH and temperature optima of 7.2 and
47 °C. The maximum rate of hydrolysis of triglycer-
ides was observed on tripropionin (C3:0), followed in
order by tributyrin (C4:0), tricaproin (C6:0) and tri-
caprylin (C8:0). Hydrolysis of substrates in solution
was low in comparison to hydrolysis of emulsified sub-
strates, suggesting that the enzyme is a lipase. Dupuis
et al. (1993) screened a number of strains of propionic
acid bacteria for both esterolytic and lipolytic activities
on actetate, propionate and butyrate esters and tribu-
tyrin. Intracellular fractions prepared from strains
grown in liquid media showed both esterase and lipase
activities. In the study of Dupuis et al. (1993) evidence
was provided for the presence of an extracellular
esterase; however, the extent to which this activity
may have been due to cell lysis was not determined.
Kakariari et al. (2000) purified an intracellular
esterase from P. freudenreichii subsp. freudenreichii.
Cell-free supernatant, cell wall fractions and a soni-
cated intracellular extract were assayed for esterase
activity. However, in contrast to Dupuis et al. (1993),
esterase activity was not found in the cell-free
medium, or in the cell wall fractions. The enzyme
purified by Kakariari et al. (2000) was therefore either
cytoplasmic or cell membrane-associated.
Catabolism of Fatty Acids and Other
Reactions
In cheese, FFAs are precursors of many important
flavour and aroma compounds, such as methyl ketones,
lactones, esters, alkanes and secondary alcohols. Path-
ways for fatty acid catabolism are summarized in Fig. 1.
Methyl ketones (alkan-2-ones) are the most import-
ant flavour components in Blue cheese and are present
at very high concentrations. Heptan-2-one and nonan-
2-one are the predominant methyl ketones in Blue
cheese; their concentrations in Gamonedo increased
during the early part of ripening to a maximum at 60
days, after which levels began to decrease (Gonzalez
de Llano et al., 1990). Methyl ketones are formed in
Blue cheese by the action of Penicillium roqueforti
(Urbach, 1997). P. camemberti and G. candidum may
also produce methyl ketones (Lamberet et al., 1982;
Cerning et al., 1987; Molimard and Spinnler, 1996).
Penicillium spores, as well as vegetative mycelia, can
produce methyl ketones (Chalier and Crouzet, 1998).
Metabolism of fatty acids by Penicillium spp. involves
four main steps corresponding to the early stages of
-oxidation (Fig. 2). Initially, fatty acids are released by
lipases, followed by the oxidation of FFA to -ketoacids
and decarboxylation to alkan-2-ones, of one less carbon
atom than the parent FFA; alkan-2-ones may be reduced
to the corresponding secondary alcohol (alkan-2-ol).
It has been suggested (Dartey and Kinsella, 1973;
Kinsella and Hwang, 1976a) that FFAs are not the
 Lipolysis and Catabolism of Fatty Acids in Cheese 377

Triglyceride

O
C
O O
C
O
O
C
O

se OH O
pa
Li C OH

γ - or δ -hydroxy fatty acids

Partial glycerides
H2 O

C
O
γ - or δ -lactones
O

C OH

Fatty acids
R-SH
Thiols CH3CH2OH
Ethanol
β-Oxidation

O CO2 O

C S-R O C CH3
Thioesters C OCH2CH3 Alkan-2-ones
Ethyl esters

OH

CH CH3
Alkan-2-ols

Figure 1 Pathways for the catabolism of free fatty acids.

only precursors of methyl ketones; they can also be
formed from ketoacids naturally present at low con-
centrations in milk fat or by oxidation of monounsatur-
ated fatty acids (Kinsella and Hwang, 1976b). The rate
of production of methyl ketones in cheese is affected
by temperature, pH, physiological state of the mould
and the concentration of FFAs. Spores of P. roqueforti
can oxidize fatty acids containing 4–12 carbon atoms
to methyl ketones, with octanoic acid being most rap-
idly oxidized. Oxidation of longer chain FFAs contain-
ing 16 or 18 carbon atoms has also been reported
(Chalier and Crouzet, 1993). Mycelia oxidize FFAs
over a wide pH range, with an optimum between pH 5
and 7, which is similar to the pH of mature Blue
cheese (Dwivedi and Kinsella, 1974; Gripon, 1993).
Dumont et al. (1974a,b,c) identified 11 methyl
ketones in Camembert cheese. Methyl ketones with
even-numbered carbon chains appeared late in ripen-
ing and were present at low levels, except in very
mature cheese. Heptan-2-one is present at significant
concentrations in Parmigiano-Reggiano (Meinhart and
Schreier, 1986). In full-fat Cheddar cheese, levels of
heptan-2-one, nonan-2-one and undecan-2-one increased
for approximately 14 weeks, after which levels
decreased; concentrations of methyl ketones in low-fat
Cheddar cheeses were lower than the levels in full-fat
cheese (Dimos, 1992), perhaps due to lower levels
of FFAs in the former (Dimos et al., 1996). Engels et al.
(1997) compared the volatile compounds in the water-
soluble fraction of seven cheese varieties (Gouda,
378 Lipolysis and Catabolism of Fatty Acids in Cheese
Saturated fatty acids (C2n)
CoA-SH
β-Oxidation, –2H 2 + H2O
Keto acyl-CoA
CoA-SH
Thiohydrolase Thiolase
CoA-SH + β-Keto acid Acetyl-CoA + Acyl (C2n –2 ) -CoA
Krebs’ Cycle
β-Ketoacyldecarboxylase CO2
Methyl ketone (C2n –1 ) + CO2
Reductase
Secondary alcohol (C2n –1 )
Figure 2 Pathway for the catabolism of fatty acids by Penicil-
lium spp.
Proosdij, Gruyere, Maasdam, Edam, Parmesan and
Cheddar). Nine ketones, mostly methyl ketones, were
identified. Dirinck and De Winne (1999) reported levels
of heptan-2-one and nonan-2-one ranging from 2903
to 3210 ng g 1 and from 1841 to 1960 ng g 1, respect-
ively, in Gouda cheese; levels varied between 3332 and
3598 ng g 1 and 1768 and 1986 ng g 1, respectively, in
Emmental. In another study, pentan-2-one and heptan-
2-one were the most abundant methyl ketones in aged
ewes’ milk cheese (14 samples were analysed, nine of
which were Manchego); mean levels were 737 and 368
~or et al., 2000).
g kg 1, respectively (Villasen
Secondary alcohols can be formed in cheeses by
enzymatic reduction of methyl ketones (Engels et al.,
1997). P. roqueforti is responsible for the reduction of
methyl ketones to secondary alcohols (e.g., 2-pentanol,
2-heptanol and 2-nonanol) in Blue cheese (Martelli,
1989). Gonzalez de Llano et al. (1990) reported that
2-heptanol and 2-nonanol are the main secondary alco-
hols in artisanal Gamonedo Blue cheese. Production of
2-propanol from acetone and 2-butanol from butanone
has been reported in Cheddar cheese (Urbach, 1993),
while Thierry et al. (1999) reported an increase in the
concentrations of secondary alcohols in the aqueous
phase of Emmental during ripening.
A great diversity of esters, formed by the reaction of
a FFA with an alcohol, is present in cheese (Molimard
and Spinnler, 1996). While methyl, ethyl, propyl and
butyl esters of FFAs have been reported in various
cheese varieties (Meinhart and Schreier, 1986), ethyl
esters predominate (Arora et al., 1995). Esterification
reactions resulting in the production of esters occur
between short- to medium-chain fatty acids and
ethanol, derived from lactose fermentation or from
amino acid catabolism. Ethyl acetate may also arise
from esterification of ethanol with acetyl-coenzyme
A (Yoshioka and Hashimoto, 1983). Holland et al.
(2002) suggested that esters are formed in cheese dur-
ing ripening by the transesterification of a FFA from
partial glycerides to ethanol. Thirty eight esters were
identified by Meinhart and Schreier (1986) in Parmi-
giano-Reggiano cheese; ethyl acetate, ethyl octanoate,
ethyl decanoate and methyl hexanoate were the most
abundant. Methyl and ethyl esters have been found at
high levels in artisanal Blue cheese (Gonzalez de
Llano et al., 1990). Fourteen esters were found in
Emmental cheese by Imhof and Bosset (1994) and
Rychlik et al. (1997). Ethyl esters of fatty acids and
smaller quantities of methyl esters have also been
identified in Manchego cheese (Villasen ~or et al.,
2000).
Thioesters are formed when FFAs react with sul-
phydryl compounds (Molimard and Spinnler, 1996)
and may be formed by the action of a wide range of
microorganisms associated with cheese (Lamberet et al.,
1997).
Lactones are cyclic compounds formed by the
intramolecular esterification of hydroxy fatty acids
through the loss of water to form a ring structure
(Christie, 1983; Molimard and Spinnler, 1996) and
may be produced by heating their precursor hydroxy-
acids (Eriksen, 1976). - and -lactones are highly
reactive and unstable (Fox and Wallace, 1997) but -
and -lactones (5- and 6-membered rings, respect-
ively) are stable and have been identified in cheese
(Eriksen, 1976).
It has been reported that the mammary gland of
ruminants has a -oxidation system for fatty acid
catabolism (see Fox et al., 2000) which may produce
the precursor hydroxy acids for the production of lac-
tones, which may also be formed from keto acids after
their reduction to hydroxyacids (Wong et al., 1975).
The presence of large amounts of high molecular
weight lactones has been reported in rancid Cheddar
cheese, which has led to the suggestion that pathways
other than the release of hydroxy acids from trigly-
cerides may contribute to the formation of lactones
(Wong et al., 1975). Dodecalactone may be formed
by P. roqueforti spores and vegetative mycelia from
long-chain unsaturated fatty acids (C18:1 and C18:2)
(Chalier and Crouzet, 1992). Lactone precursors
(hydroxy FA) may also be generated by the action
of lipoxygenases and other enzymes present in mem-
bers of the rumen microflora (Dufossé et al., 1994).
 Lipolysis and Catabolism of Fatty Acids in Cheese 379
Production of lactone precursors in milk is influenced by
several factors, including feed, season, stage of lactation
and breed (see Fox et al., 2000). The sweet-flavoured
-dodecanolactone and -dodec-Z-6-enolactone occur
at much higher levels in milk from grain-fed than from
pasture-fed cows (Urbach, 1997).
-Dodecalactone and -tetradecalactone are the
principal lactones in 75 day-old Blue cheese (Jolly and
Kosikowski, 1975). In Cheddar cheese, lactone levels
increased most rapidly early in the ripening period
and were present at concentrations well above their
flavour thresholds (Jolly and Kosikowski, 1975).
Wong et al. (1975) reported levels of 1.5, 0.8, 4.9 and
8.9 g g 1 of C10, C12, C12 and C14, respectively,
in Cheddar cheese at 14 months. Several lactones have
been identified in Parmigiano-Reggiano cheese; quan-
titatively, the most important lactone is -octalactone
(Meinhart and Schreier, 1986). -Decalactone, -
decalactone, -dodecalactone and -dodecalactone have
been found in Camembert cheese (Gallois and Langlois,
1990).
Aldehydes may be formed via the catabolism of
amino acids (see ‘Catabolism of Amino Acids in Cheese
During Ripening’, Volume 1). However, some straight-
chain aldehydes, e.g., butanal, heptanal and nonanal,
may be formed by the -oxidation of unsaturated fatty
acids. Gruyere and Parmesan have high levels of FFAs
and high concentrations of straight chain aldehydes
which have ‘green grass-like’ aromas (Moio et al.,
1993).
Contribution of Lipolysis and Catabolism
of FFA to Cheese Flavour
Lipids play a major role in the quality of cheese:
• They affect cheese rheology and texture (see ‘Rheol-
ogy and Texture of Cheese’, Volume 1).
• They influence flavour by:
– Acting as a source of fatty acids which in turn may
be catabolized to other flavour compounds, e.g.,
methyl ketones, esters, thioesters and lactones.
– Acting as a solvent for sapid compounds pro-
duced from lipids or other precursors.
• Many reactions occur at the fat–water interface.
Reduced-fat cheese lacks typical flavour and con-
tains lower concentrations of FFAs than full-fat
cheese, supporting the theory that FFAs are important
to cheese flavour (Foda et al., 1974; Olson and Johnson,
1990; Dimos et al., 1996; Wijesundera et al., 1998).
Cheddar cheese manufactured from milk in which the
fat was replaced by vegetable or mineral oils has also
been reported to develop atypical flavours (Foda et al.,
1974; Wijesundera and Watkins, 2000). Foda et al.
(1974) found that cheese containing vegetable fats had
very little Cheddar flavour, and cheese containing
mineral oil had only a slight Cheddar flavour. Cheese
containing milk fat gave the best results but its flavour
was still inferior to that of cheese made from whole
milk. These results suggest that the MFGM, which is
replaced on homogenization of the milk fat, may have
important enzymes or other factors which play a role
in the development of Cheddar flavour. It is also plau-
sible that the interface between the lipid and aqueous
phases in the cheese is important for flavour develop-
ment. However, Wijesundera and Drury (1999)
reported no significant difference in Cheddar cheese
flavour intensity between cheeses made from whole
milk or from milk reconstituted from skim milk and
cream or anhydrous milk fat.
Long-chain FFAs (12 carbon atoms) are consid-
ered to play a minor role in cheese flavour due to their
high perception thresholds (Molimard and Spinnler,
1996). Short- and intermediate-chain FFAs (C4:0–C12:0)
have considerably lower perception thresholds and
each gives a characteristic flavour note. Butanoic acid
contributes ‘rancid’ and ‘cheesy’ flavours; hexanoic
acid has a ‘pungent’, ‘Blue cheese’ flavour note, while
octanoic acid has a purported ‘wax’, ‘soap’, ‘goat’,
‘musty’, ‘rancid’ and ‘fruity’ note. Depending on their
concentration and perception thresholds, volatile fatty
acids can either contribute positively to the aroma of
the cheese or to a rancidity defect. The flavour effect
of FFAs in cheese is affected by pH. In cheeses with a
high pH, e.g., smear-ripened and Blue cheeses, the
flavour impact of fatty acids may be affected due to
neutralization of the cheese (Molimard and Spinnler,
1996).
In general, the flavour threshold of methyl ketones is
quite low. Flavour threshold values determined in water
vary widely; for heptan-2-one, ranging from 0.0009 to
3 mg kg 1 and for propan-2-one ranging from 40.9 to
500 mg kg 1 (Molimard and Spinnler, 1996). Octan-
2-one, nonan-2-one, decan-2-one, undecan-2-one and
tridecan-2-one are described as having ‘fruity’, ‘floral’
and ‘musty’ notes, while heptan-2-one has a Blue cheese
note (Rothe et al., 1982). The mushroom and musty
notes of methyl ketones are important contributors to the
flavour of Camembert cheese (Molimard and Spinnler,
1996).
According to Eriksen (1976), lactones have a strong
flavour; although the aromas of lactones are not
cheese-like, they may contribute to overall cheese
flavour (see Fox et al., 1993, 2000; Fox and Wallace,
1997) and have been reported to contribute to a but-
tery character in cheese (Dirinck and De Winne, 1999).
-Lactones have low flavour thresholds compared to
other volatile flavour compounds (O’Keefe et al., 1969)
380 Lipolysis and Catabolism of Fatty Acids in Cheese
and are generally characterized by very pronounced,
fruity notes (‘peach’, ‘apricot’ and ‘coconut’) (Dufossé
et al., 1994). -Lactones have generally higher detection
thresholds than -lactones; thresholds for -octalactone,
-decalactone and -dodecalactone are 7–11 g kg 1
in water and are even lower for shorter chain lactones
(Dufossé et al., 1994).
In a survey of various cheese varieties, Engels et al.
(1997) found high concentrations of ethyl butanoate
in cheeses with a ‘fruity’ note, e.g., Gruyere, Parmesan
and Proosdij. This fruity flavour is considered undesir-
able in Cheddar cheese (Urbach, 1997; McSweeney
and Sousa, 2000).
Arora et al. (1995), who analysed the odour-active
volatiles in the headspace of Cheddar cheese, found
that most of the esters separated had a ‘buttery’/‘fruity’
aroma. However, thioesters formed by the reaction of
short-chain fatty acids with methional imparted a char-
acteristic ‘cheesy’ aroma to Cheddar cheese. According
to Lamberet et al. (1997), S-methyl thioesters con-
tribute a characteristic flavour to various smear-ripened
soft cheeses (e.g., Tilsit, Limburger and Havarti).
Secondary alcohols may contribute to cheese
flavour (Arora et al., 1995). Propan-2-ol, butan-2-ol,
octan-2-ol and nonan-2-ol are present in most soft
cheeses and are typical components of the flavour of
Blue cheeses (Engels et al., 1997). Moinas et al. (1975)
found that heptan-2-ol and nonan-2-ol represented
10–20% and 5–10%, respectively, of all aromatic com-
pounds in Camembert cheese. Oct-1-en-3-ol has the
odour of raw mushroom with a perception threshold
of 10 g kg 1, and has been proposed as one of the
key compounds in the aromatic note of Camembert
cheese (Molimard and Spinnler, 1996).
Patterns of Lipolysis in Various Cheese
Varieties
The level of lipolysis in cheese varies considerably from
moderate (e.g., Cheddar, Cheshire, Caerphilly) to
extensive (e.g., mould-ripened, hard Italian and surface
bacterially-ripened (smear) varieties) (de Llano et al.,
1992; McSweeney and Fox, 1993; Fernandez-Garcia
et al., 1994; Fox and Wallace, 1997; Fox et al., 2000;
McSweeney and Sousa, 2000). Concentrations of FFAs
reported for a number of cheese varieties are shown in
Table 1. Excessive lipolysis is considered undesirable
in many varieties (e.g., Dutch-type cheeses, Cheddar,
Emmental) and cheeses containing even a moderate
concentration of FFAs may be considered as rancid by
some consumers (Fox et al., 1993). However, limited
lipolysis is thought to be desirable in these varieties.
In Emmental cheese, moderate levels of FFAs, in the
range 2–7 g kg 1, are liberated during ripening and
make an important contribution to the characteristic
flavour and aroma of both raw and pasteurized milk
Emmental cheese (Zerfiridis et al., 1984; Steffen et al.,
1993; Chamba and Perreard, 2002). Recent data for
lipolysis in Emmental cheese manufactured from raw
or microfiltered milk, with or without various strains
of propionic acid bacteria, indicate that FFAs are
released during ripening by the lipolytic activity of
propionic acid bacteria and starter and that the con-
centration of FFAs produced in the cheese is specific
to the strain of Propionibacterium used for cheesemaking
(Chamba and Perreard, 2002).
The highest levels of lipolysis have been observed in
mould-ripened cheeses; 5–10% of total triglycerides are
hydrolysed in Camembert and up to 25% in Blue
cheeses (Anderson and Day, 1966; Gripon et al., 1991;
Gripon, 1993). Extensive lipolysis occurs in mould-
ripened cheeses without rancidity due to neutralization
of fatty acids as the pH increases during ripening
(Gripon, 1993). The most important lipolytic agents in
mould-ripened cheeses are the enzymes of Penicillium
spp., although a high proportion of free oleic acid in
Camembert has been attributed to the action of the
lipase of Geotrichum candidum (Gripon, 1993). In the
manufacture of Danablu cheese, raw milk is separated
and the resulting cream is homogenized and held before
pasteurization. This process damages its MFGM and
activates the LPL; additionally, homogenization reduces
fat globule size and increases total fat globule surface
area, providing a larger lipid–serum interface for LPL
activity (Nielsen, 1993). Extensive lipolysis is also char-
acteristic of many Italian varieties (e.g., Grana Padano
and Parmigiano-Reggiano) (Woo and Lindsay, 1984;
Bosset and Gauch, 1993). Arnold et al. (1975) found a
direct relationship between the flavour intensity of
ripened Romano-type cheese and its butyric acid con-
tent. Many Italian varieties are manufactured from raw
milk (e.g., Parmigiano-Reggiano, Grana Padano, Pro-
volone) which leads to higher levels of lipolysis in the
ripened cheese due to the action of LPL. Pregastric
esterase is responsible for extensive lipolysis in cheeses
made using rennet paste, resulting in the characteristic
‘piccante’ flavour of these varieties (Fox et al., 2000;
McSweeney and Sousa, 2000). High levels of lipolysis
occur in smear cheeses (e.g., Limburger) (Woo et al.,
1984). Brevibacterium linens is a major constituent of the
surface microflora of bacterial smear-ripened cheeses
and produces active lipolytic enzymes (Reps, 1993).
Measurement of Lipolysis
Various quantitative techniques have been developed
to monitor release of FFAs in cheese. The ‘copper
soaps’ method is a colorimetric method enabling the
 Table 1 Concentration (mg kg 1 cheese) of free fatty acids in some selected cheese varieties

Cheese
type C2:0 C4:0 C6:0 C8:0 C10:0 C12:0 C14:0 C16:0 C18:0 C18:1 C18:2 C18:3 Total Method Reference

Rennet coagulated
Internal bacterially-ripened
Extra-hard
Parmesan 1055 451 243 440 439 1540 3896 1171 3471 123 13697 GC de la Feunte
et al. (1993)
Parmesan 140 106 84 158 181 684 1750 1890* GC Woo and Lindsay
Romano 1756 843 328 942 428 448 785 1224* (1984)
Hard
Cheddar 476 952 143 175 159 571 952 1556 794 2841 635 238 9492 HPLC Kilcawley et al.
1587 952 191 159 175 619 746 1253 508 1476 413 175 8254 (2001)
1270 794 111 111 48 238 397 619 270 667 206 111 4842
15 2 6 25 37 103 285 524* 997 GC Woo et al.
111 33 38 67 68 183 397 131* 1028 (1984)
308a 144a 185a 387a 225a 423a 1148a 3259a* 6079a
10 24 28 45 117 309 131 503 41 23 GC McNeill and
5 16 20 46 110 270 123 524 40 27 Connolly
0 25 27 46 120 299 139 496 45 26 (1989)
0 6 20 37 86 231 81 200 21 0
⬃8 ⬃12 ⬃25 ⬃45 ⬃70 ⬃275 ⬃130 ⬃370 947 GC Reddy and
Marth (1993)
37 34 22 16 18 38 117 168* 450 GC Woo and Lindsay
43 26 16 29 25 61 202 299* 701 (1982)
45 35 18 16 13 27 110 169* 433
47 33 25 55 30 59 196 387* 832
56 45 29 22 35 76 216 365* 844
60 44 29 22 35 74 229 385* 878
142 68 73 134 94 175 433 1377* 2506
865 115 38 41 49 81 218 503 172 467 69 40 Titration Bills and Day
(1964)
69 105 13 12 15 48 76 61 209 36 GC Marsili (1985)
10 43 12 6 11 GC Dulley and
Grieve (1974)
⬃128 ⬃490 ⬃69 ⬃90 ⬃171 ⬃295 ⬃299 GC McSweeney
et al. (1993)
Cheshire 8 12 32 59 162 394 132 418 36 12 GC McNeill and
Connolly
(1989)
Cheshire 0 10 27 49 112 275 94 276 29 8
Roncal 505 302 377 372 392 1007 2243 620 1438 100 8178 GC de la Feunte
Idiazabal 285 118 65 578 358 667 1553 372 1299 40 5577 et al. (1993)
381

continued
382 Table 1 continued

Cheese
type C2:0 C4:0 C6:0 C8:0 C10:0 C12:0 C14:0 C16:0 C18:0 C18:1 C18:2 C18:3 Total Method Reference

Manchego 255 133 110 318 202 443 994 348 816 GC Poveda et al.
(1999)
Manchego 876 704 426 398 340 608 1925 508 1384 106 7285 GC Fernandez-
Garcia
et al. (1994)
Semi-hard
Caerphilly 18 15 31 57 150 387 127 419 36 13 GC McNeill and
Connolly
(1989)
Mahon 383 377 274 394 307 810 2107 902 2235 82 8743 GC de la Feunte
et al. (1993)
Colby 81 9 33 49 22 67 196 93* 356 GC Woo et al.,
Monterey Jack 93 3 10 37 20 110 252 211* 736 (1984)
Majorero 431 400 513 1964 1378 2327 5877 1327 4921 886 20794 GC de la Feunte
et al. (1993)
Majorero 216 250 225 723 364 663 1745 528 1400 6144 GC Martin-Hernández
and Juarez
(1992)
Cheeses with eyes
Swiss-type
Swiss 170 90 45 122 208 311 1904 1427* 4277 GC Woo et al.
345 21 25 53 88 267 930 1197* 2926 (1984)
392a 135a 150a 381a 522a 1337a 4262a 2664a* 9843a
Emmental 112 45 71 68 207 515 256 842 59 31 GC McNeill and
Connolly
(1989)
Gruyere 1037 83 6 16 13 58 193 75* 1481 GC Woo et al. (1984)
Gruyere 288 120 26 21 44 51 160 475 tr 14 50b GC Zerfiridis et al.
(1984)
Dutch-type
Edam 60 8 9 14 47 39 122 57* 356 GC Woo et al. (1984)
Pasta-Filata cheeses
Mozzarella 54 7 1 120 12 27 76 66* GC Woo and Lindsay
Provolone 782 308 81 172 122 120 199 334* (1984)
Mould-ripened
Surface mould-ripened
Camembert 35 5 14 35 43 69 270 210* 681 GC Woo et al.
(1984)
 Camembert 208 101 58 448 1028 1421 GC Lesage et al.
(1993)
Camembert 361 287 160 225 298 622 1442 303 1043 5066 GC de la Feunte
et al. (1993)
Brie 124 9 19 44 74 255 839 1314* 2678 GC Woo et al.
(1984)
Blue cheese
Roquefort 992 751 715 2104 1403 2632 6452 17404* 32453 GC Woo et al. (1984)
Roquefort 961 626 707 2280 1295 3185 6230 2241 6282 896 25969 GC de la Feunte
et al. (1993)
Blue 1146 777 546 1275 1835 4147 11416 14088* 35230 GC Woo et al. (1984)
Blue d’Auvergne
(Blue, with 105 700 815 2135 4460 GC Kinderlerer
conidia) et al. (1996)
(White, no 620 160 235 185
conidia)
Gamonedo 937 804 1040 3366 2329 7850 19768 9157 30434 75685 GC de Llano et al.
blue (1992)
Surface-ripened
Port Salut 41 4 8 54 33 86 275 199* 700 GC Woo et al. (1984)
Limburger 1475 688 24 50 92 602 565 709*
Brick 72 2 8 35 22 63 161 39* 402
62 27 68 193 153 357 952 538* 2350
995a 232a 446a 1067a 613a 1805a 2835a 5977*a 13970a
Serra da 37 34 33 89 46 98 198 106 220 12 7 GC Partidario et al.
Estrela (1998)
Serra da 35.7 34 35 97 52 102 201 103 208 11 3 GC Partidario (1999)
Estrela
Munster 163 102 66 154 206 704 2057 833 1412 59 504 GC de Leon-
Gonzalez et al.
(2000)
Concentration/crystallization
Gjetost 106 35 31 180 49 170 456 631* 4558 GC Woo et al. (1984)

* C18:0 congeners.
a Commercial samples exhibiting distinct flavour defects.
b C18:2  C18:3.
383
384 Lipolysis and Catabolism of Fatty Acids in Cheese
determination of the total level of FFAs (IDF, 1991).
Copper soaps of fatty acids are selectively transferred
to an organic phase; a coloured complex is then
formed between the copper soaps and sodium diethyl
dithiocarbamate, the intensity of which is related to
the concentration of FFAs. While the method is sensi-
tive and rapid (IDF, 1991), the total level of FFAs is
only estimated. Quantification of the total level of
FFAs by extraction and titration of FFAs with alco-
holic KOH gives an acid degree value (ADV), defined
as the number of milliequivalents of alkali required to
neutralize the FFAs in 100 g of fat.
Gas chromatography (GC) has been the method
most commonly used to quantify levels of individual
FFAs in cheese and is the dominant technique for the
routine analysis of FFAs; the flame ionization detector
is robust with a wide and dynamic range, enabling
accurate FFA quantification. In a review of the deter-
mination of FFAs in milk and milk products (IDF,
1991) methods for analysis of FFAs by GC were
grouped under three headings. In the first group, FFAs
are analysed directly and this group includes the
method of Nieuwenhof and Hup (1971). Free fatty
acids are isolated on an alkaline silica gel column, the
eluate is concentrated and FFAs are quantified directly
by GC. However, the silicic acid column used by
Nieuwenhof and Hup (1971) was later shown to
induce hydrolysis of the fat. The method of Woo and
Lindsay (1982) is also included in this group; this
method involves removal of lactic acid by a partition
pre-column, followed by isolation of FFAs on a modi-
fied silicic acid-potassium hydroxide arrestant column.
Free fatty acids are then separated in formic acid-
mobilized elutes on a glass column packed with diethyl-
ene glycol succinate (DEGS-PS) by GC. This GC
procedure enables rapid separation and quantification
of FFAs and has been used by others (Woo and Lindsay,
1984; Woo et al., 1984) to quantify individual FFAs in
various cheeses. The second group of GC methods
includes those of Iyer et al. (1967), McNeill and Con-
nolly (1989) and Fontecha et al. (1990). Solvent is ini-
tially used to extract the lipid fraction from the cheese,
followed by the preparation of methyl esters of FFAs
and the separation and quantification of methyl esters
of FFAs by GC. In general, the third group of GC
methods quoted in Table 1 involves solvent extraction
of the lipid fraction from the cheese, followed by separ-
ation of FFAs by, e.g., saponification (Martinez-Castro
et al., 1986; Martin-Hernández et al., 1990; Martin-
Hernández and Juarez, 1992; de la Feunte et al., 1993;
Poveda et al., 1999), separation using chromatographic
columns (Deeth et al., 1983; Lesage et al., 1993), or
methylation to fatty acid methyl esters (FAME) (Ha
and Lindsay, 1990; Kinderlerer et al., 1996; Partidario
et al., 1998; Partidario, 1999). The method of Dulley
and Grieve (1974) uses steam-distillation followed by
GC to quantify volatile fatty acids.
The method described by de Jong and Badings
(1990) is now a commonly used procedure for the
quantification of FFA in cheese. Accurate and rapid
determination of FFAs from C2:0 to C20:0 is achieved
by direct separation of underivatized FFAs using capil-
lary GC. In brief, the method involves extraction of
FFA using ether/heptane from a cheese paste contain-
ing anhydrous sodium sulphate and H2SO4. Extracted
FFAs are then isolated using alumina or an anion-
exchange method (aminopropyl columns) and separ-
ation of FFAs (C2:0–C20:0) is achieved by capillary GC.
Few studies have compared the different methods
of FFA quantification from the same sample. Accord-
ing to IDF (1991), isolation of FFAs from the sample
material is a vital step in any method. FFAs must be
isolated from both the aqueous phase and the fat
phase. Organic solvents are used in the extraction
procedures of the majority of the methods refer-
enced. According to IDF (1991), it is difficult to
combine good extraction of short chain FFAs from
the aqueous phase together with a good extraction
from the fat phase. While these extraction methods
commonly involve the use of internal standards of
fatty acids dissolved in an organic solvent, this does
not guarantee the completeness of the extraction
procedure.
Chavarri et al. (1997) compared two methods for
sample preparation for the determination of FFAs in
ewes’ milk cheese by GC. In method 1, after fat extrac-
tion, FFAs were separated from triacylglycerides by
aminopropyl bonded-phase chromatography. The frac-
tion containing FFAs was then injected directly onto
the gas chromatograph. In method 2, extracted fat was
treated with tetramethylammonium hydroxide and the
methyl ester derivatives were then formed in the injec-
tor. It was concluded that FFAs should be separated
from the triacylglycerides before derivatization and
chromatographic analysis, particularly for samples in
which a minor fraction of the triacylglycerides has
been hydrolysed to FFAs.
Three gas chromatographic methods for the analy-
sis of FFAs in cheese were compared by Ardö and
Polychroniadou (1999).
Method 1 included the preparation and methylation
procedures described by Martinez-Castro et al. (1986)
and Martin-Hernández et al. (1988). This involves
diethyl ether extraction of fat followed by methylation
with tetramethylammonium hydroxide, which results
in an upper layer containing methyl esters from gly-
cerides and a lower layer which holds the tetramethyl-
ammonium soaps of FFAs. The upper layer may be
 Lipolysis and Catabolism of Fatty Acids in Cheese 385
used to analyse the fatty acid composition of gly-
cerides. For FFA analysis, the bottom layer is separ-
ated, washed with ethyl ether and adjusted to pH 9.
The methyl esters are then analysed by programmed
GC, as described by Juarez et al. (1992). N2 or He was
used as carrier gas; the column was a silica capillary
column with silicone, containing 50% phenyl and 50%
cyanopropyl groups as the stationary phase. Injecting
samples at 300 °C gave the best quantitative results.
The derivitisation technique tested resulted in no loss
of the volatile components, because the FFAs were
injected in the form of soaps.
Method 2 involved the extraction of fats with
diethyl ether, fixation of FFAs onto an Amberlyst
resin, methylation of FFAs followed by quantification
of methyl esters of FFAs by GC using an FID detector.
Method 3 involves extraction of fat using heptane
with the addition of sodium sulphate and sulphuric
acid, followed by overnight storage of samples. On the
day of analysis, the sample is extracted using a mix-
ture of diethyl ether and heptane, the mixture is then
added to a NH2 Sep-Pak Vac cartridge. The cartridge is
rinsed using a chloroform/isopropanol mixture, FFAs
are eluted using a diethyl ether/formic acid mixture
and subsequently analysed by GC.
Ardö and Polychroniadou (1999) reported on the
accuracy and recovery for method 1 only. The accur-
acy of method 1 was checked using two types of
cheese; one was a fresh cheese with a low FFA content
and the other a cheese that had undergone moderate
lipolysis. Reproducibility values for the fresh cheese
were comparable to those reported by Woo and Lindsay
(1982) for Cheddar cheese; however, they were lower
than those reported by McNeill and Connolly (1989)
and Deeth et al. (1983) also for Cheddar cheese. Accur-
acy was substantially higher for cheese with a high
FFA content and there was an improvement in the
overall coefficient of variation. Recovery was examined
by adding standard mixtures of FFAs to a sample from
a cheese of known FFA content. Recoveries ranged
from 91% for butanoic acid (C4:0) to 103% for octade-
canoic acid (C18:0). It was concluded that this tech-
nique provides a fast and reliable method of FFA
analysis in cheeses with a low FFA level and particu-
larly for those with a high FFA level.
High performance liquid chromatography (HPLC)
may also be used as a method for FFA analysis (Marsili,
1985). High performance liquid chromatography can
operate at ambient temperature, ensuring relatively lit-
tle risk to sensitive functional groups (Christie, 1997).
In the method of Kilcawley et al. (2001), C2:0, C3:0 and
C4:0 were recovered by steam-distillation and quanti-
fied by ligand-exchange, ion-exclusion HPLC. C6:0 to
C18:3 are derivatized to bromophenacyl esters follow-
ing solvent extraction and separation is achieved by
reversed-phase HPLC.
Bills and Day (1964) used a silicic acid column to
separate FFAs, which were then quantified by titra-
tion with potassium hydroxide, using phenol-
phthalein as an indicator. Removal of CO2 from the air
stream when titrating is vital to maintain the stability
of the end point. In this method, the air stream was
freed from CO2 by bubbling through 20% KOH, and
IDF (1991) recommend titration under nitrogen. It
was later shown that the resin used induced fat
hydrolysis (McNeill and Connolly, 1989). When using
a titration method to quantify FFAs, the eluate of each
FFA must be titrated separately and the method
is not as accurate as more sophisticated GC or HPLC
methods.
At present, the most suitable methods for the deter-
mination of lipolysis in cheese use GC and HPLC; levels
of FFAs and FFA profiles may be determined. There is
potential for increased use of established technologies,
such as gas chromotography/mass spectroscopy (GC/MS)
and emerging technologies, e.g., liquid chromotogra-
phy/mass spectroscopy, for the measurement of FFAs.
GC/MS provides very accurate qualitative and quanti-
tative measurement of FFAs as well as the other volatile
components of dairy products (see ‘Instrumental Tech-
niques’, Volume 1).
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 Proteolysis in Cheese during
Ripening
V.K. Upadhyay, P.L.H. McSweeney, A.A.A. Magboul and P.F. Fox, Department
of Food and Nutritional Sciences, University College, Cork, Ireland
Introduction
As discussed in ‘Biochemistry of Cheese Ripening:
Introduction and Overview’, Volume 1, proteolysis is
the most complex and, in most varieties, the most
important of the three primary biochemical events
which occur in cheese during ripening. Because of its
importance, the subject has been reviewed extensively
(Grappin et al., 1985; Rank et al., 1985; Fox, 1989; Fox
and Law, 1991; Fox et al., 1993, 1994, 1995b, 1996;
Fox and McSweeney, 1996, 1997; Sousa et al., 2001).
Proteolysis contributes to:
• The development of cheese texture:
– via hydrolysis of the protein matrix of cheese;
– via a decrease in aw through changes to water
binding by the new carboxylic acid and amino
groups liberated on hydrolysis of peptide bonds.
These groups are ionized at the pH of cheese and
thus bind water;
– indirectly via an increase in pH caused by the
liberation of ammonia from amino acids pro-
duced by proteolysis.
• Flavour and perhaps the off-flavour of cheese,
– directly by the production of short peptides and
amino acids, some of which have flavours;
– indirectly by the liberation of amino acids which
act as substrates for a range of catabolic reactions
which generate important volatile flavour com-
pounds (see ‘Catabolism of Amino Acids in
Cheese During Ripening’, Volume 1);
– by facilitating the release of sapid compounds
from the cheese matrix during mastication.
Proteolysis in cheese during ripening is catalysed
by proteinases and peptidases from six sources:
• The coagulant: The enzymes involved depend on
the type of coagulant used (chymosin, pepsin, fungal
acid proteinases, plant acid proteinases). Residual
coagulant activity retained in the curd is the major
source of proteolytic activity in most cheeses except
pasta-filata varieties and those with a high cook
temperature in which enzymes from this source are
denatured extensively.
Cheese: Chemistry, Physics and Microbiology, Third edition – Volume 1: General Aspects
ISBN: 0-1226-3652-X
Set ISBN: 0-1226-3651-1
• The milk: A number of indigenous proteinases
are present in milk, the most important of which
is plasmin, which is produced from an inactive
precursor, plasminogen. The action of plasmin is of
particular importance in pasta-filata and high-cook
cheeses (since it is a heat-stable enzyme) and in
cheeses the pH of which increases during ripen-
ing (since the pH optimum of plasmin is c. 7.5).
Somatic cells, recruited into milk to flight mas-
titic infection, contain lysosomes which contain
a number of proteinases, including cathepsins
D and B.
• Starter lactic acid bacteria (LAB): The starter
LAB contain a cell envelope-associated proteinase
(CEP, lactocepin, PrtP) which contributes to ripening
principally by hydrolysing intermediate-sized and
short peptides produced from the caseins by the action
of chymosin or plasmin. The starter is the principal
source of peptidases in cheese, which are responsible
for the hydrolysis of short peptides and the liberation
of amino acids.
• Non-starter lactic acid bacteria (NSLAB): All
ripened cheeses contain an adventitious secondary
microflora which grows during ripening (see ‘The
Microbiology of Cheese Ripening’, Volume 1). The
proteinases and peptidases of NSLAB are generally
similar to those of starter LAB and contribute to
ripening in a similar fashion.
• Secondary starter: Many cheese varieties are
characterized by the development of a secondary
microflora which is added to or deliberately encour-
aged to grow (e.g., Propionibacterium freudenreichii
subsp. shermanii in Swiss-type cheese, Penicillium
roqueforti in Blue cheese, P. camemberti in Camem-
bert and Brie-type cheeses and a complex Gram-
positive bacterial microflora on the surface of smear
cheeses). Most of these microorganisms possess
potent enzyme activities, including proteinases and
peptidases, which contribute greatly to proteolysis in
these varieties.
• Exogenous proteinases and peptidases: Exogen-
ous proteolytic enzymes have been studied as a
means of accelerating ripening, accentuating flavour
Copyright © 2004 Elsevier Ltd
All rights reserved
392 Proteolysis in Cheese during Ripening
or debittering cheese. With the exception of enzyme-
modified cheese (see ‘Cheese as an Ingredient’,
Volume 2), exogenous proteinases and peptidases
are not used widely in cheese manufacture, although
the action of these enzymes can dominate patterns
of proteolysis in cheeses in which they are used.
The use of exogenous proteinases to accelerate
cheese ripening was discussed by Upadhyay and
McSweeney (2003).
The relative importance of enzymes from each of
these sources depends on the cheese variety. Techniques
used to assess the relative importance of proteolytic
enzymes from different sources were discussed by Fox
et al. (1993) and are outlined in ‘Biochemistry of Cheese
Ripening: Introduction and Overview’, Volume 1. This
chapter will focus on the principal proteolytic enzymes
found in cheese and their role in cheese during ripen-
ing, with particular emphasis on their specificity on the
caseins. Most attention will be paid to enzyme systems
common to many cheese varieties (i.e., enzymes from
the coagulant, milk and starter). Patterns of proteolysis
in individual cheese varieties and the enzymes of spe-
cific secondary microorganisms are also discussed in
some chapters of Volume 2.
Enzymes from the Coagulant
As discussed in ‘Rennets: General and Molecular
Aspects’, Volume 1, chymosin (EC. 3.4.23.4), the princi-
pal proteinase in traditional rennets used for cheesemak-
ing, is an aspartyl proteinase of gastric origin, secreted by
young mammals. The principal role of chymosin in
cheesemaking is to hydrolyse the Phe1059Met106 bond
of the micelle-stabilizing protein, -casein, as a result
of which the colloidal stability of the micelles is
destroyed, leading to gelation at temperatures ⬃20 °C
(see ‘Rennet-induced Coagulation of Milk’, Volume 1).
Most of the rennet added to cheesemilk is removed in
the whey but some is retained in the curd and plays a
major role in the initial proteolysis of caseins in many
cheese varieties. From ⬃0 to 30% of the coagulant
added to the cheesemilk is retained in the curd,
depending on enzyme type, cooking temperature, pH
at draining and the moisture content of curd. In Ched-
dar cheese, ⬃6% of the chymosin added to the milk is
retained in the curd, but the amount increases with
decreasing pH at whey draining (Holmes et al., 1977;
Creamer et al., 1985). Pepsins, especially porcine
pepsin, are more pH-sensitive than chymosin and
hence the amount of these coagulants retained in
cheese curd is very strongly dependent on the pH of
the milk at setting and shortly thereafter; in fact,
increasing the pH of the curds–whey mixture to ⬃7
after coagulation of milk by porcine pepsin is one of
the methods used to produce rennet-free cheese curd
(see ‘Biochemistry of Cheese Ripening: Introduction
and Overview’, Volume 1). Only 2–3% of Rhizomucor
rennets is retained in Cheddar cheese curd and
appears to be independent of pH (Creamer et al.,
1985). In high-cooked cheeses, e.g., Emmental, chy-
mosin is extensively denatured (Matheson, 1981;
Singh and Creamer, 1990; Boudjellab et al., 1994), and
makes relatively little contribution to ripening.
The action of chymosin on the B-chain of insulin
indicates that it is specific for hydrophobic and aro-
matic amino acid residues (Fish, 1957). Relative to
many other proteinases, chymosin is weakly proteolytic;
indeed, limited proteolysis is one of the characteristics
to be considered when selecting proteinases for use as
rennet substitutes (Fox, 1989; Dalgleish, 1993).
The primary chymosin cleavage site in the bovine
milk protein system is the Phe1059Met106 bond
in -casein which is many times more susceptible
to chymosin than any other bond in milk proteins.
s1-, s2- and -caseins are not hydrolysed during
milk coagulation but may be hydrolysed in cheese
during ripening.
A number of authors (Pelissier et al., 1974;
Creamer, 1976; Visser and Slangen, 1977; Carles and
Ribadeau-Dumas, 1984) have investigated the hydrol-
ysis of -casein by chymosin. In solution in 0.05 M Na
acetate buffer, pH 5.4, chymosin cleaves -casein
at seven sites: Leu1929Tyr193  Ala1899Phe190 
Leu1659Ser166  Gln1679Ser168  Leu1639Ser164 
Leu1399Leu150  Leu1279Thr128 (Visser and Slangen,
1977). The parameters, KM and kcat, for the action of
chymosin on the bond Leu1929Tyr193 are 0.075 mM
and 1.54 s 1, respectively, for micellar -casein and
0.007 mM and 0.56 s 1 for the monomeric protein
(Carles and Ribadeau-Dumas, 1984). NaCl inhibits the
hydrolysis of -casein by chymosin to an extent
dependent on pH; hydrolysis is strongly inhibited by
5% and completely by 10% NaCl (Mulvihill and Fox,
1978).
The primary site of chymosin action on s1-casein is
Phe239Phe24 (Hill et al., 1974; Carles and Ribadeau-
Dumas, 1985; McSweeney et al., 1993b). Cleavage of
this bond is believed to be responsible for the initial
softening of cheese texture (de Jong, 1976; Creamer
and Olson, 1982) and the small peptide ( s1-CN
f1923) is hydrolysed rapidly by starter proteinases.
The specificity of chymosin on s1-casein in solution
was studied by Pelissier et al. (1974), Mulvihill and
Fox (1979a), Pahkala et al. (1989) and McSweeney
et al. (1993b). In 0.1 M phosphate buffer, pH 6.5,
chymosin cleaves s1-casein at Phe239Phe24, Phe289
Pro29, Leu409Ser41, Leu1499Phe150, Phe1539 Tyr154,

Leu1569Asp157, Tyr1599Pro160 and Trp1649Tyr165
(McSweeney et al., 1993b). These bonds are also
hydrolysed at pH 5.2 in the presence of 5% NaCl (i.e.,
similar to the conditions in many cheese varieties),
and, in addition, Leu119Pro12, Phe329Gly35, Leu1019
Lys102, Leu1429Ala144 and Phe1799Ser180. The rate at
which many of these bonds are hydrolysed depends on
the ionic strength and pH, particularly Leu1019Lys102
which is cleaved far faster at the lower pH. The kcat
and KM for the hydrolysis of Phe239Phe24 bond of
1 and 0.37 mM,
s1-casein by chymosin is 0.7 s
respectively (Carles and Ribadeau-Dumas, 1985). The
hydrolysis of s1-casein by chymosin is influenced by
pH and ionic strength (Mulvihill and Fox, 1977,
1980).
s2-Casein appears to be relatively resistant to pro-
teolysis by chymosin; cleavage sites are restricted to
the hydrophobic regions of the molecule (sequences
90–120 and 160–207), i.e., Phe889Tyr89, Tyr959
Leu96, Gln979Tyr98, Tyr989Leu99, Phe1639Leu164,
Phe1749Ala175 and Tyr1799Leu180 (McSweeney et al.,
1994b). Although para--casein has several potential
chymosin cleavage sites, it does not appear to be
hydrolysed either in solution or in cheese (Green and
Foster, 1974). Presumably, this reflects the relatively
high level of secondary structure in -casein com-
pared to the other caseins (see Swaisgood, 1992); it
would be interesting to investigate the hydrolysis of
para--casein by chymosin or pepsin in the presence
of a high concentration of urea (pepsin is active in
8 M urea).
Good quality veal rennet contains about 10% bovine
pepsin (EC 3.4.23.1; Rothe et al., 1977) but many ‘calf
rennets’ contain up to 50% bovine pepsin. The proteo-
lytic products produced from Na-caseinate by bovine
pepsin are similar to those produced by chymosin
(Fox, 1969), although, as far as we are aware, the
specificity of bovine or porcine pepsins on bovine
caseins has not been determined rigorously. However,
Mulvihill and Fox (1979b) compared the hydrolysis of
bovine, ovine, caprine and porcine -caseins by chy-
mosins and pepsins from these species. The large pep-
tides produced (detectable by urea–polyacrylamide gel
electrophoresis) suggested generally similar specifici-
ties for chymosins and pepsins although differences
were apparent in the short (pH 4.6-soluble) peptides.
Pepsins were more proteolytic than the corresponding
chymosins.
For many years, the supply of calf rennet has been
insufficient to meet demand, and much effort has been
expended on searching for suitable rennet substitutes
for cheesemaking (see Green, 1977; Phelan, 1985).
Several proteinases have been assessed but only bovine
pepsin and proteinases from Rhizomucor pusillus,
Proteolysis in Cheese during Ripening 393
R. miehei and Cryphonectria parasitica have been used
extensively in commercial practice (Phelan, 1985; van
den Berg, 1992); blends of calf rennet and porcine
pepsin (50:50) have been used in the past with gener-
ally satisfactory results. Of these rennets, that from
R. miehei is the most widely used (under various trade
names) and gives generally satisfactory results. The
gene for R. miehei proteinase has been cloned in
Aspergillus oryzae, resulting in a rennet containing less
contaminating proteolytic enzymes. This coagulant
was found to be acceptable for the manufacture of
Cheddar cheese (Chen et al., 1994). C. parasitica pro-
teinase is considerably more proteolytic than chy-
mosin (Tam and Whitaker, 1972), especially on
-casein, and is rarely used for cheesemaking except
for high-cooked varieties (e.g., Swiss), in which the
proteinase is extensively denatured by the high cook
temperature.
The specificity of many of these enzymes on the oxi-
dized B-chain of insulin was summarized by Green
(1977). Preliminary studies on the hydrolysis of sodium
caseinate by fungal rennets were reported by Tam and
Whitaker (1972) and later by Phelan (1985). Rea
(1997) compared hydrolysates of sodium caseinate by
chymosin, R. miehei proteinase and C. parasitica pro-
teinase; the specificities of the three enzymes on the
caseins were clearly very different (C. parasitica was
particularly active on -casein). However, the bonds
cleaved by the fungal proteinases have not been
reported (except that the primary cleavage of -casein
by C. parasitica proteinase is at Ser1049Phe105, rather
than Phe1059Met106 which is cleaved by chymosin and
R. miehei proteinase (Drohse and Foltmann (1989)).
Indigenous Proteinases in Milk
Plasmin
Milk contains a number of indigenous proteinases, of
which plasmin (EC 3.4.21.7) is the most important
with respect to cheese ripening. Plasmin is a trypsin-
like serine proteinase with pH and temperature optima
of ⬃7.5 and 37 °C, respectively. Plasmin is secreted
into blood as its inactive zymogen, plasminogen,
which is then activated to plasmin, the principal func-
tion of which in blood is to degrade fibrin clots. A
complex plasminogen/plasmin system exists in blood,
comprised of plasmin, plasminogen, plasminogen acti-
vators (PA), plasmin inhibitors, and inhibitors of PA,
all of which are also present in milk. A diagrammatic
representation of the plasmin/plasminogen system
in milk is shown in Fig. 1. In milk, plasminogen,
plasmin and PA are associated with the casein micelles
while plasmin inhibitors and inhibitors of PA are in
394 Proteolysis in Cheese during Ripening

Plasminogen activators Plasminogen activator inhibitors

Plasminogen Plasmin Plasmin inhibitors

Casein Polypeptides

Figure 1 Plasmin/plasminogen system in milk (adapted from Bastian and Brown, 1996).

the serum phase. Plasmin has trypsin-like specificity,
showing preference for bonds of the type Lys9X and, to
a lesser extent Arg9X, and acts on caseins in the order
 艐 s2 >> s1, while -casein appears to be resistant to
hydrolysis by plasmin (Bastian and Brown, 1996). -
Casein has 15–17 (depending on the genetic variant)
potential plasmin-susceptible bonds, but only three are
hydrolysed at significant rates in milk, Lys289Lys29,
Lys1059His106 and Lys1079Glu108, hydrolysis of which
results in the release of 1-CN (-CN f29–209), 2-CN
(-CN f106–209), 3-CN (-CN f108–209), proteose
peptone (PP)8 fast (-CN f1–28), PP8 slow -CN
(f29–105) and (f29–107) and PP5 (-CN f1–105
and 1–107) (Fig. 2). In solution, plasmin hydrolyses
-casein at Lys979Val98, Lys1139Tyr114, Lys1699Val170,
Lys1769Ala177, Arg1839Asp184, Arg2029Gly203 as well
as at Lys289Lys29, Lys1059His106 and Lys1079Glu108
(Fox et al., 1994). s2-Casein is also a good substrate
for plasmin. In buffered systems, plasmin hydrolyses
s2-casein at Lys219Gln22, Lys249Asn25, Lys1499Lys150,
Lys1509Thr151, Lys1819Thr182, Lys1889Ala189,
Lys1979Thr198 and Arg1149Asn115 (Le Bars and
Gripon, 1989; Visser et al., 1989), but it has not yet
been determined whether peptides resulting from
cleavage at these sites are produced in milk or dairy
products, although this is likely since the concen-
tration of s2-casein, which is a poor substrate for
chymosin, decreases in cheese during ripening (Fox
and McSweeney, 1997). s1-Casein is less susceptible to
hydrolysis by plasmin than -casein (Andrews and
Alichanidis, 1983). However, Le Bars and Gripon (1993)
identified seven Lys9X and four Arg9X plasmin-
susceptible bonds in s1-casein while McSweeney et al.
(1993c) identified 12 Lys9X and 5 Arg9X plasmin-
susceptible bonds in s1-casein (Lys39His4, Lys79
His8, Arg229Phe23, Lys349Glu35, Lys369Lys37, Lys589
Gln59, Lys799His80, Arg909Tyr91, Arg1009Leu101,
Lys1029Lys103, Lys1039Tyr104, Lys1059Val106, Arg1199
Leu120, Lys1249Glu125, Gln1319Lys132, Arg1519Gln152,
Lys1939Thr194), including all those reported by Le Bars
and Gripon (1993). It is likely that -caseins originate as
a result of hydrolysis of s1-casein by plasmin (Aimutis

Lys 28 ⎯ Lys Lys105 ⎯His106 Lys113 ⎯Tyr114 Arg ⎯Asp

29 183 184

1 209

Lys107 ⎯ Glu108

Products

Proteose peptones γ-Caseins

Known Probable
PP8f β-CN f1–28 β-CN f106–113 γ1-CN (β-CN f29–209)
PP8s β-CN f29–105 β-CN f108–113 γ2-CN (β-CN f106–209)
PP8s β-CN f29–107 β-CN f114–183 γ3-CN (β-CN f108–209)
PP-T β-CN f29–113 β-CN f106–183 γ4-CN (β-CN f114–209)
PP5 β-CN f1–105 β-CN f108–183 γ5-CN (β-CN f184–209)
PP5 β-CN f1–107 β-CN f1–113 (unlikely)

Figure 2 Hydrolysis of -casein by plasmin (adapted from Fox et al., 1994).


and Eigel, 1982; O’Flaherty, 1997). The resistance of
-casein to hydrolysis by plasmin is probably due to
the carbohydrate moieties attached to its C-terminal
region (Doi et al., 1979). Since they are associated with
the casein micelles, plasmin, plasminogen and PA are
incorporated into the cheese curd but plasmin
inhibitors and inhibitors of PA are present in the
cheese whey (Bastian and Brown, 1996).
The contribution of plasmin to proteolysis varies
depending on cheese type. Farkye and Fox (1990)
found that plasmin activity in cheese was in the order
EmmentalBlarneyRomano-typeGoudaCheddar
Cheshire cheeses. Many factors (e.g., cooking tempera-
ture, pH during ripening) contribute to this difference
in plasmin activity in cheese. Plasmin is the primary
proteolytic agent in Swiss-type cheese due to the fact
that these cheeses are cooked at a high temperature
(⬃55 °C), at which most of the coagulant, chymosin, is
inactivated, while plasmin, which is a heat-stable
enzyme, survives cooking. At high cooking tempera-
tures, activation of plasminogen occurs, probably due to
heat-induced inactivation of inhibitors of PA and of
plasmin (Farkye and Fox, 1990). Hence, differences in
plasmin activity between Swiss and Cheddar cheeses are
due to differences in the cooking temperature used in
the manufacturing protocols. Plasmin also plays a major
role in ripening of mould-ripened (e.g., Camembert)
and smear-ripened (e.g., Tilsit) cheese varieties. During
ripening of Camembert-type cheese, catabolism of lactic
acid and deamination of amino acids with the produc-
tion of NH3 by the mould on the surface of the cheese
result in an increase in the pH of the surface layer to
⬃7.0. Gradually, the pH of the interior of the cheese also
increases due to the outward migration of lactic acid and
the inward migration of NH3 (see ‘Metabolism of Resid-
ual Lactose and of Lactate and Citrate’, Volume 1). The
increased pH facilitates the action of plasmin, which
contributes significantly to proteolysis in these cheeses
(Gripon, 1993). The pH of the surface layer of smear-
ripened cheeses also increases during ripening, which
facilitates plasmin action on the caseins and influences
the quality of these cheese varieties (O’Farrell et al.,
2002).
Due to the significance of plasmin to proteolysis
during the ripening of many cheese varieties, a num-
ber of attempts have been made to increase plasmin
activity in cheese using different approaches. Farkye
and Fox (1992) added exogenous plasmin to cheese-
milk for Cheddar cheese. The level of water-soluble N
in cheese enriched with plasmin was ⬃20% higher
than in the control cheeses. As expected, increased
plasmin activity did not increase the level of phospho-
tungstic acid-soluble N, which is mainly due to free
amino acids. The authors claimed superior organolep-
Proteolysis in Cheese during Ripening 395
tic quality for the plasmin-enriched cheese. Somers
et al. (2002) enriched milk for the manufacture of
Mozzarella-type cheese with plasmin; greater hydroly-
sis of -casein by plasmin was observed in the experi-
mental cheeses compared to the control, but enzyme
treatment did not affect the composition or functional-
ity of the cheese. Primary proteolysis (as measured by
levels of pH 4.6-soluble N and urea–polyacrylamide
gel electrophoresis, PAGE), was accelerated in smear-
ripened cheese made from plasmin-enriched milk
(O’Farrell et al., 2002). Farkye and Fox (1991), who
added 6-aminohexanoic acid, a plasmin inhibitor, to
stirred-curd Cheddar cheese, found slower degrada-
tion of -casein compared to control cheese.
Successful attempts have been made to accelerate
proteolysis in cheese by increasing plasmin activity
by the use of exogenous PA (e.g., urokinase) in the
manufacture of Cheddar (Barrett et al., 1999), Swiss
(Bastian et al., 1997), ultrafiltered Havarti and Saint
Paulin (Bastian et al., 1991) cheeses. Bastian et al.
(1991) manufactured Havarti and Saint-Paulin cheese
by traditional or ultrafiltration (UF) technology and
added urokinase and KIO3, individually and in
combination, to UF retentate before cheesemaking.
Addition of urokinase increased plasmin activity in
UF Havarti and Saint-Paulin cheese and increased
proteolysis. Swiss cheese manufactured from milk
containing 0.05 UL 1 or 0.5 UL 1 urokinase showed
increased plasmin activity and faster degradation of
-casein than control cheese (Bastian et al., 1997).
The use of exogenous urokinase in the manufacture
of Cheddar cheese resulted in activation of plasmino-
gen and acceleration of proteolysis compared to con-
trol cheese (Barrett et al., 1999). Streptokinase, an
extracellular protein secreted by Streptococcus uberis,
forms a 1:1 complex with plasminogen, inducing a
conformational change in plasminogen, which ren-
ders the proteinase active without prior proteolytic
cleavage. The active streptokinase–plasminogen com-
plex is then able to convert other plasminogen mole-
cules to plasmin by proteolysis (Johnsen et al., 2000).
Streptokinase activated most of the plasminogen in
milk and increased plasmin activity in experimental
Cheddar cheese compared to controls (V.K. Upadhyay,
unpublished). Proteolysis in the cheese was accel-
erated as indicated by an increased level of pH
4.6-soluble nitrogen, breakdown of -casein and the
production of hydrophobic peptides. In an alternative
approach, V.K. Upadhyay (unpublished) used a
starter strain, which had been genetically modified to
produce streptokinase, for the manufacture of Ched-
dar cheese; plasminogen was activated to plasmin,
and proteolysis in the cheese during ripening was
accelerated.
396 Proteolysis in Cheese during Ripening
All the approaches for increasing plasmin activity in
different cheese varieties have accelerated proteolysis.
However, plasmin activity is not rate-limiting for
flavour development in cheese during ripening as plas-
min is responsible for the production of large to inter-
mediate-sized peptides (Farkye and Fox, 1992), which
do not contribute directly to flavour. However, plasmin
may have an indirect role in cheese flavour by produc-
ing intermediate-sized peptides which are hydrolysed
further by lactocepin and starter peptidases, ultimately
to free amino acids, which are important precursors of
volatile flavour compounds (see ‘Catabolism of Amino
Acids in Cheese During Ripening’, Volume 1).
Other indigenous proteinases
During mastitis, there is an increase in the somatic cell
count of milk as a result of increased transfer of leuko-
cytes from blood to milk. Somatic cells contain many
active proteinases, including cathepsins B, D, G, H, L
and elastase (Kelly and McSweeney, 2003). The pres-
ence of cathepsins D and B in milk is confirmed. How-
ever, it is likely that other lysosomal proteinases are
also present in milk.
Cathepsin D is an aspartic proteinase located in
the lysosomes of mammalian cells and has pH and
temperature optima of 4.0 and 37 °C, respectively.
Cathepsin D is synthesized on the rough endoplas-
mic reticulum as its inactive form, procathepsin D.
Procathepsin D is converted autocatalytically to pseu-
docathepsin D which is then converted to mature
active cathepsin D by further proteolysis, mediated
by cysteine proteinases. Larsen and Petersen (1995)
purified five molecular forms of cathepsin D, having
apparent molecular masses of 46, 45, 43, 39 and
31 kDa, from bovine milk; the 46 and 45 kDa forms
corresponded to procathepsin D (the reason for this
variability in molecular mass is unknown; Hurley
et al., 2000a), 43 kDa to pseudocathepsin D and
39 and 31 kDa to mature single-chain and heavy-
chain cathepsin D, respectively (Fig. 3). However,
Larsen and Petersen (1995) did not detect a band that
corresponding to light chain cathepsin D (14 kDa),
probably due to lack of sensitivity of their experimental
methods. The concentration of cathepsin D in skimmed
milk and whey is estimated to be 0.4 and 0.3 g ml 1,
respectively, indicating that it is a serum protein (Larsen
et al., 1996). Therefore, little cathepsin D would be
expected in cheese. Furthermore, cathepsin D only par-
tially survives heat treatment at 55 °C for 30 min (45%
survival) or HTST pasteurization (72 °C  15 s) (8%
survival) (Larsen et al., 2000; Hayes et al., 2001).
Hydrolysis of the caseins by cathepsin D has been
studied in model systems. Kaminogawa et al. (1980)
reported that cathepsin D partially purified from milk
P1 1 46kDa 346
Procathepsin D
346
P27 1 43kDa
Pseudocathepsin D
1 39kDa 346
Cathepsin D
(Single chain)
102 31kDa 346
Cathepsin D
(Heavy chain) 1 99
14 kDa
Cathepsin D
(Light chain)
Figure 3 Molecular forms of cathepsin D present in bovine milk
(adapted from Larsen and Petersen, 1995).
hydrolyses s1-casein with the production of a peptide
with a molecular mass similar to s1-CN (f24–199),
a peptide that is produced from s1-casein by chy-
mosin (Fig. 4a) (McSweeney et al., 1993b). Bonds in s1-
casein susceptible to the action of cathepsin D include
Phe239Phe24, Phe249Val25, Leu989Leu99 and Leu1499
Phe150 (Larsen et al., 1996). Cathepsin D hydrolyses
-casein to peptides similar to those produced by
chymosin (Fig. 4b); susceptible bonds include Phe529
Ala53, Leu589Val59, Pro819Val82, Ser969Lys97,
Leu1259Thr126, Leu1279Thr128, Trp1439Met144,
Phe1579Pro158, Ser1619Val162, Leu1659Ser166,
Leu1919Leu192, Leu1929Tyr193 and Phe2059Pro206.
However, hydrolysates of s2-casein produced by
cathepsin D differ markedly from those produced
by chymosin and have very few peptides in com-
mon when analysed by urea-PAGE and reversed
phase-high performance liquid chromatography (RP-
HPLC) (McSweeney et al., 1995). Cathepsin D-sensitive
bonds in s2-casein include Leu999Tyr100, Leu1239
Asn124, Leu1809Lys181 and Thr1829Val183 (Larsen
et al., 1996). Cathepsin D hydrolyses -casein at
Leu329Ser33, Leu799Ser80 and Phe1059Met106. Two
cleavage sites for cathepsin D on -lactalbumin have
been identified (Leu529Phe53 and Trp1049Leu105),
while -lactoglobulin appears to be resistant to this
enzyme (Larsen et al., 1996).
Although the specificity of cathepsin D is similar
to that of chymosin, it coagulates milk poorly
(McSweeney et al., 1995). It is very difficult to assess
the contribution of cathepsin D to proteolysis in
cheese made with rennet due to the low level of
cathepsin D and the masking effect by the much
larger level of chymosin in cheese. Wium et al.
(1998) reported that s1-CN (f24–199) was produced
in a Feta-type cheese made without the addition of
rennet from pasteurized, homogenized, ultrafiltered
milk, acidified using gluconic acid--lactone (GDL),
 (a)

Cathepsin G

Cathepsin B

Elastase

RPKHPIKHQGLPQEVLNENLLRFFVAPFPEVFGKEKVNELSKDIGSESTEDQAMEDIKQMEAESISSSEEIVPNSVEQKHIQKEDVPSERYLGYLEQLLRLKKYK
20 40 60 80 100

Plasmin

Cathepsin D

Chymosin

Cathepsin G

Cathepsin B

Elastase

VPQLEIVPNSAEERLHSMKEGIHAQQKEPMIGVNQELAYFYPELFRQFYQLDAYPSGAWYYVPLGTQYTDAPSFSDIPNPIGSENSEKTTMPLW
120 140 160 180 199

Plasmin

Cathepsin D

Chymosin

Figure 4 Primary structure of (a) bovine s1-casein showing the cleavage sites of elastase (Considine et al., 2000), cathepsin B (Considine et al., 2004), cathepsin G (Considine
et al., 2002), plasmin (Le Bars and Gripon, 1993; McSweeney et al., 1993c), cathepsin D (Larsen et al., 1996) and chymosin (McSweeney et al., 1993b) and (b) bovine -casein
showing the cleavage sites of elastase (Considine et al., 1999), cathepsin B (Considine et al., 2004), cathepsin G (Considine et al., 2002), plasmin (Eigel et al., 1984; Visser et al.,
1989; Singh et al., 1994), cathepsin D (Larsen et al., 1996) and chymosin (Visser and Slangen, 1977).
397
398

(b)

Cathepsin G

Cathepsin B

Elastase

RELEELNVPGEIVESLSSSEESITRINKKIEKFQSEEQQQTEDELQDKIHPFAQTQSLVYPFPGPIHNSLPQNIPPLTQTPVVVPPFLQPEVMGVSKVKEAMAPKHKEMPF
20 40 60 80 100

Plasmin

Cathepsin D

Chymosin

Cathepsin G

Cathepsin B

Elastase

PKYPVEPFTERQSLTLTDVENLHLPLPLLQSWMHQPHQPLPPTVMFPPQSVLSLSQSKVLPVPQKAVPYPQRDMPIQAFLLYQEPVLGPVRGPFPIIV
120 140 160 180 200

Plasmin

Cathepsin D

Chymosin

Figure 4 continued

which was attributed to the action of cathepsin D.
Hurley et al. (2000b) made Quarg, from raw skim
milk, pasteurized skim milk or raw skim milk with
added pepstatin (a potent inhibitor of aspartyl pro-
teinases) by acidification using GDL. Reversed
phase–high performance liquid chromatography of
the water-soluble fraction of the cheeses made from
raw or pasteurized skim milk showed peptides elut-
ing at 46–48 min, but these peptides were absent
from the profiles of pepstatin-treated cheese, suggest-
ing indigenous aspartyl proteinase activity in Quarg,
presumably cathepsin D. Cathepsin D activity may
contribute to a low extent to the degradation of
s1-casein in high-cooked cheese varieties (e.g.,
Swiss-type, Parmigiano-Reggiano) in which most of
the chymosin is inactivated.
The presence of cysteine proteinase activity in
bovine milk has been reported (Suzuki and Katoh,
1990; O’Driscoll et al., 1999). Mammalian cysteine
proteinases (cathepsins B, H, L and I) are lysosomal
enzymes that are synthesized as proenzymes (MW
37–55 kDa), which are later converted to catalytically
active forms (MW 23–30 kDa) (Barrett and Kirschke,
1981; Zeece et al., 1992; Kirschke et al., 1998).
Magboul et al. (2001) resolved five fractions (f I–f V)
with cysteine proteinase activity by ion-exchange
chromatography of acid whey on Q-Sepharose. Frac-
tions fIII and fV were the most active and were capable
of hydrolysing s1- and -caseins. Immunoblotting of
fIII with antibodies to the bovine lysosomal cysteine
proteinase, cathepsin B, indicated the presence of
cathepsin B in fIII. Partially purified fIII retained 20%
of its original activity when heated at 72 °C  30 s. It
is possible that cathepsin B may contribute to cheese
ripening but further research is required to determine its
role, if any, and the distribution of this enzyme in milk.
Cathepsin G is a serine proteinase with a molecular
mass of 24–26 kDa. In solution, cathepsin G readily
hydrolyses s1- and -casein extensively, including some
sites that are very close, or identical, to chymosin cleav-
age sites; it produces s1-CN (f1–23) by cleavage of the
Phe239Phe24 (Fig. 4a) (Considine et al., 2002). Many
cathepsin G cleavage sites in s1- and -caseins are also
close to those of plasmin. Cathepsin G has broad speci-
ficity on s1- and -caseins and hence it is possible that
it may contribute to proteolysis in cheese made from
high SCC milk; however the presence of this enzyme in
milk has not been demonstrated.
Elastase, a serine proteinase with a molecular weight
of 29.5 kDa, is an important lysosomal enzyme in
somatic cells, although its presence in milk has not
been demonstrated. Elastase can hydrolyse a wide
variety of proteins, including s1- and -caseins and
has many cleavage sites in common with chymosin,
Proteolysis in Cheese during Ripening 399
plasmin and the lactocepin of Lactococcus (Considine
et al., 1999, 2000). Hence, if elastase is present in milk, it
may contribute to proteolysis in cheese during ripening.
Of all the indigenous enzymes discussed above, only
plasmin and, to a lesser extent, cathepsin D, have been
studied in detail. Research to establish the presence of
the other enzymes in milk, and to elucidate their role in
proteolysis in cheese during ripening is needed.
Proteolytic Enzymes of LAB
Lactic acid bacteria are fastidious organisms that have
complex amino acid requirements (Law et al., 1976;
Morishita et al., 1981; Chopin, 1993). The concentra-
tions of amino acids in milk are below the nutritional
requirements for the growth of the auxotrophic LAB to
high cell populations. When growing in milk, their
complex proteolytic system degrades mainly caseins
into small peptides and amino acids which fulfil their
nutritional requirements and inadvertently contribute
to the flavour of fermented dairy products (Law and
Mulholland, 1995; Steele, 1995).
Many components of the proteolytic system of LAB
have been purified and characterized and most of the
corresponding genes have been cloned and sequenced
(for reviews of the extensive literature on this subject,
see Pritchard and Coolbear, 1993; Bockelmann, 1995;
Exterkate, 1995; Poolman et al., 1995; Juillard et al.,
1996; Kunji et al., 1996; Law and Haandrikman, 1997;
Christensen et al., 1999). The best-studied proteolytic
system among the LAB is that of Lactococcus followed
by themophilic Lactobacillus spp. because of their
economic importance as starter cultures in dairy fer-
mentations. The proteolytic systems of mesophilic lac-
tobacilli, which dominate the non-starter microflora of
Cheddar, Dutch-type and probably most cheeses dur-
ing ripening (Jordan and Cogan, 1993; Williams and
Banks, 1997; ‘The Microbiology of Cheese Ripening’,
Volume 1), have received less attention.
The main components of the proteolytic system of
LAB are proteinases (mainly the cell envelope-associated
proteinase, CEP, or lactocepin; EC 3.4.21.96), although
intracellular proteinases have been reported; Muset
et al., 1989; Akuzawa et al., 1990), amino acid and pep-
tide transport systems, and a range of intracellular pepti-
dases. During the growth of LAB in milk, the initial step
in casein degradation is performed by lactocepin and the
short peptides produced are taken up by the cell via pep-
tide transport systems (Juillard et al., 1995). Further
degradation to amino acids is catalysed by a number of
intracellular peptidases (Kunji et al., 1996; Law and
Haandrikman, 1997; Christensen et al., 1999). The
starter stops growing in cheese curd soon after the
end of manufacture due to the low pH, increasing NaCl
400 Proteolysis in Cheese during Ripening
concentration, low temperature and lack of a fer-
mentable carbohydrate substrate (see ‘The Microbiology
of Cheese Ripening’, Volume 1). However, its enzymes
play a very important role in proteolysis during ripening,
particularly when intracellular enzymes are released
from the cell following lysis. The rate of secondary pro-
teolysis is higher in cheese made with fast-lysing than
that with slow-lysing starter strains (Wilkinson et al.,
1994; O’Donovan et al., 1996; Morgan et al., 1997;
Martínez-Cuesta et al., 2001; Hannon et al., 2003).
Proteinases from LAB
Immunogold-labelling and genetic studies have shown
that lactocepin is located outside the lactococcal cell
(Hugenholtz et al., 1987; de Vos and Siezen, 1994).
Calcium is necessary for stable attachment of lacto-
cepin to the cell envelope; the proteinase is released by
incubation of the cells in a calcium-free buffer, a char-
acteristic which is usually exploited as the first step in
isolation procedures (Mills and Thomas, 1978;
McSweeney et al., 1993a). Lactocepins have a molecu-
lar mass of c. 140 kDa and a pH optimum of 5.5–6.5
(Law and Haandrikman, 1997).
The lactocepins from a number of Lactococcus
strains have been characterized biochemically and
genetically; they are homologous with the subtilisin
family of serine proteinases, with similar catalytic
domains (Pritchard and Coolbear, 1993; Kok and de
Vos, 1994; Kunji et al., 1996; Law and Haandrikman,
1997; Siezen, 1999; ‘Starter Cultures: General Aspects’,
Volume 1). The lactocepins and subtilisins have a con-
served active site triad, consisting of aspartic acid, his-
tidine and serine. A region of 107 residues which
includes the active site and the substrate-binding
region is highly conserved (Law and Haandrikman,
1997). Lactocepins of Lactococcus were initially classi-
fied into two broad groups, PI- and PIII-type pro-
teinases (Tan et al., 1993). PI-Type enzymes (e.g.,
produced by Lc. lactis subsp. cremoris HP and Wg2)
degrade -casein rapidly but act only slowly on
s1-casein whereas PIII-type proteinases (e.g., AM1 and
SK11) hydrolyse -casein differently to PI-type strains
and rapidly hydrolyse s1- and -caseins (Visser et al.,
1986; Fox and McSweeney, 1996; Law and Haandrikman,
1997). Although this broad classification scheme
remains useful, it soon became apparent that the lacto-
cepin of certain strains of Lactococcus had a specificity
intermediate between PI- and PIII-type enzymes. The
nucleotide sequence of the gene encoding all lacto-
cepins is very similar (98% homology; Kok et al.,
1988; Vos et al., 1989a,b) and alteration of a few
amino acid residues in the enzyme can alter its speci-
ficity. Exterkate et al. (1993) proposed a classification
scheme for lactocepins based on their specificity on
the peptide, s1-CN (f1–23), which is released early in
cheese ripening by the action of chymosin (Fig. 5).
The specificity of the lactocepins from a number of
strains of Lactococcus on s1-, s2-, - and -caseins is
summarized in Figs 6–9.
The primary role of lactocepin is to degrade the
caseins to provide short peptides to support the
growth of the lactococcal cells in milk. However, its
role in cheese ripening is different. Peptides isolated
from Cheddar cheese, the N- or C-terminus of which
corresponds to the specificity of lactocepin, do not
contain a major chymosin or plasmin cleavage site
(Fox and McSweeney, 1996), suggesting that chy-
mosin or plasmin act first and that lactocepin then
hydrolyses the resulting intermediate-sized peptides. A
number of authors have investigated the action of lac-
tocepins on peptides produced from the caseins by the
action of chymosin (Fig. 10).
Cell envelope-associated proteinases with proper-
ties similar to the lactococcal lactocepins have also
been isolated from a number of strains of Lactobacillus
(see Kunji et al., 1996; Law and Haandrikman, 1997;
Christensen et al., 1999).
Although much less well studied than the lacto-
cepins, Lactococcus spp. also possess intracellular
proteinases. Muset et al. (1989) isolated an intracel-
lular metalloproteinase from Lc. lactis subsp. lactis
NCDO763 which was optimally active at pH 7.5 and
45 °C and exhibited thermolysin-like specificity.
Akuzawa et al. (1990) identified four intracellular
proteinases with caseinolytic activity. The enzymes
with activity on casein ranged in molecular mass
from 12 to 160 kDa and were optimally active at pH
5.5–7.0. Two enzymes were metalloproteinases, one
had a serine catalytic mechanism and one was a thiol
proteinase. The authors also obtained eight fractions
with activity on benzgloxycarbonyl-L-Phe-L-Arg-7-
(4-methyl) coumarylamide but none of the eight
fractions was able to hydrolyse casein. Three intra-
cellular proteinases (P1, dimeric, Mr 124 kDa;
P2, monomeric, Mr  64 kDa and P3, monomeric,
Mr  47 kDa) were demonstrated in the cytoplasmic
fraction of the lactocepin-negative strain, Lc. lactis
subsp. cremoris MG1363, by Stepaniak et al. (1996).
P1 was a metalloproteinase while P2 and P3 were
serine proteinases. The enzymes were optimally
active at pH 7.0 and 35 °C (P1) or pH 7.5 and 45 °C
(P2, P3).
Peptidases
While the role of lactocepin when the cell is growing
in milk is the degradation of caseins to oligopeptides,
 Figure 5 Classification of lactocepins of lactic acid bacteria according to their specificity on s1-CN (f1–23) (from Fox et al., 2000).
401
402

[1] ↓ ↓ ↓ ↓ ↓
[2]
R1 P K H P I K H Q G L P Q E V L N E N L20 L R F F V A P F P E V F G K E K V N E L40 S K D I G S E S T E50

[1] ↓ ↓ ↓ ↓
[2]
D Q A M E D I K Q M60 E A E P I P P P E E I V P N S V E Q K H80 I Q K E D V P S E R Y L G Y L E Q L L R100

[1] ↓ ↓ ↓ ↓ ↓ ↓
[2] ↓ ↓ ↓
L K K Y K V P Q L E I V P N S A E E R L120 H S M K E G I H A Q Q K E P M I G V N Q140 E L A Y P Y P E L F150

[1] ↓ ↓ ↓ ↓
[2] ↓ ↓ ↓ ↓
R Q F Y Q L D A Y P160 S G A W Y Y V P L G T Q Y T D A P S F S180 D I P N P I G S E N S E K T T M P L W199

αs1-casein

[1] Lc. lactis subsp. cremoris SK11 (Reid et al., 1991a)

[2] Lc. lactis subsp. lactis NCDO 763 (Monnet et al., 1992)

Figure 6 Reported cleavage sites for lactocepins on bovine s1-casein (from Fox et al., 1994).
 Proteolysis in Cheese during Ripening 403

K N T M E H V S S S E E S I I S Q E T Y20 K Q E K N M A I N P S K E N L C S T F C40 K E V V R N A N E E50

↓ ↓
E Y S I G S S S E E60 S A E V A T E E V K I T V D D K H Y Q K80 A L N E I N E F Y Q K F P Q Y L Q Y L Y100

Q G P I V L N P W D Q V K R N A V P I T120 P T L N R E Q L S T S E E N S K K T V D140 M E S T E V F T K K150

↓ ↓ ↓ ↓ ↓ ↓ ↓
T K L T E E E K N R160 L N F L K K I S Q R Y Q K F A L P Q Y L180 K T V Y Q H Q K A M K P W I Q P K T K V200

↓
I P Y V R Y L207

αs2-casein

Figure 7 Reported cleavage sites for lactocepins on bovine s2-casein (from Monnet et al., 1992).

the hydrolysis of these peptides (after internalization
into the cell) to amino acids is catalysed by peptidases.
Many different peptidases from LAB have been charac-
terized biochemically and genetically (see Kunji et al.,
1996; Law and Haandrikman, 1997; Christensen et al.,
1999; Siezen et al., 2002). The biochemical properties
of the peptidases from cheese-related bacteria charac-
terized to date are shown in Table 1. While the role of
some of these peptidases (e.g., endopeptidases) is the
degradation of oligopeptides to shorter peptides,
exopeptidases function to release one or two amino
acids at a time from short peptides. Based on their sub-
strate specificity, peptidases are classified into different
groups, as shown in Fig. 11.
Endopeptidases
Several endopeptidases have been reported in lacto-
cocci and lactobacilli (Table 1), most of which are
monomeric metallopeptidases. On the basis of sub-
strate specificity, LAB appear to possess three types of
endopeptidases (Monnet et al., 1994). PepO is a
monomeric metallopeptidase with a molecular mass of
⬃70 kDa. It is capable of efficiently hydrolysing Met-
enkephalin, bradykinin, substance P, glucagon, oxi-
dized B-chain of insulin and several casein fragments
but not di-, tri- or tetra-peptides. PepO was the first
endopeptidase for which the gene was sequenced
(Mierau et al., 1993). The pepO gene is located imme-
diately downstream of the genes for the oligopeptide
transport system, indicating that the two systems are
physiologically linked (Tynkkynen et al., 1993).
Another oligopeptidase, designated PepF, specifically
cleaves the Phe9Ser bond in bradykinin and was purified
from Lc. lactis subsp. lactis NDCO 763; its gene (pepF)
was cloned and sequenced (Monnet et al., 1994). This
enzyme is a monomeric metallopeptidase of ⬃70 kDa
and is capable of hydrolysing peptides containing 7–17
amino acids with broad specificity but not smaller or
larger peptides. PepF is unable to hydrolyse Met-
enkephalin, which is a good substrate for PepO.
A gene (pepE) encoding a thiol-dependent endopep-
tidase has been isolated from Lb. helveticus CNRZ32
(Fenster et al., 1997). The deduced amino acid
sequence of PepE showed high homology with PepC
from Lb. delbrueckii subsp. lactis DSM7290 (Klein
et al., 1994a), Lb. helveticus CNRZ32 (Fernandez et al.,
1994; Vesanto et al., 1994), Sc. thermophilus CNRZ302
(Chapot-Chartier et al., 1994) and Lc. lactis subsp. cre-
moris AM2 (Chapot-Chartier et al., 1993). Fenster et al.
(1997) isolated and characterized recombinant PepE;
the general properties of this enzyme indicated that it
was different from the other metallo-endopeptidases
characterized from LAB.
Di- and tripeptidases
Tripeptidases (PepT) purified from LAB are generally
di- or tri-meric metallopeptidases (Table 1) with broad
specificity, capable of hydrolysing tripeptides with
acidic, basic or neutral N-terminal amino acid
residues. A broad-specificity general dipeptidase, PepV,
which hydrolyses only dipeptides, is found in LAB
(Kunji et al., 1996; Law and Haandrikman, 1997). A
number of dipeptidases with similar properties have
been purified and characterized from strains of Lacto-
coccus and Lactobacillus (see Table 1). Most of the
dipeptidases isolated from LAB are monomers with a
molecular mass in the range 40–55 kDa (Table 1).
With the exception of a dipeptidase from Lb. helveticus
53/7, which was reported to have a thiol catalytic
mechanism (Vesanto et al., 1996), all the dipeptidases
characterized to date are metallopeptidases (Table 1).
All dipeptidases of LAB show broad specificity and are
capable of hydrolysing all dipeptides except those con-
taining a proline residue.
Carboxypeptidases
Carboxypeptidases are exopeptidases which catalyse
the hydrolysis of peptides from the C-terminal. No
carboxypeptidase activity has been detected in lacto-
cocci but some activity towards N-terminal-blocked
404

[1] ↓ ↓
[2] ↓ ↓ ↓ ↓
[3] ↓ ↓ ↓ ↓ ↓
R1 E L E E L N V P G E I V E S L S S S E20 E S I T R I N K K I E K F Q S E E Q Q Q40 T E D E L Q D K I

[1] ↓ ↓ ↓
[2] ↓ ↓ ↓ ↓ ↓ ↓
[3] ↓ ↓
[5] ↓ ↓ ↓ ↓ ↓ ↓
[6] ↓ ↓ ↓
H P F A Q T Q S L V Y60 P F P G P I P N S L P Q N I P P L T Q T80 P V V V P P F L Q P E V M G V S K V K E100

[1] ↓ ↓ ↓ ↓ ↓ ↓
[2] ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓
[3] ↓ ↓ ↓ ↓ ↓
[6] ↓ ↓ ↓
A M A P K H K E M P F P K Y P V E P F T120 E S Q S L T L T D V E N L H P L P L L140 Q S W M H Q P H Q

[1] ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓
[2] ↓ ↓ ↓ ↓ ↓ ↓
[3] ↓ ↓ ↓ ↓ ↓ ↓
[4] ↓ ↓ ↓ ↓ ↓ ↓ ↓
[5] ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓
[6] ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓
[7] ↓ ↓ ↓ ↓
P L P P T V M F P P Q160 S V L S L S Q S K V L P V P Q K A V P Y180 P Q R D M P I Q A F L L Y Q E P V L G P200

[4] (↓)
[7] ↓
V R G P F P I I V209

β -casein

[1] Lc. lactis subsp. cremoris H2 (Reid et al., 1991b) [5] Lc. lactis subsp. cremoris AC1 (Monnet et al., 1989)
[2] Lc. lactis subsp. cremoris SK112 (Reid et al., 1991b) [6] Lc. lactis subsp. lactis NCDO 763 (Monnet et al., 1989)
[3] Lc. lactis subsp. cremoris AM1 (Visser et al., 1991) [7] Lc. lactis subsp. lactis NCDO 763 (Monnet et al., 1986)
[4] Lc. lactis subsp. cremoris HP (Visser et al., 1988)

Figure 8 Reported cleavage sites for lactocepins on bovine -casein (from Fox et al., 1994).
 [1] ↓ ↓ ↓ ↓
[2] ↓ ↓ ↓
[3] ↓ ↓
E E Q N Q E Q P I R C E K D E R F F S D20 K I A K T I P I Q Y V L S R Y P S Y G L40 N Y Y Q Q K P V A L50

[1] ↓ ↓ ↓ ↓ ↓ ↓ ↓
[2] ↓ ↓ ↓ ↓
[3] ↓ ↓ ↓
[4] ↓ ↓
I N N Q F L P Y P Y60 Y A K P A A V R S P A Q I L Q W Q V L S80 D T V P A K S C Q A Q P T T M A R H P H100

[1] ↓
[2] ↓ ↓
[3] ↓
[4] ↓ ↓ ↓
P H L S F M A I P P K K N Q D K T E I P120 T I N T I A S G E P T S T P T I E A V E140 S T V A T L E A S P150

[1] ↓
[3] ↓ ↓
[4] ↓ ↓ ↓↓ ↓↓ ↓
E V I E S P P E I N160 T V Q V T S T A V169

κ-casein

[1] Lc. lactis subsp. lactis NCDO 763 (Monnet et al., 1992)
[2] Lc. lactis subsp. cremoris H2 (Ried et al., 1994)
[3] Lc. lactis subsp. cremoris SK11 (Reid et al., 1994)
[4] Lc. lactis subsp. cremoris AM1 (Visser et al., 1994)

Figure 9 Reported cleavage sites for lactocepins on bovine -casein (from Fox et al., 1994).
405
406

[1] ↓ ↓ ↓ ↓ ↓ ↓
[2] ↓ ↓ ↓
[3] ↓↓ ↓
[4] ↓
[5] ↓ ↓ ↓
[6] ↓ ↓ ↓ ↓
[7] ↓
[8] ↓ ↓↓ ↓
↓ ↓ ↓
[9] ↓ ↓ ↓
R1 P K H P I K H Q G L P Q E V L N E N L L R F23 α s1-CN f1–
23
[9] ↓ ↓ ↓
Y91 L G Y L E N L L R100 α s1-CN f91–
100
[6] ↓ ↓ ↓ ↓
Y165 Y V Y P L G T Q Y T D A P S F S D I P N P I G S E N S E K T T M P L W199 α s1-CN f165–
199
[4] ↓ ↓ ↓
[5] ↓ ↓ ↓ ↓
Y193 Q E P V L G P V R G P F P I I V209 β-CN f193–
209
[5] ↓ ↓ ↓ ↓ ↓
Y193 Q Q P V L G P V R202 G203 P F P I I V209 β -CN f193–
202, f203–
209
[4] ↓ ↓
[5] ↓ ↓ ↓ ↓
[8] ↓ ↓ ↓↓ ↓ ↓ ↓
[9] ↓ ↓↓ ↓
F1 V N Q H L C G S H L V E A L Y L V C G E R G F F Y T P K A30 β -Chain of Insulin
[6] ↓ (Oxidized)
[9] ↓
T G G F M Met Enkephalin
[8] ↓
M106 A I P P K K N Q D K T E I P T I…I E S P P E I N T V Q V T S T A V 169 κ -CN f106–
169

[1] Lc. lactis subsp. cremoris H61 (Kaminogawa et al., 1986)
[3] Lc. lactis subsp. cremoris AM1 (Exterkate et al., 1991)
[5] Lc. lactis subsp. cremoris C13 (Baankreis, 1992)
[7] Lc. lactis subsp. cremoris (Yan et al., 1987b)
[9] Lc. lactis subsp. cremoris H61 (Yan et al., 1987a)
[2] Lc. lactis subsp. cremoris HP (Exterkate et al., 1991)
[4] Lc. lactis subsp. cremoris HP (Baankreis, 1992)
[6] Lc. lactis subsp. lactis MG 1363 (Stepaniak and Fox, 1993)
[8] Lc. lactis subsp. lactis NCDO 763 (Monnet et al., 1992)

Figure 10 Reported cleavage sites for lactococcal endopeptidases (4–7, 9) and lactocepins (1–3, 8) on various oligopeptidases (from Fox et al., 1994).
 Table 1 Peptidases isolated and characterized from cheese-related bacteria

Assay Mol. mass pH Temperature

Microorganism substrate (kDa) optimum optimum (°C) Subunits Classa References

Aminopeptidases
PepN**
Lc. lactis CNRZ 267* Lys-pNA 85 6.5 35 – M Desmazeaud and Zevaco (1979)
Lc. lactis subsp. lactis AC1 Lys-pNA 36 7.0 40 1 M Geis et al. (1985)
Lc. lactis subsp. cremoris Wg2 Lys-pNA 95 7.0 40 1 M/T Tan and Konings (1990)
Lc. lactis subsp. cremoris MG 1363 – 95 – – – – van Alen-Boerighter et al. (1991)
Lc. lactis subsp. cremoris HP – 95 – – – – Exterkate et al. (1992)
Lc. lactis subsp. cremoris Wg2 – 95 – – – – Strøman (1992)
Lc. lactis subsp. cremoris Wg2 – 95 – – – – Tan et al. (1992b)
Lb. delbrueckii subsp. lactis 1183 Lys-pNA 78 6.2–7.2 47.5 1 M Eggimann and Bachmann (1980)
Lb. acidophilus R-26 Lys-pNA 38 – – – M Machuga and Ives (1984)
Lb. delbrueckii subsp. bulgaricus Lys-pNA 95 – – – M Atlan et al. (1989)
CNRZ 397
Lb. helveticus CNRZ 32 Lys-pNA 97 6.5 – 1 M Khalid and Marth (1990a)
Lb. casei subsp. casei LLG Leu-pNA 87 7 39 1 M Arora and Lee (1992)
Lb. delbrueckii subsp. bulgaricus Lys-pNA 95 7.0 50 1 M Bockelmann et al. (1992)
B14
Lb. helveticus LME-511 Lys-pNA 92 7.0 37 1 M Miyakawa et al. (1992)
Lb. helveticus ITGL1 Lys-pNA 97 6.5 50 1 M Blanc et al. (1993)
Lb. delbrueckii subsp. lactis DSM Lys-pNA 95 6.5–7.0 45–55 1 M Klein et al. (1993)
7290
Lb. delbrueckii subsp. bulgaricus Lys-pNA 98 6 40 1 M Tsakalidou et al. (1993)
ACA-DC233
Lb. helveticus CNRZ32 – 97 – – – – Varmanen et al. (1994)
Lb. helveticus CNRZ32 – 97 – – – – Christensen et al. (1995)
Lb. helveticus SBT 2171 Lys-pNA 95 6–8 50 1 M Sasaki et al. (1996)
Lb. curvatus DPC2024 Leu-pNA 95 7 40 1 M Magboul and McSweeney (1999a)
Lb. plantarum ESB5004 Lys-pNA 70 7 37 2 M Macedo et al. (2003)
Sc. thermophilus CNRZ1199 Lys-pNA 89 6.5 35 1 M Tsakalidou et al. (1993)
Sc. thermophilus CNRZ302 Lys-pNA 97 7.0 36 1 M Rul et al. (1994)
Sc. thermophilus NDCO573 Lys-pNA 96 6.9–7.0 35 1 M Midwinter and Pritchard (1994)
Sc. thermophilus A Lys-AMC 95 7.0 37 1 M Chavagnat et al. (1999)
PepS
Sc. thermophilus CNRZ302 Arg-pNA 45 7.5–8.5 55 1 M Fernández-Esplá and Rul (1999)
PepC
Lc. lactis subsp. cremoris AM2 His-NA 300 7 40 6 T Neviani et al. (1989)
Lc. lactis subsp. cremoris AM2 – 50 – – – – Chapot-Chartier et al. (1993)
Lb. delbrueckii subsp. lactis DSM – 51 – – – T Klein et al. (1994a)
7290
Lb. delbrueckii subsp. bulgaricus Leu-Gly-Gly 220 6.5–7.0 50 4 T Wohlrab and Bockelmann (1993)
B14
Lb. helveticus CNRZ32 – 50 – – – T Fernandez et al. (1994)
407

continued
408 Table 1 continued

Assay Mol. mass pH Temperature

Microorganism substrate (kDa) optimum optimum (°C) Subunits Classa References

Lb. helveticus 53/7 – 51.4 – – – T Vesanto et al. (1994)

Lb. casei subsp. casei IFPL 731 Leu-pNA 200 7.5 55 4 T Fernandez de Palencia et al. (1997)
Lb. delbrueckii subsp. lactis DSM 7.0 50–55 T Klein et al. (1997)
7290
Lb. helveticus CNRZ 32 200 6.5 45 4 T Fernandez de Palencia et al. (2000)
Sc. thermophilus CNRZ 302 Lys-NH-Ph 300 7.0 50 6 T Chapot-Chartier et al. (1994)
PepA
Lc. lactis subsp. cremoris HP Glu-pNA 130 – 50–55 3 M Exterkate and de Veer (1987)
Lc. lactis subsp. lactis NCDO 712 Glu-pNA 245 8 65 6 M Niven (1991)
Lc. lactis subsp. cremoris HP Glu-pNA 520 8 50 ⬃10 M Baankreis (1992)
Lc. lactis subsp. cremoris AM2 Asp-pNA 240 – – 6 M Bacon et al. (1994)
Lc. lactis subsp. lactis MG 1363 Glu-pNA – M I’Anson et al. (1995)
Sc. thermophilus CNRZ 302 Asp-pNA 360 8.5 62 8 M Rul et al. (1995)
PCP
Lc. lactis subsp. cremoris HP Pyr-pNA – – – – – Exterkate (1977)
Lc. lactis subsp. cremoris HP Pyr-pNA 80 8.0–8.5 37 2 S Baankreis (1992)
Leucyl aminopeptidase (PepL)
Lb. debrueckii subsp. lactis DSM7290 Leu-NA 35 – – – – Klein et al. (1995)
Lb. sake IATA115 Leu-pNA 35–36 7.5 37 1 C Sanz and Toldra (1997)
Lb curvatus DPC2024 Leu-pNA 64 7 40 2 C Magboul and McSweeney (1999b)
Proline iminopeptidase (PepI)
Lc lactis subsp. cremoris HP Pro-Gly-Gly 110 8.5 37 2 M Baankreis and Exterkate (1991)
Lb. delbrueckii subsp. lactis DSM7290 33 S Klein et al. (1994b)
Lb. delbrueckii subsp. bulgaricus 33 S Atlan et al. (1994)
CNRZ 397
Lb. delbrueckii subsp. bulgaricus Pro-pNA 100 6.0–7.0 40 3 S Gilbert et al. (1994)
CNRZ 397
Lb. helveticus LHE-511 70 6.5 35 1 T/S Miyakawa et al. (1994b)
Lb. casei subsp. casei LLG Pro-AMC 46 7.5 40 1 T Habibi-Najafi and Lee (1995)
Aminopeptidase P (PepP)
Lc. lactis subsp. lactis NCDO 763 bradykinin 43 8.0 37 1 M Mars and Monnet (1995)
Lc. lactis subsp. cremoris AM2 Leu-Pro-Pro 41 8.5 – 1 M McDonnell et al. (1997)
Lc. lactis subsp. cremoris – 43 – – 1 M Matos et al. (1998)
X-Prolyl dipeptidyl aminopeptidase (PepX)
Lc. lactis subsp. lactis H1 X-Pro-pNA 150 6.0–9.0 – 2 S Lloyd and Pritchard (1991)
Lc. lactis subsp. cremoris P8-2-47 X-Pro-pNA 180 7 45–50 2 S Kiefer-Partsch et al. (1989)
Lc. lactis subsp. cremoris NCDO 763 Arg-Pro-pNA 190 8.5 40–45 2 S Zevaco et al. (1990)
Lc. lactis subsp. cremoris AM2 Gly-Pro-AMC 117 6.0–9.0 – 2 S Booth et al. (1990b)
Lc. lactis subsp. cremoris nTR – 88 7.5 – 2 S Yan et al. (1992)
 Lc. lactis subsp. cremoris Wg2 – – – – – – Tan et al. (1992a)
Lc. lactis subsp. lactis Ala-Pro-pNA 175 – – – – Chich et al. (1995)
Lb. delbrueckii subsp. lactis Gly-Pro-AMC 165 7.0 50–55 2 S Meyer and Jordi (1987)
Lb. delbrueckii subsp. bulgaricus X-Pro-pNA 82 7.0 50 2 S Atlan et al. (1990)
CNRZ 397
Lb. delbrueckii subsp. bulgaricus B14 Ala-Pro-pNA 170–200 6.5 45 2 S Bockelmann et al. (1991)
Lb. acidophilus 357 Ala-Pro-pNA 170–200 6.5 45 2 S Bockelmann et al. (1991)
Lb. helveticus CNRZ 32 X-Pro-pNA 72 7.0 40 1 S Khalid and Marth (1990b)
Lb. delbrueckii subsp. bulgaricus Gly-Pro-pNA 270 6.5 50 3 S Miyakawa et al. (1991)
LBU-147
Lb. casei subsp. casei LLG Gly-Pro-pNA 79 7.0 50 1 S Habibi-Najafi and Lee (1994)
Lb. helveticus 53/7 Gly-Pro-pNA 165 6.5 45 2 S Vesanto et al. (1995)
Lb. helveticus ITG LH1 Gly-Pro-pNA 140 7.0 40 1 S Degraeve and Martial-Gros (2003)
Lb. delbrueckii subsp. bulgaricus Ala-Pro-pNA 95 7.0 46–50 1 S Meyer-Barton et al. (1993)
DSM7290
Lb. casei subsp. casei IFPL731 Arg-Pro-pNA 67 6.0–7.0 40 1 S Fernández-Esplá et al. (1997a)
Lb. helveticus LHE-511 Gly-Pro-pNA 93–87 6.5 50 1 S Miyakawa et al. (1994a)
Lb curvatus DPC2024 Ala-Pro-pNA 200 7.5 45 2 S Magboul and McSweeney (2000)
Sc. thermophilus Gly-Pro-AMC 165 6.5–8.2 45 2 S Meyer and Jordi (1987)
Sc. thermophilus ACA-DC 4 X-Pro-pNA 160 7.0 50 2 S Tsakalidou et al. (1998)
Sc. macedonicus ACA-DC 191 Gly-Pro-pNA 167 7.0 – 2 S Georgalaki et al. (2002)
Prolinase (PepR)
Lb. helveticus CNRZ32 Pro-Leu 35 – – – T/S Dudley and Steele (1994)
Lb. helveticus 53/7 Pro-Leu 35 – – – T/S Varmanen et al. (1996)
Lb. helveticus CNRZ32 Pro-Leu 125 6.0–6.5 45–50 4 S Shao et al. (1997)
Lb. rhamnosus 1/6 Pro-Leu 34 – – – S Varmanen et al. (1998)
Prolidase (PepQ)
Lc. lactis subsp. cremoris H61 Leu-Pro 43 6.5–7.5 – 1 M Kaminogawa et al. (1984)
Lc. lactis subsp. cremoris AM2 Leu-Pro 42 7.35–9.0 – 1 M Booth et al. (1990c)
Lb. delbrueckii subsp. lactis DSM7290 Stuckey et al. (1995)
Lb. casei subsp. casei IFPL 731 Leu-Pro 41 6.5–7.5 55 1 M Fernández-Esplá et al. (1997b)
Lb. delbrueckii subsp. bulgaricus Morel et al. (1999)
CNRZ 397
Dipeptidases
Lc. lactis CNRZ267* Leu-Leu 51 7.5–8.0 – 1 M Desmazeaud and Zevaco (1977)
Lc. lactis subsp. cremoris H61 Leu-Gly 100 8.0 – – M Hwang et al. (1981)
Lc. lactis subsp. cremoris Wg2 dipeptides 49 8.0 50 1 M van Boven et al. (1988)
Lb. delbrueckii subsp. bulgaricus Leu-Leu 51 7.0–7.5 50 1 M Wohlrab and Bockelmann (1992)
B14
Lb. delbrueckii subsp. lactis DSM7290 -Ala-Ala 52 – – – M Vongerichten et al. (1994)
Lb. sake L110 Leu-Gly 50 7.6–8.0 40–45 1 M Montel et al. (1995)
Lb. helveticus SBT2171 Leu-leu 50 8.0 55 1 M Tan et al. (1995)
Lb. helveticus CNRZ32 Leu-Leu 54 – – – – Dudley et al. (1996)
Lb. helveticus 53/7 Gly-Tyr 420 6.0 55 8 T Vesanto et al. (1996)
409

continued
410 Table 1 continued

Assay Mol. mass pH Temperature

Microorganism substrate (kDa) optimum optimum (°C) Subunits Classa References

Lb. casei subsp. casei IFPL 731 Leu-Leu 46 7.5 60–75 1 M Fernández-Esplá and
Martin-Hernandez (1997)
Lb. curvatus DPC2024 Leu-Leu 52 8.0 50 1 M Magboul and McSweeney (1999c)
Tripeptidases
Lc. lactis CNRZ267* Tripeptides 75 7.0 35 – M Desmazeaud and Zevaco (1979)
Lc. lactis subsp. cremoris Wg2 Leu-Leu-Leu 103–105 7.5 55 2 M Bosman et al. (1990)
Lc. lactis subsp. cremoris AM2 Tripeptides 105 8.6 – 2 M Bacon et al. (1993)
Lc. lactis subsp. cremoris Leu-Leu-Leu 72 5.8 33 3 T/M Sahlstrøm et al. (1993)
IMN-C 12
Lb. delbrueckii subsp. bulgaricus Leu-Gly-Gly 85 6.0 40 3 M Bockelmann et al. (1995)
B14
Lb. delbrueckii subsp. bulgaricus Leu-Gly-Gly 77 6.0 50–55 2 M Bockelmann et al. (1997)
B14
Lb. sake IATA115 Ala-Ala 55 7.0 40 1 M Sanz et al. (1998)
Lb. helveticus Met-Cly-Gly 150 – – 3 M Savijoki and Palva (2000)
Endopeptidases
Lc. lactis subsp. cremoris H61 peptides 98 7–7.5 40 1 M Yan et al. (1987a)
Lc. lactis subsp. cremoris H61 s1-CN (f1–23) 80 6.0 37 2 M Yan et al. (1987b)
Lc. lactis subsp. cremoris Wg2 Met-enkephalin 70 6–6.5 30–38 1 M Tan et al. (1991)
Lc. lactis subsp. cremoris HP s1-CN (f1–23) 180 8–9 42 2 M Baankreis (1992)
Lc. lactis subsp. cremoris C13 s1-CN (f1–23) 70 6–7 35 1 N Baankreis (1992)
Lc. lactis subsp. lactis MG 1363 s1-CN (f1–23) 70 7.5 40 1 M Stepaniak and Fox (1995)
Lc. lactis subsp. cremoris SK11 bradykinin 70 6.0 – 1 M Pritchard et al. (1994)
Lc. lactis subsp. lactis NCDO 763 bradykinin 70 8.0 40 1 M Monnet et al. (1994)
Lb. delbrueckii spp. bulgaricus Met-enkephalin 70 7.7 47 1 M Bockelmann et al. (1996)
B14
Lb. paracasei Lc01 bradykinin 140 8.0 40 4 M Tobiassen et al. (1997)
Lc. lactis subsp. cremoris P8-2-47 – 70 – – 1 M Mierau et al. (1993)
Lc. lactis subsp. lactis MG1363 Met-enkephalin 350 7.0 35 6–7 M Stepaniak et al. (1998a)
Lb. helveticus CNRZ32 Met-enkephalin 50 4.5 32–37 1 T Fenster et al. (1997)
Lb. helveticus CNRZ32 s1-CN (f1–23) – – – – – Christensen et al. (2003a)
-CN (f193–209)
Sc. thermophilus A Met-enkephalin 70 6.5 41 1 M Chavagnat et al. (2000)
P. freudenreichii ATCC 9614 bradykinin 120 6.7–7.5 40–50 4 M Stepaniak et al. (1998b)
P. freudenreichii ATCC 9614 bradykinin 44 6.5–8.0 45–50 1 M Tobiassen et al. (1996)
P. freudenreichii subsp. shermanii Ala-Pro-pNA 84 7.0 40 1 S Fernández-Esplá and Fox (1997)
NCDO 853

a Class of enzyme M: metallo and T: thiol-peptidase; NA: -naphthylamide; pNA: p-nitroanilide.

* Citrate positive strain.
** Classification of a few of these enzymes as PepN is uncertain.
 Proteolysis in Cheese during Ripening 411

Endopeptidases (PepO, PepF)

Exopeptidases

Aminopeptidases (PepN, PepA, PepC, PepL) Iminopeptidase (PepI)

Pyrolidonyl carboxylyl peptidase (PCP)

Pyro-Glu

X-Prolyldipeptidyl aminopeptidase (PepX) Carboxypeptidase

Dipeptidases (PepV, PepD) Prolidase (PepQ) Prolinase (PepR)

P P

Tripeptidase (PepT)

Figure 11 Schematic representation of the action of peptidases found in lactic acid bacteria.

peptides has been reported in strains of lactobacilli
(Abo-Elnaga and Plapp, 1987; El Soda et al., 1987a,b).
There are no reports on the purification and character-
ization of a carboxypeptidase from Lactobacillus or
other LAB.
Aminopeptidases
The most thoroughly studied exopeptidase from LAB
is the general aminopetidase, PepN. In most strains
studied, this enzyme is a monomeric metallopeptidase
of 85–98 kDa. PepN is a broad specificity aminopepti-
dase; in addition to p-nitroanilide (pNA) derivatives of
amino acids, the enzyme is capable of hydrolysing a
wide range of peptides differing in both size and
amino acid composition (Arora and Lee, 1992;
Miyakawa et al., 1992; Tan et al., 1992a,b; Niven et al.,
1995; Sasaki et al., 1996). Substrates with a hydropho-
bic or basic amino acid residue at the N-terminal are
hydrolysed preferentially. The ability to hydrolyse pep-
tides containing hydrophobic amino acids suggests its
potential as a debittering enzyme. The addition of
PepN from Lc. lactis subsp. cremoris Wg2 was found to
be effective in reducing the bitterness of tryptic digests
of -casein (Tan et al., 1993). The manufacture of
cheese using PepN-negative mutants resulted in
increased bitterness (Baankreis, 1992). Generally,
PepN does not hydrolyse substrates with Glu, Asp or
Pro at the N-terminal or dipeptides containing Pro
412 Proteolysis in Cheese during Ripening
(Tan et al., 1991; Arora and Lee, 1992; Miayakawa
et al., 1992; Tan et al., 1993). However, PepNs from
Lb. delbrueckii subsp. bulgaricus B14 (Wohlrab and
Bockelmann, 1993) and Lb. helveticus SBT 2171
(Sasaki et al., 1996) hydrolysed Pro-containing sub-
strates. Specificity studies indicated that the PepN
from Lc. lactis subsp. cremoris Wg2 was active on
oligopeptides with a preference for peptides with six
amino acid residues (Niven et al., 1995).
PepC in LAB is a metal-independent general
aminopeptidase (Kunji et al., 1996; Table 1). PepCs
from Lactococcus and Lactobacillus strains character-
ized so far are multimeric thiol aminopeptidases which
are inhibited by p-chloromercuribenzoate and iodo-
acetamide (Neviani et al., 1989; Wohlrab and Bockel-
mann, 1993; Fernandez de Palencia et al., 1997). In
both cases, the subunit molecular mass of the enzyme
is ⬃40–50 kDa. PepC shows broad specificity, with
particularly high activity on synthetic substrates con-
taining a hydrophobic amino acid but exhibits little
activity on peptides with positively charged amino acid
residues (Neviani et al., 1989; Wohlrab and Bockelmann,
1993; Fernandez de Palencia et al., 1997; Mistou and
Gripon, 1998).
A gene (pepG) encoding a novel cysteine aminopep-
tidase and with a high degree of similarity to PepC has
been identified in Lb. delbrueckii subsp. lactis DSM7290
by Klein et al. (1997). These authors over-expressed
the pepG gene in E. coli and compared the enzyme to
PepC; although both enzymes were structurally related,
they had different substrate specificities.
Lactococcal glutamyl/aspartyl aminopeptidase
(PepA) is a multimeric metallopeptidase with a subunit
molecular mass of 38–43 kDa (Table 1). PepA is a nar-
row-specificity peptidase which releases only an N-
terminal Glu or Asp from di-, tri- and oligo-peptides
with up to ten amino acid residues (Exterkate and de
Veer, 1987; Niven, 1991; Bacon et al., 1994). Glutamate is
a well-recognized flavour enhancer and therefore the
role of PepA in the development of flavour in cheese
may be of great importance. Studies on mature Ched-
dar cheese have shown that glutamate is important for
Cheddar cheese flavour (McGugan et al., 1979; Aston
and Creamer, 1986; Engels and Visser, 1994; Fox et al.,
1994). However, the precise role of PepA in the devel-
opment of cheese flavour is unclear.
Under certain conditions, the N-terminal glutamyl
residue of a peptide can undergo spontaneous intramole-
cular cyclization, forming an N-terminal 2-pyrrolidone-
5-carboxylic acid (PCA; pyroglutamate residue) (Law
and Haandrikman, 1997). An N-terminal PCA residue
has been found in bitter peptides produced from -
casein by the lactocepin of Lc. lactis subsp. cremoris HP
(Visser et al., 1983). Pyrrolidone carboxylyl peptidase
(PCP) is an aminopeptidase capable of releasing a PCA
residue from peptides and proteins (Kunji et al., 1996).
This enzyme is present in lactococcal strains and has
been partially characterized from Lc. lactis subsp. cre-
moris HP (Baankreis, 1992). Two serine peptidases with
a molecular mass of 25 and 80 kDa and PCA-
p-nitroanilide hydrolase activity were identified in Lc.
lactis subsp. cremoris HP using non-denaturing gel elec-
trophoresis (Baankreis, 1992).
The presence of more than one leucyl aminopepti-
dase in LAB has been reported (Atlan et al., 1989;
Blanc et al., 1993; Banks et al., 1998). A gene encoding
a specific leucyl aminopeptidase (pepL) in Lb. del-
brueckii subsp. lactis DSM 7290 has been cloned and
sequenced (Klein et al., 1995). PepL has a molecular
mass of 35 kDa (Table 1) and it preferentially hydroly-
ses dipeptides (and some tripeptides) with an N-terminal
leucyl residue. Sequence alignments of PepL with pro-
linases from Lb. helveticus and B. coagulans and an
iminopeptidase from Lb. delbrueckii subsp. lactis and
Lb. delbrueckii subsp. bulgaricus showed 46, 21.5, 25.5
and 25.5% homology, respectively.
Two aminopeptidases, with characteristics similar
to PepL, were purified from Lb. sake IATA115 and Lb.
curvatus DPC2024 by Sanz and Toldra (1997) and
Magboul and McSweeney (1999b), respectively. The
former was a monomer with a molecular mass of
35–36 kDa and maximum activity at pH 7.5 and 37 °C,
while the latter was a dimer with a subunit molecular
mass of ⬃32 kDa and optimum activity at pH 7.0 and
40 °C. The 20 N-terminal amino acid residues of the
PepL from Lb. curvatus DPC2024 showed 50, 80 and
95% homology with PepL from Lb. delbrueckii subsp.
lactis DSM 7290 (Klein et al., 1995), the prolinase
from Lb. helveticus CNRZ32 (Dudley and Steele, 1994)
and the prolinase from Lb. rhamnosus 1/6 (Varmanen
et al., 1998), respectively.
Proline-specific peptidases
Caseins, the major proteins in bovine milk, are rich in
the imino acid, proline. Because of its unique struc-
ture, specialized peptidases are required to hydrolyse
peptide bonds involving proline, thus making peptides
accessible to the action of other peptidases (see review
by Cunningham and O’Connor, 1997). Several pro-
line-specific peptidases with distinct substrate speci-
ficities have been found in LAB.
X-Prolyl dipeptidyl aminopeptidase (PepX) is a
peptide hydrolase capable of releasing X-Pro and
sometimes X-Ala dipeptides from the N-terminal of
oligopeptides. Due to its unique specificity, PepX is the
best characterized of the proline-specific peptidases.
The enzyme has been demonstrated in several genera
of LAB and isolated from a number of strains and

characterized (Table 1). All PepXs purified from LAB
have a serine catalytic mechanism and most are
dimeric proteins with a native molecular mass of
117–200 kDa (Table 1); however, a high molecular
mass endopeptidase (⬃350 kDa) with PepX activity
and able to hydrolyse s1-casein was isolated and char-
acterized by Stepaniak et al. (1998a). Increasing the
proportion of pepX-negative mutants in a starter cul-
ture reduced the organoleptic quality of the resultant
cheese but did not increase bitterness (Baankreis,
1992). Meyer and Spahni (1998) studied the role of
PepX from Lb. delbrueckii subsp. lactis by using PepX-
negative mutants. This enzyme influenced proteolysis
and the sensorial characteristics of Gruyere cheese but
it was not essential for the growth of the microorgan-
ism in milk (Meyer and Spahni, 1998).
Proline iminopeptidase (PepI) catalyses the release
of an N-terminal proline residue from di-, tri- and
oligo-peptides. PepI from Lc. lactis subsp. cremoris HP
(Baankreis and Exterkate, 1991) is the only iminopep-
tidase that has been purified from Lactococcus. This
enzyme is a dimeric metallopeptidase with a native
molecular mass of 110 kDa (Table 1). In contrast, the
iminopeptidases purified from Lb. helveticus LHE-511
(Miyakawa et al., 1994b) and Lb. casei subsp. casei LLG
(Habibi-Najafi and Lee, 1995) were monomeric thiol
peptidases which were slightly inhibited by the serine
protease inhibitor phenylmethyl sulphonyl fluoride.
The molecular mass of the enzymes from Lb. helveticus
and Lb. casei was estimated as 70 and 46 kDa, respect-
ively. In addition to these two iminopeptidases, a PepI
was purified from Lb. delbrueckii subsp. bulgaricus
CNRZ 397 by amplification and expression of the gene
in E. coli (Gilbert et al., 1994). The purified enzyme
was characterized as a trimeric serine peptidase with a
subunit molecular mass of 33 kDa (Table 1).
Prolinase (PepR) is a specific dipeptidase which
hydrolyses dipeptides with the sequence Pro-X. PepR
from Lb. helveticus CNRZ32 was purified and biochemi-
cally characterized by Shao et al. (1997) and found to
have a relatively broad specificity. The PepR from Lb.
rhamnosus 1/6 (Varmanen et al., 1998), in addition to its
prolinase activity, hydrolysed the aminopeptidase sub-
strates, Pro-NA, Leu-NA and Phe-NA. Prolidase
(PepQ) is an X-Pro-specific dipeptidase. With the excep-
tion of PepQ from Lb. helveticus CNRZ32, which is a
homodimer with a subunit molecular mass of 45 kDa,
most PepQs characterized to date are monomeric metal-
lopeptidases with a native molecular mass of ⬃42 kDa.
These enzymes hydrolysed most X-Pro dipeptides with
the exception of Gly-Pro and Pro-Pro (Kaminogawa
et al., 1984; Fernández-Esplá et al., 1997b; Morel et al.,
1999). However, PepQs isolated from Lc. lactis subsp.
cremoris AM2 (Booth et al., 1990a) and Lb. delbrueckii
Proteolysis in Cheese during Ripening 413
subsp. lactis DSM7290 (Stuckey et al., 1995), hydrolysed
di- and tripeptides that did not contain Pro, in addition
to Pro-X dipeptides.
Aminopeptidase P (PepP) is a specific aminopepti-
dase that catalyses the removal of the N-terminal
amino acid from oligopeptides having the sequence
X-Pro-Pro-(X)n or X-Pro-(X)n (Kunji et al., 1996). The
enzyme has been purified from strains of Lactococcus
and is a monomeric metallopeptidase with a molecular
mass of 41–43 kDa (Table 1). Provided that the pep-
tide contains the above sequences, PepP is capable of
releasing the N-terminal amino acid from oligopep-
tides up to 11 residues long. This enzyme also hydro-
lyses peptides with Ala in the penultimate position but
at a slower rate (McDonnell et al., 1997).
Enzymes from Secondary Starter
Microorganisms
Enzymes of LAB play an important role in the second-
ary proteolysis in internal-ripened cheese varieties, and
hence contribute significantly to the development of
flavour and aroma. In mould-ripened, smear-ripened
and Swiss-type cheeses, microorganisms other than LAB
play a pivotal role in the development of characteristic
flavour and texture. The ripening of these cheese var-
ieties involves complex biochemical reactions, which are
discussed in detail in Volume 2. While the enzymes of
LAB have been well studied and characterized, there
have been fewer studies on organisms associated with
mould-ripened or smear-ripened cheese varieties or on
enzymes from Propionibacterium freudenreichii subsp.
shermanii.
The microbial flora of surface mould-ripened and
blue-veined cheese, such as Camembert and Roque-
fort, includes yeasts (e.g., Kluyveromyces lactis, Saccha-
romyces spp. and Debaryomyces hansenii), moulds
(Geotrichum candidum, Penicillium spp.), lactococci,
lactobacilli, micrococci, staphylococci, coryneform
bacteria and coliforms. Penicillium spp. are major
components of the microflora and their enzymes play
an important role in cheese ripening. Proteolytic
systems of P. camemberti and P. roqueforti are somewhat
similar; both synthesize an aspartyl proteinase, a
metalloproteinase, an acid carboxypeptidase and an
alkaline aminopeptidase (‘Surface Mould-ripened
Cheeses’ and ‘Blue Cheese’, Volume 2). The aspartyl
proteinase from P. camemberti hydrolyses s1-casein
faster than -casein or -casein (Gripon, 1993). Acid
proteinases of P. camemberti and P. roqueforti have simi-
lar action on -casein and hydrolyse Lys979Val98,
Lys999Glu100 and Lys299Ile30 bonds at a faster rate
than other bonds in -casein (Le Bars and Gripon,
1981; Trieu-Cuot et al., 1982). Metalloproteinases of
414 Proteolysis in Cheese during Ripening
both species have similar properties and have a pH
optimum in the range 5.5–6.0. Chrzanowska et al.
(1995) purified an aspartic proteinase from the culture
filtrate of P. camemberti by a two-step purification
procedure. The proteinase had a molecular mass of
33.5 kDa and an optimum pH of 3.4 on haemoglobin.
The enzyme showed specificity towards peptide bonds
containing an aromatic or hydrophobic amino acid
residue in the B-chain of insulin. Besides these pro-
teinases, P. roqueforti has a carboxypeptidase, which
has an optimum pH of 3.5 and releases acidic, basic or
hydrophobic amino acids (Gripon, 1993).
Geotrichum candidum also synthesizes extracellular
and intracellular proteinases, but the contribution of
these enzymes to cheese ripening is less than that of
enzymes from Penicillium spp. (Gripon, 1993).
The bacterial microflora of surface cheeses, such
as Tilsit, Limburger, Münster or Taleggio at the
beginning of ripening is dominated by yeasts and
moulds, which are acid and salt tolerant, but at the
end of ripening, bacteria of the genera Brevibacterium,
Arthrobacter, Micrococcus, Staphylococcus and
Corynebacterium dominate (Eliskases-Lechner and
Ginzinger, 1995; Valdés-Stauber et al., 1997; ‘Bacterial
Surface-ripened Cheeses’, Volume 2). Growth of B.
linens on the cheese surface is thought to play an
important role in the development of the characteris-
tic colour, flavour and aroma of smear surface-ripened
cheese varieties (Rattray and Fox, 1999) and hence, its
enzymes have been characterized. Extracellular
enzymes of B. linens include proteinases, aminopepti-
dases and esterases, the biochemical properties of
which vary because of wide inter-strain differences
within the species. Brézina et al. (1987) partially puri-
fied four extracellular proteinases from B. linens, with
pH and temperature optima of 5.0–8.0 and 50 °C,
respectively. Hayashi et al. (1990) purified five extra-
cellular proteinases from B. linens F (designated A, B,
C, D and E), having a molecular mass of 37, 37, 44,
127 and 325 kDa, respectively, as determined by size
exclusion chromatography (SEC). Proteinases A and
B were stable at 35 °C for 1 h and had a temperature
optimum of 40 °C, while proteinases C, D and E were
stable at 45 °C for 1 h and had a temperature optimum
of 50 °C. All five proteinases were optimally active at
pH 11.0 and were serine proteinases. The production
of multiple forms of the extracellular proteinases
by B. linens ATCC 9172 is a result of aggregation of
subunits and autocatalytic degradation (Buchinger
et al., 2001).
An extracellular serine proteinase partially purified
from a strain of B. linens (Laktoflora 200), had a
molecular mass of 52–55 kDa, as determined by SDS-
PAGE, and pH and temperature optima of 7.0–8.5 and
45 °C, respectively (Juhász and Skárka, 1990). A thermo-
stable proteinase was partially purified from B.
linens IDM 376; it had molecular mass of 18.5 kDa
and pH and temperature optima of 7.5 and 67.5 °C,
respectively, on azocasein (Clancy and O’Sullivan,
1993). An extracellular serine proteinase purified
from B. linens ATCC 9174 had a molecular mass of
126 kDa, as determined by SEC and was optimally
active at pH 8.5 and 50 °C (Rattray et al., 1995). It
hydrolysed s1-casein at His89Gln9, Ser1619Gly162 and
either Gln1729Tyr173 or Phe239 Phe24 (Rattray et al.,
1996) and -casein at Ser189Ser19, Glu209Glu21,
Gln569Ser57, Gln729Asn73, Leu779Thr78, Ala1019
Met102, Phe1199Thr120, Leu1399Leu140, Ser1429Trp143,
His1459Gln146, Gln1679Ser168, Gln1759Lys176,
Tyr1809Pro181 and Phe1909Leu191 (Rattray et al.,
1997).
One of the five extracellular enzymes of B. linens
ATCC 9172 was purified to homogeneity by Tomaschová
et al. (1998) using ion-exchange chromatography and
native preparative PAGE. The enzyme had nearly iden-
tical properties to the serine proteinase of B. linens
ATCC 9174 purified by Rattray et al. (1995). Its
molecular mass was estimated to be 56 kDa by SDS-
PAGE and pH and temperature optima were 8.0 and
50 °C, respectively.
B. linens also produces extracellular aminopepti-
dases, intracellular peptidases and proteinases. Sørhaug
(1981) reported the presence of intracellular dipep-
tidase activity in six strains of B. linens. The presence
of three extracellular aminopeptidases in B. linens
(Laktoflora 200), having pH and temperature optima of
7.0–9.0 and 30 °C, respectively, was reported by Brézina
et al. (1987). Two extracellular aminopeptidases, desig-
nated A and B, with a molecular mass of estimated to be
150 and 110 kDa, respectively, and pH and temperature
optima of 9.3 and 40 °C, respectively, were purified
from B. linens F by Hayashi and Law (1989). Ezzat et al.
(1993) reported the presence of cell wall proteinases
and dipeptidase activities in B. linens CNRZ 944. The
authors partially purified the cell wall proteinase, which
had maximum activity at pH 6.5 and 40 °C. An intracel-
lular aminopeptidase from B. linens ATCC 9174, with a
molecular mass of 59 kDa, as determined by SDS-
PAGE, and 69 kDa by SEC, was reported by Rattray and
Fox (1997). The enzyme was optimally active at pH 8.5
and 35 °C. Curtin et al. (2002) showed aminopeptidase,
dipeptidase and tripeptidase activities in brevibacteria,
corynebacteria, staphylococci and brachybacteria, isol-
ated from smear surface-ripened cheeses, Tilsit and
Gubeen.
Species of the genus Arthrobacter are major com-
ponents of the microflora of surface mould-ripened
cheeses, such as Brie and Camembert and red-smear

cheeses. However, the enzymes of Arthrobacter have
not been well studied. Smacchi et al. (1999a) purified
two extracellular serine proteinases from A. nicotianae
9458, with molecular masses of about 53–55 and
70–72 kDa, as determined by SDS-PAGE. The enzymes
were optimally active at 55–60 and 37 °C, respectively.
Both enzymes were optimally active in the pH range of
9.0–9.5 and preferentially hydrolysed -casein over
s1-casein. An extracellular PepI from A. nicotianae
9458 with a molecular mass of about 53 kDa, was
purified and characterized by Smacchi et al. (1999b).
The enzyme was optimally active at 37 °C and 8.0.
Some Micrococcus spp. are very proteolytic and pro-
duce extracellular proteinases and intracellular pro-
teinases and peptidases (Fox et al., 1993). Nath and
Ledford (1972) reported that extracellular proteinases
from certain micrococci preferentially hydrolysed s1-
casein; production of extracellular proteinase was also
reported by Garcia de Fernando and Fox (1991).
Bhowmik and Marth (1989) purified and characterized
an aminopeptidase, with broad substrate specificity,
from M. freudenreichii ATCC 407.
Propionibacterium spp. are weakly proteolytic, but
they are highly peptidolytic, especially on proline-con-
taining peptide bonds, thus contributing to the charac-
teristic flavour of Swiss-type cheeses (see ‘Cheese with
Propionic Acid Fermentation’, Volume 2). Biochemical
characteristics of peptidases from propionic acid bac-
teria have been reviewed by Gagnaire et al. (1999). A
PepX with a molecular mass of 84 kDa and pH and
temperature optima of 7.0 and 40 °C, respectively,
was purified and characterized from P. freudenreichii
subsp. shermanii NCDO 853 by Fernández-Esplá and
Fox (1997). Endopeptidases have been isolated from
P. freudenreichii subsp. shermanii and characterized
(Table 1) (Tobiassen et al., 1996; Stepaniak et al.,
1998b).
Patterns of Proteolysis in Cheese
The pattern of proteolysis in many varieties may be
summarized as follows: the caseins are hydrolysed ini-
tially by residual coagulant activity retained in the
curd and by plasmin (and perhaps other indigenous
proteolytic enzymes) to a range of large and intermedi-
ate-sized peptides which are hydrolysed by proteinases
and peptidases from the starter LAB, NSLAB and per-
haps secondary microflora to shorter peptides and
amino acids.
However, the pattern and extent of proteolysis varies
considerably between varieties due to differences in
manufacturing practices (particularly cooking tempera-
ture), which cause differences in moisture content,
residual coagulant activity, activation of plasminogen to
Proteolysis in Cheese during Ripening 415
plasmin, and possibly the development of a highly
proteolytic secondary microflora and ripening time. The
extent of proteolysis (i.e., the degree to which the
caseins and peptides therefrom are hydrolysed and
measured by the development of water- or pH 4.6-
soluble N) in cheese varies from very limited (e.g.,
Mozzarella) to very extensive (e.g., Blue) and is summa-
rized for many varieties in Table 2. The pattern of pro-
teolysis (i.e., the relative concentrations of different
peptides and amino acids) is very variable and is essen-
tially unique to a particular variety. The differences in
soluble N content are due to differences in moisture
content, temperature and pH, length of ripening, cook-
ing temperature and pH at draining (Fox and
McSweeney, 1996) and is mainly due to the action of
chymosin and to a lesser extent of plasmin (Fox and
McSweeney, 1997). A short ripening period (⬃3 weeks)
and extensive denaturation of chymosin during the
high temperature (⬃70 °C) stretching step during the
manufacture of Mozzarella cheese explain the low level
of soluble N, whereas extensive proteolysis is character-
istic of Blue cheese and some smear-ripened varieties,
caused by the action of chymosin, plasmin and pro-
teinases from their characteristic secondary microflora.
In addition, differences in the action of these proteolytic
agents cause differences in peptide profiles.
Primary proteolysis is similar during the ripening of
most cheeses; chymosin hydrolyses the Phe239Phe24
bond of s1-casein (Hill et al., 1974; Carles and Rib-
adeau-Dumas, 1985) except in cheeses that are cooked
at a high temperature (⬃55 °C, e.g., Swiss cheese), in
which plasmin is the principal proteolytic agent. In
blue-veined cheeses, after sporulation, enzymes from
P. roqueforti hydrolyse s1-CN (f24–199) and other
peptides, changing the peptide profile (Gripon, 1993).
Analysis of the water-insoluble fraction of various
cheeses by urea-PAGE gives insight into the differ-
ences in peptide profile between cheeses (Fig. 12). In
many cheeses, s1-casein is hydrolysed faster than
-casein (Sousa et al., 2001). In Blue-veined cheeses,
both s1- and -caseins are completely hydrolysed at
the end of ripening. In Swiss-type cheeses, -casein is
hydrolysed faster than s1-casein, with concomitant
increases in -caseins, indicating a role of plasmin and
denaturation of chymosin during cooking. However,
s1-CN (f24–199) is produced slowly in Swiss cheese,
indicating either the survival of some chymosin during
cooking or the activity of indigenous milk acid pro-
teinase, cathepsin D (Gagnaire et al., 2001), which has
specificity similar to chymosin (Hurley et al., 2000a).
In the case of Camembert-type cheese, about ⬃20% of
total N is soluble at pH 4.6 (Khidr, 1995) (Table 2)
and the pattern of proteolysis is similar to Cheddar
cheese (Fig. 12). During the ripening of Mozzarella
416 Proteolysis in Cheese during Ripening

Table 2 Soluble N as % of total nitrogen in different cheese varieties

Cheese Age SN/TN % References

Mozzarella 25 days 4–5 Somers et al. (2002)

O’Reilly et al. (2002)
Guinee et al. (1998)
Quarg 4 weeks ⬃12 Mara and Kelly (1998)
Gouda 6 weeks 12–13 Messens et al. (1999)
24 weeks 23–25 Exterkate and Alting (1995)
Camembert 1 month Surface 15–17 Michalski et al. (2003)
Core 9–12 Sousa and McSweeney (2001)
1 month Surface ⬃20 Khidr (1995)
Core ⬃12
Swiss 16 weeks 16–17 Cooney et al. (2000)
Feta 2–6 months 17–20 Michaelidou et al. (2003)
Sarantinopoulos et al. (2002)
Katsiari et al. (2000)
Moatsou et al. (2002)
Mahon 2 months 19–20 Taborda et al. (2003)
Cheddar 4–6 months 20–25 Shakeel-Ur-Rehman and Fox (2002)
Barrett et al. (1999)
Shakeel-Ur-Rehman et al. (1998)
Lane et al. (1997)
Tilsit 28 weeks Surface 24–25 Churchill et al. (2003)
Core 22–23
Parmesan 24 months 31–35 Careri et al. (1996)
Gorgonzola – 43–46 Zarmpoutis et al. (1997)
Danablu – 50–53

cheese, s1-CN (f24–199) is produced slowly and
-caseins more rapidly, indicating weak chymosin activ-
ity and fairly high plasmin activity (Kindstedt, 1993).
Plasmin and Lactobacillus proteinases are responsible
for extensive proteolysis in Parmigiano-Reggiano
cheese that is ripened for a long period (⬃24 months)
at an elevated temperature (⬃18–20 °C) (Battistotti
and Corradini, 1993). The high cooking temperature
used during the manufacture of Parmigiano-Reggiano
cheese denatures most of the chymosin.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Figure 12 Urea-polyacrylamide gel electrophoretograms water-
insoluble fraction of a selection of cheese varieties. Lane 1 Na
caseinate, lane 2 Cheddar, lane 3 extra-mature Cheddar, lane 4
Cheshire, lane 5 Red Leicester, lane 6 Double Gloucester, lane 7
Emmental, lane 8 Leerdammer, lane 9 Jarlsberg, lane 10 Vorarl-
berger Bergkase, lane 11 Edam, lane 12 Gouda, lane 13 Norve-
gia, lane 14 Parmesan, lane 15 Parmesan (from McGoldrick,
1996).
Several peptides from Cheddar, Parmigiano-Reggiano,
Blue, Swiss and Feta cheeses have been isolated and
characterized. Of these varieties, the peptide profile of
Cheddar cheese is the best characterized and is summa-
rized in Figs 13 and 14. All the principal water-insoluble
peptides in Cheddar cheese are produced either from
s1-casein by chymosin or from -casein by plasmin
(McSweeney et al., 1994a). After cheese manufacture,
residual chymosin acts on Phe239Phe24 of s1-casein to
produce the large C-terminal peptide s1-CN (f24–199)
and a small peptide s1-CN (f1–23) (Fox and
McSweeney, 1997). s1-CN (f24–199) is hydrolysed by
chymosin at Leu1019Lys102 (Fig. 15) and more slowly
at Phe329Gly33, Leu1099Glu110, Phe289Pro29 and
Leu409Ser41; s1-CN (f24–199) is also hydrolysed
slowly by plasmin at Lys1039Tyr104 and Lys1059Val106
of s1-CN (f24–199). The large C-terminal peptides s1-
CN (f24–199), s1-CN (f33–199), s1-CN (f102–199),
s1-CN (f110–199), s1-CN (f99–199), s1-CN
(f104–199) and s1-CN (f106–199) have been identi-
fied in the water-insoluble fraction of Cheddar cheese
(McSweeney et al., 1994a; Mooney et al., 1998). The
bond Trp1649Tyr165, which is hydrolysed rapidly in
solution by chymosin (McSweeney et al., 1993b), does
not appear to be hydrolysed in cheese, perhaps due to
intermolecular interactions. Peptide s1-CN (f1–23) is
hydrolysed at the bonds Gln99Gly10, Gln139Glu14,
Glu149Val15 and Leu169Asn17 by lactocepin (Fox and

Figure 13 Principal water-insoluble peptides derived from s1-casein (A) and -casein (B) isolated from Cheddar cheese by
McSweeney et al. (1994a) and Mooney et al. (1998) (from Sousa et al., 2001).
McSweeney, 1996). The peptides s1-CN (f1–9),
s1-CN (f1–13) and s1-CN (f1–14) accumulate and
dominate the RP-HPLC chromatogram of water-soluble
fraction of Cheddar cheese (Fig. 15).
Although the bond Leu1929Tyr193 of -casein in
solution is very susceptible to chymosin, it is hydrolysed
very slowly in cheese, probably due to the effect of ionic
strength which promotes hydrophobic interactions
between susceptible regions of -casein molecules (Fox
and McSweeney, 1997). The cleavage of Leu1929Tyr193
in cheese is undesirable, as -CN (f193–209) is very
hydrophobic and bitter (Visser et al., 1983). Plasmin
preferentially hydrolyses -casein at Lys289Lys29,
Lys1059His106 and Lys1079Glu108, producing 1-, 2-,
3-caseins and PPs (5, 8 fast and 8 slow); -caseins are
present in the water-insoluble fraction of Cheddar
(McSweeney et al., 1994a; Lane and Fox, 1999;
McGoldrick and Fox, 1999) and many other cheeses.
No large peptides originating from s2-casein have
been identified in Cheddar cheese (Mooney et al., 1998)
and only four small peptides have been identified in the
water-soluble fractions (Singh et al., 1995, 1997).
Water-soluble peptides are characteristic of particular
cheese varieties and are related to the specificity of the
starter and non-starter proteinases and peptidases (Fox
Proteolysis in Cheese during Ripening 417
and McSweeney, 1996). In terms of number, most of
the peptides in the water-soluble fraction of Cheddar
cheeses originate from N-terminal half of -casein (par-
ticularly from residues 53 to 91) and a small number
from the N-terminal half of s1-casein. However, many
of these peptides are present at low levels in cheese, and
the water-soluble fraction of Cheddar cheese is dom-
inated by a relatively small number of peptides, originat-
ing from s1-casein. Peptides in the water-soluble
fraction of Cheddar cheese do not contain intact plas-
min or chymosin cleavage sites and hence probably
arise, not directly from the caseins, but rather from
larger precursor peptides (produced by chymosin or
plasmin) by the action of lactocepin or other microbial
enzymes.
Little work has been done on pH 4.6-soluble pep-
tides in blue-mould cheese, in which extensive proteol-
ysis occurs. González de Llano et al. (1991) studied the
production and identification of phosphotungstic acid
(PTA)-soluble peptides in Gamonedo blue cheese. Low
molecular mass peptides from the PTA-soluble fraction
were isolated and their amino acid composition was
determined. The isolated peptides contained 7–10
amino acids and the major amino acids were Ser, Glu,
Gly/Thr, Ile and Leu (González de Llano et al. 1991).
418 Proteolysis in Cheese during Ripening

Figure 14 Water-soluble peptides derived from s1-casein (A), s2-casein (B) and -casein (C) isolated from Cheddar cheese by
Singh et al. (1994, 1995, 1997), Breen et al. (1995) and Fernandez et al. (1998). The principal chymosin, plasmin and lactocepin
cleavage sites are indicated (from Sousa et al., 2001).
 Proteolysis in Cheese during Ripening 419

Chymosin
1 199

Chymosin
1 23 199
24

Lc-CEP
102 199

f1-9, f-1-13

Further hydrolysis products

Figure 15 Schematic representation of the early proteolysis of s1-casein during the ripening of many cheeses and the location of
peptides produced on a urea-polyacrylamide gel electrophoretogram and a reverse-phase HPLC elution profile.

Low molecular weight peptides formed in Parmi-
giano-Reggiano cheese during ripening were isolated
and identified by Addeo et al. (1992, 1994, 1995)
using fast atom bombardment–mass spectrometry.
Oligopeptides originating from regions 1–20 and 6–28
of -casein, five phosphopeptides originating from the
region 64–84 of s1-casein, three phosphopeptides
from the region 1–21/24 of s2-casein and one peptide
from C-terminal part of s2-casein were identified.
Several water-soluble peptides in Feta were identi-
fied by Michaelidou et al. (1998), including s1-CN
(f1–14), (f4–14), (f24–30), (f24–32), (f40–49),
(f91–98), (f102–109), -CN (f164–180), (f191–205)
and -CN (f96–105). Gagnaire et al. (2001) identified
91 peptides in aqueous phase of Emmental cheese, 52
of which originated from s1-casein, 29 from -casein,
9 from s2-casein and 1 from -casein.
Significant concentrations of amino acids, the final
products of proteolysis, occur in all cheeses that have
been investigated. Levels of free amino acids in a num-
ber of cheese varieties are given in Table 3. Relative to
the level of water-soluble N, Cheddar contains low
concentrations of amino acids; the principal amino
acids are Glu, Leu, Arg, Lys, Phe and Ser. Parmigiano-
Reggiano contains a very high concentration of amino
acids which contribute to the characteristic flavour of
this cheese (Resmini et al., 1988). Many amino acids
have characteristic flavours (see McSweeney et al.,
1997); although none has a cheese-like flavour, it is
believed that they contribute to the savory taste of mature
cheese. However, the principal role of amino acids in
flavour development is as precursors of volatile flavour
compounds produced by the range of catabolic reactions
as discussed in ‘Catabolism of Amino Acids in Cheese
During Ripening’, Volume 1.
Methods for Monitoring Proteolysis in
Cheese
A range of analytical techniques have been developed to
study proteolysis in cheese and have been reviewed (IDF,
1991, 1999; McSweeney and Fox, 1993, 1997; Fox et al.,
1995a; Wallace and Fox, 1998). These methods for
assessment of proteolysis in cheese can be classified in
two categories, namely non-specific and specific meth-
ods. Non-specific methods, which give information
about the extent of proteolysis and the activity of prote-
olytic agents, include determination of the nitrogen sol-
uble in, or extractable by, various solvents or buffers (see
Christensen et al., 1991; McSweeney and Fox, 1997;
Ardö, 1999) or the measurement of reactive groups (e.g.,
NH2-groups) (McSweeney and Fox, 1997; Wallace and
Fox, 1998). Soluble nitrogen is usually determined by
the macro-Kjeldahl method, which is a highly repeat-
able, but time-consuming and potentially dangerous
technique (Wallace and Fox, 1998). A number of tech-
niques for quantifying proteolysis are based on cleavage
of the peptide bond, which results in the formation of a
new amino group which can react with several chro-
mogenic (e.g., 2,4,6-trinitrobenzenesulphonic acid or
ninhydrin) or fluorogenic (e.g., o-phthadialdehyde or
fluorescamine) reagents (McSweeney and Fox, 1997;
Wallace and Fox, 1998).
Non-specific methods give a general idea of prote-
olysis, but give no information about the specific
peptides produced or degraded during ripening. Specific
420 Proteolysis in Cheese during Ripening

Table 3 Concentration of amino acids (mg kg 1 cheese) in different cheese varieties (based on Fox and Wallace, 1997)

Parmigiano-
Amino acid Cheddar Edam Emmental Reggiano Gorgonzola Danablu

Cys 44.7 – – – 1380 1160

Asp 1532.8 20.5 166.5 3241.9 1020 300
Thr – 144.5 688.5 4033.3 530 190
Ser 416.3 71.1 548.9 4459.5 1570 1020
Glu 3144.1 351.7 2680.5 14489.0 3940 1730
Pro 336.2 153.8 2535.2 – 2320 530
Gly 306.86 34.6 430.8 2115.6 390 160
Ala 356.2 68.8 568.2 2260.2 1140 340
Val 1096.4 167.4 1561.5 6011.9 2220 610
Met 434.8 60.3 502.7 2351.5 780 500
Ile – 48.1 1051.1 5205.2 1300 300
Leu 2774.1 426.6 1794.9 7290.4 2910 1530
Tyr 464.1 89.2 285.9 2054.7 850 520
Phe 1472.6 291.9 1279.1 4314.9 1590 680
His – 49.7 868.2 – 800 610
Lys 1127.2 245.5 2219.8 10091.0 3050 1540
Arg 1096.4 130.5 19.2 791.4 280 510

techniques (i.e., electrophoresis and chromatography)
have been used extensively to resolve, isolate and
identify the peptides that are produced during cheese
ripening (Fox et al., 1995a; McSweeney and Fox,
1997; Otte et al., 1999; Singh et al., 1999). In addition,
these techniques are used to determine peptide pro-
files of cheese extracts; data obtained using these tech-
niques are often analysed using multivariate statistical
techniques (e.g., Pripp et al., 1998, 1999, 2000a,b;
Molina et al., 1999; Shakeel-Ur-Rehman et al., 1999).
Urea-PAGE is a powerful tool for monitoring prote-
olysis during the early stages of cheese maturation and
for comparing casein hydrolysis patterns in cheeses
manufactured from the milk of different species (Mar-
cos et al., 1979; Sousa and Malcata, 1997). Urea-PAGE
is widely used to monitor proteolysis as it resolves
proteins based on a combination of charge and mass
while sodium dodecylsulphate (SDS)-PAGE, which is
used more widely in biochemistry, is less suitable for
studying proteolysis in cheese because this technique
resolves proteins based on size and the caseins have
similar molecular masses. Peptides separated by SDS-
or urea-PAGE can be isolated by excision of the bands
or by electroblotting (McSweeney et al., 1994a; Singh
et al., 1995; Sousa and Malcata, 1998a,b) and the
N-terminal sequence of isolated peptides determined.
Isolation of peptides by electroblotting has been used
widely to study proteolysis in cheese (e.g., Singh et al.,
1995, 1997; Gouldsworthy et al., 1996; Ferranti et al.,
1997; Broadbent et al., 1998).
Capillary electrophoresis (CE) is reported to be an
excellent technique for resolving the caseins (including
different genetic variants), peptides derived therfrom
and whey proteins (Otte et al., 1997). Peptide profiles
obtained by CE supplement the information obtained by
reversed-phase high performance liquid chromatography
(RP-HPLC) (Otte et al., 1997; Molina et al., 1998).
Capillary electrophoresis has been used to study:
• proteolysis in Cheddar (Strickland et al., 1996),
Mozzarella, Feta and Danbo (Otte et al., 1997),
Tilsit (Bockelmann et al., 1998), Roncal (Irigoyen
et al., 2000), Danbo cheeses (Sørensen and Benfeldt,
2001) and Serpa, a raw ewes’ milk cheese (Roseiro
et al., 2003);
• the effect of the amount of rennet on proteolysis and
texture in Feta cheese made from ultrafiltered milk
(Wium et al., 1998);
• the effect of different strains of Penicillium roqueforti on
the ripening of blue-veined cheese (Larsen et al., 1998);
• the effect of added proteinases and level of starter
cultures on the formation of biogenic amines in
Manchego cheese made from raw milk (Fernández-
García et al., 1999).
A number of chromatographic techniques, such as
ion-exchange chromatography, SEC and RP-HPLC have
been used to fractionate milk proteins or to fractionate
cheese extracts for the purification of peptides, or less
commonly, as analytical techniques to generate peptide
profiles (see McSweeney and Fox, 1997). Ion-exchange
and SEC are suitable for the fractionation of large casein-
derived peptides. High performance ion exchange chro-
matography and HP-SEC have the advantage of speed
and reproducibility. RP-HPLC is a very good method for
resolving water-soluble peptides and has been used to
characterize and compare the degree of proteolysis in

cheeses of various ages and quality, and to study the
effect of various cheesemaking parameters on proteolysis
(Singh et al., 1999). Reversed phase-high performance
liquid chromatography has been used extensively to
characterize peptides in casein hydrolysates (e.g., Le
Bars and Gripon, 1989, 1993; McSweeney and Fox,
1993) as well as in studies on proteolysis of the caseins
in cheese during ripening (e.g., González de Llano et al.,
1991; Addeo et al., 1992, 1994; McSweeney et al., 1994a;
Lynch et al., 1997; Sousa and Malcata, 1998a;
McGoldrick and Fox, 1999; Shakeel-Ur-Rehman et al.,
2000; Katsiari et al., 2001; Trujillo et al., 2002; Poveda
et al., 2003). Numerous peptides from cheese have been
purified by a combination of chromatographic proced-
ures and subsequently identified, usually by Edman
degradation and mass spectrometry, leading to a better
knowledge of the proteolytic pathways in cheese during
ripening (Singh et al., 1999; Gagnaire et al., 2001).
Free amino acids in cheese have been analysed
using amino acid analysers based on ion-exchange
chromatography, with post-column ninhydrin derivat-
ization and photometric detection at 570 nm and
440 nm for primary and secondary amino acids, respec-
tively. This method is relatively simple, accurate and
quantitative and requires little sample preparation
(Bütikofer and Ardö, 1999). Alternatively, fluores-
cent amino acid derivatives (e.g., dansyl, OPA or N-
(9-fluorenylmethoxycarbonyl)) can be prepared and
separated and quantified by RP-HPLC; amino acids
can also be quantified by gas chromatography but this
method is rarely used (McSweeney and Fox, 1997).
In addition to the above methods, new techniques
have been evaluated for indirect measurement of proteo-
lysis. During ripening, the texture of cheese undergoes
major changes due to proteolysis, and in recent years,
the use of new techniques such as fluorescence spec-
troscopy and ultrasound have been investigated to moni-
tor changes in protein structure in different types of
cheese during maturation. Fluorescence spectroscopy
has the advantages of high sensitivity and rapidity for
the characterization of molecular interactions and reac-
tions. Tryptophan can be used as an intrinsic probe for
monitoring changes in protein structure during cheese
ripening as all the major proteins of bovine milk contain
at least one tryptophan residue, which has a characteris-
tic excitation in the region 280–295 nm and broad emis-
sion spectra (Hebert et al., 2000). Fourier transform
infrared spectroscopy (FTIR) has been used to measure:
• the levels of protein, fat and moisture in different
cheeses (McQueen et al., 1995);
• differences between soft cheese varieties (Herbert
et al., 2000) and different Emmental-type cheeses
(Picque et al., 2002);
Proteolysis in Cheese during Ripening 421
• changes in protein structure during cheese ripen-
ing (Mazerolles et al., 2001), protein/protein and
protein/fat interactions and their relation to the
texture of soft cheeses (Dufour et al., 2001);
• cheese melting and its correlation with rheological
properties (Karoui et al., 2003);
• the stability of processed cheese (Christensen et al.,
2003b).
Mazerolles et al. (2001) investigated changes in the
amide I and amide II regions of the FTIR spectra and
in the tryptophan fluorescence spectra of 16 experi-
mental semi-hard cheeses, varying in moisture, pro-
tein, fat and degree of maturity. The data obtained
from mid-infrared and fluorescence spectral data were
analysed by PCA, and correlations between spectral
data and chemical composition as well as correlations
between mid-infrared and fluorescence spectral data
were found by canonical correlation analysis (CCA)
methods. PCA and CCA of data helped to discriminate
between samples at different stages of ripening.
Application of low intensity ultrasonics in the food
industry has increased during the last decade because it
is non-destructive, rapid and cost-effective (McClements,
1997). Ultrasonics have been used in the dairy industry
to monitor milk coagulation during cheesemaking
(Gunasekaran and Ay, 1996; O’Donnell et al., 1996), to
determine the maturity of Mahon, a Spanish semi-soft
cheese variety (Benedito et al., 2000a), to determine
structural defects in Parmesan cheese (Orlandini and
Annibaldi, 1983) and to determine the physical proper-
ties of Cheddar cheese (Cho et al., 2001). The use of
ultrasonic devices to monitor the maturity of Cheddar
cheese non-destructively has been reported. Benedito
et al. (2000b) ripened blocks of Cheddar cheeses at 5 or
12 °C; the ultrasonic velocity increased during matur-
ation and decreased with increasing ripening tempera-
ture. Cho et al. (2001) measured ultrasound velocity
and relative attenuation in Cheddar cheese using a non-
contact piezoelectric ultrasound system and correlated
results with the physical properties of Cheddar cheese
(such as failure strain, failure stress, Young’s modulus
and toughness) using multi-linear neutral network
analysis. Besides the above applications, ultrasonics can
also be used to detect cracks in cheese and to assess eye
distribution and size in Emmental cheese (Benedito
et al., 2002). Research done so far has been limited to a
few varieties of cheeses; research is required for other
cheese varieties with different physical properties and
ripening behaviour (e.g., mould-ripened cheeses, smear-
ripened cheeses or cheese with eyes) for a clearer
understanding of the interaction of ultrasound and
cheese and subsequent application of ultrasound as a
tool for monitoring ripening.
422 Proteolysis in Cheese during Ripening
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This Page Intentionally Left Blank
 Catabolism of Amino Acids
in Cheese during Ripening
Á.C. Curtin and P.L.H. McSweeney, Department of Food and Nutritional Sciences,
University College, Cork, Ireland
Introduction
Catabolism of amino acids plays a major role in flavour
development in cheese during ripening (McSweeney
and Sousa, 2000; Yvon and Rijnen, 2001; Smit et al.,
2002; ‘Sensory Character of Cheese and its Evaluation’
and ‘Cheese Flavour: Instrumental Techniques’, Volume 1).
In particular, the catabolism of sulphur-containing
amino acids (principally methionine), aromatic amino
acids and branched-chain amino acids to flavour, or
perhaps off-flavour, compounds has received consider-
able attention. The major aroma compounds produced
from these amino acids are listed in Table 1.
Recently, many authors have studied amino acid
catabolism by cheese-related bacteria, including lacto-
cocci, non-starter lactic acid bacteria (NSLAB) and
smear bacteria. However, it is important to remember
that results obtained under laboratory conditions may
not be representative of actual activity under cheese-
ripening conditions, as cheese constantly undergoes
changes which may not be replicated in laboratory
experiments.
The pathways for amino acid catabolism remain to be
characterised fully although much work has been done
recently on LAB and Brevibacterium linens (see Yvon and
Rijnen, 2001). There appear to be two major pathways
by which amino acids are catabolised (Yvon and Rijnen,
2001; Fig. 1). The first series of reactions is initiated by
the action of an aminotransferase which transfers the
amino group from amino acid A to an -keto acid B
(usually -ketoglutaric acid) and results in the produc-
tion of an -keto acid corresponding to amino acid A
and a new amino acid corresponding to -keto acid B
(usually glutamic acid). -Keto acids produced by the
transamination of aromatic amino acids, branched-chain
amino acids and methionine may be degraded further to
other compounds by enzyme-catalysed reactions or by
chemical reactions. The second major series of reactions
by which amino acids are catabolised is initiated by the
action of amino acid lyases which cleave the side chains
of amino acids. These pathways are particularly import-
ant for the catabolism of aromatic amino acids and
methionine. Other pathways by which amino acids may
Cheese: Chemistry, Physics and Microbiology, Third edition – Volume 1: General Aspects
ISBN: 0-1226-3652-X
Set ISBN: 0-1226-3651-1
be catabolised include the production of amines by
decarboxylases and the production of NH3 by deamin-
ases. There are also specific pathways for the metab-
olism of threonine, aspartic acid, glutamic acid and
arginine. Because of their importance to cheese flavour,
the catabolism of methionine, branched-chain and aro-
matic amino acids will be discussed in separate sections
below.
Transamination of Amino Acids
Aminotransferases (EC 2.6.1.x) catalyse the transfer
of the amino group from an -amino acid to an -keto
acid (Hemme et al., 1982). The enzymes have broad
substrate specificity and can catalyse reversible transam-
ination reactions (Weimer et al., 1999). Transamin-
ation is the first step in the degradation of amino acids
by lactococci. In LAB, catabolism of aromatic amino
acids, branched-chain amino acids and methionine is
initiated by transamination since degradation occurs
only in the presence of an -keto acid which acts as an
amino group acceptor (see Yvon and Rijnen, 2001). In
the context of cheese-related microorganisms, amino-
transferases of LAB and B. linens have been researched
most intensively; however, enzymes of Propionibac-
terium freudenreichii have also been studied (Thierry
et al., 2002). In a study on the enzyme activities of a
range of bacteria present on the surface of smear
cheese (brevibacteria, corynebacteria, staphylococci
and brachybacteria), Curtin et al. (2002) found
methionine aminotransferase activity in only a strain
of Staphylococcus equorum.
Aminotransferases are pyridoxal-5 -phosphate (PLP)-
dependent enzymes (Hemme et al., 1982; Weimer et al.,
1999; McSweeney and Sousa, 2000). The transamin-
ation reaction occurs in two steps. The first step
involves transfer of the amino group of the amino acid
to PLP to yield an -keto acid and an enzyme-bound
pyridoxamine-5 -phosphate. In the second step, the
amino group is transferred from pyridoxamine-
5 -phosphate to an -keto acid to produce an amino
acid and to regenerate PLP. The amine acceptor is
Copyright © 2004 Elsevier Ltd
All rights reserved
436 Catabolism of Amino Acids in Cheese during Ripening

Table 1 Name and chemical nature of the major aroma compounds derived from methionine, branched-chain amino acids and
aromatic amino acids (adapted from Yvon and Rijnen, 2001)

Amino acid Aldehydes Alcohols Carboxylic acids Thiols/Misc.

Leucine 3-Methylbutanal/ 3-Methylbutanol 3-Methylbutanoic acid/

isovaleraldehyde isovaleric acid
Isoleucine 2-Methylbutanal 2-Methylbutanol 2-Methylbutanoic acid
Valine 2-Methylpropanal/ 2-Methylpropanol 2-Methylpropanoic acid/
isobutyraldehyde isobutyric acid
Phenylalanine Phenylacetaldehyde, Phenylethanol Phenylacetic acid
benzaldehyde
Tyrosine OH-phenylacetaldehyde, OH-phenylethanol OH-phenylacetic acid p-Cresol, phenol
OH-benzaldehyde
Tryptophan Indole-3-acetaldehyde, Tryptophol Indole-3-acetic acid Skatole, indole
Methionine 3-Methylthiopropanal/methional 3-Methylthiopropanol 3-Methylthiopropionic acid Methanethiol

usually -ketoglutaric acid. Tamman et al. (2000) appears that the activity of transaminases may also rely
observed that nine lactobacilli isolated from a 3-year- on the presence of other compounds. For example,
old Cheddar were able to transaminate amino acids Amarita et al. (2001) found that the methionine
only if exogenous -ketoglutarate was supplied. It aminotransferase activity of 29 Lactobacillus strains

Aromatic amino acids

Branched-chain amino acids
TRANSAMINATION

Methionine

Aminotransferase

ELIMINATION

Met Tyr Trp

HA-DH α-Keto acid
n
io
at α-KADC
Hydroxyacid xid MGL
O ? CBL TPL TIL
α-KADH CGL
?
Aldehyde Acyl-CoA

Benzaldehyde Alcohol DH Methanethiol Indole

(Phe) aldDH
Hydroxybenzaldehyde Alcohol Phenol
(Tyr ) Carboxylic acid
Indole-3-acetate
(Trp)

Ester Methyl thioester

Dimethyldisulphide
Dimethyltrisulphide
Cresol, skatole
(Tyr, Trp)

Figure 1 Schematic diagram of pathways for amino acid catabolism found in different microorganisms and some chemical reac-
tions (dotted lines) occurring in cheese during ripening (modified from Yvon and Rijen, 2001). AT: aminotransferase, HA-DH: hydroxy-
acid dehydrogenase, -KADH: -keto acid dehydrogenase, -KADC: -keto acid decarboxylase, aldDH: aldehyde dehydrogenase,
alcohol DH: alcohol dehydrogenase, MGL: methionine--lyase, CGL: cystathionine--lyse, CBL: cystationine--lyase, TPL: tyrosine-
phenol lyase, TIL: tryptophan-indole lyase.
 Catabolism of Amino Acids in Cheese during Ripening 437
increased in the presence of glucose in the reaction
mixture. Williams et al. (2002) studied the effects of
rate and stage of growth, amino acid type and the pres-
ence of glucose on aminotransferase activity in strains
of NSLAB from Cheddar cheese. Aminotransferase
activity of the cell-free extract of two strains of Lb.
paracasei was maximal at, or close to, pH 6.0 and
30 °C, but activity was detected under conditions simi-
lar to those in cheese during ripening. The effect of
NaCl on activity at pH 8 and 30 °C differed with
leucine or phenylalanine as substrate. Glutamic acid
was formed most efficiently from -ketoglutarate
using aromatic, branched-chain or sulphur-containing
amino acids as substrate. Martinez-Cuesta et al. (2002)
found that aminotransferase activity was increased in
cells treated with a bacteriocin, lacticin 3147, to increase
the permeability of the cell wall to amino acids.
Aminotransferases have been studied in several
cheese-related bacteria. An aminotransferase from Lc. lac-
tis subsp. cremoris NCDO763, with a pH optimum of
6.5–8, was found to be PLP-dependent but metal ion-
independent (Yvon et al., 1997). The enzyme acted on
leucine, methionine and aromatic amino acids. One of
the two substrates of the reaction it catalyses (amino
acid or keto acid) must have a hydrophobic group
attached to the -carbon of the compound. This
enzyme was responsible for the biosynthesis of phenyl-
alanine and tyrosine, but it also had a catabolic role
when high concentrations of aromatic amino acids were
present. Yvon et al. (1997) suggested that the enzyme is
involved in the catabolism of leucine, methionine,
phenylalanine, tyrosine and tryptophan, and also in the
synthesis of the aromatic amino acids.
Engels (1997) purified two aminotransferases from
Lc. lactis subsp. cremoris B78. The enzymes were active
on methionine, leucine, isoleucine, valine and phenyl-
alanine. Both transaminases were dimeric proteins, had
a high temperature optimum and an alkaline pH opti-
mum. The enzymes were found to catalyse the conver-
sion of methionine to L-methylthio-2-ketobutyric acid.
Two aminotransferases were also isolated from Lc.
lactis subsp. lactis S3 by Gao and Steele (1998). The
enzymes were PLP-dependent methionine amino-
transferases. Both also had activity on aromatic amino
acids and leucine. The aminotransferases were active
under cheese-ripening conditions and produced com-
pounds that may be precursors of off-flavour com-
pounds in cheese, e.g., p-hydroxyphenylpyruvic acid
produced from tyrosine can breakdown chemically to
p-cresol.
Dias and Weimer (1998a) detected high levels of
methionine aminotransferase activity in several strains of
lactococci but found only slight activity in two strains
of B. linens. This is in contrast to earlier findings of Lee
et al. (1985) who observed aromatic aminotransferases
in 23 coryneforms isolated from cheese. These authors
reported that the aminotransferase(s) of B. linens were
inducible while the enzymes of other coryneform
strains tested were constitutive. The aromatic amino-
transferase of B. linens 47 was studied by Lee and
Desmazeaud (1985). The inducible enzyme was respon-
sible for removal of the amino group of amino acids for
their use as sole nitrogen sources by the bacterium;
-ketoglutarate was preferred as the amino group accep-
tor over pyruvate.
An aminotransferase from Lc. lactis LM0230 which
acts on branched-chain amino acids was cloned and
sequenced by Atiles et al. (2000). The enzyme was
active on methionine and phenylalanine in addition to
the three branched-chain amino acids. Sequence analy-
sis showed high homology with other branched-chain
aminotransferases. Transamination by Lb. helveticus
was studied by Klein et al. (2001). The bacterium was
incubated with a mixture of amino acids (phenylala-
nine, tyrosine, methionine, leucine, valine, isoleucine) in
ratios similar to those in Emmental cheese. The
authors concluded that transamination was the first
and main step in the conversion of amino acids by Lb.
helveticus, with phenylalanine and tyrosine being con-
verted most efficiently. The transamination of branched-
chain amino acids by Lb. paracasei has also been studied
(Hansen et al., 2001).
It is clear that many bacteria found in cheese are
capable of amino acid transamination but what role do
these reactions play in cheese flavour development? It
has been suggested that the rate-limiting step in
flavour development is the conversion of free amino
acids to aroma compounds. Yvon et al. (1998) added
-ketoglutarate to St Paulin-type cheese in an effort to
accelerate flavour development. The level of glutamate
in cheese increased when -ketoglutarate was added
but the level of some amino acids, including leucine,
phenylalanine, tyrosine and valine, decreased. The
addition of -ketoglutarate increased the degradation
of methionine. However, after six weeks of ripening,
large amounts of the resulting -keto acids, produced
by transamination, remained in the cheese. Thus,
these authors suggested that aminotransferase activity
was not limiting, but that subsequent steps in the for-
mation of aroma compounds may have been limiting.
Banks et al. (2001) supplemented Cheddar cheese
with -ketoglutarate to enhance amino acid catabolism
during ripening. -Ketoglutarate (20 g kg 1 milled
curd) was added as a mixture with the salt. It was
observed that the levels of leucine, phenylalanine,
valine, threonine, methionine, alanine and isoleucine
decreased on addition of -ketoglutarate. Sensory
analysis showed that the addition of -ketoglutarate
caused statistically significant changes in aroma inten-
sity, creamy character and fruity notes. In fact, the
438 Catabolism of Amino Acids in Cheese during Ripening
aroma intensity of the 12-week-old cheese supple-
mented with -ketoglutarate was equal to that of a
24-week-old control Cheddar. Supplementation of
Cheddar cheese with -ketoglutarate caused statistically
significant effects on the production of certain volatile
flavour compounds.
Shakeel-Ur-Rehman and Fox (2002) supplemented
Cheddar cheese with -ketoglutarate, pyruvate or
PLP and reported a beneficial effect of added
-ketoglutarate and pyruvate on flavour. Cheese
supplemented with 1 g -ketoglutaric acid per kg of
curd was considered to be as mature at 60 days as
90-day-old commercial Cheddar cheese. Addition of
-ketoglutaric acid to cheese also promoted synere-
sis during pressing of the curd but did not affect
plasmin or chymosin activity.
In an effort to overcome limitations in the concen-
tration of -ketoglutarate, Rijnen et al. (2000) inves-
tigated the possibility of using lactic acid bacteria
capable of producing -ketoglutarate from glutamate
in cheese, which can be converted to -ketoglutarate by
glutamate dehydrogenase. The authors cloned the glu-
tamate dehydrogenase gene (gdh) from Peptostrepto-
coccus asaccharolyticus into Lc. lactis, and followed the
conversion of amino acids to aroma compounds in the
Ch-Easy model. It was observed that the gdh strain
produced -ketoglutarate from glutamate under
cheese-ripening conditions and this allowed transamin-
ation of aromatic amino acids and branched-chain
amino acids. The gdh strain could be used as an alter-
native to the addition of exogenous -ketoglutarate to
cheese to increase amino acid catabolism.
A number of groups are now working on the genet-
ics of amino acid-catabolising enzymes with a view to
developing strategies for controlling the development of
cheese flavour. Rijnen et al. (1999a) characterised the
gene (araT) encoding the lactococcal aromatic amino-
transferase. It was shown that araT is transcribed as a
single gene. This enzyme is essential for the catabolism
of aromatic amino acids and is involved in the conver-
sion of leucine and methionine. It also plays a role in
the biosynthesis of phenylalanine and tyrosine. Subse-
quently, Rijnen et al. (1999b) inactivated the lactococcal
araT gene and studied the possibility of controlling
flavour development by directing the degradation of
amino acids by starter bacteria in cheese. These authors
followed the production of aroma compounds and the
degradation of amino acids in St Paulin-type cheese
using radiolabelled amino acids and quantified volatile
products by GC–MS. -Ketoglutarate was added to half
the cheeses to enhance transamination. It was observed
that inactivation of the lactococcal araT gene did not
significantly affect the levels of volatile compounds pro-
duced from branched-chain amino acids or methionine
during ripening, perhaps because lactococci produce
more than one aminotransferase with overlapping
specificities (Engels, 1997; Gao and Steele, 1998).
Cheeses containing added -ketoglutarate were described
as ‘more odorous’ than the control. In cheeses supple-
mented with -ketoglutarate and made with an araT-
negative starter, the formation of aroma compounds was
lower than in control cheeses. Since many of the aroma
compounds produced from aromatic amino acids, such
as phenethanol, phenol and indole, contribute to off-
flavours in cheese, the use of araT-negative starter
strains may prevent the development of certain off-
flavours in cheese.
Amino acids react chemically with carbonyl com-
pounds to form azomethines; if the carbonyl com-
pound has an electron-withdrawing group adjacent to
the carbonyl group (e.g., a dicarbonyl), then transami-
nation and decarboxylation occur. This process is
known as the Strecker degradation, through which
aldehydes are formed (Belitz and Grosch, 1987).
O
C O
+ H2N CHR COOH
C N CHR C
C O
C O
O H
Dicarbonyl Amino acid
CO2
H2O
H
NH2 C N CHR
C
+ O CHR
C O C OH
Strecker
aldehyde
Transamination via the Strecker degradation forms
the same volatile aldehydes as formed by enzyme-
catalysed transamination, although by a different
reaction mechanism. Strecker degradation plays an
important role in flavour development of many foods
since dicarbonyls are produced by the Maillard reac-
tion. There have been reports of Strecker aldehydes
in cheese (e.g., Dunn and Lindsay, 1985) but the
extent to which this chemical reaction occurs in
cheese is unclear since the same aldehydes are pro-
duced from a pathway initiated by the action of
aminotransferases.
The keto acids produced as a result of transamin-
ation of methionine, branched-chain amino acids or
aromatic amino acids serve as precursors of aroma
compounds (Yvon and Rijnen, 2001), which can be
formed by enzymatic or chemical reactions. Four main
 Catabolism of Amino Acids in Cheese during Ripening 439
degradation pathways for -keto acids are used by
cheese-related microorganisms (Fig. 1):
• -Keto acids may be reduced to the corresponding
hydroxyacid by the action of 2-hydroxyacid dehy-
drogenases, which have been found in LAB (see
Yvon and Rijnen, 2001). Although hydroxyacids are
not important flavour compounds, their production
reduces the levels of -keto acids available for other
reactions.
• -Keto acids derived from branched-chain amino
acids, aromatic amino acids and methionine may
also be decarboxylated to the corresponding alde-
hydes, although this pathway is not important in
most LAB. Aldehydes produced by this pathway
may be oxidised to the corresponding carboxylic
acid by aldehyde dehydrogenases or reduced to
alcohols by alcohol dehydrogenases.
• -Keto acids produced by transamination may also
be oxidatively decarboxylated to carboxylic acids by
the action of -keto acid dehydrogenases, generating
acyl-CoAs which are hydrolysed, releasing car-
boxylic acids (Yvon and Rijnen, 2001). However, this
pathway does not appear to be common in micro-
organisms found in cheese.
• -Keto acids may degrade chemically. Phenyl pyru-
vate and hydroxyphenyl pyruvate (produced from
Phe and Tyr, respectively) may be converted to ben-
zaldehyde and hydroxybenzaldehyde, respectively.
Likewise, indole-3-pyruvate, which is produced from
Trp, is quite unstable and degrades to indole acetic
acid, indole-3-aldehyde and skatole. Non-enzymatic
degradation of -heto--methylthio butyrate, pro-
duced from methionine, to methanethiol has also
been reported (Gao et al., 1998).
Production of Volatile Sulphur Compounds
by Amino Acid Catabolism
Volatile sulphur compounds are found in most cheeses
and are important components of flavour (Fox and
McSweeney, 1996). Since methionine is present in the
caseins at a higher concentration than cysteine, sulphur
compounds in cheese presumably originate principally
from methionine. The pathways for the production of
various flavour compounds from methionine are shown
in Fig. 2.
Weimer et al. (1999) discussed some of the sulphur
compounds in cheese and their importance to flavour.
They reported that dimethyldisulphide does not con-
tribute to flavour, while dimethyltrisulphide is a flavour
compound. The occurrence of these compounds is
related to methanethiol content and the low redox poten-
tial of the cheese. Dimethylsulphide and dimethyldisul-
phide can be produced from methanethiol but it is
unclear how dimethyltrisulphide is produced in cheese
(McSweeney and Sousa, 2000). Methional (-methyl
mercaptopropionaldehyde), which is considered to be
part of Cheddar cheese aroma (Weimer et al., 1999), can
be degraded to methanethiol either spontaneously or by
decarboxylation.
Methanethiol is a volatile compound with a ‘putrid
faecal-like’ aroma at high concentrations. However, at
low concentrations, it contributes to the characteristic
aroma of cheese (Weimer et al., 1999). It is also a pre-
cursor of other volatile sulphur compounds which
contribute to the garlic aroma of smear-ripened
cheese (Hemme et al., 1982). Starter cultures, flavour
adjunct bacteria and non-starter bacteria may form
methanethiol in cheese from methionine (Fig. 2).
Researchers continue to study the production of
methanethiol and its role in cheese flavour. Dias and
Weimer (1999) investigated the production of volatile
sulphur compounds in Cheddar cheese slurries. It was
observed that the production of total volatile sulphur
compounds was four times higher in slurries acidified
by Lc. lactis subsp. cremoris S3 than in those chemically
acidified with gluconic acid--lactone. Addition of brevi-
bacteria and methionine--lyase (ML) to the slur-
ries increased the production of volatile sulphur
compounds. They concluded that adjunct cultures and
enzymes are required to produce volatile sulphur com-
pounds above their flavour threshold. Molimard and
Spinnler (1996) also believed that coryneform bacteria,
especially B. linens, are key agents in the production of
sulphur compounds in surface mould-ripened cheese.
The production of sulphur compounds by LAB was
investigated by Seefeldt and Weimer (2000). Lacto-
cocci are auxotrophic for methionine while lactobacilli
are auxotrophic for both cysteine and methionine.
In addition, it was observed that lactococci possess
greater cystathionine lyase activity than lactobacilli.
The cell-free extract of both lactococci and lacto-
bacilli was able to produce volatile sulphur com-
pounds, including methanethiol, dimethyldisulphide
and dimethyltrisulphide.
Five bacteria from cheese were analysed by Bonnarme
et al. (2001a) for enzymes involved in the production of
methanethiol. B. linens had the highest demethiolating
activity. S. equorum and M. luteus showed demethiolating
activity but they produced only trace amounts of volatile
sulphur compounds; all bacteria examined formed
methanethiol.
The ability of 7 Lb. casei and 22 Lb. plantarum
strains to produce flavour compounds from methion-
ine was investigated by Amarita et al. (2001). Several
enzyme activities were studied: methionine amino-
transferase, hydroxyacid dehydrogenase, methionine
440
O

H 2N CH C OH

CH2

CH2

O
S
O
H2N CH C OH
CH2
H2N CH C OH
CH2
H 2N CH C OH
Cystathionine-β-lyase
CH2 Other reactions
Cystathionine-γ-lyase
Cystathionine-β-synthase CH2
O
SH SH

Cysteine Cystathionine Homocysteine

methyltransferase
Homocysteine
O O NH2
O

Aromatic aminotransferase H2N CH C OH

O C C OH H2N CH C OH N
or N
transaminase B CH2
CH2 CH2
Methionine adenosyltransferase N
CH2 N
CH2 CH2
Amino acid oxidase
S CH2
S S O

CH3 H H
CH3 CH3
H H
Methionine OH OH
α-Keto-γ-thiomethylbutyrate
S-Adenosylmethionine

Methionine-γ-lyase
α-Keto-γ-thiomethylbutyrate
demethiolase
H3 C S S CH3
H3C SH

Chemical conversion Dimethyldisulphide

Methanethiol
Other products
H 3C S S S CH3

Dimethyltrisulphide

Figure 2 Metabolic pathways for the interconversion of methionine and other sulphur compounds (modified from Dias and Weimer, 1998a; Bonnarme et al., 2000).
 Catabolism of Amino Acids in Cheese during Ripening 441
lyase and amino acid decarboxylase. About 25% of the
strains were capable of transaminating methionine to
-keto--methylthiobutyrate. Dehydrogenase activity
was observed to increase in the presence of glucose,
the influence of which may have been due to the sup-
ply of energy by the proton motive force generated by
lactate efflux. No methionine lyase or amino acid
decarboxylase activities were detected.
Amarita et al. (2002) studied the production of
methional in a cheese slurry system by Lc. lactis
IFPL730. The authors reported a decrease in the con-
centration of methional, and a corresponding increase
in 3-methylthiopropanol. It was concluded that the
conversion of methional to other volatile compounds,
including 3-methylthiopropanol, contributes to cheese
flavour.
Catabolism of methionine by five cheese-ripening
bacteria and five yeasts was investigated by Bonnarme
et al. (2001b). For both yeasts and bacteria,
dimethyldisulphide was the major sulphur compound
produced. In addition, the microorganisms produced
different amounts of methanethiol. G. candidum pro-
duced a low concentration of S-methyl thioacetate,
and was the only yeast with the ability to produce
S-methyl thioesters.
Berger et al. (1999) observed that G. candidum, a
fungus found commonly on the surface of smear-
ripened cheese, can produce sulphur-containing
flavour compounds; dimethyldisulphide was the main
sulphur compound produced by the ten strains stud-
ied. The majority of the strains also produced
methanethiol and dimethyltrisulphide. Four strains
which produced sulphides could generate S-methyl
thioesters. The authors concluded that G. candidum
may play an important role in development of flavour in
smear-ripened cheese. The pathways for the production
of flavourful sulphides by G. candidum were subse-
quently studied by Demarigny et al. (2000). Two path-
ways were used to produce sulphides from methionine.
The first pathway, which operated at high methio-
nine concentrations, was initiated by the action of
-demethiolase and resulted in the production of
methanethiol, dimethyldisulphide and dimethyltrisul-
phide. The other pathway operated at low methionine
concentrations and resulted in the production of
dimethylsulphide. Bonnarme et al. (2001a) found that
G. candidum cultures produced three S-methylthioesthers
(S-methylthioacetate, S-methylthiopropionate and
S-methylthiobutyrate) from methionine. It was also
found that G. candidum metabolised L-methionine
using different pathways; L-methionine demethiolating
activity was constitutive while L-methionine amino-
transferase and 4-methylthio-2-ketobutyrate demethiol-
ating activities were not.
Methanethiol can be formed from methionine by a
number of enzyme-catalysed reactions. Methanethiol-
producing capacity (MTPC) was found to varying
degrees in LAB and brevibacteria (Weimer et al.,
1997). B. linens BL2 had the highest MTPC. Cell-free
extracts had no measurable MTPC under cheese-
like conditions. Dias and Weimer (1998a) examined
the conversion of methionine to thiols by lactococci,
lactobacilli and brevibacteria. Again, MTPC was not
detected under cheese-ripening conditions (pH 5.2,
5% NaCl), using methionine as a substrate, but had
activity on cystathionine was found under such
conditions. The authors found that lactococci and
lactobacilli catabolise methionine using cystathionine
- and -lyases while Brevibacterium spp. use an
ML. According to the authors, the cystathionine
lyases from lysed lactococci make an insignificant
contribution to the production of volatile sulphur
compounds from methionine in cheese during
ripening.
Amarita et al. (2001) did not detect methionine
lyase activity in 29 strains of Lactobacillus. No cys-
tathionine lyase or methionine lyase activity was
detected in the strains of Lactococcus investigated by
Gao et al. (1998). Methionine aminotransferase activ-
ity was found to be responsible for initiating methio-
nine catabolism. The production of methanethiol
from methionine by B. linens CNRZ918 was studied
by Ferchichi et al. (1985), who found greatest
methanethiol production at pH 8 in the case of both
the rod and coccus forms of this microorganism.
MTPC decreased as the proportion of coccoid forms
increased.
Amarita et al. (2002) used a model system to study
the ability of Lc. lactis to produce methional and other
compounds from methionine. Slurries containing resting
cells and the intracellular fraction from Lc. lactis
IFPL730 showed the highest production of methional at
the outset of incubation, with a concomitant increase in
the production of 3-methylthiopropanol. Sensory analy-
sis of slurries indicated the characteristic aroma of
methional (cooked potato-like) in samples containing
-keto--methylthiobutyrate and the intracellular frac-
tion from Lc. lactis IFPL730. On extended incubation,
the intensity of methional aroma decreased but samples
developed a cheese-like aroma.
S-methylthioesters are important flavour compounds
in surface-ripened cheese. Their specific flavour depends
on their chain length and configuration (Weimer et al.,
1999). B. linens GC71 is capable of esterifying acetic,
propionic and methyl branched-chain acids with
methanethiol to produce thioesters (Lamberet et al.,
1997a). Lamberet et al. (1997b) compared the S-methyl
thioester-synthesising ability of some cheese-related
442 Catabolism of Amino Acids in Cheese during Ripening
bacteria, including coryneforms, Micrococcaceae, Lacto-
coccus and Leuconostoc. The strains of B. linens tested
had a tendency to produce branched-chain thioesters,
which was not observed in other coryneforms tested.
Lactococci were shown to have poor ability to synthesise
these compounds.
S-methyl thioacetate was produced by G. candidum,
Saccharomyces cerevisiae, Debaryomyces hansenii,
Kluyveromyces lactis and Yarrowia lipolytica (Arfi et al.,
2002). These authors also reported that L-methionine
aminotransferase, L-methionine and 4-methylthio-
2-oxobutyrate decarboxylase activities were present in
all the yeasts studied.
Lyases involved in the catabolism of methionine
Methionine--lyase or methioninase (EC 4.4.1.11) is a
PLP-dependent enzyme which catalyses the conver-
sion of methionine to -ketobutyrate, methanethiol
and ammonia (Soda et al., 1983; Tanaka et al., 1985).
The enzyme plays an important role in the bacterial
metabolism of methionine.
O Dwivedi et al. (1982) isolated a CL from E. coli
H2N CH C OH O which had a pH optimum of 9–10 and contained 1 mol
CH2 O C C OH
+ H3C SH + NH3
CH2 CH2
S CH3
CH3
Methionine α-Ketobutyrate Methanethiol Ammonia
Methionine--lyase have been isolated from several
bacteria, including Pseudomonas ovalis (Tanaka et al.,
1976), Aeromonas sp., Ps. putida (Nakayama et al.,
1984), B. linens CNRZ918 (Ferchichi et al., 1985),
B. linens NCDO739 (Collin and Law, 1989), Trichomonas
vaginalis (Lockwood and Coombs, 1991) and B. linens
BL2 (Dias and Weimer, 1998b). The optimum pH for
activity is generally 7.5–8. The enzyme is PLP-dependent
and usually composed of four identical subunits, apart
from the enzyme of Ps. ovalis which is composed of two
non-identical subunits (Tanaka et al., 1976).
The ML of Ps. ovalis can also degrade various - and
-substituted amino acids in addition to L-methionine,
and can perform both , and , elimination reactions.
The enzymes from Ps. ovalis and Aeromonas spp. can act
on derivatives of L-methionine and L-cysteine in addition
to L-methionine (Tanaka et al., 1976; Nakayama et al.,
1984). The ML of Ps. putida ICR3460 performed ,-
and -replacement reactions of L-homocysteine and its
S-substituted derivatives (Nakayama et al., 1984). The
enzyme from Trichomonas vaginalis catabolised homo-
cysteine, ethionine and methionine by ,-elimination
(Lockwood and Coombs, 1991).
The ML of B. linens BL2 is active under cheese-
ripening conditions (Dias and Weimer, 1998b). Unlike
the release of intracellular proteolytic and lipolytic
enzymes, the release of which has been shown to be
necessary for their activity in cheese, the influence of
amino acid catabolic enzymes on ripening may be
linked to the ability of the cells to resist lysis and
remain metabolically active during ripening. However,
the role of enzyme release by cell lysis in the produc-
tion of flavour compounds by amino acid catabolism is
unclear and requires more study.
The reactions catalysed by cystathionine--lyase
(CL) and cystathionine--lyase (CL) are shown in
Fig. 3. CL (EC 4.4.1.8) catalyses the conversion of cys-
tathionine to homocysteine, pyruvate and ammonia
while the products of CL are cysteine, ammonia and
-ketobutyrate (Weimer et al., 1999). Aubel et al. (2002)
studied the genetics of the CL of Lb. delbrueckii subsp.
bulgaricus NCDO1489 and concluded that its physiolog-
ical role is probably in the biosynthesis of methionine.
of PLP per enzyme subunit. The gene coding for cys-
tathionine lyase (metC) of E. coli was cloned by Laber
et al. (1996) who also constructed a strain of E. coli
which over-produced this enzyme.
The CL of Lc. lactis subsp. cremoris B78 is
reported to be a tetramer of identical ⬃40 kDa sub-
units (Alting et al., 1995). The enzyme catalyses the
,-elimination reaction but is able to catalyse the
,-reaction also. It was active at the pH and salt con-
centration of normal Gouda cheese. Unlike the ML of
B. linens BL2, lysis of cells was required for full activ-
ity. Fernandez et al. (2000) cloned and characterised
the metC gene encoding CL from Lc. lactis strains
B78 and MG1363. The proteins encoded by the
genes were similar and had high homology to other
PLP-dependent enzymes. Enzyme activities were
determined in strains which overproduced CL activ-
ity or in which this activity was deleted. Results
showed that the product of the gene metC is essential
for the degradation of cystathionine but that at least
one other lyase contributes to methionine degradation
by an , elimination. Dobric et al. (2000) reported
the nucleotide sequence of the gene for the C/L of
Lc. lactis subsp. cremoris MG1363. The enzyme was
unique as it could perform either ,- and ,-
elimination reactions on the same substrate.
Yamagata et al. (1993) studied the CYS3 gene encod-
ing the CL of Saccharomyces cerevisae. No detectable
 Catabolism of Amino Acids in Cheese during Ripening 443

O O

H2N CH C OH O C C OH NH3

Cystathionine-γ-lyase CH2 CH2

O

SH CH3
H2N CH C OH

Cysteine α-Keto butryate Ammonia

CH2

CH2
γ
S
O O
β
CH2
H2N CH C OH H3C C C OH NH3

H2N Cystathionine-β-lyase
CH C OH
CH2 O

O
CH2

Cystathionine SH

Homocysteine Pyruvate Ammonia

Figure 3 The reactions catalysed by cystathionine -lyase and cystathionine -lyase.

homology was found between the CYS3 of S. cerevisae
and the cysE gene of E. coli. H2S and methanethiol were
the only volatile sulphur compounds found during the
degradation of L-cysteine and L-methionine by CL of
Lc. lactis subsp. cremoris SK11 (Bruinenberg et al., 1997).
Lb. fermentum DT41 was isolated from the starter
for traditional Parmesan cheese. A PLP-dependent
CL was isolated from this strain by Smacchi and
Gobbetti (1998). It was composed of four identical
⬃35 kDa subunits and was optimally active at 37 °C
and pH 8. The enzyme was reported to retain activity
under cheese-ripening conditions.
A 160 kDa homotetrameric CL was purified
from Lb. reuteri DSM20016 by de Angelis et al.
(2002). The enzyme was optimally active at pH 8
and 35 °C and catalysed the conversion of a range of
amino acids, including methionine. This organism,
together with other lactobacilli, was used as an
adjunct in the manufacture of Canestrato Pugilese-
type cheese and cheeses containing an adjunct com-
posed of Lb. fermentum DT41, and Lb. reuteri DSM
20016 had the highest levels of methanethiol,
dimethyl sulphide, dimethyl disulphide and dimethyl
trisulphide.
Catabolism of Aromatic Amino Acids
Tryptophan
Pathways for the catabolism of tryptophan are shown
in Fig. 4. Gummalla and Broadbent (1999) studied
tryptophan catabolism by lactobacilli used as adjunct
cheese cultures to investigate the contribution of lacto-
bacilli to off-flavour development. The main mechan-
ism for tryptophan catabolism by Lb. casei involved its
conversion to indole lactic acid (ILA) by a series of
transamination and dehydrogenation reactions with
indole-3-pyruvic acid as the sole intermediate. The
results suggest that non-starter and adjunct lactobacilli
may have important roles in secondary reactions involv-
ing indole-3-pyruvic acid and other aromatic metabolites.
Tryptophan catabolism by B. linens BL2, under both
optimum (pH 6.5, 25 °C with agitation) and cheese-
like (pH 5.2, 15 °C, 4% NaCl) conditions, was studied
by Ummadi and Weimer (2001). At optimum tempera-
ture and pH, B. linens BL2 could degrade tryptophan
by various routes simultaneously but under cheese-
like conditions, the bacterium did not catabolise it.
Cells grown under either conditions did not show
tryptophanase, tryptophan decarboxylase or trypto-
phan 2-monooxygenase activities.
Gao et al. (1997) investigated the catabolism of aro-
matic amino acids in lactococci. They reported that the
first step in the catabolism of tryptophan by eight lacto-
coccal strains is due to aminotransferase activity. Amino-
transferase activity on L-tyrosine and L-phenylalanine
was also found.
Tyrosine
Tyrosine is a precursor of several compounds in cheese
– tyramine formed by decarboxylation, p-cresol and
phenol formed by an atypical Strecker degradation and
p-hydroxyphenyl pyruvate formed by aminotransferase
444 Catabolism of Amino Acids in Cheese during Ripening

NH3 CO2
H H
N H
N
N

Deaminase Decarboxylase
CH2 CH2
CH2
CH2 NH2
CH Tryptamine CH2
Indole-3-propionate C
C HO NH2
HO
O
O
Tryptophan

α-Keto acid
Aminotransferase
Amino acid
H
N

CH2
H
N C
C O
HO
O

Indole-3-pyruvate
CH2

CH
C
HO OH
O H
N
Indole-3-lactate

CH2

C
HO O

Indole-3-acetate

H
N

CH3
Skatole
(3-methyl-1H-indole)

Figure 4 Pathways for the catabolism of tryptophan.

activity (McSweeney and Sousa, 2000), as shown in
Fig. 5. Tyramine, a biogenic amine, can cause mon-
amine intoxication (see ‘Toxins in Cheese’, Volume 1).
Phenylalanine
Phenylmethanol, phenylethanol, phenylpropane,
methylphenyl hydroxyacetate, phenylacetaldehyde,
phenylpyruvate and phenylethyl acetate are flavour
compounds derived from phenylalanine and have been
found in cheese or model systems (Adda et al., 1982;
Dunn and Lindsay, 1985; Jollivet et al., 1992). Enzymes
involved in the breakdown of pheylalanine are amino-
transferases, L-amino acid oxidases, L-pheylalanine
ammonia lyases, L-aromatic amino acid decarboxylases
and phenylalanine dehydrogenases. Phenylpyruvate is
 Catabolism of Amino Acids in Cheese during Ripening 445

NH3 CO2
OH OH OH

Deaminase Decarboxylase
CH2 CH2 CH2
CH2 H2N CH C OH CH2
C OH O NH2
O Tyrosine
Tyramine
p -Hydroxy phenylpropionate
α-Keto acid
Aminotransferase
Amino acid

OH

CH2
O C C OH
O
p -Hydroxy phenylpyruvate
OH

CH2
HO CH C OH
OH
O OH
p -Hydroxy phenyl lactate
OH

C H
CH2 O
C OH p-Hydroxy benzaldehyde
H3 C
O
p -Hydroxy phenyl acetate p-Cresol

Figure 5 Pathways for the catabolism of tyrosine.

formed by the action of an aminotransferase and it can
be degraded to phenethanol and benzaldehyde (Fig. 6).
During the first week of Camembert ripening,
actively growing yeast cells produced phenethyl
alcohol from L-phenylalanine (Lee and Richard,
1984). Several compounds with a phenyl group were
also identified when the ability of B. linens to pro-
duce volatile compounds in liquid cultures was
examined (Jollivet et al., 1992). The compounds
included phenylmethanol, phenylethanol, phenyl-
propane and methylphenylhydroxyacetate.
The formation of benzaldehyde from phenylalanine
by Lb. plantarum is initiated by an aminotransferase
(Nierop Groot and de Bont, 1998). Phenylpyruvic acid
was an intermediate in the reaction and its conversion
to benzaldehyde was catalysed by Mn (II) which was
present at high levels in these cells (Nierop Groot and
de Bont, 1999) (Fig. 7). The low pH, low oxygen con-
centration and low temperature of cheese during ripen-
ing would not favour this chemical reaction. However,
the authors pointed out that the reaction may still make
a significant contribution because of the long ripening
time of certain cheeses. Klein et al. (2001) also
attributed the production of benzaldehyde from indole
pyruvate, which was formed by transamination of
phenylalanine by Lb. helveticus, to a chemical reaction.
446 Catabolism of Amino Acids in Cheese during Ripening

CO2
NH3

Decarboxylase
Deaminase
CH2 CH2 CH2
CH2 H2 N CH C OH CH2
C OH O NH2
O Phenylalanine Phenylethylamine
Phenylpropionate
α-Keto acid
Aminotransferase
Amino acid

CH2
O C C OH
O
Phenylpyruvate

CH2
HO CH C OH
O
Phenyl lactate
C H CH2

O CH2 CH2
Benzaldehyde
C OH OH

O
Phenylethanol
Phenylacetate

Figure 6 Pathways for the catabolism of phenylalanine.

An inducible aromatic amino acid aminotransferase
was isolated from B. linens 47 by Lee and Desmazeaud
(1985), who detected several metabolites of aromatic
amino acid catabolism, including phenylpyruvate,
phenylacetate, 4-hydroxyphenyl acetate, indolepyru-
vate and indoleacetate, in the growth medium using
HPLC. Hayashi et al. (1993) characterised an aromatic
amino acid aminotransferase from E. coli. The halo-
enzyme contained 1 mol PLP per mol and could catalyse
the transamination of a variety of neutral amino acids,
in addition to aromatic amino acids.
Two aromatic amino acid aminotransferases have
also been purified from Lc. lactis subsp. lactis S3 and
characterised (Gao and Steele, 1998). Both were active
under cheese-ripening conditions. The authors
observed that p-hydroxyphenylacetic acid (pHPA)
could be produced from tyrosine; pHPA can degrade
chemically to p-cresol. The enzymes could also pro-
duce indole-3-acetic acid from tryptophan which
could be converted to skatole. It was concluded that
further research is required to confirm the role of aro-
matic amino acids in the development of off-flavours
in cheese. An aminotransferase from Lc. lactis
NCDO763 catalysed the transamination of L-amino
acids only (Yvon et al., 1997). Using -ketoglutarate as
an amino group acceptor, the resting cells of this
organism produced phenylpyruvate, phenylacetate and
phenylactate from phenylalanine. The authors suggested
 Catabolism of Amino Acids in Cheese during Ripening 447

O
O O

HO C C OH
H2N CH C OH O C C OH

CH
CH2 Aminotransferase CH2

Metal ion/alkaline pH

Phenylalanine Phenylpyruvate Enol tautomer

Chemically
O2

COOH COOH COOH

CHO CH2 HC OH C O

Benzaldehyde Phenylacetic acid Mandelic acid Phenylglyoxylic acid

Figure 7 Proposed mechanism for benzaldehyde production by both enzymatic and chemical steps (modified from Nierop Groot
and De Bont, 1998).

that the aminotransferase could have an important
role in flavour development during ripening as it
transaminates amino acids (leucine, phenylalanine,
tyrosine, tryptophan, methionine) which are precur-
sors of aroma compounds.
Gummalla and Broadbent (2001) studied the
catabolism of tyrosine and phenylalanine by Lacto-
bacillus adjuncts. It was observed that in the two
Lb. casei and two Lb. helveticus strains examined, the
specific activity of phenylalanine aminotransferase
was higher than that of tyrosine aminotransferase.
The strains were incubated under conditions similar
to those in cheese during ripening to assess catabol-
ism. No decarboxylase activity was detected during 20
days of incubation but aminotransferase and dehydro-
genase activities were observed. Micellar electokinetic
capillary chromatography was used to show that the
four lactobacilli catabolised tyrosine to produce
p-hydroxyphenyl lactic acid and p-hydroxyphenyl
pyruvic acid under conditions similar to those in
ripening cheese. Phenyl lactic acid, phenyl acetic acid
and benzoic acid were produced from phenylalanine
under conditions similar to those in cheese.
Catabolism of Branched-Chain Amino
Acids
Branched-chain amino acids are degraded by amino-
transferases to -keto acids (Fig. 8). The aminotrans-
ferase of Lc. lactis subsp. cremoris NCDO 763 was most
448 Catabolism of Amino Acids in Cheese during Ripening

O O

H2N CH C OH O C C OH

CH2 CH2

CH CH3
A CH CH3
M
CH3 CH3

Leucine
I
α-Ketoisocaproate
N
O
O O
T
H2 N CH C OH C C OH
R O

CH CH3
A CH CH3

CH2
N CH2

CH3
S CH3

α-Keto-β-methylvalerate
Isoleucine
F
E
O R O

H2N CH C OH A O C C OH

CH CH3
S CH CH3

CH3
E CH3

α-Keto-isovalerate
Valine

Figure 8 Transamination of the branched-chain amino acids to their corresponding -keto acids.

active on leucine, although it was also, but less, active on
the aromatic amino acids (Yvon et al., 1997), while that
of Lc. lactis subsp. cremoris B78 catalysed the transam-
ination of valine, isoleucine and leucine (Engels,
1997). A branched-chain aminotransferase from Lc. lac-
tis subsp. cremoris NCDO 763 was characterised by
Yvon et al. (2000). The enzyme catalysed the transamin-
ation of the three branched-chain amino acids and was
active under cheese-ripening conditions, although it
had pH and temperature optima of 7.5 and 35–40 °C,
respectively. Since the enzyme has a role in the degrada-
tion of isoleucine and valine, as well as in the transamin-
ation of leucine and methionine and was active under
conditions similar to those found in cheese during
ripening, the authors concluded that the enzyme was
involved in flavour development. The branched-chain
aminotransferase of Lc. lactis LM0230 has been cloned
and sequenced (Atiles et al., 2000). The enzyme has
broad specificity, being active on isoleucine, leucine,
valine, methionine and phenylalanine.
Lb. paracasei subsp. paracasei LC01 produces at least
one aminotransferase, capable of transaminating
branched-chain amino acids, which was most active on
isoleucine and leucine (Hansen et al., 2001). Response-
surface methodology showed that leucine concentration
had a negligible effect on aminotransferase activity, while
too high a concentration of -ketoglutarate could inhibit
the enzyme.
Ayad et al. (2001a) studied the effects of combining
selected lactococci on flavour formation in milk. A
chocolate-like flavour was produced by a combination of
Lc. lactis subsp. cremoris NIZO131157 and Lc. lactis
subsp. cremoris SK110. The authors speculated that this
flavour was due to branched-chain aldehydes produced
from branched-chain amino acids. Subsequently, Ayad
et al. (2001b) studied the flavour-generating ability of
wild lactococci isolated from artinsanal and non-dairy
sources (fermented raw goats’, sheep’s and cows’ milk, as
well as from soil, grass, silage and the udder) in milk and
in a cheese model. The authors believed that these wild
 Catabolism of Amino Acids in Cheese during Ripening 449
strains may be able to produce more flavour compounds
in cheese than the industrial strains currently used in
cheesemaking. The majority of wild strains produced dif-
ferent flavours from industrial strains. Methylated alco-
hols and methylated aldehydes, probably produced from
branched-chain amino acids, were the main volatile com-
pounds formed. It was concluded that wild strains could
be used for the development of new cheeses or to alter
the flavour of existing types of cheese. However, since
the non-dairy wild strains had no proteolytic activity,
they would be unable to grow in and acidify cheese milk
and would have to be combined with industrial starters.
The catabolism of leucine by propionic acid bacteria
was investigated by Thierry et al. (2002). P. freudenreichii
catabolised leucine to -ketoisocaproic acid, but only if
-ketoglutarate was present. The bacterium also con-
verted -ketoisocaproic acid to isovaleric acid via oxida-
tive decarboxylation by -ketoacid dehydrogenase
activity yielding an acyl-CoA derivative which was then
converted to the acid. The authors noted that the catab-
olism of branched-chain amino acids by P. freudenreichii
was different to the catabolism of branched-chain
amino acids by lactococci.
Deaminases
There are two types of deamination involving redox
reactions (Hemme et al., 1982), differing according to
the nature of hydrogen acceptor:
• Dehydrogenases (EC 1.4.1) which utilise NAD as
the co-enzyme. The general reaction catalysed by
these enzymes is:
L-amino acid  H2O  NAD : -keto acid
 NH4  NADH
These reactions can produce compounds such as
pyruvic acid and -ketoglutaric acid from alanine
and glutamic acid, respectively.
• Oxidases which use oxygen as hydrogen acceptor.
L-amino acid oxidases (EC 1.4.3.2) produce -keto
acids according to the following reaction:
L-amino acid  O2 : -keto acid  NH3  H2O2
L-amine oxidases (EC 1.4.3.6) form aldehydes
according to the reaction:
Amine  O2 : aldehyde  NH3  H2O2
Ammonia, a product of these deamination reac-
tions, is an important constituent of the flavour of
cheeses such as Camembert, Gruyère and Comté and
contributes to an increase in pH during ripening
(McSweeney and Sousa, 2000). Microorganisms from
the smear surface have deaminating ability, e.g., G. can-
didum (see Fox and Wallace, 1997), while B. linens
produces large quantities of ammonia from serine, glu-
tamine, asparagine and threonine. However, most
strains of coryneform bacteria from smear cheese were
found to have low deaminating activity except on ser-
ine, glutamine and asparagine (Hemme et al., 1982).
Williams et al. (2001) studied the deaminating ability
of LAB isolated from mature Cheddar. Deaminase
activity was not widespread in the isolates but this
may have been due to the insensitivity or lack of speci-
ficity of the assay method used.
Decarboxylases
Decarboxylation is the conversion of an amino acid to
the corresponding amine with the removal of CO2.
Decarboxylases generally have an acid pH optimum
(⬃pH 5.5) and usually require PLP as a coenzyme
(Hemme et al., 1982). Amines generally have strong and
often unpleasant aromas, as evident in certain smear-
ripened cheese types (Fox and McSweeney, 1996). In
addition, many amines (e.g., tyramine, histamine, trypt-
amine, putrescine, cadaverine and phenylethylamine)
cause adverse physiological effects (‘biogenic amines’;
see ‘Toxins in Cheese’, Volume 1). The relative concen-
tration of amines in cheese depends on the type of
cheese and its microflora (McSweeney and Sousa,
2000). The relative concentration of some amines does
not compare with that of the parent amino acid, which
may be due to differences in the rates of conversion of
amino acids (Adda et al., 1982). Most amines in cheese
can be formed by decarboxylation, as is the case with
the production of tyramine from tyrosine and hista-
mine from histidine. However, the formation of sec-
ondary and tertiary amines cannot be explained readily
(Fox and McSweeney, 1996).
Joosten (1988) studied factors that affect the con-
centrations of biogenic amines formed in cheese. It
was observed that in Gouda cheese, a higher pH, com-
bined with a storage temperature of 21 °C, caused an
increase in concentration of histamine, as did low salt-
in-moisture. Starter type and pasteurisation of milk
did not appear to affect the formation of histamine.
The role of non-starter bacteria in the formation of
biogenic amines in cheese was examined by Joosten
and Northolt (1987) who investigated the decarboxy-
lase activity of bacteria including lactobacilli, entero-
cocci, enterobacteriaccae and pediococci. Some strains
of lactobacilli could form biogenic amines in cheese.
Since the number of enterococcal cells required to pro-
duce significant amounts of tyramine is rarely reached
450 Catabolism of Amino Acids in Cheese during Ripening
in cheese, these bacteria are not important for amine
formation in Dutch cheese, although this may not be
true for certain artisanal cheeses for which enterococci
are a major part of the starter. The authors concluded
that non-starter lactobacilli were the most important
agents in Dutch cheese for the formation of biogenic
amines. This is in agreement with the findings of
Broome et al. (1990) who reported that the concentra-
tions of tyramine and histamine in cheeses inoculated
with lactobacilli were twice as high as in control
cheeses, indicating that decarboxylases of lactobacilli
have a role in their production.
Novella-Rodríguez et al. (2002a) studied the effect
of defined-strain starters on the production of amines
in goats’ milk cheese during ripening. The main
amines found were tyramine (94.59 mg kg 1),
putrescine and tryptamine. The effect of high hydro-
static pressure on the production of amines in goats’
milk cheese was studied by Novella-Rodríguez et al.
(2002b) who found maximum production of amines
when the cheeses were treated at 50 MPa for 72 h;
rates of production were lower when cheeses received
higher pressure treatments (400 MPa for 5 min or 400
MPa for 5 min followed by 50 MPa for 72 h) and in
the untreated control cheeses.
In addition to being involved in the production of
amines, B. linens is able to reduce the amounts of his-
tamine and tyramine in cheese during ripening
(Leuschner and Hammes, 1998). During the four
weeks of ripening of Münster cheese, B. linens reduced
the histamine and tyramine content by 55–70%.
Degradation of amines occurs at the surface of the
cheese but the concentration of amines on the surface
and interior differed only slightly after inoculation
with B. linens LTH456. It was suggested that the
concentration gradient was removed by diffusion of
amines, leading to a decrease in the concentration of
biogenic amines in the interior of the cheese.
Lactobacilli used as cheese starter adjuncts were
incubated by Gummalla and Broadbent (1999) in a
defined medium containing L-tryptophan under carbo-
hydrate starvation (CS), or under near-cheese ripening
conditions (a chemically defined medium containing 4%
salt, at pH 5.2). The specific activity of the tryptophan
decarboxylases from Lb. casei strains was lower than
those of the corresponding enzymes from Lb. helveticus
strains. Generally, activity in either strain did not vary
significantly with time or incubation conditions.
Twenty-two Lb. plantarum strains and seven strains of
Lb. casei had no decarboxylase activity on methionine
(Amarita et al., 2001).
The combined effects of temperature, pH and salt on
the growth of E. faecalis EF37, its proteolytic activity and
its ability to produce biogenic amines were studied by
Gardini et al. (2001) who observed that 2-phenylethyl-
amine accounted for more than half of the total content
of biogenic amines. The production of biogenic amines
was found to be independent of the incubation tempera-
ture and in general, was very low at the higher NaCl
concentration and was increased by lower pH.
Roig-Sagues et al. (2002) studied the ability of 694
strains of bacteria isolated from Spanish artisanal
cheeses to produce histamine and tyramine. Tyramine-
forming activity (mainly by enterococci and some
other LAB) was found more frequently than hista-
mine-forming activity, which was formed mainly by
enterobacteria, but also by small numbers of other
LAB. Most of the tyramine-forming strains of LAB
were isolated from cheeses containing the highest levels
of tyramine. However, histamine-forming LAB were
generally isolated from samples with a low level of his-
tamine. The amount of tyramine found in the samples
was significantly higher than that of histamine.
The distribution of aromatic L-amino acid decar-
boxylases in 326 bacteria (four species of E. coli,
Erwinia herbicola, Serratia plymuthicum, two species of
Proteus, Alcaligenes faecalis, Bacillus natto, Achrombac-
ter hartlebii, 11 species of Micrococcus, one Staphylococ-
cus, three Sarcina spp., Brevibacterium ammoniagenes,
Bacterium cadaveris and three Pseudomonas spp.) was
studied by Nakazawa et al. (1977). Micrococcaceae
were observed to have the highest decarboxylase
activity on L-tryptophan, S-hydroxy-L-tryptophan and
L-phenylalanine. The amino acid decarboxylase of
M. percitreus was reported by this author to be involved
in synthesis of aromatic amines such as dopamine and
tyramine.
A histidine decarboxylase, which did not require
PLP as a coenzyme, has been purified from Lactobacil-
lus 30a (Chang and Snell, 1968). The substrates of the
enzyme were found to have a heterocyclic nitrogen
atom at the same position relative to its alanyl side
chain which may be important in the formation of
the enzyme–substrate complex. Jetten and Sinskey
(1995) studied a decarboxylase isolated from a strain
of Corynebacterium glutamicum with activity on
oxaloacetate. The enzyme, which catalysed the decarb-
oxylation of oxaloacetate only, a key intermediate in
carbon metabolism, had optimum activity between pH
7.0 and 7.5. A glutamate decarboxylase was isolated
from Lb. brevis IFO 12005 by Ueno et al. (1997) and
was found to be a dimer. Temperature and pH optima
were 30 °C and 4.2, respectively. The enzyme could
not decarboxylate any other amino acid assayed.
Lucas and Lonvaud-Funel (2002) purified the tyro-
sine decarboxylase of Lb. brevis IOEB 9809. The enzyme
had features typical of pyridoxal phosphate-dependent
amino acid decarboxylases although this enzyme was
 Catabolism of Amino Acids in Cheese during Ripening 451

not related by sequence homology to any known tyro- Lactic acid bacteria isolated from wine can
sine decarboxylase. catabolise arginine by at least two pathways (Arena
et al., 1999). The arginine deiminase pathway pro-
duces orthinine, CO2 and NH3 via three enzymatic
Catabolism of Other Amino Acids
reactions. The enzymes involved in this pathway
Goux et al. (1995) investigated aspartate catabolism in are arginine deiminase (EC 3.5.3.6), catabolic ornithine
an effort to understand ammonia generation by E. coli. transcarbamoylase (EC 2.1.3.3) and carbamate kinase
It was reported that arginine may be an intermediate (EC 2.7.2.2) (Champomier Verges et al., 1999). Alter-
in aspartate catabolism, and may also be an intermedi- natively, the arginase–urease pathway leads to the pro-
ate for ammonia production from aspartate during duction of urea. Arginine deiminase, ornithine
nitrogen-limited growth. Hayashi et al. (1993) com- transcarbamoylase and carbamate kinase from the sour-
pared an aspartate aminotransferase with the aromatic dough microorganism, Lb. sanfranciscencis CB1, were
amino acid aminotransferase of E. coli. Both enzymes isolated by de Angelis et al. (2003). The enzymes had
were composed of two identical ⬃43.5 kDa subunits, acidic pH optima and were optimally active at 30–37 °C.
and contained one molecule of PLP per subunit. An Interestingly, arginine has been proposed as a pos-
aspartate aminotransferase isolated from Lc. lactis sible growth substrate for the secondary microflora of
LM0230 was cloned and characterised by Dudley and Swiss cheese (Laht et al., 2002); calculations showed
Steele (2001). It was determined using homologous that ATP available from the metabolism of arginine
recombination that a mutation in the Asp biosynthetic to ornithine was theoretically sufficient to support
pathway prevented this strain from growing in milk. the growth of non-starter bacteria to populations of
According to Kaneoke et al. (1993), at least seven 108 cfu g 1.
L-arginine degradation pathways are known, and in The catabolism of threonine, asparagine, arginine and
some species, more than one of these pathways can be glutamate in cheese has attracted some study. Amino-
operational. These authors studied the arginine oxygen- transferase or dehydrogenase activities catabolise gluta-
ase pathway in two coryneforms, Arthrobacter globi- mate to produce -ketoglutarate, while -aminobutyrate
formis IFO 12137 (ATCC 8010) and B. helvolum IFO is formed from glutamate by the action of a decarboxy-
12073. This pathway involves four enzymes and pro- lase. Threonine is converted to acetaldehyde and
duces succinate from L-arginine (Fig. 9). This pathway glycine (McSweeney and Sousa, 2000). The specific
in the coryneforms studied is not identical to other pathways for the catabolism of other amino acids (e.g.,
pathways reported, e.g., the pathways of Pseudomonas glycine, alanine and serine) by cheese-related micro-
aeruginosa and Streptococcus faecalis. E. coli utilises the organisms have attracted little attention.
ammonia-producing succinyl transferase pathway for
arginine catabolism, and to a lesser degree, the argin-
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 Sensory Character of Cheese
and its Evaluation
C.M. Delahunty, Department of Food and Nutritional Sciences, University College
Cork, Ireland
M.A. Drake, Department of Food Science, Southeast Dairy Foods Research Center,
North Carolina State University, Raleigh, North Carolina, USA
Introduction
A remarkable variety of cheeses are made in all parts of
the world where milk is produced. Cheeses are con-
sumed for their highly regarded nutritional value, and
enjoyed for their complex and varied eating quality. The
sensory characteristics of cheeses, which determine their
eating quality, are properties that are perceived by the
human senses, predominantly during consumption.
These properties can be described as appearance charac-
teristics, flavour characteristics and texture character-
istics. However, cheeses are complex foods, produced
using milk from different animals, by many different
techniques, and are presented in a variety of sizes,
shapes, packages or coatings. Some cheeses are pro-
duced in small quantities, such as farmhouse types, sold
in local markets and consumed by a relatively small
number of people. Others are produced in large quanti-
ties in very large automated facilities, may find their way
to markets in many different countries and are con-
sumed by very many people. Some cheeses are ripened
or matured for years before they are consumed; others
are consumed young or unripened. Cheeses may have
moulds of different types growing on their surface, may
be pierced to allow blue moulds grow within the cheese,
or include ingredients such as herbs and/or spices. This
considerable diversity in cheesemaking practice, and the
number of stages that any single cheese undergoes dur-
ing its production, results in a wide variety of cheeses
each of which has complex sensory characteristics. Sens-
ory evaluation of cheese is absolutely necessary to
determine the relative merits of cheesemaking proced-
ures and the influence of measured composition on
specific sensory characteristics of cheese. Sensory evalu-
ation is also needed to determine the influence of sens-
ory characteristics on the eating quality of cheese and
its consumer acceptability. However, the complexity of
cheese presents a considerable challenge for its sensory
evaluation. This chapter will focus on human perception
of sensory characteristics, on the advantages and disad-
vantages of sensory evaluation methods, on the intensity
Cheese: Chemistry, Physics and Microbiology, Third edition – Volume 1: General Aspects
ISBN: 0-1226-3652-X
Set ISBN: 0-1226-3651-1
and quality of the sensory characteristics of cheeses, and
on the relationships between cheesemaking, cheese
composition, cheese sensory characteristics and con-
sumer acceptability of cheese.
A Definition of Sensory Character
Sensory characteristics of cheeses are human
responses to perceptions of stimuli that are experi-
enced with the cheeses, and can generally be described
using terms defined within the categories of appear-
ance, flavour and texture. Sensory characteristics
result from interactions of the human sensory modali-
ties of vision, touch, olfaction, gustation and mouth-
feel with stimuli induced by rheological, structural
and chemical components of the cheese. Sensory
characteristics are perceived by consumers when they
observe, manipulate, smell and take cheese into the
mouth for consumption, and are subsequently
expressed as a behavioural response using actions or
descriptive terms. A majority of sensory characteristics
are complex and are stimulated by the association of
many different properties of the cheese, with different
sensory modalities acting together. It is this complex-
ity, or component balance, that hinders attempts to
adequately represent cheese sensory character using
instrumental or chemical analyses. In addition, and
unfortunately from the sensory scientists’ point of
view, consumers differ from one another. Sensory
perception, and particularly its communication, differs
between individuals as a result of physiological, psy-
chological, social and cultural differences.
Sensory Characteristics and Cheese
Preferences
Cheese quality has been defined for many years by
manufacturers as cheese produced reliably and econom-
ically (Muir et al., 1995a). In the past, limited choices
were available to consumers and as a result of this
Copyright © 2004 Elsevier Ltd
All rights reserved
456 Sensory Character of Cheese and its Evaluation
limited experience, the consumer’s palate was less dis-
cerning. Today, cheese markets are international, and
cheesemakers compete openly for consumers, offering
them an ever-widening choice. Cheese consumers are
more affluent and many have tasted or regularly con-
sume a diversity of cheese types, leading them to
become increasingly discerning. These consumers
now define the quality standard for cheeses, which is
ultimately determined by eating quality.
The eating quality of cheese, or a consumer’s liking
for cheese, is an integrated response. The stimuli are the
sensory characteristics, perceived before and during
consumption. However, the response is influenced by
other individual consumer-related factors that include
sensory abilities, past experiences with cheese, what is
expected from a cheese and when and where it will be
consumed. Expectations are based on experience, but
are created for a specific product by marketing, packag-
ing and familiarity. Finally, liking for, and satisfaction
with, a cheese is determined by the context in which it
is consumed, and its appropriateness for that context
(e.g., would one wish to consume Epoisses for break-
fast?). Eating quality determines consumer acceptability
and willingness to repeat purchase. Highly regarded eat-
ing quality is not that found in a cheese with no defects,
but that which offers unique and appealing characteris-
tics consistently. The producer of the cheese with the
most acceptable sensory characteristics, if he is aware of
this and can ensure that the market presentation of his
cheese matches its sensory character, will have an
advantage in the market.
The concepts of ‘healthy eating’ and ‘conscientious
eating’ (e.g., vegetarian and vegan diets) and environ-
mentally friendly eating are now increasingly important
to consumers. To meet these consumers’ expectations,
cheese producers are challenged to produce new,
wholesome products that taste as good as traditional
alternatives. This task is proving difficult as dietary
guidelines for healthy eating may recommend reducing
the intake of ingredients that provide desirable sensory
character, such as fat or salt. The production of
reduced- and low-fat cheeses to replace traditional
types is an example of such consumer-driven product
development. However, the majority of new low-fat
cheeses do not meet the sensory quality requirements of
discerning consumers (Mistry, 2001). This is because
fat is not just a provider of desirable sensory character,
but it is also important for cheese texture and body, for
the development of compounds responsible for flavour
and for the release of flavour compounds during con-
sumption. It will be difficult to improve the eating
quality of these cheeses unless eating quality is under-
stood better.
Cheesemaking and the Variety of Sensory
Character
The sensory characteristics of a cheese at the time of its
consumption reflect the milk from which it was pro-
duced (e.g., a goats’ milk cheese is distinct from a cows’
milk cheese), the processes used in its production and
the physical and the chemical changes that occurred
during maturation (e.g., proteolysis breaks down pro-
teins to amino acids during cheese maturation, which
may subsequently act as substrates for the formation of
volatile compounds (see ‘Catabolism of Amino Acids in
Cheese during Ripening’, Volume 1)). Milk from the
cow, sheep, goat, buffalo or other animals can be used as
raw material, and its qualities are determined by breed,
diet and stage of lactation. Treatment of milk before
cheese production, particularly pasteurisation, can kill
micro-organisms and reduce enzyme activity that could
otherwise contribute to the development of sensory
character during maturation. During cheese production,
the coagulant used to form curds, the amount of salt
added, the type of starter culture and the use of adjunct
cultures will determine sensory characteristics. Finally,
the maturation time and the temperature of maturation
may be varied. The sensory characteristics of different
types of cheese, and the potential variety that may be
achieved, are determined by the choices the producer
makes at each of the stages in production.
Sensory characteristics of many different cheeses
are described in the literature and in specialist cheese
books. However, the sensory characteristics of rela-
tively few types have been defined, standardised and
measured objectively using sensory science methods.
Lack of objective knowledge makes it difficult to com-
pare accurately the sensory characteristics of different
cheese types, but more importantly, as the cause of sens-
ory characteristics is only partially known, it is diffi-
cult to compare accurately cheese appearance, texture
and flavour research carried out in different laborator-
ies. Tables 1 and 2 present terms used to describe the
appearance, texture and flavour characteristics of
cheeses that have been defined and standardised in an
objective way. Table 3 presents terms used for descrip-
tive sensory analysis by other researchers, but that
have not been defined and standardised adequately.
Similar terms are used in many cases even though each
descriptive language referenced was developed inde-
pendently by different research groups. In addition, in
many cases, similar terms have been used to describe
dominant characteristics of different cheese types. This
comparison suggests that even though a remarkable
variety of cheese types are produced, that potentially
exhibit a wide variety of sensory characteristics, it should
 Sensory Character of Cheese and its Evaluation 457

Table 1 Terms used to describe the appearance and texture of cheese using descriptive analysis methods. Terms in this list were
developed and defined by trained panels, and in many cases standard materials that help to illustrate the term are provided.
Cheeses studied were low-fat, full-fat and smoked Swiss, Cheddar and Gouda (Adhikari et al., 2003), natural and processed
cheeses (Drake et al., 1999a; Gwartney et al., 2002), ten different types of cheese (Lawlor and Delahunty, 2000), Cheddar and
Camembert (Cooper, 1987) and Mozzarella cheeses (Brown et al., 2003)

Term Definition a

Appearance
Chalky Resembling chalk in appearance
Colour/colour intensity The colour of Cheddar ranging from pale yellow to orange, the palest yellow representing
the start of the scale
The colour of cheese ranging from white to orange
Mottling The evenness of colour shading within the cheese sample, with the most uniform coloured
cheese being free from mottling, marbling or any other deficiencies in colour
Mouldy The degree of mouldiness/visible mould growth in the cheese structure
Open/openness The extent to which the interior of the cheese (that is the cut surface) is open, this
encompasses cracks, pinholes, irregular-shaped holes and any other openings
Shiny The extent to which the surface of the cheese is shiny, glossy, moist or sweaty-looking, as
opposed to looking matt or dull
Texture
Adhesiveness The degree to which the chewed mass sticks to mouth surfaces, evaluated after five chews
Chewy Requiring a good deal of mastication, toffee-like texture. Degree of chewing needed
to break up the cheese
Cohesiveness The degree to which the chewed mass holds together, evaluated after five chews
Creamy/creaminess The extent to which the texture has broken down to a creamy semi-liquid texture, assessed
between tongue and palate during mastication
The feeling associated with heavy whipping cream (e.g., 30% fat content)
Crumbly/crumbliness The extent to which the cheese structure breaks up in the mouth, assessed during the
first 2–3 chews
The feeling in the mouth when the sample falls apart quickly in mouth during mastication
Crustiness The force required to break through the crust of the cheese when taking the first bite, assessed
using the front teeth
Curdiness The extent to which a curdy or mealy texture is perceived in the mouth during mastication
Degree of breakdown The amount of breakdown that occurs in the sample as a result of mastication, evaluated
after five chews
Dry The degree of dryness or moistness sensed in the mouth during mastication
Firm/firmness Ranging from soft to firm. The extent of resistance offered by the cheese, assessed during
the first five chews using the front teeth
The force required to squeeze a cube (1.5  1.5  1.5 cm) of cheese flat between the first
finger and thumb
The amount of force required to take the first bite of cheese, assessed using the front teeth
The amount of force required to completely bite through the cheese, assessed using the molars
First-bite sticky Sticky sensation experienced during the first bite
Fracturability at first bite Completely bite through the sample with the molars and evaluate the degree to
which the sample fractures
Grainy The extent to which granular structures are formed as the sample breaks down, perceived
in the second half of chewing
The feeling of coarse particles in the mouth during mastication
Hardness The force required to bite the sample (first bite)
Mealy The feeling in mouth when the sample breaks down in small pieces and it is difficult to
gather them for swallowing
Moist The perceived moisture content of the cheese. Ranging from dry to moist
The extent to which the cheese has a moist or wet texture around the palate during mastication
Mouth-coating The extent to which the cheese coats the palate and teeth during mastication
The degree of coating on the tongue and the palate during mastication
Oily Oily, fatty, greasy mouth-feel of any kind
Rate of recovery Depress sample between thumb and first finger 30%, evaluate the speed or rate at which
the sample returns to its original shape
Residual mouthfeel The degree of ‘bittiness’ or graininess in the mouth just before swallowing
Residual smoothness The degree of smoothness felt in the mouth after expectorating the sample
of mouth coating

continued
458 Sensory Character of Cheese and its Evaluation

Table 1 continued

Term Definition a

Rubbery/rubberiness The extent to which the cheese returns to its initial from after biting, assessed
during the first 2–3 chews
The degree of springiness experienced while biting the sample
Slimy Of the nature of slime, soft, glutinous or viscous substance, soft, moist and sticky
Smooth/smoothness The smoothness of the cheese against the palate as it breaks up during mastication
The degree to which the chewed mass surface is smooth, evaluated after five chews
Softness Yielding easily to pressure, easily moulded, pliable, easily spreadable
Springiness Depress sample between thumb and first finger 30%, evaluate the total amount
of recovery of the sample
Sticky/stickiness The stickiness of the cheese against the palate and around the teeth during mastication
Overall sensation of stickiness during mastication
Viscous The mouthfeel associated with consuming very viscous fluids like heavy whipping
cream or honey

a The precise wording of some definitions has been changed to allow the use of consistent language in this table. However, the
meaning of each definition is unchanged.

Table 2 Terms used to describe the flavour of cheese using descriptive analysis methods. Terms in this list were developed,
defined and referenced using standard materials by trained panels. Cheeses studied were: Cheddar (Murray and Delahunty, 2000b;
Drake et al., 2001), low-fat, full-fat and smoked Swiss, Cheddar and Gouda (Adhikari et al., 2003), aged natural cheese of many
types (Heisserer and Chambers, 1993), ewes’ milk cheese (Bárcenas et al., 1999) and cheese flavours (Stampanoni, 1994)

Term Definition a Standard b, c

Acid/yoghurt, acidic The taste on the tongue associated with
acids (citric, lactic... )
A sour, tangy, sharp, citrus-like taste. The
fundamental taste sensations of which
lactic and citric acids are typical
Age Flavours indicating age in Cheddar cheese
Ammonia – Ammonia solution (0.25% in water)
Animal, animalic The combination of aromatics reminiscent
of farm animals and barnyards
Astringent The complex of drying, puckering, shrinking
sensations in the oral cavity causing
contraction of the body tissues
A mouth-drying and harsh sensation
Balanced Mellow, smooth, clean. In equilibrium, well-
arranged or disposed, with no constituent
lacking or in excess
Bell pepper Aroma associated with freshly cut
green peppers
Biting The slightly burning, prickling and/or
numbness of the tongue and/or
mouth surface
Bitter Fundamental taste sensation of which
caffeine or quinine are typical
A chemical-like taste
Blue – Octan-2-one (1% in PG)
Brine The combination of aromatics associated
with the saturated brine used during
traditional ewes’ milk cheesemaking
Brothy Aromatics associated with boiled meat or
vegetable stock soup
0.35–0.86 g lactic acid/100 g Ricotta
Fermented milk
Natural yoghurt
Citric acid (0.2% in water)
Aged Cheddar cheese (1 yr or older)
4-Methyl-octanoic acid (2% in PGd)
1-Phenyl-2-thiourea (5000 mg/kg in PG)
Alum (0.1% in water)
Tea, six bags soaked in watere for 3 h
Tannic acid (0.05% in water)
Mild Cheddar
Methoxy pyrazines (5 g/kg)
Freshly cut bell pepper
Horseradish sauce
Caffeine (0.02, 0.06 or 0.08% in water)
Tonic water, quinine (0.01% in water)
Ewes’ milk cheese brine at room temperature
Canned potatoes
Low-sodium beef broth cubes
Methional (20 mg/kg)
 Sensory Character of Cheese and its Evaluation 459

Table 2 continued

Term Definition a Standard b, c

Butter milk – Pasteurised butter milk

Buttery Fatty, buttery tasting, of the nature of, Unsalted butter
or containing butter Lightly salted butter
The aromatics commonly associated with Pasteurised cooking butter
natural, fresh, slightly salted butter Diacetyl (1% in PG)
Aroma rising from butter at room temperature Diacetyl in vaseline oil (several
concentrations)
Butyric, butyric acid Sour flavour, similar to baby vomit Butyric acid, 2500 mg/kg in vaseline oil SSf.
The aromatics reminiscent of baby 2 ml SS  cotton in 60-ml flask
vomit; is sour and cheesy Butyric acid (10 000 mg/kg in PG)
Butyric acid (1% in PG)
Capric acid – Capric acid (pure)
Caramel The taste and aromatics associated Condensed milk
with burnt sugar or syrup; toffee 3-Hydroxy-2-methyl-4-pyrone (2% in PG)
made from sugar that has been
melted further
Caseinate – Sodium caseinate powder
Catty Aroma associated with tom-cat urine 2-Mercapto-2-methyl-pentan-4-one (20 mg/kg)
Cheddary The taste and aromatics associated Processed cheese
with typical Cheddar Mature Cheddar cheese
Typical aroma and taste of sharp/mature
Cheddar cheese
Cheese rind – Cheese rind (Tilsit mild, pasteurised full fat)
Cooked, cooked milk Aromatics associated with cooked milk Skim milk heated to 85 °C for 30 min
The combination of sweet, brown flavour Evaporated milk
notes and aromatics associated with UHT milk 3.6% fat, cooked for 10 min
heated milk
Cottage cheese – Cottage cheese 25% fat
Cowy/phenolic Aromas associated with barns and p-Cresol (160 mg/kg), bandaids
stock trailers, indicative of animal
sweat and waste
Creamy Fatty, creamy tasting, of the nature of, Mascarpone cheese
or containing cream -Decanolactone (0.1% in PG)
UHT Cream 35% fat
Dairy fat The oily aromatics reminiscent of milk Whipping cream
or dairy fat Unsalted butter
Dairy sour The sour aromatics associated with dairy- Sour cream
soured products
Dairy sweet The sweet aromatics associated with Vitamin D milk
fresh dairy products
Decaying animal The aromatics reminiscent of decaying Dimethyl disulfide (bottom notes only;
animal material 10 000 mg/kg in PG)
Diacetyl Aromatics associated with diacetyl Diacetyl (20 mg/kg)
Earthy – Geosmin (0.001% in PG)
Fatty – Palm kernal fat
Faecal Aroma associated with complex Indole, skatole (20 mg/kg)
protein decomposition
Fermented – Fermented milk, 12% fat
Fermented fruity / winey The combination of aromatics Burgundy cooking wine
reminiscent of red wine in general; it
is sweet, slightly brown, overripe
and somewhat sour
Flavour intensity The overall intensity of flavour in the sample,
from mild to strong
Free-fatty acid Aromatics associated with short chain Butyric acid (20 mg/kg)
fatty acids
Fresh fish The aromatics associated with fresh fish Elodea (an aquatic plant) growing in water

continued
460 Sensory Character of Cheese and its Evaluation

Table 2 continued

Term Definition a Standard b, c

Fruity The taste and aromatic blend of
different fruity identities
The aromatics associated with
different fruits
Goaty The aromatics reminiscent of wet animal
hair; it tends to be pungent, musty
and somewhat sour
Green-grass – cis-3-Hexenol (1% in PG)
Methyl ketone / blue Aroma associated with blue-vein cheeses
Milkfat / lactone Aromatics associated with milkfat
Milky The aromatics commonly associated
with ewes’ raw milk
Mouldy, mouldy/musty The combination of tastes and aromatics
generally associated with moulds; they
usually are earthy, dirty, stale, musty
and slightly sour
Aromas associated with moulds and/or
freshly turned soil
Mushroom The taste and aromatics associated with
raw mushrooms
Musty Aroma of a damp room or very old book
Nutty The aromatics reminiscent of several
dry fruits such as pecans, walnuts
and hazelnuts
The non-specific nut-like taste and
aromatics characteristic of several
different nuts, e.g., peanuts,
hazelnuts and pecans
The nut-like aromatic associated with
different nuts
Overall intensity Strength of the stimuli perceived
by the nose
Strength of global stimuli originated
by the volatiles released during masti-
cation and perceived on the olfactory
receptors via the retronasal route
Oxidised Aroma associated with
oxidised fat
Pineapple The fruity aromatic associated
with pineapple
Canned fruit salad (in syrup)
trans-2-Hexenal (10 000 mg/kg in PG)
Canned fruit cocktail juice
Fruit of the forest yoghurt
Ethyl butyrate (0.1% in PG)
trans-2-Hexenal. 300 mg/kg in vaseline
oil  SS. 3 ml SS  cotton in 60-ml flask
Fresh pineapple
Ethyl hexanoate (20 mg/kg)
Hexanoic acid (5000 mg/kg in PG)
2-Octanone (40 mg/kg)
Fresh coconut meat
Heavy cream
-Dodecalactone (40 mg/kg)
Ewes’ milk raw
Pasteurised milk, 3.6% fat
2-Ethyl-1-hexanol (10 000 mg/kg in PG)
2-Ethyl-1-hexanol (40 mg/kg)
Stilton cheese
2,4,6 Trichloroanisole (1% in PG)
Button mushroom (raw)
Brown mushrooms (chopped, raw)
1-Octen-3-ol (0.5% or 1% in PG)
3-Octanol (10 000 mg/kg in PG)
3-Octanol. 5–10 mg/kg in vaseline
oil  SS. 3 ml SS  cotton in 60 ml flask
Cola infusion in ethanol (pure)
Damp room
Very old book
Wheat germ
2 g Walnuts  2 g hazelnuts, minced in 60-ml
flask (mixed particulates to be sampled)
Mixed crushed nuts
2-Acetyl-pyridine (0.01% in PG)
Lightly toasted unsalted nuts
Unsalted wheat thins
Roasted peanut oil extract
Roasted peanuts, ground hazelnuts,
ground almonds, 1:1:1
1000–73 nut base by Givaudan-Roureg
(10% in PG)
4 g cheese aroma/100 ml of pasteurised
ewes’ milk
0.5-3.5 g cheese aroma/100 g Quark
91549-24 by Givaudan Roureg
91483-24 by Givaudan Roure
91428-24 by Givaudan Roure
91125-73 by Givaudan Roure
10418-71 by Givaudan Roure
2,4 Decadienal, 20 mg/kg
4-Pentenoic acid (10 000 mg/kg in PG)
Canned pineapple chunks
 Sensory Character of Cheese and its Evaluation 461
Table 2 continued
Term Definition a
Prickle/bite Chemical feeling factor of which the sensation
of carbonation on the tongue is typical
Processed A bland, shallow and artificial taste.
Made by melting, blending and
frequently emulsifying other cheeses
Propionic acid – Propionic acid (1% in PG)
Pungent A physically penetrating sensation
in the nasal cavity. Sharp smelling
or tasting, irritating
Irritative, burnt and/or penetrating
sensation in the interior of the mouth
Rancid The taste and aroma associated
with sour milk and oxidised fats. Having
the rank unpleasant aroma or taste
characteristic of oils and fats when
no longer fresh
Rennet The aromatics associated with
natural lamb rennet
Rosy/floral Aroma associated with flowers
Salty Fundamental taste sensation of which
sodium chloride is typical
Fundamental taste sensation
elicited by salts
Fundamental taste sensation produced
by aqueous solutions of several
products such as sodium chloride
Sauerkraut The aromatics associated with
fermented cabbage
Scorched Aroma associated with extreme heat
treatment of milk proteins
Sharp The total impression associated with the
combination of aromatics that are sour,
astringent and pungent
Total impression of penetration into
the nasal cavity
The perception associated with aged and
ripened cheeses, from flat to sharp
Smokey, smoky The penetrating, dark brown, acrid aromatic
of charred wood
Aroma and taste of hickory-smoked ham
The penetrating smoky taste and aromatics,
similar to charred wood
Tainted by exposure to smoke
Perception of any kind of smoke odour
(hickory, apple, cherry, mesquite or
artificial flavouring)
Soapy A detergent-like taste and smell. Similar
to when a food is tainted with a
cleansing agent
Sour Fundamental taste sensation elicited by acids
Fundamental taste sensation of which
lactic and citric acids are typical
Soya sauce The aromatics that are reminiscent of soy
sauce; they are sour, slightly
brown and pungent
Standard b, c
Soda water
Cheese strings (a processed cheese
snack)
A ratio of 1 part sour cream to
0.68 parts horseradish sauce
Danish blue cheese
Ammonia (1% in PG)
0.5 g cayenne/100 ml water,
boiled in water for 5 min, 1.5 ml
of filtration/10 g Quark
Cheese stored at 21 °C for 4 days
Butyric acid (0.1% in PG)
Natural lamb rennet (33% NaCl)
2-Phenethylamine, 20 mg/kg
Sodium chloride (0.25, 0.5, 0.75 or
1% in water)
Pecorino Romano sheep cheese,
1200 mg NaCl/100 g Quark
Dimethyl disulfide (top notes only;
10 000 mg/kg in PG)
Milk heated to 121 °C for 25 min
Propionic acid (100 000 mg/kg in PG)
5000 mg/kg of propionic acid in Vaseline
oil  SS. 2 ml SS  cotton in 60 ml flask
Oil of cade
Hickory smoked ham
Applewood cheese
Guaiacol (0.5% in PG)
Guaiacol in vaseline oil (several
concentrations)
Liquid smoke flavouring. 40 l
 cotton in 60-ml flask
Lauric acid (pure)
Mellow processed Cheddar
Citric acid (0.08% in water)
Lactic acid (0.05 and 0.085% in water)
Soya sauce
continued
462 Sensory Character of Cheese and its Evaluation

Table 2 continued

Term Definition a Standard b, c

Spicy/pungent – Valeric acid (1% in PG)

Strength The overall intensity of aroma and flavour, English blue Stilton cheese
the degree of mildness and maturity
Sulfur Aromatics associated with sulphurous Boiled mashed egg. H2S bubbled through
compounds water; struck match
Sweaty The aromatics-associated reminiscent Isovaleric acid (10 000 mg/kg in PG)
of perspiration-generated foot odour; Isovaleric acid (0.1% in PG)
sour, stale, slightly cheesy and is found in Isobutyric acid (5% in PG)
unwashed gym socks and shoes Cheese stored at 30 °C for 3 h
Sweet Fundamental taste sensation of which Sucrose (1, 3, 4 or 5% in water)
sucrose is typical Condensed milk
Fundamental taste sensation 1.2 g sucrose/100 g Quark
elicited by sugars
Fundamental taste sensation produced by
aqueous solutions of several products such
as sucrose or fructose
Toasty The combination of sweet aromatics Cooked condensed milk
produced after food toasting or cooking Ciclotene (several concentrations in water)
Umami Chemical feeling factor elicited by certain Monosodium glutamate (1% in water)
peptides and nucleotides
Vinegary Aroma described as acidic, fermented Combination of acetic, butyric and
and sweaty by the panelists propionic acids
Waxy, waxy/crayon The sweet aromatic that is associated Decanoic acid (pure)
with waxed paper or wax candles Capric acid, lauric acid or decanoic
Aromatics associated with medium chain acid (100 mg/ml)
fatty acids
Whey Aromatics associated with Cheddar Fresh Cheddar whey
cheese whey Whey powder
Yeasty Aromatics associated with fermenting yeast Raw yeast dough
Yeast in 3% warm sucrose water
Yoghurt – Yoghurt, 3.2% fat

a The precise wording of some definitions has been changed to allow the use of consistent language in this table. However, the
meaning of each definition is unchanged.
b Units of measurement are changed to a standard format where possible.
c Publications referenced often provided brand names of food standards used. Brand names are not provided in this table as it is
recognised that many of these will only be of interest to readers in their country of origin. In addition, as some publications refer-
enced are now more than 10 years old, products may have changed.
d Propylene glycol.
e Volume of water not given in publication referenced.
f Stock solution.
g Codes refer to commercially available flavour mixtures that can be provided by Givaudan Roure, Switzerland.

be possible to develop and standardise a terminology
that can be used universally, and for all cheese types,
eventually leading to a much-improved understanding
about the eating quality of cheese.
The Human Senses and the Sensory
Properties of Cheese
Cheese appearance
Humans are highly visual creatures and allow vision to
dominate other sensory modalities. Vision is the percep-
tion of shape and texture, size and distance, brightness,
colour and movement. Appearance characteristics of
cheese are assessed visually, usually prior to consuming
the cheese, or when preparing the cheese for consump-
tion by cutting or spreading. Appearance characteristics
include colour, presence of eyes or holes (or openness),
mould, rind, and visual texture (Tables 1 and 3). In
addition, appearance includes a cheese’s market image
(e.g., size, shape, packaging), as most cheese is pur-
chased in this form (Murray and Delahunty, 2000a).
Appearance characteristics create sensory expectations,
or expectations of how the cheese will ‘taste’, and as
vision can dominate other sensory modalities, visual
aspects of cheese can often have a strong influence on
 Table 3 Studies of cheese sensory character that have used descriptive sensory analysis. Cheese types studied in each case and vocabularies used are listed to allow comparisons

References and cheeses studied Descriptive vocabularies

Adhikari et al., 2003 Aroma: Smokey, vinegary, cheddary, buttery, musty, pungent, other (Swiss)
Low-fat, full-fat and smoked Swiss, Flavour: Smokey, salty, sweet, bitter, acidic, cheddary, sharp, flavour intensity
Cheddar and Gouda Texture: Grainy, hardness, first-bite sticky, sticky, creamy, mouth-coating, viscous, rubbery, dry, crumbly, mealy
Antoniou et al., 2000 Texture: Hardness, elasticity, fracturability, cohesiveness, adhesiveness, chewiness
Münster, Emmental, Roquefort, Beaufort,
Camembert, Reblochon, Pont l’Eveque,
Brie de Meaux, Tomme de Savoie, Valencay,
Saint Nectaire, Pyrenees Brebis, Bleu d’Auvergne,
Comte Vieux, Fourme de Salers
Bárcenas et al., 1999, 2001 Odour: Overall intensity, sharp, milky, brine, rennet, buttery, toasty, smoky, mushroom
Castellano, Idizábal, Manchego, Roncal (ewes’ milk Flavour: Overall intensity, fruity, butyric, nutty, buttery, acid/yoghurt, sweet, salty, pungent, rennet, smoky
cheeses), Garrotxa (goats’ milk), Tetilla (cows’ milk) Texture: Surface rugosity, surface moisture, elasticity, firmness, friability, adhesiveness, solubility, moisture, granularity
Drake et al., 2001, 2002, 2003 Flavour: Cooked, whey, diacetyl, milkfat/lactone, fruity, sulphur/eggy, sulphur/match, free fatty acid, brothy, nutty, catty,
Truong et al., 2002 cowy/phenolic, age, yeasty, mouldy/musty, methyl ketone/blue, oxidised, waxy/crayon, faecal, bell pepper,
Gwartney et al., 2002 rosy/floral, scorched, bitter, salty, sweet, sour, umami, prickle/bite
Cheddar, processed cheese Texture: Elasticity (evaluated by hand), hardness, cohesiveness, elasticity, adhesiveness between teeth,
adhesiveness to teeth, cohesiveness of the mass (mass evaluated after 3–5 chews), adhesiveness of the
mass, smoothness of the mass, smoothness of film (evaluated after swallow)

Heisserer and Chambers, 1993
Asiago, Bel Paese, Blue Cheese, Bond-ost, Brisk,
Brie, Butter Cheese, Camembert, Cheddar, Chevre,
Colby, Danish Cream Havarti, Edam, Emmentaler,
Feta, Fontina, Gorgonzola, Gouda, Gruyere, Jarlsberg,
Kreme Kase, Limburger, Manchego, Mozzarella,
Monterey Jack, Parmesan, Port du Salut, Provolone,
Romano, Roquefort, Sap Sago, Stilton, Swiss
Hort and Le Grys, 2001
Cheddar
Hough et al., 1996
Reggianito grating cheese
Flavour: Buttery, cooked milk, dairy fat, dairy sour, dairy sweet, animalic, butyric acid, decaying animal,
fresh fish, fish oil, goaty, sweaty, waxy, fermented/fruity/winey, nutty, pinapple, sauerkraut, smokey, soy sauce,
mouldy, mushroom, astringent, biting, pungent, sharp, bitter, salty, sour, sweet
Texture: Creaminess, crumbliness, firmness, hardness, springiness, graininess
Visual texture: Grain uniformity, grain size, number round openings, gloss of openings, number cracks, fracturability
Manual texture: Crumbly grain, elasticity, fracturability, resistance to cut, resistance to press, ball hardness, ball
fracturability, ball cohesiveness
Oral texture: Hardness, fracturability, cohesiveness, roughness, water absorption, cohesiveness of mass,
adhesiveness to teeth, crystals
Aroma: Total intensity, sweet, sour, lipolysis, milky-creamy
Flavour: Total intensity, cheese, salty, sweet, bitter, acid, lipolysis, milky-creamy, tongue-tingling, hot, residual intensity

continued
463
464
Table 3 continued

References and cheeses studied Descriptive vocabularies

Lawlor et al., 2001, 2002, 2003
Appenzeller, Ambassedeur, Bleu d’Auvergne,
Blue Shropshire, Blue Stilton, Cambozola, Cashel
Blue, Chaumes, Danish Blue, Dubliner, Emmental,
Fontina, Gabriel, Gruyère, Huntsman, Mahón,
Old Amsterdam, Raclette, Tête de Moine,
Tetilla, Wensleydale
Madsen and Ardö, 2001
Danbo cheese
Odour: Pungent, caramel, mushroom, silage, sweaty/sour, fruity, mouldy, Cheddar dairy-sweet, sweet, creamy
Flavour: Buttery, caramel, dairy sweet, rancid, mushroom, oily, mouldy, nutty, smoky, soapy, silage, processed,
sweet, salty, acidic, bitter, pepper, burnt-aftertaste, strength, balanced
Appearance: Colour intensity, crumbly, mottling, mouldy, softness, openness, shiny
Texture: Firmness, rubbery, crumbly, smooth, moist, oily, chewy, slimy, grainy, mouth-coating
Texture: Elasticity, firmness, deformability, friability, adhesivity

McEwan et al., 1989 Odour: Strength, creamy/milky, sour, rindy, manure

Cheddar Flavour: Creamy/milky, strength, sour, manure, salty, acid, smoky, rindy
Texture/mouthfeel: Tongue tingling, soft-firm, rubbery, mouth-coating, graininess
Muir and Hunter, 1992a,b,c Odour: Intensity, creamy, sulphur, fruity, nutty, rancid, other
Banks et al., 1993 Flavour: Cheddar intensity/overall intensity, creamy/milky, sour/acid, sulphur/eggy, fruity/sweet, nutty, rancid,
Muir and Banks, 1993 bitter, cowy, unclean/manurial, salty, other
Muir et al., 1995a,b,c,d Texture: Firmness, rubbery, crumbly, pasty, grainy, mouth-coating
Muir et al., 1996
Muir et al., 1997a,b
Cheddar, Farmhouse Cheddar

Murray and Delahunty, 2000a,b,c Aroma: Pungent, caramel, sweaty/sour, sweet, creamy, fruity
Delahunty and Murray, 1997 Flavour : Pungent, caramel, sweaty, creamy, fruity, buttery, rancid, cheddary, mushroom, mouldy, nutty, smoky, soapy
Bogue et al., 1999 processed, sweet, salty, acidic, bitter, astringent, strength, balanced
Fenelon et al., 2000 Appearance: Colour intensity, mottled, uniformity, open, shiny
O’Riordan and Delahunty, 2003b Texture: Firm, rubbery, crumbly, smooth, moist, grainy, mouth-coating
Hannon et al., 2003
Irish farmhouse and Cheddar cheese

Neilsen and Zannoni , 1998 Smell: Strength/intensity, creamy, yoghurt, fruity/citrus fruit/other fruit/nutty, grass, animal/cowshed, caramel,
Hunter and McEwan, 1998 acid/sour, ammonia, hay/grass
Caerphilly, Cheddar, Comté, Danbo, Edam, Emmental, Aroma/taste: Strength/intensity, creamy/yoghurt, grass, fruity/citrus fruit/other fruit/nutty, animal/cowshed,
Fontina, Gouda, Jarlsberg, Parmigiano-Reggiano, toasted/caramel, sour, pungent, ammonia, sweet, salty, acid, bitter
Sbrinz, Svenbo Texture: Rubbery/elastic, hardness/firmness, crumbly/crumbliness, coating/adhesiveness, dryness,
melting/solubility, grainy
Ordoňez et al., 1998 Odour: pungent, acid, sweet, characteristic, others
Idiazábal cheese (ewes’ milk cheese) Taste: pungent, acid, sweet, salty, bitter, characteristic, others
Aftertaste: pungent, acid, bitter, others, persistent
Texture: elasticity, creaminess, firmness, grainy, others
Appearance: paste colour (internal), eyes (internal), shape (external), rind (external)
 Papademas and Robinson, 2001
Halloumi
Piggott and Mowat, 1991
Delahunty et al., 1996a,b
Jack et al., 1994
Cheddar
Roberts and Vickers, 1994
Cheddar
Stampanoni, 1994
Cheese flavours: Cheese
general, fresh cheese, soft
cheese, hard cheese,
goat/sheep cheese
Wendin et al., 2000
Cream cheese
465
Taste and flavour: Salty, bitter, acidity, creamy, milky, minty, intensity
Texture: Springy, moist, chewy, crumbly
Appearance: Colour, body
Appearance: White to orange
Flavour: Milky, buttery, cheesy, mouldy, rancid, pungent, sour (aroma), sweet (aroma), salty (taste),
sour (taste), bitter (taste), processed, strength, maturity, aftertaste
Texture: Dry, hard/soft to firm, coarse, creamy, moist, smooth, sticky, grainy, crumbly, rubbery, chewy,
pasty, mouth-coating
Aroma: Buttery, fatty, fruity, fermented, mouldy, nutty, sweaty/sour, pungent, rancid, smoky, spoiled dairy, vinegary
Flavour: Acid, acid bite, astringent, barny, bitter, buttery, cardboard, chemical, fatty, fruity, metallic, milky,
mouldy, peppery, sweaty/sour, rancid, salty, sharp, smoky, soapy, diacetyl (yoghurt), sweet
Aftertaste: Acid, bitter, milky, smoky, fishy, mouldy, peppery, soapy, sweaty/sour
Texture: Chalk, chewy, creamy, crumbly, firm, grainy, moist, greasy, pasty, squeaky, waxy
Flavour: Milky, cooked milk, fatty, buttery, creamy, nutty, butter milk, yoghurt, cottage cheese, caseinate,
whey, soapy, fermented, mushroom, earthy, musty, spicy-pungent, blue, ammonia, green-grass, cheese rind,
propionic acid, capric acid, butyric acid, fruity, sweaty, animal
Appearance: Yellow colour, granularity, watery, compact
Flavour/taste: Sourness, butter, saltiness
Texture/mouthfeel: granularity, fat/creamy
Texture by hand: Spreadability
466 Sensory Character of Cheese and its Evaluation
the perception of other characteristics that, general
experience has taught us, are related (even if they may
not be physically related). For example, many con-
sumers believe that a coloured cheese is more intensely
flavoured than its uncoloured equivalent (Bogue et al.,
1999).
Cheese texture
Texture can be defined as the attribute of a cheese
resulting from a combination of physical properties,
including size, shape, number, nature and conformation
of the constituent structural elements, that are per-
ceived by a combination of the senses of touch (tactile
texture), vision (visual texture) and hearing (auditory
texture). For example, the ‘softness’ of a cream cheese
can be assessed visually upon cutting the cheese, by
proprioceptive sensations when spreading the cheese,
and finally by tactile sensations in the mouth during
consumption. During mastication and consumption,
texture perception occurs in the superficial structures
of the mouth, around the roots of the teeth and in the
muscles and tendons. Cheese texture characteristics
frequently described include firmness, rubberiness,
crumbliness, graininess, cohesiveness and adhesiveness
(Tables 1 and 3).
Cheese flavour
Flavour is most often defined as the integrated percep-
tion of olfactory, taste and chemesthesis (or trigeminal)
stimuli. Flavour perception begins prior to consump-
tion when a consumer can smell a cheese, but is finally
perceived during consumption when compounds that
stimulate the olfactory system in the nose, the taste
system in the mouth and the trigeminal system in the
mouth and nose are released from the cheese and
become available to receptors. A large number of
flavour characteristics have been described in cheese.
Some that have been defined and standardised for
application in descriptive sensory evaluations are listed
in Table 2.
Smell or aroma is usually the first aspect of flavour
encountered by a consumer. The stimuli for smell are
air-borne compounds of volatile substances that allow
them to travel from their source to the olfactory recep-
tors, where perceptions are created that are endowed
with distinctive smells. Volatile stimuli are released from
cheese into the air, and may be delivered to the nose
orthonasally, often consciously, by sniffing (e.g., when
one opens a cheese package or removes a trier from the
cheese for evaluation). Volatile compounds may also be
released into the buccal cavity air during consumption,
where they are delivered to the nose retronasally without
any conscious effort. Many hundreds of different volatile
compounds, each with a distinctive aroma character,
have been identified in cheese, and these provide the
largest contribution to the diversity of cheese flavours.
Compounds identified in cheeses include fatty acids,
methyl, ethyl and higher esters, methyl ketones,
aliphatic and aromatic hydrocarbons, short- and long-
chain alcohols, aromatic alcohols, aldehydes, amines,
amides, phenols and sulphur compounds (Maarse and
Visscher, 1996). Much of what we commonly refer to as
‘taste’ is incorrectly localised smell detection. The signifi-
cant contribution of aroma to flavour can be easily
demonstrated if one pinches the nose shut whilst eating,
effectively blocking air circulation through the nasal pas-
sages. Then, a familiar cheese, e.g., Cheddar, will not be
recognised, and can easily be confused with one that
would otherwise be easily distinguished, e.g., Gruyère.
Taste is another aspect of flavour. Tasting occurs in
the oral cavity, primarily on the tongue, but also on the
soft palate. The primary stimuli for taste are non-
volatile compounds, and these must make contact with
the taste receptors. This contact creates perceptions
that endow four distinctive taste qualities, referred to
as sweet, salty, sour and bitter. A fifth taste, ‘umami’,
has been accepted more recently, particularly in Japan
and other cultures where it is the most familiar and the
most easily perceived. Compounds that contribute
directly to cheese taste include lactic acid (sour),
sodium chloride (salty), mineral salts of potassium,
calcium and magnesium (salty) and free amino acids
and peptides of varying types (sweet, bitter, umami)
(Warmke et al., 1996; Engel et al., 2000).
The last aspect of flavour is chemesthesis. This
term is used to describe the sensory system respon-
sible for detecting chemical irritants. Detection is
more general than that of taste and smell and occurs
primarily in the eyes, nose and mouth. The perception
is closely related to the somato-sensory characteristics
of pain and temperature change, and provokes a
strong behavioural response. The fizz of carbon dioxide
(CO2), the cooling sensation of menthol and the burning
sensation of chilli are perhaps the best examples of
how chemical irritation can provide additional charac-
ter that is very much desired in a wide range of food
products. With regard to cheese, the pungency, the
prickle/bite and the sharpness of mature Cheddar are
examples of perceived chemical irritation (Table 2).
Sensory interactions
Cross-modal sensory interactions also occur, adding
complexity to the perception and description of sensory
character. Consumers rarely distinguish between stimuli
of different sensory modalities (unless trained to do so),
and generally describe the integrated sensation as ‘taste’.

However, the factors that cause apparent cross-modal
sensory interactions are not always the same and can
be difficult to comprehend. A first cause of apparent
sensory interactions when perceptible components of a
cheese are studied together can be interactions between
the components of the cheese prior to introduction to
the senses. For example, changing the fat content or salt
content of a cheese can influence the physical chemistry
of the cheese matrix dramatically, changing the partition
coefficients of volatile compounds, and therefore releas-
ing volatiles from the cheese matrix (Delahunty and
Piggott, 1995). As a cheese matures, the protein compo-
sition changes significantly due to proteolysis, and this
may change the binding ability of the cheese for specific
volatile compounds (Delahunty and Piggott, 1995). A
second cause of sensory interaction is termed a halo
effect, and is caused by learning to place greater reliance
on one sensory modality over another to make behav-
ioural decisions. This effect was referred to in the
context of appearance, as it is most obvious by the domi-
nance, or bias, of the visual sense over the taste or olfac-
tory sense. It can be demonstrated by confusing familiar
colour and flavour combinations, or by varying colour
intensity beyond expectation (Clydesdale, 1993). With
regard to cheese, an influence of added colour on con-
sumers’ perception of flavour has been reported (Bogue
et al., 1999). A true cross-modal sensory interaction is
one where the function of one sense (e.g., threshold
measures, concentration-response functions) is changed
by stimulation of another sense. This type of interaction
can occur at receptor level, where one component
blocks access to the receptor by another (e.g., increasing
viscosity may coat the tongue and reduce access of tas-
tants to taste receptors (Lynch et al., 1993)), or where
stimulation by both components results in neural con-
vergence as receptor sites are in close proximity and are
served by the same nerve (e.g., capsaicin desensitisation
reduces perceived taste intensity (Karrer and Bartoshuk,
1995)). The extent of these types of interactions in
cheese, and their effect, is not known.
Taste–aroma interactions are also observed and
appear to be true interactions even though the physiol-
ogy of the senses of olfaction and taste is independent.
In this case, interaction is believed to occur centrally at
a cognitive level where stimulus integration takes place
(Stevenson et al., 1999). Taste–odour interactions have
been observed in many different types of food and
are easily demonstrated in model food studies (Noble,
1996). When volatile compounds are introduced to the
oral cavity in the absence of taste-active compounds,
they are generally perceived to be of low intensity and
are described as bland in character. In cheese, it is most
likely that the flavour impact of specific volatile com-
pounds will be pronounced (and become familiar) only
Sensory Character of Cheese and its Evaluation 467
when perceived in combination with appropriate taste-
active compounds, such as lactic acid, mineral salts,
free amino acids and bitter peptides typically present in
cheese (Frank and Byram, 1988). In addition, varia-
tions in taste quality and intensity, for example an
increase in sourness (i.e., acidity), or an increase in bit-
terness, will affect how aroma is perceived (although
volatile composition may be unchanged) and give the
impression that overall flavour has changed considerably.
Flavour–texture interactions are also observed widely.
The precise nature of many of these interactions is not
known, although structural components, such as pro-
teins, can bind volatile compounds; rheology and struc-
ture can also influence mass transfer of non-volatile and
volatile compounds to the surface of a cheese bolus
where they will be released and become available for
perception; fat can coat the receptor surface of the
tongue, effectively blocking taste transduction (Lynch
et al., 1993) and finally, interactions may occur at a cog-
nitive level during perception integration in a way simi-
lar to taste–odour interactions (Weel et al., 2002).
Texture–flavour interactions can also be influenced by
individual consumer physiology, such as mastication
behaviour and saliva flow rate and volume.
Sensory Methods Used to Evaluate Cheese
Many reported studies on cheesemaking, cheese compos-
ition and cheese microbiology had the objective of con-
trolling or improving sensory characteristics such as
appearance, flavour and texture. However, it is difficult
to compare the success of these studies as the final sens-
ory character was often measured inappropriately. In
many studies, judgements of overall sensory quality (i.e.,
a grade of ‘good’ or ‘bad’), rather than objective measure-
ments of the perceived intensity of specific sensory char-
acteristics, were carried out to determine the influence
of cheese composition, counts of micro-organisms, or
control of a cheesemaking variable on flavour or texture
quality. Although standard procedures may be followed,
e.g., International Dairy Federation standards (IDF,
1997), quality judgements are biased by the individ-
ual(s) who makes them. In addition, and of greater
importance, traditional quality judgements do not allow
the application of statistical analyses that would enable
relationships between cheese study variables and specific
sensory characteristics to be determined. The unaware
reader of the literature can very easily confuse meas-
urements of overall sensory quality with descriptions of
sensory difference, as it is often reported, for example,
that a specific cheesemaking procedure produced cheeses
that ‘tasted’ similar, when in fact they were judged to be
of similar quality (i.e., had no defects). Cheeses judged
to be of similar quality by the same judge may differ
468 Sensory Character of Cheese and its Evaluation
significantly in sensory characteristics (Delahunty and
Murray, 1997).
The American Society for Testing and Materials
(ASTM) Committee E-18 on Sensory Evaluation of
Materials and Products has defined sensory evaluation as
‘a scientific discipline used to evoke, measure, analyse
and interpret reactions to the characteristics of foods and
materials as they are perceived by the senses of sight,
taste, touch and hearing’. A key distinction between sens-
ory evaluation and other chemical and instrumental ana-
lytical techniques, is that different techniques can be
used to evoke, measure and interpret sensory character-
istics that have very different objectives and outcomes.
Sensory evaluation can be carried out to determine
whether cheeses exhibit defects or other undesirable
characteristics, whether a difference in overall sensory
character can be detected between two or more cheeses,
whether specific differences in sensory characteristics
can be perceived, to quantify the intensity of one or
more sensory characteristics, to quantify the onset, max-
imum intensity and decline of a sensory characteristic,
and to determine whether consumers find the cheeses to
be acceptable or not, based on their sensory characteris-
tics. The distinctions in sensory evaluation methodology
can be broadly classified as quality scoring, discrimin-
ation testing, descriptive testing, time–intensity testing
and consumer acceptability testing, respectively. There
are some excellent texts that outline sensory tests in
detail (Piggott, 1988; Stone and Sidel, 1993; Lawless and
Heymann, 1998; Meilgaard et al., 1999).
Grading and quality scoring
The manufacture of cheese of consistent quality is
extremely difficult due to the number of production
factors that ultimately contribute to eating quality (see
‘Factors that Affect the Quality of Cheese’, Volume 1).
In addition, cheeses are susceptible to a large number
of defects that can originate in milk, transfer to the
cheese curd during making and storage, result from
microbial contamination or develop during maturation
if the composition at manufacture is not controlled.
However, to maintain consumer confidence and loyalty
towards a cheese, it is very important to control its
quality. In addition, as consumers are becoming more
brand-conscious, they become less-accepting of varia-
tions in sensory characteristics that traditionally would
not be considered defects, and expect to find a cheese
with near-identical appearance, flavour and texture in
the package each time. To test instrumentally for all
possible flavours and structural properties that con-
tribute to eating quality would be an extremely labori-
ous task, and may not achieve success. For example,
many compounds that contribute to flavour are present
in concentrations below the detection limit of even the
most sophisticated instruments.
Quality scoring, grading or judging against speci-
fied defects on standardised scorecards (Bodyfelt et al.,
1988) is the traditional and still most widely used type
of formal sensory evaluation in the cheese industry.
Cheese grading is carried out to classify the potential
of a cheese to develop a satisfactory character during
maturation, and to maintain quality at the point of
sale. Grading standards generally specify a scoring sys-
tem, where top grade is awarded a maximum score,
and points are deducted when defects are found. For
example, the IDF provides standard scorecards for
cheese, and specifies a scale that ranges from 5, repre-
senting the highest possible quality, to 0, representing
the lowest possible quality (IDF, 1997). Each point
deducted from the scale is supported by a list of
defects that merit the deduction. The defect list that
accompanies each score on the scale aims to provide
objectivity to the evaluation. The US cheese grading
system and the American Dairy Science Association
(ADSA) cheese-judging ballot operate in a similar
manner (Bodyfelt et al., 1988). Tables 4 and 5 show
the United States Department of Agriculture (USDA)
standards for grades of Cheddar cheese, effective since
1956, which provide guidelines for the award of four
grades – AA, A, B or C. Table 6 shows the ballot used
by the ADSA to judge Cheddar cheese quality. McBride
and Muir (1999) recently reviewed grading systems
used for Cheddar cheese in Australia, United Kingdom,
United States, Canada, the IDF and New Zealand. In
addition, chapters in recent textbooks by Kosikowski
and Mistry (1997) and Robinson and Wilbey (1998)
review in detail methods of cheese grading and defects
found in cheese.
Kosikowski and Mistry (1997) described the
sequence of cheese quality judgement. One or more
expert evaluators, who have detailed product know-
ledge built up over many years and maintain a stand-
ard in memory of what the ideal product is in terms of
sensory characteristics, carry out this evaluation.
These experts have the ability to relate their recogni-
tion of specific defects to the cause of that defect and
to weight the influence of each defect at different levels
of severity and how they detract from overall product
quality. The overall exterior of a cheese is first judged
to determine if it appears deformed or soiled in any
way. The rind or surface is judged next as it may be
discoloured, cracked or irregular. Internal appearance is
judged following cutting, or directly from a cheese
trier, as it may have holes, cracks, spots or other open-
ing defects, and colour may be uneven, mottled or
dull. Odour, which may be uncharacteristic in many
ways, is judged prior to placing a cheese in the mouth,

Table 4 Specifications for Grade AA and Grade A Cheddar cheese (United States Department of Agriculture, Agricultural Marketing
Service, Dairy Division)
Detailed specifications for US Grade AA
Fresh or current
(a) Flavour :
Fine and highly pleasing. May be
lacking in flavour development
or may possess slight characteristic
Cheddar cheese flavour. May possess
a very slight feed flavour, but shall be
free from any undesirable
flavours and odours.
(b) Body and texture:
A plug drawn from the cheese shall
be firm, appear smooth, compact,
close and should be slightly
translucent, but may have a few
small mechanical openings. The texture
may be definitely curdy or may be partially
broken down if more than 3 weeks old.
Shall be free from sweet holes, yeast
holes and gas holes of any kind.
(c) Colour :
Shall have a uniform, bright attractive
appearance; practically free from white
lines or seams. May be coloured or
uncoloured but if coloured it should be a
medium yellow-orange.
(d) Finish and appearance:
Bandaged and paraffin-dipped.
Shall possess a sound, firm rind
with a smooth bandage and paraffin
coating adhering tightly but may
possess soiled surface to a very
slight degree. The cheese shall be
even and uniform in shape.
Rindless. The wrapper or covering
shall be practically smooth,
properly sealed with adequate
overlapping at the seams or by
any other satisfactory type of
closure. The wrapper or covering
shall be neat and adequately and
securely envelop the cheese. May
be slightly wrinkled but shall be of
such character as to protect fully
the surface of the cheese and not
detract from its initial quality. Shall
be free from mould under wrapper
or covering and shall not be huffed
or lopsided.
Sensory Character of Cheese and its Evaluation 469
Medium cured
Fine and highly pleasing.
Possesses a moderate degree
of characteristic Cheddar
cheese flavour. May possess
a very slight feed flavour
but shall be free from any
undesirable flavours and odours.
A plug drawn from the cheese shall
be firm, appear smooth, waxy,
compact, close, flexible and
translucent, but may have a few
mechanical openings if not large
and connecting. May be slightly
or not entirely broken down. May
possess not more than one sweet
hole per plug but shall be
free from other gas holes.
Shall have a uniform, bright
attractive appearence; practically
free from white lines or seams. May
be coloured or uncoloured, but
if coloured it should be medium
yellow-orange.
Bandaged and paraffin dipped.
Shall possess a sound, firm
rind with a smooth bandage and
paraffin coating adhering tightly
but may possess very slight
mould under bandage and paraffin,
and the following other character-
istics to a slight degree: Soiled
surface and surface mould.
The cheese shall be
even and uniform in shape.
Rindless. Same as for current,
except very slight mould
under wrapper or covering
permitted.
Cured or aged
Fine and highly pleasing characteristic
Cheddar cheese flavour showing
moderate to well-developed degrees
of flavour or sharpness. May
possess a very slight feed flavour
but shall be free from any
undesirable flavours and odours.
A plug drawn from the cheese
shall be firm, appear smooth, waxy,
compact, close, and translucent
but may have a few mechanical
openings if not large and connecting.
Should be free from curdiness and
possess a cohesive velvet-like
texture. May possess not more than
one sweet hole per plug but shall be
free from other gas holes.
Shall have a uniform, bright attractive
appearance; practically free from
white lines or seams. May show
numerous tiny white specks. May
be coloured or uncoloured, but if
coloured it should be medium
yellow-orange.
Bandaged and paraffin dipped.
Shall possess a sound, firm rind
with a smooth bandage and
paraffin coating adhering
tightly but may possess the
following characteristics to a
slight degree: Soiled surface
and mould under bandage
and paraffin; and surface mould
to a definite degree. The cheese
shall be even and uniform in shape.
Rindless. Same as for medium.
continued
470 Sensory Character of Cheese and its Evaluation
Table 4 continued
Detailed specifications for US Grade A
Fresh or current
(a) Flavour:
Shall possess a pleasing flavour.
May be lacking in flavour
development or may possess
slight characteristic Cheddar cheese
flavour. May possess very slight
acid, slight feed but shall not possess
any undesirable flavours and odours.
(b) Body and texture:
A plug drawn from the cheese shall
be firm, appear smooth, compact,
close and should be slightly
translucent but may have a few
mechanical openings if not large
and connecting. May possess
not more than two sweet holes
per plug but shall be free from
other gas holes. May be definitely
curdy or partially broken down
if more than 3 weeks old.
(c) Colour:
Shall have a fairly uniform,
bright attractive appearance.
May have slight white lines or
seams or be very slightly wavy.
May be coloured or uncoloured
but if coloured, it should be a
medium yellow-orange.
(d) Finish and appearance:
Bandaged and paraffin dipped. Shall
possess a sound, firm rind with the
bandage and paraffin coating
adhering tightly, but may possess the
following characteristics to a very
slight degree: Soiled surface and
surface mould; and to a slight degree:
Rough surface, irregular bandaging,
lopsided and high edges.
Rindless. The wrapper or covering
shall be practically smooth,
properly sealed with adequate
overlapping at the seams or by
any other satisfactory type of closure.
The wrapper or covering shall be
neat and adequately and securely
envelop the cheese. May be
slightly wrinkled but shall be of
such character as to fully protect
the surface of the cheese and not
detract from its initial quality. Shall be
free from mould under the wrapper
or covering and shall not be huffed
but may be slightly lopsided.
Medium cured
Shall possess a pleasing
characteristic Cheddar cheese
flavour and aroma. May
possess a very slight bitter
flavour and the following
flavours to a slight degree:
Feed and acid.
A plug drawn from the cheese
shall be reasonably firm, appear
reasonably smooth, waxy, fairly
close and translucent but may have
a few mechanical openings if not
large and connecting. May be slightly
curdy or not entirely broken down.
May possess not more than two sweet
holes per plug but shall be free from
other gas holes. May possess the
following other characteristics to a
slight degree: Mealy, short and weak.
Shall have a uniform, bright
attractive appearance. May
have slight white lines or seams.
May be coloured or uncoloured
but if coloured, it should
be a medium yellow-orange.
Bandaged and paraffin dipped.
Shall possess a sound, firm
rind with the bandage and
paraffin coating adhering
tightly but may possess
very slight mould under bandage
and paraffin and the following
other characteristics to a
slight degree: Soiled surface,
surface mould, rough surface,
irregular bandaging, lopsided
and high edges.
Rindless. Same as for current, except
very slight mould under wrapper or
covering permitted.
Cured or aged
Shall possess a pleasing
characteristic Cheddar cheese
flavour and aroma with moderate
to well-developed degrees of flavour
or sharpness. May possess the
following flavours to a slight degree:
Bitter, feed and acid.
A plug drawn from the cheese should
be fairly firm, appear smooth,
waxy, fairly close and translucent
but may have a few mechanical
openings. Should be free from
curdiness. May possess not
more than two sweet holes
per plug but shall be free from
other gas holes. May possess the
following other characteristics to
a slight degree: Crumbly, mealy,
short, weak and pasty.
Shall have a uniform, bright
attractive appearance. May have
slight white lines or seams and
numerous tiny white it should be a
medium specks. May be coloured or
uncoloured, but if coloured, it
should be a medium yellow-orange.
Bandaged and paraffin dipped.
Shall possess a sound, firm
rind with the bandage and
paraffin coating adhering
tightly but may possess the
following characteristics to a
slight degree: Soiled surface,
rough surface, mould under
bandage and paraffin, irregular
bandaging, lopsided and high
edges; and surface mould to a
definite degree.
Rindless. Same as for medium.

Table 5 Specifications for Grade B and Grade C Cheddar cheese (United States Department of Agriculture, Agricultural Marketing
Service, Dairy Division)
Detailed specifications for US Grade B
Fresh or current
(a) Flavour:
Should possess a fairly pleasing
characteristic Cheddar cheese flavour, but
may possess very slight onion and the
following flavours to a slight degree: Acid,
flat, bitter, fruity, utensil, whey-taint, yeasty,
malty, old milk, weedy, barny and lipase;
feed flavour to a definite degree.
(b) Body and texture:
A plug drawn from the cheese may
possess the following characteristics
to a slight degree: Coarse, short, mealy,
weak, pasty, crumbly, gassy, slitty and
corky; the following to a definite degree:
Curdy open, and sweet holes.
(c) Colour :
May possess the following characteristics
to a slight degree: Wavy, acid-cut, mottled, surface; and the following characteristics
salt spots, dull or faded; and definitely
seamy. May be coloured or uncoloured but
if coloured, may be slightly unnatural
(d) Finish and appearance:
Bandaged and paraffin dipped. Shall
possess a reasonably firm sound rind,
but may possess very slight mould
under bandage and paraffin. The following
characteristics to a slight degree: Soiled
surface, surface mould, defective coating,
checked rind, huffed, weak rind, and sour
rind; and to a definite degree: Rough
surface, irregular bandaging, lopsided
and high edges.
Rindless. The wrapper or covering shall be
fairly smooth and properly sealed
with adequate overlapping at the
seams or by other satisfactory
type of closure. The wrapper or covering
shall be fairly neat and adequately and
securely envelop the cheese.
May be definitely wrinkled but shall
be of such character as to protect
the surface of the cheese and not detract
from its initial quality. Shall be free from
mould under wrapper or covering but may
be slightly huffed and slightly lopsided.
Sensory Character of Cheese and its Evaluation 471
Medium cured
Should possess a fairly pleasing character-
istic Cheddar cheese flavour and aroma.
May possess very slight onion and the
following flavours to a slight degree: Flat,
yeasty, malty, old milk, weedy, barny and
lipase; the following to a definite degree:
Feed, acid, bitter, fruity, utensil,
and whey-taint.
A plug drawn from the cheese may possess
the following characteristics to a slight
degree: Curdy, coarse, gassy, slitty,
and corky; the following to a definite
degree: Open, short, mealy, weak,
pasty, crumbly, and sweet holes.
May possess a very slight bleached
to a slight degree: Wavy, acid-cut,
mottled, salt spots, dull or faded and
definitely seamy. May be coloured or
uncoloured but if coloured, may be
slightly unnatural.
Bandaged and paraffin dipped. Shall
possess a reasonably firm sound rind,
but may possess the following
characteristics to a slight degree: Surface
mould, mould under bandage and paraffin,
checked rind, huffed, weak rind, and sour
rind; the following to a definite degree:
Soiled surface, rough surface, irregular
bandaging, lopsided, high edges and
defective coating.
Rindless. Same as for current, except slight
mould underwrapper or covering permitted.
Cured or aged
Should possess a fairly pleasing
characteristic Cheddar cheese
flavour and aroma, with moderate
to well-developed degrees of flavour
or sharpness. May possess very
slight onion and the following
flavours to a slight degree: Flat,
yeasty, malty, old milk, weedy,
barny, lipase and sulfide; the
following to a definite degree:
Feed, acid, bitter, fruity, utensil,
and whey-taint.
A plug drawn from the cheese may
possess the following character-
istics to a slight degree: Gassy,
slitty, the following to a definite
degree: Open, sweet holes, short,
mealy, weak, pasty and crumbly.
May possess the following
characteristics to a slight degree:
Wavy, acid-cut, mottled, salt spots,
dull or faded; and definitely seamy.
May be coloured or uncoloured but
if coloured, may be slightly
unnatural.
Bandaged and paraffin dipped.
Shall possess a reasonably
firm sound rind, but may possess
the following characteristics
to a slight degree: Checked rind,
huffed, weak rind, and sour rind;
the following to a definite degree:
Soiled surface, surface mould,
mould under bandage and
paraffin, rough surface, irregular
bandaging, lopsided, high
edges and defective coating.
Rindless. Same as for medium.
continued
472 Sensory Character of Cheese and its Evaluation
Table 5 continued
Detailed specifications for US Grade C
Fresh or current
(a) Flavour :
May possess the following flavours
to a slight degree: Sour, metallic,
onion; and to a definite degree: Acid,
flat, bitter, fruity, utensil, whey-taint,
yeasty, malty, old milk, weedy, barny,
and lipase; feed flavour to a
pronounced degree.
(b) Body and texture:
A plug drawn from the cheese may
possess the following characteristics
to a definite degree: Curdy, coarse,
corky, crumbly, mealy, short, weak,
pasty, gassy, slitty, pinny; and to a
pronounced degree: Open and
sweet holes. The cheese shall be
sufficiently compact to permit
the drawing of a plug.
(c) Colour :
May have a slight bleached surface
and possess the following other
characteristics to a definite degree:
Wavy, acid-cut, mottled, salt spots,
dull or faded; and seamy to a
pronounced degree. May be coloured
or uncoloured but if coloured, may be
definitely unnatural. The colour shall
not be particularly unattractive.
(d) Finish and appearance:
Bandaged and paraffin dipped.
May possess the following
characteristics to a slight
degree: Cracks in rind, soft spots
and wet rind; and mould under
bandage and paraffin; and to a
definite degree: Soiled surface,
surface mould, defective coating,
checked rind, weak rind, sour rind,
and huffed; and to a pronounced
degree: Rough surface, irregular
bandaging, lopsided and high edges.
Rindless. The wrapper or covering
shall be fairly smooth and
properly scaled with adequate
overlapping at the seams
or by other satisfactory type
of closure. The wrapper or covering
shall adequately and securely
envelop the cheese. May be
definitely soiled and wrinkled but
shall be of such character as to
protect the surface of the cheese
and not detract from its initial
quality. May have slight mould under
the wrapper or covering and may be
definitely huffed and lopsided.
Medium cured
May possess the following flavours to a
slight degree: Onion and sulfide; and to
a definite degree: Flat, sour, metallic,
sour, metallic, yeasty, malty, old milk,
weedy, barny and lipase; and to a
pronounced degree: Feed, acid, bitter,
fruity, utensil, and whey-taint.
A plug drawn from the cheese may be
slightly curdy and may possess the
following other characteristics to a
definite degree: Coarse, corky, gassy,
slitty and pinny; and to a pronounced to
a pronounced degree: Open, sweet holes,
short, weak, pasty, crumbly and mealy.
The cheese shall be sufficiently compact
to permit the drawing of a plug.
May possess the following
characteristics to a definite degree:
Wavy, acid-cut, mottled, salt spots,
bleached surface, dull or faded;
and seamy to a pronounced degree.
May be coloured or uncoloured but if
coloured may be definitely unnatural.
The colour shall not be particularly
unattractive.
Bandaged and paraffin dipped.
May possess very slight rind
rot and the following other
characteristics to a slight degree:
Cracks in rind; soft spots and
wet rind; and to a definite degree:
Surface mould, mould under bandage
and paraffin, huffed; and to a
pronounced degree: Checked rind,
weak rind, sour rind and
huffed; and to a pronounced
degree: Soiled surface, rough
surface, defective coating, irregular
bandaging, lopsided and high edges.
Rindless. Same as for current, except
definite mould under the wrapper or
covering permitted.
Cured or aged
May possess slight onion and the
following flavours to a definite degree:
Flat, yeasty, malty, old milk, weedy,
barny, lipase and sulfide; and to a
pronounced degree: Feed, acid, bit-
ter, fruity, utensil and whey-taint.
A plug drawn from the cheese may
possess the following characteristics
to a definite degree: Gassy, slitty,
pinny; and to a pronounced degree:
Open, sweet holes, short, weak,
pasty, crumbly and mealy. The
cheese shall be sufficiently compact
to permit the drawing of a plug.
Same as for medium.
Bandaged and paraffin dipped. May
possess the following characteristics
to a slight degree: Rind rot, cracks
in rind; and to a definite degree:
Checked rind, weak rind, sour rind,
wet rind, soft spots and huffed; and
to a pronounced degree: Rough
surface, soiled surface, surface mould,
mould under bandage and paraffin,
defective coating, irregular bandaging,
lopsided and high edges.
Rindless. Same as for medium.
 Sensory Character of Cheese and its Evaluation 473

Table 6 American Dairy Science Association ballot for judging the quality of Cheddar cheese. A score of 10 is awarded if the judge
cannot find fault with the flavour of the cheese. A score of 5 is awarded if a judge cannot find fault with the body and texture of the
cheese. When scores of 9 or less, or 5 or less, for flavour or body and texture, respectively, are awarded, the cause for deduction of
marks should be indicated
474 Sensory Character of Cheese and its Evaluation

and usually immediately upon opening a packed
cheese, cutting a coated cheese or removing a trier
plug from a cheese. Flavour judgement is made next,
when a sample of cheese is placed in the mouth,
chewed and moved around and then expectorated. As
for odour, numerous uncharacteristic flavours may be
detected in defective cheese, and in addition a cheese
that is over-salty or very bitter may be considered
defective. Finally, but sometimes simultaneously, body
and texture are judged. Defects such as over-hardness,
crumbliness, mealy and sticky are judged, most often
by working a cheese between the thumb and the fin-
gers. Table 7 presents a list of cheese sensory quality
characteristics, which are mostly defects recognised
internationally and are described in the IDF standard
(IDF, 1997). It should be noted that a characteristic
considered to be a defect in one cheese type may be
very much desired in another (e.g., the acceptable
hardness of Parmigiano-Reggiano would be considered
a defect in Cheddar), and therefore judges must take
this into account, and evaluate based on their experi-
ence of each cheese type individually. In addition, it
may be that a characteristic found in the same cheese
type produced in two different countries may be con-
sidered defective in one country, but acceptable in
another. This will be related to the experience of the
cheese consumer in each country, which can be very
different. However, cheeses produced in automated
facilities today are much less likely to suffer from sig-
nificant defects due to improved hygiene practices at
all stages of milk handling and cheesemaking, begin-
ning on the farm. In addition, control over cheesemak-
ing has improved significantly in recent years.
Cheese grading or quality scoring provides a rapid
and simple way quickly to assess overall sensory qual-
ity, but does not adequately take into account so-called
‘non-quality’ related differences in sensory characteris-
tics that give the cheese of individual producers, or
regions of production, a distinctive taste. Traditional
‘quality criteria’ are changing as product ranges expand
(e.g., to include low-fat cheeses); variety of cheeses is
much greater, and differentiation is increasingly made
by purposely developing distinctive sensory character-
istics, such as those now given to cheeses by the use of
adjunct cultures. Sensory characteristics that are not
traditionally considered defects, but which can also dif-
fer from one cheese to another, are now also important
in determining eating quality for the discerning con-
sumer. What is a negative attribute to one consumer
may be a desirable attribute to another consumer. Also,
although the characteristics that expert judges seek are
those that their market demands, their assessments do
not always coincide with those of consumers. It is now
well documented that the consumer and the expert
opinions of quality often differ. For example, McBride
and Hall (1979) found that consumers’ preferences
among twelve cheeses, ranging from poor to good qual-
ity, were not correlated with their official grade scores.
Finally, the current cheese-grading practice does not
measure accurately the intensity of a given defect, and

Table 7 Terms used by cheese graders to describe sensory characteristics of cheeses that determine quality with particular
emphasis on defects (IDF, 1997; Robinson and Wilbey, 1998)

Exterior appearance
Rind/surface
Appearance interior: Openings
Appearance interior: Colour
Consistency, body and texture
Flavour, odour and taste
Concave, convex, deformed (bulged), dirty, oblique, soiled, too flat, too high, vaulted (blown)
Corroded, cracked, discoloured, dry, fatty, holes, incorrect mould, irregular mould, mould
under covering, rotten, rough, smear under covering, smeary, speckled, spots of mould,
thick, thin, too little mould, too little smear, too much smear, wet, wrinkled
Blown, close, collapsed, cracks, distorted, foreign material, foreign mould, glossy openings,
hoop side mould, many holes near the surface, nesty openings, no holes, not typical,
pin-holed, spots of putrification, too few, too large, too many, too small, uneven, unevenly
mouldy
Bleached near the surface, bright, brownish, dirty, discoloured, grey, marbled, mottled,
natural, pale/dull, red colour near the surface, speckled, streaky, strong, two-coloured,
unevenly coloured, weak, yellow
Brittle, chalky, close, coarse, crumbly, curdy, dripping, dry, elastic, firm, flaky, friable, gassy,
granular (grainy), greasy, gritty, gummy, hard, harsh, hoop side sift, layered, leathery, long,
lumpy, mealy, pasty, runny, rough, short, smeary, smooth, soft, soggy, spongy, springy,
squeaky, sticky, stringy, thin (watery), tight, tough, uneven, wet
Acid, alcoholic, ammoniacal, aromatic, bitter, bland, burnt, buttery, butyric acid, chemical,
clove, cooked, cowy, creamy, ethereal, feedy, fermented, fishy, flat, flowery, foreign flavour,
foul, foetid, fruity, garlic, goaty, harsh, malty, metallic, mild, mouldy, musty, musty-flat, nutty,
off, oily, oniony, over-ripe, pale, peardrop, putrid, rancid, resinous, rich, ripe, sandy, salty,
sharp, soapy, sour, spicy, stale, strong, superfine, sulphide, sweaty, sweet, tangy, tallowy,
uncharacteristic, unclean, weedy, yeasty

therefore further statistical analyses that determine the
extent to which cheeses differ, and that mathematically
relate composition to defect intensity, are not appropri-
ate. It is important to note that there are still industry
situations where grading or quality scoring may be
appropriate due to a large number of products that
must be assessed in a short period of time. However,
these sensory tools were not designed to be quantita-
tive or representative of the entire cheese sensory pro-
file and are not ideal tools for research or marketing.
Discrimination tests
Sensory discrimination tests differ from quality scoring
tests in that they involve direct comparisons of cheeses
to determine whether there is either an overall differ-
ence between them or whether they differ for a specific
and designated characteristic. The most commonly
used discrimination tests include the Paired Compari-
son (ISO, 1983a), Duo-Trio (ISO, 1991), Triangle
(ISO, 1983b) and Ranking tests (ISO, 1988). In the
Paired Comparison test, two cheeses are presented for
comparison with one another and assessors are asked
whether they differ; generally, a difference for one spe-
cific sensory characteristic is tested. In the Duo-Trio
test, assessors are asked which of the two products is
the most similar to a third reference product, allowing
a common reference to be used again and again. This
test has obvious advantages for quality control, although
it is not possible to maintain a consistent cheese refer-
ence over time. In the Triangle test, assessors are pre-
sented with three cheeses and asked to choose which
is the most different from the other two. In the Rank-
ing test, four to six cheeses are generally presented for
comparison of a single-designated attribute, and the
assessor is asked to rank them in order of increasing
intensity of that attribute. In best practice, the asses-
sors are forced to make a choice each time for all dis-
crimination tests, thus eliminating response bias.
Whether a difference exists or not is determined statis-
tically, based on the number of choices a panel of
assessors makes for each cheese in the test, using
binomial tables or Chi-squared tests. Therefore, dis-
crimination tests are the most objective and the most
sensitive of sensory tests. An additional advantage of
these tests is that they do not require well-trained
assessors. The only requirement is that all assessors
are reasonably sensitive and recognise and understand
the designated attribute in a common way. When com-
pared with the traditional quality scoring methods,
these discrimination procedures are by far better
suited to application to research problems, they follow
good sensory evaluation principles and do not
encounter problems in scaling and statistical analyses.
Sensory Character of Cheese and its Evaluation 475
For this reason, their principles should now be added
to quality scoring methods in an attempt to introduce
comparability between the scores of one judge and
another. It is also common practice to carry out dis-
crimination tests on cheeses to determine whether a
difference exists prior to further testing by more costly
methods that aim to describe and quantify differences.
Descriptive analyses
A majority of scientists who study cheese are inter-
ested in understanding the fundamental reasons why a
cheese ‘tastes’ as it does, not just whether the cheese is
acceptable, and for this purpose quality control sens-
ory methods are of little value. Descriptive sensory
analysis refers to a collection of techniques that seek
not only to discriminate between the sensory charac-
teristics of a range of cheeses, but also to determine a
quantitative description of all the sensory differences
that can be identified. For example, Figs 1a and 1b
illustrate quantitative differences in perceived flavour,
measured using descriptive analysis, between two hard
Swiss cheeses and two Blue cheeses, respectively. All
cheeses may be profiled in this way, providing object-
ive and reproducible sensory descriptions of cheeses
and providing a basis for determining what character-
istics are influenced by changes in cheesemaking prac-
tice or composition, and also what characteristics are
important for consumer acceptance. The most com-
monly used descriptive analysis methods for all food
types include the Flavour Profile Method (Cairncross
and Sjöstrom, 1950), Texture Profile Method
(Brandt et al., 1963), Quantitative Descriptive Analysis
(QDA)™ (Stone et al., 1974), the Spectrum™ method
(Meilgaard et al., 1999), Quantitative Flavour Profiling
(Stampanoni, 1993a,b), and Free-Choice Profiling
(Langron, 1983; Thompson and MacFie, 1983).
A review of descriptive sensory analysis, which details
advantages, disadvantages and applications of each of
the methods referred to above was published recently
by Murray et al. (2001).
Each descriptive method has three stages to its
implementation. The first involves selecting a panel to
conduct the sensory evaluations, the second, establish-
ing terminology or a vocabulary, by which to describe a
products’ sensory characteristics and the third, quan-
tifying these sensory aspects. However, for each method,
the process is somewhat different. In the cheese indus-
try, as there is a strong tradition of judging that is
linked to extensive knowledge of cheese, then it is a
wise approach that seeks to build on this knowledge
rather than to reinvent the wheel. If the investment in
descriptive sensory testing is for the long term, then
the Spectrum™ method, or a similar one, is preferable.
476

(a) (b)
Pungent Pungent
Caramel odour
Balanced 50 80
Silage Balanced Mushroom
Strength 45
Astringent 40 Sweaty/sour
60
35
Burnt-aftertaste Fruity
30 Strength Fruity

25 40
Pepper Dairy-sweet odour
20
15 20
Bitter Sweet
10
Acidic Mouldy odour
5
Acidic 0 Creamy 0

Salty Buttery

Salty Cheddar
Sweet Caramel flavour

Processed Dairy-sweet flavour

Processed Sweet
Silage Rancid
Soapy Mushroom
Mouldy flavour Oily
Smoky Oily Appenzeller Blue Shropshire
Nutty
Gruyere Danish Blue

Figure 1 Comparison of the flavour profiles of two Swiss cheeses, Appenzeller and Gruyère (1a) and two Blue cheeses, Blue Shropshire and Danish Blue (1b) (Lawlor
et al., 2002, 2003). Flavour characteristics were measured using descriptive sensory analysis by a trained sensory panel.

Using this method, a group of cheese experts develop
and define a descriptive language using a series of uni-
versal intensity scales upon which assessors score their
perceptions (Drake et al., 2001; Drake and Civille,
2003). The sensory panels that will use the method,
often at more than one research site, are then exten-
sively trained. When trained, individual assessors must
be able to discriminate between cheeses using each
attribute in the descriptive language, repeat their
assessments and agree with other panel members on
the size and the direction of differences in cheese
attributes. The advantages of this descriptive analysis
technique are that one panel can be trained readily on
several cheese types since one intensity scale is used,
different types of cheese can be compared directly and
panel scaling is less prone to drift with time (Drake and
Civille, 2003). In addition, this approach is objective
and allows comparison of results between panels,
between laboratories, and from one time to another.
For example, if one wishes to study the maturation of a
cheese over time, then one must ensure that the differ-
ences observed in the results between 3, 6 and 9
months are related specifically to changes that occur in
the cheese and not to changes in the performance of
the sensory panel.
If a cheese type is to be evaluated not very often, or
the sensory panel available will not specialise in
cheese only, or resources are limited, then the QDA™
approach may be preferable. Using this method, the
panel of assessors develop and define the language
themselves whilst tasting a wide range of the test
cheeses (Murray and Delahunty, 2000b). Assessors
must agree with other panel members on the meaning
of terms in the descriptive vocabulary and repeat their
assessments, but are not required to agree on how to
use the attribute scales to rate intensity. When this
method is used instead of the Spectrum™ method, it
is more difficult to compare the results from one study
with those from another in absolute terms.
Free-Choice Profiling (FCP) is another useful
descriptive analysis method (Williams and Langron,
1984). This method allows the use of untrained asses-
sors, or consumers, to profile the sensory characteris-
tics of cheese. Each assessor may use an individual
descriptive vocabulary that they have developed them-
selves, and which they then readily understand, and
data are analysed using Generalised Procrustes Analysis
(GPA; Arnold and Williams, 1986). Free Choice Profil-
ing has been used to describe Cheddar cheese (Jack et
al., 1993; O’Riordan et al., 1998), Parmigiano-Reggiano
(Parolari et al., 1994) and ewes’ milk cheeses (Bárcenas
et al., 2003). The advantages of FCP are that accurate
discrimination between cheeses in terms of perceived
sensory characteristics can be achieved in a very short
Sensory Character of Cheese and its Evaluation 477
time and at a relatively little cost, and that discrimin-
ation is based on a large selection of informative words
that consumers use and with which they are familiar
with. The main disadvantage is that it is difficult to
correlate perceived intensity of sensory characteristics
obtained in this way, as they are too numerous and
imprecise, and there is no consensus vocabulary.
To obtain improved accuracy, sensory panels used
for descriptive analyses generally comprise of 10–12
assessors instead of a smaller number of experts (with
the exception of FCP where 15–20 assessors are
needed). These assessors are screened for sensory acu-
ity and relative interest (Stone and Sidel, 1993). A
panel or group of individuals is used as factors such as
age, saliva flow and onset of fatigue vary between
assessors. Assessors also vary in sensitivity to particu-
lar stimuli, and it is highly probable that they also vary
in their concentration-response functions (Lawless et al.,
1994; Williams, 1994). In addition, temporary illness
or psychological bias can cause day-to-day changes in
sensory ratings. The key point of objective descriptive
analysis is that it should be reproducible and inde-
pendent of consumer preferences. Unlike traditional
quality methods that use scorecards, there is no judg-
ment of ‘good’ or ‘bad’ as this is not the purpose of the
evaluation. The trained sensory panel operates as an
instrument and generates quantitative data analogous
to instrumental data. As with any instrument, replica-
tion is required.
Two guidelines have been published dealing with
cheese texture (Lavanchy et al., 1994) and the aroma
and flavour of cheese (Bérodier et al., 1997a). These
guidelines are very valuable as they define descriptive
vocabularies, and then detail a procedure for evaluation
of each characteristic, including the use of universal
scales that are standardised at a number of points with
common food references. In addition, they provide
translations of many descriptive characteristics of
cheese in Spanish, French, Italian, English and German.
However, it is important to note that sensory lexicons
or languages are not finite and will continue to evolve
with time, usage and application.
Time–intensity sensory analyses
The sensory methods discussed above do not account
for the dynamics of flavour release from cheeses that
occurs during their consumption. Nor do they account
adequately for changes to cheese texture, which occur
progressively during mastication and breakdown of a
cheese in the mouth. When using conventional sensory
procedures, particularly descriptive analyses, assessors
‘time-average’ their responses to arrive at a single inten-
sity value. This looses much useful information such as
478 Sensory Character of Cheese and its Evaluation
rate of onset of stimulation, time and duration of max-
imum intensity, rate of decay of perceived intensity, time
of extinction and total duration of the entire process
(Lee and Pangborn, 1986). To determine most details
about sensory characteristics, changes in sensory char-
acter that occur during cheese consumption (which
can take up to 30 s for a ‘bite-sized’ piece) can be
measured using time–intensity methodology (Lee and
Pangborn, 1986), or in the case of texture, using pro-
gressive profiling (Jack et al., 1994). Time–intensity
methods are useful for the study of new cheese types,
such as low-fat cheeses, as the reduction in fat content
not only influences sensory character development, but
also the breakdown of the cheese in the mouth during
consumption and the rate of release of compounds that
contribute to flavour. For example, in a study of Ched-
dar cheese flavour, the time taken to reach maximum
intensity for ‘sharpness’, ‘bitterness’ and ‘astringency’
was consistently longer in reduced-fat than in full-fat
Cheddar and, more importantly, the rate of flavour
release was greater (Shamil et al., 1991/92). Temporal
differences in perception indicate an altered flavour
balance, caused by reducing the fat content of the
cheese, which may be important in consumer accept-
ability. Delahunty et al. (1996a) showed that a ‘fruity’
note, which might be considered an off-flavour (Aston
et al., 1985; Urbach, 1993), became a dominant flavour
characteristic sooner during consumption and at a
much greater intensity in a low-fat Cheddar-type
cheese than in the full-fat equivalent. Delahunty et al.
(1996b) also demonstrated that improved relation-
ships between volatile composition and perceived sens-
ory characteristics could be achieved by relating
time–intensity sensory data with dynamic volatile com-
pound release data. Jack et al. (1994) found that the
texture of Cheddar cheese was perceived to be relatively
coarse and crumbly earlier in the chewing sequence,
but became increasingly smooth and creamy as chewing
progressed. In addition, other more subtle or specific
cheese-dependent changes occurred as breakdown in
the mouth progressed. It was hypothesised that know-
ledge of these dynamic changes in texture character is
important for understanding consumer acceptability.
Consumer acceptability testing
Trained sensory panels should not be asked to express a
preference as their expert knowledge will introduce bias.
To determine the eating quality of cheese, a naive con-
sumer panel or subjective assessors are used. Ideally,
these assessors will be regular consumers of the prod-
uct type under test or represent the target market for
the product. Such consumers bring their subjective
experience to this test, for although their preferences
will be based on the sensory characteristics tested,
they will be referring to past eating experience. In
addition, when one considers that the target market
may be children, elderly consumers, consumers in
another country or consumers from a culture virtually
unknown to the producer, then it becomes clear that
the internal expertise in a company or organisation
cannot hope to predict acceptability adequately. Con-
sumer acceptability testing makes use of rating scales
that measure relative dislikes and likes (e.g., the nine-
point hedonic scale (Peryam and Girardot, 1952)),
discrimination tests based on preference (e.g., paired
preference, ranked preference) or just right scales that
ask a consumer how they feel about the designated sens-
ory characteristic. It is recommended that a minimum
of 50–60 targeted consumers be used for consumer-
sensory testing, and a greater number than this if
one expects segmentation of preferences (MacFie and
Hedderly, 1993).
One of the biggest challenges in consumer research
is the clarification of consumer language. Consumers
may use terms that are ambiguous, have multiple
meanings, are associated with ‘good’ or ‘bad’ or are
combinations of several terms. Integrated terms, such
as ‘creamy’, are often used by consumers to represent a
combination of positive attributes. Determining
exactly what attribute or attributes ‘creamy’ refers to
(flavour or texture or mouthfeel) have been the sub-
ject of many studies relating consumer and trained
sensory panels (Mela, 1988; Elmore et al., 1999; Bom
Frost et al., 2001). Dacremont and Vickers (1994a,b),
who used concept matching to clarify consumer per-
ception of Cheddar cheese flavour, found that the con-
cept of Cheddar cheese flavour is a consumer concept
and probably varies widely among consumers, as does
Cheddar cheese flavour itself. However, the number of
consumers questioned was small and further studies
with larger consumer groups, and with demographic
information, including types (brand, age) of Cheddar
cheese normally consumed, would provide additional
clarification.
Influence of Cheesemaking Variables on
Sensory Character
During the past ten years or so, there have been numer-
ous reports of the application of descriptive sensory
analysis to determine accurately the influence of cheese-
making variables, e.g., maturation time and temperature,
starter culture or use of adjunct cultures, on the sensory
characteristics of cheese (Table 3).
Studies of Cheddar cheese maturation have found
that, overall, the intensity of odour, flavour and after-
taste is determined by the length (Piggott and Mowat,
 Sensory Character of Cheese and its Evaluation 479

1991; Muir and Hunter, 1992a) and the temperature of 6 0.5

cheddary
maturation (Hannon et al., 2003). However, flavours
such as milky/buttery and creamy decrease in intensity,
while flavours such as sour, bitter, rancid and pungent balanced
increase in intensity (Piggott and Mowat, 1991; Muir
strength S2
and Hunter, 1992a; Hannon et al., 2003). Some textural 39
mushroom
S3 S2
changes, e.g., firmness, are controlled by the cheese- S3 39 39
making procedure and cheese composition, whereas astringent39 S2 S2 S2

PC2 (17%)
–0.5 S339 39 39 0.5
mouth-coating character is related to maturation time 39S2 39
S3
39 S1
S339 39 S3 sweet
(Piggott and Mowat, 1991; Muir and Hunter, 1992a). pungent S1 S1 39 S2
–6 S3 acidic S1 6
Hort and Le Grys (2001), who also studied Cheddar, 39
39
38
38

found that springiness decreased, and crumbliness and S1

S137 processed
creaminess increased as maturation progressed. bitter
39

Banks et al. (1993) and Fenelon et al. (2000) used salty

descriptive analysis to determine the sensory proper-
ties of low-fat Cheddar cheese, and to compare these
with the sensory properties of full-fat Cheddars.
Fenelon et al. (2000) found that there were some dif-
ferences in flavour characteristics related to fat content
that were present regardless of the age of cheese. Full-
fat cheeses were consistently more buttery, creamy and
caramel-like. Adhikari et al. (2003) found that low-fat
and full-fat Swiss cheeses, and low-fat Cheddar
cheeses were dry and crumbly. Factory and farmhouse
Cheddars have also been compared using descriptive
sensory analysis (Muir et al., 1997a; Murray and
Delahunty, 2000c); farmhouse cheeses were found to
have a greater diversity in sensory characteristics. In
addition, cheeses produced from pasteurised milk
were found to be clearly different from those produced
from unpasteurised milk, with the unpasteurised milk
cheeses being more diverse in sensory character and
more intensely flavoured (Grappin and Beuvier, 1997;
Muir et al., 1997a; Murray and Delahunty, 2000c).
Numerous studies have used descriptive sensory
analysis to address the role of specific adjunct cultures
or starter culture enzyme systems in Cheddar cheese
flavour (Drake et al., 1996, 1997; Muir et al., 1996;
Delahunty and Murray, 1997; Lynch et al., 1999; Banks
et al., 2001; Broadbent et al., 2002). Muir et al. (1996)
demonstrated that starter culture type and adjunct
determined the sensory character of cheese. However,
they also found direct and interactive effects of com-
position. More recently, O’Riordan and Delahunty
(2003a,b) found that starter culture type led to consist-
ent differences in sensory characteristics between
Cheddar cheeses, but that composition led to signifi-
cant variation within batches of cheese made using the
same starter culture. Delahunty and Murray (1997)
also demonstrated differences between Cheddar
cheeses based on starter culture type, although these
cheeses were awarded the same grade score (Fig. 2).
Descriptive sensory analysis has been used to determine
the impact of yeast extract and milk standardisation
creamy S1
S1 rancid 37
37 mouldy
buttery
–6 –0.5
PC1 (43%)
Figure 2 Two-dimensional representation of the result of Prin-
cipal Components Analysis of descriptive analysis sensory data
for Cheddar cheese produced using three different starter cul-
tures (coded S1–S3). Grade scores for flavour, awarded by an
expert cheese grader, are also illustrated close to each cheese
code (range 37–39).
with milk protein concentrate on reduced-fat Cheddar
cheese flavour (Shakeel-ur-Rehman et al., 2003a,b,c)
and of smoking parameters on cheese flavour (Shakeel-
ur-Rehman et al., 2003d).
There have been many studies of cheese types other
than Cheddar, and to discuss them all would be imposs-
ible within the scope of this chapter. Of most interest are
studies of Comté cheese using a flavour-descriptive
vocabulary developed by Bérodier et al. (1997b; pub-
lished in French). This lexicon has been used to identify
naturally existing cheese geo-regions within France
(Monnet et al., 2000). In addition, Virgili et al. (1994)
used descriptive analysis to study the sensory–chemical
relationships in Parmigiano-Reggiano cheese. Descrip-
tive analysis of cheese texture has been conducted
recently on a variety of cheeses, on cheeses of different
fat contents and on fat replacers (Drake and Swanson,
1996; Drake et al., 1999a; Lobato-Calleros et al., 2001;
Madsen and Ardo, 2001; Gwartney et al., 2002). In these
studies, descriptive sensory analysis was used to differ-
entiate cheeses and/or the impact of various treatments.
A sensory texture language, like a cheese flavour lan-
guage, is also not necessarily finite. The language will
continue to be refined, particularly as additional cheeses
are studied or as additional instrumental studies are con-
ducted. The texture languages used by Drake et al.
480 Sensory Character of Cheese and its Evaluation
(1999a) and Gwartney et al. (2002) were merged into
one complete language by Brown et al. (2003).
Towards a Universal Cheese-Sensory
Language
As mentioned previously, some of the key advantages
of using descriptive sensory vocabularies with defin-
itions and references are the ability to communicate
accurately results between multiple research groups or
to reproduce research results at different sites. Hirst et al.
(1994) compared the evaluation of cheese between
trained British and Norwegian panels using independ-
ently developed sensory languages. Cross-cultural dif-
ferences were attributed to the observed discrepancies
in term usage and sample differentiation. More recently,
ring trials at seven sites across the European Union
were conducted and a core sensory language for evalu-
ation was developed (Hunter and McEwan, 1998;
Nielsen and Zannoni, 1998). While similar patterns of
differentiation among samples by panels that use dif-
ferent languages are expected (particularly if the
vocabularies are comprehensive and the panellists
highly trained), standardised language with definitions
and references improves communication, cross panel
validation and subsequent application of descriptive
analysis results to instrumental or consumer data.
Further, other sources of variation potentially exist in
comparing panel results at different sites within the
same country using the same language. Drake et al.
(2002) reported on the performance of three descriptive
panels trained at different sites by different panel leaders
on a previously developed and standardised cheese
descriptive language (Drake et al., 2001). Panels were
able to communicate accurately attribute differences
between cheeses. However, differences were observed
between these panels in scale usage and attribute recog-
nition. These differences were attributed to the differ-
ences in panel leadership and the duration of panellist
training. In a similar study, Martin et al. (2000) com-
pared odour profile results of two panels. Language,
scale and method of presentation were standardised.
Results obtained from the two panels were similar. How-
ever, differences between attribute intensities were
reported and were attributed to differences in the experi-
ence and/or perception of individual panellists. As
with the conclusions of Drake et al. (2002), strong
panel–leader interaction was recommended as a means
of rectifying these differences, along with regular feed-
backs between the two panels.
As referred to previously, Tables 1, 2 and 3 present
terms used for descriptive sensory analysis by different
research groups for a wide variety of cheeses. In many
cases similar terms have been used to describe dominant
characteristics of different cheese types, suggesting that
it could be possible to develop and standardise a termin-
ology that can be used universally and for all cheese
types. The will to achieve this objective is much needed.
Relating Sensory Characteristics to
Consumer Preferences
Preference mapping is a generic term given to a collec-
tion of techniques, which have emerged in recent years
to quantify, analyse and interpret consumer preferences
for products. A premise can be made that the prefer-
ences of a group of consumers of sufficient size (60 or
more) will discriminate between comparable products
based on intrinsic sensory differences, and that the
degree and direction of discrimination will reflect the
number and the intensity of sensory differences that
can be perceived. Therefore, by simply quantifying and
analysing preference, or acceptance for the range, a
preference map reflecting sensory differences can be
drawn. The preferences of individual consumers can be
represented as a map loading, and areas of minimum
and maximum preference can be identified. In addition,
segmentation techniques, when used in tandem, can
illustrate opportunities for a selection of optimised prod-
ucts within the same range (or sensory space). Analysis
of consumer preference data in this way is referred to as
internal preference mapping (McEwan, 1995; Schlich,
1995).
When consumer preference evaluation of a set of
cheeses is followed by the application of descriptive
analysis to the same set of cheeses, this allows multi-
variate statistical analysis, e.g., using Partial Least
Squares Regression (PLSR; Martens and Martens, 1986),
and relation of descriptive properties that describe
exactly what attributes are perceived and at what levels
with the extent and direction of consumer preferences.
This additional analysis facilitates interpretation of the
internal preference map, and is referred to as external
preference mapping (McEwan, 1995; Schlich, 1995).
These techniques provide a powerful research tool for
market analysis and new product development. One
can extend the preference map by seeking technical
extensions, or relationships between preferences, sen-
sory characteristics and physical and chemical proper-
ties of products. One can also extend the preference
map by seeking behavioural extensions, or by deter-
mining characteristics of the consumers and how they
have developed their preferences and make their choice
decisions.
Preference mapping has been conducted with many
products, including cheese (McEwan et al., 1989;
Lawlor and Delahunty, 2000; Murray and Delahunty,
2000a,c; Bárcenas et al., 2001). Recently, Young et al.

(2003) conducted preference mapping of Cheddar
cheeses using consumers at two different locations
(Oregon and North Carolina, USA). Seven Cheddar
cheeses with distinct descriptive sensory properties
were selected. Six distinct consumer clusters were
identified, indicating a wide variability in consumer
preferences even among one cheese type. Analysis of
the consumer concept of ‘aged cheese flavour’ and
‘young cheese flavour’ indicated that consumers could
differentiate between young and aged Cheddar cheeses
and that these concepts were consistent with descrip-
tive panel language. However, the consumer concept of
‘Cheddar flavour’ varied widely and was not pinpointed
to specific descriptive cheese flavour terms. Lawlor and
Delahunty (2000) conducted preference mapping with
a diverse range of cheese types, and also found wide
variability in consumer preferences. Although a Blue
Shropshire cheese, described as coloured, mouldy and
crumbly, was the least liked overall (162 consumers), it
was preferred by two of seven segments of the con-
sumer sample, representing 50% of the total questioned.
On the other hand, a Gruyère cheese, described as
fruity, sweet and firm, was preferred overall, but was the
first choice of only one segment with 10 consumers.
Relating Sensory Perception to Chemical
Components and Instrumental
Measurements
Relating defined sensory flavour and/or texture to spe-
cific instrumental tests or chemical compounds is an
important and expanding area of research. Cheese
flavour chemistry and texture analyses are addressed
in detail in ‘Cheese Flavour: Instrumental Techniques’
and ‘Rheology and Texture of Cheese’ of Volume 1, but
sensory characteristics of cheeses cannot be addressed
without brief attention to this subject. Relating
sensory perception to instrumental measurements is
important because in certain cases an instrumental test
would be more cost-effective and/or convenient than
sensory testing. However, more importantly, establish-
ment of key relationships between sensory perception
and flavour chemistry or rheology provides the poten-
tial to link cheese flavour or texture to the technology
of cheese production; this is a key issue in providing a
consistent and high-quality product to the discerning
consumer. Relating sensory language and chemical
volatile compounds represents a challenge for several
reasons. The relative concentration of a compound in a
cheese is not necessarily a measure of its sensory impact
due to different sensory thresholds and the effects of the
food matrix on retention and release. The sensitivity
and selectivity of the extraction technique must also
be taken into account (Delahunty and Piggott, 1995).
Sensory Character of Cheese and its Evaluation 481
Finally, only a small percentage of the volatile compon-
ents in a food are odour-active (Friedrich and Acree,
1998; see also ‘Cheese Flavour: Instrumental Tech-
niques’, Volume 1). Establishing these relationships is
time-consuming and tedious. To use flavour as an
example, extensive and relevant instrumental volatile
analysis must be conducted, followed by gas chro-
matography–olfactometry (GC–O) and quantitative
analysis to pinpoint volatiles of interest. On the sensory
side, descriptive analysis with a defined and anchored
language is required. Sensory threshold testing to con-
firm that key volatile compounds are above detection
thresholds must be conducted, followed by descriptive
sensory analysis of compounds in model systems
across the concentration range found in the cheese to
confirm the sensory response (Drake and Civille,
2003). It should also be noted that the perception of
the cheese flavour is an integrated response to numer-
ous mixed stimuli, including volatile compounds, non-
volatile compounds and structural properties. The
perception of this stimulation is multi-modal, but
simultaneous, and therefore very complex.
Panelists tasted water-soluble extracts of Comté
cheese to identify fractions, which had particular
tastes, in an attempt to clarify the effect of peptides
and amino acids on flavour (Salles et al., 1995).
Preininger et al. (1996) used an unripened cheese
matrix to evaluate both volatile and non-volatile
flavour components of two Swiss cheese samples. A
similar study was conducted on Emmental cheese and
reduced-fat Cheddar cheeses (Rychlik et al., 1997;
Suriyaphan et al., 1999). Suriyaphan et al. (2001)
identified key chemical volatile components of
cowy/barny and earth/bell pepper sensory perceived
flavours in selected aged British Farmhouse cheeses.
In this study, sensory properties were identified by
descriptive sensory analysis, aroma volatiles were
quantified by gas chromatography–mass spectrometry
(GC–MS) and aroma properties described by GC–O.
Suspected key volatiles were selected from GC–O data
based on aroma properties and flavour dilution values.
The selected aroma components were subsequently
incorporated into mild (bland) cheese across the con-
centration range encountered in the Farmhouse
cheeses and evaluated by descriptive analysis. Studies
such as these provide convincing evidence of the con-
tribution of particular compounds to flavour. Model
systems have not as yet provided insights into the role
of compound mixtures and the role of compounds at
sub-threshold levels. These are complex issues and
will require extensive future research.
An alternative approach to determining the influence
of composition on sensory character is to use multivari-
ate statistical techniques, such as PLS, to determine
482 Sensory Character of Cheese and its Evaluation
relationships between compositional data and quantita-
tive descriptive sensory data. This technique has the
advantage of enabling comparison of all mathematically
possible combinations of compositional variables with
perceived intensity of one or more sensory characteris-
tics, following theoretically the principle of the compon-
ent balance theory (Mulder, 1952). The validity and
value of relationships determined in this way will
depend on the amount and type of compositional data
collected, and the accuracy of both the compositional
and the sensory data. Lawlor et al. (2001, 2002, 2003)
determined predictive models using this technique for
numerous flavour and texture attributes described in a
wide variety of cheese types.
Many studies have also been conducted to explore
the relationships between sensory properties, compos-
itional measurements and instrumental measurements
of cheese texture (Wium et al., 1997; Bachmann et al.,
1999; Drake et al., 1999b; Antoniou et al., 2000;
Benedito et al., 2000; Truong et al., 2002) and to
devise instrumental methods to assess more accurately
or predict sensory properties of cheese (Sorensen and
Jepsen, 1998; Breuil and Meullenet, 2001; Meullenet
and Finney, 2002). Lawlor et al. (2001, 2002, 2003),
using PLS, determined relationships between gross
composition and perceived texture for a wide variety
of cheeses, and found a number of consistent relation-
ships. In particular, it was found that firmness was
positively correlated with protein and mineral salt con-
tent, and negatively correlated with moisture and pH.
Both hand and mouth terms can be used for sensory
analysis of cheese texture (Drake et al., 1999c). In gen-
eral, empirical texture tests and large-strain tests (com-
pression) have been shown to correlate well with sensory
bite terms (firmness, elasticity) although the correlation
varies with cheese type, instrumental test and specific
sensory term and definition. More recently, Brown et al.
(2003) demonstrated specific knowledge gaps in relating
sensory chewdown terms to rheological tests. Sensory
rigidity and resiliency terms were correlated with rheo-
logical tests. However, chewdown terms such as ‘degree
of breakdown’, ‘cohesiveness’, ‘adhesiveness’, ‘smoothness
of mass’ and ‘smoothness of mouth coating’ were not
related to instrumental tests. Additional work is needed
to investigate the role that fundamental rheological tests
can play in differentiating and relating to these important
sensory texture parameters in cheese.
Conclusions
The sensory characteristics of cheese determine the
eating quality of cheese and consumer acceptability.
The appearance, flavour and texture of cheese are
extremely complex, not simply due to the very wide
diversity of cheese types that are produced, but also
the many stages that any cheese goes through during
its production and ripening. The complex compos-
ition and structure of cheese stimulate each of the
human sensory modalities at approximately the same
time, resulting in an integrated perception that a con-
sumer responds to during and after cheese consump-
tion. The dairy industry, including cheese production
and marketing, has relied on outdated grading and
judging methods for quality control and product
development for many years. While these methods
still have use, objective descriptive analysis tech-
niques are increasingly being applied in cheese quality
research in parallel with innovative studies of cheese-
making, cheese composition and consumer accept-
ability of cheese. Advances in the application of
objective sensory science techniques have improved
understanding of the relationships between these fac-
tors and the sensory attributes of cheese. However,
direct comparison of research findings between differ-
ent laboratories working with the same cheese type,
and between studies on different types of cheese, will
not be possible until such time as a universal lan-
guage to describe cheese sensory character is defined
and standardised.
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This Page Intentionally Left Blank
 Cheese Flavour: Instrumental
Techniques
J.-L. Le Quéré, Institut National de la Recherche Agronomique (INRA), Unité Mixte de
Recherche sur les Arômes (UMRA), Dijon, France
Introduction
The sensory properties of food are important determin-
ants in the choice of foodstuffs by the consumer, and
flavour plays a prominent role in this context. Flavour
may be defined as the combination of taste and odour,
sensations of pain, heat and cold (chemesthesis or
trigeminal sensitivity), and tactile sensation. Sensory
analysis is clearly the most valid means of measuring
flavour characteristics. Applied to cheese flavour, sen-
sory evaluation is a prominent descriptive tool which
is used widely in dairy science and industry (Issanchou
et al., 1997; see also ‘Sensory Character of Cheese
and its Evaluation’, Volume 1). However, determining
flavour also means analysing volatile compounds that
are sensed in the nose at the olfactory receptors either
via the orthonasal (odour) or retronasal (aroma)
routes when foods are eaten, non-volatile compounds
sensed on the tongue (taste), and compounds per-
ceived as mouthfeel and texture.
Instrumental analyses of flavour have been used pri-
marily to analyse volatile components. The main reason
for this is the major importance of aroma in the overall
flavour of a food, as is easily demonstrated by the diffi-
culties encountered by subjects attempting to identify a
particular flavour if the air flow through the nose is pre-
vented, and the fact that volatile components are more
amenable to conventional instrumental analysis than
non-volatile compounds. Therefore, since the early stud-
ies published in the 1960s and the 1970s (Dumont and
Adda, 1972, and references cited therein), instrumental
methods have concentrated on identification of aroma
compounds (Mariaca and Bosset, 1997). Only recently,
some significant efforts have been made to develop
instrumental procedures to characterise non-volatile
components in cheese which are responsible for cheese
taste (Salles et al., 1995a; Salles and Le Quéré, 1998;
Engel et al., 2000a,b; Le Quéré and Salles, 2001).
Instrumental analysis of aroma volatiles has been
the subject of important specialised treatises (for the
most recent literature on the subject, see Ho and Manley,
1993; Marsili, 1997; Mussinan and Morello, 1998;
Stephan et al., 2000; van Ruth, 2001a; Reineccius,
2002, and specifically for instrumental analysis of
Cheese: Chemistry, Physics and Microbiology, Third edition – Volume 1: General Aspects
ISBN: 0-1226-3652-X
Set ISBN: 0-1226-3651-1
volatiles in milk and dairy products see Delahunty and
Piggott, 1995; Mariaca and Bosset, 1997). Therefore,
the part of this chapter that will be devoted to the
analysis of cheese volatiles will focus on particular
techniques adapted to the particular characteristics of
cheese. Cheese flavour components result from the
principal biochemical degradation pathways: glycoly-
sis, lipolysis and proteolysis (see ‘Biochemistry of
Cheese Ripening: Introduction and Overview’, ‘Metab-
olism of Residual Lactose and of Lactate and Citrate’,
‘Lipolysis and Catabolism of Fatty Acids in Cheese’,
‘Proteolysis in Cheese during Ripening’ and ‘Catabol-
ism of Amino Acids in Cheese During Ripening’, Vol-
ume 1). The aroma compounds produced are mainly
hydrophobic, or lipophilic, and consequently they
tend to concentrate in the cheese fat according to their
water/fat partition coefficient. Instrumental analysis of
cheese volatiles must, therefore, consider, as a first
step, an extraction method suitable for separating
these volatiles from the cheese fat matrix. However, no
single method yields a ‘true’ picture of a food aroma
(Reineccius, 2002), and isolation and analysis of aroma
remain challenging (Teranishi, 1998). Moreover, not
only may the extraction step lead to artefacts, but the
total volatile content in most cases is very difficult to
relate to the flavour profile determined by a panel in
sensory evaluation. Therefore, it appears much more
efficient to concentrate efforts on the identification of
those compounds that are really relevant to flavour. As
no universally suitable extraction method exists, it
appears essential to choose a method that yields an
extract representative of the sensory properties of the
food (Abbott et al., 1993; Etiévant et al., 1994; Etiévant
and Langlois, 1998). Once this extraction method has
been chosen, the next steps involve various forms of
gas chromatography among which gas chromatogra-
phy–olfactometry (GC–O) plays a prominent role in
determining the key volatile compounds that con-
tribute significantly to the flavour of the food (Leland
et al., 2001), and gas chromatography–mass spectrom-
etry (GC-MS), which is essential for the identification
of those key odorants.
Water-soluble extracts (WSE) from cheese have
strong flavours (Biede and Hammond, 1979; McGugan
Copyright © 2004 Elsevier Ltd
All rights reserved
490 Cheese Flavour: Instrumental Techniques
et al., 1979; Aston and Creamer, 1986). Such extracts
contain some volatile compounds (Le Quéré et al.,
1996; Engels et al., 1997; Le Quéré and Salles, 2001),
partly extracted by water according to their water/fat
partition coefficient, although flavour compounds are
generally more lipophilic than hydrophilic. However,
the water extract mainly contains non-volatile com-
pounds. This non-volatile, water-soluble fraction is
composed of mineral salts, lactic acid, lactose, amino
acids and peptides and has characteristic taste proper-
ties (Salles et al., 1995a). Amino acids and small pep-
tides are considered to be mainly responsible for the
taste characteristics of water-soluble extracts (McGugan
et al., 1979; Aston and Creamer, 1986), their flavour
impact being modulated by interaction with calcium
and magnesium ions (Biede and Hammond, 1979).
Moreover, it has been recognised for a long time that
water-soluble, low molecular weight and mainly
hydrophobic peptides, which accumulate during ripen-
ing as a result of proteolysis, are responsible for bitter-
ness in cheese (Lowrie and Lawrence, 1972; Schalinatus
and Behnke, 1975; Furtado, 1984; Lemieux and Simard,
1992). Some fundamental studies on model com-
pounds have characterised the tastes of amino acids
and low molecular weight peptides (Salles et al., 1995a
and references cited therein); studies conducted on
tastes in casein hydrolysates were reviewed by Roudot-
Algaron (1996). However, until recently and apart
from bitterness, no clear sensory data were obtained
on water-soluble extracts from cheese. Although sev-
eral hundred peptides have been isolated and identi-
fied from various types of cheese, only a few small
peptides, that are suspected to be responsible for par-
ticular tastes, were isolated from the water-soluble
fractions of various cheeses and identified (Salles et al.,
1995a and references cited therein). However, no
direct correlations between these peptides and the
organoleptic properties of the fractions have been
demonstrated, apart from bitterness. In fact, the water-
soluble fraction of cheese generally has a very complex
composition, and separation and identification of indi-
vidual compounds are difficult. Moreover, most ana-
lytical techniques require the use of non-food-grade
solvents or buffers that make sensory evaluation of
sub-fractions difficult or impossible.
Part of this chapter will focus on recent advances
made to study and identify the taste-active compon-
ents present in the water-soluble fraction of cheese. A
general procedure for the preparation of fractions
involves an extraction of grated cheese by water fol-
lowed by a fractionation scheme, generally adapted
from the fractionation protocol used to isolate cheese
nitrogen fractions in the study of proteolysis in cheese
during ripening (Fox et al., 1994; McSweeney and
Fox, 1997). However, as sub-fractions have to be evalu-
ated sensorially to assess their relative sensory impact
and try to link it to their chemical composition, a suit-
able eluent has to be used in the chromatographic
steps. Water (Roudot-Algaron et al., 1993; Salles et al.,
1995a; Molina et al., 1999) or water-food-grade ethanol
mixtures (Lee and Warthesen, 1996a,b) have been
used for this purpose in combination with gel perme-
ation chromatography (GPC) or high-performance liq-
uid chromatography (HPLC). The final identification
step generally involves mass spectrometry (MS) and
tandem mass spectrometry (MS/MS) of nitrogenous
compounds isolated using HPLC, either in a stand-
alone mode or coupled with a mass spectrometer
(HPLC–MS) (Roudot-Algaron et al., 1993, 1994b;
Sommerer et al., 2001). A specific method for the isol-
ation of small peptides from cheese has been described
(Sommerer et al., 1998a).
As already outlined for cheese aroma, the relation-
ships between all flavour compounds identified in a
food and sensory perception experienced by con-
sumers when eating this food are still not entirely
clear. In fact, it is particularly difficult to predict a
flavour perception as it is still not known how the var-
ious components combine to produce an overall sen-
sory impression. Moreover, interactions between taste
and aroma (Noble, 1996) and interactions of trigemi-
nal sensations with taste and aroma (Green, 1996)
occur and play an important role in overall flavour
perception. However, methods that allow direct analy-
sis of flavour molecules released in the mouth during
consumption have been developed in recent years
(Taylor and Linforth, 1996; Roberts and Taylor, 2000).
Development of instrumental techniques and data
obtained recently for volatile and non-volatile flavour
compounds in cheese will be presented which may
explain the link between flavour perception and
cheese composition.
Finally, specific instrumental techniques have been
developed for the analysis of the complete flavour of
cheese. The methods currently used in the quality
control of food flavour are still usually based on sen-
sory evaluation by a panel of experts. These panels are
able to monitor the quality of a particular food, to
detect defects and to compare samples for classifica-
tion purposes. Nevertheless, obtaining results rapidly
at low cost using instruments could be desirable. The
so-called ‘electronic noses’ based on gas sensor tech-
nology, despite some important drawbacks for some of
them (Schaller et al., 2000a), are theoretically able to
perform some classification tasks (Schaller et al.,
1998), and some applications for the analysis of
cheese have been developed (Mariaca and Bosset,
1997; Schaller et al., 1999). However, two other global

analysis methods based on mass spectrometry seem
more powerful and reliable for purposes of classifica-
tion. One of these methods analyses total headspace
using a mass spectrometer, without any prior GC separ-
ation (Vernat and Berdagué, 1995). This method is
often referred to as a mass-based electronic nose. Alter-
natively, headspace sampling may be replaced by solid-
phase microextraction (SPME) of food volatiles
(Marsili, 1999). Both sampling methods, followed
directly by mass spectrometry, have found applications
for the rapid characterisation of cheese (Schaller et al.,
2000b; Pérès et al., 2001, 2002a). The second method
is pyrolysis mass spectrometry (Aries and Gutteridge,
1987), where a small food sample is pyrolysed at up to
500 °C. The resulting volatile fraction, characteristic of
the flavour but also of the matrix composition, is
analysed by a mass spectrometer. As with the other
rapid instrumental methods for classification, a pat-
tern or fingerprint is obtained for each sample, and
extensive data treatment, either by conventional multi-
variate statistics or artificial neural networks, allows
the construction of maps useful for classification and
quality control purposes (Pérès et al., 2002b).
Characterisation of Aroma (Volatiles)
Sample treatment
Volatile aroma compounds in cheese, like in other
foodstuffs, are hydrophobic, generally distributed in a
heterogeneous manner throughout the matrix and
present at low or even traces (10 g/kg) concentra-
tions. Their analysis in cheese requires homogenisa-
tion of the sample prior to extraction, where isolation
procedures adapted to lipophilic material dispersed in
trace amounts in a high-fat food are required. A prac-
tice commonly used for cheese is freezing the sample
in liquid nitrogen, followed by grating to a fine pow-
der with a blender at low temperature. The rind is
generally removed before sample homogenisation. The
powder is then used for subsequent steps as such, or
after dispersion and homogenisation in water, with
possible pH adjustment, if necessary.
Extraction methods
As already outlined, all the extraction procedures used
to isolate the aroma fraction from the cheese matrix
should be adapted to the analysis of trace levels of
lipophilic material dissolved in a fatty phase, while
minimising losses of highly volatile molecules and pre-
venting modification of compounds or the formation
of artefacts.
Many techniques have been proposed for the extrac-
tion of volatile compounds from cheese, amongst
Cheese Flavour: Instrumental Techniques 491
which, the most traditional and popular methods,
based on the volatility of aroma compounds, involve
distillation. Although steam distillation methods, and
particularly the simultaneous steam distillation/solvent
extraction (SDE) technique (Chaintreau, 2001) are
still used for dairy products, they have several draw-
backs. Highly volatile compounds are recovered
poorly, thermally sensitive compounds may disappear
and artefacts may appear unless distillation is per-
formed under a reduced pressure, with tight control of
temperature. In either approach, at atmospheric pres-
sure or under vacuum, the quality of the aroma extract
finally obtained is dependent on the volatility of the
aroma compounds and on their solubility in the sol-
vent used. If steam distillation (also called hydrodistil-
lation) is used without simultaneous solvent extraction,
it is necessary to add a large quantity of water to the
grated cheese to obtain a homogeneous slurry (c. 1 l
for 100 g of cheese). The distillate obtained is in fact
a dilute aqueous solution of volatile compounds
(Dumont and Adda, 1972). A subsequent extraction
with large amounts of a suitable solvent, followed by a
concentration step, is required.
High-vacuum distillation techniques, on the con-
trary, produce small volumes of concentrated aqueous
distillates (cheese moisture content only) that can be
extracted with tiny volumes of an organic solvent. A
typical experiment involves two steps. In the first step,
the frozen grated cheese is transferred to a cone-shaped
flask that is connected to a static vacuum (c. 10 Pa)
renewed from time to time. Using sub-ambient tem-
perature and rotation of the flask in order to break the
continuously dehydrating surface of the sample, the
volatiles are condensed with most of the cheese water
in traps maintained at the temperature of liquid nitro-
gen (Dumont and Adda, 1972; Le Quéré and Molimard,
2002). In the second step, the flask containing the
dehydrated cheese powder is connected to a molecular
distillation apparatus operating under a high vacuum
(c. 10 2 Pa). In this step, also called ‘cold-finger
molecular distillation’, the remaining water and volatiles
are transferred directly to the surface of a cold con-
denser maintained at the temperature of liquid nitro-
gen and situated at a very short distance from the
surface of the sample (Fig. 1). The condensed ice layer
on the surface of the condenser contains less volatile
and more lipophilic compounds. This fraction is com-
bined with the aqueous distillate obtained in the first
step. Since early workers (see Dumont and Adda, 1972
and references cited therein), high-vacuum distillation
techniques have been applied to the extraction of
aroma compounds from a large variety of cheeses (see
Mariaca and Bosset, 1997 and references cited therein;
Moio and Addeo, 1998; Moio et al., 2000). Working at
492 Cheese Flavour: Instrumental Techniques
Figure 1 Apparatus used for cold-finger molecular distillation. A,
I, traps for volatiles cooled with liquid nitrogen; E, round-bottom
flask containing dehydrated cheese powder, equipped with a
‘cold-finger’ cooled with liquid nitrogen; F, H, J, K, L, high-vacuum
stopcocks; M, connection to the high vacuum pumping system; N,
guard trap cooled with liquid nitrogen.
ambient or even at sub-ambient temperature, the tech-
niques prevent thermal degradation, but need a sub-
stantial amount of sample (c. 50–250 g) and are very
time-consuming (a long distillation period up to a few
hours). Moreover, the aqueous distillates have to be
extracted with a suitable solvent (e.g., dichloromethane
or diethyl ether) before performing further analysis. A
chemical fractionation by controlling the pH of the
aqueous distillate results in separation of the organic
extracts into acid, neutral and basic fractions which
may be analysed separately (Mariaca and Bosset, 1997;
Reineccius, 2002). A nice example on Swiss Gruyère
cheese was published 20 years ago (Liardon et al.,
1982; Bosset and Liardon, 1984, 1985; Bosset et al.,
1993). Specific methods for the analysis of volatile free
fatty acids may be found in the literature (see for
example Ha and Lindsay, 1990).
Cheese volatiles may be extracted directly from
samples by a solvent (e.g., diethyl ether). However,
further steps are required to separate the aroma from
the lipids that are also extracted very efficiently by the
solvent. While a simple solvent extraction introduces
some bias into an aroma profile, the following steps,
which are necessary, may add more bias (Reineccius,
2002). For instance, separation of volatile and non-
volatile compounds that have dissolved in the solvent
by distillation under high vacuum has been used by
Grosch and co-workers in the study of Swiss (Preininger
and Grosch, 1994; Preininger et al., 1994; Rychlik et al.,
1997) and Camembert (Kubickova and Grosch, 1997)
cheese and more recently by Qian and Reineccius
(2002a) in a study on Parmigiano-Reggiano cheese.
The risk in this case is that only the most volatile com-
ponents are selected from an oil-rich phase (Reineccius,
2002). Therefore, direct solvent extraction methods
that necessitate a subsequent distillation step under
vacuum do not offer a significant advantage compared
to other vacuum distillation methods.
Dialysis techniques, that are based on molecular size
differences and which separate molecules according to
their ability to diffuse through a specific membrane at
room temperature, may seem a good alternative.
Reineccius and co-workers have used this technique
(Benkler and Reineccius, 1979, 1980) and compared it
to other methods for the isolation of volatiles from
Cheddar cheese (Vandeweghe and Reineccius, 1990).
More recently, Spinnler and co-workers reinvestigated
the technique (Molimard and Spinnler, 1993) and
applied it to the extraction of aroma volatiles in the
study of the impact of the microflora on the aroma of
Camembert-type cheese (Molimard, 1994; Spinnler
et al., 1995). In order to eliminate adsorption and arte-
fact formation, these authors used 1% water in the sol-
vent, diethyl ether, to inactivate acidic sites on the
perfluorosulphonic acid membrane. They also improved
the dialysis yield by recycling the solvent using a distil-
lation device to recycle the solvent attached to the dialy-
sis cell (Fig. 2) in order to maintain a maximum
concentration gradient between the two compartments
of the cell (Molimard, 1994). Nevertheless, the method
is time-consuming (72 h dialysis time), and its effi-
ciency decreases dramatically as the number of carbon
atoms of the aroma molecule increases (n  10), modu-
lated by their hydrophobicity (Molimard and Spinnler,
1993).
A high-performance size-exclusion chromatographic
method has also been described for the purification of
aroma compounds from organic extracts of fat-containing
food (Lübke et al., 1996). The method was applied
Figure 2 Dialysis cell with solvent recycling device. A, B, cell
compartments; C, round-bottom flask containing solvent to distil;
D, condenser; E, magnetic stirrers; F, dialysis membrane.

successfully to the clean-up of a dichloromethane extract
from goat cheese (Lübke et al., 1996). The main interest
in this size-exclusion chromatographic method is the
limited number of injections necessary and the reduced
final volume of the fractions, which in terms of final use-
ful concentration, appeared significantly quicker and
gave rise to less thermally induced artefacts and to
reduced losses of the most volatile components than any
other distillation method (Lübke et al., 1996).
Headspace methods, either static or more often
dynamic, also called ‘purge-and-trap’ methods, are
popular techniques used to isolate volatiles from
cheese. Although direct analysis of the equilibrium
headspace would appear to be an ideal method to
study aroma compounds, in terms of sensory represen-
tativeness and ease of use, static headspace techniques
have severe limitations in terms of sensitivity, being
restricted to the most volatile and abundant compon-
ents (Mariaca and Bosset, 1997; Reineccius, 2002).
Dynamic headspace, or ‘purge-and-trap’, methods are
basically pre-concentration and enrichment techniques.
They use stripping of the volatiles from the cheese
samples, sometimes dispersed in water, with an inert
gas. The volatiles are concentrated in a cold trap or
adsorbed onto an inert support (adsorbing polymer,
generally of the Tenax® type) and analysed by
subsequent thermal desorption or elution by a suitable
solvent (Mariaca and Bosset, 1997; van Ruth, 2001a;
Reineccius, 2002). Although dynamic headspace methods
minimise artefacts developed or introduced during
sampling (van Ruth, 2001a), distortion of the aroma
profile may result from the trapping of aromas
(Reineccius, 2002), especially when polymeric adsor-
bents are used. However, despite the drawback of rela-
tively poor sensitivity compared to other extraction
methods, the main advantages of dynamic headspace
techniques are the small amount of sample needed to
perform the analysis (c. 20 g) and its speed (Le Quéré
and Molimard, 2002). The technique, even though
it favours the isolation of the most volatile flavour
compounds (Reineccius, 2002), has been applied widely
to the analysis of cheese volatiles (see for example
Arora et al., 1995; Canac-Arteaga et al., 1999a,b, 2000;
Larrayoz et al., 2001; Rychlik and Bosset, 2001a,b).
Recent comprehensive reviews on the technique
include Wampler (1997) and Pillonel et al. (2002). A
comparative study on the advantages of the use of
dynamic headspace with cheese samples in the ‘dry’
form or in ‘dispersed suspension’ in water has been
published recently (Larrayoz et al., 2001). The ‘dry’
method allowed the extraction of a greater number of
compounds and in larger quantities, but a few com-
pounds were extracted better using the ‘suspension’
technique (Larrayoz et al., 2001). Simultaneous distil-
Cheese Flavour: Instrumental Techniques 493
lation extraction (SDE) was also used in this study and
compared to dynamic headspace analysis. As expected,
the authors concluded that the techniques were com-
plementary; dynamic headspace extracted more highly
volatile compounds and SDE was more efficient for
phenols, free fatty acids, lactones and heavier alde-
hydes, ketones, alcohols and esters (Larrayoz et al.,
2001). Interference from water in dynamic headspace
that could be detrimental to the efficiency of the tech-
nique has been discussed in detail by Canac-Arteaga
et al. (1999a,b, 2000) and Pillonel et al. (2002).
Solid-phase microextraction, first developed for
the extraction of volatile organic compounds in water,
has been applied recently to the isolation of aroma
compounds from food (Harmon, 1997; Pillonel et al.,
2002; Reineccius, 2002). Solid-phase microextraction
partitions analytes between a liquid or a vapour phase
and a thin solid-phase adsorbent, of which there are
several choices in terms of polarity and film thickness,
coated on inert fibres, generally associated with a
syringe which serves as a direct injection device
(Harmon, 1997). The method, which is an equilibrium
one, can be performed either in the direct extraction
mode (immersion of the fibre in the sample matrix,
generally in an aqueous solution or suspension) or in
a headspace configuration. It can be automated very
easily, but the extraction of the solutes depends on
polarity, volatility, partition coefficients, sample
volume, temperature and the nature of the adsorbent-
coating material. Therefore, the technique exhibits a
certain degree of selectivity, but with the advantages of
sensitivity, ease of use, no solvent and small sample
volume (Harmon, 1997; Pillonel et al., 2002; Reinec-
cius, 2002). Solid-phase microextraction, used for the
first time for the analyses of cheese volatiles by Chin
et al. (1996), has since been used in some significant
applications on cheese aroma (Dufour et al., 2001;
Pillonel et al., 2002 and references cited therein).
Analysing volatiles directly by immersing the fibre in
highly complex matrices (as cheese) could damage
the fibre, and SPME is, therefore, used almost always
in the headspace mode. Comparison of direct SPME
and headspace SPME of Camembert volatiles obtained
after cryo-trapping of the aqueous phase under
vacuum showed only a slight reduction in sensitivity
using headspace SPME compared to direct SPME
( Jaillais et al., 1999).
The water-soluble extract (WSE) of cheese has been
described for a long time as possessing a strong flavour
(Biede and Hammond, 1979; Aston and Creamer,
1986; Engels and Visser, 1994; Salles et al., 1995a).
Besides non-volatile materials responsible for taste,
WSE also contains volatile compounds responsible for
its intense aroma. Thus, water-soluble extracts of various
494 Cheese Flavour: Instrumental Techniques
cheeses, obtained by direct extraction with water fol-
lowed by various centrifugation steps (Le Quéré et al.,
1996; Engels et al., 1997; Engel et al., 2002c) or by
pressing to obtain an aqueous phase called ‘cheese
juice’ (Salvat-Brunaud et al., 1995; Thierry et al.,
1999), have been investigated for their volatile compo-
nents. To be analysed using gas chromatography, WSEs
were either extracted with a suitable solvent (Le Quéré
et al., 1996), submitted to dynamic headspace analysis
(Engels et al., 1997; Thierry et al., 1999) or fraction-
ated using nanofiltration as the final membrane-filtration
step (Engel et al., 2002c).
Representativeness
As already outlined, because there is no universally
applicable method, none of the extraction techniques
described above yields an aroma isolate that truly repre-
sents either qualitatively or quantitatively the aroma
profile of a food (Reineccius, 2002). This fact explains
the frequently observed discrepancies between aroma
analysis of a food extract and sensory analysis of the
food itself. Therefore, the flavour analyst must choose
the isolation procedure best suited to address the prob-
lem faced: determination of the complete aroma profile,
identification of key odorants or off-flavours, monitor-
ing aroma changes with time in foods or prediction of
sensory properties (Reineccius, 2002). When the ulti-
mate aim of a particular study is the identification of the
compounds that are important for flavour (the key
odorants), the most reliable results will be obtained if
the odour of the extract resembles closely that of the
food itself (Etiévant et al., 1994; Etiévant and Langlois,
1998). Different sensory methods, which necessitate a
trained sensory panel, can be used to check the sensory
representativeness of the food extract odours (Etiévant
et al., 1994). When an estimation of the relative impor-
tance of key constituents in a single sample is required,
a similarity test is preferred. The panellists are asked to
score the similarity of the odour of the extracts obtained
by different methods to the odour of the food itself used
as reference on an unstructured 10 cm scale. This
approach was applied to three French and Swiss hard-
type cheeses by Etiévant et al. (1994) and Guichard
(1995). It was shown that the distillates obtained at a
pressure in the range 10–100 Pa had odours more simi-
lar to those of the cheeses than the distillates obtained
at a lower pressure (10 mPa). This result means that
strongly absorbed and less volatile flavour compounds,
extracted only at lower pressure, may not be important
for the odour of these cheeses. Similar results were
obtained for extracts of Camembert cheese, showing
clearly that the second step (molecular distillation oper-
ated under a high vacuum) is not necessary to obtain a
representative distillate of the cheese odour.
When applied to goat milk cheese, this approach
indicated that the best extract was obtained by a direct
water extraction of the cheese volatiles (Le Quéré et al.,
1996). This result could perhaps be explained by the
chemical and hydrophilic nature of the free fatty acids
identified as key odorants of goat milk cheese (Le Quéré
et al., 1996; Salles and Le Quéré, 1998; Le Quéré and
Salles, 2001). A key point in these evaluations of rep-
resentativeness is the choice of a suitable matrix for
testing the olfactory character of the extracts. For
cheese, the best results have been obtained when the
extracts are added to an emulsion, i.e., a matrix similar
to cheese in terms of fat composition (Etiévant et al.,
1994). Since, generally, a combination of techniques
should be used to obtain a reasonably complete view
of an aroma profile (Reineccius, 2002), it is notewor-
thy that sensory evaluation of headspace or SPME
extracts by ‘direct GC-olfactometry’ (i.e., without a chro-
matographic column) has been demonstrated recently
(Lecanu et al., 2002; Rega et al., 2003).
Identification of volatile aroma compounds using
hyphenated GC techniques
As aroma molecules are essentially volatile, the tech-
niques used to analyse them are usually based on separ-
ation using high resolution gas chromatography
(HRGC). Substantial progress has been made in this
field during the last 20 years and several stationary
phases are available which allow almost all separation
problems to be overcome. Combined with universal or
selective detectors, HRGC is clearly a fundamental tech-
nique, essential for all aroma identification studies. A
comprehensive review on the use of HRGC for the
analysis of milk and dairy products is available (Mariaca
and Bosset, 1997). Other interesting comments on qual-
itative, including multidimensional GC (Wright, 1997),
and quantitative aspects may be found in Marsili
(1997), van Ruth (2001b) and Reineccius (2002).
Among the hyphenated techniques that are coupled
to HRGC, the one that uses the human nose as a detec-
tor and known as gas chromatography–olfactometry
(GC–O, sometimes referred to as ‘GC-sniffing’), has
received considerable attention during the past 20
years in aroma research (see for example Blank, 1997;
Leland et al., 2001; Reineccius, 2002). The selectivity
of this specific detector is based only on the odorous
properties of the individual compounds separated by
HRGC. As the most abundant volatiles may have little,
if any, odour of significance in a food (Mistry et al.,
1997), GC-sniffing has been an invaluable tool for
identifying target compounds in aroma extracts that
are always very complex.
The primary aim of this technique is to discrimi-
nate the odorous compounds from the many back-
ground volatile components. The so-called ‘aromagram’

constructed from the chromatogram obtained by sim-
ply smelling a GC effluent (Blank, 1997; Reineccius,
2002) constitutes an interesting interface with sensory
analysis, as odour descriptors sensed at the GC sniff-
ing port can be compared to the descriptors generated
by a sensory panel. This method is particularly effi-
cient for identifying off-flavours. Selection of key
odorants or character-impact compounds in a food is
another objective of GC-sniffing. Quantitative
approaches (the true GC-olfactometry) based on
odour detection thresholds or on odour intensity have
been developed and are the subject of specialised trea-
tises (Mistry et al., 1997; Leland et al., 2001; van Ruth,
2001b; Reineccius, 2002).
Three different methods have been developed for
GC–O: dilution analyses based on determination of
detection thresholds, detection frequency methods and
intensity measurement methods. Original dilution
methods, CHARM (for Combined Hedonic Aroma Meas-
urement) analysis developed by Acree and co-workers
(Acree et al., 1984) and Aroma Extract Dilution Analy-
sis (AEDA) developed by Grosch and co-workers
(Ullrich and Grosch, 1987) are essentially screening
methodologies since the methods, based only on detec-
tion threshold determinations, violate certain sensory
rules and psychophysical laws (Reineccius, 2002 and
references cited therein). They can be used to deter-
mine those odorous compounds that are most likely to
contribute to the odour of a food. Originally developed
by McDaniel et al. (1990), the odour-specific magni-
tude estimation (OSME) method is basically a cross-
modal technique aimed at measuring the perceived
odour intensity of eluting volatiles. In OSME and other
cross-modality matching methods (Guichard et al.,
1995; Etiévant et al., 1999), results are not based on
odour detection thresholds, and only one concentra-
tion of the extract is evaluated by a panel, unlike dilu-
tion methods where several dilutions of the extract are
evaluated. Results can be subjected to statistical analy-
sis and more consistent results are obtained when pan-
ellists are trained (Callement et al., 2001). The
detection frequency methods, originally developed by
Roozen and co-workers (Linssen et al., 1993), and
referred to as nasal impact frequency (NIF) or surface
nasal impact frequency (SNIF) since the work of
Chaintreau and co-workers (Pollien et al., 1997), also
use a group of assessors who simply have to note
when they detect an odour in a single GC run (i.e.,
also at only one concentration). Those GC peaks being
detected as odorous by the greatest number of asses-
sors are considered to be the most important. Not
being based on real odour intensities, the method has
important drawbacks, especially when all the odorous
compounds are present above their sensory threshold
for all the assessors (Reineccius, 2002).
Cheese Flavour: Instrumental Techniques 495
There is no perfect GC-sniffing method for finding
key odorants in foods. Each of the methods described
above has its advantages and weaknesses. Only two
studies have compared the methods in terms of perform-
ance (Le Guen et al., 2000; van Ruth and O’Connor,
2001). In both cases, the results obtained with the dif-
ferent techniques were found to be very similar and
well correlated. Finally, the choice of a GC–O method
depends on the objective of the study, on the quality of
the panel and on the time scheduled for the analyses
(Le Guen et al., 2000). Dilution techniques are clearly
time-consuming, intensity methods require a trained
panel (Le Guen et al., 2000; Callement et al., 2001) while
detection frequency methods are the least demanding
but also the least precise (Le Guen et al., 2000).
The aim of any GC–O experiment is to determine
the relative odour potency of volatiles present in an
aroma extract or fraction and to prioritise compounds
for identification. This identification step is done mainly
through the use of another hyphenated technique that
couples HRGC to mass spectrometry (GC–MS). For
difficult identifications, GC coupled with Fourier trans-
form infrared spectroscopy (GC/FTIR) provides an
interesting complement to GC–MS (Le Quéré, 2000).
Mass spectrometry is also used for quantification pur-
poses through the use of a stable isotope dilution assay
(Milo and Blank, 1998; Blank et al., 1999 and refer-
ences cited therein). Such a precise quantitation is
required for the determination of odour activity values
(OAVs) generally calculated when using AEDA
(Grosch, 1994). Odour activity values, calculated as
the ratio of concentrations to odour thresholds,
despite their limitations in terms of psychophysical
validity (Mistry et al., 1997), give a good indication of
the respective contributions of key odorants to the
aroma of foods. They are the basis of the first attempts
at using recombination studies to validate impact
odorants sensorially in model cheeses (Grosch, 1994).
Aroma-recombination studies are the important last
step in sensorially verifying the analytical data
obtained by GC–O and for quantification of key odor-
ants of food (Mistry et al., 1997). Either bland
unripened cheese (Grosch, 1994; Preininger et al.,
1996; Kubickova and Grosch, 1998a) or specially
designed odourless model cheese systems (Smit et al.,
1995; Salles et al., 1995b) have been used to incorpor-
ate potential key odorants. Thus, the importance
of methional, 4-hydroxy-2,5-dimethyl-3(2H)-furanone
and 2-ethyl-4-hydroxy-5-methyl-3(2H)-furanone, acetic
acid and propionic acid was confirmed as key com-
pounds for the aroma of Emmental-type cheese
(Preininger et al., 1996). The branched-chain volatile
fatty acids, 4-methyloctanoic and 4-ethyloctanoic acids,
were confirmed to be essential for the typical goaty
note of goat cheese (Le Quéré et al., 1996) and their
496 Cheese Flavour: Instrumental Techniques
retronasal aroma thresholds were determined in a
cheese model (Salles and Le Quéré, 1998; Le Quéré
and Salles, 2001; Salles et al., 2002). Finally, the odour
profile of the aroma model built with a set of 11 potent
odorants identified in a GC–O study of an extract from
Camembert cheese (Kubickova and Grosch, 1997,
1998b), with four additional volatile compounds iden-
tified by headspace–GC–O, has been found to resem-
ble closely the aroma of genuine French Camembert
cheese (Kubickova and Grosch, 1998a; Grosch et al.,
2001).
The GC–O methods that have been developed dur-
ing the past 20 years, combined with either aroma
extracts, headspace or even SPME (Dufour et al., 2001),
have facilitated the identification of potent odorants in
various cheeses, including Swiss (Preininger and
Grosch, 1994; Rychlik et al., 1997; Rychlik and Bosset,
2001a,b), Cheddar (Arora et al., 1995; Christensen
and Reineccius, 1995; Dufour et al., 2001), Parmigiano-
Reggiano (Qian and Reineccius, 2002a,b), Blue (Le
Quéré et al., 2002; Qian et al., 2002), Mozzarella (Moio
et al., 1993), Grana Padano (Moio and Addeo, 1998)
and Gorgonzola (Moio et al., 2000) cheeses.
Characterisation of Sapid (Non-Volatile)
Flavour Compounds
Water-soluble extracts (WSE) of cheese
The water-soluble extract (WSE) of cheese has been
reported to possess a strong flavour (Biede and
Hammond, 1979; McGugan et al., 1979; Aston and
Creamer, 1986). Apart from some water-soluble volatile
components responsible for aroma, a WSE of cheese con-
tains mainly non-volatile components that have been
considered to be responsible for the taste of cheese
(McSweeney, 1997). It has been recognised for a long
time that bitterness, which can limit cheese acceptabil-
ity if too intense, is due to an excessive concentration of
low molecular weight and mainly hydrophobic pep-
tides, which accumulate during ripening as a result of
proteolysis (Lemieux and Simard, 1992; McSweeney,
1997). Amino acids and small peptides were hypothe-
sised to be mainly responsible for the basic taste of
cheese (McGugan et al., 1979; Aston and Creamer,
1986; Engels and Visser, 1994), their flavour impact
being supposedly influenced by their interaction with
calcium and magnesium ions (Biede and Hammond,
1979). However, the exact role of medium- and small-
size peptides and free amino acids in cheese flavour
has not been clearly demonstrated, although it is likely
that they contribute to the background flavour of cheese
(McSweeney, 1997). In fact, until recently and apart from
bitterness, no clear sensory data have been available for
WSEs of cheese and no direct correlations between spe-
cific nitrogen-containing compounds and organoleptic
properties of fractions have been demonstrated.
Among the mineral salts present in the WSE of
cheese, the compound responsible for the salty taste is
almost always supposed to be NaCl (McSweeney, 1997).
The taste of most high molecular weight salts is
known to be bitter rather than salty (McSweeney, 1997
and references cited therein).
Acid taste is caused by H3O and the principal acid
in cheese is lactic acid. However, total lactate concen-
tration does not seem to be a good index of cheese
acidity as the pH may increase during ripening caused
by the production of ammonia (McSweeney, 1997).
Moreover, the perception of acidity in cheese was
hypothesised to be influenced by the concentration of
NaCl (Stampanoni and Noble, 1991), and no correl-
ation between acid taste and either cheese pH or the
amount of lactic acid was found for the flavour of
Swiss cheese (Biede and Hammond, 1979), while the
acid flavour correlated positively with the levels of tri-
and tetra-peptides and with amino acids (Biede and
Hammond, 1979). It has also been hypothesised that
short- and medium-chain fatty acids might con-
tribute to the acid taste of cheese (McSweeney, 1997).
Although this assumption seems reasonable for short
chain acids (e.g., formic, acetic or propionic), their prin-
cipal contribution to cheese flavour is to its aroma in the
unionised form (RCOOH) (Le Quéré et al., 1996; Salles
and Le Quéré, 1998; Qian and Reineccius, 2002b).
Extraction, separation, identification of sapid
compounds in relation to their sensory properties
The study of taste-impact compounds in cheese, or
more precisely in its water-soluble fraction, involves
the study of soluble low molecular weight material
(i.e., small peptides, amino acids, organic acids, min-
erals, etc.) dispersed in a very complex mixture. As it is
necessary to assess the relative sensory impact of poten-
tial taste-active compounds, a fractionation scheme suit-
able for subsequent sensory evaluation is needed, and
non-food-grade solvents or buffers must be rejected.
Commonly used procedures involve extraction of
grated cheese with water, possibly completed by
precipitation of caseins and large peptides at pH 4.6,
leading to edible fractions with good recovery of
nitrogenous compounds (Kuchroo and Fox, 1982).
The fractionation scheme that follows is generally
adapted from the fractionation protocol used for isolat-
ing cheese nitrogen fractions for the study of proteolysis
(Fox et al., 1994; McSweeney and Fox, 1997). The fol-
lowing steps (Fig. 3) involve ultrafiltration using
membranes with 1, 3 or 10 kDa molecular weight cut-
off or precipitation with 70% ethanol (Cliffe et al.,

Figure 3 Possible fractionation schemes used to isolate and evaluate non-volatile compounds from cheese.
1993). The ultrafiltered water-soluble or 70% ethanol-
soluble extracts are then subjected to gel filtration
chromatography (Fig. 3). Sephadex G10 (Engels and
Visser, 1994; Roudot-Algaron et al., 1994a; Engels et al.,
1995; Molina et al., 1999), G15 (Roudot-Algaron et al.,
1993; Warmke et al., 1996; Kubickova and Grosch,
1998a), G25 (Cliffe et al., 1993; Salles et al., 1995a), or
Toyopearl HW-40S (Salles et al., 1995a, 2000; Sommerer
et al., 1998a, 2001) media have been used for this pur-
pose, using pure water (generally), 0.01 M NaCl
(Engels and Visser, 1994), or aqueous 0.5 M acetic
acid (Warmke et al., 1996; Kubickova and Grosch,
1998a) as eluent. The fractions obtained by gel perme-
ation chromatography may be evaluated sensorially
(Fig. 3) after freeze-drying and re-dissolution in water,
possibly with pH adjustment. Alternatively, liquid
chromatographic methods involving Sep Pak C18 car-
tridges eluted with a stepwise water–ethanol gradient
(Engels and Visser, 1994; Engels et al., 1995) or HPLC
Cheese Flavour: Instrumental Techniques 497
using a water/food-grade ethanol gradient (Lee and
Warthesen, 1996a,b) have been used instead of gel fil-
tration. This fractionation scheme was developed
originally in order to identify small hydrophobic pep-
tides supposedly responsible for taste characteristics
such as bitter or umami (Mojarro-Guerra et al., 1991;
Cliffe et al., 1993; Roudot-Algaron et al., 1993, 1994a).
A dedicated liquid chromatographic purification method
has been developed to isolate and identify oligopeptides
from the WSE of goat milk cheese (Sommerer et al.,
1998a, 2001). Systematic sensory evaluation of the
final fractions allows target fractions to be determined
that possess interesting tastes, and physicochemical
assessment of these key fractions should permit the
identification of those compounds that are really rele-
vant to the flavour of cheese (Engels and Visser, 1994;
Salles et al., 1995a).
Using this approach, some recent studies have been
dedicated to the taste of the WSE of various cheeses.
498 Cheese Flavour: Instrumental Techniques
Low molecular weight peptides, with two to four
amino residues, were identified in Vacherin Mont d’Or
(Mojarro-Guerra et al., 1991). As there was not enough
natural material available for sensory evaluation, com-
mercially available analogous synthetic peptides were
used in sensory experiments. The dipeptides tested
were dissolved in tap water at a rather high concentra-
tion (50 mg/100 mL) and were found to be essentially
bitter. However, neither quantitative nor threshold data
were estimated and the importance of these peptides
for the overall taste of the cheese was only an hypoth-
esis (Mojarro-Guerra et al., 1991). In a study on Ched-
dar cheese, Cliffe et al. (1993) found bitter fractions in
material thought to be large hydrophobic peptides
while lower molecular weight fractions with savoury
notes were thought to be small, more hydrophilic pep-
tides and amino acids. The flavour of the WSE of
Comté cheese was the subject of substantial efforts in
the early 1990s. A great variety of small peptides was
identified in these extracts (Roudot-Algaron et al.,
1993, 1994a,b). Some of them were found to be essen-
tially bitter (Roudot-Algaron et al., 1993), -glutamyl
dipeptides were found to be sour (Roudot-Algaron
et al., 1994a), but all the identified compounds,
including non-peptide material (Roudot-Algaron et al.,
1993; Salles et al., 1995a), were found at a concentra-
tion much lower than their threshold values. Although
possible synergistic effects between several molecules
found at concentrations below individual threshold
values cannot be a priori eliminated, these observa-
tions suggest that these components alone could not
affect cheese flavour (Salles et al., 1995a). Umami taste
was clearly identified in a fraction and easily explained
by a substantial amount of monosodium glutamate
which was found at a concentration ten times above its
threshold value, while the concentrations of the other
amino acids were all well below their thresholds
(Salles et al., 1995a).
Following the same methodology, Grosch and
co-workers evaluated the taste compounds of Emmen-
tal cheese (Warmke et al., 1996). The contribution of
individual free fatty acids, free amino acids, minerals,
biogenic amines, lactic and succinic acids, and ammo-
nia was estimated on the basis of taste activity values
(TAVs), a concept analogous to the odour activity val-
ues (OAVs), and defined as the ratio of concentration
to taste threshold. From these results, acetic and pro-
pionic acids were confirmed to be important contribu-
tors to the taste of Emmental cheese. Glutamic acid
was the major taste compound in the fraction contain-
ing free amino acids while all the ions investigated
might be involved in the taste of Emmental, as were
also biogenic amines (tyramine and histamine), ammo-
nia, lactic and succinic acids (Warmke et al., 1996).
However, taste evaluation of mixtures of compounds
conducted in tap water suggested that the character-
istic taste compounds of Emmental are acetic, propi-
onic, lactic, succinic and glutamic acids, each in the
undissociated form and/or as ammonium, sodium,
potassium, magnesium and calcium salts, as well as
chlorides and phosphates analogues (Warmke et al.,
1996). A study conducted on a model based on
unripened Mozzarella-type cheese confirmed the
importance of acetic, propionic, lactic, succinic and
glutamic acids, and sodium, potassium, calcium,
magnesium, ammonium, phosphate and chloride
ions to the taste of Emmental cheese (Preininger et al.,
1996).
The same approach applied to Camembert led to
the conclusion that the important taste contributors
for Camembert are acetic, butyric, 3-methylbutyric,
caprylic and succinic acids, monosodium glutamate,
ammonia and NaCl (Kubickova and Grosch, 1998a). It
was also found that the biogenic amine, cadaverine,
and the rare amino acids, ornithine and citrulline,
when present, are likely to contribute to the bitter
taste of Camembert (Kubickova and Grosch, 1998a).
The above results clearly indicated that only low
molecular weight compounds found in the WSE con-
tribute significantly to the taste of cheese, while small
peptides do not seem to be key flavour compounds, as
was previously hypothesised. A study on goat milk
cheese led to the same conclusions (Salles and Le Quéré,
1998; Salles et al., 2000; Le Quéré and Salles, 2001).
The taste of the various goat milk cheeses investigated
was essentially due to mineral salts and lactic acid.
Fractions rich in small peptides and free amino acids
were found to be essentially tasteless when evaluated
either in water (Salles et al., 2000) or in a model cheese
(Salles and Le Quéré, 1998; Le Quéré and Salles, 2001).
In a comparative study on cheeses made from cows’,
ewes’ or goats’ milk, Molina et al. (1999) concluded
that, even though differences were found in the inten-
sity and predominance of individual tastes in the frac-
tions of the cheeses made from the milk of the three
species, it was difficult to correlate the peptide pattern
and the free amino acid content of cheese with the
sensory evaluation of the fractions.
However, synergistic effects on taste have been
demonstrated between peptides, amino acids and min-
eral salts (Wang et al., 1996) and interactions between
tastes in mixtures may exist (Breslin, 1996). Therefore,
it appeared interesting to generalise the evaluation of
model mixtures of compounds that have been identi-
fied and quantified in the WSE of cheese (Warmke
et al., 1996; Kubickova and Grosch, 1998a). Moreover,
fractionation of the WSE by gel filtration has two
main limitations: poor resolution and the necessity of

tedious repetitive steps in order to obtain sufficient
peptide material for sensory evaluation. To clarify the
putative effect of the small water-soluble peptides on
the taste of cheese, it was therefore necessary to develop
a new isolation procedure. Nanofiltration using ionis-
able membranes with a molecular weight cut-off of
500 Da was used by Sommerer et al. (1998b). A nanofil-
trate was prepared from the 1-kDa permeate obtained
by ultrafiltration of the WSE (Fig. 3). A large propor-
tion of mineral salts and a substantial proportion of
amino acids were thus eliminated from the nanofiltra-
tion retentate in which the majority of small peptides
were concentrated (Sommerer et al., 1998b). This rela-
tively pure and edible peptide-containing fraction
could be used in sensory analysis, after incorporation
into a bland model cheese system (Salles et al.,
1995b), on its own or with the addition of putative
synergistic effectors such as mineral salts or amino
acids (Sommerer et al., 1998b). Using omission tests
(see Engel et al., 2002a,b, and references cited therein
for a comprehensive review), it was shown that small
peptides have no effect on the taste of goat milk
cheese, and no additive or synergistic effects were
found between those peptides and salts or amino acids
(Sommerer et al., 1998b). This unexpected result has
been confirmed after complete physicochemical
assessment of the WSE from goats’ milk cheese has
allowed the development of a model mixture that was
validated sensorially (Engel et al., 2000a). Using omis-
sion tests, the relative impact of WSE components on
goat cheese taste has been determined (Engel et al.,
2000b). Among the main taste characteristics of the
WSE from goats’ milk cheese (salty, sour and bitter),
saltiness was explained by additive effects of Na, K,
Ca2 and Mg2, sourness was due to synergism
between NaCl, phosphates and lactic acid, and bitterness
resulted entirely from CaCl2 and MgCl2. Amino acids,
lactose and peptides had no significant impact on the
taste properties of the WSE of goats’ milk cheese
(Engel et al., 2000b). The same procedure was applied
recently to a specially selected bitter Camembert
cheese (Engel et al., 2001a,b,c) and confirmed that the
WSE from cheese contained taste-active compounds,
the impact of which could be modulated by an effect
of the cheese matrix (Engel et al., 2001a). Sourness of
Camembert WSE was explained by an enhancing
effect of NaCl on the acid taste due to the concentra-
tion of H3O, saltiness was due to NaCl whereas
bitterness was mainly due to the bitter peptides found
in the fraction with a molecular weight in the range
500–1000 Da (Engel et al., 2001b). The intense prote-
olytic activity of the strain of Penicillium camemberti,
specially selected to develop bitterness in this case, has
been demonstrated to be responsible for the accumula-
Cheese Flavour: Instrumental Techniques 499
tion of small (MW  1000 Da) bitter peptides during
ripening (Engel et al., 2001c).
Dynamic Methods for Flavour
Characterisation
Even if the ‘best’ extraction and identification methods
are used, poor correlations are often found between
the overall levels of flavour components (volatile and
non-volatile) and sensory perception experienced by a
consumer. In other words, it is not enough to know
the exact composition of food in terms of flavour com-
pounds to understand perfectly the perception of its
flavour. In fact, the perception of flavour is a dynamic
process (Piggott, 2000). During the consumption of
food, the concentration of aroma compounds at the olfac-
tory epithelium and of sapid compounds at the taste
buds varies with time. Flavour components are released
progressively from the food matrix during chewing.
Kinetics of the release of flavour depends on the
nature of the food matrix composition and of individ-
ual mastication pattern. Sensory methods, such as
time-intensity, have been used to study the dynamic-
and time-related aspects of flavour perception (Piggott,
2000).
Release of volatiles in vivo
Techniques which measure volatiles directly in the
mouth or in the nose have been developed to obtain
physico-chemical data that reflect the pattern of aroma
molecules released from food and that are effectively
present at the olfactory receptors during consumption
(Linforth and Taylor, 1993; Taylor and Linforth,
1994). Among the various approaches aimed at sam-
pling aroma from the nose (nose-space), the collection
of expired air samples on Tenax® traps (Fig. 4) pro-
vided the first robust results (Linforth and Taylor,
1993; Taylor and Linforth, 1994). When applied to
Tenax trap GC
MS
Analysis
Pump
Sampling
Figure 4 Collection and analysis of expired air by Tenax trap-
ping and GC–MS (reproduced from Roberts and Taylor (2000),
with permission from the American Chemical Society).
500 Cheese Flavour: Instrumental Techniques
Cheddar cheese (Delahunty et al., 1994), the ‘buccal
headspace’ method demonstrated that, despite a simi-
lar composition of volatiles found with conventional
headspace analysis, some cheeses, depending on their
fat content, released a different balance of volatiles
during consumption (Delahunty et al., 1996a). Gas
chromatography–olfactometry of buccal headspace
showed a number of volatile compounds which have
been suspected to contribute primarily and most likely
to Cheddar cheese flavour (Delahunty et al., 1996b). It
was presumed that the buccal headspace extract was
representative of the aroma compounds that a con-
sumer perceives during consumption (O’Riordan and
Delahunty, 2001).
By overlapping the sampling time periods, release
curves can be constructed and temporal changes
reflecting relative concentrations of volatiles at a par-
ticular moment during consumption can be deter-
mined (Linforth et al., 1996). When applied to Cheddar
cheese, ‘temporal buccal headspace’ results, obtained
on an accumulated ‘time-concentration’ basis (four
time periods: 15, 30, 45 and 60 s of cheese consump-
tion), were correlated with sensory time–intensity data
(Delahunty et al., 1996c). Time-course data confirmed
the results of conventional analysis while providing
improved sensory predictions from the instrumental
results (Delahunty et al., 1996c). Mastication behav-
iour using electromyography and saliva production
rates of individuals have also been measured during
consumption of Cheddar cheese (Delahunty et al.,
1998; O’Riordan et al., 1998). Combined to nose-space
analysis and sensory evaluation using free choice pro-
filing, these authors demonstrated that although there
were differences in chewing styles and saliva produc-
tion rates, the assessors’ individual nose-space profiles
were very similar for all Cheddar cheeses examined
(Delahunty et al., 1998). Partial least-squares regres-
sion analysis allowed the most important flavour
differences between cheeses to be predicted from
the volatiles released during consumption (O’Riordan
et al., 1998).
Recently, atmospheric pressure ionisation–mass
spectrometry (API–MS) has been developed to monitor
aroma release during chewing (Taylor et al., 2000). Air
from the nose (nose-space) is sampled directly into the
API-MS source through an interface (Fig. 5), making
real time breath-by-breath analysis possible (Linforth
et al., 1996; Taylor and Linforth, 1996). Therefore,
by combining time–intensity sensory studies with
nose-space analysis, it is now possible to relate tem-
poral parameters of aroma release to perception (Linforth
et al., 2000). The method, reviewed in detail in
specialised treatises (Roberts and Taylor, 2000; Taylor,
2002), has been applied recently to soft French
Nitrogen 10 l/min Cone
Venturi
Quad
Corona
Breath flow pin
Figure 5 Principle of the API-MS interface used for breath by
breath analysis (reproduced from Roberts and Taylor (2000),
with permission from the American Chemical Society).
cheeses (Salles et al., 2003). Three French mould-
ripened soft cheeses (Brie made from pasteurised milk,
Camembert made from pasteurised milk and from raw
milk) were evaluated by a panel of 15 assessors (Salles
et al., 2003). Retronasal aroma profiles made by cita-
tion frequency of attributes revealed four main
descriptors for the three cheeses. The sulphury note
(cabbage/cauliflower/vegetable) was particularly intense
for the Camembert cheeses, while the buttery/creamy
note was important for the three cheeses studied;
the mushroom attribute was less intense in the
Camembert cheeses, and ammonia was perceived in all
cheeses but was found particularly difficult to score by
the panellists (Salles et al., 2003). Therefore, the three
main aroma notes (sulphury, buttery and mushroom)
were selected for subsequent time–intensity (TI) scor-
ing (15 assessors evaluated each attribute, with three
replicates of each cheese). Gas chromatography–olfac-
tometry of the dynamic headspace sampling of the
three cheeses allowed odour-active compounds to be
identified, amongst which sulphur compounds
(methanethiol, dimethylsulphide (DMS), S-methylthio-
acetate, dimethyldisulphide (DMDS), 2,4-dithiapentane,
dimethyltrisulphide, 2,3,5-trithiahexane and dimethyl-
tetrasulphide) could be related to the sulphury attribute
scored by the panellists. However, API-MS nose-space
experiments allowed the detection of only six com-
pounds of which three contained sulphur ones (DMS,
S-methylthioacetate and DMDS). Simultaneous TI
scoring of the sulphury note allowed a perfect super-
position of the time–intensity curve with the release of
the sulphur compounds (Fig. 6). The most significant
perception and flavour release parameters allowed the
three cheeses to be well discriminated by principal
component analysis (PCA), showing a good agreement
between perception scored by assessors and consist-
ency in their release of aroma compounds while eating
cheeses (Salles et al., 2003). Another PCA analysis
showed a positive correlation for the sulphury note
between the perception parameters derived from the
TI curves and parameters derived from the aroma
 Cheese Flavour: Instrumental Techniques 501

Perceived intensity
10
9
sampling technique using a motor-driven ribbon placed
8 T-I
7 across the tongue while a panellist chews a food sam-
6
5 Sulphury/cabbage attribute ple has also been described (Davidson et al., 2000). At
4
3
2
the end of the eating process, the ribbon was cut into
1
0
5 cm lengths after estimation of the saliva weight
25 adsorbed on the ribbon, each piece representing a cer-
20
tain time. Non-volatile components were extracted
I (m/z 90.8)

S-methyl thioacetate
15
10
m/z 90.8 from the pieces of ribbon with a solvent and their con-
5 centration determined by direct liquid phase API- or
0
electrospray (ES)–MS (Davidson et al., 2000). Tem-
600
500
poral release of sucrose and glucose from biscuits, of

API-MS
DMDS
I (m/z 93.7)

400 sodium from potato crisps, of sucrose, glucose and

300 m/z 93.7
200
fructose, citric and malic acids from fresh orange and
100 finally minerals (sodium, calcium and potassium)
0
400
from Cheddar cheese was monitored successfully
350 (Davidson et al., 2000). The cotton bud technique has
I (m/z 62.9)

300 DMS
250
200 m/z 62.9
150
100
50
0 with literature data had been incorporated (Pionnier
0 1 2 3
Time in min
Figure 6 Flavour release from Camembert cheese for one
assessor. Simultaneous time–intensity scoring of the sulphury/
cabbage attribute and API-MS analysis of S-methyl thioacetate,
dimethyldisulphide (DMDS) and dimethylsulphide (DMS) in the
nose-space.
release curves (Salles et al., 2003), as suggested by the
characteristic curves presented in Fig. 6 for one panel-
list within one session.
Non-volatiles in vivo
Development of methods to study flavour release has
concentrated mainly on the volatile fraction, while
only a limited number of studies have been devoted to
the release of non-volatile compounds in the mouth.
Conductivity measurements have been used to relate
the release of salt during chewing to Cheddar cheese
texture (Jack et al., 1995), and a similar approach with
additional in-mouth measurement of pH has been
used with a variety of foodstuffs, including Cheddar
cheese (Davidson et al., 1998). However, in these
approaches, the sensors available for in vivo measure-
ments only give the best estimate for salt (non-specific
to sodium) and acid release. Saliva sampling using cot-
ton buds coupled to a direct liquid mass spectrometry
procedure has been described to study the rate of
release of sucrose (Davidson et al., 1999). Panellists
were instructed to take a swab from a specific location
on the tongue at different times during mastication
using a cotton bud. The weight of saliva swabbed was
measured and sucrose concentration was monitored
using liquid-API–MS after extraction by a methanol–
water solution (Davidson et al., 1999). A continuous
been applied recently to a model processed cheese in
which aroma and non-volatiles compounds consistent
4 5
et al., 2003). As it was demonstrated that with certain
foodstuffs the increased frequency of sampling affected
the chewing pattern (Davidson et al., 2000), each pan-
ellist produced only one saliva sample per mastication,
at a time-consuming cost, however. Using ES–MS in
negative ionisation mode, time-course release curves
for minerals (sodium, calcium, magnesium and potas-
sium), amino acids (leucine, phenylalanine, glutamic
acid), organic acids (citric, lactic, propanoic and butyric)
and phosphoric acid have been obtained (Pionnier et al.,
2003). As a typical example, Fig. 7, shows release
curves from cheese for phenylalanine, glutamic acid,
leucine, phosphoric and lactic acids obtained for one
assessor. The first conclusion that could be stressed
from the analyses of the release curves is that individ-
ual physiological parameters (mainly mastication
behaviour and salivation rate) are related more closely
to the temporal release of taste compounds than to
their physico-chemical properties (Pionnier et al., 2003).
Model mouth systems
A number of mechanical devices which mimic in more
or less detail the processes that occur in the mouth
during eating ‘model mouths’ have been developed
(Piggott, 2000 and references therein). These are often
variants of dynamic headspace analysis, but their aim
is to obtain time-resolved samples containing volatiles
as similar as possible to those present during actual
eating. The various parameters like temperature, air
flow, mastication rate and addition of artificial saliva
can be varied to study their effects on volatile flavour
release. The main advantages of model mouths are the
large quantities of food samples that can be handled,
overcoming some sensitivity problems encountered
502 Cheese Flavour: Instrumental Techniques
0.6
Concentration (g/100 g saliva)
0.5
0.4
0.3
0.2
0.1
0.0
0 10
Figure 7 Flavour release from a flavoured model cheese for one assessor. In mouth, time-course release of non-volatile com-
pounds (concentration in g/100 g saliva) using the cotton bud technique. Data points measured in electrospray-MS in negative ion-
isation mode are the mean of three replicates.
when monitoring volatiles at low concentrations (Taylor,
2002), and the suppression of inter-individual vari-
ations, always encountered when working with a
panel, that can be detrimental to a robust interpreta-
tion of the data. Recently, using an imitation cheese
preparation, the release of volatile flavour compounds
from the Retronasal Aroma Simulator (RAS), originally
developed by Roberts and Acree (1995), has been
compared with flavour release in vivo using API-MS
detection in both cases (Deibler et al., 2001). While
delivering higher concentrations of volatiles than from
human breath, the RAS gave a good approximation of
time-averaged flavour release in the mouth, with
volatile compounds present at similar ratios (Deibler
et al., 2001). Volatiles in the RAS effluent from Ched-
dar, Brie and vanilla ice cream were measurable
(Deibler et al., 2001). The model-mouth device origin-
ally developed by Roozen and co-workers (van Ruth
et al., 1994) has been used to investigate the relation-
ships between the gross, non-volatile and volatile com-
positions and the sensory attributes of eight Swiss-type
cheeses (Lawlor et al., 2002). Eight flavour attributes
were found to be correlated with subsets of volatiles,
amino acids, free fatty acids and gross compositional
constituents with, for instance, the nutty flavour of
Emmental that was positively correlated with the concen-
trations of propionic acid, ethyl acetate and 2-pentanone
(Lawlor et al., 2002).
Flavour release and flavour perception are dynamic
processes and must be studied using dynamic methods
(Piggott, 2000). Dynamic physico-chemical methods
have been developed to study the parameters of flavour
release from foods. Parallel increased applications of
dynamic sensory methods provide a better understand-
ing of food flavour, with important results obtained for
Phenylalanine
Glutamic acid
Leucine
Phosphoric acid
End of mastication
Lactic acid
20 30 40 50 60 70 80 90 100
Time (s)
cheese flavour. However, further work is needed to
improve our knowledge of various interactions arising
at different levels in the process of food consumption:
e.g., interactions between food ingredients (Delahunty
and Piggott, 1995; Pionnier et al., 2002; Taylor, 2002),
and interactions at the perceptual levels such as taste-
aroma interactions (Noble, 1996; Given and Paredes,
2002; Hollowood et al., 2002; Taylor, 2002), or trigem-
inal interferences (Green, 1996; Given and Paredes,
2002), as these play a fundamental role in overall
flavour perception.
Global and Fast Assessment of Cheese
Flavour
The methods currently used to evaluate and control
the quality of cheese flavour are still essentially based
on sensory evaluation by a panel of experts. These
trained panels are able to handle such difficult tasks
like quality monitoring through descriptive analysis,
off-flavour detection and comparison of samples for
classification purposes. It could be interesting for such
tasks to substitute humans by instruments that could
give quicker answers at a reduced cost.
Electronic nose
Evaluation of the complete aroma emitted from food
using gas sensors, the so-called ‘electronic noses’, is
now theoretically feasible (Hodgins, 1997; Schaller et al.,
1998). Electronic noses are composed of arrays of
non-specific gas sensors which are based on different
physical principles (Hodgins, 1997; Schaller et al.,
1998). The most common sensors are semiconducting
metal oxides and conducting organic polymers, but

they all give rise to a response with a typical pattern.
Therefore, pattern recognition software, using either
standard statistics or artificial neural network technol-
ogy, must be used for data treatment and final presen-
tation of the results (Hodgins, 1997; Schaller et al.,
1998). The electronic nose is particularly attractive for
quality control applications where conformity/non-
conformity answers are expected. Some discrim-
inative studies have been conducted on cheese
samples (Schaller et al., 1998 and references cited
therein). Using metal oxide semiconductors, it was
possible to distinguish between five Swiss cheese vari-
eties (Mariaca and Bosset, 1997). However, some prob-
lems occurred with the repeatability of the system that
could be possibly related to the product itself, the sam-
pling technique or the moisture content of the air used
for sampling, precluding its use in routine tests
(Schaller et al., 1998). Samples of Swiss Emmental
cheese at different stages of ripening have been evalu-
ated using different technologies over a period of one
year (Schaller et al., 1999). The metal oxide semicon-
ductors technology has given the best discriminative
results. However, the sensors seemed to be damaged
by short-chain fatty acids released from cheese. Con-
ducting organic polymer sensors showed poor sensi-
tivity to volatile components of cheese, the main
problem being that these sensors are unstable (Schaller
et al., 2000a). The other technologies tested were not
sensitive enough to cheese volatile compounds and
electronic noses containing these sensors showed poor
discriminative power (Schaller et al., 1999). However,
recently, the ripening of Danish Blue cheese was moni-
tored by means of an electronic nose which contained
14 conducting polymer (polyaniline) sensors; results
were found to be highly correlated to those of sensory
analysis and GC–MS analysis of volatile compounds
during a 5–12-week ripening period (Trihaas et al.,
2003). The close control of the experimental sampling
conditions (quality of dry air with a humidity 0.5%
and equilibration time at controlled temperature)
might explain this success (Schaller et al., 1998). Nev-
ertheless, despite some success in some classification
tasks when using perfectly controlled sampling condi-
tions, electronic noses hardly meet the requirements of
the food industry in terms of precision, reproducibility,
sensitivity and stability. Of particular importance, the
sensors are known to deteriorate over time or can be
poisoned, therefore changing their response. Even
with frequent calibration, the inherent weaknesses of
the technique make the general applicability of the
databases problematic. Moreover, these instruments
cannot be used to identify single odorants or to differ-
entiate samples with subtle differences in distinctive
sensory attributes. Therefore, in off-flavour studies,
Cheese Flavour: Instrumental Techniques 503
where identification of the off-flavour compound is a
pre-requisite and in quality control assessment, they
may be used successfully only after recognising their
inherent weaknesses (Reineccius, 2002).
Mass spectrometry-based systems
For classification purposes, two other global and fast
analytical methods, based on mass spectrometry, have
been used for dairy products and seem more powerful
and reliable than electronic noses. The first consists of
a global analysis of a headspace sample by a mass
spectrometer operated in electron ionisation mode,
without GC separation (Vernat and Berdagué, 1995).
The feasibility of the method was originally demon-
strated for rapid classification of four rather different
French cheeses (Vernat and Berdagué, 1995). This
method is often described as a ‘MS-based electronic
nose’ (Schaller et al., 2000b). The mass patterns
obtained, considered as fingerprints of the food prod-
ucts analysed, also need data treatment, either by con-
ventional statistics or artificial neural networks. The
technique has been used successfully to discriminate
four Swiss Emmental cheeses differing in age (Schaller
et al., 2000b), and Camembert-type cheeses according
to their origin, manufacturing process or ripening
stage (Pérès et al., 2002a).
Solid-phase microextraction may be used as a pre-
concentration technique instead of dynamic headspace
analysis (Marsili, 1999). Applied to rapid characterisa-
tion of cheeses, SPME has been demonstrated to be a
very efficient pre-concentration technique (Schaller
et al., 2000b). In the task of discriminating Swiss
Emmental cheeses ripened for different times, SPME
has been found to be superior to dynamic headspace
analysis in terms of repeatability, simplicity and com-
patibility with an autosampler (Schaller et al., 2000b).
However, when applied to the characterisation of
Camembert-type cheese (Pérès et al., 2001), SPME
yielded less satisfactory results than those obtained by
dynamic headspace analysis (Pérès et al., 2002a). The
better performance of the dynamic headspace method
in that case was attributed to the absence of signal
drift (ageing of the SPME fibres causes drift, as
demonstrated by Pérès et al., 2001) and to automation
of the injection of sample into the mass spectrometer.
According to the authors, the protocol chosen for the
analysis by dynamic headspace-MS was more efficient
than SPME in terms of extraction yield, and reduced
thermal, mechanical and chemical modification of the
samples (Pérès et al., 2002a).
Developed in the 1980s for food applications, direct
pyrolysis-MS is another method that delivers ‘finger-
prints’ which can be used for classification/authentication
504 Cheese Flavour: Instrumental Techniques
purposes (Aries and Gutteridge, 1987). With this
method, a tiny sample is pyrolysed rapidly at up to
530 °C and the resulting volatile fraction, characteris-
tic of the flavour but also of the matrix breakdown, is
analysed immediately by a mass spectrometer operated
in low energy electron ionisation mode. Here again a
mass pattern, this time rather complex, is obtained for
each sample and several data pre-processing steps are
often necessary to select a reduced number of mass
fragments that allow satisfactory classification. Curie-
point pyrolysis–mass spectrometry with associated
multivariate data analysis techniques is considered as a
powerful classification tool in microbiology for the
recognition of micro-organisms (Talon et al., 2002 and
references cited therein) and food science (Aries and
Gutteridge, 1987; Pérès et al., 2002b and references
cited therein). However, when applied to the discrimin-
ation of five Camembert-type cheeses, it appeared less
competitive than SPME–MS or dynamic headspace–MS
in terms of sample preparation and analysis time
(Pérès et al., 2002b). The main advantage of the method
is that it provides a specific fingerprint of the cheese
matrix which could be potentially related to textural
parameters (Pérès et al., 2002b). Recently, in a similar
approach, the proton transfer reaction mass spectra
(PTR-MS, another atmospheric pressure ionisation
mode MS source) of the static headspace of Mozzarella
cheese have been found to display comparable dis-
crimination power to sensory descriptive analysis
(Gasperi et al., 2001).
Concluding Remarks
Cheese is a biochemically active product that under-
goes many changes during ripening. The development
of flavour is one of the consequences of these bio-
chemical changes that occur over the entire ripening
period. Modern instrumental methods allow for detailed
analyses of volatile compounds, and some pertinent
complementary sensory information can be obtained
by combining gas chromatography with olfactometry.
Recent developments have allowed the identification
of the role of non-volatile components in the overall
flavour of cheese. Nevertheless, the relationship between
flavour–aroma and sapid compounds present in a food-
stuff and sensory perception of that food by a consumer
is not so easy to establish. It is still not well under-
stood how the various flavour-active components
combine to produce a particular sensory perception.
Recent developments in dynamic instrumental meth-
ods that can follow the in vivo sequential release of the
flavour molecules are valuable tools that can account
for the balance of flavour compounds released, a bal-
ance that changes with time. With more complete and
accurate information, combined flavour chemistry and
sensory evaluation should help understand the relation-
ship between flavour stimuli and perceived flavour and
explain the mechanisms of flavour perception.
Authentication of cheese (for instance, varieties
with protected designations of origins) is another chal-
lenge. Tools developed recently that combine analyt-
ical instrumentation for global assessment of flavour
with multivariate data analyses have demonstrated
their usefulness for classification purposes.
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 Rheology and Texture of Cheese
D.J. O’Callaghan and T.P. Guinee, Dairy Products Research Centre, Teagasc, Ireland

Introduction – Overview of Cheese 3. the use of cheese as an ingredient, as they influence

Rheology and Texture its behaviour when subjected to different size reduc-
tion methods (such as shredding, grating or shear-
Rheology of materials, e.g., cheese, may be defined
ing) and how it interacts and blends with other
simply as the study of their deformation and flow
ingredients in foods in which cheese is an ingredient.
when subjected to a stress or strain. The rheological
4. its ability to retain a given shape at a given tempera-
properties of cheese are those that determine its
ture or when stacked;
response to stress or strain, as applied, for example,
5. its ability to retain gas and hence to form eyes or
during compression, shearing or cutting. In practice,
cracks or to swell.
such stresses and strains are applied to cheese during
processing (e.g., portioning, slicing, shredding and
Hence, the rheological properties of cheese are sig-
grating) and consumption (slicing, spreading, masticat-
nificant quality attributes of importance to the manu-
ing and chewing). The rheological properties include
facturer, pre-packer, distributor, retailer, industrial user
intrinsic characteristics such as elasticity, viscosity and
and consumer.
viscoelasticity that are related primarily to the compos-
The rheology of cheese is a function of its compos-
ition, structure and the strength of attractions between
ition, microstructure (i.e., the structural arrange-
the structural elements of the cheese. The rheological
ment of its components), the physico-chemical state
characteristics of cheese are quantified by rheolog-
of its components, and its macrostructure, which
ical quantities that are measured in tests involving the
reflects the presence of heterogenities such as curd
application of stress or strain under defined experi-
granule junctions, cracks and fissures. The physico-
mental conditions. The output variables from these
chemical properties include parameters such as the
tests (e.g., creep, stress relaxation, compression tests),
level of fat coalescence, ratio of solid-to-liquid fat,
which may include change in dimensions over time,
degree of hydrolysis and hydration of the para-
the ratio of stress-to-strain for certain strain levels,
casein matrix, and the level of inter-molecular
stress or strain required to induce fracture, enable
attractions between para-casein molecules. Hence,
the determination of quantities such as shear modulus,
the rheological characteristics differ markedly with
fracture stress and firmness. In lay terms, the behav-
the cheese variety and age. The effect of variety on the
iour of the cheese when subjected to these stresses
rheological properties is readily apparent on compari-
and strains is referred to by descriptive terms such
son of an almost-flowable mature Camembert with
as hardness, firmness, springiness, crumbliness or
a firm, brittle Parmesan or of a crumbly Cheshire
adhesiveness.
cheese or with an elastic springy Swiss-type cheese
Owing to the variations in manufacturing condi-
or String cheese (Table 1). Similarly, the influence of
tions and composition, different cheese varieties exhibit
age is clear on comparison of a young (e.g., 1–2
a wide range of rheological behaviour, ranging from
months) rubbery Cheddar with a fully mature pli-
the viscous behaviour of soft cheese to the elastic
able Cheddar (Table 1).
behaviour of hard cheeses at low strain.
Cheese rheology and factors that affect it have been
The rheological properties of cheese are of consid-
reviewed extensively (Sherman, 1969; Eberhard, 1985;
erable importance as they affect:
Visser, 1991; van Vliet, 1991a; Rao, 1992; Prentice et al.,
1. its handling, portioning and packing characteristics; 1993; Ustunol et al., 1995; Beal and Mittal, 2000; Fox
2. its texture and eating quality, as they determine the et al., 2000; Madsen and Ardö, 2001; Guinee, 2003).
effort required to masticate the cheese or alterna- In this chapter, the basic rheological characteristics of
tively the level of mastication achieved for a given cheeses in general and the methods for their quantifi-
level of chewing. The degree of chewing required cation will be examined. The effects of compositional
may, in turn, influence the flavour/aroma properties and biochemical factors on the rheological properties
and the suitability of the cheese for different con- of cheese are discussed in ‘Cheese as an Ingredient’,
sumer groups (e.g., children, aged); Volume 2. For detailed information on cheese texture,
Cheese: Chemistry, Physics and Microbiology, Third edition – Volume 1: General Aspects Copyright © 2004 Elsevier Ltd
ISBN: 0-1226-3652-X All rights reserved
Set ISBN: 0-1226-3651-1
512 Rheology and Texture of Cheese

Table 1 Rheological properties of raw cheese and their definitionsa

Cheese type displaying

Rheological property Definition property

Elasticity (rubberiness) Tendency of cheese to recover its original shape and Swiss-type cheese, low-moisture
dimensions upon removal of an applied stress Mozzarella
Springiness Tendency to recover from large deformation (strain) Swiss-type cheese, low-moisture
after removal of deforming stress Mozzarella
Elastic fracturability Tendency of hard cheese to crack, with very limited Parmesan, Romano, Gruyere
flow (confined to vicinity of crack); after fracture,
the broken surfaces can be fitted to each other
Brittleness Tendency of hard cheese to fracture at a relatively Romano, Parmesan
low permanent deformation
Firmness (hardness) High resistance to deformation by applied stress Cheddar, Swiss-type cheese,
Romano, Parmesan, Gouda
Longness The resistance of cheese to fracture until a relatively Mozzarella, Swiss
large deformation is attained
Toughness (chewiness) A high resistance to breakdown upon mastication Mozzarella, String cheese,
Halloumi
Softness Low resistance to deformation by applied force Blue cheese, Brie, Cream cheese
Plastic fracturability The tendency of cheese to flow on fracture Mature Cheddar, Blue cheese,
Chaumes, Raclette
Shortness The tendency to plastic fracture at a small deformation; Camembert, Brie
low resistance to breakdown upon mastication
Adhesiveness (stickiness) The tendency to resist separation from another material Mature Camembert
with which it makes contact (e.g., another ingredient
or a surface such as a knife blade or palate)
Crumbliness The tendency to break down easily into small, irregularly Cheshire, Wensyledale, Blue
shaped particles (e.g., by rubbing) cheese, Stilton, Feta
Shear thickening The tendency to increase in apparent viscosity when Cream cheese (when heated),
subjected to an increasing shear rate (especially ‘creaming’ of processed cheese
upon heating) products
Shear thinning The tendency to exhibit a decrease in apparent viscosity Quarg (especially at low
when subjected to an increasing shear rate temperatures, i.e., 4 °C)

a Definitions modified from Szczesniak (1963a), van Vliet (1991a) and Fox et al. (2000).

the reader is referred to ‘Sensory Character of Cheese
and its Evaluation’, Volume 1 and the following
reviews: Szczesniak (1963a,b, 1998), Brennan (1988)
and Rosenthal (1999).
Terminology of Rheology and Texture
General rheological terminology
The general terminology used to describe the rheology
of materials has been discussed extensively (Sherman,
1983; Rao and Steffe, 1992; Whorlow, 1992; Collyer
and Clegg, 1998; Sharma et al., 1999). The terms most
commonly applied to the rheology of cheese are
described in Table 1.
Deformation and strain
Any rheological measurement involves deforming a
sample of material by applying a force, e.g., by compres-
sion or by shear (Fig. 1). The displacement in response
to the force at the point of application is known as
deformation. The term ‘deformation’ used in this sense
does not imply permanent deformation but rather a
change in shape (i.e., form) which may be temporary,
permanent or partly recoverable. A series of instantan-
eous measurements of force and associated displace-
ment describe the rheological characteristics of a material
under the measurement conditions. The conditions
which affect the force–displacement response include
temperature, type of deformation (compression, exten-
sion, shear or pressure), level of deformation in relation
to the elastic limit and fracture point of the material,
rate of deformation, previous history of deformation.
Strain may be defined as the fractional displacement
that occurs under an applied stress.
Stress
Stress is defined as the distribution of force over an area
of a material. The ‘area’ over which a force is distributed
may be a surface (e.g., the surface of a cylindrical sample
exposed to a compression plate) or an imaginary section
within a material (e.g., an internal fracture plane). The
force applied at a surface is distributed throughout the
material and is borne by the structural elements, e.g., in
 Rheology and Texture of Cheese 513

Table 2 Rheological properties derived from stress/strain curves obtained from large strain deformation of cheese

Textural characteristic to
Rheological Property Abbreviation Interpretation which parameter is related

Elastic, or compression, modulus E Measure of elasticity at low strain Elasticity

Apparent elastic modulus, or / Ratio of stress to strain in a Elasticity
Deformability Modulus viscoelastic region below the
fracture point
Fracture stress f Stress required for fracture and collapse Strength of cheese matrix
of cheese mass beyond point of
recovery.
Fracture strain f Deformation required to induce fracture brittleness and “shortness” or
“longness” of cheese
Firmness (maximum stress) max Stress to required to compress firmness or hardness
cheese sample to a given deformation
Fracture work Wf The energy required to fracture the cheese toughness

the case of cheese, the casein strands of the matrix where A is the cross-sectional area over which the force
and the occluded fat globules. The rheological behaviour (F) is applied (Fig. 1). Normal stress can occur in ten-
of the material is effected by the response of the struc- sion or compression. In a large strain compression situ-
tural elements to the applied stress. The initial response ation, two expressions are used for normal stress,
of a cheese sample to an applied stress is determined differing in respect of the calculation of area. Apparent
mainly by the para-casein matrix. At larger deform- stress is the applied force divided by the original cross-
ations, the moisture and fat phases, which are occluded sectional area of the sample, while true (or, more
in the matrix, contribute to the rheological response. strictly, corrected) stress is the applied force divided by
the instantaneous area of the sample, allowing for the
Shear and normal modes of stress and strain fact that the sample spreads as it is compressed. When
Two modes of stress can be applied on a surface, true stress is plotted against strain, a more distinct peak
namely shear or normal. Shear stress is created when a is observed around the fracture point (Fig. 2).
force is applied parallel to the plane of a surface elem- However, the instantaneous area is not easily meas-
ent, whereas normal stress is created by a force applied ured and is often approximated by a calculation based
perpendicularly to a surface element. Normal stress, , on constant volume (Ak and Gunasekaran, 1992).
is defined as: Shear stress, , is defined as:
F F
 
A A

F kPa

ΔL 450
400
F
A 350
A
300 σ
ΔL 250
200
Lo 150
Lo σt
100
50
0
(a) (b) 0 0.2 0.4 0.6 0.8
ε
Figure 1 Deformation of a solid material by the application of a
force, F, (a) in a direction normal to the surface (area, A), result- Figure 2 Apparent stress, , and true stress, t, plotted against
ing in a compression deformation, or (b) tangential to the sur- strain, ␧, from force–displacement data obtained in the compres-
face resulting in shear deformation. The stress is calculated as sion of Cheddar cheese on a texture analyser, Stable Micro Sys-
F/A; strain is calculated as L/Lo , where Lo is the original length tems, model TA.HDi, showing a more distinct peak near the
of the sample of material. fracture point in the t plot.
514 Rheology and Texture of Cheese

where A is the cross-sectional area over which the
shear force (F) is distributed (Fig. 1).
Two alternative expressions for normal strain have
been used in tensile or compression situations, namely
Cauchy strain and Hencky strain. Cauchy strain ( ),
also referred to as strain, apparent strain or engineer-
ing strain, is defined as the deformation relative to the
original sample dimension, i.e.,
L
 where L is the shear (tangential) displacement on the
Lo
where Lo is the original height of the sample and L
is the displacement under applied stress,  (Fig. 1).
Hencky strain, sometimes referred to as natural strain
or true strain, is defined as the natural logarithm of
the ratio between the sample length upon application
of force and the original length, i.e.,
impractical, as the transition from elastic to vis-
cous behaviour is gradual. Throughout this text, strain
is used in the engineering or Cauchy sense, unless
otherwise stated.
Shear strain,  is defined as:
L

Lo
application of shear stress, .
Compression testing is generally used for the rheolog-
ical evaluation of cheese because of the relatively low
tensile strength of most cheeses, e.g., compared to its
compression strength. Exceptions include members of
the pasta-filata family of cheeses, such as Mozzarella and
Haloumi (see ‘Pasta-Filata Cheeses’, Volume 2), which
when heated are able to undergo a high degree of stretch-

冢 LL 冣,
ing when pulled. As discussed in ‘Pasta-Filata Cheeses’
t  ln and ‘Cheese as an Ingredient’, Volume 2, this characteris-
o
tic is associated with the presence of para-casein fibres
where Lo is the original sample length and L is the which are formed during the exposure of the curds to
length under load. Hencky strain is thought to be more high temperatures (e.g., 58–60 °C) at a low pH (e.g.,
relevant than engineering strain in describing fluid (or 5.1–5.4) during manufacture and stretching.
non-recoverable) behaviour, e.g., squeeze flow patterns In practice, normal and shear stresses occur simultan-
of deformation, as occur in large strain compression eously during testing, size reduction at industrial level
and in spreading cheese on a cracker. However, for (e.g., comminution, shredding, grating) and consump-
small strains, the Hencky strain approaches the Cauchy tion (mastication). In a compression test, a normal
strain (Fig. 3). The relationship between Cauchy and force is applied but fracture generally occurs as a con-
Hencky strain can be derived as: sequence of shear stresses built up in the sample. Like-
wise, in a torsion test, a normal force must be applied
 ln(1 ). to maintain sufficient contact between the sample and
t
the plate delivering the shear stress. In general, the
Ideally, engineering strain should be used for recover- simplest fundamental rheological properties (e.g.,
able (elastic) deformations and true strain for non- Young’s modulus, shear modulus) are defined for one
recoverable (viscous) deformations. Obviously, this is mode of stress in one dimension, and for this reason,
rheological measurements often attempt to confine
stresses to one mode and one dimension. However, this
is possible only in some low deformation situations,
Initial height, since stresses in one dimension tend to produce struc-
Lo = 25 mm tural displacements, and hence stresses, in other
dimensions and modes. Thus, it is not possible to cre-
Displacement, Cauchy Hencky
ate large deformations in one dimension in isolation, as
ΔL (mm) strain strain for example during compression testing when deform-
0 0 0.00 ations exceed the linear viscoelastic limit (see ‘Funda-
5 0.20 0.22 mental Measurements: Oscillatory Rheometry for
10 0.40 0.51 Linear Viscoelastic Measurements in Cheese’). How-
15 0.60 0.92
20 0.80 1.61
ever, cheese generally undergoes relatively large deform-
25 1.00 ation during handling and consumption, and hence it
8

is necessary to describe its rheological characteristics

Figure 3 Deformation of a sample of cheese, originally 25 mm
under large deformation conditions, e.g., during com-
high (L o), under axial compression, showing equivalence between pression by the molar teeth (⬃70%). It is difficult to
displacement (L), Cauchy strain ( ) and Hencky strain ( t ). measure shear and compression stresses simultaneously

in all dimensions under large strain deformation, and,
consequently, much use is made of empirical or semi-
empirical methods to describe the rheology of cheese or
other foods under large strain deformation conditions
to which cheese is subjected in practice. In contrast,
low strain linear viscoelastic tests, while giving precise
rheological quantities (i.e., storage and loss moduli)
and indirect information on structure, tell little about
the expected rheological behaviour of the cheese
during processing and eating. Bagley and Christianson
(1987) suggested a generalised approach to the meas-
urement and interpretation of the rheological proper-
ties of foods aimed at dealing with the difficulties in
describing behaviour that is highly viscous and highly
elastic at the same time. With this approach, constants
can be derived which enable the rheological property
being measured in a given test, e.g., compression modu-
lus at low strain during compression testing, to be
related to a rheological property measured in another
test, e.g., shear modulus in a torsion test or shear test.
In practice, inhomogeneities and graininess in cheese
can confound such comparisons and it is difficult to
interpret the significance of such results (Bagley and
Christianson, 1987).
The relationship between stress and strain
Stress and strain at a micro level result from an exter-
nally applied force at a macro level, the displacement
at the point of application being the cumulative effect
of a strain at every point along the length of the sam-
ple (Fig. 4). The relationship between stress and strain
is characteristic of the material but depends on tem-
perature, and for viscoelastic materials, on other fac-
tors including the time over which the stress is applied
and the pre-test stress–strain history and the rate of
strain (see ‘Uniaxial Compression’).
Force
Displacement
Stress
Original
height
X
(a)
Figure 4 The application of an external force over a surface area results in stress and strain throughout the sample, as illustrated
in (a). The displacement at the surface of application is the cumulative effect of a strain at every point, e.g., X, along the length of the
sample. For low strain deformations there is a linear relationship between stress and strain, and a modulus, equal to the ratio
between them, may be determined (b).
Rheology and Texture of Cheese 515
Under low , solids, including some cheeses (e.g.,
a young – medium-aged, low-moisture, part-skim Moz-
zarella cheese), exhibit a simple linear relationship
between  and which can be expressed in terms of var-
ious moduli. In compression or tensile testing, Young’s
Modulus (E) may be defined as:

E
where  is the normal stress and is the strain on the
material. In shear tests, the shear modulus (G) is given by:

G

where  is the shear stress and  is the shear strain.
The above elastic moduli are intrinsic rheological
characteristics of the material, that are independent of
sample dimensions, time and strain rate. However, for
most cheeses, the elastic region is small (e.g., 0.006;
Guinee et al., 2000a,b) and of little consequence
because most strains applied in practice are 0.05.
Bulk modulus and compressibility of cheese
Isotropic stress, or pressure, is sometimes described as a
third mode of stress. This usually occurs in fluid mater-
ials and is really a normal force applied equally in each
of three dimensions. In general, the application of
isotropic stress to a material results in a slight reduction
in volume, or shrinkage. From the volume reduction, a
bulk modulus (K) may be determined and is defined as:
PV
K
V
Sample
surface ---- Sample interior -----
Area
Force Stress
Modulus
Strain
L Strain
Displacement
(b)
516 Rheology and Texture of Cheese

where P is the applied pressure, V is the initial volume
of material and V is the change in volume.
There are simple relationships between compress-
ibility, usually expressed in terms of bulk modulus
(K), Young’s modulus (E) and Poisson’s ratio ()
(Whorlow, 1992; Rosenthal, 1999):
E  3K(1 2)
and
E  2G(1  )
Since  ⬃0.5 for most cheeses (see Poisson effect in
Glossary), these relationships simplify further to:
Rheological concepts applied to cheese
Cheese structure
Cheese is essentially a concentrated protein gel, which
occludes fat and moisture. Gelation is brought about
by either of the following mechanisms (see ‘Rennet-
induced Coagulation of Milk’ and ‘Formation, Struc-
tural Properties and Rheology of Acid-coagulated
Milk Gels’, Volume 1):
1. slow quiescent acidification (e.g., using a starter
culture or food-grade acid and/or acidogen), at a
temperature of 20–40 °C, to the isoelectric pH of
casein, i.e., ⬃4.6;
2. sensitisation of the casein to calcium via the hydroly-
sis of the principal micelle-stabilising casein, -casein,
by added acid proteinases (i.e., rennets); or

E  3G
(a) (b)

However, all of the above relationships apply only in Force, F

the linear (i.e., elastic) region. velocity, v
The volume reduction up to the point of fracture at depth, y
is about 9% for Cheddar cheese (Calzada and Peleg,
1978). Cheese with a large vacuole volume (e.g.,
where eyes occupy a significant proportion of the Figure 5 Two situations where viscous forces are at work:
(a) flow between parallel plates which move relative to each
volume, as in Swiss cheese), are more compressible
other; (b) flow in a pipe.
and therefore have lower K values than cheeses with-
out eyes. However, unlike their behaviour under
normal or shear stresses, most cheeses are relatively
non-compressible under isotropic stress. Conse-
Strain, γ

quently, from a practical point of view, their bulk e

rat
ear
modulus is of little interest, but may be of interest in = sh
the calculation of true stress in uniaxial compression pe
Slo
(see ‘Uniaxial Compression’).
Time
Viscous deformation
Figure 6 Ideal viscous (Newtonian) response to constant .
Flow is normally the result of shear displacement. applied stress, i.e., strain () increases at a constant rate ( ).
Shear forces occur when a liquid flows inside a pipe
or when a molten mass (e.g., melted cheese) flows
along a surface (Fig. 5). In a fluid, strain is not 10
Shear rate, γ arbitrary units

recoverable and applied stress results in a continu-

ously changing strain. A viscous material behaves as 8
a fluid and responds to shear (stress), in terms of
6
strain, in a time-dependent manner (Fig. 6). For an .

ideal viscous material, i.e., a Newtonian fluid, the 4

rate of strain is proportional to applied stress (Fig. 7).
The relationship between stress () and rate of strain 2
.
( ), or shear rate, is described by the coefficient of
viscosity (): 0
0 2 4 6 8 10
Stress, σ, arbitrary units

 . Figure
. 7 Relationship between shear stress (s) and shear rate
 ( ) for an ideal (Newtonian) liquid.

3. a combination of acid and heat, e.g., heating milk
to ⬃90 °C at ⬃pH 5.6.
The micro-structure of milk gels and cheeses has
been studied extensively (Hall and Creamer, 1972;
Kalab and Harwalkar, 1974; Kimber et al., 1974; Kalab,
1977, 1979; de Jong, 1978; Green et al., 1981a,b, 1983;
Green, 1990a,b; Kiely et al., 1992, 1993; Mistry and
Anderson, 1993; Bryant et al., 1995; Desai and Nolting,
1995; Everett et al., 1995; Guinee et al., 2000a). The
physico-chemical properties of the para-casein matrix
and occluded components may be deduced from
micro-structural observations, compositional analyses
and theoretical considerations of the chemistry of the
conversion of milk to cheese and partition of compon-
ents (e.g., milk salts) between the whey and the cheese
curd (Walstra and van Vliet, 1986).
Natural rennet-curd cheese is essentially a particu-
late calcium phosphate–para-casein matrix, composed
of interconnected and overlapping strands of partially
fused para-casein aggregates (in turn formed from
fused para-casein micelles). The integrity of the matrix
is maintained by various intra- and inter-aggregate
hydrophobic and electrostatic attractions. In young
cheese, the matrix has an ‘internal’ structure consisting
of a relatively loose network of clearly recognisable
particles (para-casein micelles and aggregates of para-
casein micelles) which are in contact with neighbour-
ing particles over part of their surfaces. Ongoing fusion
of para-casein particles during maturation leads to a
gradual reduction in the extent of internal matrix
structure, as reflected by the disappearance of interpar-
ticle boundaries and the formation of a more homogen-
eous mass (Kimber et al., 1974; de Jong, 1978).
The para-casein network is essentially continuous,
extending in all directions, although some discontinuities
exist in the matrix at the micro- and macro-structural
levels. Micro-structural observations made using
transmission electron microscopy (TEM) suggest that
hydrolysis of para-casein (e.g., by rennet) to water-
soluble peptides results in parts of the matrix losing
contact with the main para-casein network, an occur-
rence that leads to discontinuities or ‘breaks’ in the
para-casein matrix at the micro-structural level (de Jong,
1978). Hence, it is noteworthy that ageing of Moz-
zarella for 50 days results in the degradation of ⬃50%
s1-casein to s1-CN f 24–199 and an increase in the
porosity of the defatted para-casein matrix, as observed
using scanning electron microscopy (SEM) (Kiely et al.,
1993). Discontinuities at the macro-structural level
exist in the form of curd granule junctions or curd
chip junctions (in Cheddar and related dry-salted var-
ieties) (Kalab and Harwalkar, 1974; Kalab, 1979;
Lowrie et al., 1982; Paquet and Kalab, 1988). Curd
Rheology and Texture of Cheese 517
granule junctions in low-moisture Mozzarella are well
defined, ⬃3–5 m wide and appear as veins running
along the perimeters of neighbouring curd particles
(Kalab, 1977).
Unlike the interior of the curd particles, the junc-
tions are comprised mainly of casein, being almost
devoid of fat. Factors that contribute to the formation
of these junctions include leaching of the fat from the
surface of the curd particles and dehydration of sur-
face protein, during the cutting, acidification, cooking
and pressing stages of cheese manufacture. Chip junc-
tions in Cheddar and related dry-salted varieties are
clearly discernible on examination of the cheese by
light microscopy and, like curd granule junctions,
have a higher casein-to-fat ratio than the interior. The
difference in cheese composition at junctions, com-
pared to the interior of the curd particles, probably
leads to differences in the molecular attractions
between contiguous para-casein layers in the interior
and exterior of curd particles, and thus to differences
in structure–function relationships. From a rheological
viewpoint, the occurrence of structural discontinuities
may result in the lack of tensile strength in many
cheeses which in practical terms may be reflected as
crumbliness, shortness, fracturability, e.g., Feta, Stilton
and Cheshire. Discontinuities probably also contribute
to poor replication of rheological measurements.
The matrix encases fat globules (in varying degrees of
coalescence), moisture, dissolved solutes and enzymes
within its pores (Kimber et al., 1974; Laloy et al.,
1996; Guinee et al., 2000a). Clumping and coales-
cence of fat globules occur during manufacture due to
the combined effects of shear stress on the fat globule
membrane and shrinkage of the surrounding para-
casein matrix which forces the occluded globules into
close contact. Evidence for fat clumping is provided by
scanning electron micrographs which show fissures, or
irregular-shaped openings, in the para-casein matrix,
which remain after removal of fat during sample
preparation (Mistry and Anderson, 1993; Bryant et al.,
1995; Fig. 8). The frequency of these fissures
decreases as the fat content is reduced, e.g., from 33.2
to 8.2%, w/w, fat (Mistry and Anderson, 1993; Guinee
et al., 2000b).
Major physico-chemical changes occur in the pro-
tein and fat phases of cheese during maturation. These
include partial hydrolysis of the matrix comprising
para-casein, increase in hydration of the para-casein,
and coalescence of fat globules, resulting in the forma-
tion of fat pools (Fox et al., 1996, 2000; Guinee and
Law, 2001; Guinee, 2002). These changes are mediated
by the residual rennet, micro-organisms and their
enzymes, and changes in mineral equilibrium between
the serum and para-casein matrix. The type and level
518 Rheology and Texture of Cheese
(a) (b)
5 μm 1 μm
Figure 8 Scanning electron micrographs of Cheddar cheese,
showing the continuous para-casein matrix (arrow heads) perme-
ated by holes and fissures, corresponding to discrete, clumped
or coalesced fat globules (solid arrows). Bar, i.e., 5 m in (a) and
1 m in (b) (from Guinee et al. (1998), reproduced with permis-
sion from the society of Dairy Technology).
of physico-chemical changes depend on the variety
and composition of cheese and ripening conditions.
These changes assist in the conversion of fresh ‘green’
curd to a mature cheese and markedly influence its
rheological, textural, functional and flavour character-
istics (see ‘Biochemistry of Cheese Ripening: Introduc-
tion and Overview’, ‘Metabolism of Residual Lactose
and of Lactate and Citrate’, ‘Lipolysis and Catabolism
of Fatty Acids in Cheese’, ‘Proteolysis in Cheese dur-
ing Ripening’, ‘Catabolism of Amino Acids in Cheese
During Ripening’ and ‘Sensory Character of Cheese
and its Evaluation’, Volume 1). Thus, a storage period
is generally required before rennet-coagulated cheeses
attain the desired rheological and textural attributes
(e.g., fracturability, firmness, spreadability, brittleness)
associated with the particular variety.
Creep and stress relaxation in cheese
The time-dependent rheological behaviour of cheese
has been studied (Visser, 1991; Ma et al., 1996; Pereira
et al., 2001; Venugopal and Muthukumarappan, 2001).
Creep is the time-related change in strain on application
of a constant stress to a material such as cheese. Practi-
cal examples of creep occur when curd or cheese is
compressed gradually under its own weight (e.g.,
Camembert), is pressed or stacked, e.g., during retail-
ing. Creep (J) may be expressed in terms of strain or
compliance, which is the ratio of strain to applied
stress. When a constant stress, , applied for a time, t,
results in a strain, (t), then the creep compliance is:
(t)
J(t) 

A creep curve for Cheddar cheese is shown in Fig. 9.
Three characteristic regions can be identified. In the
elastic region (A–B),  is instantaneous and fully
reversible; in this region, the creep compliance is elastic
(J0). Viscoelastic deformation occurs in region B–C,
where the material is partly elastic and partly viscous;
the creep compliance is retarded elastic (JR) and the
recovery of the elastic component of  on the removal
of  is delayed. In the viscous region (C–D), 
increases linearly with time and permanent deforma-
tion occurs; the creep compliance is referred to as
being Newtonian ( JN).
On removal of the stress at point D, the strain
recovery curve shows three identifiable regions: an
instantaneous elastic recovery (D–E), a delayed recov-
ery (E–F), and an eventual flattening. The vertical dis-
tance from the flat portion of the recovery curve to the
time axis is the non-recoverable  per unit , which is
related to the amount of structural damage to the sam-
ple during the test.
In the elastic region of the creep curve, the strands
of the cheese matrix absorb and store the stress energy,
which is instantly released on removal of , enabling
the cheese to regain its original dimensions. The
extent and duration of the elastic region depends on
the magnitude of  and the structural and compos-
itional characteristics of the cheese. At   critical
strain, the structure of the cheese is altered via the
breaking of bonds between structural elements, which
are stressed beyond their elastic limit. Eventually,
when the stress-bearing structural casein matrix has
fractured, the cheese is said to flow. At short time
scales and low , most hard cheese varieties are essen-
tially elastic, whereas after a long time, they flow,
albeit very slowly, and do not recover to their original
shape on removal of the stress. Failure to appreciate
this characteristic can often lead to loss of shape (e.g.,
manifested by bulging, inclined surfaces) during stor-
age, distribution and retailing, especially if cheeses of
different consistencies are laid haphazardly upon each
other.
A stress relaxation test generally entails the instant-
aneous application of a constant deformation or strain,
(typically 0.10–0.20), by compression of the cheese
sample between two parallel plates of a texture
analyser (e.g., TA HDi Texture Analyser, Stable Micro
Systems, Godalming, England; Instron Universal Testing
Instrument (UTM); Instron Corporation, Massachusetts,
USA.). On the application of ,  increases instantan-
eously to o (i.e., zero-time value) but decays expo-
nentially with time (t) (Shama and Sherman, 1973).
The resultant -time curve is used to determine the
stress relaxation time, t, which may be defined as the
time required for  to decrease to a fraction of o, e.g.,
t at which   o/e, where e is the base of the natural
logarithm. In a variation on such a test, Emmons et al.
(1980) compressed full-fat (35%) and reduced-fat
(17%) Cheddar cheeses, having a common level of
moisture-in-non-fat-substance, at a constant speed to
a strain of 0.2 and held the strain for 1 min. They
 Rheology and Texture of Cheese 519

Sustained constant Recovery after

0.3 removal of stress
stress

0.2

Strain, ε
C E

0.1 F
B
Non-recoverable
A strain
0
0 50 100 150 200 250 300
Time, s

Figure 9 Creep–relaxation curve for mature Maasdammer cheese (fat, 29%, w/w, protein, 28%, w/w). A stress of 3700 Pa was applied
to a cheese disc (diameter, 40 mm; height, 2.27 mm), placed between the parallel plates of a controlled strain rheometer (TA Carrri-Med
csl2500) at 20 °C, and removed after 180 s. The curve is divided into regions indicating elastic, viscoelastic and viscous behaviour.

showed that the initial compression slope (or modulus
of deformability), the relaxation slope and the residual
force (after 1 min) were much higher for reduced-fat
cheese, made from milk with or without homogenisa-
tion, than for full-fat cheese.
Mechanical models of cheese rheology
From its creep and stress-relaxation behaviour (Fig. 9),
it can be inferred that cheese is a viscoelastic material.
It exhibits elastic and viscous characteristics, but
unlike true elastic or viscous materials, the relationship
between stress and strain depends on the magnitude
and the duration of the applied stress or strain. On the
application of a low stress, that is sufficiently small so
as not to induce permanent damage or fracturing
(breaking of bonds between the structural elements)
of the microstructure, for short times, cheese behaves
as an elastic solid. However, a low stress applied over a
relatively long time scale results in an increasing
strain, a gradual failure of the structure and an even-
tual flow. Hence, the relationship between  (or ) and
 (or ) is linear only at very low  and short time
scales. The  at which linearity between  and  is lost
is referred to as the critical strain (i.e., at the end
of the linear viscoelastic range), which for most solid-
like foods, including cheese, is relatively small, e.g.,
0.02–0.05 (Walstra and van Vliet, 1982).
The modelling of cheese rheology begins with
simple relationships such as Hooke’s Law for small
displacements in the elastic region. In the region
beyond the elastic limit, sometimes referred to as the
elastoplastic region (i.e., where recovery following
deformation is partial on removal of stress), modelling
the rheology of cheese requires more complex models.
Mechanical models have been used to simulate creep
and relaxation effects in materials (Rao, 1992; Tanner,
2000). The viscoelastic behaviour of cheese may be
simulated by various mechanical models that contain
different arrangements of dashpots (representing the
fluid element) and springs (representing the elastic
element) in series and/or in parallel.
A simple model consisting of a spring in parallel with
a dashpot is referred to variously as a Kelvin or Voigt ele-
ment (Whorlow, 1992) or Kelvin-Meyer solid (Tanner,
2000) (Fig. 10). In contrast, a Maxwell element consists
of a spring in series with a dashpot, which gives an
exponentially decaying response to a suddenly applied
constant strain (Fig. 11). Several models have been
based on multiple Kelvin bodies in series, or Maxwell
bodies in parallel, to simulate creep and stress relax-
ation, respectively, in viscoelastic solids (Whorlow,
1992); elements with a spectrum of time constants are
employed in these models to approximate viscoelastic
σ
γ
γ
Time
Figure 10 Kelvin model and its response to constant applied
stress.
520 Rheology and Texture of Cheese
Applied
strain
σ
Time
Figure 11 A Maxwell model and its stress relaxation response
to a constant applied strain.
behaviour (Fig. 12). Subramanian and Gunasekaran
(1997b) showed that a model consisting of eight
Maxwell elements could simulate the shear modulus
over a wide dynamic range in low amplitude oscillation
(0.1–20 Hz). Ma et al. (1996) showed that a six-element
Kelvin model could simulate creep compliance in full-fat
and reduced-fat Cheddar cheese. The Burgers body,
which consists of a combination of Maxwell and Kelvin
elements in series (Fig. 13), affords a close approxima-
tion to both the creep and stress relaxation behaviour of
cheese. The mechanical representation of these models
provides an intuitive guide to the nature of viscoelastic-
ity and a simulation of rheological behaviour based on
(a) (b)
τ1 τ2 τ3
τ1 Applied
strain
τ2
τ3
Figure 12 (a) Series of three Kelvin elements with a spectrum
of time constants, which may be used to simulate creep and
(b) A combination of Maxwell elements with a spectrum of time
constants, which may be used to simulate relaxation behaviour
in a viscoelastic solid.
Applied
strain
Strain, ε
Time
Figure 13 Burgers four element model, which simulates creep
and relaxation behaviour of cheese.
the force–displacement equations of these models
(Whorlow, 1992; Steffe, 1996).
Large strain deformation
Definitions and terminology
Large strain measurement implies permanent deforma-
tion and measurement of non-linear rheological char-
acteristics which are related to deformation of the
microstructure. In contrast to linear viscocelastic
deformation where applied strains are generally
0.05, large strain deformation may be defined as that
which occurs at strains in the range of ⬃0.1–0.9 dur-
ing compression, and even at higher strains in the case
of shear deformation (e.g., 1).
Consideration of the forces that are applied to cheese
from manufacture to consumption, indicates a very
broad range of deformation. In some situations, the
strains are of a relatively low magnitude and do not
result in visible damage (e.g., during ripening, transport,
retailing), while in others the strain results in fracture
(e.g., during portioning) or complete disintegration
of the cheese mass (e.g., comminution, as in shredding,
grating, grinding, as for example in the preparation
of cheese ingredients and in the manufacture of
processed cheese products and cheese powders). Hence,
in the current context, large strain deformation is arbi-
trarily subdivided into two regions, i.e., large strain
deformation-elastoplastic (LSD-E; e.g., strains ⬃0.1–0.5;
Fig. 14), where deformation does not result in fracture
and the structure can partially recover, and large strain
deformation-fracture (LSD-F; 0.3–0.9), where the cheese
mass undergoes fracture or disintegrates and cannot
recover. In the following discussion, the LSD-E and
LSD-F regions will be treated jointly (Fig. 14).
Measurement using texture analyser
Large strain deformation testing of cheese usually
involves the application of strains (e.g., ⬃ 0.8) that
result in fracture, by compression of the cheese sample
Typical linear Typical extent of
visco-elastic limit compression to which
cheese is subjected in
chewing and in
Typical fracture compression testing
point
0.05
0 0.40 0.80 1.0
Strain, ΔL/Lo
Figure 14 Range of strain in compression tests on cheese.

between two parallel plates of a texture analyser (Culioli
and Sherman, 1976; Dickinson and Goulding, 1980;
Creamer and Olson, 1982; Tunick et al., 1991; Guinee
et al., 1996; Fenelon and Guinee, 2000; Truong et al.,
2002). The cheese sample is placed on a base plate and
is compressed at a fixed rate (typically 20 mm/min 1)
to a pre-determined level (e.g., 75% of its original
height) by the mobile plate (cross-head). However, the
rate of compression used in various studies has dif-
fered widely, e.g., 5–500 mm/min 1 (Table 3). The
force (F) developed during compression is recorded as
a function of distance (or displacement); alternatively,
the force may be converted to  and the displacement
to . The resultant  versus curves for a range of hard
rennet-curd cheeses (Fig. 15) typically show a number
of distinct regions and enable the determination of a
number of rheological parameters:
• A–B;  increases proportionally with . The slope of
this linear region defines the compression modulus, E
(i.e., E = / ), which is of little practical significance
in relation to cheese behaviour during processing or
consumption, where strains are 0.05. However, in
the commercial grading of cheese, E may be an indi-
cation of springiness (e.g., where a grader sensori-
cally monitors the resistance to small deformation, as
in pressing the thumb into the outside of the cheese
block; the force applied during this hand deformation
is typically 18 N or  ⬃ 40 kPa).
• B–C,  increases less than proportionally with . The
slightly lower slope of the curve in this region com-
pared to that in A–B is probably due to the formation
of microcracks that do not spread throughout the
sample but which allow some stress to be dissipated;
• C–D, the slope of the / curve decreases markedly.
The cheese begins to fracture at C, as cracks grow
and spread throughout the entire sample at an
increasing rate. Eventually, at D the rate of collapse
of the stress-bearing para-casein matrix overtakes
the build-up of  within the matrix through further
compression and a peak , denoted as the fracture
stress, is reached. The fracture stress, f, and strain,
f, are measures of the stress and strain, respectively,
required to cause complete fracture of the sample.
Strength, or fracturability, is defined as the stress
required to fracture the sample (at D), while tough-
ness, or fracture work, is defined as the area under
the curve up to the point of fracture.
• D–E,  decreases with further compression due to
the collapse of the stress-bearing structure. The
decrease in  may be attributable to: (i) shattering
of the samples into pieces that spread over the
base plate, resulting in an increased surface area
and (ii) the probable loss of contact between some
Rheology and Texture of Cheese 521
of the pieces of cheese and the base plate which
results in dissipation of stress energy stored within
the individual pieces.
• E–F,  increases as the cross-head begins to com-
press the fragmented pieces of cheese. The  at the
end of the compression (point F) is a measure of
firmness, as judged in the first bite of mastication
(Sherman, 1969; van Vliet, 1991a).
The various quantities obtained from the – curve
and their interpretation are given in Table 2.
The application of a strain to a segment of cheese
(e.g., cube or cylinder) and monitoring the resultant 
by a texture analyser, as above, is a typical method for
measuring the large strain deformation behaviour of
cheese. However, many variations of both the procedure
of stress or strain application, and the levels, are possible.
A so-called apparent elastic modulus can be calculated
at a strain well below the fracture point, e.g., ⬃ 0.1, as
the ratio between  and . A preferred term for this
parameter is modulus of deformability, as the deforma-
tion in question may include some plastic flow (Ak and
Gunasekaran, 1995; Johnston, 2000). However, such a
parameter needs to be interpreted with caution as some
apparent initial deformation may occur before complete
contact is made between the compression plate and the
sample surface, an occurrence that could lead to erro-
neous values.
Fracture and work of fracture. Rheological behav-
iour over such a range of in the form of shear or
compression, can be explored in several ways, such as
applying a gradually increasing , a fixed , a defined
 followed by its removal, a gradually increasing up
to a point followed by its reversal. Stress–strain cycles,
often referred to as bites (analogous to compression
between the molar teeth during mastication), may be
repeated at interval(s) or applied in a given sequence
(e.g., pre-test compression). Depending on the level
of applied strain, cheese exhibits a combination of
rheological behaviours, such as non-linear elastic (e.g.,
region B–C, Fig. 15), sometimes referred to as vis-
coelastic, or inelastic (e.g., region D–E, Fig. 15),
sometimes referred to as plastic behaviour.
Rheological Measurements in Cheese:
Sensoric Methods
The methods used to assess the rheological character-
istics of cheese may be broadly classified as sensoric or
instrumental, where instrumental methods can be cat-
egorised further as empirical or fundamental.
The aim of sensoric methods, which are performed
routinely by cheese graders, is to acquire an impression
522 Table 3 Test conditions used with uniaxial compression of cheese

Sample
dimensionsa Speed Maximum Tc
Cheese type (mm) (mm/min) strain ( ) Instrument b (°C) Reference

Gouda 15   15 25–500 0.80 UTM 20–21 Culioli and Sherman (1976)

Cheddar 20   20 10–100 0.50 UTM not stated Calzada and Peleg (1978)
Cheddar, Cheshire, Leicester 29   30 5–1000 0.50 UTM 0–40 Dickinson and Goulding (1980)
Cheddar, Mozzarella, processed cheese spread 57   20–40 5 0.75 UTM 22 Casiraghi et al. (1985)
Italico, Montasio, Sbrinz, Grana Padana, Pecorino 20  20  20 5–200 0.80 UTM 20 Casiraghi et al. (1985)
Cheddar, Cheshire 17   15 50 0.80 UTM 21 Green et al. (1985); Green
et al. (1986)
Processed cheese analogue 17   15 5–500 0.80 UTM 21 Marshall (1990)
Brie 15   19 33 0.80 Alwetron TCT5 5 and 20 Molander et al. (1990)
Gouda 15   20–30 1–200 ⬃0.75 Overload 20 Luyten et al. (1991a,b)
Dynamics
Cheddar 19   19 (or 27) 2.5–125 UTM 22 Ak and Gunasekaran (1992)
Munster, Carré de l’Est, Camembert, Coulommiers 16   17 10 0.70 UTM 15 Hennequin and Hardy (1993)
and three speciality soft cheeses
Mozzarella 67   12 5–500 0.70–0.75 30–60 Ak and Gunasekaran (1995)
Cheese analogs 10   10 250 0.80 TA.XT2 20 Lobato-Calleros et al. (1997)
Munster, Emmental, Roquefort, Beaufort, Camembert, 21   21 10 0.80 UTM 20 Antoniou et al. (2000)
Reblochon, Pont l’ Eveque, Brie de Meaux, Tomme
de Savoie, Valencay, St. Nectaire, Pyrenees Brebis,
Blue d’Auvergne, Comte Vieux, Fourme de Salers
Cheddar cheese with varying fat content (6–33%, w/w) 30   29 50 0.70 UTM 8 Fenelon and Guinee (2000)
Cheddar 30   29 50 0.75 UTM 8 Guinee et al. (2000b)
Cheddar, Swiss, Romano, Havarti 10  10  10 TA.XT2 20 Halmos (2000)
Cheddar 19   26 50 0.75 UTM 20 Hort and LeGrys (2000)
Cheddar 18   24 20 0.75 UTM 20 Johnston (2000); Johnston
et al. (2002)
Tilsit 20   20 10 0.70 UTM 15 Weinrichter et al. (2000)
Queso Fresco 20   15 100 0.30 TA.XT2 RTd Hwang and Gunasekaran
(2001)
1250 0.90 UTM
Gruyere, processed Mozarella 20   5–20 3.6–14 0.52 UTM RT Charalambides et al. (2001)
Commercial cheeses: Swiss, Colby, Edam, Cheddar, 12.7  12.7  12.7 60 0.70 TA.XT2 7 Breuil and Meullenet (2001)
Gouda, Mozzarella, Romano,
White American, Yellow American 21   10 12–600 0.90 TA.XT2 4 Xiong et al. (2002)
Montasio 25   15 50 0.40 or 0.80 UTM 22 Innocente et al. (2002)
Cheddar 15  15  15 24 0.80 TA.XT2 25 Truong et al. (2002)

a , diameter of cylinder.
b UTM, Universal Testing Machine (Instron); TA.XT2, texture analyser (Stable Micro Systems).
c T, Temperature.
d RT, Room temperature.
 Rheology and Texture of Cheese 523

kPa
400
350
F

Stress, σ
300
250
200 D E
150
100 C
50 B
A
0
0.0 0.2 0.4 0.6 0.8
Strain, ε

Figure 15 Large strain deformation test: typical stress–strain curve of a 6-month-old mature Cheddar cheese sample compressed
to 25% of its original height; several regions of the curve are identifiable, based on slope variation (see text for details).

of how the texture of the cheese is perceived during con-
sumption. Cheese texture may be defined as a composite
sensory attribute resulting from a combination of phys-
ical properties that are perceived by the senses of touch
(including kinaesthesis and mouthfeel), sight and hear-
ing. The test conditions are arbitrary, frequently involv-
ing deformation which results in visual fracture, e.g., as
when rubbing cheese between the fingers until it
becomes pliable, three (finger) point bending of a cylin-
drical cheese plug or slice, and mentally gauging the
force required to bend or break it. Alternatively, the
cheese may be assessed by the application of forces or
deformations which cause no visible fracture, e.g., press-
ing the ball of the thumb into the surface of a whole
cheese and mentally assessing the degree of indentation
or the force exerted on the fingers. In all cases, a sensory
impression is formed and the grader assigns a score,
based on one or more criteria, such as test conditions
and response. The sensory properties of cheese, includ-
ing texture, are discussed comprehensively in ‘Sensory
Character of Cheese and its Evaluation’, Volume 1.
Rheological Measurements in Cheese:
Empirical Instrumental Methods
A wide range of instrumental techniques is used for
characterising the rheology of cheese (Table 4). Instru-
mental methods may be arbitrarily classified as empiri-
cal or fundamental. In general, the nature of the
stresses and strains in empirical methods is less well
defined than in fundamental methods. Moreover,
unlike fundamental methods, the measurements
obtained with some empirical methods are on an arbi-
trary scale (e.g., the ball compressor).
Empirical instrumental measurements
Many textural studies have involved rheological meas-
urements to imitate the sensory evaluation of cheese
texture. The aim of empirical tests is to measure a
parameter, which experience indicates, or suggests, is
related to the textural characteristics of the cheese.
Hence, while the test conditions are arbitrary and the
stresses and strains involved may not be well defined,

Table 4 Typical rheological testing techniques applied to cheese

Typical sample shape

Type of instrument and dimensionsa
Test used (mm) Reference

Oscillatory shear Rheometer Cylinder: 30   3 Ma et al. (1996)

(parallel plate)
Uniaxial compression Texture analyser or UTM Cube: 25  25  25 (see Table 3)
Cone penetration Texture analyser or Instron, Cube: 12.7  12.7  12.7 Breuil and Meullenet (2001)
30° cone
Puncture Texture analyser or Instron, Cube: 12.7  12.7  12.7 Hennequin and Hardy (1993);
2–5 mm diameter needle Breuil and Meullenet (2001)
Bending test Texture analyser Finger: 25  25  50 Rosenthal (1999)
Wire cutting test Texture analyser Finger: 25  25  50 Green et al. (1986)
Torsion test Torsion gelometer Capstan: 19 max  27.8 Truong and Daubert (2000)

a , diameter of cylinder.
524 Rheology and Texture of Cheese
a value is obtained which gives some indication of the
textural characteristics of the cheese and differentiates
one sample from another. However, they provide only
single datum values that are an overall measure of the
many different facets of rheological behaviour.
In these tests, a sample is compressed or penetrated
in one or more bites, thereby simulating the compres-
sive and penetrative actions of the teeth on cheese dur-
ing mastication. Likewise, the action of a cheese grader
who presses the ball of the thumb into the cheese is
imitated by the ball-compressor test. Some empirical
instrumental tests are discussed briefly below.
Imitative tests
Imitative instruments include the bite tenderometer
and the denture tenderometer which measure the
forces involved in chewing using strain gauges, and
typically involve compression to 60% of the original
height. In the Volodkevich bite tenderometer, which
was designed to simulate the motions of mastication, a
pair of tooth-like jaws, or wedges, compress a sample
of about 6 mm thickness, imitating the squeezing and
biting action of teeth (Szczesniak, 1963b). Later
instruments used plungers to penetrate a sample, or
parallel plates to compress a sample, e.g., to ⬃20–30%
of its original height (Szczesniak, 1963b). Early
devices for evaluating the hardness of cheese involved
compression by a ball, in an instrument known as the
ball-compressor, where deformation resulting from
applying a fixed force for a specified time was meas-
ured (Szczesniak, 1963b). The action simulated that of
a thumb pressing against cheese when making a sen-
sory evaluation of the product.
The General Foods Texturometer was designed to
simulate the biting of food by the jaws and teeth
(Friedman et al., 1963; Bourne, 1978). A food sample
(⬃12.6 mm high) was loaded onto a fixed plate and
then subjected to a deforming force by a tooth-shaped
plunger, which was mounted on a hinge and actuated to
simulate the vertical action of a human jaw. The area
of the samples is at least that of the plunger base,
which is available in sizes from 16 to 50 mm . The
instrument compresses samples to a height of 3.2 mm,
i.e., 75% compression. When the plunger deforms the
sample, strain gauges detect the movement of the
plunger and a force–time trace is recorded and is
known as a texture profile. The sample is subjected to
two successive deformations (referred to as bites). The
Texturometer has been superceded by uniaxial com-
pression instruments, such as the Instron UTM, for
the purpose of texture profile analysis. One distinction
between the Texturometer and other instruments is
that the Texturometer simulates the action of the
human jaw, whereby the plunger decelerates as it
reaches the end of the compression stroke, and then
accelerates upward as it withdraws. The usual practice
with other instruments is compression at constant
speed.
Cutting tests
Cutting tests measure the resistance to the passage of a
knife or a wire through a cheese (e.g., Cherry-Burell
Curd tension meter). As wire-cutting tests tend to be
more fundamental, they are discussed in more detail in
‘Fundamental Measurements: Large Strain Deformation’.
Penetration tests
Penetration tests involve measurement of the force
required to insert a probe (cone or cylinder) a given
distance into cheese, or alternatively the depth of pene-
tration of a probe under a constant load for a given
time. As the probe penetrates the sample, the cheese in
its path is fractured and forced apart. The progress of
the probe is retarded to an extent depending on the
hardness of the cheese in its path, the adhesion of the
cheese to its surface (which depends on the depth of
penetration into the cheese and the thickness of the
needle, or angle of the cone, used). Hennequin and
Hardy (1993) used a cylindrical probe (5 mm diameter
at a speed of 10 mm/min to a depth of 10 mm) to pene-
trate soft cheeses (e.g., Camembert, Coulommier,
Munster) and found that the force at 10 mm penetra-
tion gave a high correlation with sensory firmness
(r  0.94, n  19). They concluded that the technique
is suitable as a rapid method for texture measurement
in soft cheese. Breuil and Meullenet (2001) found a sig-
nificant correlation between measurements obtained
using a cone penetrometer (30°), or a 2-mm needle,
and textural characteristics of a wide range of commer-
cial cheeses (e.g., Colby, Edam, Cheddar, Mozzarella
and Cream cheese) as measured by a sensory panel.
Fundamental Measurements: Oscillatory
Rheometry for Linear Viscoelastic
Measurements in Cheese
Elastic shear (G ) and loss modulus (G)
As discussed in ‘General rheological terminology’,
there is a range of strain, typically 0.05, over which
cheese recovers fully, although not instantaneously,
when stress is removed. This behaviour is referred to
as viscoelastic. Even at these low strains, ideal elastic
(Hookean) or viscous (Newtonian) behaviour, repre-
sents two extremes, and cheeses, like most organic
materials, show some characteristics of both. Hence,
the accurate characterisation of cheese rheology
requires the measurement of both elastic and viscous
responses. In the elastic region, where there is a linear
 Rheology and Texture of Cheese 525

relationship between dynamic stress and strain, the
behaviour is described as linear viscoelastic.
Linear viscoelastic measurements are typically
made by applying torsion, using oscillatory rheometry
(van Vliet, 1991b). This involves using a precision
actuator to apply a low oscillating strain to a sample
(which could be a liquid, a ‘soft’ solid, or a rigid solid),
and the measurement of the resultant stresses within
the sample, using a sensitive transducer. Alternatively,
a small stress is applied to the sample and the resultant
strain is measured. The former is referred to as con-
trolled strain rheometry, whereas the latter is known as
controlled stress rheometry.
Several geometries are possible for applying torsion,
but the most convenient for a solid material, like cheese,
are parallel circular plates. Parallel plates have the advan-
tage that the sample can be easily prepared to fit the
plates; moreover, plates with serrated surfaces minimise
the risk of slippage, associated with fat liquefaction
(Subramanian and Gunasekaran, 1997a; Guinee et al.,
1999). With parallel plate geometry, the cheese sample,
which is disc-shaped, is gripped between the plates, one
of which is fixed, while the other applies a low-amplitude
torsional harmonic motion (Fig. 16). At any time t, the
angle of rotation, &, of the oscillating plate is defined by:
&  a sin t
where a is the maximum angle of rotation and  is the
angular velocity. The shear applied by the plate varies
throughout the sample, from zero at the central axis to a
maximum at the edge. At a point on the oscillating plate,
at a radial distance, r, from the axis, the displacement due
to rotation by an angle & is r&, and the shear strain  is:
r&

w
where w is the thickness of the sample. This displace-
ment results in a strain, (t), at any radius r:
ar
冢
w冣
sin t  o sin t
where o is the amplitude of (t). (In this notation,
(t) is used interchangeably with .) In general, the
resultant oscillating stress is out of phase with the
applied shear by a phase angle . The stress wave can
be reconstructed as the sum of two sine waves, one
in-phase with, and the other out-of-phase (by 90°)
with, the strain wave (Fig. 17). Thus,
   '  "  o' sin t  o" cos t
where o' and o" indicate the stress components
which are in-phase and out-of-phase with the strain ,
and are related by the phase angle  (Fig. 18):
o"
tan  
o'
The stress wave has an amplitude o, defined by:
o  √(o')2 (o")2
Two dynamic moduli, elastic shear modulus (or
storage modulus), G , and viscous modulus (or loss
modulus), G, may be defined from the relationship
between  and , where,
o'
G' 
o

(a)

δ
(c) Strain
Stress

ωt
Shear

(a)
(b) 90° Elastic, or in-phase,
component, τo' sin ω t

(b) Radial distance from

centre of sample
Figure 16 Schematic showing viscoelastic deformation of
cheese during low-amplitude oscillation rheometry: (a) sample
before test; (b) sample being subjected to a torsional shear dis-
placement; (c) shear displacement as a function of radial dis-
tance from the central axis.
Loss, or out-of-
phase, component,
τo'' cos ω t
Figure 17 Stress and strain traces in dynamic oscillation,
showing phase delay, , between stress and strain (a), and reso-
lution of in-phase and out-of-phase components of stress (b).
526 Rheology and Texture of Cheese

(a) (b) Complex viscosity

, τo Oscillatory shear also implies an oscillatory shear rate

ess cos ω t .
Str Loss component, (), since
τo″ sin (ωt + δ )

δ . d .
sin ω t   (o sin t)  '  cos t  o cos t
Elastic component, τo′ dt
Strain, γo . .
where  o is, by definition, the amplitude of . This
Figure 18 Trigonometrical representation of phase relationship enables a complex viscosity to be calculated, with vis-
between dynamic strain and dynamic stress in oscillatory meas- cous and elastic components, by dividing the appropriate
urement in viscoelastic region, (a), with vector direction indicat- component of shear stress by the shear rate. The ‘viscous’
ing phase, as in (b).
component of viscosity (in-phase with shear rate) is:

and  '" G" .

o"
G" 
o
Therefore,
tan  
G"
G'
Consideration of a spring-dashpot mechanical model,
e.g., a Kelvin element, indicates that the same relation-
ship between stress and strain applies (Fig. 19). How-
ever, it is noteworthy that the magnitudes of G and G
for the spring and dashpot components of the mechani-
cal model in the viscoelastic region differ markedly from
those which characterise large strain deformation.
Ma et al. (1996) reported decreases (of ⬃50%) in G
and G for full-fat and reduced-fat cheeses on increasing
stress by a factor of 10. The tendency of  to approach a
constant value as the strain was reduced towards zero
shows that cheese behaves in a viscoelastic manner
rather than an elastic manner at very low strains.
'  .  since o  o 
o 
The ‘elastic’ component of viscosity (out-of-phase with
shear rate) is:
'' G"
"  . 
o 
Thus, complex viscosity equals the complex (shear)
modulus divided by the angular frequency, , with
the in-phase and out-of-phase components inter-
changed (Fig. 20). According to the Cox-Merz rule,
the complex viscosity determined by dynamic
rheometry is virtually identical to the (steady) vis-
cosity when compared at numerically equal values of
shear rate and . Dynamic rheometry is often used
for this purpose since it is easier to perform than vis-
cometry on viscoelastic materials. Cylindrical geom-
etry may be used in the case of liquids or gels, or
cone-and-plate or parallel plate geometry in the case
of more solid samples. It is preferable to operate in a
shear-rate control mode to avoid the occurrence of

Stress τ

Oscillatory strain γο sin ω t Shear rate, (a) (b)

.
γo
τo"
τo η' = .
e ss, γο
Str Loss component,
η
Spring Dashpot τo"
G' G''
δ

Elastic component, τo' τ '

η" = .o
γo

Figure 19 Kelvin element and stress response () to oscillatory Figure 20 Trigonometrical representation of phase relationship
strain ( ). The spring represents the elastic, or in-phase, compon- between shear rate and dynamic stress in oscillatory measure-
ent, G . The dashpot represents the loss, or out-of-phase com- ment in viscoelastic region (a), with corresponding components
ponent, G. of complex viscosity illustrated in (b).

unpredictably large shear. The strain is kept low
if one wishes to measure without structural damage
to the sample but may be increased if one wishes to
determine a fracture stress or to simulate a practical
situation.
Ma et al. (1996) found that elastic and loss moduli,
G and G, respectively, increase only to a second order
with increasing frequency, i.e., by a factor of about 3
for a 1000-fold increase in frequency. This implies a
300-fold decrease in the viscosity components,  and
, respectively (i.e., multiplying by 3/1000 ⬃ dividing
by 300), showing that the complex modulus is much
more stable with respect to frequency for cheese than
is complex viscosity.
Various studies on different cheese varieties have
indicated that G and G decrease, while  increases,
with increasing temperature in the range 4–40 °C, the
range normally encountered during consumption and
mastication (Taneya et al., 1979; Horne et al., 1994;
Guinee et al., 2000b; Guinee, 2002). These changes,
mark a transition from a cheese which is largely elastic
in character at low temperature ( ⬃ 12–16°), to one
which is more viscous in character at the higher tem-
perature ( ⬃ 40°) and may be attributed to fat lique-
faction. Tunick et al. (1990) made shear measurements
on Cheddar and Cheshire cheeses in the range 20–40 °C
and fitted the Arrhenius equation to the complex vis-
cosity in the form:
*  Avisc exp 冢 ERT 冣
visc
where Avisc is a pre-exponential factor, Evisc is the acti-
vation energy, R is the gas constant and T is absolute
temperature. A logarithmic form of this equation gives
a linear plot:
冢 冣
A (a)
log10 *  B
T
where A  Evisc/(2.3 R) and B  log10 Avisc. Cheddar
cheese had a higher activation energy (137 000 J/mol)
than Cheshire (84 800 J/mol) at 60 weeks of age,
implying a greater sensitivity to temperature for the
rheological properties of Cheddar cheese. However,
the viscosity–temperature curves for the two cheeses
crossed around 25 °C, i.e., Cheddar had a higher vis-
cosity than Cheshire below 25 °C and vice versa above
25 °C (Fig. 21). The sensitivity of viscosity to tempera-
ture was found to decrease with age for Cheshire
cheese (Fig. 21).
Rheology and Texture of Cheese 527
+ Cheshire at 20 weeks
6.5
6.0 Cheshire at 60 weeks
5.5
log η∗
5.0 Cheddar at 60 weeks
4.5
4.0
20 30 40
Temperature, °C
Figure 21 Variation of complex viscosity of Cheddar and
Cheshire cheese with temperature in the range 20–40 °C. Ched-
dar at 60 weeks, ; Cheshire at 20 and 60 weeks, O, , respect-
ively. Data taken from Tunick et al. (1990).
Fundamental Measurements: Large Strain
Deformation
Large strain deformation measurements on cheese are
usually undertaken using uniaxial compression, shear
(or torsion), wire-cutting or bending. The methodology
and instrumentation used for these measurements, and
factors affecting the measurements, are discussed below.
Uniaxial compression
The most common types of rheological measurement in
cheese involve linear (uniaxial) displacement, e.g., using
Instron UTM (Lee et al., 1978; Weaver et al., 1978; Imoto
et al., 1979; Creamer and Olson, 1982; Visser, 1991; van
Vliet, 1991b; Guinee et al., 1996; Pons and Fiszman,
1996; Fenelon and Guinee, 2000), the Stevens Response
Compression Analyser (Stevens Group Ltd, Blackburn,
UK) (Brennan, 1984), the TA.XT2 texture analyser or its
derivatives from Stable Micro Systems (Truong et al.,
2002; Xiong et al., 2002) (Table 3; Fig. 22).
(b)
(c)
Figure 22 TA. HDI texture analyser (Stable Micro Systems)
(a), and sample of Cheddar cheese before (b) and after (c) uni-
axial compression to 20% of original height.
528 Rheology and Texture of Cheese
Measurements are generally made in compression
mode rather than in tensile mode because (a) com-
pression behaviour is relevant to sensory perception
arising from chewing and mastication, (b) it is difficult
to grip cheese samples to carry out a tensile test and
(c) hard cheese has inhomogeneities which occur in
random positions and directions with respect to sam-
ple dimensions, making it difficult to obtain repro-
ducible results for tensile fracture. A compression
measurement involves compression of a rectangular or
cylindrical sample between parallel plates. Compres-
sion testing is more suited to large strain deformation
than to linear viscoelastic deformation (low strain).
This is because initial contact between the parallel plates
and the sample usually involves some realignment of the
sample surface due to imperfections in the surface
smoothness, as a result of intrinsic macrostructural
characteristics of the cheese (e.g., veins, cracks, open-
ness). Moreover, the difficulty in fine precision cutting
can give rise to lack of accuracy in low strain measure-
ments (up to 0.5 mm of deformation). Ideally, the cheese
sample may be conditioned by applying one or more
low compression deformations (e.g., ⬃ 0.05) prior to
testing. A typical force–displacement curve, obtained
by compression of a sample of Cheddar cheese at con-
stant velocity to a strain of 0.8 (i.e., final height of the
sample is 20% of original height), is shown in Fig. 23.
Compared to other rheological methods for evaluat-
ing cheese, large-strain deformation compression
offers several advantages: the strains applied are in the
range of those applied to cheese during size-reduction
operations as applied commercially; it is a dynamic
method for which the calculated parameters (e.g., f,
f) depend on a range of stress–strain data accumu-
250
200
fracture point
150
Force, N
100
50
0
0 5 10 15 20
Displacement, mm
Figure 23 Force–displacement curve obtained from uniaxial
compression of 6-month-old Cheddar cheese at 4 °C, on a
TA.HDi texture analyser, showing the fracture point. The shaded
area represents the fracture work or toughness.
lated during the test; sample preparation does not
require specialised cutting equipment; all cheeses,
apart from soft cheeses with a very high f, e.g.,
mature Camembert with an almost-runny consistency,
can be prepared easily and tested; and the test method
is simple and rapid. However, for reproducible results,
sample shape and dimensions need to be precise,
which can be difficult where cylindrical samples are
acquired by pushing a cylindrical borer into a portion.
As is common to all large strain deformation methods,
a serious limitation is the difficulty in obtaining results
for cheeses with eyes.
Relationship between shear and normal stresses in
uniaxial compression
A proper interpretation of uniaxial compression tests
requires that the complexity of internal forces in a
sample be understood. A sample undergoing uniaxial
compression is distorted in various directions at any
one time, e.g., vertically, horizontally and diagonally
(Fig. 24). Even though the applied force is uniaxial, a
combination of compressive and shear forces is created
in different planes within the sample (Fig. 25). A frac-
ture is more likely to be caused by shear rather than
compression forces, since cheese is relatively incom-
pressible. Thus, large strain uniaxial compression indi-
rectly measures shear behaviour. Mohr’s circle, a
construction widely used in the analysis of the
strength of materials, enables the shear and compres-
sion stresses (up to the fracture point) on an inclined
plane, at any angle, &, to the horizontal to be calcu-
lated (Fig. 26). Analysis shows that the maximum
normal stress (max  F/A) is twice the maximum
shear stress (i.e., max  F/(2A)).
(a) (b)
θ1 θ2
Figure 24 Illustration of strains in relation to the principal axes
of strain (vertical, y and horizontal, x ) in a sample undergoing
uniaxial compression. Compression along the y-axis results in a
simultaneous extension along the x-axis. This coincides with a
reduction in the angle of inclination, from &1 to &2 of a diagonal
line implying shearing along an inclined plane.

(a) Compression forces (b) Shear forces
Frictional force
Figure 25 Compression (a) and shear forces (b) within a sam-
ple during a uniaxial compression test.
Effect of pre-test strain history
Fracture stress decreases significantly if the sample has
been cycled through successive compressions. This was
shown for Cheddar, Cheshire and Leicester cheeses at
20 °C by Dickinson and Goulding (1980). The effect
was noticeable even when the previous strains were rela-
tively low, e.g., f in Cheddar and Cheshire cheeses fell
by ⬃ 30% after 50 cycles of ⬃ 0.1; the reduction in f
in Leicester cheese was about 12% under the same con-
ditions. However, pre-test strain history (50 cycles at
⬃ 0.2) produced no significant effect on f. This indi-
cates that a history of recoverable deformation causes
some internal structural weaknesses, which reduce the
subsequent strength of the cheese, but the flow condi-
tions under which fracture occurs are not affected.
Effect of sample-machine interface conditions and
sample dimensions
The expression of compression characteristics in the form
of stress–strain curves rather than force–displacement
F At low compression plate speeds (20 mm/min),
Shear
stress
τ
F/A
σ τ
2θ Normal
σ stress
θ
Figure 26 Mohr’s circle, for computing normal and shear
stresses on any plane in a sample under uniaxial compression.
Each point on the circle represents the normal (, x-axis) and
shear (, y-axis) stresses which occur on a plane inclined at an
angle &. It can be inferred from the diagram that maximum 
occurs at &  45° and equals half of the uniaxially applied ,
i.e., F/A, at &  0°, where F is the applied force and A is the
cross-sectional area of the sample.
Rheology and Texture of Cheese 529
curves is meant to remove the effect of sample dimen-
sions. At low values, the effect of sample dimensions
may be eliminated in this way. However, for large
(0.1), especially  f, the distribution of stress and
strain within the sample depends on sample dimensions,
as the sample may be deformed into an irregular shape,
due to fracturing, barreling and squeezing. Squeezing
flow is an intrinsic aspect of large strain uniaxial com-
pression of cheese, i.e., as sample height is reduced,
the cheese spreads in a lateral direction (Fig. 24). This
implies that shearing takes place within the sample and
that frictional shear forces occur at the points of contact
between the sample and the compression plates. Friction
can be reduced by lubrication of the contact surfaces
with mineral oil or grease; in contrast, surface friction
can be increased by the use of emery paper, or the sur-
faces can be bonded using glue, both of which eliminate
slippage as a result of cheese ‘sweating’. Because lubrica-
tion allows lateral movement at the contact surfaces dur-
ing compression, the sample shows a slight tendency
towards an hour-glass shape, as opposed to the relatively
large barreling effect. Lubrication can reduce the stresses
in squeezing flow by as much as 50% and increase the
observed f from ⬃0.45 to 0.55, in the case of Gouda
cheese at 20 °C with an aspect ratio (i.e., height/width)
of unity and a cross-head speed of 500 mm/min (Culioli
and Sherman, 1976). However, the frictional effect
increases with cross-head speed. At a low cross-head
speed (5 mm/min), lubrication decreased f by ⬃20% in
Cheddar cheese where the aspect ratio was 0.35, with
the effect becoming more pronounced (of the order of
20–30%) at  f (Casiraghi et al., 1985). In contrast,
the bonding of the cheese surfaces to the compression
plates (e.g., using cyanoacrylate ester adhesive) had rela-
tively little effect on f, f and max. A similar trend was
found for Mozzarella and processed cheese spread
(Casiraghi et al., 1985).
friction had only a negligible effect in Gruyere and
processed Mozzarella cheese with aspect ratios near
unity (Charalambides et al., 2001). However, at aspect
ratios 0.5,  at a given was slightly higher when
the samples were not lubricated. Similar findings were
reported by Ak and Gunasekaran (1992), using min-
eral oil as a lubricant on Cheddar cheese with aspect
ratios of 0.65 and 1.0. Hence, accurate – curves can
be obtained from unlubricated testing provided the
aspect ratio is sufficiently high, e.g., 1.
Effect of deformation rate
Due to viscous effects,  at a given (e.g., 0.1) and f
depend on rate of deformation and increase by
⬃40–50% per 10-fold increase in compression plate
speed in cheeses at 4–20 °C (Luyten et al., 1991a; Ak
530 Rheology and Texture of Cheese
and Gunasekaran, 1992; Pons and Fiszman, 1996;
Xiong et al., 2002). f is not significantly affected by
deformation rate.
Influence of shape
Cylindrical and rectangular-shaped samples have been
used in uniaxial compression (Table 3). For both
shapes, only slight differences have been reported in
the – characteristics up to the fracture point (⬃40%
compression). However, at  f, compression of
cubic samples resulted in significantly greater forces
(and ) than cylindrical samples (Culioli and Sherman,
1976). For a given shape (e.g., cylindrical) and aspect
ratio (e.g., unity), doubling the absolute dimensions
had little effect on  at a given deformation (Culioli
and Sherman, 1976).
Large strain shear measurements
A rheometer or a viscometer may be used also, in addi-
tion to a texture analyser, to apply large strain shear to
cheese. In the rheometer, the parallel plate geometry
already described (see ‘Fundamental Measurements:
Oscillatory Rheometry for Linear Viscoelastic Measure-
ments in Cheese’) for linear viscoelastic deformation,
becomes applicable for large shear strain (i.e., torsion
testing, where a shear is applied in rotational mode). A
large shear strain may be applied also with a rheometer
using samples of cheese with capstan geometry (Truong
and Daubert, 2000, 2001; Truong et al., 2002). The effect
of cutting the cheese into a capstan shape is that the
greatest shear stress occurs at the cross-section of mini-
mum radius (Fig. 27). Bowland and Foegeding (1999)
used this technique to determine the fracture properties
of model processed cheese. Torsion shear tests, as above,
have been applied recently to cheese and offer few if any
distinct advantages above large strain deformation in
(a) (b)
2r
2R 2R
Figure 27 Illustration of sample shapes used in torsion tests,
using parallel plate geometry (a), and capstan geometry (b). The
capstan shape is obtained by milling a cylindrical sample using
a purpose-built milling machine.
compression/extension mode. However, for highly
deformable cheeses, e.g., fresh low-moisture part-skim
Mozzarella and young reduced-fat cheeses, e.g., Cheddar,
which generally do not undergo elastic fracture (i.e.,
where the sample breaks into distinct pieces), but rather
plastic fracture, on compressing to strains of 0.7–0.8
(Fenelon and Guinee, 2000; Guinee et al., 2002), torsion
shear may be useful in determining fracture stress and
fracture strain. The latter parameters may be important
in some cheeses, e.g., in the formation of cheese strings
containing two different-coloured cheeses in a twisted
helical (rope-like) configuration. However, preparation
of capstan-shaped samples requires specialised milling
equipment and is time-consuming.
Vane rheometry has been used for large strain shear
deformation tests in processed and natural cheeses,
including Cheddar and Mozzarella. In this method a
probe, typically with four blades or vanes, is inserted
into a sample and rotated slowly at a constant rate
(e.g., 10 cycles per minute), while the torque is meas-
ured (Fig. 28). The technique produces a shear stress
versus strain characteristic with a distinct peak at the
point of failure, i.e., fracture. Fracture stress is shear
rate-dependent in both cases, being smaller by a factor
of 2–3 with the vane technique than with the torsion
technique. Using vane rheometry and torsion, it was
possible to separate different cheese types on fracture
stress/fracture strain ‘texture maps’, which were similar
in both cases (Truong and Daubert, 2001).
The vane method, which is rapid and does not require
sample preparation, as in cutting, has been found to give
trends comparable to those obtained using capstan
geometry for processed cheese, Cheddar and Mozzarella
(Truong and Daubert, 2001). However, disadvantages of
vane rheometry may include the difficulty of inserting
the vane without cracking the cheese mass (e.g., hard
cheeses such as Romano or Parmesan, with a low frac-
ture strain and soft cheeses with a low fracture strain,
such as Feta), and variability of results for cheeses, such
as Blue or Gouda, with macrostructural heterogeneities,
such as cracks, veins, small openings and openness.
Bending tests
Hard cheeses can be subjected to bending tests which
may involve three- or four-point loading (Whorlow,
1992; Rosenthal, 1999); a schematic for three-point
bending of a ‘finger-shaped’ cheese sample is shown
in Fig. 29. Such tests produce compression and ten-
sion on alternate sides of a neutral axis, with the max-
imum tensile strain occurring on the bottom surface
of the sample and the maximum compressive strain
on the top surface (Fig. 29). A force–displacement
curve obtained from three-point bending of a cheese
sample, e.g., Cheddar cheese (Fig. 30), enables the
 Rheology and Texture of Cheese 531

(a) (b)

Figure 28 Vane rheometer probe before (a) and during (b) shear test on process cheese. Photos courtesy of Truong and Daubert
(2000) Gel Consultants Inc. (See Colour plate 11.)

estimation of fracture stress (f). With the assumption

that the sample deforms into an arc shape on bending,
the tensile strain ( ) at any point on the bottom sur-
(a) W H face may be estimated for any displacement (y) as:

4Hy

L2  4y2
L

(b) compression where y is the displacement at any point of contact

E
along the axis where the sample makes contact with
neutral axis
the moving beam, H is the sample height and L is the
C E1 tension span between the supporting beams (Fig. 29).
D
Assuming that  is proportional to , the tensile or
Figure 29 Schematic of a bending test with three-point load- compressive stress (M) at any point on either sur-
ing: (a) Geometry with cheese sample in place prior to testing, face of the sample can be approximated, subject to
resting on the two support beams, C and D; (b) cheese sample
assumptions about linearity, as:
being deformed by mobile beam E during testing.

3FL
M ⬇
2WH2
50
where F is the force (obtained from the force-displace-
40 ment curve) and W is the width of the sample (Fig. 29).
f
Even though the – behaviour departs from linearity
Force, N

30
before the point of fracture, the formula can be used
20 to approximate the fracture stress (f). Since fracture
is more likely to occur in tension than in compres-
10
sion, fracture behaviour in tensile mode can be com-
0 pared more easily using bending tests than by using
0 5 10 15 tensile tests, which are difficult to carry out because
Displacement, y, mm
of the difficulty of grabbing a sample without deform-
Figure 30 Force–displacement curve for three-point bending of ing it. The fracture strain ( f) obtained during three-
a 180-day-old Cheddar cheese sample (25  25  50 mm), point bending may give a better estimation of the
ripened at 4 °C. The sample was deformed at a rate of cutting behaviour of cheese than f obtained from
20 mm/min (by the mobile beam) on a TA.HDi texture analyser, compression testing (see ‘Uniaxial Compression’), as
using a three-point bend rig (model HDP/3PB) with 40 mm span
between the supporting beams. A distinct fracture point (f) at point
cutting involves a combination of tensile and shear
E1 (Fig. 29) on the bottom surface of the sample coincides with strains. In compression testing, fracture is due to
the maximum extension. shear displacement.
532 Rheology and Texture of Cheese
Wire-cutting
Fracture energy during cutting is quantified by measur-
ing the force required to push wires of different diam-
eters at constant velocity through a cheese mass (Green
et al., 1986; Marshall, 1990). Luyten et al. (1991b)
investigated the fracture properties of Gouda cheese
using wire-cutting. A typical force–time curve shows an
initial rise in force, which reaches a maximum as the
wire penetrates the sample surface. Once the surface
has been broken, the force rapidly drops to a constant
level, Fc, as the wire ‘ploughs’ through the sample. Fc
increases somewhat with cutting speed (⬃ doubling for
a 20-fold increase in speed) and with wire diameter (by
3–4 fold for 300 m compared with 25 m diameter).
Since fracture develops around a crack, a specific
fracture energy ( J/m2), Rf, can be defined as the energy
needed per unit area (of crack) to cause a fracture to
spread. While it is not possible to determine specific
fracture energy precisely, because of the inherent het-
erogeneity in cheese structure (see ‘Cheese structure’),
its order of magnitude can be determined by measuring
cutting force using wires with a series of diameters and
extrapolating to a diameter of zero. The specific frac-
ture energy is calculated as:
Fc0
Rf 
d ture. The discrepancy between the latter studies in
where Fc0 is the cutting force, extrapolated to cutting
with a wire of zero diameter, and d is the sample width,
i.e., the length of wire in contact with the cheese
(Luyten et al., 1991b). The fracture energy obtained
with the wire-cutting method may give a more accurate
prediction of the behaviour of cheese during cutting
(e.g., portioning, slicing) than that obtained using
large-strain shear or compression deformation tests.
Measurement of Time-Dependent
Rheological Characteristics
As has been stated (see ‘Terminology of Rheology
and Texture’ and ‘Fundamental Measurements: Large
Strain Deformation’), the rheological behaviour of
viscoelastic materials, like cheese, is generally influ-
enced not only by instantaneous stress or strain, as
in the case of ‘ideal’ materials, but also by rheolog-
ical history of the material, i.e., the stresses and
strains which have already been experienced. This is
verified by natural occurrences, such as a gradual
deformation of a pane of glass under its own weight
in a window of an old building. Indeed the compres-
sion of rock or ice under gravitational force results
in flow, albeit very slow, analogous to a creep experi-
ment. Similarly, hard cheeses can eventually bulge,
especially if stacked. Such time-dependent behaviour
may be measured in creep and stress-relaxation tests,
and may be carried out by means of compression,
tension or torsion in the viscoelastic region (e.g.,
0.1; cf. Fig. 9).
Effect of Sample Temperature on Large
Strain Deformation Characteristics in
Cheese
Early research showed that increasing the temperature
of Gouda cheese in the range 10–20 °C reduced the
value of f, f and max, as measured by compression
to 80% using the Instron UTM (Culioli and Sherman,
1976). While f in Cheshire and Leicester cheeses
decreased exponentially as the temperature was
increased from 0 to 40 °C, the effect on fracture strain
depended on the type of cheese; fracture strain for
Cheshire cheese increased by ⬃2 over the range of
temperature, while fracture strain for Leicester cheese
was not affected by temperature (Dickinson and
Goulding, 1980).
Molander et al. (1990) reported a similar trend for f
and max in 4-week-old Brie between 5 and 20 °C; how-
ever, in contrast to the results of Culioli and Sherman
(1976), f increased slightly on raising the tempera-
relation to strain may be attributable to differences in
the degree of fat coalescence, proteolysis and therefore
fat separation and slippage. On heating cheese to a tem-
perature (30–60 °C) greater than those (e.g., 4–25 °C)
normally associated with retailing, domestic refrigera-
tion and consumption, compression results in squeez-
ing flow behaviour (Ak and Gunasekaran, 1995), i.e.,
stress increases with strain as the cheese is squeezed
between the plates and no fracture point is observed.
The deformability modulus (initial slope of the
stress–strain curve) showed an Arrhenius type of char-
acteristic, decreasing exponentially with temperature
from 18 kPa at 30 °C to 3 kPa at 60 °C. Such a trend is
expected, as milkfat is essentially fully liquid at 30 °C
(Norris et al., 1973). Indeed, heating cheese to 60 °C
in the absence of an applied stress generally results in
flow of the part-molten cheese mass to an extent
dependent on cheese type and heating time.
Techniques for Measurement of Viscosity
In some situations, cheese products may occur in ‘liq-
uid’ form, either in the course of processing or in their
usage. Typical examples are processed cheese, cheese
dips and cheese sauces. The viscosity of these products
may be measured by a number of instruments, e.g., the

Bostwick consistometer, which has been used to give
an empirical measurement of viscosity of a soft
processed cheese spread (Rosenthal, 1999). In the lat-
ter instrument, a sample of the material being tested is
placed in a cell and released by opening a simple guil-
lotine slide gate, allowing the product to flow horizon-
tally across a scale marked in centimeters. The length
of flow in a given time period (usually 30 s), known as
the Bostwick number, is taken as a measure of viscos-
ity. Alternatively, viscosity can be measured under
defined shear or low amplitude stress or strain in a
rheometer, using different geometries such as concen-
tric cylinders, a cone and plate, or parallel plates.
Online measurements of viscosity of cheese prod-
ucts may be important, e.g., as an early measure of
indicating the susceptibility of a formulation to ‘cream-
ing’ (see ‘Pasteurized Processed Cheese and Substi-
tute/Imitation Cheese Products’, Volume 2). A range of
commercial on-line viscometers are available for meas-
uring viscosity in a continuous flow situation.
Terminology Applied to Cheese Texture
Cheese texture may be defined as a composite sensory
attribute resulting from a combination of physical
properties that are perceived by the senses of touch
(including kinaesthesis and mouth-feel), sight and hear-
ing (Brennan, 1988). Thus, cheese texture is directly
measurable only by sensory analysis. Sensory analysis
requires definition and classification of textural attri-
butes or descriptors.
Descriptors applied to cheese texture have been
grouped into mechanical, geometrical and other char-
acteristics (Fig. 31). The mechanical characteristics are
sensed as forces on the teeth, tongue and the mouth
Initial perception
(before placing in
mouth)
Primary characteristics
Initial perception
on palate
Secondary characteristics
Mastication
Tertiary characteristics
(high shearing
stress)
Residual
masticatory
impression
Figure 31 Classification of food texture into primary, secondary or tertiary characteristics, based on Sherman (1969).
Rheology and Texture of Cheese 533
generally during eating, and by hearing in the case of
fracture, whereas geometrical characteristics are mostly
sensed visually but may also be partly sensed by touch.
The other characteristics are ‘mouth-feel’ qualities,
described subjectively by terms such as hard, soft, firm,
springy, crumbly, adhesive, moist or dry. These terms
are thought to have significance in relation to con-
sumer appeal and satisfaction (Szczesniak, 1963a).
The mechanical characteristics, in turn, have been
divided into five primary parameters and three sec-
ondary parameters (Table 5, Fig. 31). The secondary
parameters are considered to be composed of various
intensities of hardness and cohesiveness. The geomet-
rical parameters are divided into two classes, i.e., those
related to particle size and hardness, and those related
to particle shape and orientation. Experience shows
that panelists found hardness relatively easy to sense
but that adhesiveness was much more difficult to
judge (Halmos, 2000).
Sensory texture terms, as distinct from rheological
terms, have linguistic boundaries, i.e., they are suscep-
tible to different interpretation in different languages
(Lawless et al., 1997; Bourne, 2002). Some texture-
related characteristics can be measured by machines
and these are not bound by language. These character-
istics include hardness, cohesiveness, adhesiveness,
elasticity, viscosity, brittleness, chewiness and gummi-
ness, definitions for which are given in Table 5. The
measurements give objective quantifiable data, pro-
vided the measurement conditions are well defined.
Relationships between cheese texture and rheology
The Texture Profile Analysis (TPA) method, involving
instrumental measurement using double bite compres-
sion, was developed to imitate the compressing action
• visual appearance
• sampling and slicing characteristics
• spreading, creaming characteristics, pourability
• analytical characteristics
• particle size, shape and size distribution
• oil content; size, shape and size distribution of oil
particles
• elasticity, cohesion
• viscosity
• adhesion (to palate)
• hard, soft
• brittle, plastic, crisp, rubbery, spongy
• smooth, coarse, powdery, lumpy, pasty
• creamy, watery, soggy
• sticky, tacky
• greasy, gummy, stringy
• melt down properties on palate
534 Rheology and Texture of Cheese

Table 5 Classification of the mechanical characteristics of cheese into primary and secondary parametersa

Primary parameters Secondary parameters

Hardness – the force necessary to attain a given deformation
Cohesiveness – strength of internal bonds making up the
body of the product
Elasticity – the rate at which a deformed material returns to
its original form after the deforming force is removed
Viscosity – rate of flow per unit force
Adhesiveness – the work necessary to overcome the
attractive forces between the surface of a food and surface
of other materials with which it comes in contact, e.g., the
teeth, palate and tongue
Brittleness – the force at which the material fractures
Chewiness – the energy required to masticate a solid food,
e.g., some cheeses such as Mozzarella, to a state ready
for swallowing
Guminess – the energy required to disintegrate a semi-solid
food, e.g., some cheeses such as ripe Camembert, to a
state ready for swallowing

a Modified from Szczesniak (1963a), Bourne (1978).

of molar teeth while masticating food in the mouth Texture profile analysis (TPA)
(Szczesniak, 1963a; Peleg, 1976; Bourne, 1978).
A system of rheological parameters (e.g., firmness,
Classification of the mechanical attributes of cheese
elasticity) related to texture and known as TPA was
texture, as described above, was designed with the aim
developed (Fig. 32; Table 6; Friedman et al., 1963).
of integrating sensory data for foods evaluated by
The rheological measurements were originally carried
trained panels, with texture-profile data obtained on
out using the General Foods Texturometer (see ‘Imita-
the same foods using compression testing. For this
tive tests’), using double-bite compression (Bourne,
purpose, objective rheological parameters, some of
1978). Texture profile analysis parameters were later
which correspond in name to the sensory-determined
calculated from measurements using uniaxial double-
parameters, were defined (Table 5) and are known as
bite compression at constant speed, using texture
TPA parameters (see ‘Texture profile analysis’).
analysers including the Instron UTM (Breene, 1975;
While this classification system has been modified,
Bourne, 1978; Lee et al., 1978) and the texture
the textural descriptors and their interpretation as
analyser (TA series from Stable Micro Systems) (Halmos,
devised by this classification scheme (Table 5) are still
1997; Meullenet and Gross, 1999).
widely used in textural evaluation of food (Brennan,
1988; Drake et al., 1999). Sherman (1969) proposed Use of TPA to evaluate cheese texture
an alternative classification of food texture (Fig. 31).
The characteristics contributing to the texture of cheese, Szczesniak (1963b) found a curvilinear relationship
and other foods, during eating have been classified as between TPA hardness and an organoleptic rating of
primary, secondary (e.g., adhesiveness) or tertiary hardness. Casiraghi et al. (1989), working with five
(e.g., firmness) (Fig. 31). The primary characteristics, different Italian cheese varieties, including Grana
from which all others are derived, include the food’s Padano and Italico, showed that sensory hardness was
composition, its micro- and macro-structure, and its highly correlated with instrumental hardness.
molecular properties. The secondary and tertiary cate-
gories of textural properties include many characteris- 1st compression
tics which are directly related to the rheological stroke
properties as it is subjected to various stresses and
strains during eating, e.g., hardness, brittleness and 2nd compression
stroke
adhesiveness (Sherman, 1969). According to this clas-
Stress, σ

sification, the secondary characteristics are associated

H1
with initial perception in the mouth, i.e., upon contact
with tongue, palate and teeth prior to mastication. A1 A2
0
Sherman (1969) claimed that the main characteristics A3
sensed at this stage are elasticity (E), viscosity () and
adhesion to the palate, where elasticity is understood
as the tendency to recover its shape after removal of
the stress. Two of those characteristics, namely elastic- Time
ity and viscosity, can together be represented by the Figure 32 Typical stress trend during a double-bite compression
Burgers mechanical model (see ‘Cheese texture’). test, from which TPA parameters are calculated (see Table 5).
 Rheology and Texture of Cheese 535

Table 6 Texture profile analysis (TPA) parameters and physical definitionsa

Terminology b Physical definition (TPA term) Units

Fracturability Stress (or sometimes, force) to fracture point, H1 (Fig. 32) Pa, kPa
Firmness Stress (or sometimes, force) at a given deformation Pa or kPa
Springiness (or Percentage of deformation which is recovered between the first and second bites –
elasticity)
Cohesiveness Area of second bite over area of the first bite (A2/A1) in Fig. 32 –
Gumminess Hardness  Cohesiveness Pa, kPa
Chewiness Hardness  Cohesiveness  Springiness Pa, kPa
Adhesiveness Work necessary to pull the plunger (or compression plate) away from the sample J/m3
(Area 3 in Fig. 32)

a Sources: Bourne (1978), van Vliet (1991a), Szczesniak (1963a), Yang and Taranto (1982).
b Fracturability was originally known as brittleness (Bourne, 1978), and firmness as hardness (Szczesniak, 1963a).

Green et al. (1985) found significant correlations
between five sensory attributes (firmness, springiness,
crumbliness, graininess and stickiness) and instru-
mental parameters (f and f ). Hennequin and Hardy
(1993) reported that TPA-hardness, i.e., force at 70%
compression, also had a high correlation with sensory
hardness (r  0.78, n  19, P  0.001) for four soft
cheeses. Halmos (2000) compared sensory and instru-
mental measurements of hardness, cohesiveness and
adhesiveness of six cheeses with a wide range of tex-
ture (including Havarti, Swiss and Romano). The sen-
sory measurements increased with the corresponding
instrumental readings, apart from one parameter for
Romano cheese, for which the cohesiveness as meas-
ured instrumentally was ranked higher than the corres-
ponding sensory measurement. The significant
correlations, which were characteristic of the overall
study, confirm the value of objective measurements in
support of sensory measurements. However, the devia-
tion in the trend for the Romano cheese highlights the
complexity of textural (i.e., tactile sensory) character-
istics as compared with instrumental measurements.
Antoniou et al. (2000) performed sensory and TPA
analyses on 15 French cheeses (Munster, Emmental,
Roquefort, Beaufort, Camembert, Reblochon, Pont
l’Eveque, Brie de Meaus, Tomme de Savoie, Valencay,
St Nectaire, Pyrenees Brebis, Blue d’Auvergne, Comte
Vieux and Fourme de Salers). The cheeses fell into
three compositional groups on the basis of moisture
(means 34, 45 and 51%, w/w). This grouping carried
through to the results of mechanical and sensory
analysis. The mechanical (TPA) terms which were
most significant in differentiating the groups were:
force at 10% deformation, relaxation force (after hold-
ing sample for ⬃12 s at 10% compression), force at
80% deformation (hardness), fracture force and adhe-
siveness. The most significant sensory terms were:
hardness, fracturability and chewiness. Some of the
mechanical parameters were highly correlated with
each other (e.g., force at 10% deformation, fracture
force and hardness). Likewise, some of the sensory
parameters were inter-correlated, e.g., hardness with
adhesiveness. In agreement with previous studies
(Green et al., 1985; Casiraghi et al., 1989; Hennequin
and Hardy, 1993; Halmos, 2000), sensory parameters
were highly correlated with mechanical parameters,
e.g., mechanical hardness with sensory hardness. It is
noteworthy that the 10% compression measurements
(a level of deformation that is mostly recoverable) pre-
dicted cheese texture (i.e., as judged in sensory terms)
better than the 80% compression tests.
Despite the significant correlations between some
sensory textural parameters and rheological measure-
ments, instrumental analysis of texture, e.g., using tex-
ture analysers, is not considered a complete substitute
for sensory evaluation (see Halmos, 2000), because of
several factors: complexity of mastication, differences
between individuals in the perception of texture, effect
of time of day upon perception of texture, and others.
While instrumental methods alone cannot be relied
upon to determine consumer acceptance, their value
resides in their ability consistently to enable small
changes in physical characteristics, which contribute
to texture, to be quantified.
Use of instrumental shear deformation to evaluate
cheese texture
Three techniques for large strain shear deformation
testing have been described (see ‘Large strain shear
measurements’). Truong et al. (2002) compared instru-
mental textural measurements on Cheddar cheese, as
obtained using vane rheometry (shear), uniaxial
compression (single bite) or TPA (double bite), with
the corresponding sensory texture measurements.
Instrumental texture maps of ten commercial Cheddar
cheeses, generated by the vane method and by com-
pression testing, clearly separated the cheeses and
showed similar distribution patterns. Highly significant
536 Rheology and Texture of Cheese
correlations were found between vane parameters and
TPA parameters (i.e., by uniaxial compression), and
between TPA parameters and sensory texture parameters
(by mouth). Correlations between vane parameters and
sensory parameters were significant, but not as highly
significant as between sensory and TPA parameters. The
higher correlation between sensory texture and TPA
texture could be due to the fact that TPA parameters
were developed in conjunction with compression (i.e.,
General Foods Texturometer), while no corresponding
texture-related parameters have been developed for tor-
sional techniques, such as the vane method.
Conclusions
The rheological properties of cheese have a large influ-
ence on its texture and behaviour during size reduc-
tion, and hence, its suitability as an ingredient (see
‘Cheese as an Ingredient’, Volume 2). Many factors
influence the rheological properties, including manu-
facturing procedure, variety, composition and bio-
chemical changes during ripening. The latter
parameters have a major influence on the degree of
hydration, or aggregation, of the para-casein matrix,
which is the major structural element controlling
deformation on the application of a stress.
Many methods are available for measuring the rheo-
logical properties of cheese; some measure within the
linear viscoelastic range to yield precise rheological
quantities. In contrast, rheological measurements
made under large strain or stress yield quantities
which are more empirical in nature, but which are
typically related to the stresses and strains experienced
during consumption and size reduction.
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Rheology and Texture of Cheese 539
Glossary
Cauchy strain. See Engineering strain.
Compliance. Symbol J, is the ratio of strain to stress.
In the elastic region, J  1/G , where G is shear (or
storage) modulus.
Cox-Merz rule. This rule states that the (steady) vis-
cosity versus shear rate curve is virtually identical to
the viscosity versus frequency curve, determined by
dynamic oscillation.
Creep. The response to a constant applied (normal or
shear) stress. Creep can be expressed in terms of strain
or compliance.
Creep compliance. The ratio of strain, (t), resulting
from an applied constant stress, c, to the stress, i.e.,
(t)/c.
Creep modulus. The inverse of creep compliance, i.e.,
c/(t).
Deformability modulus. Slope of the stress–strain
curve in an approximately linear region, typically up
to a strain of ⬇0.10.
Elastic material behaviour. An elastic deformation is
one where the material recovers fully upon removal
of applied stress without time dependency, i.e., recov-
ery is instantaneous and complete upon removal of
stress.
Elastoplastic material behaviour. When the stress in
the material exceeds a certain limit, irreversible defor-
mation results with negligible time dependency, i.e.,
partial recovery is instantaneous upon removal of
stress; also known as elastoplastic deformation.
Engineering strain. Deformation relative to original
sample dimension, i.e., L/Lo, is called engineering
strain, or Cauchy strain, or strain.
Engineering stress. The ratio between applied force, F,
and original sample area, Ao, is known as engineering
stress or stress.
Fracture work. See Toughness.
Kelvin element. Also known as a Voigt element or a
Kelvin-Meyer solid. This is a mechanical model con-
sisting of a spring in parallel with a dashpot. A num-
ber of such elements in series, with a spectrum of time
constants, can be used to simulate creep compliance.
Kinematic viscosity. This is the ratio between dynamic
viscosity and density. Units: m2/s  104 stokes.
Linear behaviour. If one measured parameter varies in
proportion to another, e.g., stress in proportion to a
range of applied strain, their behaviour is described as
linear and a modulus may be defined as the ratio
between the parameters, e.g., Young’s modulus.
Linear viscoelastic deformation. Cheese and other
organic materials exhibit a combination of elastic and
viscous behaviour at low strains, i.e., they recover their
shape upon removal of applied stress, but not instantly.
The elastic and viscous effects can be determined using
540 Rheology and Texture of Cheese
low-amplitude oscillatory rheometry. At low ampli-
tudes of oscillation there is a constant relationship
between the elastic and viscous components of com-
plex modulus. Consequently, displacements of this
type are referred to as linear viscoelastic deformation.
Loss modulus. The ratio between the out-of-phase
component of shear stress and shear strain (G   /)
in a dynamic oscillatory measurement; also referred to
as viscous modulus.
Maxwell element. This is a mechanical model consist-
ing of a spring in series with a dashpot. A combination
of such elements in parallel, with a spectrum of time
constants, may be used to simulate relaxation behav-
iour in a viscoelastic material.
Modulus of deformability. See Deformability modulus.
Poisson effect. When a sample is compressed it bulges
in the lateral direction, i.e., the cross-section increases
with compression; this is the Poisson effect. The ratio
between lateral strain and longitudinal strain is known
as Poisson’s ratio. Poisson’s ratio equals 0.5 in the
absence of a volume change, and is less than 0.5 for a
compressible material.
Shear modulus. The ratio between the in-phase com-
ponents of shear stress and shear strain (G   /) in
a dynamic oscillatory measurement; also referred to as
storage, elastic, or in-phase, modulus.
Storage modulus. See Shear modulus.
Strength. The maximum stress a material withstands
before it breaks (i.e., fractures) or flows (i.e., becomes
plastic).
Stress. See Engineering stress.
Stress relaxation modulus. The stress that is required
to maintain a constant deformation is observed, as
a function of time (i.e., in a stress relaxation test).
The ratio of shear stress to strain is known as stress
relaxation modulus, or relaxation modulus. The relax-
ation modulus depends on the applied strain if the
strain exceeds the limit of linear viscoelasticity. Thus,
G(t, )  (t)/.
Stress relaxation test. This test involves an initial appli-
cation of (a normal or shear) strain at a constant rate up
to a pre-determined level of strain and then measuring
the decay of stress as a function of time while holding
the sample at constant strain; also known as a step
strain transient test.
Toughness. The work required to fracture; this is
measured as the area under a force–deformation curve
up to the point of fracture (Fig. 23).
True strain. The accumulated strain during the applied
loading,  ln (L/Lo), where ln is the natural loga-
rithm, L is the sample length under load, and Lo is the
original sample length, is known as the true strain,
Hencky strain or natural strain. This is applicable where
the strain is large and sample cross-section changes
appreciably under the load. True strain is not used
very much in cheese rheology. True strain can be
related to engineering strain, , using,  ln (1  ).
True stress. The ratio between applied force, F, and
actual area of cross-section, A , is termed true stress.
Thus, true  F/A , where A is the actual area, taking
the Poisson effect into account.
Uniaxial compressive strength. The apparent stress at
fracture, i.e., Fo/Ao, where Fo is the compression force
at fracture and Ao is the initial cross-sectional area of
the sample.
Viscoelastic material behaviour. Where rheological
behaviour can be resolved into elastic and viscous
components, e.g., as represented by a Maxwell model.
Viscoplastic material behaviour. In contrast to elastic
behaviour, this is a time-dependent and irreversible
deformation that occurs when a certain stress level has
been exceeded, i.e., strain does not respond instantan-
eously to applied stress, but instead strain keeps on
growing while the stress is applied and does not return
to zero upon removal of stress; also referred to as vis-
cous material behaviour.
Viscosity or dynamic viscosity. Coefficient of dynamic
viscosity, , is the ratio between shear stress and shear
rate.
  /" where  is shear stress and " is shear rate.
Units: Pa.s or N.s/m2  10 poise.
Viscous modulus. See Loss modulus.
Young’s modulus. The ratio between normal stress and
engineering strain (E   / ).
 Growth and Survival of Microbial
Pathogens in Cheese
C.W. Donnelly, Department of Nutrition and Food Science The University of Vermont,
Burlington, USA
Introduction
Cheesemaking evolved centuries ago as a means of pre-
serving raw milk via fermentation. Selection of the
beneficial natural flora in milk, such as lactobacilli,
streptococci and lactococci, or direct addition of these
as starter cultures, preserves products and in many
instances allows competition with bacterial pathogens.
However, cheeses can become contaminated with
pathogens as a result of their presence in the raw milk
used for cheesemaking and subsequent survival dur-
ing the cheesemaking process. Alternatively, bacterial
pathogens can contaminate cheese via post-processing
contamination if sanitation and other measures in
the processing plant are not sufficient to prevent
re-contamination (Linnan et al., 1988; Johnson et al.,
1990a). The characteristics of the specific cheese variety
will dictate the potential for growth and survival of
microbial pathogens, with ripened soft cheeses present-
ing a higher risk for growth and survival of pathogens
than aged hard cheeses where a combination of factors,
including pH, salt content and aw, interact to render
cheeses microbiologically safe. Although cheeses have
been linked with documented outbreaks of food-borne
illness, epidemiological evidence collected from around
the world confirms that this occurs infrequently
( Johnson et al., 1990a; Altekruse et al., 1998; De Buyser
et al., 2001). This chapter will provide an overview of
factors which affect growth and survival of microbial
pathogens in cheese.
Factors that Influence the Safety of Cheese
The pathogens, Salmonella enterica, listeria monocyto-
genes, Staphylococcus aureus and enteropathogenic
E. coli (ETEC) pose the greatest risk to the safety of
cheese (Johnson et al., 1990a; De Buyser et al., 2001;
Leuschner and Boughtflower, 2002). If active lactic
acid starter cultures are used, Staph. aureus is con-
sidered to be a low-risk pathogen (Johnson et al.,
1990a). However, in traditional cheeses where active
starter cultures are not used, Staph. aureus may pose a
significant risk for toxin production in cheese if num-
bers are sufficiently high. The factors that contribute to
Cheese: Chemistry, Physics and Microbiology, Third edition – Volume 1: General Aspects
ISBN: 0-1226-3652-X
Set ISBN: 0-1226-3651-1
the safety of cheese with respect to pathogenic bacteria
include milk quality, starter culture or native lactic acid
bacterial growth during cheesemaking, pH, salt, con-
trol of aging conditions and chemical changes that
occur in cheese during aging (Johnson et al., 1990c).
Other technologies (e.g., use of starter cultures that
produce substances inhibitory to pathogens) may pro-
vide opportunities to add additional barriers to the
growth of bacterial pathogens. It is particularly import-
ant for the producers of raw milk cheeses to have a
documented and systematic approach to ensure prod-
uct safety.
Pathogens in raw milk
S. enterica, L. monocytogenes, Staph. aureus and ETEC
are associated with raw milk. E. coli 0157:H7 can
readily contaminate raw milk on the farm with con-
tamination levels of 4.2–10% and 2% reported in the
US and Canada, respectively (D’Aoust, 1989; Padhye
and Doyle, 1991). Over 70 cases of E. coli infection,
characterized by bloody diarrhea, haemolytic uremic
syndrome (HUS) and kidney failure, have been traced
to the consumption of raw milk (Martin et al., 1986;
Borczyk et al., 1987; Bleem, 1994) with a few add-
itional cases in England linked to yoghurt (Morgan
et al., 1993). E. coli 0157:H7 was first characterized in
1982 during epidemiological investigations of two out-
breaks which occurred in North America. Cattle are
thought to be the principal reservoir for this important
human pathogen, and in investigations where food has
been identified as the vehicle of transmission, ground
beef is the product most frequently linked to human
illness. Shere et al. (1998), in a longitudinal study of
E. coli dissemination on four Wisconsin dairy farms,
identified contaminated animal drinking water as the
most probable vehicle for infection of animals and a
potential intervention point for on-farm control of
dissemination of this pathogen. Since shedding of this
pathogen by cattle is intermittent, re-inoculation from
an environmental source rather than colonization of
the pathogen is the more likely explanation than inter-
mittent shedding.
Copyright © 2004 Elsevier Ltd
All rights reserved
542 Growth and Survival of Microbial Pathogens in Cheese
S. enterica serovars Enteritidis, Typhimurium and
Dublin have been associated with food-borne disease
outbreaks involving raw milk and milk products
(Maguire et al., 1992; Cody et al., 1999; Villar et al.,
1999; De Valk et al., 2000). A 1987 FDA survey
revealed the presence of salmonella in 32 of 678 (4.7%)
samples of raw milk obtained from bulk-tank trucks in
Wisconsin, Michigan and Illinois, with 10 of 16
(62.5%) collection sites also testing positive (McManus
and Lanier, 1987). Salmonella spp. were isolated from
26 of 292 (8.9%) of farm bulk tank samples collected in
eastern Tennessee and southwest Virginia (Rohrbach
et al., 1992). Wells et al. (2001) examined recovery of
salmonella from faecal samples obtained from dairy
cows in 91 herds from 19 US states. Salmonella spp.
were recovered from 5.4% of the samples. Recovery lev-
els from cows on farms with less than 100 animals were
much lower (0.6%) than those from farms with over
100 cows, where recovery levels were 8.8%. The inci-
dence of Salmonella spp. in milk samples would be
expected to occur at a much lower frequency than in
faecal samples. Most farmstead cheesemakers maintain
small dairy herds, where the lower incidence data
would apply. S. enterica serotype Typhimurium defini-
tive type (DT) 104 emerged in the UK as an important
source of human infection in the late 1980s (Threlfall
et al., 1996). Subsequent outbreaks of human illness
traced to dairy sources were reported in Vermont
(Friedman et al., 1998), Nebraska, California (Cody et al.,
1999) and Washington State (Villar et al., 1999). This
organism is notable because it is resistant to multiple
antibiotics. Two outbreaks of S. enterica subsp. enterica
serotype Typhimurium DT104 infection were recently
linked to the consumption of Mexican-style soft cheese
manufactured from raw milk (Cody et al., 1999; Villar
et al., 1999). Aceto et al. (2000) conducted a survey to
assess the herd prevalence of S. enterica subsp. enterica
serotype typhimurium DT 104 in Pennsylvania dairy
herds. Of 51 farms surveyed, 11 were positive for sal-
monella species and 4 for S. typhimurium, 2 of which
were DT-104 positive. S. enterica serovar Dublin is pres-
ent in dairy cattle and was identified as the most inva-
sive of the salmonella bacteria for humans in studies
conducted in Denmark (Lester et al., 1995).
Beckers et al. (1987) and Lovett et al. (1987) esti-
mate that extremely low levels of L. monocytogenes
(0.5–1.0 ml) exist in commercial bulk-tank raw milk.
Listeria is inactivated by pasteurization, and contam-
ination of processed dairy products is therefore most
likely a function of post-pasteurization contamination
from the dairy plant environment. In fact, numerous
surveys document the presence of listeria within the
dairy plant environment, including floors in coolers,
freezers, processing rooms, particularly entrances, cases
and case washers, floor mats and foot baths and the
beds of paper fillers (Charlton et al., 1990; Klausner
and Donnelly, 1991). Pritchard et al. (1994), in a study
of dairy processing facilities, found that processing
plants near a farm had a significantly higher incidence
of listeria contamination than those without an on-site
dairy farm. Arimi et al. (1997) demonstrated the link
between on-farm sources of listeria contamination
(dairy cattle, raw milk and silage) and subsequent con-
tamination of dairy-processing environments. These
investigators subjected listeria strains collected from
farms and dairy processing environments over a 10-year
period to strain-specific ribotyping using the auto-
mated Riboprinter™ microbial characterization system.
A total of 388 listeria isolates from 20 different dairy
processing facilities were examined along with 44 silage,
14 raw-milk bulk tank and 29 dairy cattle isolates.
These 475 isolates included 93 L. monocytogenes, 362
L. innocua, 11 L. welshimeri, 6 L. seeligeri, 2 L. grayii
and 1 L. ivanovii strains. Thirty-seven different listeria
ribotypes (RTs) comprising 16 L. monocytogenes
(including five known clinical RTs responsible for
food-borne listeriosis), 12 L. innocua, 5 L. welshimeri, 2
L. seeligeri, 1 L. ivanovii and 1 L. grayii were identified.
Greatest diversity was seen among the isolates from
dairy-processing facilities with 14 of 16 (87.5%) L. mono-
cytogenes RTs (including 5 clinical RTs), and 19 out
of 21 (90.5%) non-L. monocytogenes RTs detected.
Sixty-five of the ninety-three L. monocytogenes isol-
ates belonged to the group of the five clinical RTs,
which included one RT unique to dairy-processing
environments, two RTs common to dairy-processing
environments and silage, and one RT common to dairy-
processing environments, silage, raw milk and dairy
cattle with the last RT appearing in dairy-processing
environments, silage, raw-milk bulk tanks and dairy
cattle. The finding of eight L. monocytogenes and twelve
non-L. monocytogenes RTs common to both dairy-
processing and farm environments clearly implicates the
farm as a natural reservoir for listeria RTs capable of
entering dairy-processing facilities. These findings, which
support the link between on-farm sources of listeria con-
tamination (dairy cattle, raw milk and silage) and subse-
quent contamination of dairy-processing environments,
stress the importance of farm-based Hazard Analysis and
Critical Control Points (HACCP) programmes for con-
trolling listeria. This work also showed that two import-
ant clinical L. monocytogenes ribotypes which were
previously identified as RT 19092 and RT 19161 and epi-
demiologically linked to listeriosis cases involving pas-
teurized milk and turkey frankfurters were recovered
from dairy-processing facilities A and B for 12 and 3
months, respectively, with L. innocua RT 19094 also pres-
ent in these same two facilities for at least five years.
 Growth and Survival of Microbial Pathogens in Cheese 543
Abou-Eleinin et al. (2000) analysed 450 goats’ milk
samples obtained from the bulk tanks of 39 goat farms
for listeria spp. over a 1-year period. Modified versions
of the USDA-FSIS (McClain and Lee,1989) and FDA
(Lovett et al., 1987) protocols were used for recovery
of listeria. Overall, 35 (7.8%) samples yielded listeria,
with L. monocytogenes identified in 17 of the 35 (3.8%)
listeria-positive samples and L. innocua in 26 (5.8%) of
samples. Eight milk samples contained both L. monocyto-
genes and L. innocua. Milk samples from 18 of the 39
(46.2%) farms were positive for listeria at least once
during the year-long study. Five different listeria RTs
were identified from 34 selected L. monocytogenes isol-
ates, 2 of which were deemed to be of clinical import-
ance. Isolation rates of listeria were markedly higher
during the winter (14.3%) and spring (10.4%), com-
pared to autumn (5.3%) and summer (0.9%). Similar
trends have been previously reported for cows’ milk
(Rea et al., 1992; Ryser, 1999).
Milk quality
Raw-milk quality is important in producing all cheeses,
but particularly for those made from raw milk. Low
bacterial counts and low somatic cell counts are the
key indicators of milk quality, and as their numbers
increase, there is a higher risk for contamination of
milk and cheese with pathogens. Monitoring and con-
trolling bacteria and somatic cell counts in milk should
be components of a HACCP programme to ensure
product safety. As rapid, cost-effective methods become
available for detection of bacterial pathogens in raw
milk, the use of specific pathogen testing could become
part of a HACCP programme. In general, when raw-
milk bacteria and somatic cell counts are high, there
will be other negative impacts on cheese quality that
may reduce consumer acceptability and cheese yield. In
most artisanal cheesemaking, the time from milking to
cheesemaking is very short and in some cases the milk
is made into cheese immediately on the farm without
cooling. Minimizing the time from milk collection to
the initiation of cheesemaking reduces the opportunity
for the growth of undesirable bacteria in raw milk.
Conversely, when milk is cooled and held in transport,
the opportunity for pathogen growth, particularly
growth of psychrotrophic pathogens, is increased.
The European Community Directives 92/46 and
92/47 (Anonymous, 1992) contain regulations for the
hygienic production and placing on the market of raw
milk, heat-treated milk and milk-based products. These
regulations establish hygienic standards for raw-milk
collection and transport that focus on issues such as
temperature, sanitation and microbiological standards,
enabling the production of raw milk of the highest
possible quality. Raw cows’ milk must meet quality
standards, e.g., a standard plate count at 30 °C of
100 000 cfu/ml and somatic cell counts of #400 000
per ml of milk. To meet these and other established
standards, countries employ HACCP principles in the
production of fluid dairy products. This involves iden-
tification of sites to be monitored and evaluated to
ensure that products are produced under the correct
conditions, as well as the development of critical limits
established by valid and verifiable parameters. In the
case of fluid milk products, many processors have
identified length of shelf-life as a critical limit. Shelf-life
is influenced by a number of factors, including clean-
ing and sanitizing of pipelines and milking equipment,
condition of raw milk used to produce product and
storage temperature. Pasteurization will eliminate some
of the indigenous microflora in the raw milk, including
pathogenic bacteria; however, thermoduric organisms
survive pasteurization. Post-pasteurization contamin-
ation of milk is problematic if the processing/packaging
environment is not maintained. Moreover, many con-
taminants, including listeria, are able to form biofilms
which protect them from cleaning and sanitizing
agents. Some regulations, such as those of the EU, have
established microbiological limits at the sell-by-date for
products such as cheeses. With respect to regulations
which govern the use of raw milk for cheesemaking,
limits have been established for Staph. aureus in raw
milk. Finished cheeses must meet specific hygienic
standards, in which case the presence of Staph. aureus
and E. coli indicates poor hygiene.
Heat treatment of milk
Milk contains heat-labile compounds (e.g., lactoferrin,
lysozyme and lactoperoxidase) that are inhibitory to the
growth of some pathogens. Recent work by Pitt et al.
(2000) has demonstrated that the growth of Staph.
aureus, S. enteritidis and L. monocytogenes was slower in
raw milk held at 37 °C for 72 h than in pasteurized milk
held under the same conditions. During the first 16 h of
incubation, the number of organisms increased in both
raw and pasteurized milks, but after 16 h, the number of
recoverable viable pathogenic organisms in the raw milk
began to decrease; an overall decrease of 2–5 logs was
found. The inhibitory effect of raw milk on the survival
of the above three pathogens in milk, reported by Pitt
et al. (2000), is probably of great importance for cheese-
making from raw milk, and additional research needs to
be undertaken. The lactoperoxidase system (LPS) is a
naturally occurring inhibitory system in raw milk and
comprises three components, lactoperoxidase, thio-
cyanate and hydrogen peroxide. All three components
are required to exhibit maximum antimicrobial effects.
544 Growth and Survival of Microbial Pathogens in Cheese
Gram-negative psychrotrophs, such as pseudomonads,
are extremely sensitive to this system. Approximately
0.5–1.0 g/ml lactoperoxidase is needed for effective
inhibition, and bovine milk typically contains 30 g/ml
(Björck, 1978). Quantities of thiocyanate and hydrogen
peroxide in milk are variable. For a full inhibitory effect,
about 10 g/ml of hydrogen peroxide is required, and
bovine milk normally contains 1–2 g/ml hydrogen per-
oxide. The thiocyanate levels in milk range from 0.02 to
0.25 mM but 8–25 mM is needed for optimum activity.
Pitt et al. (2000) hypothesized that the inhibitory effect
of raw milk in their study was due to activation of the
lactoperoxidase system by hydrogen peroxide-producing
lactic acid bacteria naturally present in the raw milk,
which grew at 37 °C. The authors postulated that the
reduction in growth of these organisms in the raw milk
could result from inhibitory products produced by acti-
vation of the LPS. However, it is highly unlikely that
pasteurization inactivates lactoperoxidase in milk, and
so there must be an alternative explanation for the much
greater inhibition of Staph. aureus, S. enteritidis and
L. monocytogenes by the raw milk.
Some cheeses are made from milk that has been given
a sub-pasteurization heat treatment at the farm, but are
technically classified as raw-milk cheeses. This process
can be beneficial when milk has to be transported and
stored at refrigeration temperature at a cheesemaking
facility and when there will be a time delay before cheese
manufacture. These near-pasteurization thermal treat-
ments are often called thermization and they help
to reduce the gowth of psychrotrophic bacteria that
cause quality defects in cheese. However, the thermiza-
tion process may partially inactivate some indigenous
antimicrobial milk components that were mentioned
previously.
Comprehensive studies conducted by the US Food
and Drug Administration (FDA) and the US Depart-
ment of Agriculture (USDA; Bunning et al., 1986,
1988) and by Health and Welfare Canada (Farber et al.,
1992) have shown that listeria are unable to survive
normal pasteurization conditions. Knabel et al. (1990)
found that growing L. monocytogenes at 43 °C prior to
heat-inactivation caused an increase in thermo-
tolerance, but a study conducted by Farber et al.
(1992) demonstrated that even under worst-case sce-
nario conditions, which included cultivation of L. mono-
cytogenes populations at 43 °C prior to inactivation,
pasteurization would render a 4.5–6.2-D process.
Therefore, while populations of L. monocytogenes have
been shown to survive minimum pasteurization
(71.1 °C/16 s) in various laboratory studies, survival
under actual conditions of commercial milk pasteuriza-
tion and processing is unlikely. Studies which define
the impact of commercial heat treatment of raw milk,
either naturally or artificially contaminated with bacter-
ial pathogens, are still relatively scarce. However,
L. monocytogenes is generally regarded as being more heat
resistant than salmonella or E. coli 0157:H7 (D’Aoust
et al., 1987; Line et al., 1991). Using raw milk inoculated
to contain various pathogens at a level of 105 cfu/ml,
D’Aoust et al. (1987) concluded that salmonella were
inactivated in milk after heating to 64.5 °C (148.2 °F)
or above for 16.2 sec, except for S. senftenberg which sur-
vived until the treatment exceeded 67.5 °C (153.5 °F)
for 16.2 sec. Heating at 63 °C (145.4 °F) for 16.2 sec
reduced populations of S. senftenberg by 3 orders of
magnitude. Thermal inactivation of E. coli 0157:H7
was complete at temperatures 64.5 °C (148.2 °F) for
16.2 sec (Line et al., 1991) which is similar to that
required for most salmonella except S. senftenberg.
Much of the aged raw milk cheese produced in the US
is subjected to some form of heat treatment, generally
thermization. This treatment generally consists of heat
treatment at 55 °C for a period ranging from 2 to 16
sec. The specific impact of this heat treatment com-
bined with the interactive effects of salt and pH during
subsequent ripening on pathogens such as listeria, sal-
monella and E. coli has not been well explored.
Extrinsic and intrinsic parameters in cheese which
dictate microbial growth
Growth of microbial pathogens in cheese is dictated by
extrinsic and intrinsic parameters. The important intrin-
sic parameters include moisture content, pH and acidity,
nutrient content, redox potential, presence of antimicro-
bial compounds, either those occurring naturally or
those which are added as food preservatives, e.g., NO3,
and the presence of competitive microflora (ICMSF,
1986). All of these factors dictate the potential for
bacterial pathogens to grow, persist or decline in cheeses.
Extrinsic parameters include factors such as type of
packaging/packaging atmosphere, time and temperature
of storage and holding conditions, processing steps,
product history and traditional use. The interaction of
these factors dictates the potential for microbial growth
in cheese.
Depending on the cheese variety, intrinsic parameters
such as pH may serve to enhance or inhibit the growth
of bacterial pathogens. Ryser and Marth (1987a) studied
the behaviour of L. monocytogenes in Camembert cheese.
The high moisture content and the neutral pH of this
surface-ripened cheese facilitate growth and survival of
pathogens such as listeria. Growth of listeria in Camem-
bert cheese was found to parallel the increase in cheese
pH during ripening and reached a final population of
106–108 per g. This contrasts with Blue cheese, where
listeria failed to grow and decreased in number during
 Growth and Survival of Microbial Pathogens in Cheese 545
56 days of storage (Papageorgiou and Marth, 1989).
These authors suggested that Penicillium roqueforti may
produce bacteriocins against L. monocytogenes. In hard
cheese varieties like Colby and Cheddar, L. monocyto-
genes populations decline during aging, with survival
strongly influenced by the moisture content and the pH
(Ryser and Marth, 1987b; Yousef and Marth, 1990).
Cheeses such as Camembert and Feta have nearly identi-
cal composition in terms of moisture content, water
activity, % salt-in-water and ripening temperature. How-
ever, fully ripened Camembert has a pH of 7.5 versus
Feta which has a pH of 4.4 that prevents the growth of
listeria.
Cheeses made from raw milk
In the US and other parts of the world, the manufacture
of cheese from raw milk is a topic which is being revis-
ited from the perspective of microbiological safety. Pas-
teurization of milk prior to cheesemaking is but one step
that may reduce the risk of the presence of pathogenic
bacteria in cheese. Current US regulations which govern
the use of raw, heat-treated and pasteurized milk for
cheesemaking were promulgated in 1949 (Anonymous,
1950; 21 CFR Part 133). One of the two options can be
selected by cheesemakers to assure the safety of cheese –
pasteurize milk destined for cheesemaking or hold
cheese at a temperature of not less than 1.7 °C (35 °F)
for a minimum of 60 days. Recent research has shown
that S. typhimurium, E. coli 0157:H7 and L. monocyto-
genes can survive well beyond the mandatory 60-day
holding period in Cheddar cheese prepared from pas-
teurized milk (Reitsma and Henning, 1996; Ryser,
1998). In a referral to the National Advisory Committee
on Microbiological Criteria for Foods in April 1997, the
FDA asked if a revision of policy requiring a minimum
60-day aging period for raw-milk hard cheeses was nec-
essary. The FDA, in its communication, noted that such
a duration may be insufficient to provide an adequate
level of public health protection. The FDA cited numer-
ous studies and outbreak investigations documenting
the presence of listeria, salmonella and E. coli 0157:H7
in raw milk. Of particular concern was the report by
Reitsma and Henning (1996) detailing the survival of
E. coli 0157:H7 in aged Cheddar cheese. The FDA did
note, however, that there was ‘limited epidemiological
evidence that food-borne illness results from consump-
tion of raw-milk hard cheeses that have been aged for 60
days’, citing work by Fabian (1947), D’Aoust et al.
(1985) and Johnson et al. (1990b) in support of this
claim. Groups outside of the US have recently expressed
concern about the safety of raw-milk cheeses. The Insti-
tute of Food Science and Technology (IFST, 2000) in the
UK issued a position statement drawing attention to the
potential public health hazards posed by pathogenic
bacteria in cheeses made from raw milk. The IFST indi-
cates that these hazards apply particularly to soft and
semi-soft cheeses (IFST, 2000). Codex Alimentarious
is presently recommending a ‘combination of control
measures’ (including pasteurization) to achieve the
appropriate level of public health protection (Groves,
1998).
In a comprehensive review of all outbreaks of human
illness associated with the consumption of aged raw-
milk cheese, in the majority of instances, confounding
parameters other than use of raw milk contributed to
pathogens being present in the product at the time of
consumption (Donnelly, 2001). Further, in challenge
studies which examine the fate of pathogens in aged
cheese, confounding factors can also explain the appear-
ance of pathogens following 60 days of aging. Such con-
founding parameters in actual outbreaks or challenge
studies involve the use of pasteurized versus raw milk in
cheesemaking trials, inadequate development of acidity
during cheesemaking, a low salt level, contamination by
ill employees during manufacture, temperature abuse of
milk designed for cheesemaking and environmental con-
tamination during cheesemaking.
Previous Reviews on the Safety of Raw
Milk Cheeses
Two comprehensive reviews have been published regard-
ing outbreaks of human illness linked to consumption of
cheese. Johnson et al. (1990b) conducted a comprehen-
sive review of the epidemiological literature during the
40-year period, 1948–1988. These authors identified
only six outbreaks of illness transmitted by cheese pro-
duced in the US during this period. Post-pasteurization
contamination was the most frequent causative factor in
these outbreaks. Improper pasteurization equipment
and/or procedures were implicated in only one outbreak
each in the US and Canada, and use of raw milk was a
factor in one outbreak in each of these countries. No
outbreaks were linked to hard Italian cheese varieties
such as Parmesan, Romano and Provolone. In rare
instances, Swiss and Cheddar cheeses were linked to
food-poisoning outbreaks. Factors other than pasteuriza-
tion cited by Johnson et al. (1990b) as contributors to
cheese safety include milk quality and management, lac-
tic starter management, pH, salt, controlled aging condi-
tions and natural inhibitory substances in the raw milk.
These authors proposed three actions to improve the
safety of raw milk cheeses: (1) Establish a guideline for
minimum heat-treatment of milk for cheesemaking,
e.g., 64.4 °C (148 °F) for 16 sec or equivalent with
adequate process control, (2) Evaluate current safety
546 Growth and Survival of Microbial Pathogens in Cheese
technology and practices used for cheese manufacture
and (3) Evaluate technologies not currently used in
cheese manufacture for safety potential (Johnson et al.,
1990c).
Altekruse et al. (1998) reviewed all cheese-associated
outbreaks reported to the Centers for Disease Control
and Prevention (CDC) during the period 1973–1992.
These authors noted the infrequency of large, cheese-
associated outbreaks reported during this period and
suggested that improvement of cheesemaking methods
and process control have resulted in cheese being a safer
product. There were 32 cheese-associated outbreaks,
11 of which could be attributed to contamination at the
farm, during manufacturing or during processing. Of the
11 outbreaks attributed to contamination prior to distri-
bution, 5 were associated with the consumption of
Mexican-style soft cheese versus only one outbreak
linked to Cheddar cheese. It is notable that no outbreaks
reported to the CDC during 1973–1992 were associated
with raw milk cheese that was aged for a minimum of
60 days. The authors indicated that salmonella, E. coli
0157:H7 and L. monocytogenes may survive the aging
process. However, the literature reference for survival of
listeria points to Camembert cheese (Ryser and Marth,
1987a), and the authors failed to note the rapid decline
of listeria populations in aged Cheddar cheese as docu-
mented by Ryser and Marth (1987b). Altekruse et al.
(1998) suggest that aging alone may not be a sufficient
pathogen control step to eliminate salmonella, listeria
and E. coli 0157:H7 from cheese.
Outbreaks involving Cheddar cheese
In 1976, seven lots of Cheddar cheese manufactured
from pasteurized milk were contaminated with
S. heidelberg and were responsible for 339 confirmed
cases of illness and an additional 28 000–36 000 cases of
illness (Fontaine et al., 1980). The cheese involved was
aged for less than 60 days, and improper pasteurization
was cited as the cause of the outbreak. Follow-up with
the first few patients led epidemiologists to suspect
cheese eaten in Mexican-style restaurants as the vehicle
of infection. Seven lots of Cheddar cheese produced
from pasteurized milk by a Kansas manufacturer and
purchased from a single Denver distributor were identi-
fied as the potential sources of contamination. The epi-
demic began in July in two widely separated Colorado
cities, Denver and Pueblo. Levels of S. heidelberg in these
cheeses were estimated to be 0.36–1.8 per 100 g. The pH
of contaminated cheese was 5.6, which may have been a
factor in this outbreak. Poor manufacturing practices
coupled with inadequate control programmes at the
cheese plant were cited as causative factors in this
outbreak. The Kansas State Health Department had
recorded 25 instances of non-compliance with good
manufacturing practices by that particular food-processing
plant. The Kansas Board of Agriculture required that raw
milk contain 3 000 000 organisms/ml. Routine micro-
bial analysis of the grade B or surplus grade A milk used
at the plant revealed that counts greatly exceeded this
standard. In the production of cheese, raw milk was
stored for 1–3 days in an insulated but unrefrigerated
holding tank prior to pasteurization at 71.6 °C for
15 sec. The milk was filtered after pasteurization, which
is a violation of FDA guidelines for pasteurization.
Salmonella outbreaks in Ontario, Canada, during the
period 1980–1982 occurred in raw-milk Cheddar
cheese. S. muenster was identified in the cheese and
traced to a single farm where one cow was shedding the
organism (Wood et al., 1984). Subsequent trials using
milk from this infected cow were conducted to deter-
mine potential for survival during commercial prepar-
ation of raw milk cheese. Curd tested positive in 11 of
181 vats. During curing, one lot was negative after 30
days but one lot was positive after 125 days. It would be
unlikely for this scenario to be repeated as cheese is
rarely manufactured from milk from a single cow. Milk
is co-mingled, and the dilution effect with milk from
other animals and other farms reduces the level of
pathogens, if present.
A large Canadian outbreak of salmonellosis linked
to the consumption of Cheddar cheese was reported in
four Canadian Atlantic provinces (Newfoundland, New
Brunswick, Prince Edward Island and Nova Scotia)
between January and July 1984. This outbreak proved
to be the largest single epidemic of salmonellosis ever
to occur in Canada, ultimately involving more than
2700 cases of illness (Bezanson et al., 1985; Johnson
et al., 1990b). Production of the cheese, which was
manufactured from either pasteurized (73.8 °C
(165 °F) for 16 sec) or heat-treated (66.7 °C (152 °F)
for 16 sec) milk, was traced to a single plant on Prince
Edward Island. Testing of the raw milk supply identi-
fied two cows in separate herds, one which shed
S. typhimurium and one which shed S. heidelberg.
D’Aoust et al. (1985) reported on the distribution and
survival of S. typhimurium phage type 10 isolated from
Cheddar cheese in this outbreak. Levels of salmonella
ranged from 0.36 to 9.3 per 100 g. The pH of the
cheese ranged from 4.97 to 5.40, consistent with nor-
mal Cheddar, which has a pH range of 5.0–5.5.
S. typhimurium phage type 10 was found to survive
in Cheddar cheese for up to 8 months at 4 °C. The
data provided by D’Aoust et al. (1985) is very inter-
esting. The authors compare salmonella recovery
as a function of whether mild Cheddar cheese was
manufactured from heat-treated (16 s at 66.7 °C; not
pasteurized) or pasteurized (16 sec at 73.8 °C) milk.
 Growth and Survival of Microbial Pathogens in Cheese 547
Tested samples of mild Cheddar manufactured from heat-
treated milk were found to contain 0.36–9.3 salmo-
nella/100 g. However, four lots of mild Cheddar
manufactured from pasteurized milk were also found to
contain 0.36–4.3 salmonella/100 g. Certain lots of cheese
contained Staph. aureus at high levels (105 per g),
which may indicate poor starter activity (Johnson et al.,
1990b) or contamination through handling. It is difficult
to understand how D’Aoust et al. (1985) could support
their concluding statement in this article ‘Although pas-
teurization of milk used in cheesemaking increases the
safety of the finished product, use of heat-treated (unpas-
teurized) milk in the manufacture of medium and old
Cheddar cheese and survival of salmonella during pro-
longed periods of refrigerated storage raises legitimate
doubts of the safety of current manufacturing practices.’
In the data presented, pasteurization did not result in the
unequivocal safety of mild Cheddar cheese.
An evaluation of the pasteurization process, described
by Johnson et al. (1990b), indicated that the employee
in charge of the process manually overrode the elec-
tronic controls, which shut down the pasteurizer while
milk continued to flow through the unit and into the
vat. The pasteurizer was shut down after filling three
vats and later restarted to fill the next three vat series.
The first and the third vats of each three vat sequence
tested positive for salmonella, except for the first vat of
the day and the middle vat of each three vat series which
consistently tested negative. This pattern only occurred
when raw milk which included milk from the cow shed-
ding S. typhimurium was used. Bezanson et al. (1985)
subsequently subjected outbreak strains to molecular
analysis by biotyping, antibiotic resistance patterns, plas-
mid restriction and endonuclease analyses and revealed
that two genetically distinct organisms were the aetio-
logic agents in this outbreak. These studies revealed the
existence of a double infection, indicating that the
incriminated cheese likely had two sources of contam-
ination. S. typhimurium phage type 10 subgroup I strains
were identified among cultures from raw milk and cattle
associated with the incriminated dairy. S. typhimurium
phage type 10 subgroup I and II strains were recovered
from individuals employed at the dairy along with their
family members. S. typhimurium subgroup I and II
strains were present in cheese curd samples obtained
from the plant as well as from a consumer pack obtained
from a distributor. Cheese plant workers from whom
both subgroup I and II strains were cultured were
involved in the production and/or packaging of Cheddar
cheese, raising questions about the possibility of con-
tamination of the cheese by ill workers. Salmonella were
confirmed in a cheese-trim bucket. Plant inspections
revealed that employees used their bare hands to transfer
cheese to a forming machine, and an employee tested
positive for S. typhimurium. It is likely that this incrim-
inated cheese was also responsible for an outbreak of ill-
ness reported at the same time in Ontario linked to
S. typhimurium phage type 10 biotype 4 (D’Aoust et al.,
1985).
Hedberg et al. (1992) reported on a multi-state out-
break of S. javiana and S. oranienburg linked to the
consumption of contaminated Mozzarella cheese and
shredded cheese products. Cases were more likely to
have consumed cheese manufactured at a single cheese
plant or cheese shredded at processing plants that also
shredded cheese from the single plant, than matched
controls. The outbreak strains were isolated from 2 of 68
unopened 16-oz blocks of Mozzarella cheese. Inspec-
tions revealed deficiencies in plant sanitation and clean-
ing, and equipment was not routinely cleaned and
sanitized between shredding different types of cheese
from different manufacturers. However, no deficiencies
in pasteurization were identified. Cheese-manufacturing
equipment was found to be susceptible to environmental
contamination and contamination by aerosols. Investi-
gators believed that the contaminated Mozzarella cheese
sent to four processing plants for shredding, cross-
contaminated other cheese products at those plants. It is
most likely that the cheese was contaminated from
environmental sources or from infected production
workers.
Four outbreaks occurring in the late 1990s were
reported in the UK, although detailed epidemiologic
data on these outbreaks is lacking. An outbreak of E. coli
0157:H7 (phage type 8, Verotoxin gene 2) infection
involving 22 cases was reported in Scotland in 1994.
This outbreak was associated with the consumption of
raw-milk cheese (Anonymous, 1997a). A December
1996 outbreak of salmonella gold-coast which occurred
in England and Wales was linked to the consumption of
a brand of mild, coloured, Cheddar cheese produced in
August and September 1996 in Somerset, England.
Phosphatase tests and examination of recording chart
records from the pasteurizer indicated that pasteuriza-
tion had failed at the plant on several occasions
(Anonymous, 1997b). An outbreak of infection caused
by E. coli 0157:H7 (phage type 21/28 VT2) was reported
in 1999 in north-east England (Anonymous, 1999a,b).
The vehicle of infection was Cotherstone cheese, a raw-
milk cheese, manufactured in small quantities and dis-
tributed to specialty cheese shops in England. Samples
from the dairy herd, slurry and environmental samples
from the cheese manufacturing facilities were negative
for E. coli 0157:H7. In March of 1999, a large outbreak
of infection was reported in England and Wales due to
consumption of contaminated milk from a single dairy.
An outbreak of E. coli 0157:H7 infection was reported
which was linked to the consumption of fresh cheese
548 Growth and Survival of Microbial Pathogens in Cheese
curd, which was held for 60 days, from a dairy plant in
Wisconsin (Durch et al., 2000). Nineteen of 55 labora-
tory-confirmed patients had purchased cheese curds
from an unrefrigerated display at the cheese plant. To be
legal, cheese curds must be manufactured from pasteur-
ized milk. Vats of raw-milk Cheddar cheese were
inadvertently used to make fresh curds, which were
incorrectly labelled as ‘pasteurized’ Cheddar cheese curd.
A comprehensive risk assessment would consider,
among other factors, the degree to which the consuming
population is exposed to risks associated with the con-
sumption of aged raw-milk cheeses. Cheddar cheese is
produced worldwide and is therefore considered an
important variety of hard cheese. The USDA, National
Agricultural Statistics Service, reports that Cheddar
cheese was the most popular variety of cheese produced
and consumed in the US in 1999, with a production
level of 2.8 billion pounds (1.2 million tonnes) or 35.4%
of the total cheese produced (Anonymous, 1999c).
Given that a large amount of this cheese is produced
from raw or heat-treated milk, the high degree of expos-
ure (consumption) of this product coupled with the low
incidence of disease outbreaks attests to the safety of
aged cheese made from raw and heat-treated milk.
Table 1 summarizes outbreaks involving Cheddar
cheese which have occurred since 1976. Listed in this
table are confounding parameters which contributed to
the presence of pathogens in the finished product and
the subsequent onset of human illness.
Challenge Studies
Reitsma and Henning (1996) examined the survival of
E. coli 0157:H7 during the manufacture and ripening of
Cheddar cheese. E. coli 0157:H7 was inoculated at two
levels into pasteurized milk, 1  103 cfu/ml and
1 cfu/ml. The organism showed a sharp decrease in
numbers over the 158-day testing period. Treatment
1 (1000 cfu/ml) showed a 2-log CFU/g reduction after
60 days of ripening; however, E. coli 0157:H7 was still
present even after 158 days of ripening when viable cells
were detected in four of five replicates. Treatment
2 (1 cfu/g) showed a reduction to 1 cfu/g in 60 days,
with no viable E. coli 0157:H7 detected at 158 days.
As the authors state, ‘the results of this study cannot
predict the behaviour of heat-injured cells which could
result from the pasteurization of naturally contaminat-
ing E. coli.’ Further, the low salt-in-moisture content
(SM) and absence of natural inhibitors present in raw
milk create an artificially protective environment for
E. coli 0157:H7 in pasteurized milk. The SM deter-
mines the water activity, which, in turn, dictates the
potential for growth of a micro-organism in the cheese
environment. The SM in that study ranged from 2.75 to
3.76% with a mean of 3.25%, whereas in normal Ched-
dar, the SM ranges from 4 to 6%. The low SM could
have affected the results in the study of Reitsma and
Henning (1996) and the authors recommend further
research with Cheddar containing a higher SM to
determine if similar results would be obtained with an
SM more commonly encountered in Cheddar cheese.
NaCl is an important inhibitor of microbial growth in
cheese. The major roles of NaCl in Cheddar cheese are
to check lactic acid fermentation after an optimum
peak has been attained, reduce moisture through
syneresis of the curd, suppress the growth of spoilage
micro-organisms and create physical changes in cheese
proteins which influence cheese texture, protein solu-
bility and protein conformation (Fox et al., 2000; ‘Salt
in Cheese: Physical, Chemical and Biological Aspects’,
Volume 1). While there are no state or federal stand-
ards for the amount of salt added to Cheddar cheese,
variations in salt content from 0.8 to 2% are common.
The minimum aw (adjusted with NaCl) for the growth
of E. coli is 0.950 (Fennema, 1985). Further, most raw
milk receives some form of heat treatment, albeit sub-
pasteurization. The combination of heat, salt and nat-
ural inhibitors could provide barriers to the survival of
E. coli 0157:H7. The experimental design used by
Reitsma and Henning (1996) failed to consider these
potential safeguards. It is plausible that the use of pas-
teurized milk for cheesemaking provides E. coli
0157:H7 with a more protective environment than raw
milk, thus heat treatment could create more of a prob-
lem to food safety. The authors state ‘The low number
of outbreaks seem to indicate that pathogens in cheese
are not a major problem.’ The authors further state
‘treatment 1 (1000 cfu/ml) would not likely be encoun-
tered in industry because of co-mingling of milk from
several or many farms, thus creating a dilution effect.’
Concern is expressed about the authors’ concluding
statement ‘The current requirement for ripening of
Cheddar cheese will not assure consumers of a safe
product if the cheese is made from raw milk and a
pathogen such as E. coli 0157:H7 is present in the
cheese at the beginning of ripening.’ This statement is
contradicted by the authors’ own data which show that
E. coli 0157:H7 present at 60 cfu/g in curd after salting
was reduced to 1 cfu/g after 60 days, even in the arti-
ficially low SM of cheeses in the study.
A subsequent study by Zhang and Henning (1999)
described mathematically the decline of E. coli popula-
tions during cheese ripening. The authors inoculated
pasteurized whole milk with E. coli biotype 1 at popu-
lations of 100–1000/ml. The authors used a complete
factorial design to investigate the effects of high- and
low-level environmental factors such as moisture
(34–40%), pH (5.1–5.6), temperature (4–13 °C) and salt
 Growth and Survival of Microbial Pathogens in Cheese 549

Table 1 Data from outbreak investigations involving aged raw milk cheese and confounding parameters which contribute to the
presence of pathogens

Number of Cheese Confounding

Date Location Isolate cases type parameter Reference

1976 Colorado Salmonella 339 confirmed; Cheddar 1. Raw milk Fontaine

heidelberg 28 000– made from did not meet et al., 1980
36 000 pasteurized standards
suspected milk 2. Raw milk
stored 1–3 days
in holding tank – no
refrigeration
3. Milk filtered
after pasteurization
4. Cheese pH, 5.6
5. 25 instances of non-
compliance with GMP
1980–1982 Ontario Salmonella Raw-milk Milk traced to Wood et al.,
muenster Cheddar single farm; lack of 1984
co-mingling
1984 4 Canadian Salmonella 2700 Cheddar 1. Employee Bezanson
Atlantic typhimurium confirmed made from manually shut et al.,1985;
Provinces phage type cases pasteurized down D’Aoust
and Ontario 10, group I and/or heat- pasteurizer et al., 1985
and II treated milk 2. Group II type
shed by workers
1989 Multistate Salmonella 164 Shredded 1. Deficiencies Hedberg
(Minnesota, javiana and cheese in cleaning and et al., 1992
Wisconsin, Salmonella sanitation
Michigan, oranienburg 2. Equipment
New York) not routinely
cleaned and
sanitized between
shredding of
different cheese
types from
different makers
3. Cheese
equipment
susceptible to
contamination from
environment/aerosols
4. Cheese
contaminated by
infected workers
5. No deficiencies
in pasteurization
1999 E. coli Fresh cheese Incorrectly Durch et al.,
0157:H7 curd held for labelled as a 2000
60 days pasteurized product

concentration (0.8–1.7%) on survival of E. coli. Tempera-
ture and pH were found to have the most significant
impact on survival, and there was no significant interac-
tion among the four parameters studied. Salt concentra-
tion within the ranges used in this study (0.8–1.7%) was
found to have no impact on survival of E. coli.
Teo and Schlesser (2000) examined the survival of
three groups of bacteria in raw-milk Cheddar cheese
during cheesemaking and ripening; naturally occurring
coliforms, a streptomycin-resistant strain of E. coli K12
(ATCC 35695) and E. coli 0157:H7. Populations of nat-
urally occurring coliforms present at levels of ⬃105
cfu/ml experienced a 1-log reduction after 60 days of
aging at 7 °C, and a further 3–4-log reduction after 180
days (Teo and Schlesser, 2000). In contrast, E. coli K12
populations exhibited a less than 1-log reduction dur-
ing 60 days of aging, and only a 1–2-log reduction by
90 days. Similar results were recorded with a five-strain
550 Growth and Survival of Microbial Pathogens in Cheese
cocktail of E. coli 0157:H7 where populations declined
by 1 log following 60 days at 7 °C, and by 1–2-logs fol-
lowing 90 days at the same temperature.
A number of questions are raised by the data pre-
sented by Teo and Schlesser (2000). The coliform levels
used were extremely high, and, in practice, such levels
would raise concerns about raw milk quality. The FDA
has set standards for ETEC and E. coli in cheese at lev-
els of 103 and 104 cells/g, respectively (Anonymous,
1998). Thus the cheese produced by Teo and colleagues
did not comply with these standards, and further,
exceeded these standards by 103–104/ml as shown in
their Figure 6 where initial populations of E. coli
0157:H7 exist at approximately 5  107/ml. This study
has a biased objective, to ‘confirm prior work that sug-
gests 60-day aging inadequate to protect public health.’
Figure 2 presented in the paper documents a decline in
populations over time, thus if a reasonable starting
population of 1–10 E. coli 0157:H7 were used, no viable
cells should be present following aging.
Studies by Ryser and Marth (1987a,b,c) examined the
fate of L. monocytogenes during the manufacture of
Cheddar, Camembert and Brick cheeses. Rapid growth
to populations of 5  107 cfu/ml is observed in Camem-
bert cheese, in which the pH normally increases during
ripening, thereby creating a favourable growth environ-
ment for listeria (Ryser and Marth, 1987a). In contrast,
listeria populations show a marked decline in viable
population levels during the ripening of Cheddar cheese.
However, population levels do not decline to undetect-
able levels. Current US regulations call for cheese made
from raw or sub-pasteurized milk to be ripened at 1.7 °C
(35 °F) for at least 60 days prior to sale. Ryser and Marth
(1987b) have shown that aging alone will not ensure the
production of listeria-free Cheddar cheese. This stated, it
is clear, that the greatest threat posed to the safety of
cheese is due to post-process environmental contamin-
ation with listeria. While outbreaks of illness have
resulted from the presence of L. monocytogenes in soft-
ripened and Hispanic-style cheeses (Linnan et al., 1988),
no outbreaks of listeriosis have been reported as a result
of survival of listeria in cheese aged for a minimum of 60
days. Genigeorgis et al. (1991) evaluated the ability
of 24 types of market cheeses to support the growth of
L. monocytogenes. Cheeses able to support growth
included soft Hispanic-type cheeses, Ricotta, Teleme,
Brie, Camembert and Cottage cheeses (pH range,
4.9–7.7). Cheeses which did not support growth, and
which resulted in the gradual death of L. monocytogenes,
included Cotija, Cream, Blue, Monterey Jack, Swiss,
Cheddar, Colby, String, Provolone, Münster, Feta and
Kasseri (pH range, 4.3–5.6). A correlation was observed
between the growth of listeria in cheeses having a pH of
greater than 5.5, and in cheeses which were manufac-
tured without a starter culture.
Approximately 80% of the cheeses made in Switzerland
are manufactured from raw milk. However, the term
‘raw milk cheese’ as applied to Swiss cheese is a mis-
nomer because Swiss cheese receives an extensive heat
treatment during manufacture. Bachman and Spahr
(1995) assessed the safety of Swiss hard and semi-hard
cheeses made from raw milk. These authors inoculated
Aeromonas hydrophila, Campylobacter jejuni, E. coli,
L. monocytogenes, Pseudomonas aeruginosa, Salmonella
spp., Staph. aureus and Yersinia enterocolitica into raw
milk at levels ranging between 104 and 106 cfu/ml for
the manufacture of hard (Swiss-type) and semi-hard
(Tilsit-type) cheese. In the hard cheese, no pathogens
were detected beyond 1 day. This was attributed to the
curd-cooking temperature of 53 °C (127.4 °F) for 45 min
and 42 °C (107.6 °F) for 15 min for Swiss hard and
semi-hard cheeses. Further, the rapid decrease of the
redox potential of Swiss cheese is likely to impart
additional inhibitory effects. Pathogens were found to
survive longer in the semi-hard than in the hard
cheese. After 90 days of aging at 11–13 °C, when
ripening was complete, all pathogens except L. mono-
cytogenes were below detectable limits. Growth of L. mono-
cytogenes was not observed in the interior of the cheese,
but they grew well on the cheese surface. Thus, manu-
facturing parameters used in the production of semi-
hard cheese are bacteriostatic, not bacteriocidal, for
L. monocytogenes. Based upon these studies, the Swiss
dairy industry has adopted a listeria-monitoring pro-
gramme for cheese and other dairy products. The syn-
ergistic effects of active antimicrobial enzyme systems
in raw milk coupled with antagonistic effects of starter
cultures, fast acidification, inhibitory effects of lactic
acid and high curd-cooking temperatures render a
microbiologically safe hard cheese when produced
under good manufacturing practices. Spahr and
Schafroth (2001), in studies which examined the fate
of Mycobacterium avium subsp. paratuberculosis, recorded
pH values associated with Swiss hard and semi-hard
cheese manufacture. After 24 h, cheeses manufactured
under these curd-cooking conditions reached a pH
value of 5.3 in hard cheese and 5.2 in semi-hard cheese,
and these pH conditions remain for 10 days for hard
cheese and 25 days for semi-hard cheese. Further, the
rapid decrease of the redox potential of Swiss cheese
likely imparts additional inhibitory effects. The synergis-
tic effects of active antimicrobial enzyme systems in raw
milk coupled with antagonistic effects of starter cultures,
fast acidification, inhibitory effects of lactic acid and
high curd-cooking temperatures render a microbiologic-
ally safe hard cheese when produced under good manu-
facturing practices.
Pellegrino and Resmini (2001) examined the safety of
the Italian hard cheeses, Grana Padano and Parmigiano
Reggiano. The authors noted several parameters
 Growth and Survival of Microbial Pathogens in Cheese 551
associated with these cheeses which contribute to their
microbiological safety; (1) cooking of cheese curd to a
temperature between 53 and 56 °C for 15–20 min, with
a total holding time of up to 85 min at these tempera-
tures, (2) moulding of the cheese, whereby it is held at
temperatures of 52 °C (126 °F) and 56 °C (133 °F) for
at least 10 h at pH 5.0, (3) brine-salting of the cheese
which lowers the aw to 0.9 and (4) extended ripening
for periods of 9 months (Grana Padano) to 12 months
(Parmigiano Reggiano) which promotes a further
decrease in the aw to levels inhibitory for growth of
bacterial pathogens. Resmini and Pellegrino (1996)
demonstrated that the high-temperature–low-pH condi-
tions occurring within Grana cheeses, which they
describe as self-pasteurization, result in the inactiva-
tion of alkaline phosphatase, except within the outer-
most 3–4-cm layer. However, in this outer layer the
SM ranges between 8 and 24% in the ripened cheese
and the aw is close to 0.8. Staph. aureus, which is more
tolerant of low aw, cannot survive below an aw of 0.86
and can produce toxins only above an aw of 0.90
(Sperber, 1983). Pecorari et al. (2001) examined the
fate of pathogens during the production and ripening
of Parmigiano Reggiano cheese. E. coli, S. typhimurium,
Staph. aureus and L. monocytogenes were inoculated
into raw milk at levels ranging between 104 and
106 cfu/ml. None of the inoculated pathogens were
detected 24 h after cheesemaking, confirming that the
cheesemaking conditions of Grana cheeses do not sup-
port pathogen growth or survival. These results are
consistent with those obtained by Yousef and Marth
(1990) who reported a rapid decline of L. monocyto-
genes from an initial level of ⬃104 g of Parmesan
cheese to undetectable levels within 14–112 days of
ripening. These authors attributed the decline of L.
monocytogenes viability in Parmesan cheese to the fol-
lowing parameters – addition of lipase (for US Par-
mesan) for flavour development, heat treatment of the
curd and reduction in moisture content (aw) during
ripening. Battistotti (1995), in an analysis of more
than 100 samples of mature Italian Grana cheeses,
failed to detect salmonella, Staph. aureus, L. monocyto-
genes, coliforms or enterococci, further confirming the
microbiological safety of hard Italian cheeses.
The results of the aforementioned challenge studies
are summarized in Table 2. Most studies, which show
the survival of pathogens, have been based on the use
of pasteurized milk rather than raw milk in the experi-
mental design. The growth rate of listeria (and pre-
sumably other pathogens) in milk is a function of the
degree and extent of heat treatment. The fastest rate of
growth is observed in UHT milk, followed in turn by
HTST, heat-treated and raw milks (Northolt et al.,
1988; Rajikowski et al., 1994). Therefore, challenge
studies, which assess the survival of pathogens when
inoculated into pasteurized milk, may overestimate
survival during 60 days of aging. The UK Institute of
Food Science and Technology (IFST) has stated that
the total health risk to the consumer is less from
cheese made from pasteurized milk than from cheese
of similar composition made from unpasteurized milk
(IFST, 2000). Alternative hypotheses could be offered,
including consideration that the use of raw milk pro-
vides protective effects from pathogens in milk and
that post-pasteurization environmental contamination
poses a far greater threat to the safety of cheese. As a
result, the use of pasteurized milk in cheesemaking
may provide an environment, which provides for opti-
mal growth of pathogens whereas, in raw milk, the
normal flora and natural inhibitors provide a margin
of control over pathogen growth. In fact, a study con-
ducted by Rudolf and Sherer (2001) showed a higher
incidence of L. monocytogenes in cheeses made from
pasteurized milk (8%) than in cheese made from raw
milk (4.8%). Phage typing of isolates revealed persist-
ent listeria contamination within dairy plant environ-
ments for periods of weeks to several months and
documented cross-contamination within the plant
environment as a significant factor associated with the
contamination of cheese. The recommendation for
mandatory pasteurization may ultimately lead to use
of milk of inferior quality for cheesemaking. Patho-
gens harboured in this inferior quality milk can be
transported to a processing facility and become estab-
lished as environmental pathogens. A wiser strategy
may involve routine testing of incoming lots of raw
milk and working with producers when infected ani-
mals are identified to allow treatment and confinement
of animals to control infectious disease. There is no
evidence in the literature to support the view that
cheese made from raw milk where pathogens are not
present is a dangerous food. Thus, raw milk screening
coupled with the use of good manufacturing prac-
tices to control environmental contamination during
cheesemaking may be the most effective control strat-
egy to improve the safety of aged cheese. The US FDA
has recently stated ‘a review of the literature relating to
the potential for growth of pathogens in hard cheeses
that are aged for at least 60 days shows that such
growth is not likely to occur because of the combined
effect of decreased pH, decreased water activity, and
possibly other factors inherent to these cheeses’
(Anonymous, 1999d). Although survival during aging
is possible, the FDA cited a considerable body of
evidence which showed that certain cheeses do not
support the growth of pathogens during the aging
process and subsequent storage.
Both facultative and obligate heterofermentative lac-
tobacilli have been isolated from Cheddar cheese, such
as Lactobacillus casei, Lb. paracasei, Lb. plantarum, Lb.
552 Growth and Survival of Microbial Pathogens in Cheese

Table 2 Results of selected challenge studies which examine the fate of pathogens in raw milk cheeses and parameters which
promote survival/decline of pathogens

Cheese Milk Factors promoting

Reference type/pathogen inoculation levels 60 days of aging survival/decline

Reitsma and Cheddar/E. coli 1 cfu/ml and No survival at Cheese

Henning, 1996 0157:H7 1000 cfu/ml 1 cfu/ml; Survival at manufactured
1000 cfu/ml from pasteurized
milk; low salt levels
Ryser and Cheddar/ 5  102 cfu/ml Survival during Decline in
Marth, 1987b L. monocytogenes aging at 6 or 13 °C populations after
35 days of storage
Teo et al., 2000 Cheddar/E. coli 105/ml 1-log decrease E. coli populations in
0157:H7 cheese exceeded
FDA standards
Bachman and Swiss hard/ 104–106 cfu/ml No detection of Cook at 53 °C;
Spahr, 1995 semihard pathogens redox potential
Aeromonas, beyond 1 day
Campylobacter,
E. coli, L. monocyto-
genes, P. aeruginosa,
Salmonella,
Staphylococcus,
Yersinia
Pellegrino and Italian Grana Curd cooked at
Resmini, 2001 53–56 °C,
brine-salting,
extended ripening to
lower aw
Yousef and Parmesan/ 104–105 cfu/ml Undetectable Addition of lipase,
Marth, 1990 L. monocytogenes heat treatment of
curd, reduction of aw

SMP, Skim Milk Powder.

casei subsp. pseudoplantarum, Lb. curvatus, Lb. brevis, Lb.
rhamnosus and unclassified strains (Broome et al.,
1990; Jordan and Cogan, 1993; McSweeney et al., 1993;
Fitzsimons et al., 1999; Fox et al., 2000; Tammam et al.,
2000). These are usually termed the non-starter lac-
tic acid bacteria (NSLAB; see ‘The Microbiology of
Cheese Ripening’, Volume 1). Lactobacilli are usually
present at low numbers (50/g) in cheese immediately
after manufacture, but grow at a temperature-dependent
rate during ripening and eventually become the domin-
ant viable micro-organisms in cheese, reaching a popu-
lation of 107 cfu/g in 10–60 days. The NSLAB
population decreases with storage and usually approach
5  106 cfu/g after one year (Prentice and Brown,
1983). Conditions, which dictate the rate and extent
of growth of NSLAB in cheese, include pH, moisture
content, salt concentration and ripening temperature
(Martley and Crow, 1993). In commercially produced
cheese, NSLAB may originate from raw milk, post-
pasteurization environmental contamination and/or
ingredients. These organisms may well offer a protect-
ive effect against the growth of pathogens and this role
should be studied as this may be a positive contribu-
tion of raw milk to the safety of raw-milk Cheddar
cheese, consistent with competitive exclusion theories
which have helped to advance the safety of products
such as poultry. A lower total bacterial load in raw milk
entering the pasteurizer results in a lower total bacter-
ial count in pasteurized milk, but after 10–16 h, an
increase in the number of NSLAB still occurs.
Growth and Survival of Bacterial
Pathogens in Soft and Semi-Soft Cheeses
Legitimate concerns can be raised regarding the safety
of soft and semi-soft cheeses manufactured from raw
milk, as well as high-moisture, low-salt aged cheeses.
An outbreak of food-borne listeriosis linked to cheese
was reported by Bille et al. (1992). This outbreak
occurred in Vaud, Switzerland, and was linked to the
consumption of Vacherin Mont D’Or cheese. A total of
122 cases during the period 1983–1987 were reported.
The normal endemic rate of listeriosis in Switzerland
is 5–10 cases/million persons. During the outbreak
 Growth and Survival of Microbial Pathogens in Cheese 553
period, the rate of listeriosis rose to 50 cases/million
persons. Sixteen cases were reported in 1983, 24 in
1984, 13 in 1985, 28 in 1986 and 41 in 1987. A mortality
rate of 28% was associated with these cases. Of the clini-
cal isolates available from the epidemic period, 111 of
120 (93%) were serotype 4b of two unique phage
types, and 85% of these strains matched the epidemic
phage types isolated from Vacherin Mont D’Or cheese.
L. monocytogenes in Mexican-style cheese has been
responsible for two major outbreaks of food-borne dis-
ease in the US. Mexican-style cheeses comprise a range
of cheese products which include Queso Blanco,
Quesco Fresco, Panela Ranchero, Queso de Hoja and
soft Hispanic cheese (Bolton and Frank, 1999). These
cheeses do not have a standard of identity, and most are
coagulated and using rennet, may have added organic
acids (citric, acetic and lactic); usually a lactic starter
culture is not used (Bolton and Frank, 1999). The first
documented link between cheese consumption and an
outbreak of listeriosis was reported in California in
1985. Jalisco brand Mexican-style cheese was impli-
cated as the vehicle of infection (Linnan et al., 1988). A
total of 142 cases involving 93 pregnant women or their
offspring and 49 non-pregnant, immuno-compromised
adults were documented in Los Angeles County, CA.
Forty-eight deaths were recorded, giving a mortality
rate of 33.8%. The majority of afflicted individuals
(62%) were pregnant Hispanic women. Although an
additional 160 cases occurred in other parts of California,
for logistical reasons, the study reported by Linnan
et al. (1988) was limited to Los Angeles County. In
this outbreak, the cheese was most likely manufac-
tured from a combination of raw and pasteurized
milks, and the cheese plant that manufactured the
incriminated cheese was found to harbour listeria as
an environmental contaminant. The epidemic strain
in this outbreak was a serotype 4b, and this serotype
was recovered from unopened packages of Queso
Fresco and Cotija Mexican-style cheese.
An outbreak of listeriosis associated with home-
made, Mexican-style, fresh, soft cheese occurred in
North Carolina between October 2000 and January
2001 (Boggs et al., 2001). The outbreak involved 12
cases, consisting of 10 pregnant women, 1 post-partum
female and a 70- year-old immuno-compromised male.
The 11 women, upon hospital admission, reported
symptoms of fever, chills, headache, abdominal cramps
and vomiting. The cheese implicated in the outbreak was
purchased from door-to-door vendors. L. monocytogenes
isolates obtained from nine patients, three cheese sam-
ples from two stores, one cheese sample from a patient’s
home and one raw milk sample from a dairy all had
indistinguishable PFGE patterns, indicating a common
link. It is important to note that the manufacturing con-
ditions in this outbreak would not be those encountered
in a licensed, inspected commercial cheese-processing
facility.
Microbiological surveys of raw milk conducted in the
US have shown the presence of L. monocytogenes in
1.6–7% of samples. This incidence is similar to that in
Canadian (1.3–5.4%) and Western-European (2.5–6.0%)
raw milks. In the recently released Health and Human
Services (HHS) and USDA listeria Risk Assessment and
listeria Action Plan, USDA and FDA advise pregnant
women, older adults and people with weakened immune
systems that ‘Cheeses that may be eaten include hard
cheeses, semi-soft cheeses such as Mozzarella, pasteur-
ized processed cheeses such as slices and spreads, Cream
cheese and Cottage cheese.’ However, persons in these
risk groups are advised ‘do not drink raw (unpasteur-
ized) milk or eat foods that contain unpasteurized milk.’
This advice may be ambiguous with respect to aged raw-
milk cheeses (Anonymous, 2001).
Stress Adaptation of Pathogens and Impact
upon Cheese Safety
Over the past several years, microbiologists who study
stress adaptation in bacterial pathogens are aware of
genetic mechanisms which allow a number of Gram-
positive and -negative bacteria to adapt to hostile envir-
onments. Rpos is a sigma factor which is thought to
allow induction of specific stress-related components in
tolerant isolates. The rpos-regulated proteins enhance
acid tolerance and cross-protect E. coli 0157:H7 against
subsequent heat and salt challenges. The acid-tolerance
response (ATR) gene encodes for the ability to with-
stand lethal pH conditions following adaptation to sub-
lethal pH in L. monocytogenes, S. typhimurium, E. coli
and A. hydrophila. These mechanisms play a role in pre-
dicting the fate of pathogens in acidic foods. Acid adap-
tation increases the general resistance, including acid
tolerance, of L. monocytogenes, S. typhimurium and
E. coli, so that they survive better in both acidic and fer-
mented foods than unadapted cultures. These findings
have important implications for the safety of hard
cheeses that are aged for at least 60 days where the
combined effects of pH, salt and decreased aw dictate
potential for pathogen survival.
Leyer and Johnson (1992) inoculated the surfaces of
commercially produced cheeses with adapted and non-
adapted S. typhimurium at an initial level of 104/ml.
Acid-adapted salmonella survived in Cheddar cheese
through 74 days of storage at 5 °C under aerobic storage
compared with non-adapted salmonella, which were
not detected after 14 days. In Swiss cheese, end prod-
ucts such as propionate and acetate produced by propi-
onic acid bacteria were found to inhibit salmonella.
554 Growth and Survival of Microbial Pathogens in Cheese
Dineen et al. (1998) examined the persistence of
E. coli 0157:H7 in fermented dairy products (yoghurt).
The authors concluded that post-processing contam-
ination of fermented dairy products with E. coli 0157:H7
represents the greatest potential health hazard to
humans. Those strains of E. coli 0157:H7 which pos-
sessed the rpos system appeared to contribute most
effectively to bacterial survival under moderately lethal
conditions, but did not appear to play much of a role in
survival under sub-lethal conditions. The authors
offered the following recommendations: (i) coliforms,
including E. coli 0157:H7, may be present in raw milk,
(ii) coliforms are destroyed by pasteurization, (iii) the
primary objective of a comprehensive sanitation pro-
gramme should be to prevent recontamination of pas-
teurized products and (iv) the presence of active starter
cultures may help minimize the presence of bacterial
pathogens in finished products.
L. monocytogenes is able to withstand low pH follow-
ing sub-lethal exposure to acidic conditions (O’Driscoll
et al., 1997). According to Chawla et al. (1996) and
Chen et al. (1997), temperature and acidity had a sig-
nificant effect on the fate of acid-injured L. monocyto-
genes, with complete repair occurring at pH 6.6. At
pH values 5.6 (which are typically found in Cheddar
cheese), refrigeration temperatures were bacteriostatic,
whereas higher temperatures were bacteriocidal. These
findings are consistent with those reported by Ryser
and Marth (1987b) where L. monocytogenes was inacti-
vated faster in Cheddar cheese ripened at 13 °C versus
6 °C. Since repair of sub-lethal injury requires optimal
conditions, decreased survival of sub-lethally injured
bacteria in Cheddar cheese would be expected due to
low pH and high salt conditions.
Mathew and Ryser (2002) assessed the ability of sub-
lethally heat-injured L. monocytogenes cells to compete
with a commercial mesophilic lactic acid starter culture
during fermentation of UHT milk. L. monocytogenes
strains were heat-injured by two treatments (low heat-
injured (LHI) and high heat-injured (HHI)) to yield
greater than 99% injury. The UHT milk was inoculated
to contain 104–106 LHI, HHI or untreated L. monocyto-
genes together with 0, 0.5 or 2% of a commercial Lacto-
coccus lactis subsp. lactis/Lc. lactis subsp. cremoris
starter. While listeria populations grew to levels of
approximately 109 cfu/ml after 8 h of fermentation, after
24 h, 93% of the non-injured control population became
injured. In starter-free controls, 80% of both HHI and
LHI cells were repaired within 10 h of incubation.
These findings document the suppression of listeria
growth by the starter culture which causes microbial
injury, resulting in cells which are unable to grow or
express pathogenicity. The potential of listeria and other
pathogens to become inactivated and/or sub-lethally
injured during cheesemaking should be investigated.
The combined effects of acid production by starter cul-
tures, salt and mild heat alone or in combination all
have the potential to injure bacterial pathogens such as
L. monocytogenes, E. coli and salmonella. These inter-
active effects could provide an explanation for the
remarkable safety record of aged raw-milk cheese.
Improvement in Cheese Safety
Utilization of more sensitive methods for the detection
of pathogens existing at low levels in Cheddar and aged
raw-milk cheeses could do much to assure cheese safety.
Baylis et al. (2000) compared the Oxoid Ltd SPRINT
salmonella system (Oxoid, Ltd) against the ISO
6579:1993, Qualicon BAX PCR (Wilmington, DE,
USA), bioMerieux VIDAS (Hazelwood, MO, USA) and
Tecra Unique methods (Willoughby, NSW, Australia).
The SPRINT system was developed for the rapid detec-
tion of low levels of injured salmonella in foods. This
system utilizes an enrichment broth that contains a
specifically developed peptone that allows consistent
and rapid recovery of injured salmonella cells, coupled
with a Recovery Supplement which contains an
Oxyrase® Enzyme System that assists recovery through
reduction in oxidative stress of the medium. After 5 h of
incubation, selective agents are added to the medium.
When tested with ice cream and skimmed milk powder
containing low levels of heat-injured S. typhimurium,
the SPRINT method was superior (61% confirmed posi-
tive samples) to the ISO (37% positive), BAX (36% pos-
itive), VIDAS (30% positive) and Tecra (25% positive)
methods. Similar improvements have been advanced by
Pritchard and Donnelly (1999) for recovery of injured
listeria in dairy products, where continuing work on
enrichment of dairy environmental samples at the
University of Vermont (UVM) and listeria Repair Broth
(LRB) has shown that combining these two primary
enrichment media into a single tube of Fraser broth for
secondary enrichment yields a significantly higher
(p0.05) percentage of listeria-positive samples than
when either LRB or UVM is used alone.
Altekruse et al. (1998) stated that ‘Because of inher-
ent problems of statistical sampling of foods for micro-
bial pathogens (ICMSF, 1986), end-point testing may
not assure the safety of cheese. These problems are
amplified when organisms are present in small num-
bers below the sensitivity of the test or when there is
intermittent contamination and the tested specimens
do not contain pathogens.’ Raw ingredient testing, i.e.,
screening of the raw milk supply, may overcome these
shortcomings.
In recent years, cheese and cheese products have
been recalled due to the presence of pathogenic bacteria
 Growth and Survival of Microbial Pathogens in Cheese 555
such as salmonella, L. monocytogenes and E. coli. In
some instances, cheeses, both domestic and imported,
have been linked to outbreaks of human illness. In
November 1998, the FDA issued the Domestic and
Imported Cheese and Cheese Products Food Compli-
ance Program (Anonymous, 1998). The objectives of
this programme are for the FDA to conduct inspections
of domestic cheese firms, to examine samples of
imported and domestic cheese for microbiological con-
tamination, the presence of phosphatase, and filth and
to take appropriate regulatory action when violations
are encountered. Target pathogens for analysis include
L. monocytogenes, salmonella, E. coli (ETEC), enterohem-
orrhagic E. coli 0157:H7 and Staph. aureus. Under this
initiative, direct reference seizure or detention of cheese
based on the presence of L. monocytogenes is authorized.
It should be noted that ETEC analysis is performed
only if E. coli is present at 104 cfu/g. A review of the
FDA’s Product Recalls, Alerts and Warnings Archive
(http://www.fda.gov/oc/po/firmrecalls/archive.html) for
the calendar years 1999, 2000 and 2001 revealed sev-
eral recalls due to the presence of L. monocytogenes in
cheese, one recall involving E. coli contamination of
Blue and Gorgonzola cheeses, and one recall involving
salmonella contamination of Mexican White cheese.
The strain of E. coli identified was not 0157:H7. Listeria-
contamination appears to be a function of post-process
contamination. In no instance during this period were
aged cheeses made from raw milk the subject of a
recall.
Future Research and Conclusions
A number of gaps in the scientific literature have been
identified as a result of this review. Future research is
suggested in a number of areas:
1. Fully explore the impact of pasteurization of milk
on the microbial ecology of cheeses aged for more
than 60 days. Does pasteurization increase the sus-
ceptibility of cheese to the growth of pathogens
introduced via post-processing contamination?
2. Evaluate the potential for survival of S. typhimurium
DT 104 intentionally added at low levels (10 and
100 cfu/ml) to raw milk destined for aged raw-milk
cheesemaking.
3. Evaluate improved microbiological methods for raw-
milk screening and aged raw-milk cheese analysis.
4. Understand the contributions of microbial injury to
the interactive effects of salt, pH and mild heat in
the suppression of growth of listeria, E. coli and
salmonella. Do acid-adapted cultures of these
microbial species show enhanced ability to persist
in aged raw-milk cheese by withstanding salt, aw
and mild heat conditions encountered during aged
raw-milk cheesemaking?
5. Develop microbiological criteria for raw milk des-
tined for aged cheesemaking by setting tolerance
limits for coliforms, E. coli, ETEC, enterohemor-
rhagic E. coli 0157:H7, salmonella, L. monocytogenes
and Staph. aureus. Explore the impact of utilization
of raw milk with stringent microbiological standards
on the safety of raw-milk cheese.
6. Explore the development of risk reduction proced-
ures and practices at both the primary production
level (milk screening) and the cheese production
level to improve the safety of aged raw-milk cheese.
Aged cheeses made from raw milk are microbiologic-
ally safe when manufactured under conditions that use
milk-screening procedures, GMPs and HACCP. The raw
and heat-treated aged cheese issue is not unlike the lis-
teria and potential for survival during pasteurization
issue, which confronted the dairy industry in the
1980s. There was a body of scientific evidence which
indicated that listeria was able to survive the pasteur-
ization process. Through careful research and analysis
over a period of years, it was established that pasteur-
ization, in fact, offered adequate public health protec-
tion and that the greatest risk from listeria posed to
dairy products was the threat of post-processing con-
tamination. Careful investigation of the safety of aged,
raw-milk cheeses may indicate that raw milk provides
protective effects from pathogens in milk and that
environmental contamination poses a far greater threat
to the safety of cheese. This issue deserves the benefit
of full study, careful evaluation of published research
information and new research to fully assess all poten-
tial risks and benefits.
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This Page Intentionally Left Blank
 Toxins in Cheese
N.M. O’Brien and T.P. O’Connor, Department of Food and Nutritional Sciences,
University College, Cork, Ireland
J. O’Callaghan and A.D.W. Dobson, Department of Microbiology, University College,
Cork, Ireland
Biogenic Amines and Mycotoxins
In this chapter, we will discuss the formation of toxic
compounds, such as biogenic amines and mycotoxins,
in cheese. Both these classes of compounds have been
reported to be present in cheese and are produced as
a result of the activity of micro-organisms, both
fungi and bacteria, either in the raw materials used in
cheese manufacture or during the production and
storage/ripening process. The factors that affect the
formation of biogenic amines and mycotoxins will be
discussed, together with their occurrence and poten-
tial toxic effects.
Biogenic Amines
Biogenic amines are non-volatile, low molecular mass
aliphatic, alicyclic or heterocyclic organic bases which
cause physiological effects (Davidek and Davidek,
1995). Typically, they originate in foods from the
decarboxylation of specific amino acids. Decarboxy-
lation can occur due to indigenous decarboxylases
in foods or to decarboxylases produced by micro-
organisms in the food. Biogenic amines are found in
a variety of foodstuffs, most commonly fish of the fam-
ilies Scombridae and Scombereoscidae, but also in
cheese (Maga, 1978; Smith, 1981; Chang et al., 1985;
McCabe, 1986; Joosten, 1988; Lopez-Glaria et al., 2001;
Innocente and D’Agostin, 2002).
In cheese, biogenic amines are produced by
decarboxylation of amino acids during ripening
(see ‘Catabolism of Amino Acids in Cheese during
Ripening’, Volume 1). Levels produced vary as a
function of ripening period and microflora (Renner,
1987; Leuschner et al., 1998). High levels of biogenic
amines are most likely to be detected in cheeses
heavily contaminated with spoilage micro-organisms
( Joosten, 1987). The principal biogenic amines
detected in cheese are histamine, tyramine, trypta-
mine, putrescine, cadaverine and phenylethylamine
(El Sayed, 1996; Roig Sagues et al., 1998; Vale and
Gloria, 1998; Novella-Rodriguez et al., 2000; Finoli
et al., 2001a). Roig Sagues et al. (1998) reviewed the
literature on the concentrations of histamine and
Cheese: Chemistry, Physics and Microbiology, Third edition – Volume 1: General Aspects
ISBN: 0-1226-3652-X
Set ISBN: 0-1226-3651-1
tyramine and other biogenic amines reported in
cheese (Table 1).
The ingestion of biogenic amine-containing foods
may cause adverse toxic reactions (Stratton et al.,
1991). Some of the biogenic amines have vasoactive
properties (e.g., histamine, tyramine, phenylethylamine,
tryptamine) while others act primarily by inhibiting
histamine-detoxifying enzymes, e.g., the putrefactive
amines, putrescine and cadaverine (Hui and Taylor,
1985).
Histamine
Histamine has been reported to exert a wide range of
effects in the body (Taylor et al., 1984). It stimulates
both the sensory and the motor nerves, modulates gas-
tric secretion and stimulates both vascular and
extravascular smooth muscle. Histamine toxicity can
result in a wide variety of symptoms such as rash,
urticaria, inflammation, nausea, vomiting, diarrhoea,
abdominal cramping, hypotension, tingling sensations,
flushing, palpitations and headache (Taylor, 1986;
Bartholomew et al., 1987). In general, toxic symptoms
are relatively mild and many patients may not need
medical attention. Thus, the exact prevalence of hista-
mine toxicity worldwide is unclear. The prevalence of
cheese-related toxicity is also unclear although, as dis-
cussed below, several incidences have been reported in
the literature.
Taylor (1986) comprehensively reviewed the toxi-
cological and clinical aspects of histamine toxicity. He
noted that the most common effects of histamine are
on the cardiovascular system, causing dilation of
peripheral blood vessels, capillaries and arteries with
resultant hypotension, headache and flushing. Abdom-
inal cramping, vomiting and diarrhoea may be related
to histamine effects on H1 receptors. Urticaria may
also be related to the interaction of histamine with H1
receptors, resulting in sensory and motor neuron
stimulation.
However, for most individuals, ingestion of even
large concentrations of biogenic amines, such as his-
tamine, does not elicit symptoms of toxicity since
they are rapidly converted to aldehydes by monoamine
Copyright © 2004 Elsevier Ltd
All rights reserved
562 Toxins in Cheese
Table 1 Concentration (mg/kg) of biogenic amines in different cheeses*
Cheese Histamine Tyramine Tryptamine
Emmental 69–650 0–917
Blue cheese 3–910 40–1100 nd–1100
Camembert nd–480 10–210 nd–60
Dutch (Edam nd–450 0.1–670 nd–200
and Gouda)
Cheddar nd–2120 nd–1530 nd–300
Parmesan nd–293 85–280
* Adapted from Roig Sagues et al. (1998).
nd, not detected.
oxidase (MAO) and diamine oxidase (DAO) and then
to carboxylic acids by oxidative deamination
(Edwards and Sandine, 1981). These enzymes, pres-
ent in the gastrointestinal tract, may prevent/reduce
the absorption of unmetabolised histamine into the
bloodstream (Taylor and Lieber, 1979; Lyons et al.,
1983; Hui and Taylor, 1985). However, if the activities
of MAO and DAO are impaired due to a genetic defect
or the presence of potentiators such as food-borne
putrefactive amines (e.g., putrescine, cadaverine) or
pharmacologic agents (e.g., isoniazid), adverse reac-
tions may occur on ingestion of biogenic amines (Rice
et al., 1976; Diamond et al., 1987; Joosten, 1988).
Putrescine and cadaverine have been reported to
inhibit two histamine-detoxifying enzymes, DAO and
histamine N-methyltransferase (HMT) (Hui and
Taylor, 1985). Taylor and Sumner (1986) noted that
many bacteria, especially Enterobacteriaceae, are cap-
able of producing putrescine and cadaverine as they
possess ornithine decarboxylase and lysine decarboxy-
lase. Stratton et al. (1991) noted that putrefactive
amine potentiators of histamine toxicity are usually
formed by bacteria other than those responsible for
histamine production since only relatively few bacteria
possess histidine decarboxylase. Tyramine, tryptamine
and phenylethylamine can also act as potentiators.
Tyramine inhibits MAO, tryptamine inhibits DAO and
phenylethylamine inhibits both DAO and HMT.
Joosten (1988) reported that tyramine is the only
inhibitor, of MAO and DAO, present in significant
quantities in cheese.
The anti-tuberculosis drug, isoniazid, inhibits his-
tamine-metabolising enzymes and has been reported
to result in histamine poisoning in conjunction
with cheese consumption (Smith and Durack, 1978;
Lejonc et al., 1979; Uragoda and Lodha, 1979).
Other drugs administered as antidepressants, anti-
histamines or antimalarials can sometimes inhibit
histamine-metabolising enzymes (Stratton et al.,
1991).
Phenylethylamine Putrescine Cadaverine
0.1 0.5 16
10 44 42
0.1 7–20 17–48
nd–300
Histamine is a normal constituent of the body;
it is formed from histidine by a pyridoxal phosphate-
dependent decarboxylase and modulates several import-
ant bodily functions (Douglas, 1980). The concentration
of histamine in the blood is strictly regulated. Orally
administered histamine causes poisoning only when
regulatory mechanisms fail to counteract the ingested
dose, i.e., caused by consumption of a very high dose
or inhibition of histamine-metabolising enzymes. Oral
ingestion of up to 1 mmol (⬃100 mg) of histamine
does not elicit toxic symptoms in normal individuals
(Motil and Scrimshaw, 1979). However, vasodilation
and increased heart rate result following intravenous
administration of 0.07 mol histamine, demonstrate
the important detoxifying role of intestinal histamine-
metabolising enzymes.
Factors influencing formation of histamine and other
biogenic amines
The presence of histamine-producing bacteria in foods
such as cheese is a key factor in histamine formation.
Enterobacteriaceae is the main family implicated in
histamine production. However, Clostridium, Lacto-
bacillus and some strains of Klebsiella, Morganella and
Hafnia have also been reported to possess histidine
decarboxylase, and hence are potential histamine pro-
ducers (Sakabe, 1973; Taylor et al., 1978, 1979; Taylor,
1986; Marino et al., 2000). Low concentrations of free
histidine are present in milk. However, proteolysis
during cheese ripening can liberate large amounts of
histidine (Hinz et al., 1956).
Histamine formation can be controlled primarily by
good hygienic practices and by low storage tempera-
tures. Joosten (1988) reported that lactobacilli play a
significant role in histamine formation in Gouda
cheese; he reported that ripening temperature, pH and
salt concentration influence the ability of Lactobacillus
to produce histamine in cheese. Ripening for one year
at 21 °C resulted in 6.8 mmol histamine/kg cheese
compared to 2.2 mmol/kg cheese after 1 year at 9 °C;

6.5 mmol histamine/kg cheese was detected after 2
weeks of ripening when the pH was 5.39 whereas only
3.4 mmol/kg was detected when the pH was 5.19. A
high salt concentration in the Gouda (salt-in-moisture,
4.8%) resulted in 3.5 mmol histamine/kg cheese while
a salt-in-moisture of 2.6% resulted in 2.1 mmol hista-
mine/kg cheese.
Chambers and Staruszkiewicz (1978) reported that
higher levels of biogenic amines are formed in cheese
made from pasteurised milk than in raw milk cheese.
It appears that bacteria responsible for biogenic amine
formation are present in milk prior to processing
rather than as post-processing contaminants. Thus,
adherence to high standards of cleanliness during milk
production can play a role in reducing the formation
of biogenic amines in cheese.
Storage temperature also appears to play a role in
histamine formation in cheese. Elevated storage tem-
perature increases the potential for histamine forma-
tion in cheese, particularly if significant numbers of
bacteria with decarboxylase activity are present
(Sumner et al., 1985). As noted earlier, increasing the
storage temperature for Gouda cheese from 9 to 21 °C
results in higher histamine levels (Joosten, 1988).
Enhancing proteolysis during cheese ripening by
addition of proteolytic enzymes has been reported to
increase the concentration of biogenic amines in
cheese (Leuschner et al., 1998; Fernandez-Garcia et al.,
2000).
Biogenic amines in cheese
Only a few cases of histamine poisoning due to cheese
consumption have been reported in the literature.
Gouda containing 85 mg histamine/100 g cheese was
implicated in an outbreak in Holland (Doeglas et al.,
1967). Salt-tolerant lactobacilli, which contaminated the
rennet, were considered the most likely factor respons-
ible for the high levels of histamine (Stadhouders and
Veringa, 1967). Cheese-related histamine poisoning has
also been reported in the United States. In 1978, 38 peo-
ple exhibited symptoms of toxicity following consump-
tion of Swiss cheese containing more than 9 mmol/kg of
histamine (Chambers and Staruszkiewicz, 1978), and in
1980, 6 people aboard a naval ship were poisoned by
Swiss cheese containing 16.8 mmol/kg of histamine
(Taylor et al., 1982).
An individual in Canada being treated with ison-
iazid exhibited toxicity after consuming Cheddar con-
taining 40 mg histamine/100 g (Kahana and Todd,
1981). Similar reactions to histamine-containing
cheese by individuals taking isoniazid have been
reported by Uragoda and Lodha (1979) and Taylor
(1986).
Toxins in Cheese 563
Swiss cheese was implicated in an outbreak of his-
tamine poisoning reported by Sumner et al. (1985). A
strain of Lactobacillus buchneri, which possessed histi-
dine decarboxylase activity, was isolated from the
cheese. However, other strains of Lb. buchneri did not
possess this enzyme activity and were incapable of
producing histamine.
Recsei and Snell (1982) reported that a strain
known as Lactobacillus 30a, which closely resembled
Lactobacillus delbrueckii, is capable of producing large
amounts of histamine. Sumner et al. (1985) reported
that the ability to produce histamine appears to be
limited to a few strains of lactobacilli, making them
difficult to characterise. Other organisms such as
Enterococcus faecium, Streptococcus mitis, Lb. delbrueckii
subsp. bulgaricus, Lb. plantarum, Lb. casei, Lb. acid-
ophilus and Lb. arabinose have been shown to possess
histidine decarboxylase activity (Stratton et al., 1991).
Joosten and Northolt (1989) isolated five histamine-
producing strains similar to Lb. buchneri from Gouda
cheese.
Tham (1988) reported that enterococci are probably
irrelevant in cheese-related histamine toxicity. How-
ever, Gardin et al. (2001) reported that Enterococcus
faecalis produced 2-phenylethylamine and also sub-
stantial amounts of tyramine in skim milk. They noted
that the main biological feature influencing the forma-
tion of biogenic amines was the extent of growth of
micro-organisms, such as Ec. faecalis, characterised by
decarboxylase activity. In traditional and artisanal
cheeses produced from raw milk, enterococci often
reach levels of 107 cfu/g. Gardin et al. (2001) cautioned
that it is important that the presence of biogenic
amines due to the activity of these micro-organisms is
maintained within safe levels, without affecting the
positive effects of enterococci on the final organoleptic
characteristics of the cheese.
In addition to histamine, tyramine in cheese has
also been reported to induce adverse reactions, such as
headache and hypertension, in patients taking MAO
inhibitors (Blackwell, 1963; Smith and Durack, 1978;
Lejonc et al., 1979). Tyramine is found at levels rang-
ing from non-detectable to 70 mg/100 g in cheese
(Voigt et al., 1974). These workers detected tyramine in
81 of 85 samples of Cheddar cheese tested. Ingles et al.
(1985) reported high levels of tyramine (625 g/g)
as well as histamine (490 g/g) in Danish Blue cheese.
Voigt and Eitenmiller (1978) concluded that many
organisms may be responsible for generating biogenic
amines in cheese but that most are adventitious rather
than part of the starter culture population. The
build-up of amines is influenced by the availability
of substrate, pH, salt concentration and temperature
( Joosten and van Boekel, 1988).
564 Toxins in Cheese
Mycotoxins
Mycotoxins are a group of secondary metabolites pro-
duced by various filamentous fungi which can cause a
toxic response, termed a mycotoxicosis, when ingested
at low concentrations by higher vertebrates and other
animals (Fig. 1). The biosynthetic pathways for many
of these mycotoxins have been extensively charac-
terised, particularly the aflatoxin biosynthetic pathway
(Fig. 2). Ingestion of mycotoxins can lead to the
deterioration of liver or kidney function. Some myco-
toxins are neurotoxins, while others produce effects
ranging from skin sensitivity or necrosis to extreme
immunodeficiency. This, coupled with the fact that
aflatoxin B1 (AFB1) is regarded as the most potent
liver carcinogen known for a wide variety of animal
species, makes contamination of the human food
chain, including dairy produce, with mycotoxins a sig-
nificant problem in global food safety.
The presence of mycotoxins in cheese is fundamen-
tally due to three main reasons: (1) the presence of
aflatoxin M1 (AFM1) in fresh or reconstituted milk
(Blanco et al., 1998) used in cheese production, as a
consequence of feed contaminated with AFB1 eaten by
dairy cattle (Lund et al., 1995), which is often termed
indirect contamination, (2) synthesis of mycotoxins by
fungi such as Penicillium and Aspergillus species,
which grow on cheese, termed direct contamination
and (3) the production of mycotoxins by fungi which
are used in the manufacture of mould-ripened cheeses.
Indirect contamination
It is now well-established that the intake by dairy cows
of feedstuffs contaminated with either aflatoxin B1
(AFB1) or aflatoxin B2 (AFB2) results in the excretion
of the monohydroxylated AFM1 and AFM2 (Fig. 1)
derivatives in their milk within a few hours (Allcroft
and Carnaghan, 1963). It has been calculated that if
cows ingest AFB1 in their diet at a level of 300 ng/g
feed, they will produce milk containing 1–3 ng/ml
AFM1 24 h later (Smith et al., 1994). According to two
other studies (Veldman et al., 1992; Chopra et al.,
1999), normal carry-over is about 0.4–0.6% and a
daily intake of AFB1 70 g by cows results in greater
than the regulatory limit (0.05 g/l of AFM1) in milk
accepted in most countries. The amount of AFM1
formed depends on the individual cow, with the excre-
tion of AFM1 in the milk decreasing markedly about
1 day after the feeding of AFB1 had ceased, although
small amounts are found for a further 2–3 days. The
conversion ratio of AFB1 to AFM1 varies from 1:100 to
1:300. There is some evidence to suggest seasonal vari-
ations in the level of AFM1 in milk, with higher levels
being observed in Albanian farm milk in winter than
in summer (Panariti, 2001), while in a Greek study, no
seasonal effects were observed on AFM1 levels in milk
(Markaki and Melissari, 1997).
While AFM1 is much less toxic, less mutagenic and
less carcinogenic than AFB1, it is nonetheless classified
as a possible human carcinogen (Group 2B) and as
such its presence in milk-derived products, such as
cheese, must be a cause for concern. In addition, it
is important to note that the consumption of AFM1-
contaminated infant formula and other milk products
by infants is to be avoided and very low limits have
been set (0.01–0.05 g/kg) for infant foods, owing to
the relatively high consumption level of these prod-
ucts by infants, their low body weight and the possibly
greater susceptibility of younger children to aflatoxins
(Aksit et al., 1997).
The indirect contamination of milk with other
mycotoxins, such as sterigmatocystin, T-2 toxin (van
Egmond and Paulsch, 1986), fumonisins (Maragos and
Richard, 1994) or cyclopiazonic acid (CPA; Dorner
et al., 1994), has been reported. However, it is widely
believed that these toxins do not represent a significant
public health risk (Prelusky et al., 1990; Charmley
et al., 1993), even though CPA can potentially be car-
ried over into processed milk products (Prasongsidh
et al., 1997). There is some evidence that ochratoxin A
(OTA) can be present in cows’ milk. A Swedish study
showed OTA in 14% of 36 cows’ milk samples at a level
ranging from 10 to 40 ng/ml (Breitholtz-Emanuelsson
et al., 1993).
The results of quantitative surveys of the level of
AFM1 in milk and milk products carried out in the late
1960s and 1970s in a number of countries were sum-
marised by Smith et al. (1994). When compared with
the results of surveys undertaken in the 1980s, it
appears that the incidence of AFM1-contaminated
milk in general decreased but this trend did not occur
in all countries surveyed. This lowering of the inci-
dence of AFM1 contamination may be as a result of the
effect of legislation implemented in many countries on
the contamination of feedstuffs with aflatoxins.
In a recent review by Pittet (1998), including data
from the Czech Republic, Slovakia, Czechoslovakia,
France, Greece, Germany, Iran, Japan, Switzerland, Syria,
the USA and the Netherlands, the incidence of AFM1 in
cheese appears to be very varied. In another study in the
south of Spain in which 35 samples of local cheese were
analysed, AFM1 was detected in 16 samples (44.7%) at a
concentration between 20 and 200 g/g cheese (Barrios
et al., 1996). In a survey in the Bursa Province in Turkey,
the level of AFM1 in 7 of 57 samples of full-fat white
cheese analysed exceeded 250 ng/kg (Oruc and Sonal,
2001). In other studies, the level of AFM1 was low, with
only 4 of 204 samples of pasteurised milk, powdered
 Toxins in Cheese 565

CH2
COOH O OH O O

NH
N O CH3
N
N
H H
H O
H HN
Cl
N

Ochratoxin A Roquefortine C
O O
O O

O
O

O O OCH3
O O OCH3

Aflatoxin B1 Aflatoxin B2

O O
O O

O O
O O

O O OCH3
O O OCH3

Aflatoxin G1 Aflatoxin G2

CH3
O
O OH H N
H
O CH3

CH3
O O Me
OH O

NH

acid

OH O
O O
COOH O
O O
CH3 CHO
CH3COO
O H OH
O CH3 CH3 CH3
CH3
Citrinin Patulin PR Toxin

Figure 1 Structure of some toxins produced by fungi.

566 Toxins in Cheese

(a) (b) HO O HO O
Acetate
+ Hexanoyl-X
Malonyl-CoA
HO OH (c)
O
Norsolorinic acid
OH O OH HO O HO OH OH HO O HO OH

O
HO HO HO OH
O OHMe OH
O O
O
Averufanin 5-Hydroxyaverantin
Averantin

OH O OH O HO O OH
OH O OH
(d) O
O
HO HO HO O
O Me O OH OH O Me
O O
O Versiconal acetate
Averufin 1′-Hydroxyversicolorane
(e)
HO O OH O
HO O OH
O O OH
HO (f1) (f2)
O O
OH O OH OH
O HO O OH
Versicolorin A O
Versicolorin B
Versiconal

O OH O OH
O O
OO OH O O OH (h)
(i)
Demethylsterigmatocystin Dihydromethlysterigmatocystin

O OH
O OH

O O
OMe O O OMe
O O
Dihydrosterigmatocystin
Sterigmatocystin
(j)
(j)
OMe O OMe
O
O O
OMe O O OMe
O O
O-methylsterigmatocystin Dihydro-O-methylsterigmatocystin

O O O O
O O

O O OMe O O OMe

Aflatoxin B1/G1 Aflatoxin B2/G2

Figure 2 Aflatoxin biosynthetic pathway. Enzymes involved: (a) fatty acid synthase, (b) polyketide synthase, (c) norsolorinic acid
reductase, (d) versiconal hemiacetal reductase, (e) esterase, (f1) versicolorin B synthase, (f2) versiconal cyclase, (g) desaturase, (h)
O-methyltransferase (MT-II), (i) O-methyl-transferase, (j) O-methyltransferase (MT-I) (compiled from Trail et al., 1995; Bennet et al.,
1997; Minto and Townsend, 1997).

milk and cheese analysed in Campinas, Brazil, being posi-
tive (De Sylos et al., 1996) and only 2 of 50 cheese sam-
ples tested in Argentina being positive, with levels of 0.33
and 0.20 g/l (Lopez et al., 1998).
Fate of AFM1 in cheese during manufacture and
ripening
Initially, it was believed that the processing of milk
reduced the level of AFM1 present. However, later it
became clear that the AFM1 content of milk is not
reduced by heat treatments such as pasteurisation or
sterilisation (Yuosef and Marth, 1989). The fate of
AFM1 in milk during cheese manufacture is affected
by the principal manufacturing steps. Contradictory
data have been reported for the recovery of AFM1
after cheese production. Some early studies showed
variable losses of AFM1 during cheese manufacture,
e.g., Purchase et al. (1972) reported that Cottage cheese
made by acid coagulation of naturally contaminated
milk contained no AFM1, which was present in the
whey. However, a number of other studies have indi-
cated that AFM1 in milk partitions between the curds
and the whey in both acid-coagulated and rennet-
coagulated cheeses. A number of studies have shown
that AFM1 is stable during cheesemaking, and that
40–57% of total AFM1 is found in the curd
(Stubblefield and Shannon, 1974; Stubblefield et al.,
1980). Considering the partition coefficient of AFM1
in water, it would be expected that most of the toxin
should partition into the whey. However, a greater
than expected proportion of the toxin ends up in the
curd, possibly due to the fact that AFM1 binds to
casein (Brackett and Marth, 1982). Thus, the presence
of AFM1 in cheese may be due to the fact that, on the
one hand, this toxin binds to casein and, on the other
hand, that a part of the whey remains in the curd.
An examination of different types of cheese showed
high stability of AFM1 during maturation and storage
(Applebaum and Marth, 1982; Yuosef and Marth,
1989) and that while fluctuations in the level can
occur during cheese maturation and storage, it appears
that little if any of the AFM1 is lost during the cheese-
making process. Therefore, the presence of AFM1 in
cheese and indeed in other casein-containing products
is to be expected if contaminated milk is used as the
starting material. The best way to control the presence
of AFM1 in milk and cheese is to restrict its presence
in the feed. With this in mind, the European Union
has established an acceptable limit for AFB1 in animal
feed of 10 g/kg (Moss, 1998).
Production of toxic metabolites in cheese
The moulds, Penicillium camemberti and P. roqueforti,
have long been used in the manufacture of mould-
Toxins in Cheese 567
ripened cheeses which are eaten throughout the
world. P. roqueforti is an essential component of the
microflora of a number of cheeses such as Roquefort
(France), Stilton (UK), Tulum (Turkey), Gorgonzola
(Italy), Blauschimmelkase (Switzerland) and Danish
Blue (Denmark). P. camemberti produces cyclopiazonic
acid while P. roqueforti produces at least three toxins,
PR toxin, roquefortine and patulin (Fig. 1), and some
strains can also produce mycophenolic acid, penicillic
acid, cyclopiazonic acid, penitrem A, isofumigaclavine
A and B, festuclavine and chaetoglobosin A.
Cyclopiazonic acid (CPA) is produced by all strains
of P. camemberti, and screening of P. camemberti isolates
has failed to identify an isolate incapable of producing
toxin (Leistner and Eckardt, 1979; Fig. 1). Cyclopia-
zonic acid has been reported in samples of commercial
Blue cheese, at a level ranging from 0.05 to 1.5 g/g
(Le Bars, 1979). It appears to be found predominantly
in the rind of the cheese but has also been reported to
migrate to the core in Taleggio-type cheese (Finoli
et al., 1999). Cyclopiazonic acid is produced by P. camem-
berti in cheese usually after 5 days at 25 °C, but not
during normal storage at refrigeration temperatures
(Still et al., 1978). The low level of CPA found in
cheese (Le Bars, 1979), coupled with the relative
instability of the toxin (Noroozian et al., 1998) and its
low toxicity make consumption of these cheeses safe
for the consumer.
Finoli et al. (2001b) reported the presence of roque-
forine C in cheese ranging from 0.05 to 1.47 mg/kg,
but PR toxin was not found. In any case, PR toxin has
been reported to be unstable in Blue cheese (Scott and
Kanhere, 1979). Roquefortine C has also been reported
to be present in Valdeon, a naturally ripened Blue
cheese from Spain (Lopez-Diaz et al., 1996), while
mycophenolic acid has been reported in Manchego
cheese (Lopez-Diaz et al., 1996). Thus, while there are
reports of mycotoxin contamination of mould-ripened
cheese, the low levels present, coupled with the fact
that large quantities are seldom eaten, suggest that they
are not a hazard to human health. In any case, there is
no evidence to date of human toxicity resulting from
the consumption of mould-ripened cheeses.
Direct contamination of cheese with mycotoxins
Cheese is very susceptible to mould growth and is nor-
mally kept under refrigeration conditions; many retail
packs are either vacuum-packed or flushed with an
inert gas. Therefore, spoilage generally results from
psychrotolerant moulds that can grow at low oxygen
tensions. Mould growth during ripening and storage
often necessitates trimming. Moulds have been reported
to cause spoilage of vacuum-packaged Cheddar cheese
during maturation (Hocking and Faedo, 1992). This
568 Toxins in Cheese
defect occurs sporadically in Cheddar blocks which
are matured for up to 9–12 months at 8–12 °C and
is caused by the growth of fungi in folds and wrinkles
of the plastic film in which the Cheddar is packaged.
In one study, 195 fungi were isolated from vacuum-
packaged Cheddar, about 27% of which were Penicil-
lium species, with P. commune and P. glabrum being
dominant (Hocking and Faedo, 1992). Given that P. com-
mune can produce CPA, the presence of this fungus
must be a concern. Indeed, the potential for myco-
toxin production by mycotoxigenic fungi which con-
taminate cheese is a constant concern for both the
manufacturer and the consumer.
The most important spoilage organisms in hard,
semi-hard and semi-soft cheeses from several countries,
made without added preservatives, are P. commune and
P. nalgiovense (Lund et al., 1995). Less-important
species include P. verrucosum, P. solitum, P. roqueforti,
P. discolor, P. crustosum, P. palitans and Aspergillus versi-
color (Filtenborg et al., 1996; Kure et al., 2001). Cheese
has been reported to contain mycotoxins that are terato-
genic (OTA, AFB1), nephrotoxic (OTA, citrinin), neuro-
toxic (penitrem A, CPA) or carcinogenic (AFB1, AFG1,
OTA, sterigmatocystin; Filtenborg et al., 1996; Creppy,
2002). Others, including patulin, penicillic acid and PR
toxin have also been reported but are known not to per-
sist in cheese (Stott and Bullerman, 1976). A number of
other secondary metabolites produced by different Peni-
cillium species have also been reported to be present in
cheese. These include novel metabolites such as
cyclopeptin, viridicatol, rugulovasine A, meleagrin,
chaetoglobosin A, compactin, viridic acid, PC-2, verru-
colone, diportinic acid, anacine, verrucine A and scle-
rotigenin, which had not previously been reported from
cheese (Larsen et al., 2002). A known metabolite
of Penicillium associated with blue-veined cheese is
mycophenolic acid, which exhibits antibiotic proper-
ties. Since mycotoxin-producing moulds are obligate
aerobes, the appropriate packaging of cheese is import-
ant. For example, growth of potential mycotoxigenic
moulds in cheese can be prevented by modified atmos-
phere packaging (Taniwaki et al., 2001). In addition, it
appears that the production of roquefortine C and CPA
by P. roqueforti and P. commune can be prevented or at
least reduced considerably in cheese if adequate modi-
fied atmosphere packaging is used; atmospheres of
20–40% CO2 and 1% O2 reduce CPA production to very
low levels (Taniwaki et al., 2001).
Work has also been undertaken on the incidence of
mycotoxins in cheese contaminated with Aspergillus
spp. The ability of toxigenic aspergilli to produce afla-
toxin during growth on Cheddar cheese was first
demonstrated by Lie and Marth (1967), who demon-
strated that aflatoxin can penetrate into cheese to a
depth of up to 4 cm from the surface. Aflatoxin has
also been shown to be produced in Manchego-type
cheese, at a level up to 130 g/g cheese, following
ripening at 15 °C for 60 days (Blanco et al., 1988).
Aflatoxin was also detected in both the outer 10-mm
layer and in the second 10-mm layer following incuba-
tion at 28 °C for 30 days. Sterigmatocystin was found
in Ras cheese inoculated with A. versicolor; toxin was
detected after 45 days of ripening and reached a maxi-
mum after 90 days (Abde Alla et al., 1996).
The presence of mould growth on the surface of the
cheese does not automatically imply that mycotoxins
are present in cheese, as the minimum water activity
(aw) for growth and toxin production can be quite dif-
ferent in mycotoxigenic fungi (Moss, 1991; Sweeney
and Dobson, 1998). Therefore, even if mould growth
does occur on cheese, the level of mycotoxin contam-
ination is likely to be low, based on research findings to
date. In any case, it is recommended that if cheese is
visually contaminated with mould growth, the contam-
inated portion of the cheese be removed to a depth of
a least 2.5 cm. As previously stated, there is no direct
evidence of human toxicity resulting from the con-
sumption of cheese contaminated with mycotoxins.
However, this might simply reflect a lack of suitable
human-related assay techniques rather than the actual
absence of toxins.
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This Page Intentionally Left Blank
 Nutritional Aspects of Cheese
N.M. O’Brien and T.P. O’Connor, Department of Food and Nutritional Sciences,
University College, Cork, Ireland
Introduction
Milk and dairy products are important components of
our food supply. On average, these foods contribute
4% of total energy intake worldwide and approxi-
mately 10% of total energy intake in Europe, North
America and Australia (FAO Food Balance Sheets,
1995–1999).
Cheese is a nutritious, versatile dairy food. A wide
variety of cheese types are available to meet specific
consumer requirements and allow convenience of use.
Per caput consumption of cheese in the European
Union has been reported to be 15.2 kg/year. Greece
has the highest per caput consumption of 23.5 kg/year
(Burrell, 1996).
Cheese contains a high concentration of essential
nutrients relative to its energy level. Its precise nutri-
ent content is influenced by the type of milk used
(species, stage of lactation, full-fat, low-fat, skim), the
manner of manufacture and, to a lesser extent, the
degree of ripening. As outlined in detail elsewhere in
this book, the water-insoluble nutrients of milk (coagu-
lated casein, colloidal minerals, fat, fat-soluble vita-
mins) are retained in the cheese curd whereas the
water-soluble milk constituents (whey proteins, lac-
tose, water-soluble vitamins and minerals) partition
into the whey. However, loss of water-soluble B vita-
mins in the whey may be compensated to a certain
extent by microbial synthesis during ripening.
Milk and dairy products, including cheese, contain
components which may increase the risk of certain
chronic diseases but reduce the risk of others (Norat
and Riboli, 2003). Cholesterol and saturated fat are
potential risk factors for atherosclerosis. A recent
paper (Moss and Freed, 2003) has suggested that
non-fat constituents of milk, particularly the calcium–
magnesium ratio, lactose and milk fat globule mem-
brane antigens, have specific coronary atherogenic
effects. However, other components may reduce risks,
e.g., conjugated linoleic acid (CLA) which may have
antioxidant and anticancer properties, calcium which
may protect against hypertension and osteoporosis,
and folic acid, vitamin B6 and vitamin B12 which may
exert beneficial effects on plasma homocysteine levels
(an independent risk factor for atherosclerosis). The
Cheese: Chemistry, Physics and Microbiology, Third edition – Volume 1: General Aspects
ISBN: 0-1226-3652-X
Set ISBN: 0-1226-3651-1
epidemiological evidence for an association between
dairy products, including cheese, and colorectal cancer
has been reviewed by Norat and Riboli (2003); no sig-
nificant association between cheese consumption and
colorectal cancer was noted. Epidemiological studies
which attempt to investigate the effect of a specific
food item (e.g., cheese) on disease risk are fraught
with difficulty in interpretation as it is more likely that
it is the overall dietary profile, made up of a balance of
a wide variety of different foods, that may influence
risk of chronic disease.
Protein
Cheese contains a high content of biologically valuable
protein. As shown in Table 1 (Holland et al., 1989),
the protein content of cheese ranges from approxi-
mately 4–40%, depending upon the variety. The pro-
tein content of different cheese varieties tends to vary
inversely with the fat content.
During traditional cheese manufacture, most of the
whey proteins pass into the whey. Whey proteins rep-
resent only 2–3% of the total protein in cheese, the
remainder being casein, which is slightly deficient in
sulphur amino acids. Thus, the biological value of
cheese protein is slightly less than that of total milk
protein. If the essential amino acid index of total milk
protein is given a value of 100, then the corresponding
value of the proteins in cheese varieties ranges from 91
to 97 (Renner, 1987).
Cheese protein is almost 100% digestible, as the
ripening phase of cheese manufacture involves a pro-
gressive breakdown of casein to water-soluble peptides
and free amino acids. Hence, a significant degree of
breakdown of cheese protein has occurred before it is
consumed and subjected to the effects of gastrointesti-
nal proteolytic activity.
Milk proteins are a key source of a range of bioac-
tive peptides (BP) which can exert hormone-like regu-
latory effects in the human body (Meisel, 1998;
Gobbetti et al., 2002; Pihlanto-Leppala, 2002; Fitzgerald
and Meisel, 2003). These peptides may be released
from their parent protein by proteolysis in products
such as cheese. The production of BP is influenced by
Copyright © 2004 Elsevier Ltd
All rights reserved
574

Table 1 Composition of selected cheeses, per 100 g (Holland et al., 1989)

Energy

Cheese type Water (g) Protein (g) Fat (g) Carbohydrate (g) Cholesterol (mg) kcal kJ

Brie 48.6 19.3 26.9 Tr 100 319 1323

Caerphilly 41.8 23.2 31.3 0.1 90 375 1554
Camembert 50.7 20.9 23.7 Tr 75 297 1232
Cheddar (normal) 36.0 25.5 34.4 0.1 100 412 1708
Cheddar (reduced-fat) 47.1 31.5 15.0 Tr 43 261 1091
Cheshire 40.6 24.0 31.4 0.1 90 379 1571
Cottage cheese 79.1 13.8 3.9 2.1 13 98 413
Cream cheese 45.5 3.1 47.4 Tr 95 439 1807
Danish blue 45.3 20.1 29.6 Tr 75 347 1437
Edam 43.8 26.0 25.4 Tr 80 333 1382
Emmental 35.7 28.7 29.7 Tr 90 382 1587
Feta 56.5 15.6 20.2 1.5 70 250 1037
Fromage frais 77.9 6.8 7.1 5.7 25 113 469
Gouda 40.1 24.0 31.0 Tr 100 375 1555
Gruyere 35.0 27.2 33.3 Tr 100 409 1695
Mozzarella 49.8 25.1 21.0 Tr 65 289 1204
Parmesan 18.4 39.4 32.7 Tr 100 452 1880
Processed cheese* 45.7 20.8 27.0 0.9 85 330 1367
Ricotta 72.1 9.4 11.0 2.0 50 144 599
Roquefort 41.3 19.7 32.9 Tr 90 375 1552
Stilton 38.6 22.7 35.5 0.1 105 411 1701

Tr, trace.
* Variety not specified.

the starter culture and ripening conditions. An import-
ant class of BP are peptides that inhibit the activity of
angiotensin I-converting enzyme (ACE), inhibition of
which mainly gives rise to antihypertensive effects but
may also modulate immuno-defense and nervous sys-
tem activity (Meisel, 1993). Angiotensin I-converting
enzyme-inhibitory peptides have been reported in sev-
eral ripened cheeses (Stepaniak et al., 1995; Meisel
et al., 1997; Smacchi and Gobbetti, 1998). It appears
the BP liberated by starter proteolytic enzymes during
cheese ripening can be degraded further to inactive
fragments, as the ripening progresses. For example, an
antihypertensive peptide derived from s1-casein was
observed in 6-month-old Parmesan cheese but was not
detected in 15-month-old cheese (Meisel et al., 1997).
Anticancer effects have been reported for peptides
derived from a slurry of cheese made using Lc. lactis
subsp. lactis as a starter culture (Kim et al., 1995).
Bioactive peptides have potential as ingredients in
functional foods and pharmaceuticals.
Carbohydrate
Most of the lactose, the principal carbohydrate in milk,
is lost in whey during cheese manufacture and hence
most cheeses contain only trace amounts of carbo-
hydrate (Table 1). Furthermore, the residual lactose in
cheese curd is usually fermented to lactic acid by the
starter bacteria. Thus, cheeses can be consumed with-
out ill-effects by lactose-intolerant individuals who are
deficient in the intestinal enzyme, -galactosidase.
Fat and Cholesterol
The fat content of cheese varies considerably, depend-
ing on the milk used and the method of manufacture
(Table 1). Fat affects firmness, adhesiveness, mouth-
feel and flavour of cheese (see ‘Rheology and Texture
of Cheese’, Volume 1). In some varieties of cheese, free
fatty acids and their catabolites are important flavour
constituents (see ‘Lipolysis and Catabolism of Fatty
Acids in Cheese’, Volume 1).
From a nutritional point of view, the digestibility of
the fat in different varieties of cheese is in the range
88–94% (Renner, 1987). Most cheeses are potentially
significant dietary sources of fat. For example, a 50-g
serving of Cheddar provides 17 g fat (Table 1) which
is a significant amount when compared with typical
intakes of fat in affluent Western societies. A typical
Western diet providing 2000 kcal with 40% energy
derived from fat contains approximately 88 g fat.
Cheese fat generally contains ⬃66% saturated, 30%
monounsaturated and 4% polyunsaturated fatty acids.
Thus, cheese represents a significant dietary source of
Nutritional Aspects of Cheese 575
both total fat and saturated fatty acids. Of the many
saturated fatty acids in milk, only C12:0, C14:0 and
C16:0 have the property of raising blood cholesterol
and palmitic acid, C16:0, is relatively ineffective (Hayes
et al., 1991).
Many sets of dietary guidelines issued by expert
panels worldwide have recommended reductions in
the intake of both total and saturated fat in Western
societies. In large measure, these recommendations are
based on evidence that increased intakes of some
saturated fatty acids elevate both total and low-density
lipoprotein cholesterol in blood, which is associated
with an increased risk of coronary heart disease. While
some nutritionists dispute this hypothesis, the over-
whelming body of medical opinion worldwide sup-
ports the concept of dietary guidelines. Market forces
and consumers have responded to these guidelines
and the market for food products low in fat, choles-
terol and sodium has expanded significantly. The
cheese industry has responded by developing ‘light’
cheese products with a reduced fat content (Olson and
Johnson, 1990).
The cholesterol content of cheese is a function of its
fat content (Table 1) and ranges from approximately
10–100 mg/100 g, depending on the variety. Despite
considerable consumer confusion and the widespread
prevalence of misinformation, dietary cholesterol has
much less influence on blood cholesterol level than
dietary saturated fat (Keys, 1984). Thus, the choles-
terol content of cheese is of lesser importance than its
saturated fat content. The majority of individuals
show little or no response in blood cholesterol level to
increased dietary cholesterol intake in the range
250–800 mg/day. However, a minority (approximately
20%) of adults do exhibit an increased level of blood
cholesterol in response to increased dietary intake
(McNamara, 1987).
In recent years, there has been widespread interest
in the potential role of oxidation products of choles-
terol on the aetiology of atherosclerosis (Brown and
Jessup, 1999; Leonarduzzi et al., 2002). However, chol-
esterol oxides are formed to a negligible extent in
cheese under normal conditions of manufacture,
ripening and storage (Stanton and Devery, 2002).
Conjugated linoleic acid (CLA) is a potentially
beneficial component of milk products, including
cheese (MacDonald, 2000). Conjugated linoleic acid
is a mixture of positional and geometric isomers of
linoleic acid (C18:2) that contain conjugated unsatur-
ated double bonds. The principal isomer is cis-9,
trans-11-octadecadienoic acid which accounts for
more than 82% of total CLA in dairy products (Chin
et al., 1992). Conjugated linoleic acid has been
reported to have antioxidant and anticarcinogenic
576 Nutritional Aspects of Cheese
properties in vitro and in animal models (Ha et al.,
1987, 1990; Ip et al., 1991). However, these suggested
anticarcinogenic properties of CLA could not be con-
firmed in a recently published epidemiological study
on humans (see Voorrips et al., 2002). These authors
noted that cheese contributed approximately 21% of
total CLA intake in their study group. Conjugated
linoleic acid may also be anticholesterolaemic and
antiathrogenic (Lee et al., 1994; Mougios et al., 2001).
On average, the concentration of CLA in milk and
dairy products ranges from 0.2 to 1.6 g/100 g fat (Lin
et al., 1995; Fritsche and Steinhart, 1998). Fritsche
and Steinhart (1998) have estimated that the intake of
CLA in Germany is 0.35 g/d for women and 0.43 g/d
for men. Zlatanos et al. (2002) reported that Greek
Feta and hard cheeses contain 1.9 (average of 0.8) g
CLA/100 g fat. These authors reported higher levels of
CLA in Greek cheese derived from sheep’s and goats’
milk than the level of CLA reported by others (Lin
et al., 1995; Jiang et al., 1997; Ma et al., 1999) in
cheese derived from cows’ milk.
Vitamins
The concentration of fat-soluble vitamins in cheese
is influenced by the same factors that affect its fat
content. Most of the fat-soluble vitamins in milk are
retained in the cheese fat. The concentration of
water-soluble vitamins in cheese is generally lower
than in milk due to losses in the whey. The loss of
some of the B vitamins is offset, to a certain extent,
by microbial synthesis during cheese ripening. In
particular, propionic acid bacteria synthesize signifi-
cant levels of vitamin B12 in hard cheeses such as
Emmental (Renner, 1987). In general, most cheeses
are good sources of vitamin A, riboflavin, vitamin
B12 and, to a lesser extent, folate. The vitamin con-
tent of a range of cheeses is shown in Table 2 (Holland
et al., 1989). Cheese contains negligible levels of
vitamin C.
Minerals
Cheese is an important dietary source of several min-
erals, in particular calcium, phosphorus and magne-
sium (Table 3). A 100-g serving of hard cheese
provides approximately 800 mg calcium. However,
acid-coagulated cheeses, e.g., Cottage, contain consid-
erably less calcium than rennet-coagulated varieties
(Renner, 1987).
Bioavailability of the calcium from cheese is equiva-
lent to that from milk. Recker et al. (1988) reported that
22.9, 26.7 and 25.4% of total calcium was absorbed from
cream cheese, whole milk and yoghurt, respectively.
While the aetiology of osteoporosis is very com-
plex, it appears that adequate calcium intake during
childhood and in the teenage years is important in
assuring the development of high-peak bone mass.
Maximizing bone mass early in life is considered to be
a major preventative factor against the development of
osteoporosis in later years (Heaney, 1991). Cheese has
a potential role in supplying extra, highly bioavailable,
calcium.
Dairy products, including cheese, contribute little
dietary iron (Table 3). Iron deficiency is commonly
observed in both developing and developed countries.
Hence, there has been interest in fortifying dairy prod-
ucts with iron to enhance their nutritional value.
Cheddar and processed cheese have been successfully
fortified with iron (Zhang and Mahoney, 1989a,b,
1990, 1991).
As discussed elsewhere in this book, NaCl serves
several important functions in natural and processed
cheeses (see ‘Salt in Cheese: Physical, Chemical and
Biological Aspects’, Volume 1). A wide range of sodium
levels are found in cheese due to different amounts of
salt added during cheesemaking (Table 3). In general,
the salt content of natural cheeses tends to be lower
than that of many processed cheeses.
There is considerable evidence that high sodium
intake contributes to hypertension in a susceptible
minority (20%) of individuals who are genetically salt-
sensitive. Unfortunately, there is no simple diagnostic
test to identify salt-sensitive individuals. Hence, dietary
guidelines for the general public usually recommend
that salt intake be restricted. However, it is important
to note that even in countries with a high consump-
tion, cheese contributes only about 5–8% of total
sodium intake (Renner, 1987).
Cheese and Dental Caries
The aetiology of dental caries involves metabolism of
sugars by oral micro-organisms to acids which gradu-
ally dissolve tooth enamel. However, it is now recog-
nized that a number of dietary factors and nutrient
interactions can modify the expression of dental caries
(Herod, 1991; Kashket and DePaola, 2002). The cario-
genic potential of food is influenced by its composi-
tion, texture, solubility, retentiveness and ability to
stimulate saliva flow (Morrissey et al., 1984). Dental
caries has been acknowledged as a ‘silent epidemic’
that represents a serious threat to children and adults
(Surgeon General, 2000).
A considerable body of research has been published
on the cariostatic effects of cheese (see reviews by
O’Brien and O’Connor, 1993; Kashket and DePaola,
2002). Early work (Shaw et al., 1959; Dreizen et al.,
 Table 2 Vitamin content of selected cheeses, per 100 g (Holland et al., 1989)

Cheese Retinol Carotene Vitamin D Vitamin E Thiamine Riboflavin Niacin Vitamin B6 Vitamin B12 Folate Pantothenate Biotin
type (g) (g) (g) (mg) (mg) (mg) (mg) (mg) (g) (g) (mg) (g)

Brie 285 210 0.20 0.84 0.04 0.43 0.43 0.15 1.2 58 0.35 5.6
Caerphilly 315 210 0.24 0.78 0.03 0.47 0.11 0.11 1.1 50 0.29 3.5
Camembert 230 315 0.18 0.65 0.05 0.52 0.96 0.22 1.1 102 0.36 7.6
Cheddar 325 225 0.26 0.53 0.03 0.40 0.07 0.10 1.1 33 0.36 3.0
(normal)
Cheddar 165 100 0.11 0.39 0.03 0.53 0.09 0.13 1.3 56 0.51 3.8
(reduced-
fat)
Cheshire 350 220 0.24 0.70 0.03 0.48 0.11 0.09 0.9 40 0.31 4.0
Cottage cheese 44 10 0.03 0.08 0.03 0.26 0.13 0.08 0.7 27 0.40 3.0
Cream cheese 385 220 0.27 1.0 0.03 0.13 0.06 0.04 0.3 11 0.27 1.6
Danish blue 280 250 0.23 0.76 0.03 0.41 0.48 0.12 1.0 50 0.53 2.7
Edam 175 150 0.19 0.48 0.03 0.35 0.07 0.0 2.1 40 0.38 1.8
Emmental 320 140 N 0.44 0.05 0.35 0.10 0.09 2.0 20 0.40 3.0
Feta 220 33 0.50 0.37 0.04 0.21 0.19 0.07 1.1 23 0.36 2.4
Fromage frais 100 Tr 0.05 0.02 0.04 0.40 0.13 0.10 1.4 15 N N
Gouda 245 145 0.24 0.53 0.03 0.30 0.05 0.08 1.7 43 0.32 1.4
Gruyere 325 225 0.25 0.58 0.03 0.39 0.04 0.11 1.6 12 0.35 1.5
Mozzarella 240 170 0.16 0.33 0.03 0.31 0.08 0.09 2.1 19 0.25 2.2
Parmesan 345 210 0.25 0.70 0.03 0.44 0.12 0.13 1.9 12 0.43 3.3
Processed 270 95 0.21 0.55 0.03 0.28 0.10 0.08 0.9 18 0.31 2.3
cheese*
Ricotta 185 92 N 0.03 0.02 0.19 0.09 0.03 0.3 N N N
Roquefort 295 10 N 0.55 0.04 0.65 0.57 0.09 0.4 45 0.50 2.3
Stilton 355 185 0.27 0.61 0.03 0.43 0.49 0.16 1.0 77 0.71 3.6

N, the nutrient is present in significant quantities but there is not reliable information on the amount; Tr, trace.
* Variety not specified.
577
578 Nutritional Aspects of Cheese

Table 3 Mineral content of selected cheeses, mg/100 g (Holland et al., 1989)

Cheese type Na K Ca Mg P Fe Zn

Brie 700 100 540 27 390 0.8 2.2

Caerphilly 480 91 550 20 400 0.7 3.3
Camembert 650 100 350 21 310 0.2 2.7
Cheddar (normal) 670 77 720 25 490 0.3 2.3
Cheddar (reduced-fat) 670 110 840 39 620 0.2 2.8
Cheshire 550 87 560 19 400 0.3 3.3
Cottage cheese 380 89 73 9 160 0.1 0.6
Cream cheese 300 160 98 10 100 0.1 0.5
Danish blue 1260 89 500 27 370 0.2 2.0
Edam 1020 97 770 39 530 0.4 2.2
Emmental 450 89 970 35 590 0.3 4.4
Feta 1440 95 360 20 280 0.2 0.9
Fromage frais 31 110 89 8 110 0.1 0.3
Gouda 910 91 740 38 490 0.1 1.8
Gruyere 670 99 950 37 610 0.3 2.3
Mozzarella 610 75 590 27 420 0.3 1.4
Parmesan 1090 110 1200 45 810 1.1 5.3
Processed cheese* 1320 130 600 22 800 0.5 3.2
Ricotta 100 110 240 13 170 0.4 1.3
Roquefort 1670 91 530 33 400 0.4 1.6
Stilton 930 130 320 20 310 0.3 2.5

* Variety not specified.

1961) showed that the incorporation of dairy products
in the diet greatly reduced the development of dental
caries in rats. Reynolds and Johnson (1981) confirmed
these findings. Later work (Jenkins and Ferguson,
1966; Weiss and Bibby, 1966) indicated that if enamel
is treated with milk in vitro and subsequently washed,
the solubility of the enamel is greatly reduced. This
effect was attributed to the high levels of calcium and
phosphate in milk ( Jenkins and Ferguson, 1966) or to
casein adsorption onto enamel surfaces (Weiss and
Bibby, 1966).
Reynolds and del Rio (1984) reported that both
casein and whey proteins significantly reduced the
extent of caries, with the former exerting the greater
effect. Further evidence for the protective effect of
casein was provided in a study on rats fed with casein-
enriched chocolate (Reynolds and Black, 1987). Cal-
cium and phosphate appear to become available under
the acidic conditions of the plaque and reduce dem-
ineralization of enamel (Reynolds, 1997; Reynolds
et al., 1999). Concentrates containing various levels of
whey protein, calcium and phosphate but negligible
amounts of casein, significantly reduced the incidence
of dental caries in rats (Harper et al., 1987). Thus,
there is evidence that milk proteins, calcium and phos-
phate all exert an anticariogenic effect. Guggenheim
et al. (1999) reported that micellar casein inhibits oral
colonization by the cariogenic Streptococcus sobrinus
and dental caries in rats. Vacca-Smith et al. (1994)
demonstrated that -casein can reduce the adherence
of the cariogenic Sc. mutans to hydroxyapatite (the
mineral of enamel).
Rugg-Gunn et al. (1975) provided the first evidence
that the consumption of cheese had an anticariogenic
effect in humans. They showed that the consumption
of Cheddar cheese after sweetened coffee or a sausage
roll increased plaque pH, possibly due to increased
salivary output. Similar effects were reported by Imfeld
et al. (1978) who used a more sophisticated continu-
ous wire telemetry procedure to monitor variations in
plaque pH.
The effect of eating patterns on dental caries in rats
was investigated by Edgar et al. (1982). Rats fed addi-
tional meals of cheese while on a high-sucrose diet,
developed fewer smooth surface carious lesions and
exhibited increased salivary output (which buffers acid
formed in plaque) and a reduction in the number of
Sc. mutans. Harper et al. (1983) suggested that the car-
iostatic effect of cheese in rats is due to its calcium
phosphate and/or casein; the fat or lactose appeared to
exert little influence. Further work by Rosen et al.
(1984) on the effect of cheese, with or without
sucrose, on dental caries and the recovery of inocu-
lated Sc. mutans in rats indicated beneficial cariostatic
effects of cheese consumption but little effect on Sc.
mutans numbers. These data suggest that the cario-
static effects of cheese may not be directly related to
effects on Sc. mutans. Work on the protective effects of
four cheese varieties in an in vitro demineralization
system suggested that most, but not all, of the protective

effects of cheese could be explained by mass action
effects of soluble ions, particularly calcium and phos-
phate (Jenkins and Harper, 1983).
The effect of Cheddar cheese on experimental
caries in humans was investigated by Silva et al.
(1986) using an ‘intraoral cariogenicity test’ (ICT).
Demineralization and consequent reduction in the
hardness of enamel monitored in this test is assumed
to be typical of the early stage of the development of
caries. Enamel slabs were polished and their initial
micro-hardness determined using a Knoop Indenter.
The slabs were then wrapped in Dacron and fastened
on a prosthetic applicance made specifically for each
subject to replace a missing lower first permanent
molar. The subjects chewed 5 g of cheese immediately
after rinsing their mouths with 10% (w/v) sucrose.
Chewing cheese immediately after sucrose rinses
resulted in a 71% reduction in demineralization of the
enamel slabs, raised plaque pH but caused no signifi-
cant change in the microflora of plaque compared with
controls. Silva et al. (1987) investigated the effects of
the water-soluble components of cheese on human
caries using the ICT procedure and an experimental
protocol which avoided salivary stimulus caused by
chewing cheese. An average reduction of 55.7% in the
cariogenicity of sucrose was reported, indicating the
presence of one or more water-soluble anticariogenic
components in cheese. Further evidence that cheese
may inhibit dental caries in the absence of saliva was
provided by Krobicka et al. (1987); rats that had their
saliva-secreting glands surgically removed developed
fewer and less-severe lesions when fed with cheese in
addition to a cariogenic diet when compared to appro-
priate controls.
Trials on human subjects have confirmed that the
consumption of hard cheese leads to significant
rehardening of softened enamel surfaces (Jenkins and
Hargreaves, 1989; Gedalia et al., 1991). Jensen and
Wefel (1990) showed that processed cheese was both
antiacidogenic and enamel-protective in human sub-
jects fed with processed cheese four times a day for
one month. Saliva flow is greatly reduced in individu-
als who receive head and neck irradiation for malig-
nancies. These individuals are at high risk of
developing dental caries. Sela et al. (1994) reported
that hard cheese consumption by these individuals
was effective in controlling caries. Moynihan et al.
(1999) noted that the concentration of calcium in
plaque was significantly higher in human subjects fed
with cheese-containing meals than in control subjects
fed with meals without cheese. The beneficial effects
of cheese were observed even when it was incorpor-
ated into other foods, e.g., pasta with cheese sauce.
Epidemiological studies (Pappas et al., 1995a,b) indi-
Nutritional Aspects of Cheese 579
cate that high intake of cheese is negatively associated
with root caries in elderly populations, many of whom
are at high risk for such lesions.
While more research is needed to define the precise
mechanism(s) involved in the cariostatic effects of
cheese, there is ample evidence to support the con-
sumption of cheese as an anticaries measure (Herod,
1991; Kashket and DePaola, 2002). The most plausible
mechanisms for the protective effect of cheese appear
to be related to the mineralization potential of the
casein–calcium phosphate of cheese, to the stimula-
tion of saliva flow induced by its texture and/or
flavour, the buffering effects of cheese proteins on acid
formation in dental plaque and the inhibition of cario-
genic bacteria.
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This Page Intentionally Left Blank
 Factors that Affect the Quality
of Cheese
P.F. Fox, Department of Food Science, Food Technology and Nutrition,
University College, Ireland
T.M. Cogan, Dairy Products Research Centre, Teagasc, Fermoy, Ireland
Introduction
As discussed in ‘Cheese: An Overview’, Volume 1 and
‘Diversity of Cheese Varieties: an Overview’, Volume 2,
the manufacture of cheese exploits either of two prop-
erties of the casein system: precipitation/coagulation at
the isoelectric pH (4.6), which is exploited in the pro-
duction of fresh, acid-coagulated cheeses (⬃25% of
total cheese production), or by limited proteolysis
using rennets which specifically hydrolyse the micelle-
stabilizing protein, -casein, following which the rennet-
altered micelles coagulate in the presence of Ca2 at a
temperature 20 °C, usually 30–35 °C (⬃75% of
cheese production). Most acid-coagulated cheeses are
consumed fresh (unripened) whereas the vast majority
of rennet-coagulated cheeses are ripened for a period
ranging from ⬃3 weeks to 2 years. Although there
are recognizable differences between the unripened
curds for different cheeses, mainly with respect to
moisture content and texture, the characteristic differ-
ences between the 1000 or so varieties of cheese
develop during ripening. The quality of acid-coagulated
cheeses is subject to some variation but the fact that
they are consumed fresh and that no modifications are
required after manufacture, makes them relatively easy
to produce with consistent quality (see ‘Formation,
Structural Properties and Rheology of Acid-coagulated
Milk Gels’, Volume 1 and ‘Acid- and Acid-Rennet-Curd
Cheeses: Part A Quark, Cream Cheese and Related Var-
ieties’, Volume 2). In contrast, the characteristic quality
of rennet-coagulated cheeses develops mainly during
ripening and frequently depends on the growth of a sec-
ondary microflora, which are not readily reproducible.
During ripening, a complex array of microbiological,
biochemical and chemical reactions occur and therefore
there are many opportunities for problems to develop.
In this chapter, the quality aspects of rennet-coagulated
cheeses will be considered. Some of the principal areas
of cheese science through which cheese quality can be
improved through research will be discussed.
Cheese is the quintessential convenience food – it
can be consumed as it is without preparation, can be
Cheese: Chemistry, Physics and Microbiology, Third edition – Volume 1: General Aspects
ISBN: 0-1226-3652-X
Set ISBN: 0-1226-3651-1
used as a sandwich filler, grated or diced and used as a
condiment or as a component of several cooked
dishes. At least 50% of cheese is used, at home or in
the factory, as an ingredient or component of other
foods. The principal applications of cheese as an ingre-
dient are discussed in ‘Cheese as an Ingredient’, Vol-
ume 2. The use of cheese as a food ingredient is
increasing and for many of these applications, the
principal criterion of quality depends on the function-
ality of the cheese, which depends mainly on the
physico-chemical properties of the proteins. When
used as an ingredient in food applications, cheese is
expected to perform one or more functions and there
is considerable commercial interest in producing
cheese products with functional properties tailor-made
for particular applications. Cheese may be used as an
ingredient in several forms:
• natural cheese: sliced, diced or grated;
• processed cheese products;
• dried cheese products:
– dried, grated natural cheese (a traditional product);
– cheese powders.
• enzyme-modified cheese products, which may be
produced from mild cheese or fresh cheese curd or
from blends of enzyme-treated casein and fats and
are used mainly as high-intensity cheese flavours.
There are several aspects to the quality of cheese;
some are applicable to all cheese products and applica-
tions, others are significant for specific cheeses. Prob-
ably the most important aspects of cheese are:
• safety from a public health viewpoint
• nutritional
• flavour
• texture
• functionality
• appearance, e.g.,
– Cheese must conform to the expected characteris-
tics of the variety. There are obvious differences
between the principal families of cheese but it may not
be so easy to differentiate between cheeses claimed to
Copyright © 2004 Elsevier Ltd
All rights reserved
584 Factors that Affect the Quality of Cheese

belong to similar varieties, e.g., Gouda and Edam, Milk

Cheddar and Gloucester, Brie and Camembert. The
borderlines are blurred and within each variety, a cer- Selection
Pre-treatment
tain degree of variability is tolerated and acceptable. Standardization
– Reproducibility/reliability – consumers expect
that a product will be reproducible and consistent
between batches and over time with respect to Cheesemilk
flavour, texture, appearance and functionality, espe- Addition of:
cially for the principal, ‘mass-produced’ varieties; starter culture (acidification)
some variability is tolerated, perhaps even expected, colour (optional)
CaCl2 (optional)
in artisanal cheeses. Coagulation [rennet or acid (produced in situ or
pre-formed) or heat/acid]

Production of Rennet-Coagulated Cheese

Coagulum (gel)
The production of rennet-coagulated cheese can be
divided into two phases: Cut coagulum
Stir
• Conversion of milk to curd Heat
• Conversion of curd to cheese Acidification (rennet-coagulated cheeses)
Separation of curds from whey
The key operations are summarized in Fig. 1.
The production of good-quality cheese depends on
Curds
a good milk supply from the chemical and microbio-
logical viewpoints. Raw milk is a rather variable com- Acidification
modity and is subjected to a range of processes aimed Special operations
at modifying, standardizing and optimizing the cheese- (e.g., cheddaring, stretching)
Salting (some varieties)
making properties of milk. Given a good milk supply, Moulding
the first objective is to produce curd of the desired Pressing (some varieties)
chemical composition with the desired microflora.
Unless these criteria are met, the curd will not evolve
Fresh cheese
into cheese with the characteristic flavour, texture and
functionality during ripening. Salting (most varieties)
The ripening of cheese, and hence its quality, is due Ripening (most rennet-coagulated cheeses)
to the activity of micro-organisms and enzymes from
four or five sources: Mature cheese

• Milk Figure 1 General protocol for cheese manufacture (from Fox

• Rennet
• Primary starter
• Secondary cultures
• Adventitious (non-starter) bacteria
It is reasonable to expect that it should be possible
to produce premium-quality cheese consistently by
controlling these agents; however, in spite of consider-
able research and quality control efforts, it is not yet
possible to do so.
A very wide and diverse range of factors interact
to affect the composition of cheese curd and hence
the quality of the final cheese; an attempt to summar-
ize these is shown in Fig. 2, which follows the same
sequence as Fig. 1. Some of these factors/agents can
be manipulated easily and precisely while others are
more difficult, or perhaps impossible, to control. Indeed,
the precise influence of many of the factors included
et al., 2000).
in Fig. 2 on cheese ripening and quality are not known
precisely and many of the factors are interactive. It is
possible to apply the principles of Hazard Analysis
Critical Control Point (HACCP) analysis to cheese
production and it is hoped that this article may stimu-
late efforts to apply HACCP principles to cheesemak-
ing. The principal items in Fig. 1 and 2 will be discussed
individually in the following sections.
Milk Supply
It is well recognized that the quality of the milk supply
has a major impact on the quality and consistency of
the resultant cheese. Three aspects of quality must be
considered: microbiological, enzymatic and chemical.
 Species
Composition
Breed
Stage of lactation
Somatic cell count
Plane of nutrition
Animal health

Composition
Microbiological quality - casein
- fat
RAW MILK Public health and safety - calcium
- pH
natural creaming - enzymes
Thermization centrifugal
On-farm hygiene Standardization milk powder
Pasteurization UF
Transport temperature
CHEESE MILK Selection criteria
In-factory time Acid production
Colour Phage sensitivity
CaCl2 Ripening properties
GDL Rate of lysis
Starter culture
Type
Secondary/adjunct culture
Rennet
Type
Gel strength Amount
subjective/objective assessment COAGULUM
- cheese yield - cheese yield
Cut
- curd composition - curd composition

- curd syneresis
Cheese quality CURDS/WHEY
- curd composition
- acidification
- Cook - retention of coagulant
- syneresis
- curd composition - Agitate
- curd structure Acidification - curd syneresis
- retention of coagulant - Drain curd composiiton
- solubilization of CCP
Whey
CURDS
- Acidification
- Dehydration
- Texturization?
- Salting?
- Moulding
- Pressing?

UNRIPENED/FRESH CHEESE
- Salting
- Special secondary cultures
Rennet - Coating/packaging
Milk enzymes Moisture
Starter enzymes RIPENING pH
Secondary culture NaCl
Adventitious microflora - Composition Fat
- Temperature
- Humidity
- Time
- Proteolysis
- Lipolysis
- Glycolysis
- Secondary changes MATURE CHEESE Flavour

Texture

Functional properties

Figure 2 Interaction of compositional and technological factors that affect the quality of cheese.

585
586 Factors that Affect the Quality of Cheese
Microbiological
Three aspects of the microbiology of cheese milk are of
interest/concern:
• Public health
• Off-flavours and spoilage
• Desirable bacteria
Public health aspects
As a product of animal origin, milk may become con-
taminated with pathogenic micro-organisms, and this,
clearly, is of great concern. Previously, the principal
pathogens of concern in milk were Mycobacterium
bovis and Brucella abortus, but in developed dairying
countries today, these pathogens have been largely or
entirely eliminated from the dairy herd. Today, a wide
range of pathogens are of concern, mainly Listeria
monocytogenes, enterotoxigenic strains of E. coli, e.g.,
E. coli O157 H7, Shigella, Erwinia, Campylobacter,
Staphlyococcus, Salmonella spp. and M. paratuberculosis.
Many of these micro-organisms do not grow in milk
which simply acts as a vector. In cheese, these
pathogens die off under the rather hostile conditions
in well-made cheese which has a relatively low pH
(5.3), a relatively high salt content (5–10% salt-in-
moisture; S/M) and perhaps bacteriocins. For this rea-
son, Public Health Authorities in many counties
require that cheese made from raw milk be aged for 60
days, although this practice may not be fully effective.
Alternatively, cheese must be made from pasteurized
milk or the cheese itself must be pasteurized, as in
processed cheese. Cheese, the pH of which does not
decrease at the desired rate or to the desired extent
during manufacture (e.g., due to bacteriophage infec-
tion or contamination with antibiotics) or if the pH
increases substantially during ripening, e.g., surface
mould- or smear-ripened cheese, are most at risk.
High-moisture, fast-ripening cheeses are at a greater
risk of harbouring pathogens than low-moisture, slow-
ripening varieties.
A considerable amount of cheese is made from raw
milk, especially in France, Germany and southern
European countries, but there is increasing pressure in
northern Europe and North America to produce all
cheese from pasteurized milk. There have been very
few reported outbreaks of food poisoning from the
consumption of raw milk hard, long-ripened cheese
varieties. In all outbreaks for which adequate data are
available, mitigating circumstances, e.g., lack of a starter
culture or poor starter activity, have also been
involved. Clearly, raw milk cheese is safe if adequate
precautions are taken. However, it should be remem-
bered that most raw milk cheeses are high-cooked, hard
or extra-hard varieties – many of these cheeses, e.g.,
Parmigiano Reggiano, Grana Padano, Swiss Emmental
and Gruyère de Comté, are cooked at ⬃55 °C; the
cooked curds are transferred while hot to moulds with a
capacity of 20–60 kg curd and consequently, the curds
cool slowly – most of these ‘raw milk’ cheeses are in
fact pasteurized, as indicated by a negative alkaline
phosphatase test. High-moisture, raw-milk cheeses are
of more concern but most of these have a low initial
pH (4.6) and appear to be safe. It is probably signifi-
cant that raw-milk cheeses are made on a small/very
small scale from very fresh milk from healthy cows.
For further discussion on pathogens in cheese, see
‘Growth and Survival of Microbial Pathogens in Cheese’,
Volume 1.
It is unlikely that it will be possible to produce raw
milk guaranteed free of pathogenic bacteria. However,
milk with very low numbers of pathogens can be pro-
duced from healthy animals and any pathogens that do
enter milk can be:
• killed (pasteurization or novel alternatives);
• removed (bactofugation or microfiltration (MF); see
‘Application of Membrane Separation Technology to
Cheese Production’, Volume 1);
• prevented from growing or killed, e.g., by use of low
pH, selected bacteriocin-producing starters.
To date, efforts to remove pathogens from cheese
milk have concentrated on adequate pasteurization.
There is ongoing research on alternative methods and
it is likely that work in this area will continue and
probably expand.
Off-flavours and spoilage
A second beneficial effect of pasteurization is the killing
of spoilage micro-organisms, e.g., coliforms, pseudomon-
ads and yeasts. In countries with a developed dairy
industry, the quality of the milk supply has improved
markedly during the past 30 years – total bacterial
counts (TBC) are now usually 20 000 cfu/ml ex-farm.
The TBC probably increases during transport to and
storage at the factory, but growth can be minimized by
thermization (65 °C  15 s) of the milk on reception
at the factory, which is a standard practice in some
countries. The presence of Clostridium tyrobutyricum
poses special problems.
Although many cheeses are made from raw milk,
quantitatively, most cheese is made from milk pasteur-
ized at or close to 72 °C  15 s. If produced from good-
quality raw milk and if subsequently handled under
hygienic conditions, pasteurized milk should have a
very low TBC (a few hundred cfu/ml) and therefore

represents a very uniform raw material from a microbio-
logical viewpoint. Some alternatives to pasteurization
are likely to become industrially significant.
Desirable indigenous bacteria
Some of the adventitious micro-organisms in raw
milk, especially the non-starter lactic acid bacteria
(NSLAB), probably contribute positively to cheese
flavour – it is generally accepted that the flavour of
raw-milk cheese is more intense, although more vari-
able, than that of pasteurized milk cheese. Although
the reason(s) for the differences in flavour between
raw and pasteurized milk cheese has not been
explained to everybody’s satisfaction and is still under
investigation, there is broad support for the view that
adventitious NSLAB are mainly responsible. These
indigenous LAB are killed by pasteurization; attempts
are being made to replace them through the use of
adjunct cultures (see also ‘Secondary and Adjunct
Cultures’, ‘The Microbiology of Cheese Ripening’ and
‘Raw Mild Cheeses’, Volume 1).
While raw milk may be acceptable for cheesemak-
ing in small-scale, manually operated factories, the
cheesemaking quality of even good-quality raw milk is
too variable to be used successfully in very large,
highly automated factories such as those involved in
the manufacture of Cheddar, Gouda or Mozzarella
(Pizza) cheese. In these cases, it is highly probable that
pasteurized milk will continue to be used, with
selected adjunct cultures added to simulate the super-
ior quality of raw-milk cheese.
Alternatives to pasteurization
There are a number of alternatives to pasteurization
for the decontamination of cheese milk:
• Thermization – heat treatment at a sub-pasteurization
temperature, e.g., 65 °C  15 s; thermization is
intended to reduce the microflora of raw milk and
extend the period for which it may be held at the
factory without a risk of spoilage. Although ther-
mization does not meet the requirements for pas-
teurization from the public health viewpoint, it is
fairly widely used for cheese milk and in combin-
ation with other hurdles, e.g., cooking of the cheese
curd, low pH, high S/M, is probably adequate to ren-
der good-quality milk free of pathogens and food-
poisoning bacteria;
• Microfiltration;
• Bactofugation, which may be used as a general,
quite efficient method for the removal of bacteria
and spores from milk but which is not widely
used;
Factors that Affect the Quality of Cheese 587
• The LPO-H2O2-SCN% system, which is not used
commercially for cheese milk;
• Addition of H2O2, which is not used in developed
dairying countries;
• Pre-ripening milk with bacteriocin-producing
cultures.
Microfiltration is a very efficient method for the
removal of bacteria from milk – 99.9% of the bac-
teria can be removed, i.e., it is more efficient than pas-
teurization. Microfiltration has the added advantage
over pasteurization that no heat damage is caused to
the whey proteins. In addition to killing bacteria, MF
removes somatic cells, which are significant sources of
enzymes and are generally believed to have a negative
effect on cheese quality. However, the quality of
Cheddar (McSweeney et al., 1993) and Gruyère de
Comté (Beuvier et al., 1997) cheese made from pas-
teurized or MF milk was similar and different from
that of raw-milk cheese, suggesting that NSLAB,
rather than other factors, the distinguishing factors. At
present, MF is not used commercially for the general
removal of bacteria from cheese milk and it is not
acceptable as an alternative to pasteurization from a
public health viewpoint because it cannot guarantee
milk free from pathogens. However, MF is provision-
ally accepted in France as a suitable alternative to
HTST pasteurization for the decontamination of bever-
age milk (see ‘Application of Membrane Separation
Technology to Cheese Production’, Volume 1). If no
problems are encountered, it is likely that MF will
become an acceptable alternative to pasteurization of
cheese milk.
A serious microbiological problem in many/most
cheeses arises from the growth of Clostridium tyrobu-
tyricum in the cheese during ripening which catabolizes
lactic acid to butyric acid and H2, with the production
of off-flavours and late gas blowing. Cheddar-type
cheese is an exception owing to the rapid decrease in
pH and the rapid increase in S/M to an inhibitory level.
The principal sources of Cl. tyrobutyricum are soil and
silage; the feeding of silage to cows, the milk of which
is to be used for cheesemaking, is prohibited in
Switzerland and parts of France. However, in most
countries, the outgrowth of Cl. tyrobutyricum is pre-
vented by the use of NaNO3 or lysozyme or the spores
are removed by bactofugation or MF.
Indigenous enzymes
Milk contains about 60 indigenous enzymes (see Fox
et al., 2003), the significance of which for cheese quality
has not yet been researched adequately. Several indigen-
ous enzymes have the potential to affect cheese quality,
especially lipoprotein lipase (LPL), proteinase(s), acid
588 Factors that Affect the Quality of Cheese
and alkaline phosphatase, xanthine oxidase (XO) and
perhaps sulphydryl oxidase (SO), lactoperoxidase and
-glutamyl transpeptidase. Some of these enzymes are
active in milk prior to cheesemaking and adversely
affect the yield and/or quality of cheese. Many of the
indigenous milk enzymes survive HTST pasteurization
(72 °C  15 s) and at least some, e.g., plasmin, acid
phosphatase and XO, are active during cheese ripening.
Lipoprotein lipase has the potential to cause signifi-
cant lipolysis in milk and the resulting fatty acids are
concentrated in the cheese curd where they may cause
hydrolytic rancidity, especially in mild-flavoured
cheese. Normally, LPL has low activity in milk where it
is separated from its triglygeride substrates by the milk
fat globule membrane (MFGM). However, the MFGM
is quite susceptible to damage due to rough handling of
milk, leading to activation of the LPL, and rancidity.
Plasmin, the principal indigenous proteinase in milk,
hydrolyses s1-, s2- and, especially, -casein, produc-
ing - and -caseins, some of the proteose peptones
(PP) and other peptides. Plasmin activity reduces
cheese yield because the PPs are not incorporated into
the cheese curd and is reported to damage the quality
of the rennet-induced coagulum. Plasmin activity
increases with advancing lactation, age of cow and
mastitis and its action may result in a weak coagulum
with poor syneresis properties – the consequences are
reduced yield of cheese and a high moisture content.
The formation of -caseins in cheese during ripening
clearly indicates that plasmin is active in cheese – it is
mainly responsible for the hydrolysis of -casein in low-
cooked cheeses and for total primary proteolysis in high-
cooked varieties in which the rennet is extensively or
totally inactivated (see ‘Proteolysis in Cheese during
Ripening’, Volume 1,). Studies on the effect of the plas-
min inhibitor, 6-amino hexanoic acid, have shown that
plasmin makes a significant, but not essential, contribu-
tion to proteolysis in Cheddar cheese; addition of exogen-
ous plasmin accelerates proteolysis. A study of the effect
of plasmin inhibitors on the ripening of high-cooked
cheeses should be interesting.
Milk contains at least four times as much plasmino-
gen as plasmin. The indigenous plasminogen may be
activated by added plasminogen activators (there are
some indigenous plasminogen activators in milk), which
accelerate proteolysis in cheese (Barrett et al., 1999).
Dephosphorylation by acid phosphatase may be
responsible for some of the variability in the level of
phosphorylation exhibited by caseins but incomplete
phosphorylation may also be responsible. The signifi-
cance of the variability in the level of phosphorylation
in cheese quality is unknown but dephosphorylation of
casein-derived peptides in cheese may be significant. It
is claimed that alkaline phosphatase is active in Grana
Padano cheese during ripening and is responsible for
the dephosphorylation of casein phosphopeptides,
which is significant for proteolysis. Alkaline phos-
phatase is inactivated by HTST pasteurization.
Acid phosphatase survives pasteurization and since
it is concentrated in the MFGM, it is concentrated in
cheese curd. Many of the small water-soluble peptides
produced by primary proteolysis are phosphopeptides
and are partially dephosphorylated during ripening,
either by milk acid phosphatase or by bacterial phos-
phatases. Since phosphopeptides are resistant to the
action of proteinases and peptidases, dephosphoryla-
tion by phosphatase action is an important pre-requisite
for secondary proteolysis in cheese. However, object-
ive studies on the significance of phosphatase activity
in cheese ripening and quality have not been reported.
Xanthine oxidase reduces nitrate to nitrite which
is needed for anti-clostridial activity. Eventually, all
nitrate and nitrite are decomposed to N2, probably by
XO. Degradation of nitrate is important since it may react
with amino acids to form carcinogenic nitrosamines.
Sulphydryl oxidase oxidizes sulphydryl groups to
disulphides:
2RSH : R9S9S9R
Several small sulphydryl compounds, e.g., H2S,
methanethiol, dimethyl sulphide and dimethyl disul-
phide, are important for cheese flavour. Sulphydryl
oxidase oxidizes and protects the sulphydryl groups of
proteins and this may affect the redox potential (Eh) of
cheese and the stability of thiol compounds and hence
the quality and stability of cheese.
Somatic cells are an important source of enzymes,
especially proteinases, in milk. Somatic cell count
(SCC) is negatively correlated with cheese yield and
quality; an SCC  300 000 per ml is recommended.
As discussed above, the somatic cells in milk can be
removed by MF, which should, therefore, improve
cheese quality and reduce variability.
Although precise information is lacking, it is not
likely that indigenous enzymes in milk are a major
cause of variability in cheese quality; some of these
enzymes contribute to cheese ripening and may con-
tribute to the superior quality of raw milk cheese, a
possibility that warrants investigation.
Chemical composition
The chemical composition of milk, especially the con-
centrations of casein, fat, calcium and pH, has a major
influence on several aspects of cheese manufacture, espe-
cially rennet coagulability, gel strength, curd syneresis,
and hence cheese composition and cheese yield. When

seasonal milk production is practised, as in New Zealand,
Ireland and Australia, milk composition varies widely
but there is some variability even with random calving
patterns mainly due to nutritional factors.
The constituents of milk are influenced by several
factors, including species, breed, individuality, nutri-
tional status, health and stage of lactation of the pro-
ducing animal. Owing to major compositional
abnormalities, milk from cows in the very early or late
stages of lactation and those suffering from mastitis
should not be used for cheesemaking. Somatic cell
(leucocyte) count is a useful index of quality. Some
genetic polymorphs of the milk proteins improve
cheese yield and quality and there is an increasing
interest in breeding for these. The milk should be free
of chemical taints and free fatty acids, which cause off-
flavours in the cheese, and antibiotics which inhibit
bacterial cultures.
There is considerable information on the effects of
protein content, [Ca] and pH on the various renneting
parameters of milk in model systems and quite an
amount of information on their effects in cheesemak-
ing experiments. However, there is less information on
the effects on the simultaneous change in two or more
of these factors, especially in actual cheesemaking
experiments. Studies on the interactive effects of these
and other compositional factors on the cheesemaking
properties of milk and on the quality of the resulting
cheese are warranted.
It is possible to reduce, but not eliminate, the vari-
ability in the principal milk constituents by standard-
izing the concentrations of fat and casein, not just the
ratio (protein content can be standardized by adding
UF retentate), the pH (using gluconic acid--lactone)
and calcium content (by adding CaCl2).
Standardization of milk composition
Fat and casein
Different cheese varieties have a defined fat-in-dry
matter (FDM) content, in effect, a certain fat-to-protein
ratio, and this situation has legal status in the ‘Stand-
ards of Identity’ for many varieties. While the moisture
content of cheese, and hence the levels of fat and pro-
tein, is determined mainly by the manufacturing pro-
tocol (including size of curd particles, pH, cook
temperature, agitation, pressing), the fat to protein
ratio is determined mainly by the fat to casein ratio in
the cheese milk. Depending on the ratio required, it
can be modified by:
• removing some fat by gravity creaming, as practised
in the manufacture of Parmigiano Reggiano, or by
centrifugation;
• adding skim milk;
Factors that Affect the Quality of Cheese 589
• adding cream;
• adding milk powder or ultrafiltration retentate; such
additions also increase the total solids content of the
milk and hence cheese yield.
Calcium
Calcium plays a critical role in the coagulation of milk
and in the subsequent processing of the coagulum; it
is common practice to add CaCl2 (e.g., 0.01%) to
cheese milk, i.e., 40 mg Ca/l milk. This is small in
comparison with the indigenous concentration of Ca
in milk, 1200 mg/l. Addition of 40 mg/l Ca to milk
increases the concentrations of soluble, colloidal and
ionized Ca and reduces the pH of milk, all of which
have positive effects on the various renneting param-
eters (see ‘Rennet-induced Coagulation of Milk’ and
‘The Syneresis of Rennet-coagulated Curd’, Volume 1).
pH
The pH of milk is a critical factor in cheesemaking (see
‘Rennet-induced Coagulation of Milk’ and ‘The Syneresis
of Rennet-coagulated Curd’, Volume 1). The addition of
1–2% starter culture to milk reduces the pH of the milk
immediately by about 0.1 unit. Starter concentrates
(direct-to-vat starters; DVS), which are now used widely,
especially for small and medium factories, have no
immediate, direct acidifying effect.
Previously, it was standard practice to add the
starter to the cheese milk 30–60 min before rennet
addition. During this period, the starter began to grow
and produce acid, a process, referred to as ‘ripening’,
which served a number of functions:
• It allowed the starter bacteria to enter the exponen-
tial phase of growth and hence to be highly active
during cheesemaking; this is not necessary with
modern high-quality starters.
• The lower pH favoured rennet action and gel formation.
However, the practice increases the risk of bacterio-
phage infection of the starter; phage become distrib-
uted throughout liquid milk but after it has coagulated,
the phage cannot move through the coagulum and
hence can infect only those cells in the immediate
vicinity of an infected cell. This practice has been dis-
continued for most cheese varieties.
The pH of milk on reception at the dairy is higher
today than it was previously, owing to improved hygiene
during milking and the widespread use of refrigeration
at the farm and factory. In the absence of acid produc-
tion by contaminating bacteria, the pH of milk increases
slightly during storage due to the loss of CO2 to the
atmosphere. The natural pH of milk is ⬃6.7 but varies
somewhat (e.g., it increases in late lactation and during
mastitic infection).
590 Factors that Affect the Quality of Cheese
To off-set these variations in pH and to reduce it
as an alternative to ripening, the pre-acidification of milk
by 0.1–0.2 pH units is recommended, either through the
use of the acidogen, gluconic acid--lactone, or by lim-
ited growth of a lactic acid starter, followed by pasteur-
ization (referred to as pre-maturation). Pre-acidification
improves the uniformity of rennet-coagulated milk gels,
which is reflected in the production of cheese of more
uniform quality. Pre-acidification through the growth of
a starter culture, which is fairly widespread in France,
would appear to pre-dispose the system to the growth of
phage, which are not killed by pasteurization, and
undesirable bacteria.
O O
C C OH
HC OH HC OH
HO C H H2O HO C H
O phyll or TiO2).
HC OH HC OH
HC HC OH
CH2OH CH2OH
Gluconic acid
Gluconic acid-δ-lactone
In addition to variations in gross composition, there
are numerous minor differences and variations which
are not easily removed or standardized. Some of the
more significant of these are due to inter-species differ-
ences. Although the vast majority of cheese, world-
wide, is produced from cows’ milk, sheep’s and goats’
milk are very significant for cheese in southern Europe
and in the Middle East – many world-famous cheeses
are made from sheep’s milk, e.g., Manchego, Feta,
Roquefort and the Italian Pecorino varieties. Sheep’s
milk is used mainly for the production of cheese and
yoghurt. Goats’ milk or mixtures of sheep’s and goats’
milk are also used widely for cheese production (see
‘Cheeses Made from Ewes’ and Goats’ Milk’, Volume
2). Buffalo milk is used for the production of cheese in
southern Italy (Mozzarella di Buffala) and especially in
Egypt.
Bovine, ovine, caprine and buffalo milk differ from
each other in many respects: concentrations of fat, pro-
tein, many inorganic salts, enzymes, fatty acid profile,
types and proportions of caseins. These differences
cannot be changed and are reflected in the quality of
the cheese produced from these milks. The most obvious
difference arises from differences in fatty acids – ovine
and caprine milk-fat have considerably higher concen-
trations of hexanoic, octanoic and decanoic acids and
branched, medium-chain fatty acids than bovine milk-
fat and these are readily apparent as differences in the
flavour of the cheese. Ovine and caprine caseins are
considerably different from the bovine caseins. It is
likely that there are differences in the peptide and
amino acid profiles of cheese produced from bovine,
ovine, caprine or buffalo milk and that these affect the
flavour of the cheeses. A notable example of this is that
the rennet from the thistle, Cynara cardunculus, pro-
duces very satisfactory cheese from sheep’s milk, e.g.,
Sera de Estrala (see ‘Cheeses Made from Ewes’ and
Goats’ Milk’, Volume 2), but this rennet produces very
bitter cheese from cows’ milk.
Cows transfer high levels of carotenoids from their
feed to their milk or meat whereas sheep, goats and
buffalo do not. Consequently cheese, butter and other
dairy products produced from cows’ milk are much
more yellow than those made from milk of the other
species and may be unattractive to certain consumers.
The yellow colour can be destroyed by bleaching
(H2O2 or benzoyl peroxide) or masked (by chloro-
The milk of all species contains the same range of
enzymes but at different levels; the significance of
these differences is not known.
Several sapid compounds are transferred from the
animal’s feed to its milk and affect the flavour of
cheese made therefrom. There is a widely held view
that the milk of cows fed on unimproved pasture
yields better and more distinctive cheese than that
from cows fed a more homogeneous diet. Further
work in this area is warranted.
Coagulant (rennet)
The key and characteristic step in the manufacture of
rennet-coagulated cheeses is the coagulation of milk
through the limited proteolytic action of certain pro-
teinases, called rennets. Several proteinases can coagu-
late milk but only a few are suitable for cheese
production. Traditionally, rennets were extracts of the
gastric tissue of calves, kids or lambs, in which the prin-
cipal enzyme is chymosin. Owing to increased produc-
tion of cheese, concomitant with a reduced supply of
calfs’ stomachs, the supply of calf rennet has been
inadequate for many years. This led to a search for
‘rennet substitutes’, four of which are commercially
successful: bovine pepsin and proteinases from the
fungi, R. meihei, R. pusillus and C. parasitica (porcine
pepsin was used previously to a limited extent). All
successful rennet substitutes are aspartyl (acid) pro-
teinases. The gene for calf chymosin has been cloned
in several micro-organisms and the product (referred to
as fermentation-produced chymosin; FPC) is now
widely used for cheesemaking in many, but not all,
countries. The extract of the thistle, Cynara cardunculus,

is used in the manufacture of certain cheeses in Portugal
and Spain. The active enzyme is cardosin, which is an
acid proteinase (which are rare in plants). Thistle rennet
is unsuitable for cheesemaking in general.
The mechanism by which chymosin coagulates milk
is well established at the molecular level (see Fox and
McSweeney, 1997; Fox et al., 2000; Hyslop, 2003; ‘Rennet-
induced Coagulation of Milk’, Volume 1). Chymosin
specifically hydrolyses -casein, the protein responsible
for the stability of the casein micelles, at Phe1059Met106,
releasing the hydrophilic C-terminal peptide (referred
to as the glyco- or caseino-macropeptide) and destabil-
izing the micelles. All commercial rennet substitutes
hydrolyse the Phe1059Met106 bond except C. parasitica
proteinase. which hydrolyses Ser1049Phe105. The rennet-
altered micelles coagulate in the presence of Ca2 at a
temperature 20 °C (in cheesemaking, at 30–35 °C).
It has been proposed (Andreeva et al., 1992; Gustchina
et al., 1996) that chymosin normally exists in an inac-
tive conformation but is activated when the substrate
binds in the active site cleft of the enzyme. It has been
suggested that the sequence —H.P.H.P.H— (residues
98–102 of -casein) is responsible for this conform-
ational change. This sequence occurs in the -casein of
cow, goat, sheep and buffalo but not in the -casein of
the mare, camel, pig, rat or human, in which the corres-
ponding sequence is ..HPRPH.., ..RPRPR.., ..RPRPH..,
..HPINP.. and ..RPNLH.., respectively (Iametti et al.,
2001; Martin et al., 2003). Therefore, one would
expect that calf chymosin would not coagulate the milk
of the mare, camel, pig, human or rat. There have been
few studies on the coagulation of non-bovine milk by
calf chymosin. The commercial use of calf rennet in
cheesemaking from sheep, goat or buffalo milk indi-
cates that calf chymosin can hydrolyse the -casein in
these milks, as expected from the above hypothesis.
Calf chymosin can also coagulate porcine milk (Fox,
1975b); in fact, porcine milk is coagulated by calf rennet
at 4 °C whereas bovine milk is not, due to the nature
of the non-enzymatic secondary phase. Some investi-
gators have reported that camel milk is not coagulated
by calf rennet but Farah (1993) reported that it is
coagulated slowly to a weak gel.
The status of mares’ milk with respect to -casein
remained unclear until very recently. Ochirkhuyag et al.
(2000) reported that equine milk does not contain
-casein and that the micelle-stabilizing function is
played by -casein; however, Malacarne et al. (2002)
reported that it contains a low level (7%) of -casein
which has been isolated and sequenced (Egito et al.,
2001, 2002; Iametti et al., 2001). Equine -casein is
hydrolysed at Phe979Ile98 (Egito et al., 2001) (which
correspond to Phe1059Met106 of bovine -casein). How-
ever, equine milk does not appear (Fox, unpublished) to
Factors that Affect the Quality of Cheese 591
be coagulated by calf chymosin or Fromase (R. miehei
proteinase) at pH 6.6 (normal pH ⬃7.0 or higher). Stud-
ies on the coagulability of equine milk by different ren-
nets and under different conditions appears to be
warranted. We are not aware of studies on the coagula-
tion of human or rat milk by calf chymosin. Studies on
the action of chymosins from various species on the
caseins from a range of species would be interesting.
For a proteinase to be successful as a rennet substi-
tute, two characteristics are important:
• Specific hydrolysis of -casein at or close to
Phe1059Met106; if other bonds in any of the caseins
are hydrolysed, the resulting peptides may be lost in
the whey, causing a reduction in cheese yield.
• Its general proteolytic specificity during cheese
ripening must be low and similar to that of chy-
mosin (see below).
It is generally accepted that calf chymosin produces
the best quality cheese. An adequate supply of chy-
mosin from genetically engineered micro-organisms is
now available (although its use is not permitted in all
countries) and therefore rennet quality should not be a
cause of variability in cheese quality.
In the presence of Ca2, the rennet-altered micelles
in bovine milk coagulate to form a gel at a temperature
20 °C; this is referred to as the secondary phase of
rennet coagulation. Renneted bovine milk does not
coagulate ⬃18 °C, above which coagulation has a
Q10 of 16. The very high temperature dependence of
the secondary phase of coagulation has not been
explained fully. Presumably, hydrophobic interactions
are involved; perhaps the temperature-dependent
dissociation of -casein from the casein micelles is a
contributory factor. The temperature dependence of
the coagulation of rennet-altered micelles is reduced by
reducing the pH and increasing the [Ca2] or casein
concentration, e.g., by UF. As mentioned above,
porcine milk is coagulated by rennet at 4 °C. The
reason(s) for the difference between bovine and
porcine milk in this regard has not been explained. In
spite of many studies on the mechanism of coagulation
of rennet-altered casein micelles and kinetics thereof, a
generally applicable model of the phenomenon has not
been developed. The subject is comprehensively
reviewed in ‘Rennet-induced Coagulation of Milk’,
Volume 1. Further research on various aspects of the
secondary phase of rennet coagulation of, and the
effect of low temperatures on, bovine milk and that of
other species appear warranted.
Some of the added rennet is retained in cheese
curd. The amount retained varies with rennet type,
cook temperature and pH at draining; these variables
592 Factors that Affect the Quality of Cheese
should be standardized if cheese of consistent quality
is to be produced. The proportion of rennet retained in
the curd is proportional to its moisture content,
reflecting the presence of rennet mainly in the aqueous
phase of cheese. However, everything else being equal,
more chymosin than other rennets is retained in the
curd, suggesting that chymosin is adsorbed more
strongly on the caseins. The amount of chymosin and
pepsin retained in low-cooked cheeses increases
strongly as the pH of the curds-whey is reduced. In the
case of chymosin, this is due to increased adsorption,
for unknown reasons, onto the casein; for pepsin,
which is very pH-sensitive (irreversibly denatured at
pH 7), greater stability at a lower pH is also a major
factor. Surprisingly, pH has no effect on the retention
of fungal rennets, a lower proportion of which is
retained in the curd than chymosin (see Fox and
McSweeney, 1997). Obviously, cook temperature has
a major effect on the level of residual rennet in the
curd – chymosin and bovine pepsin are extensively or
totally denatured in high-cooked cheese, e.g., Parmi-
giano Reggiano or Emmental; porcine pepsin is exten-
sively denatured even in low-cooked cheeses due to its
sensitivity to pH  6.5. Low-cooked, low-pH, high-
moisture cheese, e.g., Camembert, retains ⬃30% of the
added chymosin activity; Cheddar retains ⬃6% and
Emmental ⬃0%.
Everything else being equal, increased retention of
the coagulant in cheese curd results in greater initial
hydrolysis of s1-casein; however, the significance of this
variable on the flavour and texture of cheese has not
been studied thoroughly. It has been suggested that the
activity of chymosin in cheese curd is the limiting factor
in cheese ripening; however, excessive rennet activity
leads to bitterness. Proteolysis in cheese during ripening
is discussed later; there have been relatively few studies
on the significance of chymosin activity to cheese qual-
ity, an aspect which appears to warrant further research.
Considering the importance of proteolysis in the
ripening and quality of cheese and the significance of
the coagulant thereto, studies on various factors that
affect the retention of the coagulant in cheese curd
appear warranted, e.g.,
• the adsorption of chymosin on casein micelles and
the apparent lack of adsorption of fungal proteinases;
• stability of various rennets under various conditions
of temperature, pH and other factors.
Starter
The second key reaction in cheesemaking is acidifica-
tion – the pH of all rennet-coagulated cheeses should
decrease to a value in the range 4.6–5.2 within a few
days of manufacture, or in some varieties, at the end of
curd manufacture (5–6 h). Acidification at the appro-
priate rate and time is an essential and characteristic
feature of cheesemaking – it is, in fact, a sine qua non.
Among the important consequences of acidification are:
• activity of the coagulant;
• survival and retention of coagulant in the curd;
• firmness of the coagulum, which affects the loss of
fat and protein in the whey on cutting and hence
reduces the yield of cheese;
• syneresis of the curds and hence the composition of
the cheese;
• solubilization of colloidal calcium phosphate (CCP),
which has a major effect on the texture, meltability
and stretchability of the cheese;
• inhibition of the growth of undesirable bacteria,
most importantly pathogenic and food poisoning
bacteria;
• the activity of various enzymes in the cheese during
ripening and consequently the rate of ripening and
the quality of the cheese.
Originally, acidification was due to the production
of lactic acid from lactose by adventitious LAB. Acidi-
fication of some cheese varieties still depends on the
activity of the adventitious microflora but most
cheeses now are acidified using selected LAB added to
the cheesemilk as a culture (starter). The idea of
using starter cultures was introduced in ⬃1870 in
Denmark.
The cultures used today in cheesemaking can be
divided into two groups:
1. Mesophilic – with an optimum growth temperature
of ⬃28 °C;
2. Thermophilic – which grow optimally at ⬃42 °C.
Mesophilic cultures are used for cheese curds which
are cooked at a temperature 40 °C while cheeses in
which thermophilic cultures are used are cooked at
50–55 °C. Mesophilic cultures contain strains of Lacto-
coccus lactis subsp. lactis and/or Lc. lactis subsp. cremoris.
Starters used for some cheeses, e.g., Gouda, Edam,
Danbo, also include citrate-utilising strains of Lc. lactis
subsp. lactis and/or Leuconostoc subsp., the principal
function of which is the production of CO2 and certain
flavour compounds. Thermophilic cultures contain
species of thermophilic lactobacilli, e.g., Lb. helveticus
and Lb. delbreuckii subsp. bulgaricus or Lb. delbreuckii
subsp. lactis, alone or with Streptococcus thermophilus.
It is possible to simulate the acid-producing func-
tion of the starter LAB by using acid or acidogen (usu-
ally GDL). Fresh acid-curd cheese (e.g., Cottage,

Quarg, Cream) of satisfactory quality may be produced
by direct acidification and some is produced commer-
cially. Some rennet-coagulated cheeses are also pro-
duced by direct acidification, usually when the flavour
is very mild or masked by other components or is less
important than physico-chemical functional proper-
ties; examples include Feta-type and Mozzarella-type
cheeses. Chemically acidified Mozzarella has better,
more consistent and stable functionality than the bio-
logically acidified product. However, most ripened
rennet-coagulated, chemically acidified cheese does
not develop a flavour typical of the variety. Possible
explanations for this situation are:
• the high redox potential (Eh) of chemically acidified
cheese (c. 150 mV compared with c. 450 mV for
biologically acidified cheese);
• a high concentration of lactose which may result in
a high count of NSLAB.
Both mesophilic and thermophilic cultures may by
mixed-strain or defined-strain. Mixed-strain cultures
contain an unknown number of strains of the same or
different species. Several such systems were in use
20–50 years ago – the cultures were selected based
on cheesemaking characteristics and various propaga-
tion procedures were used. These cultures were cap-
able of producing very good quality cheese but they
were susceptible to phage infection because many of
the strains in the culture were sensitive to the same
phage. The maintenance and propogation of mixed-
strain cultures is laborious and not very reproducible.
With the objective of improving resistance to phage,
work commenced in New Zealand in the 1930s on the
development of defined-strain cultures. The principal
criterion for selecting strains for these cultures was
phage unrelatedness, i.e., phage which infects one strain
does not affect other strains in the culture; while a phage
infection may kill off one strain, the other strains grow
normally. Other essential criteria are acid-producing
ability and strain compatibility; the ability to produce
good quality cheese was assessed from experience and
undesirable strains were removed from the culture. Ori-
ginally, blends of 5–6 strains were usually used but blends
of 2–3 strains are more common today. The defined-
strain approach was introduced initially for Cheddar
cheese and is now very widely used in New Zealand,
Australia, Ireland and the USA. Although a different
approach was used in the Netherlands to select defined-
strain cultures for Gouda cheese, the essential outcome
was similar. Defined-strain thermophilic cultures are
now used also but less widely than mesophilic cultures.
The use of defined-strain, phage-unrelated cultures
has greatly reduced the risk of phage infection but to
Factors that Affect the Quality of Cheese 593
reduce the risk even further, various techniques have
been introduced to improve starter activity and ensure
cheese quality. For discussions on starter technology,
see Cogan and Hill (1993), Cogan and Accolas (1996)
and ‘Starter Cultures: General Aspects’, Volume 1.
There have been very considerable advances in the
genetics of lactococci during the last 10 years and the
complete sequence of the genome is known (see ‘Starter
Cultures: General Aspects’ and ‘Starter Cultures: Genet-
ics’, Volume 1). The genes for many of the important
cheesemaking characteristics of lactococci, e.g., lactose
metabolism, proteolysis and phage resistance, are car-
ried on plasmids and hence are easily manipulated.
Many genetically engineered strains of Lactococcus
have been constructed but are not used in practice.
However, lactococci can be genetically modified by nat-
ural mating (conjugation) and such genetically modi-
fied strains are being used commercially. The principal
limitation with engineering superior starters is the lack
of knowledge on the key enzymes in cheese ripening.
The availability of the complete chromosome
sequence of Lc. lactis (see ‘Starter Cultures: General
Aspects’ and ‘Starter Cultures: Genetics’, Volume 1)
opens up new avenues for research on cheese starter
cultures. For example, five potential or rudimentary
prophages were identified, suggesting that starter cul-
tures are the ultimate source of phage. Examination of
the chromosome for putitive proteinases, peptidases
and lipases, especially those with unique activities,
should be useful. Since 1999, the chromosome of 20
other LAB and other cheese-related bacteria have been
or are being sequenced. These include other strains of
Lactococcus, Lb. delbruckii, Lb. helveticus, Sc. thermophilus
and B. linens. Comparative genomics of these different
bacteria should be useful in delineating the differences
that occur between them. However, the gene
sequences are of little value unless the protein prod-
ucts (enzymes) are produced. Identifying how to turn
on those genes which encode enzymes with potential
cheese ripening properties, but which are normally not
expressed, could be rewarding in studies on flavour
development in cheese.
Defined-strain cultures give very reproducible
results in terms of acid production and overall cheese
quality. However, the flavour of the cheese is consid-
ered to be rather bland, probably because of a lack of
microbial diversity, both in the starter and also in the
modern milk supply, which, if pasteurized, is essen-
tially sterile. Attempts to overcome the lack of flavour
will be discussed later.
Traditionally, cheese starters were produced at the
cheese factory from mother-cultures obtained from a
culture supplier; this is still the usual practice in larger
factories. However, the proper maintenance of starters is
594 Factors that Affect the Quality of Cheese
technically demanding and expensive. Consequently,
starter concentrates, referred to as direct-to-vat starter
(DVS) or direct vat inoculum (DVI), produced by culture
suppliers have become quite widespread among small
to medium cheese factories or as a back-up starter system
for larger manufacturers.
Another starter system warrants mention, i.e., arti-
sanal or natural cultures. These cultures are produced
in-house by the cheesemaker, who incubates some warm
whey under conditions that select bacteria with desirable
cheesemaking characteristics. Today, such cultures are
usually used for high-cooked cheeses – hot whey, per-
haps at ⬃55 °C, is transferred to insulated containers in
which it cools slowly; these conditions are selective for
thermophilic bacteria and by the time the whey has
cooled sufficiently to allow mesophilic bacteria to grow,
the pH has become inhibitory. These cultures are very
complex and their composition is not known, certainly
at the strain level, and probably not at the species level.
Although the primary function of the starter culture
is to produce acid at the appropriate rate and time, the
starter bacteria or their enzymes also play an essential
role during cheese ripening – a typical and desirable
flavour does not develop in starter-free cheese and
many flavour defects, e.g., bitterness and fruitiness, are
related to characteristics of the starter. The microbiol-
ogy and biochemistry of cheese ripening will be dis-
cussed in a later section.
In view of the significance of the starter LAB, both
for the acidification and ripening of cheese curd, it is
not surprising that the starter LAB have been the sub-
ject of extensive research since the very early days of
microbiology and consequently, LAB are now very well
characterized at the cellular, molecular and genetic
Cultures: genetics levels (see ‘Starter Cultures: General
Aspects’, ‘Starter Cultures: Genetics’ and ‘Starter Cultures:
Bacteriophage’, Volume 1).
Research on these very industrially important bac-
teria will very probably continue for the immediate
future. Among the areas likely to attract attention are:
• Characterization of Lactobacillus and Streptococcus,
which at present are less well characterized than
Lactococcus;
• Further work on the protection of LAB against
phage;
• Selection and improvement of starter strains,
through genetic engineering techniques with
respect to acid production and especially ripening
attributes;
• Selection of LAB strains with probiotic properties
for cheese production;
• The existing trend towards DVI starters will probably
continue; success will depend on economic factors;
• It is likely that the lack of diversity caused by the
use of such highly defined starter systems will be
offset by the use of adjunct starters, mainly Lacto-
bacillus spp., but possibly also Streptococcus spp., and
perhaps Enterococcus, for some varieties.
Post-Coagulation Operations
A rennet-coagulated milk gel is quite stable if left undis-
turbed but if cut or broken, it synereses strongly, thereby
creating the possibility of removing water and concen-
trating fat and protein. When the coagulum has reached
the desired degree of firmness, usually 30–60 min after
the addition of rennet, the gel is ready for further pro-
cessing. The firmness of the gel at cutting should be
optimized so as to reduce the loss of fat and protein from
the curd particles into the whey. If the coagulum is too
soft, extensive shattering will occur with high losses of
fat and protein in the whey. If the coagulum is too firm,
the coagulum may be difficult to cut using the usual
equipment; it may run before the cutting knives and
shattering may occur. Excessively firm curd is particu-
larly problematic when using UF retentate. A uniform
gel firmness at cutting also results in curd particles of
more uniform size, leading to a cheese curd of more uni-
form composition and ultimately in cheese of more
uniform quality.
Traditionally, the point at which the gel was consid-
ered to be ready for cutting was determined subject-
ively by the cheese-maker but several devices are now
available which permit objective assessment of gel
firmness (see Fox et al., 2000; ‘General Aspects of
Cheese Technology’, Volume 2). Some of these devices
can be used to activate the cutting knives.
The size of the curd particles affects the extent of
syneresis – the coagulum for low-moisture cheeses is
cut into small pieces while the gel for high-moisture
varieties is cut into large pieces or is not cut at all but
is scooped directly into moulds. Fat globules are lost
from the cut surfaces; hence, finely cutting the coagu-
lum increases the fat loss. If it were possible to achieve
the same degree of syneresis by other means, it may be
possible to increase cheese yield by using a larger cut.
Contrary to what one might first think, the fat lost
from the cut surfaces is mainly in large globules
because large globules are more exposed on the cut
surface on cutting. Would a very low degree of
homogenization improve fat retention?
The curd can be sedimented from a rennet-induced
milk gel by a low gravitational force. Would it be tech-
nically feasible to coagulate milk by some form of cen-
trifuge and to prepare curd of the desired moisture
content by centrifuging the coagulum at an appropri-
ate centrifugal force? It might be possible to eliminate

cooking of the curds and all variability associated with
syneresis of the curds.
Once the gel has been cut, the pieces begin to
synerese and expel whey. In addition to the size of the
curd pieces, the rate and extent of syneresis are
affected by several factors through which the cheese-
maker can control the composition of cheese. The
syneresis of rennet-induced milk gels is discussed in
‘The Syneresis of Rennet-coagulated Curd’, Volume 1.
As discussed below, the composition of cheese has a
major influence on the microbiology and biochemistry
of cheese ripening and ultimately on the quality of the
cheese. Syneresis is promoted by:
• increasing the temperature (cooking);
• reducing the pH;
• vigorous stirring during cooking;
• removing some or most of the whey and continuing
to stir.
The relative importance of these factors depends on
the variety – syneresis of some varieties depends
mainly on cook temperature, e.g., Emmental, Parmigiano
Reggiano and Cheddar, while others depend mainly on
pH, e.g., Camembert. The cook temperature ranges
from ⬃30 °C (no cooking) to 55 °C; the extent of
syneresis is related directly to the cook temperature.
For reproducible cheese composition and quality, it is
critical to control the rate and extent of syneresis. The
cook temperature, pH, curd particle size and the
extent of agitation are characteristic of the variety and
were probably introduced/applied emperically long
ago by artisanal cheesemakers. It seems reasonable to
suggest that the manufacture of many varieties could
be improved by changing the combination of the
above parameters; research in this area appears to be
warranted.
The composition of the curd affects syneresis and
this is a major reason for standardizing the composi-
tion of the cheese milk. The most important composi-
tional factors are:
• Fat, which has a negative effect, i.e., a high fat con-
tent causes poor syneresis, because fat is essentially
an inert filler in the gel.
• Protein, increasing the protein content, up to a cer-
tain value, promotes syneresis, but if the protein
content is too high, the gel is too firm and synereses
poorly.
• Calcium, like other aspects of renneting, the extent
of syneresis is directly affected by the concentration
of calcium.
• pH, syneresis is promoted by decreasing pH.
The syneresis of rennet-induced milk gels has not
yet been fully described at the molecular level. While
Factors that Affect the Quality of Cheese 595
the effects of processing parameters (cutting, cooking,
stirring, etc.) on syneresis are fairly well described,
the effects of compositional factors, such as amount
and type of casein, genetic polymorphs, casein micelle
structure and size, effect of plasmin and other
proteinases, fat globule size and integrity, milk salts,
pre-treatments of cheese milk (e.g., heat treatment,
high pressure treatment), have not been studied in
detail. The deficiency is explained partly by a lack of
good and appropriate analytical methods for the meas-
urement of syneresis (see ‘The Syneresis of Rennet-
coagulated Curd’, Volume 1).
When the moisture content (as assessed subject-
ively by the cheesemaker based on evaluation of the
texture of the curd) and the pH of the curd have
reached desired values, the curds are separated from
the whey and subjected to one of several treatments
designed to regulate the composition, and in some
cases the texture, of the curd. These include:
• washing of the curd to reduce its lactose content
(and thereby control its final pH) and perhaps to
increase its moisture content, which occurs on wash-
ing with cold water, e.g., Monterey Jack, washed-
curd Cheddar, low-fat cheeses;
• replacing some of the whey by warm water, e.g.,
Gouda; this practice was probably used initially as a
means of cooking the curds on farms lacking jacketed
cheese vats and the ability to generate steam, but it
has become a standard method for reducing the lac-
tose content and acidity of the curd for some varieties;
• cheddaring the curd, e.g., Cheddar, Mozzarella (Pizza
cheese), which affects the texture of the curd but its
main effect is probably to allow the pH of the curds to
fall and the CCP to dissolve, thereby affecting texture;
• kneading and stretching, as used for pasta-filata
varieties, mainly to give the cheese a fibrous texture
which affects the meltability and stretchability, i.e.,
the functionality, of the cheese;
• moulding, applied to all varieties to give a character-
istic shape and size, which are not simply cosmetic
but are significant for the ripening of the cheese,
e.g., if smear-ripened cheeses are too large, the sur-
face will become over-ripe while the core is still
immature; on the other hand, Swiss-type cheeses
must be large so as to retain a substantial portion of
the CO2 necessary for eye development; some
cheeses have a characteristic open texture, with
many mechanical openings, e.g., Samsoe, Havarti
and blue varieties; in the latter, the openings are
necessary for good mould development throughout
the cheese;
• pressing, applied to semi-hard and hard cheeses with
the objective of removing some moisture but mainly
596 Factors that Affect the Quality of Cheese
to consolidate the cheese mass which is important for
texture and the retention of gas for eye development;
• salting, which is discussed further below.
In large-scale factories, all the above operations are
mechanized and/or automated which improves the con-
sistency of the product if executed properly; however, it
is not possible for the cheesemaker to make ad hoc
adjustments if the process is not progressing as planned.
Very large cheese vats (e.g., 30 000 l capacity) are
used in large modern factories producing Cheddar,
Gouda or Mozzarella, and possibly other varieties. The
use of such large vats eliminates certain causes of vari-
ation in cheese but introduces others. About 30 min is
required to empty these large vats and separate the
curds from the whey; as a result, the curds at the start
of draining differ from those at the end in several
respects, e.g., moisture content (due to extra synere-
sis), pH and calcium content. These differences are
probably reflected in the quality of the cheese but
definitive studies have not been reported.
Salting
Salting, one of the classical methods for food preserva-
tion, operates by reducing the water activity, aw, of the
product. Most, probably all, cheeses are salted by one
of four methods:
• mixing dry salt with milled or chipped curd, e.g.,
for Cheddar-type cheese;
• brine salting of the moulded/pressed cheese; NaCl
diffuses into the cheese in response to the difference
in osmotic pressure between the brine and the aque-
ous phase of the cheese;
• surface application of dry salt to the surface of
pressed cheese, e.g., Blue cheeses;
• salting of cheese milk – for a few varieties, e.g.,
Domiati, a substantial amount of salt is added to the
milk before renneting, traditionally, to control the
microflora of the milk.
There is a substantial literature on the technology,
physics and significance of salting, which has been
reviewed by Guinee and Fox (1993), Fox et al. (2000)
and in ‘Salt in Cheese: Physical, Chemical and Biolog-
ical Aspects’, Volume 1. The principal effects of salt in
cheese are:
• A major inhibitory and selective effect on the
microflora.
• A significant effect on the activity of many enzymes.
• Through its effects on the microflora and enzymes,
salt has a major indirect effect on the ripening,
flavour and quality of cheese.
• A direct effect on flavour.
• An excessive dietary intake of NaCl has several undesir-
able effects, e.g., hypertension and osteoporosis.
Although cheese makes a relatively small contribution
to dietary NaCl intake, there is an economic incentive
to reduce the NaCl content of cheese, which may
adversely affect its quality, or partially replace it by KCl.
The significance of salt, and especially the uniform-
ity of salt concentration, on the quality of cheese is
well recognized. The physics of salt diffusion in
cheese, the effect on various micro-organisms and the
technology of salting are well known. Thus, it should
be possible to achieve a very reproducible level of salt
in cheese. However, this is not always achieved in
practice and variations in salt concentration are prob-
ably a significant but avoidable cause of variations in
cheese quality.
Use of UF in Cheese Production
Ultrafiltration (UF) has many applications in cheese
technology, as discussed in ‘Application of Membrane
Separation Technology to Cheese Production’, Volume 1.
Some problems with cheese quality persist but the
potential for UF in cheese technology is great and
research in its application will continue.
Ripening
Fresh rennet-coagulated cheese curd is suitable for
consumption and a little is consumed, e.g., Burgos
cheese, but most is ripened (matured) for a period
ranging from ⬃3 weeks (e.g., Mozzarella/Pizza cheese)
to 2 or more years (e.g., Parmigiano-Reggiano, extra-
mature Cheddar). During ripening, the characteristic
flavour, texture, appearance and functionality develop
along lines pre-determined by the microbiology and
composition of the curd, as established during the
manufacturing stage. However, the cheesemaker can
influence the rate and, to some extent, the pattern of
ripening by controlling the temperature and, for some
varieties, the humidity of the environment. Many
cheeses develop a characteristic microflora (bacteria,
yeasts, moulds) during ripening and this microflora has
a major effect on the sensory qualities of the cheese.
Traditionally, this secondary microflora was adven-
titious, acquired from the milk and/or environment,
and the growth of certain, desirable, contaminating
micro-organisms was promoted by selecting certain
environmental conditions such as pH, temperature,
humidity, oxygen concentration, salt concentration
and moisture level. However, the adventitious microflora
was likely to be variable, leading to inconsistencies in

cheese quality. In modern cheese technology, the
adventitious microflora is replaced by selected second-
ary cultures, although adventitious micro-organisms
may still grow, and even dominate in some cases. The
principal secondary micro-organisms are:
• Mesophilic lactobacilli, probably in all varieties but
especially in internal bacterially ripened varieties,
e.g., Cheddar and Gouda. Traditionally, these NSLAB
were adventitious, probably variable and uncon-
trolled; today, it is becoming increasingly common to
inoculate cheese milk with selected NSLAB (see
below and ‘Secondary and Adjunct Cultures’ and
‘The Microbiology of Cheese Ripening’, Volume 1).
• Propionic acid bacteria, characteristic of Swiss-type
cheese (see ‘Metabolism of Residual Lactose and of
Lactate and Citrate’, Volume 1 and ‘Cheese With Pro-
pionic Acid Fermentation’, Volume 2).
• Penicillium camemberti and P. roqueforti in surface-
mould and blue-mould varieties, respectively. The
inoculation of some mould-ripened cheeses with
mould spores is adventitious but increasingly, the
cheeses are inoculated with selected strains. The
metabolic activity of the mould dominates the ripen-
ing, and hence the quality, of these cheeses (see ‘Sur-
face Mould-ripened Cheeses’ and ‘Blue Cheese’,
Volume 2). Therefore, ensuring the optimum growth
of the mould is paramount.
• Coryneform bacteria, e.g., Brevibacterium, Arthrobac-
ter and Corynebacterium spp. are the characteristic
microflora of surface smear-ripened cheeses and are
responsible for their characteristic appearance,
aroma and taste. Traditionally, surface smear-ripened
cheeses acquired their secondary microflora from the
environment and from older cheeses via smearing.
However, for hygienic and consistency reasons, it is
becoming increasingly common to inoculate the sur-
face of the cheeses with selected coryneform bacteria
(see ‘Secondary and Adjunct Cultures’, Volume 1 and
‘Bacterial Surface-ripened Cheeses’, Volume 2).
Several species of yeast, e.g., Debaryomycos hansenii
and Yarrowia lipolytica, have been isolated from cheese.
These yeasts are adventitious contaminants on many
varieties; since they are aerobic and acid-tolerant, they
grow mainly on the surface of all cheeses but their
growth on many varieties is prevented through pack-
aging or rind formation. The growth of yeasts is essential
on surface smear-ripened cheeses because they catabo-
lize lactic acid, increase the pH of the curd and enable
the corynebacteria, which cannot grow at pH 5.8, to
grow. Apart from their significance in the deacidifica-
tion of smear-ripened cheese, their precise contribution
to ripening has not been quantified. However, since
yeasts are metabolically active, it is likely that their
Factors that Affect the Quality of Cheese 597
contribution is considerable. With the objective of
improving the consistency of cheeses in which they are
a significant part of the microflora, the inoculation of
such cheeses with selected strains of yeasts is becoming
increasingly common. Geotricum candidum is part of
the adventitious surface microflora of many cheeses.
A further variable through which the cheesemaker
can influence the pattern of ripening and the quality of
the final cheese is by preventing the loss of moisture
from the cheese surface by appropriate packaging (rind-
less cheese) or by controlling its loss to form a rind.
Scientific work on the significance of cheese packag-
ing on cheese quality is lacking. The main focus has
been on the prevention of mould growth on the surface
and the loss of cheese yield. Undoubtedly, the changing
composition of cheese (through evaporation of mois-
ture) and the loss of gases and probably other volatile
compounds affect cheese microflora and enzyme activity
and consequently cheese quality. Although the techno-
logical advantages accruing from the packaging of cheese
are great and perhaps cannot be off-set by other factors, a
scientific comparison of various aspects of rindless and
rinded cheese, e.g., Cheddar, may be interesting.
Cheese ripening involves a very complex set of bio-
logical, biochemical and chemical reactions which can
be classified into four groups:
• Glycolysis and the catabolism of lactic and citric acids.
• Lipolysis and the catabolism and modification of
fatty acids.
• Proteolysis and catabolism of amino acids.
• Interactions between the products of the previous
reactions.
These reactions are catalysed by living micro-organ-
isms or enzymes derived from four or five sources:
• Milk
• Coagulant
• Primary starter
• Secondary starter (for most varieties)
• Adventitious microflora
The biochemistry of cheese ripening has been
studied quite intensely in recent years and an extensive
literature has accumulated, which has been the subject
of several reviews, including Fox and McSweeney
(1997), Fox et al. (1996a, 2000), Fox and Wallace
(1997), and ‘Biochemistry of Cheese Ripening: Intro-
duction and Overview’, ‘Metabolism of Residual Lac-
tose and of Lactate and Citrate’, ‘Lipolysis and
Catabolism of Fatty Acids in Cheese’, ‘Proteolysis in
Cheese during Ripening’, ‘Catabolism of Amino Acids
in Cheese During Ripening’, Volume 1. The principal
features of glycolysis, lipolysis and proteolysis are sum-
marized in Figs 3, 4 and 5, respectively. The principal
598 Factors that Affect the Quality of Cheese

Lactose

Sc. thermophilus

Glu. + Gal
Lc. lactis
Lc. cremoris
Lactobacillus

Sc. thermophilus

D,L-Lactate

Swiss Propionate,
L-Lactate
Propionibacterium acetate, CO2

Mould and
Surface smear
Cheddar
Dutch
CO2, H2O
NSLAB
D,L-Lactate Acetate
Pediococcus
(lactobacilli)

Figure 3 Summary of lactose metabolism in cheese.

products of the various reactions have been character- Triglycerides

ized and include numerous large, medium and small
peptides, amino acids, amines, ammonia, fatty acids
and partial glycerides, other organic acids, aldehydes
and ketones, thiol compounds, CO2, H2, hydrocarbons,
pyrazines and furanones. Several hundred sapid and
aromatic compounds have been isolated from sev-
eral cheese varieties and identified (see ‘Sensory Char-
acter of Cheese and its Evaluation’ and ‘Instrumental
Techniques’, Volume 1). The spectrum of these com-
pounds is generally similar between the varieties that
have been studied in detail but the varieties differ with
respect to their concentration and proportions.
There are four aspects to the quality of cheese; the
relative importance of these depends on the variety
and application of the cheese:
• Appearance
• Flavour (aroma and taste)
• Texture
• Functionality
The appearance of cheese includes such features as:
• Depth and uniformity of colour
• Presence or absence of mechanical opening or eyes
due to gas production
• Presence or absence of mould
δ-Keto acid
n-Fatty acids δ-Hydroxyacids
CH3SH
Alcohols
Lactones
Methyl ketones
Thioesters
Esters
Figure 4 Summary of lipolysis in cheese.
Usually, the appearance of cheese is the only attri-
bute by which the purchaser can assess the quality of
cheese and hence is of the utmost importance. Today, it
is unlikely that cheese produced by large manufacturers
and sold through reputable outlets will be defective in
appearance although the appearance of artisanal
cheese may vary.
For cheese consumed as a table cheese, flavour is
probably its most important quality attribute
although flavour and texture are strongly interactive.
The flavour of cheese is due to a subtle balance
between several hundred compounds. It has been the
subject of research since the beginning of the twenti-
eth century, especially since the development of gas
chromatography (GC) in the 1950s and the interfacing

Rennet,
Rennet Plasmin
Casein Ca-para-casein
Deaminases
Carbonyls
es
Esters Acids
as
in
m
sa
Alcohols
an
α-Keto acid
Tr
Amino acid
α-Keto acid
Figure 5 Summary of proteolysis and amino acid catabolism in cheese.
of GC with mass spectrometry (MS). Considerable
progress has been made on the characterization of
cheese flavour by instrumental methods (see ‘Instru-
mental Techniques’, Volume 1). However, commer-
cially, cheese quality is usually assessed by subjective
sensory evaluation (see ‘Sensory Character of Cheese
and its Evaluation’, Volume 1).
It is still not possible to describe completely the
flavour of cheese, especially more highly flavoured
varieties. More research is needed in this area and it is
very likely that it will continue. An objective method
for grading cheese would be very useful. Analysis of
cheese volatiles by GC–MS is probably the best
approach at present but the present instruments are
not capable of handling large numbers of samples and
are too expensive. The electronic nose appears promis-
ing but considerably more work is required.
The texture of cheese is important, both directly
and indirectly. It is important directly because such
important functional attributes as sliceability, grate-
ability, crumbliness and eye development are, in fact,
related to texture. To the consumer, texture is an
indirect index of cheese flavour and general quality.
In spite of its undeniable importance, the texture of
cheese has received much less research attention than
cheese flavour although as described in ‘Rheology
and Texture of Cheese’, Volume 1, the texture of
cheese can be described quite accurately by certain
rheological parameters. The influence of various
compositional parameters and the changes that occur
during ripening have been described in rheological
terms. It should be possible to develop some of the
present rheometers based on the principle of com-
pression, penetration or cutting to take whole cheese
Factors that Affect the Quality of Cheese 599
Large peptides
Lactococcal CEP
Oligopeptidases
Aminopeptidases
Small peptides
Aminopeptidases
Dipeptidases
Amino acids
C C and C S
Lyases
CO2 Decarboxylase
Amines Various products,
including sulphur
compounds
samples and hence could be used in a factory envir-
onment – it would appear that research in this area is
warranted. Further work on the molecular basis of
cheese texture and rheological properties is also
required.
All cheeses are expected to exhibit certain physico-
chemical or functional properties when cold, e.g.,
sliceability or crumbliness or grateability or when
heated, e.g., meltability or stretchability. Most cheese
(⬃70% in the USA) is used as an ingredient in other
foods, either domestically or in a factory context. The
flexibility and ease of use of cheese as a food compon-
ent or ingredient are among its main attributes and are
discussed in detail in ‘Cheese as an Ingredient’, Vol-
ume 2. Being able to provide the user, especially the
industrial user, of cheese with a product with the cor-
rect functionality is a challenge to the cheese manu-
facturer. Considerable progress has been made but
further work is required.
The quality of cheese is determined in the first
instance by the composition of the curd both directly
and indirectly by its influence on the various ripening
agents. The significance of principal ripening agents is
described in the following sections.
Indigenous Enzymes
Milk contains 60 indigenous enzymes (see Fox et al.,
2003). Some of these enzymes, including LPL, acid
phosphatase, alkaline phosphatase and XO, are located
on the fat globule membrane, some, including plas-
min, are adsorbed on the casein micelles while others
are in the serum (whey) phase. Since the fat globules
and casein micelles are concentrated in the cheese
600 Factors that Affect the Quality of Cheese
curd, cheese is enriched with many enzymes. Several
enzymes in milk are quite heat stable and survive HTST
pasteurization. The most significant of the indigenous
enzymes as far as cheese ripening is concerned are:
• Plasmin, which fully survives HTST pasteurization
(in fact, its activity is increased due to inactivation
of inhibitors of plasmin and plasminogen activators)
and makes a significant contribution to primary
proteolysis, especially in high-cooked cheeses in
which the coagulant is extensively or totally inacti-
vated; it is mainly responsible for the hydrolysis of
-casein in most cheese varieties. Although the level
of plasmin activity in milk is variable, it is unlikely
that this variability causes significant variations in
cheese quality although it may affect cheese yield
and composition (through retarded syneresis) and
functionality which is strongly influenced by the
integrity of the casein network.
• Cathepsin D, an acid lysozomal proteinase with a
specificity similar to chymosin, occurs mainly in the
serum phase of milk and therefore most of it is lost
in the whey; furthermore, it is relatively heat labile.
Therefore, there is probably little cathepsin D activity
in cheese and, in any case, it is probably overshad-
owed by the much greater activity of chymosin.
• Lipoprotein lipase, which is extensively or totally
inactivated by HTST pasteurization, has little or no
impact on the quality of cheese made from pas-
teurized milk although it probably contributes to
lipolysis in raw-milk cheese; it is associated with the
casein micelles and is incorporated into cheese curd.
• Acid phosphatase is probably active in cheese but its
significance has not been established. Many of the
small peptides produced from casein by chymosin or
plasmin are phosphopeptides and are partially
dephosphorylated during ripening, indicating the
action of an acid phosphatase. Work is needed to
establish the contributions and significance of the
indigenous and bacterial acid phosphatases in cheeses.
One of the important nutritional features of cheese is
its anticariogenic property due to the Ca-binding
properties of the caseins and casein phosphopeptides
(see ‘Nutritional Aspects of Cheese’, Volume 1). In this
regard, the activity of acid phosphatase may be nega-
tive but dephosphorylation is necessary to enable the
further degradation of phosphopeptides which may be
important for flavour and texture development.
• Xanthine oxidase reduces nitrate to nitrite, which is
the active agent against clostridia and coliforms in
cheese and contributes to the eventual disappearance
of NO3 and NO2 : this is important from a toxico-
logical viewpoint since nitrate may lead to the for-
mation of nitrosamines.
It is possible that other indigenous milk enzymes
are active in cheese during ripening and affect its qual-
ity; their activity may contribute to the differences in
flavour between raw and pasteurized milk cheese but
definitive studies have not been reported. Research on
the significance of indigenous milk enzymes in cheese
quality is warranted.
Coagulant
The coagulant is mainly responsible for primary prote-
olysis in low-cooked cheese and for the desirable tex-
tural changes during the early stages of ripening. The
peptides normally produced by rennet are too large to
affect flavour but they serve as substrates for microbial
proteinases and peptidases which produce small pep-
tides and amino acids which contribute to background
flavour. Amino acids serve as substrates for various
catabolic reactions, the products of which (amines,
NH3, acids, carbonyls, sulphur compounds) are major
sapid compounds in cheese (see ‘Proteolysis in Cheese
during Ripening’ and ‘Catabolism of Amino Acids in
Cheese During Ripening’, Volume 1). However, exces-
sive rennet action or incorrect specificity may lead to
bitterness. The C-terminal region of -casein is very
hydrophobic and peptides released from this region,
which contains the primary chymosin cleavage sites
on -casein, are very bitter. The hydrolysis of -casein
by chymosin is inhibited by NaCl and it is not nor-
mally hydrolysed in Cheddar but is hydrolysed to
some extent in surface-salted cheese, in which the
concentration of NaCl is sub-inhibitory for a consider-
able period.
Chymosin is generally regarded as the best rennet;
since there is now an unlimited supply of fermentation-
produced chymosin, there is no excuse for rennet-
related problems in cheese. The natural function of
chymosin is to coagulate milk in the stomach of the
neonate, delay its discharge into the small intestine
and thereby increase the efficiency of digestion. Chy-
mosin is the most efficient milk coagulant known but
it was not intended for cheesemaking although it is
the best for this task also. However, it is likely that
through genetic engineering, chymosin could be
modified to improve its cheese-ripening properties,
i.e., to increase its action on certain peptide bonds to
yield desirable peptides or reduce it on other bonds to
avoid defects such as bitterness. Some mutant chy-
mosins have been produced (see ‘Rennets: General and
Molecular Aspects’, Volume 1) but we are not aware of
their use in cheesemaking trials.
C. parasitica proteinase is much more active on
-casein in cheese than chymosin, pepsins or Rhizomucor
proteinases but it does not cause bitterness. Probably, it

preferentially hydrolyses in the N-terminal region of
-casein, which is hydrophilic. Characterization of the
specificity of C. parasitica proteinase on caseins in cheese
and in model systems is warranted. Perhaps a combin-
ation of chymosin and C. parasitica proteinase might
produce cheese with interesting characteristics.
The texture and functionality of cheese are affected
strongly by even a low level of proteolysis, e.g., the
stretchability of biologically acidified Mozzarella
deteriorates after ⬃2 weeks at 4 °C due to proteolysis.
Thus, it is important to control the level and activity of
rennet in cheese. As discussed earlier, the amount of
rennet retained in cheese curd is affected by the mois-
ture content of the cheese, the pH and temperature of
cooking; activity during ripening is affected by pH,
moisture, S/M and temperature.
Starter
In addition to its essential role in the production of
acid in the manufacture of cheese curd, the starter LAB
also play a key role in cheese ripening. Experiments
with chemically acidified Cheddar and Gouda cheese
have shown that the starter is essential for normal
flavour development. Even inoculation of chemically
acidified cheese with NSLAB, which reached 108 cfu/g,
did not produce good-flavoured cheese (Lynch et al.,
1997). The starter LAB reduce the redox potential (Eh)
of cheese curd to about 250 mV and this may be of
major significance in flavour development. There is
very little information on the development and signifi-
cance of Eh in cheese, possibly because it is very diffi-
cult to measure the Eh of cheese accurately; research in
this area would appear to be highly desirable.
The precise route and mechanism for flavour gener-
ation by the starter have not been elucidated fully but
considerable progress has been made (see Fox and
Wallace, 1997; McSweeney and Sousa, 2000; Yvon and
Rijen, 2001; ‘Biochemistry of Cheese Ripening: Intro-
duction and Overview’, ‘Metabolism of Residual Lactose
and of Lactate and Citrate’, ‘Lipolysis and Catabolism of
Fatty Acids in Cheese’, ‘Proteolysis in Cheese during
Ripening’ and ‘Catabolism of Amino Acids in Cheese
During Ripening’, Volume 1, and Volume 2, on the
principal families of cheese).
The growth of the starter ceases at the end of the curd
manufacturing stage and the cells die at a rate charac-
teristic of the strain. Therefore, it seems reasonable to
conclude that starter enzymes rather than viable cells
are involved in ripening and that differences in the
enzyme profile of starter strains affect cheese quality.
Modern defined-strain starters produce acid very
reproducibly and, if properly selected and managed,
show good resistance to phage. Lactococcus strains
Factors that Affect the Quality of Cheese 601
have been selected mainly on the basis of acid-producing
ability, phage resistance and compatibility. Based on
pilot-scale studies and commercial experience, strains
that produce unsatisfactory, especially bitter, cheese
have been identified and excluded from commercial
usage. However, there are substantial and recognizable
differences in flavour quality and intensity between
cheeses made using different defined-strain cultures,
which presumably reflect differences in the enzyme
profile of the component strains; systematic studies on
strains are lacking. This probably reflects the lack of
information on precisely what attributes of a starter
are desirable from a flavour-generating viewpoint.
Studies on genetically engineered strains that super-
produce proteinase and/or the general aminopeptidase,
PepN, showed that, although proteolysis was acceler-
ated, cheese quality was not improved. Lactococcus
strains lacking one or more peptidases in various com-
binations are available; mutants lacking any one or
two peptidases can grow in milk but strains lacking
three or more peptidases cannot. Published studies on
the use of these peptidase-deficient mutants in cheese
are lacking.
Since all lactococcal enzymes, except the cell wall-
associated proteinase, are intracellular and since the
cells do not grow in cheese, the cells must lyse before
these enzymes can participate in ripening; therefore,
the rate of lysis of Lactococcus strains is being studied
with the objective of selecting strains with improved
cheesemaking properties. A bacteriocin-producing strain
of Lactococcus has been isolated which accelerates lysis
of the starter and consequently accelerates cheese
ripening (Morgan et al., 1995).
The enzymes of the glycolytic and proteolytic sys-
tems of Lactococcus are very well studied at the molecu-
lar, biochemical and genetic levels (see ‘Biochemistry of
Cheese Ripening: Introduction and Overview’, ‘Metabol-
ism of Residual Lactose and of Lactate and Citrate’,
‘Lipolysis and Catabolism of Fatty Acids in Cheese’,
‘Proteolysis in Cheese during Ripening’ and ‘Catabolism
of Amino Acids in Cheese During Ripening’, Volume 1).
Although less thoroughly studied, the phosphatase and
lipase/esterase of a few strains have been isolated and
characterized. During the past few years, there has been
increasing awareness of the significance of the amino
acid-catabolizing enzymes of the starter and a number
of reports on lactococcal deaminases, transaminases,
decarboxylases and lyases have been published (see
McSweeney and Sousa, 2000; Yvon and Rijnen 2001;
‘Catabolism of Amino Acids in Cheese During Ripen-
ing’, Volume 1). It is very likely that work on the lipo-
lytic and amino acid-catabolizing enzymes of Lactococcus
and their significance in cheese ripening and quality
will be intensified in the immediate future.
602 Factors that Affect the Quality of Cheese
The enzymes of Lactobacillus and Streptococcus
strains have been studied less thoroughly than those of
Lactococcus but the systems of the three genera appear to
be generally similar. Thermophilic lactobacilli lyse very
rapidly and are more proteolytic than lactococci.
Although considerable information is available on
the individual enzymes of Lactococcus and to lesser
extent of Lactobacillus, especially on the glycolytic and
proteolytic systems, there have been few comparative
studies on the different enzyme activities in starter
strains. There have been even fewer studies on the
relationship between different starter enzyme profiles
and cheese quality. It would appear to be highly desir-
able that studies should be undertaken to relate cheese
quality to the enzyme profile of natural starter strains
or genetically engineered cultures. The availability of
starter strains deficient in or over-producing one or
more enzymes will facilitate such studies.
It is very likely that the desirable cheesemaking
properties of starters are due to a balance between cer-
tain, perhaps secondary, enzymatic activities, which
have not yet been identified.
Non-Starter Lactic Acid Bacteria
In addition to starter bacteria, cheese curd contains
adventitious bacteria acquired from the milk and
environment. When raw milk was used widely, it was
probably the principal source of bacteria in cheese
curd, especially since it was heavily contaminated dur-
ing milking and was not cooled; counts 106 cfu/ml
were common and 90% of the bacteria in milk are
retained in the cheese curd. However, in the modern
dairy industry, the microbial quality of the raw milk
is very high and the milk is usually pasteurized;
typically, the milk entering the cheese vat contains
1000 cfu/ml. In large factories, the cheese is made in
enclosed vats, with very little contamination from the
environment.
In all ripened cheeses, a NSLAB flora, which varies
within and between cheeses made in the same plant,
develops (see ‘The Microbiology of Cheese Ripening’,
Volume 1).
Cheese is quite a hostile environment due to:
• a low pH
• a relatively high S/M
• anaerobic conditions
• lack of a fermentable carbohydrate
• the presence of bacteriocins and other inhibitory
substances produced by the starter.
Hence, relatively few species of bacteria can grow,
or even survive, in the centre of a well-made cheese.
Recent studies have shown that the non-starter
microflora of Cheddar cheese is dominated by
mesophilic lactobacilli, especially Lb. casei, Lb. paraca-
sei, Lb. plantarum and Lb. curvatus. In cheese produced
from good quality pasteurized milk in a modern plant
these NSLAB typically grow from a few hundred per
gram at the end of manufacture to 107–108/g within
2–3 months. Thus, while the population of starter LAB
declines, the number of NSLAB increases and domin-
ates the viable microflora of long-ripened cheese after
2–3 months (see ‘The Microbiology of Cheese Ripen-
ing’, Volume 1).
Although less well studied that Cheddar, the
NSLAB in Gouda, Emmental and Grana-type cheeses
are also predominantly mesophilic lactobacilli and this
is probably the normal situation in long-ripened
cheeses.
The significance of NSLAB for Cheddar and Dutch
cheese quality is controversial. Many researchers consider
their contribution to be negative (in the Netherlands,
a maximum of 2  106 NSLAB/g is specified for
Gouda). Although there are several studies on cheese
with a controlled microflora, the ripening and quality
of NSLAB-free cheese do not appear to have been com-
pared with ‘control’ cheeses containing ‘wild’ NSLAB.
Several comparative studies on cheese made under
aseptic or non-aseptic conditions using Lactococcus
starter alone or with selected Lactobacillus adjuncts
indicate that inoculation of cheesemilk with selected
strains of Lactobacillus improves cheese flavour and
possibly accelerates ripening. However, a typical but
mild flavour develops in Cheddar, Gouda and Emmen-
tal free of NSLAB, i.e., NSLAB do not appear to be
essential for cheese ripening although they do affect
the ripening pattern and cheese quality.
Since the numbers and strains of NSLAB in cheese
are uncontrolled, it is likely that they contribute to
variability in cheese quality. Therefore, it appears
worthwhile to determine what factors affect their
growth. The number of NSLAB in Cheddar is strongly
influenced by the rate at which the curd is cooled and
subsequently ripened. Rapid cooling of the curd after
moulding and pressing is the most effective way of
retarding the growth of NSLAB, although they will
grow eventually to ⬃107 cfu/g in cheese ripened at 4 °C.
The growth of NSLAB can be prevented by ripening
at ⬃1 °C but all ripening reactions are retarded
to an unacceptably slow rate. The growth of NSLAB
does not appear to be influenced by the composition
of cheese (moisture, salt or pH) within the ranges
normally found in commercial cheese.
Non-starter lactic acid bacteria grow mainly after
the lactose in the cheese curd has been metabolized
by residual starter activity. Although the growth sub-
strates in cheese for Lactobacillus are not known, they

can derive energy from the sugars of glycoproteins of
the MFGM (Diggin, 1999). It is likely that available,
suitable substrates are limiting (NSLAB normally
plateau at ⬃107–108 cfu/g) and hence it might be pos-
sible to out-compete wild NSLAB by adding selected
strains of Lactobacillus to cheese milk, thereby offering
better control of the ripening process. Non-starter lac-
tic acid bacteria may also be controlled by including a
broad spectrum bacteriocin-producing strain in the
starter culture (Fenlon et al., 1999).
Lactobacillus Species as Adjunct Cultures
Cheddar and Cheddar-type cheeses do not have an
intentional secondary microflora but there has been con-
siderable interest in recent years in the use of an adjunct
secondary culture (usually mesophilic lactobacilli) for
the following reasons:
• to intensify cheese flavour which is considered to
have become too mild owing to the improved
microbial quality of the cheese milk, pasteurization
of the milk, the use of enclosed vats and other
equipment (which reduce contamination from the
environment) and the use of defined-strain starters,
i.e., the cheese microflora have become too narrow;
• to accelerate cheese ripening; the ripening of cheese,
especially low-moisture, highly flavoured varieties, is
a slow, and consequently an expensive, process. Vari-
ous approaches to accelerate ripening have been
assessed, including the use of mesophilic lactobacilli
(see Fox et al., 1996b);
• to give identifiable flavour characteristics to cheese
produced by a particular manufacturer or sold by a
particular retailer;
• to improve the flavour of reduced-fat cheese, which
generally lacks flavour;
• inoculation of cheese with mesophilic lactobacilli
which suppresses the growth of adventitious lacto-
pH 4.85–5.20 FDM 52–55%
pH 4.95–5.10 FDM 52–55%
Premium
quality
MNFS 52–56% S/M 4.0–6.0%
MNFS 50–57% S/M 4.0–6.0% Moisture < 38%
Gilles and Lawrence (1973) Fox (1975a)
Composition of cheeses was determined at 14 days Relationship between the quality and composition
and related to quality of mature Cheddar cheese. of 10-week-old Cheddar cheese.
Figure 6 Relationships between composition (determined at various stages during ripening) and the quality of mature Cheddar
cheese (moisture-in-non-fat substances (MNFS); fat-in-dry-matter (FDM), and salt-in-moisture (S/M)).
Factors that Affect the Quality of Cheese 603
bacilli (NSLAB). Since the NSLAB are uncontrolled
(they are the only really uncontrolled component of
cheese), they probably contribute at least to some
extent to variability. In this case, the adjunct lacto-
bacilli need not contribute to the biochemistry of
ripening, just suppress the growth of adventitious
NSLAB.
A considerable amount of research on the signifi-
cance of mesophilic lactobacilli in cheese has been
reported during the past 10 years and the results seem
promising. Further research in this area is warranted.
Thermophilic Lactobacillus spp. are more effective as
adjuncts than mesophilic lactobacilli (Tobin, 1999),
probably because they die rapidly in cheese, lyse and
release intracellular enzymes. Both mesophilic and
thermophilic lactobacilli and Sc. thermophilus are
being used commercially as adjunct cultures for Ched-
dar cheese, and possibly for other varieties. Sc. thermo-
philus is used mainly to improve the phage resistance
of the culture (since it is resistant to lactococcal
phage) and to permit the use of a higher cook temper-
ature, facilitating better control over cheese composi-
tion and hence ripening and quality.
Cheese Composition
The quality of cheese is influenced by its composition,
especially moisture content, NaCl concentration
(preferably expressed as % S/M), pH, moisture-in-non-fat
substances (MNFS; essentially the ratio of protein to
moisture) and % fat-in-dry matter. At least five studies
(O’Connor, 1971; Gilles and Lawrence, 1973; Fox,
1975a; Pearce and Gilles, 1979; Lelievre and Gilles,
1982) have attempted to relate the quality of Cheddar
cheese to its composition. These authors agree that
moisture content, % S/M and pH are the key deter-
minants of cheese quality but they disagree as to the rel-
ative importance of these parameters (see Fig. 6).
Salt > 1.4% pH 4.95–5.15
Premium Premium
quality quality
pH < 5.4 MNFS 52–54% S/M 4.2–5.2%
Pearce and Gilles (1979)
Composition of cheeses was determined at 14 days
and related to quality of Cheddar cheese.
604 Factors that Affect the Quality of Cheese
O’Connor (1971) found that the flavour, texture
and total score of Cheddar were significantly correl-
ated with % NaCl and particularly with pH; moisture
content had less effect on cheese quality. Salt content
and pH were strongly correlated with each other, as
were salt and moisture.
Based on the results of a study on experimental
and commercial cheeses in New Zealand, Gilles and
Lawrence (1973) proposed a grading (selection)
scheme which has since been applied commercially in
New Zealand to young (14 day) Cheddar cheese. The
standards prescribed for Premium grade cheese were:
pH: 4.95–5.10; % S/M: 4.0–6.0%; MNFS: 52–56%;
FDM: 52–55%. The corresponding values for First
Grade cheeses were: 4.85–5.20%, 2.5–6%, 50–57% and
50–56%; young cheeses with a composition outside
these ranges were considered unlikely to yield good-
quality mature cheese. Quite wide ranges of FDM are
acceptable; Lawrence and Gilles (1980) suggested that
since relatively little lipolysis occurs in Cheddar
cheese, fat content plays a minor role in determining
cheese quality but if FDM is below about 48%, the
cheese is noticeably more firm and less attractive in
flavour. Pearce and Gilles (1979) reported that the
grade of young (14-day-old) cheeses produced at the
New Zealand Dairy Research Institute was most highly
correlated with moisture content; the optimum com-
positional ranges were: MNFS: 52–54%; S/M:
4.2–5.2%; pH: 4.95–5.15.
Fox (1975a,b) reported a weak correlation between
grade and moisture, salt and pH for Irish Cheddar
cheeses but a high percentage of cheeses with compos-
itional extremes was downgraded, especially those
with low salt (1.4%), high moisture (38%) or high
pH (5.4). Salt concentration seemed to exercise the
strongest influence on cheese quality and the lowest
percentage of down-graded cheeses can be expected in
the salt range 1.6–1.8% (S/M: 4.0–4.9%); apart from
the upper extremes, pH and moisture had little influ-
ence on quality in the sample studied. High salt levels
tend to cause a curdy texture, probably due to insuffi-
cient proteolysis; a pasty body, often accompanied by
off-flavours, is associated with low salt and high mois-
ture levels. In the same study, the composition of
extra-mature Cheddar cheeses was found to vary less
and the mean moisture content was 1% lower than
that of regular cheeses.
A very extensive study of the relationship between
the composition and quality of nearly 10 000 cheeses
produced at five commercial New Zealand factories
was reported by Lelievre and Gilles (1982). As in pre-
vious studies, considerable compositional variation
was evident but was less for some factories than
others. While the precise relationship between quality
and composition varied between plants, certain gener-
alizations emerged:
• within the compositional range suggested by Gilles
and Lawrence (1973) for ‘premium’ quality cheese,
composition does not have a decisive influence on
grade, which decreases outside this range;
• composition alone does not provide an exclusive
basis for grading;
• MNFS was again found to be the principal factor
affecting quality;
• within the recommended compositional bands,
grades declined marginally as MNFS increased from
51 to 55% and increased slightly as S/M decreased
from 6 to 4; pH had no consistent effect within the
range 4.9–5.2 and FDM had no influence in the
range 50–57%.
• there were specific intra-plant relationships between
grade and composition; therefore, each plant should
determine the optimum compositional parameters
pertinent to it.
The results of the foregoing investigations indicate
that high values for moisture and pH and a low salt
content lead to flavour and textural defects. The
desired ranges suggested by Gilles and Lawrence
(1973) appear to be reasonable, at least for New
Zealand conditions, but within the prescribed zones,
composition is not a good predictor of Cheddar cheese
quality. Presumably, several other factors, e.g., starter,
NSLAB, activity of indigenous milk enzymes, relatively
small variations in cheese composition and probably
other unknown factors, influence cheese quality but
become dominant only under conditions where the
principal determinants, moisture, salt and pH, are
within appropriate limits.
Although the role of calcium concentration in
cheese quality has received occasional mention, its
significance has been largely overlooked. Lawrence
and Gilles (1980) pointed out that the concentration
of calcium in cheese curd determines the cheese
matrix and, together with pH, indicates whether
proper procedures were used to manufacture a spe-
cific cheese variety. As the pH decreases during cheese
manufacture, CCP dissolves and is removed in the
whey. The whey removed after cooking comprises
90–95% of the total whey expressed during cheese-
making and under normal conditions contains ⬃85%
of the calcium and ⬃90% of the phosphorus lost from
the cheese curd. Thus, the calcium content of cheese
reflects the pH of the curd at whey drainage; there are
strong correlations between the calcium content of
cheese and the pH at 1 or 14 days and the amount of
starter used (see Lawrence et al., 1984). Since the pH
of cheese increases during ripening, the pH of mature

cheese may be a poor index of the pH of the young
cheese. Therefore, calcium concentration is probably
a better record of the history of a cheese with respect
to the rate of acidification than the final pH. Reduc-
tion in calcium phosphate concentration by exces-
sively rapid acid development also reduces the
buffering capacity of cheese and hence the pH of the
curd will fall to a lower value for any particular level
of acid production. No recent work on the level and
significance of calcium in Cheddar cheese appears to
be available.
The calcium content of cheese has a major effect
on its meltability and stretchability, e.g., pasta-filata
cheese does not stretch well, or not at all, until the
pH falls below ⬃5.4. Biologically acidified Mozzarella
has poor stretchability and meltability immediately
after manufacture but these properties improve dur-
ing the early stages of ripening and are optimal after
about 2–3 weeks; functionality deteriorates on con-
tinued ripening due to proteolysis. In contrast,
directly acidified cheese is functional immediately
after manufacture. The difference in behaviour is due
to the lower calcium concentration in the directly
acidified cheese owing to the faster decline in pH
to ⬃5.6. Under such conditions, much of the CCP
dissolves and is removed in the whey at drainage; the
concentration of calcium per unit of protein, which is
very important for cheese functionality, in biologic-
ally and chemically acidified Mozzarella cheese
was 27.7 and 21.8 mg/g, respectively (Guinee et al.,
2002).
There is little published information on the rela-
tionships between composition and quality for other
cheese varieties. However, it is very likely that similar
factors affect the quality of all cheeses more or less to
the same extent.
Ripening Temperature
Ripening temperature has a major influence on the
rate of ripening and quality of cheese. Traditionally,
cheese was ripened in caves or cellars at a relatively
constant temperature. This practice is still widespread
for some varieties but artificially refrigerated rooms
are now used by large-scale manufacture. The ripen-
ing temperature is fairly characteristic of the variety,
e.g., Cheddar, 6–8 °C; Gouda, 12–14 °C; Parmigiano-
Reggiano, 18–20 °C; Emmental, 6 °C for ⬃2 weeks,
then at 22 °C for 4–6 weeks to allow the propionic
acid bacteria to grow rapidly and produce adequate
CO2 for good eye development, then at ⬃4 °C for sev-
eral months to complete ripening; Camembert, 14 °C
for ⬃2 weeks to induce the growth of P. camemberti,
then at 4 °C for 2–4 weeks.
Factors that Affect the Quality of Cheese 605
Ripening can be accelerated by increasing the
ripening temperature but all reactions, desirable and
undesirable, are accelerated and an unbalanced flavour
or off-flavour may develop. Ripening at an elevated
temperature is normally considered with the objective
of accelerating ripening (see Fox et al., 1996b).
Cheese flavour can probably be modified by
manipulating temperature; however, this is rarely
practised except for Swiss-type cheeses. The rate at
which the curd is cooled after moulding has a major
effect on the growth of starter LAB and NSLAB. The
curds for most cheeses are moulded immediately after
cooking and acidification occurs mainly in the
moulds. Hence, the rate at which the curd cools in the
moulds has a major effect on starter growth and rate of
acid development, and is strongly affected by the size
of the cheese and ambient temperature. The effect of
cooling on starter growth is particularly noticeable for
high-cooked cheeses, e.g., Swiss and Grana types. The
thermophilic starters used for these cheeses do not
grow at the cook temperature but begin to grow as the
curd cools in the moulds. For consistency, it is import-
ant to control the ambient temperature.
For Cheddar-type cheeses, acidification is almost
complete at moulding. Traditionally, the moulded
cheeses were pressed overnight at ambient tempera-
ture and the cheeses cooled close to ambient during
this period, although ambient temperature probably
varied significantly with season. In modern practice,
the cheeses exit the Wincanton tower at ⬃36 °C and
are packaged and stacked on pallets (5  10 cheeses
⬃1 tonne) and transferred to ripening rooms. The
cheeses at the centre of the pallet do not decrease to
ambient (store) temperature for about 4 weeks and
this causes considerable variation in the number
and probably the type of NSLAB, and hence in the
quality of the cheese. Many factories now cool the
packaged cheese in a cooling tunnel overnight before
stacking on pallets. If the cheese is cooled to 1 °C
and ripened at this temperature, the cheese will be
free of NSLAB but the rate of ripening will be very
slow.
The humidity of the environment must be con-
trolled, at 85–90% RH, for the ripening of many var-
ieties, mainly those with a surface microflora, which
will not grow if the cheese develops a rind. Tradition-
ally, rind development was encouraged on internal
bacterially ripened cheeses by reducing the RH slowly.
The rind serves to protect the cheese against undesir-
able surface growth and the loss of moisture (weight).
Today, many varieties, e.g., Cheddar and Gouda, are
coated or wrapped in plastic, i.e., rindless cheese, to
prevent weight loss and to protect the surface of the
cheese against undesirable bacterial growth.
606 Factors that Affect the Quality of Cheese
Conclusions
Through increased knowledge of the chemistry, bio-
chemistry and microbiology of cheese, it should be
possible to produce cheese of a very high quality con-
sistently, although this is not always achieved owing to
failure to control one or more of the key parameters
that affect cheese composition and ripening. Milk is a
variable raw material and although it is possible to
eliminate major variations in the principal milk con-
stituents, some variations persist. Variability in milk
composition can also be compensated by manipulating
some process parameters in the cheesemaking process.
Most large factories operate on a strict time schedule
and hence subtle manipulation of the process on an
individual vat basis may not be possible. Therefore,
strict control of milk composition and starter activity
is critical.
From a microbiological viewpoint, the milk supply
to modern cheese factories is of very high quality and
after pasteurization contains only a few hundred bac-
teria per ml. In modern factories where enclosed vats
and other equipment is used, the level of contamin-
ation from the environment is very low; cheese curd
containing 103 NSLAB/g at 1 day is normal. How-
ever, these adventitious NSLAB grow to c. 107–108 cfu/g
and dominate the microflora of cheese after about
3 months. Since the adventitious NSLAB grow slowly
in cheese, they are most significant in long-ripened
cheese. Although the significance of the adventitious
NSLAB in long-ripened cheese is unclear, it would
appear to be desirable to control them, either by elimin-
ating them or standardizing their number and type. In
industrial-scale manufacture of cheese, it is not pos-
sible to eliminate NSLAB. It is possible to prevent their
growth by ripening at ⬃1 °C but the overall ripening
process is also reduced to an unacceptable rate. Out-
competing indigenous NSLAB by an adjunct Lacto-
bacillus culture, which does not have to contribute to
ripening, is a possibility but this approach has not
been investigated.
Although it is now possible to avoid major defects
in cheese produced using modern technology, fur-
ther research on the biochemistry of cheese ripening
is required to enable the process of cheese manufac-
ture and ripening to be refined to an extent that will
allow the consistent production of premium quality
cheese.
The key to successful cheesemaking is a good reli-
able starter, both from the viewpoint of reproducible
acid production and subsequent ripening. If properly
managed, modern starters are generally satisfactory
and their performance is being improved progres-
sively.
The use of starter adjuncts, usually mesophilic
lactobacilli, for some varieties, especially Cheddar, is
increasing, with the objective of intensifying and
modifying flavour, accelerating ripening and perhaps
controlling adventitious NSLAB and thus standardiz-
ing quality.
Basically, cheesemaking is a relatively simple
process, consisting of two phases: conversion of milk
to cheese curd and transformation of the curd to
mature cheese; both phases involve a number of
steps. The key steps in curd manufacture are: acidifi-
cation, coagulation, syneresis/dehydration and salt-
ing. With the knowledge currently available on the
mechanism of these processes and the scale and qual-
ity of the cheesemaking equipment, it should be pos-
sible to produce cheese curd of consistently premium
quality from chemical and microbiological view-
points. Unfortunately, this is not the case in practice.
Undoubtedly, variability in the composition and
microflora of the milk contribute to the variability of
cheese curd but there is variability in curd produced
during the course of a single day from a single large
batch of bulked milk using the same rennet and
starter. One factor likely to be responsible for this
variability is the time-lag in performing certain
cheesemaking operations, e.g., it requires ⬃30 min to
separate the curds and whey in the very large (30
000 l) vats now used for Cheddar, Gouda or Moz-
zarella. This time-lag continues during later opera-
tions, e.g., cheddaring, milling, salting and pressing.
The solution to this problem is the development of a
continuous curd production system, such as the
ALPMA system, but this is not used for hard cheeses.
Work in this area appears warranted.
The objective of cheesemaking is to consistently
manufacture cheese with the desired, characteristic
flavour, texture and functionality in the highest yield
possible, as cheaply and as quickly possible. The clos-
est we have come to achieving that objective is the
production of enzyme-modified cheeses, which do not
resemble closely the flavour, texture or functionality of
any natural cheese but are used successfully to replace
natural cheese in some applications (see ‘Sensory
Character of Cheese and its Evaluation’, Volume 2).
With improved knowledge of the biochemistry of
cheese ripening, it may be possible to produce some of
the milder, less complex cheese using the EMC
approach – research in this area is warranted.
Cheese ripening is a very complex biological, bio-
chemical and chemical process which is determined
and directed by the composition and microflora of the
cheese curd; if these are reproducible and consistent, it
should be possible to produce cheese of excellent
quality consistently.

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Index

Abondance, 199 control of, 164

Acid-coagulated milk gels, 6, 77–8, 105 engineered phage resistance systems, 179
casein micelles in, 105–106 antisense mRNA, 180
effect of compositional/processing parameters on textural bacteriophage-triggered defence, 181–2
properties: current status/future perspectives, 182
fat content/homogenization, 118 gene replacement/insertional mutagenesis, 180–1
heat treatment, 116–17 phage-encoded resistance (Per), 179–80
inoculation/gelation temperature, 115–16 recombinant superinfection exclusion/
pH/calcium content, 118–19 immunity, 181
rennet addition, 117 epidemiology, 167
solids non-fat (SNF) content, 117–18 Lactobacillus phage, 170–1
mechanisms: Sc. thermophilus phage, 170
physico-chemical aspects, 106–109 genome organisation/evolution, 171–6
theoretical models, 106 lysogenic life cycle, 174
physical properties: maintenance of lysogeny, 175–6
appearance, 113–14 site-specific recombination, 174–5
microstructure, 112 superinfection exclusion, 176
permeability, 112–13 lytic life cycle:
rheological, 109–11 bacteriophage lysis, 174
texture/sensory, 111–12 DNA packaging, 173
whey separation/syneresis, 114–15 DNA replication, 172–3
study by electron microscopy (EM)/ confocal scanning lysogenic/lytic switch, 172
laser microscopy (CSL), 112 phage adsorption/DNA injection, 171–2
syneresis, 114–15 structural proteins, 173–4
see also Coagulation; Rennets multiplication, 163
Adjunct cultures see Secondary/adjunct cultures natural resistance systems in lactic acid
Amino acids, catabolism of see Catabolism of amino acids bacteria, 176–7
Appenzell, 199 abortive infection, 178–9
Aroma, compounds: adsorption inhibition, 177
extraction methods, 491 injection blocking, 177
dialysis, 492 restriction/modification, 177–8
headspace, 493 proteolytic enzymes, 131
high-performance size-exclusion chromatography, raw milk cheeses, 321
492–3 resistance systems:
high-vacuum distillation, 491–2 engineered phage, 179–82
solid-phase microextraction, 493 natural bacteriophage, 176–9
solvent, 492 starter cultures, 129–38
steam distillation, 491 Bavarian Blue, 193
water-soluble extract (WSE), 493–4 Beaufort, 197
identification using hyphenated GC techniques, 494–6 Bel Paese, 193, 195
representativeness, 494 Biogenic amines, 201, 561–3
sample treatment, 491 Bleu d’Auvergne, 193
see also Sensory characteristics of cheese; Flavour Blue cheese, 123
Arzua, 323 contamination, 550
Aspartic proteinases see Chymosin/aspartic proteinases fatty acids, 378
Autolysis, 126, 289 mesophilic starters, 149
Avenato, 323 ultrafiltration, 269
use of salt, 211–12, 219
Bacteriocins, 136, 153 Brevibacterium linens, 149, 192
Bacteriophage, 154, 163–70, 287, 351, 375 Brie:
classification, 165 flavour, 502
DNA homology, 167 lipolytic agents, 376
host range, 165 moulds, 193
morphology, 165 rheology/texture, 535
serology, 165–6 Brine-salted cheese see Salt
structural protein profiles, 166–7 Buffalo milk cheese, 5
610 Index

Cabrales, 193, 306 reduced sodium, 226–7

Camembert, 123 rheology/texture, 516, 517, 518–19, 520, 527, 529,
contamination, 550 530, 535
coryneform bacteria, 195, 197 ripening, 348
fatty acids, 377 sensory characteristics, 478–9
flavour, 492, 498, 499, 500 starter cultures, 126, 128
lipolytic agents, 376 use of salt, 208–209, 211, 216–19, 240
Listeria growth, 544 Cheddar curd:
mesophilic starters, 149 salt uptake:
microbial growth, 544–5 curd depth during holding, 238
moulds, 193 degree of mixing, 238
raw milk, 321 level of salting, 237–8
rheology/texture, 528, 535 mellowing period, 238
starter cultures, 126 method, 237
ultrafiltration (UF), 271 moisture content of curd, 238–9
use of salt, 219, 240 other factors, 239
yeast flora, 306 temperature, 238
Canestrato, 5, 323 Cheese:
Casein, 7, 48–50, 55, 60, 71–2, 105–106, 351, 413, composition, 7–8, 603–605
415, 417, 589, 591 fat-in-dry matter (FDM), 7, 589
activity of cell envelope proteinase, 132 moisture in non-fat substances (MNFS), 7
chemistry, 48 pH, 8
micelle assembly, 49 history of, 1–5, 261, 605
micelle properties, 49–50, 105–106 Cheshire, 5, 208, 527
micelle stability, 50 Chymosin/aspartic proteinases, 19–33, 351, 392,
self-assembly, 49 600–601
structures, 48–9 active site, 23–7
chymosin activity on, 52, 392–3 catalytic mechanisms, 27–9
effect of NaCl, 212–13, 214, 220–3 inhibitors, 33
enrichment of cheese milk by MF, 277–8 natural sources, 19–20
gel formation/properties of, 71–4, 78–81, 91, 94, physical properties/stability, 20–1
106–109, 111, 115, 116 structure, 21–4
hydration in cheese, 220–5 substrate-binding pockets/specificity, 30–3
hydrolysis by cathepsin D, 396 zymogen activation, 29–30
mares’ milk, 591 crystals, 23
quality of cheese, 591–2 fermentation-produced chymosin, 51–2
raw milk cheese, 324, 336, 339 milk clotting mechanism, 33–4
use of capillary electrophoresis for analysis of mutant chymosins, 32–3
casein, 420 physical properties/stability:
Catabolism of amino acids, 152–3, 201, 302–303, enzyme solubility, 20–1
350–2, 435, 451 enzyme stability, 20
aromatic: molecular weight/isoelectric point, 20
phenylalanine, 444–7 recombinant calf chymosin:
tryptophan, 443 eukaryotic expression, 35–6
tyrosine, 443–4 prokaryotic expression, 34–5
branched-chain: ripening process, 351, 356
deaminases, 449 stability (solubility), 20
decarboxylases, 449–51 structure:
production of volatile sulphur compounds: active site, 24–7
lyases involved in methionine catabolism, 442–3 catalytic mechanisms, 27–9
transamination, 435–9 gene sequences, 21
Catabolism of fatty acids, 376–80 inhibitors, 33
Catabolism of lactate, 348–9 primary, 21
Cathepsins, 396, 399 secondary, 21, 23
Cauchy strain, 539 substrate-binding pockets/specificity, 30–3
Cheddar, 5, 10, 12, 14 tertiary, 23–4
contamination, 545, 546–9 zymogen activation, 29–30
dry-salted varieties, 245–6 substrate specificity, 32
fatty acids, 377 yield, 34–5
flavour, 335, 337, 492, 502, 605 see also Pepsin
lipolytic agents, 374, 375 Citrate metabolism, 130–1, 151–2, 367–8
lysis, 136 Clostridia, 7, 153, 202, 327, 328, 365–6, 562, 600
mesophilic starters, 149 Clotting see Acid-coagulated milk gels; Coagulation;
microbiology, 289, 290, 296, 297, 301 Rennets
moulds, 193 Coagulation, 10–11, 47
propionic acid bacteria, 200 Cynara cardunculus, enzymes from, 52
quality, 595, 596, 604, 605 enzymes, 47, 391, 392–3
raw milk, 323, 327, 330 influence of NaCl, 212–13
 Index 611

physico-chemical mechanisms involved in gel formation Dutch cheese, 128

from unheated milk, 106–109 lipolytic agents, 374
post-coagulation operations, 11–12, 594–6 ripening, 348
theoretical models, 106 see also Gouda; Edam
visual, 11
see also Acid-coagulated milk gels; Rennets Eastern European cheeses, 299
Comté cheese, 127 Edam:
Conjugated linoleic acid, 575–6 fatty acids, 378
Contamination see Pathogens; Safety; Toxins mesophilic starters, 149
Cooking, 224–5 starter cultures, 126
health/safety, 551 ultrafiltration, 268
Coryneform bacteria, 195, 197, 597 Elastic material behaviour, 539
antimicrobial activities, 198 Elastoplastic material behaviour, 539
form/use of adjunct culture, 198 Emmental:
lipolysis, 198 fatty acids, 378
proteolysis, peptidolysis, amino acid catabolism, 198 flavour, 502
selection as adjuncts, 197–8 propionic acid bacteria, 200
useful properties in selecting surface bacteria, 197 starter cultures, 123, 126, 127
Cottage cheese, 2, 9 use of salt, 215
acid milk gels, 105, 112, 116 whey cultures, 127
addition of rennet, 117 see also Swiss cheese
contamination, 550 Enterococci, 290, 295
heat treatment, 116 Enzymes, 19, 133, 288, 378, 448
reduced sodium, 227 coagulation, 47, 50–2, 391, 392–3
ultrafiltration, 268 indigenous, 587–8, 600
Cream cheese, 2 microbial, 214–15
acid milk gels, 105, 112 proteolytic, 19, 413–15
compliance, 539 ripening process, 352–7
modulus, 539 secondary starter microorganisms, 195, 413–15
whey removal, 114, 115 sources:
Cultures see Secondary/adjunct cultures; Starter lyosomes, 19
cultures micro-organisms, 19–20
Curd, 12, 14 plants, 19
behaviour during processing: stomach, 19
axial drainage, 95 tissues, 19
compaction of curd column, 94–6 see also Chymosin/aspartic proteinases; Lactic acid
curd fusion, 93–4, 97 bacteria; Catabolism of amino acids; Catabolism
syneresis under pressure, 93 of fatty acids; Lipolysis; Peptidases; Proteolysis;
water content of cheese, 96–8 Ripening of cheese
cooking temperature, 11 Ewe’s milk cheese, 199, 340
manufacturing process, 11–12 Exopolysaccharides, 137, 158
salt absorption/diffusion:
direct mixing with milled curd, 229, 232 Fatty acids, 349, 373–80
dry surface-salting of moulded pressed curd, 232 Feta, 5, 268–9
factors affecting uptake in Cheddar, 237–9 contamination, 550
initial moisture content, 236–7 microbial growth, 545
pH of curd/brine, 237 microbiology, 290
salt-in-moisture level/pre-salting, 208–11, 235 ultrafiltration (UF), 271
temperature of curd/brine, 235 use of salt, 219
syneresis during curdmaking: Flavour, 3–4, 129, 289, 306, 332, 347, 466, 489–91
effects of grain size, 86–7 acid milk, 112
methods of estimation, 85–8 contribution of lipolysis/catabolism of FFA, 379–80
rate equations, 85–6 dynamic methods for characterisation:
stirring, 87–8 model mouth systems, 501–502
washing, 90 release of non-volatiles in vivo, 501
Cynara cardunculus proteinase, 3, 52 release of volatiles in vivo, 499–501
fatty acids, 349
Danablu, Danish Blue, 193, 194, 306 global/fast assessment, 502
Danbo: electronic nose, 502–503
mould, 193 mass spectrometry-based systems, 503–504
ultrafiltration, 268 peptides, 352
use of salt, 220 quality, 586–7, 599, 603
yeast flora, 306 raw milk, 335–8
Debaryomyces hansenii, 191, 192, 196 sapid (non-volatile) compounds:
Deformability modulus, 539 extraction, separation, identification in relation to
Dental caries, 576, 578–9 sensory properties, 496–9
Domiati, 10, 219 water-soluble extracts (WSE), 496
Dry-salted cheese see Salt smell/aroma, 466
612 Index

Flavour – contd. oxidation, 364

taste, 466 oxidative metabolism in surface mould-ripened
see also Sensory characteristics of cheese; Aroma, varieties, 364
compounds racemization, 362–4
Free-Choice Profiling, 477 see also Lactose metabolism
Fungi, 36 Lactic acid bacteria, 399–400
aminopeptidases, 411–12
Gammelost, 306 carboxypeptidases, 403, 411
Gaziantep, 219–20 di- and tri-peptidases, 403
Gel formation see Acid-coagulated milk gels; endopeptidases, 403
Coagulation; Rennets; Syneresis of rennet-coagulated Lactobacillus delbrueckii, 290
curd Lactobacillus helveticus, 123
Genetic engineering, 155, 601 Lactococcus lactis, 123
Geotrichum candidum, 191 peptidases, 400–403
Goat cheese, 1, 5, 14, 193, 325, 498 proline-specific peptidases, 412–13
Gorgonzola, 193 proteinases, 400
Gouda, 14 see also Starter cultures, Non-starter lactic acid
fatty acids, 378 bacteria, Bacteriophage
lysis, 136 Lactobacillus spp, 123, 290
mesophilic starters, 149 see also Non-starter lactic acid bacteria
propionic acid bacteria, 199 Lactococcus lactis, 123
quality, 595, 596 chromosome, 149–50
starter cultures, 126 genetic manipulation, 155
use of salt, 223, 237 genetics of industrially important traits:
Grana, 8, 126 bacteriocins, 153–4
microbiology, 289 bacteriophage, 154–5
whey cultures, 127 lactose/citrate metabolism, 151–2
Gruyère: proteolysis/amino acid catabolism, 152–3
catabolism of fatty acids, 378 Lactose metabolism, 130, 151–2, 361–2
Coryneform bacteria in, 197 Leuconostoc, 155, 290
flavour, 492 Limburger, 195
rheology/texture, 529 Linear viscoelastic deformation, 539
starter cultures, 126, 127 Lipolysis, 198, 199–200, 201, 303, 349
whey cultures, 127 agents, 373–6
contribution of FFA to flavour, 379–80
HACCP see Hazard Analysis and Critical Control measurement of, 380, 384–5
Points patterns of, 380
Havarti, 535 raw milk cheese, 325, 327
Hazard Analysis and Critical Control Points, 542, Liquid pre-cheese (LPC), 269–73
543, 584 Listeria monocytognes, 542–4, 550, 551, 553–5
see also Safety Livarot, 195
Health see Nutrition; Pathogens; Safety; Toxins Loss modulus, 540
Heterofermentative lactobacilli, 200 Low-concentrated retentates, 267–8
form/use of adjunct cultures, 202 Lysis, 126, 289
species found in cheeses, 200–201, 291 see also Autolysis
useful properties to select as adjuncts: Lysogeny, 174
antagonistic activities, 201–202
formation of biogenic amines, 201 Maasdam, 5, 199, 378
lipolytic activities, 201 Mahon, 220
probiotic properties, 202 Manchego, 5, 335
proteolysis/amino acid catabolism, 201 Membrane processing, 261
Histamine, 561–3 applications of:
Hygiene, 321–3 liquid pre-cheeses, 269–75
medium/intermediate concentrated retentates, 268–9
Illness see Pathogens; Safety; Toxins microfiltration, 276–8
Italian cheeses, 298–9, 338 milk protein concentrates, 278–9
on-farm concentration, 275–6
Jarlsberg, 5 properties of UF retentates, 265–7
protein-standardized milk, 267–8
Kelvin element, 539 reverse osmosis, 275
Kinematic viscosity, 539 UF in cheesemaking, 267–75
Kluyveromyces, 195 APV-sirocurd process:
definitions, 262–5
Lactate: design/configuration, 261
catabolism, 348–9 hollow fibre, 262–3
changes during ripening: microfiltration (MF), 262
metabolism by Clostridium tyrobutyricum, 365–6 nanofiltration (NF), 262
metabolism by Propionibacterium, 366–7 plate and frame, 263
 Index 613

reverse osmosis (RO), 262 proteolytic activity, 194

spiral-wound, 263–4 Mozzarella, 9, 12
tubular, 262 contamination, 553
ultrafiltration (UF), 262 flavour, 498
vibrating membrane system, 264–5 quality, 595, 596
Mesophilic starters, 149–52, 597 raw milk, 321
Leuconostoc, 155–6 rheology/texture, 529, 530
plasmids, 150–1 starter cultures, 123, 126
Mexican-style cheese, 553 ultrafiltration (UF), 268, 272–3
Microbial pathogens see Pathogens use of salt, 222, 240
Micrococcus, 197, 304 Münster, 195
Microfiltration: Mycotoxins, 564, 567–8
applications:
casein enrichment of cheese milk, 277–8 Nitrogen metabolism in lactic acid bacteria, 131–2
microbial epuration of raw milk, 276–7 amino acid degradation, 133–4
modifications, 278 role of proteinase, 132
selective fractionation of globular fat, 278 transport systems/peptidases, 132–3
Milk: see also Proteolysis; Peptidases; Lactic acid bacteria
antiobiotics in, 7 Non-starter lactic acid bacteria (NSLAB), 7, 289, 291, 353
casein chemistry, 48–50 biochemical activities:
chemical composition, 7, 588–9 amino acid catabolism, 302–303
clotting mechanism, 33–4, 47 citrate utilisation, 302
composition, 91 lipolysis, 303
fat, 47 proteolysis, 302
gel formation, 106–109 enterococci, 290, 295
heat treatment, 88 growth/survival:
homogenization, 88 environmental conditions, 296
indigenous enzymes, 587–8 interactions, 297
indigenous proteinases: nutrient availability, 296–7
others, 396–9 non-starter lactobacilli, 289–90
plasmin, 213–14, 393–6, 600 pediococci, 290
microbiology: population dynamics:
desirable indigenous bacteria, 587 Cheddar, 297
off-flavours/spoilage, 586–7 Greek/eastern European cheeses, 299
public health aspects, 586 Italian cheese varieties, 298–9
pasteurized, 8, 355 Portuguese cheese varieties, 298
alternatives to pasteurization, 587 Spanish artisanal cheeses, 297–8
protein, 47–8, 573, 575 Swiss cheeses, 297
protein-standardization, 267–8 quality, 602–603
quality, 584 ripening process, 356–7
rennet-induced coagulation, 50–65 significance:
safety: adjunct as probiotics, 301–302
heat treatment, 543–4 influence on quality, 299, 301
pathogens, 541–3 use of other adjunct cultures, 301
quality, 543 source of, 295–6
standardization: see also Lactobacillus
calcium, 589 NSLAB see Non-starter lactic acid bacteria
fat/casein, 589 Nutrition, 573
pH, 589–90 carbohydrate, 575
syneresis of renneted-milk gel, 80–1 cheese and dental caries, 576, 578–9
toxins in, 564 fat/cholestrol, 575–6
various additions to, 88–9 minerals, 576
Milk gels see Acid-coagulated milk gels; Rennet protein, 573, 575
coagulation of milk vitamins, 576
Milk protein concentrates, 278–9
Modulus of deformability, 540 Parmigiano-Reggiano, Parmesan:
Morbier, 195, 199, 330 contamination, 545, 550
Moulds, 304–306, 395, 597 fatty acids, 377–8
contamination with mycotoxins, 567–8 flavour, 492
form/use of adjunct culture, 194–5 raw milk, 322
species found in cheese, 193–4 sensory characteristics, 474
useful properties in selecting as adjuncts: starter cultures, 123
appearance on/in cheese, 194 whey cultures, 127
de-acidification activity, 194 Pathogens, 7, 541
interactions with other microorganisms, 194 challenge studies, 549–52
lipolytic activity, 194 Escherichia coli, 541, 544, 545, 548, 549–50, 551, 555
production of aroma, 194 growth/survival in soft/semi-soft cheeses, 552–3
production of mycotoxins, 194 reviews on safety of raw milk, 545–6
614 Index

Pathogens – contd. Quality of cheese, factors affecting:

safety of cheese, 541 cheese composition, 603–605
extrinsic/intrinsic parameters affecting microbial coagulant (rennet), 590–2, 600–601
growth, 544–5 cultures, 592–4, 601–602
heat treatment of milk, 543–4 indigenous enzymes, 600
milk quality, 543 Lactobacillus species as adjunct cultures, 603
raw milk, 541–3, 545 milk supply:
Salmonella, 542, 544, 546–8, 551, 553, 555 alternatives to pasteurization, 584, 587
Salmonella enterica, 542, 546, 548, 553 chemical composition, 588–9
stress adaptation and impact on safety, 553–4 indigenous enzymes, 587–8
Pecorino, 5, 124, 126, 127 microbiology, 586–7
Pediococcus, 290, 292 standardization of composition, 589–90
Penicillium camemberti, 193 non-starter lactic acid bacteria, 602–603
Penicillium roqueforti, 193 packaging, 597
Pepsin, 29, 31, 354, 393 post-coagulation operations, 594–6
Peptidases, 132–3, 400, 403, 417 production parameters, 584
aminopeptidases, 411–12 ripening, 596–9, 605–606
carboxypeptidases, 403, 411 salting, 216–20, 596
di- and tri-peptidases, 403 starter, 592–4, 601–602
endopeptidases, 403 use of ultrafiltration (UF), 596
proline-specific, 412–13 Quarg, 2, 5
Phage see Bacteriophage acid milk gels, 105, 112
Pichia spp., 196 addition of rennet, 117
outbreaks involving Cheddar, 546–9 ultrafiltration, 270
Poisson effect, 540 whey removal, 115
Pont l’Eveque, 535 see also Acid-coagulated milk gels
Port Salut, 193 Queso Blanco, 5
Portuguese cheese, 298, 306
Processed cheese, 227 Raclette, 323, 328, 335, 338
Prochymosin, 20, 30, 34–6 Raw milk cheese, 8, 319–20, 545–6
Propionibacterium, 303, 449, 597 biochemical aspects:
see also Emmental; Swiss cheese lipolysis, 325, 327
Propionic acid bacteria, 198, 303, 449, 597 proteolysis, 323–5
as adjunct cultures, 200 volatile compounds, 327–35
characteristics of species found in cheeses, safety aspects:
198–9 diversity of microorganisms, 320–1
useful properties for selecting as adjuncts: hygiene, 321–3
lactate metabolism, 199 numbers of microorganisms, 320
lipolysis, 199–200 sensory aspects:
probiotic properties, 200 flavour/odour, 335–8
proteolytic activities/amino acid catabolism, texture, 338–40
199 Reconstituted skim milk, 64, 139, 208
Proteolysis, 152–3, 156, 302, 350–2 Reduced-sodium cheese, 207, 225–6
amino acids, 350–2 Cheddar, 226–7
lactocepins, 400 Cottage cheese, 227
monitoring: other cheeses, 227
amino acid analysis, 421 Rennet coagulation of milk, 2, 3, 6, 8, 10–11, 19, 584
capillary electrophoresis (CE), 420 adhesive sphere models/viscosity, 55–6
chromatographic techniques, 420–1 development of rheological properties, 56–7
fluorescent spectroscopy, 421 effect of acidification, 117
Fourier transform infrared spectroscopy fractal models/rearrangements, 62–3
(FTIR), 421 heat treatment, 64–5
tryptophan, 421 high pressure treatment, 64
ultrasonics, 421 kinetic models, 53–5
urea-PAGE, 420 measurement of clotting time/curd-cutting time, 53
patterns, 415–19 mechanisms of milk-clotting, 33–4
primary, 323–4 milk processing/gel formation, 63–5
raw milk cheese, 323–5 modelling gel-firming kinetics, 60–2
ripening, 391 post-coagulation operations, 594–6
coagulant, 391, 392–3 preparation, 52
exogenous proteinases/peptidases, 391–2 primary enzymatic phase, 11, 50–2
indigenous proteinases, 391, 393–9 production, 584
non-starter lactic acid bacteria, 200, 391 quality, 590–2, 600–601
secondary starter, 191, 391, 413–15 rennet, 354–5
starter lactic acid bacteria, 391, 399–413 ripening process, 354–5
secondary, 198, 199, 201, 324 secondary non-enzymatic phase, 11
water-soluble peptides, 416–19 substitutes, 10
Provolone, 545 theoretical basis of viscoelasticity, 57–60
 Index 615

UF retentates, 266–7 Romano, 535, 545

see also Acid-coagulated milk gels; Coagulation; Roquefort, 5
Chymosin/aspartic proteinases lipolytic agents, 376
Rennets see Chymosin/aspartic proteinases; Pepsin mould, 193, 194
Rheology, 511–33 rheology/texture, 535
cheese structure, 516–18 yeast flora, 306
compliance, 539
creep/stress relaxation, 518–19 Saccharomyces cerevisiae, 196
development of properties during rennet coagulation, Safety, 541
56–7 Cheddar, 546–9
effect of NaCl, 223–4 improvements, 554–5
empirical instrumental methods of measurement, 523 microbial growth, 544–5
exopolysaccharides, use of, 158 milk:
gel formation, 75–7, 109–10, 113–14, 117–18 heat treatment, 543–4
large strain deformation: quality, 543
bending tests, 530–1 raw, 541–3, 545–9
definition/terminology, 520 soft/semi-soft cheeses, 552–3
effect of sample temperature, 532 stress adaptation and impact of pathogens,
fracture/work to fracture, 521 553–4
measurement using texture analyser, 520–1 St Nectaire, 193–4, 306
shear measurements, 530 St Paulin, 136, 273, 323
uniaxial compression, 527–30 Salt, 10, 207–208, 348, 576, 596
wire-cutting, 532 absorption/diffusion:
mechanical models, 519–20 brine- and surface dry-salted cheeses, 244–5
oscillatory rheometry for linear viscoelastic brine concentration/concentration gradient in
measurements: brine-salted cheese, 232–3
complex viscosity, 526–7 brine-salted cheese, 228–9
elastic shear modulus, 524–6 Cheddar/dry-salted varieties, 245–6
loss modulus, 524–6 cheese geometry, 233–4, 244
overview, 511–12 concentration gradient in dry-salted cheeses,
sensoric methods, 521–3 239–40
terminology: concentration of calcium in brine, 240
bulk modulus/compressibility, 515–16 direct mixing of salt/milled curd, 229, 232
deformation and strain, 512 dry surface-salting of moulded pressed cheese
relationship between stress/strain, 515 curd, 232
shear/normal modes of stress/strain, 513–15 fat content of cheese, 243–4
stress, 512–13 initial moisture content of curd, 236–7
viscous deformation, 516 initial salt-in-moisture level of curd/pre-salting,
time-dependent measurement, 532 235
viscosity measurement, 532–3 mechanisms, 228–32
see also Texture method of brine-salting, 233
Rhizomucorprotease, 20 methods of salting, 228
Ripening of cheese, 12, 14, 347, 375, 395 moisture content of cheese, 98, 241–3
acceleration, 357 pH of curd/brine, 237
agents: salting time, 234–5
cathepsin D, 355–6 temperature of brine/cheese, 240
NSLAB, 356–7 temperature of curd/brine, 235
other indigenous enzymes, 356 uptake in Cheddar curd, 237–9
plasmin, 355 casein hydration/physical properties of cheese:
rennet, 354–5 cooking properties, 224–5
starter enzymes, 356 microstructure, 223
biochemical activities of NSLAB, 302–303 model systems, 220–3
catabolism of amino acids, 350–2, 435 rheology, 223–4
aromatic, 443–7 control of microbial growth, 208–12
branched-chain, 447–9 effect on cheese composition:
deaminases, 449 lactose content/pH, 249
decarboxylases, 449–51 moisture level, 247–9
other, 451 salt content, 249
transamination, 435–9 enzyme activity:
volatile sulphur compounds, 439–43 coagulant, 212–13
glycolysis of residual lactose/catabolism of microbial enzymes, 214–15
lactate, 347–9 milk proteinase, 213–14
lipolysis/metabolism of fatty acids, 349 gel formation, 83–4
proteolysis, 350–2, 391–2 quality, 596
quality, 596–9, 605–606 reduced-sodium cheese:
see also Proteolysis, Lipolysis; Catabolism of lactate; Cheddar, 226–7
Catabolism of amino acids; Catabolism of fatty Cottage cheese, 227
acids other cheeses, 227
616 Index

Salt – contd. exopolysaccharide production, 137–8

ripening/quality: growth, 134–5
Blue cheese, 219 lipases/esterases, 134
Camembert, 219 metabolic engineering, 135–6
Cheddar, 216–19 nitrogen, 131–4
other cheeses, 219–20 stress responses, 137
salt/moisture equilibria in brine-salted cheese sugar, 130
after salting, 244–7 mixed-strain mesophilic, 9–10, 164–5
salt/moisture equilibrium in Cheddar cheese, pH control, 139–40
246–7 phage infection, 127, 139
water activity (aw), 215–16 preparation:
Sbrinz, 127 preservation/distribution, 140–2
Secondary/adjunct cultures: propagation, 138–40
coryneform bacteria and staphylococci, 195, 197–8 time/temperature combination, 140
effect on quality, 603 quality, 592–4
heterofermentative lactobacilli, 200–202 taxonomy, 123–4
moulds, 193–5 types, 126, 128, 164
non-starter lactic acid bacteria, 7, 289, 291, 353 new sources, 129
propionic acid bacteria, 198–200 primary, 123, 124, 126, 191
yeasts, 191–3 secondary, 123, 191, 195
see also Starter cultures see also Bacteriophage
Semi-hard cheese, 273 Stilton, 193, 194, 208
Sensory characteristics of cheese, 455, 463–5 Streptococcus thermophilus, 123, 157
cheese preferences, 455–6 Stress:
consumer preferences, 14, 480–1 relaxation modulus, 540
definition, 455 relaxation test, 540
evaluation methods, 467–8 Swiss cheese, 128
consumer acceptability testing, 478 contamination, 545, 552
descriptive analysis, 475–7 flavour, 335, 338, 492
discrimination tests, 475 microbiology, 289, 296, 297
grading/quality scoring, 468–75 raw milk, 323, 327, 328
time-intensity analyses, 477–8 Syneresis of rennet-coagulated curd, 71, 114
human senses/sensory properties: during curdmaking, 84–5
cheese appearance, 462, 466 effects of other process variables:
cross-modal interactions, 466–7 acidity, 90
flavour, 466 coagulation, 89
texture, 466 heat treatment of milk, 88
universal language, 480 high-pressure treatment, 91
variety, 456–62 homogenization of milk, 88
see also Aroma, compounds; Flavour temperature, 89–90
Shear modulus, 540 ultrafiltration, 90–1
Sheep milk cheese, 1, 5, 14 various additions to milk, 88–9
Soft cheese, 270–3, 552–3 washing of curd, 90
Spanish artisanal cheeses, 297–8, 306 mechanisms, 78–9
Staphylococcus, 195, 197, 304 methods for estimating:
antimicrobial activities, 198 effects of curd grain size, 86–7
form/use of adjunct culture, 198 modelling process, 81–4
lipolysis, 198 rate equations, 85–6
proteolysis, peptidolysis, amino acid catabolism, 198 stirring, 87
selecting surface bacteria as adjuncts: renneted milk, 80–1
effect on colour of cheese surface, 197–8 review of, 91–2
growth, 197 unified approaches to gel formation/syneresis:
Starter cultures, 123, 149, 287, 288–9, 348 acid gels, 77–8
defined-strain, 164–5 behaviour during processing, 92–3
genomics, 129–30, 158–9 compaction of curd column, 94–5
mesophilic starter genetics: curd fusion, 93–4
bacteriocins, 153–4 effect of milk composition, 91
bacteriophage, 154–5 gel formation, 73–5
chromosome, 149–50 renneting, 72–3
industrially important traits, 151–5 rheological characteristics, 75–7
lactose/citrate metabolism, 151–2 under pressure, 93
Leuconostoc, 155–6 water content of cheese, 95–8
manipulation, 155
plasmids, 150–1 Texture:
proteolysis/amino acid catabolism, 152–3 terminology, 533
metabolism: evaluation:
autolysis, 136 instrumental shear deformation, 535–6
bacteriocins, 136–7 texture profile analysis (TPA), 534
citrate, 130–1 see also Rheology
 Index 617

Thermophilic starters, 56, 126 protein-standardized milk, 267–8

Lactobacillus spp.: see also Membrane processing
genetic manipulation, 157 Uniaxal compression, 527–8
important traits, 156–7 compressive strength, 540
Streptococcus thermophilus: effect of deformation rate, 529–30
genetic manipulation, 158 effect of pre-test strain history, 529
important traits, 157–8 effect of sample-machine interface conditions/sample
Tilsit, 306 dimensions, 529
Tomme, 193, 199 influence of shape, 530
Torulospora delbrueckii, 196 relationship between shear/normal stresses,
Toughness, 540 528
Toxins:
biogenic amines: Viscoplastic material behaviour, 540
formation, 562–3 Viscosity/dynamic viscosity, 540
histamine, 561–2 Volatile compounds, 327–8
in cheese, 563 alcohols, 331–2
mycotoxins: carbonyl compounds, 330–1
direct contamination of cheese, 567–8 esters, 332
fate in cheese during manufacture/ripening, 567 lactones, hydrocarbons, 334–5
indirect contamination, 564, 567 sulphur compounds, 332–4
production of toxic metabolites in cheese, 567 volatile fatty acids (VFA), 328–30

Ultrafiltration (UF), 8–9, 90–1, 265–75, 596
cheese quality:
functionality, 274–5
proteolysis/ripening characteristics, 274
texture, 273–4
liquid pre-cheeses:
fresh unripened cheeses, 269–70
other applications, 273
semi-hard cheese, 273
soft cheese, 270–3
medium/intermediate concentrated retentates:
APV-sirocurd process, 268
general considerations, 269
other cheeses, 269
structured Feta-like cheese, 268–9
properties of UF retentates:
buffering capacity, 265–6
rennet coagulation, 266–7
rheological behaviour, 266
Water-soluble extract (WSE), 493–4, 496
Whey, 105
heat treatment, 9
incubation, 9
preparation, 127
separation/syneresis, 114–15
Yeast, 36, 191, 306–307, 597
forms/use as adjunct culture, 193
interactions with other microorganisms, 192–3
species found in cheeses, 191–2
useful properties in selecting adjuncts:
effect on appearance of cheese surface, 192
lipolytic activity, 192
production of aroma, 192
proteolytic activity, 192
utilisation of residual sugars/lactate de-acidification
activity, 192
Young’s modulus, 540
This Page Intentionally Left Blank
Plate 1 An arrangement of multi-channel geometry ceramic membranes (courtesy of GEA filtration, Hudson, WI, USA). (See
page 263.)

Plate 2 Plate and frame UF system (courtesy of GEA filtration, Hudson, WI, USA). (See page 263.)
Plate 3 Spiral-wound UF membranes (courtesy of GEA filtration, Hudson, WI, USA). (See page 264.)
Plate 4 Vibrating membrane system (courtesy of Pall Corporation, Portsmouth, UK). (See page 264.)
Plate 5 Commercial UF system for the production of fresh cheese from pH 4.6 milk (courtesy of TIA, Bollene, France). (See
page 270.)
Plate 6 A selection of cheeses made by UF using the liquid pre-cheese concept. (See page 272.)
Plate 7 A UF plant for producing Feta cheese by the MMV process (courtesy of TIA, Bollene, France). (See page 272.)
Plate 8 Farm with an UF facility in Dexter, New Mexico, USA (courtesy of North American Milk Products, LLC, St Louis, Missouri, USA).
(See page 276.)
 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Plate 9 Urea-polyacrylamide gel electrophoretograms water-insoluble fraction of a selection of cheese varieties. Lane 1 Na caseinate,
lane 2 Cheddar, lane 3 extra-mature Cheddar, lane 4 Cheshire, lane 5 Red Leicester, lane 6 Double Gloucester, lane 7 Emmental,
lane 8 Leerdammer, lane 9 Jarlsberg, lane 10 Vorarlberger Bergkase, lane 11 Edam, lane 12 Gouda, lane 13 Norvegia, lane 14
Parmesan, lane 15 Parmesan (from McGoldrick, 1996). (See page 416.)

Chymosin
1 199

Chymosin
1 23 24 199

Lc-CEP
102 199
f1-9, f-1-13

Further hydrolysis products

Plate 10 Schematic representation of the early proteolysis of s1-casein during the ripening of many cheeses and the location of
peptides produced on a urea-polyacrylamide gel electrophoretogram and a reverse-phase HPLC elution profile. (See page 419.)

(a) (b)

Plate 11 Vane rheometer probe before (a) and during (b) shear test on process cheese. Photos courtesy of Truong and Daubert
(2000) Gel Consultants Inc. (See page 531.)