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ENZYMES IN FARM ANIMAL NUTRITION
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ENZYMES IN FARM ANIMAL

NUTRITION
Edited by
MICHAEL R. BEDFORD
and
GARY G. PARTRIDGE
Finnfeeds
Marlborough
Wiltshire
UK
CABI Publishing
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CABI Publishing is a division of CAB International
CABI Publishing CABI Publishing

CAB International 10 E 40th Street
Wallingford Suite 3203
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© CAB International 2001. All rights reserved. No part of this publication may be
reproduced in any form or by any means, electronically, mechanically, by photocopying,
recording or otherwise, without the prior permission of the copyright owners.
A catalogue record for this book is available from the British Library, London, UK.
Library of Congress Cataloging-in-Publication Data

Enzymes in farm animal nutrition / edited by M.R. Bedford and G.G. Partridge.
p. cm.
Includes bibliographical references.
ISBN 0-85199-393-1 (alk. paper)
1. Enzymes in animal nutrition. 2. Feeds--Enzyme content. 3. Animal feeding. I.
Bedford, M. R. (Michael Richard), 1960- II. Partridge, G. G. (Gary G.), 1953-
SF98.E58 E59 2000

636.08′52--dc21 00-044426
ISBN 0 85199 393 1
Typeset in Garamond by AMA DataSet Ltd

Printed and bound in the UK by Biddles Ltd, Guildford and King’s Lynn
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Contents
Contents
Contributors vii
Preface ix
1. The Current Feed Enzyme Market and Likely Trends 1

C. Sheppy
2. Enzymology and Other Characteristics of Cellulases and Xylanases 11

M.K. Bhat and G.P. Hazlewood
3. Enzymatic Characteristics of Phytases as they Relate to Their Use in

Animal Feeds 61
D.D. Maenz
4. Analysis of Feed Enzymes 85

B.V. McCleary
5. Maize: Factors Affecting its Digestibility and Variability in its

Feeding Value 109
J.D. Summers
6. Vegetable Protein Meals and the Effects of Enzymes 125

J. Thorpe and J.D. Beal
7. Enzyme Supplementation of Poultry Diets Based on Viscous Cereals 145

M. Choct
8. The Role and Efficacy of Carbohydrase Enzymes in Pig Nutrition 161

G.G. Partridge
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vi Contents
9. Interaction between Cereal Identity and Fat Quality and Content in

Response to Feed Enzymes in Broilers 199
S. Dänicke
10. Digestion of Phosphorus and Other Nutrients: the Role of Phytases

and Factors Influencing Their Activity 237
E.T. Kornegay
11. Enzymes in Ruminant Diets 273

T.A. McAllister, A.N. Hristov, K.A. Beauchemin, L.M. Rode and
K.-J. Cheng
12. Microbial Interactions in the Response to Exogenous Enzyme

Utilization 299
M.R. Bedford and J. Apajalahti
13. Enzymes: Screening, Expression, Design and Production 315

K. Clarkson, B. Jones, R. Bott, B. Bower, G. Chotani and T. Becker
14. Liquid Application Systems for Feed Enzymes 353

P. Steen
15. Process Stability and Methods of Detection of Feed Enzymes in

Complete Diets 377
M.R. Bedford, F.G. Silversides and W.D. Cowan
16. Future Horizons 389

R.R. Marquardt and M.R. Bedford
Index 399
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Contributors
Contributors
J. Apajalahti, Danisco-Cultor Innovation, Kantvik, FIN-02460, Finland

J.D. Beal, University of Plymouth, Seale-Hayne Faculty, Newton Abbot, Devon
TQ12 6NQ, UK
K.A. Beauchemin, Agriculture and Agri-Food Canada, Lethbridge Research Centre,
PO Box 3000, Lethbridge, Alberta, Canada T1J 4B1
T. Becker, Genencor International Inc., 925 Page Mill Road, Palo Alto, CA
94304-1013, USA
M.R. Bedford, Finnfeeds, PO Box 777, Marlborough, Wiltshire SN8 1XN, UK
M.K. Bhat, Food Materials Science Division, Institute of Food Research, Norwich
Research Park, Colney, Norwich NR4 7UA, UK
R. Bott, Genencor International Inc., 925 Page Mill Road, Palo Alto, CA
94304-1013, USA
B. Bower, Genencor International Inc., 925 Page Mill Road, Palo Alto, CA
94304-1013, USA
K.-J. Cheng, Faculty of Agricultural Sciences, University of British Columbia,
Vancouver, British Columbia, Canada V6T 1Z4: Present address; Institute of
BioAgricultural Sciences, Academia Sinica, Taipei, Taiwan
M. Choct, School of Rural Science and Natural Resources, Department of Animal
Science, The University of New England, Armidale, NSW 2351, Australia
G. Chotani, Genencor International Inc., 925 Page Mill Road, Palo Alto, CA
94304-1013, USA
K. Clarkson, Genencor International Inc., 925 Page Mill Road, Palo Alto, CA
94304-1013, USA
W.D. Cowan, Novo Nordisk, Krogshoevej 36, Bagsvaerd, Denmark 2880
S. Dänicke, Institute of Animal Nutrition, Federal Agricultural Research Centre,
Braunschweig (FAL), Bundesallee 50, D-38116 Braunschweig, Germany
G.P. Hazlewood, Finnfeeds, PO Box 777, Marlborough, Wiltshire SN8 1XN, UK
A.N. Hristov, Agriculture and Agri-Food Canada, Lethbridge Research Centre,
B. Jones, Genencor International Inc., 925 Page Mill Road, Palo Alto, CA
94304-1013, USA
vii
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viii Contributors
E.T. Kornegay (deceased), Department of Animal and Poultry Sciences, Virginia

Polytechnic Institute and State University, 3060 Litton-Reaves Hall,
Blacksburg, VA 24061, USA
D.D. Maenz, Prairie Feed Resource Centre (Canada) Inc., Department of Animal
and Poultry Science, University of Saskatchewan, Saskatoon, Saskatchewan,
Canada S7N 0W0
R.R. Marquardt, Department of Animal Science, University of Manitoba,
Winnipeg, Manitoba, Canada R3T 2N2
T.A. McAllister, Agriculture and Agri-Food Canada, Lethbridge Research Centre,
B.V. McCleary, Megazyme International Ireland Limited, Bray Business Park, Bray,
Country Wicklow, Ireland
G.G. Partridge, Finnfeeds, PO Box 777, Marlborough, Wiltshire SN8 1XN, UK
L.M. Rode, Agriculture and Agri-Food Canada, Lethbridge Research Centre,
PO Box 3000, Lethbridge, Alberta, Canada T1J 4B1: Present address; Rosebud
Technology Development Ltd, Lethbridge, Alberta, Canada T1K 3J5
C. Sheppy, Finnfeeds, PO Box 777, Marlborough, Wiltshire SN8 1XN, UK
F.G. Silversides, Denman Island, British Columbia, Canada
P. Steen, Finnfeeds, PO Box 777, Marlborough, Wiltshire SN8 1XN, UK
J.D. Summers, Poultry Industry Centre, RR#2, Guelph, Ontario, Canada
N1G 6H8
J. Thorpe, University of Bristol, Department of Clinical Veterinary Science,
Langford House, Langford, Bristol BS40 5DU, UK
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Preface
Preface
The rearing and feeding of domestic animals and the use of enzymes in processes
such as brewing have been distinct parts of human life for many thousands of
years, but it is only recently that these two disciplines have crossed paths. The first
commercial use of feed enzymes dates back to 1984 in Finland, where opportunities
existed to improve significantly the nutritional quality of barley-based rations by
inclusion of enzymes derived from the brewing industry (A. Haarisilta, Suomen
Rehu). The years since then have seen an exponential increase in the usage of many
enzyme types in rations for poultry and, to a lesser extent, swine. Significant recent
interest has also been shown by the ruminant sector. Scientific studies describing the
use of exogenous enzymes in animal nutrition dates back to the mid 1920s and
they now number in excess of 1300 papers for broilers alone (Rosen, 2000, personal
communication). This rapidly expanding field is becoming increasingly multi-
disciplinary as more is understood of the mode of action of feed enzymes. Due to
the complexity of this field it is timely to produce a single source from where the
uninitiated and well versed alike can ground themselves with the basics required to
grasp the subject. Furthermore, it is the intention of this book to provide sufficient
detail to enable an understanding of the complexities of response to such products in
all classes of farm animal livestock.
It is of interest that not only the usage of enzymes has increased but also the
scope of their use. This is likely to continue as the price of enzyme application falls,
with improvements in both enzyme efficacy and production costs making previously
uneconomic solutions more attractive. Particular attention is drawn to the ruminant
sector, where usage is in its infancy but research is demonstrating that significant
gains can be made. The challenge is to find methods of predicting enzyme response
so that enzyme application in all classes of livestock becomes increasingly a science
rather than an art.
ix
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x Preface
In memoriam
It is with great sadness that this book is produced after the death of Dr E.T.
Kornegay, a significant contributor to this publication and to the field of phytase
research in poultry and pigs. Dr Kornegay was particularly active and enthusiastic in
his enzyme phytase research and will be sadly missed.
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The Current Feed Enzyme

Market and Likely Trends
C. SHEPPY
1
Finnfeeds, PO Box 777, Marlborough, Wiltshire, SN8 1XN, UK
C. Sheppy
The
1 Current Market and Likely Trends
Global Animal Feed Production

As with any publication dealing with the application of a particular technology to a
certain industry, it is important to assess the commercial environment in which the
technology is being applied.
Despite the general euphoria and enthusiasm surrounding the dawn of a new
millennium, the challenges faced by the global animal feed industry have a familiar
feel. Human health, environmental safety and animal welfare are all issues that
continue to be to the fore of consumers’ minds and are at the top of the agenda of
the media and politicians alike. Although tending to emerge in Europe and North
America these issues are now of worldwide importance, a development that mirrors
the increasing globalization of the food chain and feed industry. Recent controversies
surrounding genetically modified crops, antibiotic growth promoters and dioxin-
contaminated feedstuffs provide some very real examples of this. A direct result
of these ‘health scares’ has been the adoption, and in some cases the imposition, of
increased regulatory, testing and quality control procedures aimed at maintaining
and improving the trust between animal producers and the ultimate consumers of
their products.
Faced with these challenges, how is the industry currently faring? Although
global compound feed production actually fell by 5% in 1998 to 575 Mt (the first
such fall in over 50 years), 1999 experienced something of a recovery, albeit small, to
586 Mt (Table 1.1).
The Asian economic crisis of 1997–1998 saw the collapse of the feed industry in
many previously booming markets and with the benefit of hindsight it is probably
fair to say that the previous rates of growth were unsustainable. As demand for meat,
milk and eggs tumbled, so did feed production: Indonesian feed output plummeted
by 40%; Korean production fell by 30%; Thai output was down nearly 25%. Even
the Chinese market appeared to stumble, with an approximate 2% decline in
industrially manufactured feed to below 55 Mt. These Asian falls were followed by a
general stagnation of the global feed market in 1998, with EU feed production falling
by 6% and the non-EU European countries witnessing a 12% decline. Only North
©CAB International 2001. Enzymes in Farm Animal Nutrition
(eds M.R. Bedford and G.G. Partridge) 1
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2 C. Sheppy
Table 1.1. Global animal feed production, 1998 and 1999. Source: Gill (1999, 2000).
1998 production 1999 production Percentage difference

(Mt) (Mt) (1999/1998)
North America 159.8 160.7 +0.6

Asia-Pacific 128.1 132.2 +3.2
European Union 114.9 116.4 +1.3
Latin America 63.3 65.4 +3.3
Non-EU Europe 47.4 48.6 +2.5
Middle East and Africa 21.6 24.0 +11.1
Other 39.9 38.7 −3.1
Total 575.0 586.0 +2.0
America (+1%), Latin America (+1%) and the Middle East and African countries
(+10%) experienced any growth in their feed industries in 1998. As clearly seen in
Table 1.1, the recovery in 1999 was global, with all major regions showing some
growth as the Asian economies improved and demand picked up in other markets
(Gill, 2000).
Despite the fall, or correction, in feed production seen in 1998, one trend within
the feed industry continues unabated – the rationalization of capacity and the
concentration and integration of production into the major food-producing
companies. Driven by the need for cost reduction, increased regulatory requirements
and hygiene and safety standards, fewer than 3800 feed mills now manufacture more
than 80% of the world’s compound feed. However, there is still plenty of room for
further consolidation and rationalization as the ten largest feed manufacturers still
only account for approximately 9–10% of global output.
Although pioneered in the poultry industry, food company integration now
extends to most of the major farmed species – poultry, swine, beef, dairy cattle and
aquatic. Swine feed is still the largest proportion of feed produced but broiler feed
production continues to grow at the expense of beef cattle feeds (Table 1.2), spurred
on by increased consumer demand for cheap, safe and healthy meat products (Gill,
2000).
So what does the future hold for the feed industry? Most informed
commentators believe that the drop in feed production seen in 1998 was a ‘one-off’
or correction caused by the dramatic collapse experienced by the Asian economies.
Muller (1999) comments that:
in the last 25 years compound feed production has always grown parallel with the
increase in world population. The decline in per capita ‘consumption’ of compound
feed from 104 kg to 97 kg in 1998 does not represent a reverse trend in global com-
pound feed production, but instead must be categorized correctly in conjunction with
the Asian crisis.
With the world population set to grow from the current 6 billion to approximately
7.8 billion over the coming two decades, the demand for food, including animal
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The Current Market and Likely Trends 3
Table 1.2. Global animal feed production by species. Source: Hoffman (1999).
Sector 1993 (%) 1998 (%)
Swine 31 31
Broiler 24 27
Beef 11 9
Dairy 17 17
Layer 8 8
Aquatic 3 5
Other 6 3
protein, is set to increase at an even greater rate as the growing populations in the
developing economies alter their dietary habits. Set against this scenario, the future
for compound feed production looks positive. However, those companies who wish
to continue in the industry and expand their businesses will need to become more
adept at operating in a market environment where many of the key influencing
factors are beyond their control.
Background to Enzyme Technology

Enzymes exist practically everywhere. They are naturally occurring and are produced
by all living organisms and, as nature’s catalysts, they speed up the chemical reactions
that enable all living things, from simple single-celled organisms, through plants and
insects to humans, to function. Without them food could not be digested.
So far over 3000 different enzymes have been discovered. Enzymes, as with all
proteins, are made from chains of amino acids. They speed up or catalyse reactions
by binding to their substrate and ‘stabilize’ the entire reaction process through to
product formation, so that far less activation energy is required to move the reaction
forwards. As a result, the rate of reaction progression is greatly increased for any given
energy status. It is the three-dimensional shape and position of the reactive amino
acids within the molecule that confer the catalytic properties of enzymes. Conditions
that significantly alter the structure of the active enzyme often result in loss of
activity. As a result, enzymes are very sensitive to the environment in which they
function and many work best in mild temperatures and mid-range pH.
Humans have made use of enzymes, often unknowingly, throughout history.
Cheese making and the use of malted barley in brewing are examples of the
harnessing of the power of enzymes. Modern enzyme technology really started in
1874 following the first documented production of a refined enzyme that was
prepared from the contents of calves’ stomachs. That enzyme, rennet, is still used
today in cheese manufacture.
Since then the technology to identify, extract and produce enzymes on a
commercial scale has progressed dramatically and they are now used in many
industrial processes. Currently enzymes are used in detergents, paper production
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4 C. Sheppy
and in leather and textile processing. In addition, they are widely used in the food
and drink industry in products ranging from bread and cheese to fruit juice, wine and
coffee. The global market value for enzymes in 1995 was estimated to be worth US$1
billion (Godfrey and West, 1996) and is forecast to rise to between US$1.7 billion
and US$2 billion by 2005.
Why use enzymes in animal feeds?
The principal rationale for the use of enzyme technology is to improve the nutritive
value of feedstuffs.
All animals use enzymes in the digestion of food, those produced either by the
animal itself or by the microbes present in the digestive tract. However, the digestive
process is nowhere near 100% efficient; for example, swine are unable to digest
15–25% of the food they eat. Therefore, the supplementation of animal feeds with
enzymes to increase the efficiency of digestion can be seen as an extension of the
animal’s own digestive process.
In many animal production systems feed is the biggest single cost and
profitability can depend on the relative cost and nutritive value of the feeds available.
Often, the limiting factor when formulating rations is the animal’s ability to digest
different constituent parts of the feed raw materials, particularly fibre. Despite recent
advances, the potential nutritional value of feedstuffs is not achieved at the animal
level. This inefficiency in the utilization of nutrients can result in a cost to the farmer,
the food company and the environment. Put simply, there are four main reasons for
using enzymes in animal feed:
1. To break down anti-nutritional factors that are present in many feed ingredients.
These substances, many of which are not susceptible to digestion by the animal’s
endogenous enzymes, can interfere with normal digestion, causing poor performance
and digestive upsets.
2. To increase the availability of starches, proteins and minerals that are either
enclosed within fibre-rich cell walls and, therefore, not as accessible to the animal’s
own digestive enzymes, or bound up in a chemical form that the animal is unable to
digest (e.g. phosphorus as phytic acid).
3. To break down specific chemical bonds in raw materials that are not usually
broken down by the animal’s own enzymes, thus releasing more nutrients.
4. To supplement the enzymes produced by young animals where, because of the
immaturity of their own digestive system, endogenous enzyme production may be
inadequate.
In addition to improving diet utilization, enzyme addition can reduce the variability
in nutritive value between feedstuffs, improving the accuracy of feed formulations.
Trials have shown that ensuring feed consistency in this way can increase the unifor-
mity of groups of animals, thus aiding management and improving profitability. The
general health status of animals can also be indirectly influenced, resulting in fewer of
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the non-specific digestive upsets that are frequently provoked by fibre components in
the feed.
Of increasing importance and relevance to the feed industry are the environ-
mental benefits of harnessing enzyme technology. Since the animal better utilizes the
feed, less is excreted. This results in manure volume being reduced by up to 20% and
nitrogen excretion by up to 15% in pigs and 20% in poultry. As significant is the
opportunity for enzymes to reduce phosphorus pollution.
How do they work and how are they used in animal feeds?
Broadly speaking, four types of enzymes currently dominate the animal feed market:
enzymes to break down fibre, protein, starch and phytic acid.
Fibre-degrading enzymes
One of the main limitations to digestion is the fact that monogastrics (pigs and poul-
try) do not produce the enzymes to digest fibre. In diets containing ingredients such
as wheat, barley, rye or triticale (the main ‘viscous’ cereals), a large proportion of this
fibre is soluble and insoluble arabinoxylan and β-glucan (White et al., 1983; Bedford
and Classen, 1992). The soluble fibre can increase the viscosity of the contents of the
small intestine, impeding the digestion of nutrients and thereby reducing the growth
of the animal. It has also been linked with the incidence of digestive disorders such as
non-specific colitis in swine, and sticky litter and hock burns in poultry.
The fibre content of wheat and barley can vary considerably according to
variety, growing location, climatic conditions, etc. This in turn means that there can
be considerable variability in the nutritional value of these ingredients and hence
diets containing them. In breaking down the fibre, enzymes (e.g. xylanase targeting
arabinoxylans, β-glucanase targeting β-glucans) can reduce this variability in
nutritional value, giving rise to improvements in the performance of the feed and the
consistency of the response. An added benefit is the reduced incidence of certain
digestive disorders.
Protein-degrading enzymes
Various raw materials contribute to the protein content in the diet and ultimately the
amino acids that fuel lean meat deposition. There is considerable variability in the
quality and availability of protein from the different raw materials typically found in
monogastric diets. Within the primary vegetable protein sources such as soybean
meal, certain anti-nutritional factors (ANFs), such as lectins and trypsin inhibitors,
can lead to damage to the absorptive surface of the gut, impairing nutrient digestion.
In addition, the underdeveloped digestive system of young animals may not be able
to make optimal use of the large storage proteins found in the soybean meal (glycinin
and β-conglycinin).
The addition of a protease can help to neutralize the negative effects of the
proteinaceous ANFs in addition to breaking down the large storage protein
molecules into smaller, absorbable fractions.
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6 C. Sheppy
Starch-degrading enzymes
To many nutritionists maize is viewed as the ‘gold standard’ of raw materials. Most
nutritionists do not consider maize digestion as being poor: in fact most would argue
that it is better than 95% digested. However, recent evidence presented by Noy and
Sklan (1994) suggests that, at the ileal level, starch digestibility rarely exceeds 85% in
broilers between 4 and 21 days of age. The addition of an amylase to animal feed can
help to expose the starch more rapidly to digestion in the small intestine, and in
doing so lead to improved growth rates from enhanced nutrient uptake.
At weaning, piglets often suffer a growth check because of changes in their
nutrition, environment and immune status. The addition of an amylase, usually in
conjunction with other enzymes, to augment the animal’s endogenous enzyme
production has been shown to improve nutrient digestibility and absorption and,
hence, growth rate for a range of diets (Close, 1995).
Phytic acid-degrading enzymes

Phosphorus is required for bone mineralization, immunity, fertility and growth and
is an essential mineral for all animals. Swine and poultry digest only about 30–40%
of the phosphorus found in feedstuffs of vegetable origin, with the remainder being
tied up in a form inaccessible to the animal – phytic acid. In many instances
additional phosphorus must be added to the diet to meet the animal’s requirement.
More than half of the phosphorus consumed from such feedstuffs is excreted in the
faeces, which can result in major environmental pollution. By adding a phytase to
the diet, the phytic acid is broken down, liberating more of the phosphorus for use by
the animal.
The two main benefits of phytase supplementation are, firstly, the reduction in
feed costs from the reduced additional supplementation of phosphorus to the diet
and, secondly, environmental from reduced excretion of waste products and the
threat of pollution.
The Current Feed Enzyme Market

It should come as no surprise that the adoption of enzyme technology by the animal
feed industry has, to a large degree, been driven by the mode of action of these four
particular types of enzymes. Taken in conjunction with the different structure of the
various segments of the animal feed industry, the background to the adoption of
the technology becomes even clearer.
The global poultry industry, particularly the broiler industry, is highly
integrated and dominated by a relatively small group of large companies. For
example, Tyson’s alone in the USA is estimated to produce some 2 billion broilers
per year, considerably larger than the entire annual production of the UK poultry
industry. The company controls every step of the production chain from the growing
of the raw materials and the breeding of the birds through to the processing of the
meat and ultimately the retailing of the finished product. This integration and
concentration of the majority of industry capacity in a relatively small number of
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hands makes the adoption of new technology a rapid occurrence, particularly when
the value of the opportunity is significant. The value of enzyme addition to broiler
feeds will typically deliver a return on investment (ROI) well in excess of 2 : 1.
By contrast the swine industry is considerably more fragmented, with many
more producers and steps in the chain. In addition, the efficacy response to enzyme
addition has been more variable and harder to measure commercially.
Although first considered in the 1950s, it was not until the 1980s that the
animal feed industry really began to understand how to harness the power of enzyme
technology properly.
As already discussed, feed grains such as wheat and barley contain relatively
high levels of fibre that monogastrics are unable to digest. If the problem element of
the fibre can be broken down, the animal has greater access to the available nutrients,
so overcoming the negative impacts of incorporating the ingredient in the diet.
Spurred on by a plentiful supply of cheap barley, European poultry nutritionists
and enzymologists investigated the opportunity to reduce the negative impact of
including barley in broiler diets by adding β-glucanase to the diet. This proved to be
a success with the ‘rule of thumb’ being adopted that ‘barley + β-glucanase = wheat’.
Encouraged by the outstanding success of this approach, wheat became the
next target for enzymatic enhancement via the use of xylanase. Work this time
concentrated on the hypothesis that ‘wheat + xylanase = maize’.
Again this approach was successful and the mid-1990s saw the rapid acceptance
of the application of enzyme technology to the animal feed industry. It is probably no
exaggeration to say that by 1996 in excess of 80% of all European broilers diets that
contained a viscous cereal (wheat, barley, etc.) would have also contained a fibre-
degrading enzyme – certainly an impressive adoption rate for a new technology in the
feed industry.
Taking a global perspective across all species and diet segments, current
estimates of market penetration suggest that approximately 65% of all poultry feeds
containing viscous cereals also contain a fibre-degrading enzyme. Given the very
different structure of the industry and the difficulties in accurately measuring an
efficacy response, it is no surprise that current penetration into the swine industry is
considerably lower, approaching 10%.
In terms of geographical spread, the utilization of fibre-degrading enzymes is
concentrated on those regions where viscous cereals are the predominant energy
source, namely Europe, Canada and Australia/New Zealand. (This is not to discount
completely the USA, South America and the rest of Asia-Pacific, where there are reg-
ular ‘window’ opportunities depending on the post-harvest price ratio between maize
and, for example, wheat.) It is for this reason that fibre-degrading enzymes are largely
seen as a ‘European’ niche product. To gain global acceptance it will be necessary for
the enzyme manufacturers to break significantly into the North American and
Asia-Pacific maize–soybean markets. Maize–soybean diets are historically regarded as
the ‘gold standard’ in terms of consistency of animal response, though most nutri-
tionists recognize that these raw materials are more variable than is often presumed.
There is increasing evidence and acceptance that this ‘gold standard’ can be
improved upon and that there is a role for enzymes in these diets that do not
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8 C. Sheppy
superficially present a problem related to fibre or viscosity. The focus of considerable

research-and-development (R&D) dollars over the last 10 years has seen the first
generation of maize–soybean enzymes for poultry diets, launched in 1996. Despite
initial mixed results the industry is now beginning to understand more fully how best
to apply this technology to reap an economic reward. Current estimates suggest that
this market segment is worth approximately US$20 million and that 5% of broiler
diets based upon maize–soybean now contain an enzyme. The combined market
for these viscous and non-viscous (carbohydrase) enzymes is currently (1999/2000)
estimated to be in excess of US$100 million.
One enzyme concept that already has both global acceptance and global
application is phytase. The type of carbohydrate source present does not drive its
mode of action, as phytic acid is present at varying levels in all vegetable feed raw
materials. Estimates suggest that the phytase market is currently worth up to US$50
million, with approximately 8% of global swine and poultry feeds containing
phytase. This again is an exceptional adoption rate for a new technology, given the
short period since the concept was launched.
The driving force behind this success has been increasing concern about the
impact of phosphorus on environmental pollution. In a number of countries
legislation has either been imposed (e.g. The Netherlands) or is being imposed (e.g.
the Delmarva Peninsula in the USA) on pig and poultry farmers. The historical
application of phytase is to replace a proportion of the supplementary calcium
and phosphorus added to the diet, but now phytase suppliers are increasingly
concentrating on the apparent potential of the product to improve the availability of
certain other nutrients (e.g. amino acids and trace minerals) in the diet. The aim is to
improve the ROI and so the uptake of this new technology.
The largest applications for phytase are currently into laying hens and swine,
given the scope to remove larger quantities of supplementary phosphorus and the
ease of application (note that many layer and swine diets are not heat treated). There
remains significant scope to increase phytase application and application uptake
in the broiler segment as the value equation improves and the enzyme industry
overcomes the issues surrounding liquid application and the thermostability of
enzymes.
Future Needs and Opportunities

The adoption of enzyme technology has forced the animal feed industry to challenge
traditional assumptions about diet formulation, ingredient selection, nutrient
requirements and the desired productive response. Ten years ago many European
nutritionists viewed wheat in the same light as North Americans view maize –
basically as an ingredient that could not be improved. As enzyme technology
becomes more accepted and widespread throughout the global animal feed industry,
so the enzyme producers must continue to develop new products and applications
to overcome the existing obstacles to growth, as well as to offer new solutions and
opportunities.
18
17 November 2000 14:57:25

The thermal stability of enzymes and their ability to survive the heat processing
steps in the manufacture of pelleted animal feed is of major concern to the industry.
This, along with concerns over enzyme assays and the need for full traceability
throughout the food chain, will force the enzyme manufacturers to increase their
efforts to discover new enzyme products that can survive the increasingly high
processing temperatures, deliver in vivo efficacy and be recovered in standard quality
control procedures. Alternatively, improved post heat treatment liquid application
systems will offer the industry short- to medium-term solutions.
The adoption of existing enzyme technology in the animal feed sector has
increased as knowledge of the interaction between the enzyme, its substrate and the
environment of the animal’s gut has evolved and improved. It is very likely that the
increased uptake of enzymes in the maize–soybean type diets will be driven by an
enhanced understanding of which ingredients will respond to enzyme treatment and
the amount of enzyme required to deliver the most economic and cost-effective
response. The ultimate scenario would see a computerized and automated ‘on-line’
process that tests the raw material as it enters the feed mill and then doses the
economically optimal amount of enzyme into the mixer as the feed is produced.
The decision by the EU authorities to ban the use of certain antibiotic growth
promoters, and the knock-on effect in other countries around the world, has forced
feed manufacturers and meat producers to explore alternatives. Enzymes are regularly
put forward as a technology that will allow producers to continue to produce safe,
nutritious and cheap meat, milk and eggs whilst meeting the ever increasing
consumer demand for these products. It is important to note here that there are many
products being promoted opportunistically on the back of the antibiotic growth
promoter ban. When searching out realistic and sustainable solutions to fill this gap,
it is important that the feed industry is rigorous and consistent in its demand for
scientifically credible technologies and products.
Despite the impressive uptake of the technology, only approximately 10% of
monogastric feeds today contain a feed enzyme, giving a total market value in excess
of US$150 million. As such, perhaps the enzyme supply industry needs to ask some
searching questions of itself as to why uptake has not been faster, particularly in those
applications where there appears to be a sound commercial case. Standardized and
more transparent quality control procedures, improved heat stability, more accurate
liquid application systems, clearer presentation of technical information and
products that deliver an even more consistent productive response are just some of
the factors raised by the feed industry to explain lack of use. It is clear that there
remains huge untapped potential for enzyme technology in the animal feed industry.
References
Bedford, M.R. and Classen, H.L. (1992) Reduction of intestinal viscosity through manipula-
tion of dietary rye and pentosanase concentration is effected through changes in the car-
bohydrate composition of the intestinal aqueous phase and results in improved growth
rate and food conversion efficiency of broiler chicks. Journal of Nutrition 122, 560–569.
19
17 November 2000 14:57:26

10 C. Sheppy
Close, W. (1995) Enzymes in action, feed mix enzymes special issue Enzymes - Nature’s
Catalyst. Misset pp. 17–20.
Gill, C. (1999) Feed International, January 1999, Watt Publishing, pp. 4–10.
Gill, C. (2000) Feed International, January 2000, Watt Publishing, pp. 4–6.
Godfrey, T. and West, S.I. (1996) Introduction to industrial enzymology. In: Godfrey, T. and
West, S.I. (eds) Industrial Enzymology, 2nd edn. Macmillan, UK, pp. 1–8.
Hoffman, P. (1999) Prospects for feed demand recovery. International Feed Markets ’99,
October 1999, Agra Europe.
Muller, A. (1999) Kraftfutter, September 1999, pp. 303–311.
Noy, Y. and Sklan, D. (1994) Digestion and absorption in the young chick. Poultry Science,
366–373.
White, W.B., Bird, H.R., Sunde, M.L., Marlett, J.A., Prentice, N.A. and Burger, W.C. (1983)
Viscosity of β-D-glucan as a factor in the enzymatic improvement of barley for chicks.
Poultry Science 62, 853–862.
20
17 November 2000 14:57:26

Enzymology and Other

Characteristics of Cellulases
and Xylanases
2
M.K. BHAT1 AND G.P. HAZLEWOOD2
1FoodMaterials Science Division, Institute of Food
Research, Norwich Research Park, Colney,
Norwich NR4 7UA, UK; 2Finnfeeds, PO Box 777,
Marlborough, Wiltshire SN8 1XN, UK
M.K. Bhat and G.P.

Characteristics
2 of Cellulases
Hazlewoodand Xylanases
Introduction
Cellulose and hemicellulose are the major plant structural polysaccharides and
account for approximately 70% of plant biomass (Ladisch et al., 1983). It has been
estimated that the amount of carbon fixed by plants during photosynthesis is over
100 billion tons per annum (Ryu and Mandels, 1980). In addition to their pivotal
role in maintaining the structural integrity of plants, cellulose and hemicellulose serve
as a major source of nutrients for herbivores and as renewable substrates for the
production of food, animal feed, paper and pulp as well as textiles (Ryu and Mandels,
1980; Gilbert and Hazlewood, 1993; Beguin and Aubert, 1994).
It has been unambiguously established that cellulose and hemicellulose can be
converted to soluble sugars by enzymes of mainly microbial origin collectively termed
cellulases and hemicellulases (Mandels, 1985; Viikari et al., 1993). Microorganisms
including fungi, bacteria and actinomycetes produce mainly three types of cellulase
components, namely endoglucanase, exoglucanase and β-glucosidase, either as
separate entities or in the form of an aggregated complex, for cellulose hydrolysis
(Wood, 1985; Lamed and Bayer, 1987, 1988; Bhat and Bhat, 1997). The hemi-
cellulose fraction of the cell walls of most species of land plants contains mainly xylan
and mannan and requires a more extensive repertoire of enzymes to effect complete
hydrolysis to soluble sugars (Biely et al., 1992; Hazlewood and Gilbert, 1998a). The
two main enzymes involved are endoxylanases (xylanases) and endomannanases
(mannanases), which attack the backbone structure (Viikari et al., 1993). Other
hemicellulases, including β-xylosidase, β-mannosidase, α-L-arabinofuranosidase, α-D-
glucuronidase, α-galactosidase, acetyl and phenyl esterases, remove side-chains and
substituents (Biely et al., 1992; Coughlan, 1992; Coughlan and Hazlewood, 1993a).
The interesting biochemical and catalytic properties of cellulases and hemi-
cellulases, coupled with their key role in the natural world and their tremendous

21
24 November 2000 15:08:54

12 M.K. Bhat and G.P. Hazlewood
potential for biotechnological applications, have stimulated research aimed at

understanding the detailed biochemistry, molecular biology and structure–function
relationships of these enzymes. This chapter summarizes current knowledge of
the enzymology and other characteristics of cellulases and xylanases from both
fundamental and applied viewpoints. Further background information can be found
in Ryu and Mandels (1980), Wood (1985, 1992a,b), Lamed and Bayer (1988),
Coughlan (1992), Coughlan and Hazlewood (1993a,b), Gilbert and Hazlewood
(1993), Bayer et al. (1994), Beguin and Aubert (1994), Bhat and Bhat (1997, 1998)
and Hazlewood and Gilbert (1998a).
Structure of Cellulose and Hemicellulose

Plant cell walls consist mainly of cellulose (40–45%), hemicellulose (30–35%) and
lignin (20–23%) (Ladisch et al., 1983). Cellulose is a linear polymer of glucose linked
by β-1,4-glycosidic bonds, having a simple primary and complex tertiary structures.
The repeating unit of cellulose is cellobiose (Fig. 2.1a).
The degree of polymerization (DP; number of glucose residues) per cellulose
chain varies from 500 to 14,000 (Marx-Figini and Schultz, 1966). In plant cell walls,
the cellulose chains are oriented in parallel with varying degrees of order. In some
regions the cellulose chains are highly ordered and strongly hydrogen bonded to form
crystallites, whereas loosely arranged cellulose molecules form the amorphous regions
(Fig. 2.1b). Native crystalline cellulose has a structure specified as type I, which can
be converted to type II by alkali treatment (Beguin and Aubert, 1994). These two
types of cellulose differ in their intrachain hydrogen bonding. In addition, native
cellulose may be composed of two slightly different forms of type I cellulose, called
Iα and Iβ, which differ in their intermolecular hydrogen bonding (Atalla and Vander
Hart, 1984).
The degree of crystallinity of cellulose varies with its origin and its treatment
after isolation (Hoshino et al., 1992). It ranges from 0% for amorphous and
acid-swollen cellulose to nearly 100% for cellulose from Valonia macrophysa (Beguin
and Aubert, 1994). Cotton cellulose is approximately 70% crystalline, while the
degree of crystallinity of commercial celluloses varies from 30 to 70% (Fan et al.,
1980; Wood, 1988). The crystalline regions of cellulose are rigid and not easily
accessible to endo-acting cellulases, while the amorphous regions are easily attacked
by either dilute acid, endoglucanases or exoglucanases (Sinitsyn et al., 1990). Thus,
for the complete hydrolysis of cellulose, either concentrated acid or a complete
cellulase system capable of attacking both amorphous and crystalline regions is
necessary.
Hemicellulose, the second most abundant plant structural polysaccharide, is
present in association with cellulose in the walls of most plant species, and can be
extracted by alkali. Based on the main sugar residues present in the polymer back-
bone, hemicelluloses can be termed xylans, glucomannans, galactans or arabinans.
The two main types of hemicelluloses are generally considered to be xylans and
22
17 November 2000 14:57:29

Characteristics of Cellulases and Xylanases 13
Fig. 2.1. Structure of cellulose and arabinoxylan. (a) Glucose and cellobiose
reprinted from FEMS Microbiology Reviews, Vol. 13, Beguin, P. and Aubert, J.-P.,
The biological degradation of cellulose, pp. 25–58, 1994, with permission from
Elsevier Science. (b) Cellulose microfibrils, showing crystalline and paracrystalline
(amorphous) regions, reprinted from Biotechnology Bioengineering Symposium,
Vol. 5, Cellulose as a Chemical and Energy Resource, Wilke, C.R. (ed.), Physical
and chemical constraints in the hydrolysis of cellulose and lignocellulosic
materials, pp. 163–181, 1975, with permission from John Wiley & Sons, Inc.,
New York. (c) A typical cereal arabino-4-O-methylglucuronoxylan, reprinted
from Biotechnology and Applied Biochemistry, Vol. 17, Coughlan, M.P. and
Hazlewood, G.P., β-1,4-D-Xylan-degrading enzyme systems: biochemistry,
molecular biology and applications, pp. 259–289, 1993, with permission from
Portland Press, London.
glucomannans (Timell, 1967; Whistler and Richards, 1970; Stephen, 1983; Puls and
Schuseil, 1993; Viikari et al., 1993).
Xylans from annual plants are more heterogeneous than xylans from perennial
plants and are designated as arabinoxylans. The two main types of arabinoxylans
are: (i) highly branched and without uronic acid substitution – found in cereal
endosperms; and (ii) much less branched and substituted with uronic acid and/or
with 4-O-methyl ether and galactose – present in lignified tissues. Arabinoxylans of
graminaceous plants contain acetic and phenolic acids (ferulic, p-coumaric) which
are esterified to the backbone xylose units and the arabinose side groups, respectively
(Fig. 2.1c) (Hartley and Ford, 1989). In addition to enzymes (endoxylanase and
23
24 November 2000 15:09:56

β-xylosidase), which cleave the backbone structure, accessory enzymes like

α-L-arabinofuranosidase, acetyl and phenyl esterases and α-D-glucuronidase are
necessary for the removal of side-chains and the complete hydrolysis of arabinoxylans
(Biely et al., 1992).
Enzymology of Cellulases and Xylanases

Source
Cellulases and xylanases are produced by a wide range of bacteria and fungi,
including aerobes, anaerobes, mesophiles, thermophiles and extremophiles. Aerobic
fungi and bacteria generally produce extracellular cellulases and hemicellulases.
Interestingly, anaerobic bacteria (Clostridium thermocellum, C. cellulovorans,
Ruminococcus albus, R. flavefaciens, Fibrobacter succinogenes, Acetivibrio cellulolyticus)
and anaerobic fungi (Neocallimastix frontalis, N. patriciarum, Piromyces equi) produce
cellulases in the form of a multienzyme aggregated complex (Groleau and Forsberg,
1981; Lamed et al., 1987; Wood, 1992a; Gilbert and Hazlewood, 1993; Beguin and
Lemaire, 1996; Bhat and Bhat, 1997).
Most of the early studies were carried out on the biochemistry and enzymology
of cellulases from aerobic mesophilic fungi, Trichoderma viride, T. reesei, Penicillium
pinophilum, Sporotrichum pulverulentum, Fusarium solani, Talaromyces emersonii and
Trichoderma koningii (Coughlan and Ljungdahl, 1988). In the past two decades,
it has been recognized that other microorganisms such as thermophilic fungi
(Sporotrichum thermophile, Thermoascus aurantiacus, Chaetomium thermophile,
Humicola insolens), mesophilic anaerobic fungi (N. frontalis, N. patriciarum,
P. communis, Sphaeromonas communis, P. equi, Orpinomyces sp.), mesophilic and
thermophilic aerobic bacteria (e.g. Cellulomonas fimi, Pseudomonas fluorescens
subsp. cellulosa, Cellvibrio sp., Microbispora bispora, Clostridium cellulolyticum and
C. cellulovorans), mesophilic and thermophilic anaerobic bacteria (A. cellulolyticus,
Bacteroides cellulosolvens, F. succinogens, R. albus, R. flavefaciens, C. thermocellum and
C. stercorarium), as well as actinomycetes (Thermomonospora fusca), produce highly
active cellulase and hemicellulase systems (Bhat and Maheswari, 1987; Aubert
et al., 1988; Beguin and Lemaire, 1996; Claeyssens et al., 1998). In addition,
hyperthermophilic microorganisms – namely, Thermotoga sp., Pyrococcus furiosus and
Thermofilum sp., which grow between 85 and 110°C – produce extremely stable
cellulolytic and hemicellulolytic enzymes (Simpson et al., 1991; Antranikian, 1994;
Winterhalter and Liebl, 1995).
In addition to many of those listed above, other microorganisms such as
Aspergillus and Cryptococcus produce xylanases and xylan debranching enzymes
(Coughlan and Hazlewood, 1993a,b; Viikari et al., 1993). The evidence currently
available suggests that the xylan- and mannan-degrading enzymes of anaerobic
microorganisms may be produced as integral components of the aggregated
multienzyme cellulase complexes that are characteristic of these species (Beguin and
Lemaire, 1996; Hazlewood and Gilbert, 1998b).
24
17 November 2000 14:58:05

Enzyme assays
The measurement of cellulase and xylanase activities is hampered by the nature of

substrates used and the complexity of the enzyme systems produced by different
microorganisms. In order to overcome these problems, numerous assays have been
developed (Wood and Bhat, 1988; Biely et al., 1992). The substrates and the
methods used to measure cellulase and xylanase activities are summarized in Table
2.1. For more detailed information on assays for accessory enzymes involved in the
degradation of substituted xylans, the reader is referred to Coughlan and Hazlewood
(1993a).
Quantitative assays for cellulases and xylanases

The cellulase system contains mainly endoglucanase (EC 3.2.1.4, 1,4-β-D-glucan
glucanohydrolase), exoglucanase or cellobiohydrolase (EC 3.2.1.91, 1,4-β-D-glucan
cellobiohydrolase) and β-glucosidase or cellobiase (EC 3.2.1.21, β-D-glucoside
glucohydrolase). Endoglucanase activity is generally determined by measuring the
reducing sugars released from either carboxymethyl (CM-) or hydroxyethyl (HE-)
cellulose (Wood and Bhat, 1988). This activity can also be measured by determining
either the decrease in viscosity of CM-cellulose, the swelling of cotton fibre in alkali
or the decrease in turbidity of amorphous cellulose. In addition, substituted,
unsubstituted, radio- and reduced end-labelled cello-oligosaccharides have been used
to characterize endoglucanases (Bhat et al., 1990). Interestingly, some endo-
glucanases catalyse transferase reactions and act synergistically with cellobiohydrolase
during the solubilization of crystalline cellulose (Wood et al., 1988; Claeyssens et al.,
1990a).
Cellobiohydrolase (CBH; exoglucanase) activity is determined by measuring the
reducing sugars released from either Avicel or H3PO4-swollen cellulose (Wood and
Bhat, 1988). Besides, CBH activity can be measured by determining either the
release of dyed cellobiose from dyed Avicel or the decrease in turbidity of amorphous
cellulose. Substituted and unsubstituted cello-oligosaccharides have been used to
characterize CBHs (Claeyssens et al., 1989).
β-Glucosidase activity is generally determined by measuring the release of
glucose and o-/p-nitrophenol from cellobiose and o-/p-nitrophenyl β-D-glucoside,
respectively (Wood and Bhat, 1988). Also, the increase in reducing power of cello-
oligosaccharides can be used as a measure of β-glucosidase activity.
Total cellulase activity, comprising endoglucanase, exoglucanase and
β-glucosidase, is measured by determining the solubilization of either cotton fibre,
filter paper or Avicel. The release of dyed soluble fragments from dyed Avicel can also
be used to measure total cellulase activity. It is generally accepted that either cotton
fibre or filter paper is the best substrate, but some consider Avicel is ideal for
measuring total cellulase activity. All three substrates contain a high proportion of
crystalline cellulose and are therefore useful for determining total cellulase activity
(Wood and Bhat, 1988).
Xylanase (EC 3.2.1.8) activity is generally determined by measuring the reduc-
ing sugars released from xylan by either the Somogyi–Nelson or the dinitrosalicylic
25
17 November 2000 14:58:06

Table 2.1. Substrates and assays used for determining the cellulase and xylanase activities. 16
Enzymes Substrate Assay

1. Quantitative assays
17 November 2000 14:58:07

Endoglucanase CM-cellulose; hydroxyethylcellulose Release of reducing sugars (Somogyi, 1952); decrease in viscosity (Wood and Bhat, 1988)
(CM-cellulase, Cotton; amorphous cellulose (Wood, 1988) Swelling in alkali (Marsh et al., 1953); release of reducing sugars (Somogyi, 1952);
endocellulase) decrease in turbidity (Nummi et al., 1981)
Substituted and unsubstituted Increase in reducing sugars (Somogyi, 1952); analysis of products by HPLC (Bhat et al.,
cello-oligosaccharides 1990)
Cellobiohydrolase Avicel; hydrocellulose and dyed Avicel Release of reducing sugars (Somogyi, 1952); dyed cellobiose (Wood and Bhat, 1988)
(exoglucanase, Amorphous cellulose Release of reducing sugars (Somogyi, 1952); decrease in turbidity (Nummi et al., 1981)
exocellulase, Substituted and unsubstituted Increase in reducing sugars (Somogyi, 1952); release of p -nitrophenol from
Avicelase) cello-oligosaccharides p -nitrophenyl-β-D-cellobioside (Deshpande et al., 1984); analysis of products by HPLC
(Bhat et al., 1990)
26
β-Glucosidase Cellobiose Release of glucose (Wood and Bhat, 1988)
(cellobiase) Cello-oligosaccharides Increase in reducing sugars (Somogyi, 1952)
o - or p -Nitrophenyl-β-D-glucosides Release of o - or p -nitrophenol (Wood and Bhat, 1988)
A3882:Bedford:AMA:First Proof: 04/09/00

Total cellulase Cotton Cellulose residue (Wood, 1969); release of reducing sugars, weight loss and loss in tensile
strength (Wood and McCrae, 1972)
Filter paper; hydro-cellulose; Avicel; Solka Floc Release of reducing sugars (Somogyi, 1952; Miller, 1959)
CHAP-2
Dyed Avicel Release of soluble dyed fragments (Wood and Bhat, 1988)

Xylanase Birchwood xylan; oat spelt xylan Release of reducing sugars (Somogyi, 1952; Miller, 1959; Bailey et al., 1992)
(endoxylanase) Insoluble xylan Decrease in turbidity (Nummi et al., 1985)
Arabinoxylan; CM-xylan Decrease in viscosity (Sengupta et al., 1987)
RBB-xylan; Ostazine Brilliant Red (OBR)-xylan Release of RBB- or OBR-dyed fragments (Biely et al., 1985)
[1-3H]-labelled, unsubstituted and substituted Analysis of products by HPLC and subsequent radioactive counting, if necessary (Bhat
xylo-oligosaccharides et al., 1990; Biely et al., 1992)

2. Qualitative assays
Endoglucanase CM-cellulose; RBB-CM-cellulose; Detection by using Congo Red and zone clearing (Coughlan, 1988)
OBR-hydroxyethyl cellulose
17 November 2000 14:58:08

Methylumbelliferyl-β-D-cellobioside (MeUmbGlc2); Release of methylumbelliferone (MeUmb) and detection by UV (van Tilbeurgh et al., 1988)
methylumbelliferyl-β-D-cellotrioside (MeUmbGlc3)
Cellobiohydrolase RBB-Avicel Zone clearing (Coughlan, 1988)
(exoglucanase) p -Nitrophenyl-β-D-cellobioside Release of p -nitrophenol (van Tilbeurgh et al., 1988)
β-Glucosidase p -Nitrophenyl-β-D-glucoside; methylumbelliferyl Detection of p -nitrophenol (Bhat et al., 1993) or MeUmb by UV (van Tilbeurgh et al., 1988)
-β-D-glucoside (MeUmbGlc)
Xylanase Soluble xylan; RBB-xylan; OBR-xylan Detection by using Congo Red and zone clearing (Biely et al., 1992)
Methylumbelliferyl-β-D-xylobioside (MeUmbXyl2); Detection of MeUmb by UV (Biely et al., 1992; Kalogiannis et al., 1996)
methylumbelliferyl-β-D-xylotrioside (MeUmbXyl3)
27
Characteristics of Cellulases and Xylanases

CHAP-2
17
acid (DNS) method (Somogyi, 1952; Miller, 1959; Bailey et al., 1992). The DNS
method tends to give a higher value than the Somogyi–Nelson method. Nevertheless,
xylanase activity determined by the DNS method, standardized with respect to a
particular xylan, is very reproducible (Biely et al., 1992). Based on a round-robin test
that involved 20 laboratories, Bailey et al. (1992) proposed a suitable procedure for
preparing xylan and measuring xylanase activity. In addition, a viscometric method
using a soluble xylan (arabinoxylan or carboxymethyl xylan) has been used for
the determination of xylanase activity in samples containing high background sugar
values (Sengupta et al., 1987). Although the above methods detect the ability of an
enzyme sample to hydrolyse xylan, they do not reveal the type of products released,
which may be important when evaluating the ability of an enzyme to degrade xylan.
Hence, the hydrolysis products should be analysed to obtain detailed information on
the mode of action of the xylanase in question.
Another specific assay, which can be used to determine xylanase activity in the
presence of either large amounts of reducing sugars or viable cells utilizing xylan
fragments (Biely, 1985), involves the use of soluble and covalently dyed xylan
(Remazol Brilliant Blue) (RBB-xylan). Although this assay is highly sensitive to
temperature and ionic strength, interlaboratory tests revealed that the release of
low molecular weight dyed fragments is a useful alternative for measuring xylanase
activity (Biely et al., 1992). A nephelometric (turbidometric) assay using insoluble
xylan has also been found to be suitable for determining xylanase activity (Nummi
et al., 1985). Furthermore, [1-3H]-labelled and substituted xylo-oligosaccharides
have been used to characterize xylanases (Biely et al., 1992; Bennett et al., 1998).
Qualitative assays for cellulases and xylanases

Qualitative assays have been developed either to select microbial strains producing
high levels of cellulases and xylanases, or to identify and/or characterize these
enzymes in a given sample. Insoluble xylan and RBB-xylan are ideal substrates
to select microorganisms producing xylanase on solid agar medium (Sprey and Lam-
bert, 1983; Farkas et al., 1985). Similarly, methods using either RBB-CM-cellulose
or CM-cellulose stained with Congo Red can be used to select microorganisms
producing endoglucanase activity (Kluepfel, 1988).
The capacity of Congo Red to complex with either polymeric xylan or CM-
cellulose, but not with small oligosaccharide products, is conveniently used to detect
xylanase and endoglucanase activities in zymograms after the fractionation of protein
mixtures by SDS/IEF-PAGE (Coughlan, 1988). RBB-xylan and RBB-CM-cellulose
can be used for similar purposes (Biely et al., 1985). The advantages of the latter
method are: (i) that the dyed fragments released from RBB-xylan/CM-cellulose
diffuse from the detection gel into the separating gel and further help to identify
the position of a xylanase or endoglucanase, which can subsequently be eluted; and
(ii) the hydrolysis of the dyed substrate can be visually followed and the reaction
terminated when necessary. The use of these substrates facilitates the identification of
multiple forms of xylanase or endoglucanase produced by different microorganisms.
Likewise, chromogenic and fluorogenic cello- and xylo-oligosaccharides have been
successfully used to identify multiple forms of cellulases and xylanases after the
28
17 November 2000 14:58:09

separation of crude enzyme mixtures by SDS/IEF-PAGE (Biely et al., 1992;

Kalogiannis et al., 1996).
Substrate specificity
Endoglucanases
Endoglucanases specifically cleave the internal β-1,4-glycosidic bonds of amorphous,
swollen and substituted celluloses as well as cello-oligosaccharides. These enzymes are
generally inactive towards crystalline cellulose and cellobiose. Some endoglucanases
attack barley glucan with mixed β-1,3 and β-1,4 linkages (Petre et al., 1986). Using
substituted, unsubstituted, [1-3H]-labelled and reduced cello-oligosaccharides, Bhat
et al. (1990) demonstrated a marked difference in the substrate specificities of
endoglucanases purified from P. pinophilum. Thus, endoglucanases III and IV were
active on cellotriose and higher cello-oligosaccharides, whereas endoglucanases II, V
and I required at least four, five and six glucose residues, respectively. Although such
variation in substrate specificity of endoglucanases was unexpected, it was speculated
that microorganisms secrete multiple endoglucanases with a wide range of substrate
specificities to effect efficient hydrolysis of complex cellulosic substrates.
CBHs (cellobiohydrolases; exoglucanases)

Like endoglucanases, the CBHs are highly active on amorphous and swollen
celluloses, but degrade crystalline cellulose and cello-oligosaccharides rather poorly
(Wood and Bhat, 1988). These enzymes are specific for β-1,4 linkages of the
cellulose chain, but are inactive on cellobiose, CM- and hydroxyethyl-celluloses. In
general, CBHs attack cellulose chains from the non-reducing end and release
cellobiose (Wood et al., 1988). Recent kinetic studies and high-resolution structural
data confirmed that there are two classes of CBHs (Barr et al., 1996). The class one
enzymes (e.g. CBH I from T. reesei and two exoglucanases E4 and E6 from T. fusca)
hydrolyse the cellulose chain preferentially from the reducing end, while class two
CBHs (e.g. CBH II from T. reesei and E3 from T. fusca) release cellobiose specifically
from the non-reducing end (Barr et al., 1996; Teeri, 1997).
b-Glucosidases
β-Glucosidases can be classified as either aryl β-D-glucosidases (hydrolysing
exclusively aryl-β-D-glycosides), cellobiases (hydrolysing diglucosides and cello-
oligosaccharides) or β-glucosidases with broad substrate specificities. Most
β-glucosidases characterized so far show broad substrate specificities and hydrolyse
aryl and alkyl β-D-glycosides and β-1,1-, β-1,2-, β-1,3-, β-1,4- and β-1,6-linked
diglucosides, as well as substituted and unsubstituted cello-oligosaccharides
(Bhat et al., 1993; Christakopoulos et al., 1994a). Interestingly, an intracellular
β-glucosidase from S. thermophile was found to be an aryl-β-glucosidase, whereas two
extracellular β-glucosidases from the same organism hydrolysed only cellobiose
(Bhat et al., 1993). Some β-glucosidases showed activity towards H3PO4-swollen
and CM-celluloses (Sadana et al., 1988), but most β-glucosidases are inactive
29
17 November 2000 14:58:10

towards these and other polymeric substrates such as Avicel, filter paper and cotton
(Woodward and Wiseman, 1982).
Xylanases
In general, xylanases are specific for the internal β-1,4 linkages of polymeric
xylan and are designated as endoxylanases. Based on their action on different
polysaccharides, endoxylanases have been classified as either specific or non-specific
(Coughlan, 1992; Coughlan et al., 1993). The specific endoxylanases are active on
xylans with only β-1,4 linkages, whereas non-specific endoxylanases hydrolyse
β-1,4-linked xylans, β-1,4 linkages of mixed xylans and other β-1,4-linked polymers
such as CM-cellulose. In fact, the determination of the kinetic constants ratio kcat/Km
for xylan and CM-cellulose, for a particular non-specific xylanase, should confirm
whether the enzyme in question is a xylanase or a cellulase. Generally, endoxylanase
activity and affinity for xylo-oligosaccharides decrease with decreasing DP (Coughlan
et al., 1993). Most endoxylanases are specific for unsubstituted xylosidic linkages
of xylans and release both substituted and unsubstituted xylo-oligosaccharides. In
contrast, some endoxylanases are specific for xylosidic linkages adjacent to
substituted groups in the main chain xylan. For example, two endoxylanases from
Aspergillus niger (pI 8.0 and 9.6) showed little or no action on xylo-oligomers or
xylans from which the arabinose substituents were removed (Frederick et al., 1985).
Furthermore, the endoxylanase with pI 9.6 cleaved the xylan chain only in the
vicinity of 4-O-methylglucuronic acid, and this substitution was reported to be an
absolute requirement for the action of the enzyme (Nishitani and Nevins, 1991).
Nevertheless, both enzymes failed to release any substituent as a free product, which
indicated that these substitutions are necessary for the proper orientation of the
substrate in the active site.
Most endoxylanases, active on mixed xylan (rhodymenan with β-1,3 and β-1,4
linkages), are specific for the β-1,4 linkages (Coughlan, 1992). Also, endoxylanases
have been grouped either as debranching or non-debranching based on their ability
to release arabinose in addition to hydrolysing the main chain (Coughlan et al.,
1993). Although some endoxylanases purified to homogeneity hydrolysed both
main-chain xylan and arabinose side-chains (Matte and Forsberg, 1992), there is
always a question as to whether the release of arabinose is due to an intrinsic property
of the enzyme or is due to the presence of a trace contaminant.
Kinetics and subsite mapping
Kinetic constants for the hydrolysis of CM-cellulose and xylan by purified

endoglucanases and xylanases have been determined (Bhat et al., 1989; Coughlan
et al., 1993). It is often difficult to determine the kinetic constants of these enzymes
using polymeric substrates. Therefore, substituted (chromogenic or fluorogenic)
and [1-3H]-labelled cello- and xylo-oligosaccharides have been prepared and
conveniently used for this purpose (Bhat et al., 1990; Claeyssens et al., 1990a,b; Biely
et al., 1992). When an enzyme attacks more than one glycosidic bond in a particular
30
17 November 2000 14:58:11

oligosaccharide, the kinetic constants (Km and kcat) for the cleavage of all glycosidic
bonds must be determined in order to understand the mode of action of the enzyme,
as well as to identify the preferred site of attack. Using this approach, the number
of glycosyl-binding sites present in different endoglucanases, CBHs and xylanases
have been determined biochemically (Claeyssens et al., 1989; Coughlan et al., 1993;
Bhat et al., 1994). Detailed information on glycosyl-binding sites and active site
architecture of the above enzymes has also been derived by determining their
three-dimensional structures with bound substrates or substrate analogues in the
active site (Davies et al., 1995; Sakon et al., 1997). In contrast, the kinetic constants
of β-glucosidases have been determined using either cellobiose or pNPC (Bhat et al.,
1993; Christakopoulos et al., 1994a), and have been shown to depend on enzyme
source and preference for alkyl or aryl-glycosides (Woodward and Wiseman, 1982;
Bhat et al., 1993).
Inhibition
Cellulases and xylanases are often inhibited by the presence of high concentrations of
their hydrolysis products. For example, most CBHs are inhibited by cellobiose (Wood
and McCrae, 1986), even though cellobiose (> 10 mM) stimulates the activity of
CBH II from P. pinophilum towards H3PO4-swollen cellulose. Similarly, endoglu-
canases are inhibited by cellobiose at or above 100 mM (Bhat et al., 1989). However,
glucose (up to 100 mM) showed little effect on many CBHs and endoglucanases
(Wood et al., 1988; Bhat et al., 1989). In contrast, β-glucosidases are inhibited by
glucose and other mono- and disaccharides, such as xylose, fucose, galactose, maltose,
lactose and meliobiose (Bhat et al., 1993). Besides, nojirimycin and gluconolactone
are known to be potent inhibitors of β-glucosidases (Bhat et al., 1993).
Like cellulases, endoxylanases are believed to be inhibited by high concentra-
tions of xylobiose but not by xylose. In general, the activity of cellulases and xylanases
is neither activated nor inhibited by metal ions and reducing agents, though the
aggregated cellulase systems of anaerobic bacteria may require divalent cations and
reducing agents for their activity towards crystalline cellulose, but not towards
swollen and substituted celluloses (Lamed and Bayer, 1988).
Transferase activity
Many cellulases and endoxylanases catalyse both hydrolysis and transferase (or
transglycosylase) reactions, especially in the presence of high concentrations of
oligomeric substrates or alcohol (Bhat et al., 1990, 1993; Christakopoulos et al.,
1994a–c). The essential difference between hydrolysis and transferase reactions is
that, in the former, water acts as an acceptor of the glycosyl moiety, whereas in the
latter the acceptor is an alcohol or a sugar molecule.
Transferase reactions have considerable significance from both fundamental and
applied viewpoints. It is generally accepted that the transferase products of cellobiose
31
17 November 2000 14:58:12

and xylobiose could be the true inducers of microbial cellulases and xylanases (Biely,
1993; Bhat and Bhat, 1997). In addition, enzymatic transglycosylation can be used
to synthesize high value alkylglycosides and oligosaccharides with desired linkages,
which have uses both for research and for commercial applications (Christakopoulos
et al., 1994a–c).
Mode of action
Fungal cellulase
All endoglucanases attack swollen and substituted celluloses and the amorphous
regions of cellulose randomly and release glucose, cellobiose and cello-
oligosaccharides (Table 2.2) (Wood, 1985; Coughlan and Ljungdahl, 1988). The
Table 2.2. Mode of action of cellulases and xylanase.
Enzyme Mode of action
Endoglucanase or endocellulase –G–G–G–G–G–G–G–G–G–G–G–

(1,4-β-D-glucan glucanohydrolase; ↑ ↑
EC 3.2.1.4) Cleaves 1,4-β linkages at random (Wood and Bhat, 1988)
Cellobiohydrolase (CBH) or G–G–G–G–G–G–G–G–G–G–G–
exocellulase (1,4-β-D-glucan ↑ (type I) ↑ (type II)
cellobiohydrolase; EC 3.2.1.91) Releases cellobiose from both reducing (type II) and
non-reducing ends (type I) (Wood et al., 1988; Vrsanska
and Biely, 1992; Barr et al., 1996; Gilkes et al., 1997)
Exoglucanase or glucohydrolase G–G–G–G–G–G–G–G–G–G–
(1,4-β-D-glucan glucohydrolase; ↑
EC 3.2.1.74) Releases glucose from the non-reducing end (McHale and
Coughlan, 1980; Wood and McCrae, 1982)
β-Glucosidase or cellobiase G–G; G–G–G–G; G–G–G–G–G
(β-D-glucoside glucohydrolase; ↑ ↑ ↑ ↑ ↑
EC 3.2.1.21) Releases glucose from cellobiose and hydrolyses short-chain
cello-oligosaccharides from both reducing and non-reducing
ends by releasing one glucose unit at a time (Wood and
Bhat, 1988; Christakopoulos et al., 1994a)
Xylanase or endoxylanase S S
(1,4-β-D-xylan xylanohydrolase; | |
EC 3.2.1.8) X–X–X–X–X–X–X–X–X–X–X–X–X–
↑ | ↑
S
Cleaves 1,4-β linkages of xylan at random with preference to
unsubstituted regions (Biely et al., 1992; Coughlan et al.,
1993; Coughlan and Hazlewood, 1993a)
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17 November 2000 14:58:13

CBHs hydrolyse H3PO4-swollen cellulose and Avicel sequentially by removing

cellobiose units from either reducing or non-reducing ends of the cellulose chain
(Wood et al., 1988; Vrsanska and Biely, 1992; Barr et al., 1996). Endoglucanase
and CBH act synergistically to effect extensive hydrolysis of crystalline cellulose.
Subsequently β-glucosidase completes the hydrolysis by converting the resultant
cello-oligosaccharides and cellobiose to glucose (Wood, 1985).
The modes of action of endoglucanase, CBH and β-glucosidase from several
sources have been extensively studied using substituted, unsubstituted and labelled
cello-oligosaccharides (van Tilbeurgh et al., 1988; Bhat et al., 1990; Claeyssens and
Henrissat, 1992). Based on these studies, it was concluded that most endoglucanases
attack internal glycosidic bonds of cello-oligosaccharides and release mainly
cellobiose and cellotriose, while CBHs hydrolyse the second glycosidic bond from
either the reducing or non-reducing end of the cello-oligosaccharides (Table 2.2).
However, β-glucosidase sequentially removes one glucose unit from either the
reducing end, the non-reducing end or both ends (Table 2.2).
The viscosity of a cellulose solution is directly related to the DP of individual
cellulose chains. An endoglucanase that attacks the cellulose chain randomly
produces the largest decrease in the viscosity of a cellulose solution per unit increase
in reducing power. Based on the relationship between viscosity reduction and
the increase in reducing end groups, the multiple endoglucanases from T. koningii
and P. pinophilum were classified as more or less randomly acting enzymes
(Wood and McCrae, 1978; Bhat et al., 1989). Using a similar approach, the mode of
action of endoglucanases and CBHs (exoglucanases) from C. fimi, P. pinophilum and
C. thermocellum has been clarified (Gilkes et al., 1984; Bhat et al., 1989, 1994; Wood
et al., 1989).
Some fungi, such as P. pinophilum and T. emersonii, produce an exoglucanase
that catalyses the removal of glucose from the non-reducing end of cellodextrins,
but does not interact cooperatively with endoglucanases during the hydrolysis of
crystalline cellulose (McHale and Coughlan, 1980; Wood and McCrae, 1982).
Anaerobic fungi such as N. frontalis, N. patriciarum and P. equi use a different
mechanism to degrade cellulose when compared with aerobic fungi (Wood, 1992a).
It has been reported that N. frontalis produces a multicomponent enzyme complex
called crystalline cellulose-solubilizing factor (CCSF), which has a molecular mass
of 700 kDa and consists of a number of subunits with molecular mass ranging
from 68 to 135 kDa (Wood, 1992a). Functionally, CCSF resembles the cellulases
from the aerobic fungi in solubilizing crystalline cellulose, since it acts synergistically
with an endoglucanase and a β-glucosidase of N. frontalis which are not part of
CCSF (Wood, 1992a). Furthermore, it was demonstrated that CCSF of N. frontalis
consists of endoglucanase, β-glucosidase and perhaps also CBH. Although the
mode of action of CCSF on cellulose is not clearly understood, it is believed that
all subunits of CCSF act cooperatively to degrade cellulose (Wood et al., 1994).
More recent molecular biology studies of individual cellulases and hemicellulases
from anaerobic fungi support the view that these organisms produce an aggregated
cellulase complex analogous to the cellulosome of C. thermocellum (Hazlewood and
Gilbert, 1998b).
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17 November 2000 14:58:14

Bacterial cellulase
Bacterial cellulases are known to adopt different mechanisms for the hydrolysis of
cellulose. For example, the aerobes Cellulomonas, Pseudomonas, Thermoactinomycetes,
T. fusca and Microbispora and the anaerobe C. stercorarium produce a cellulase system
similar to that of aerobic fungi, and degrade cellulose by the cooperative interaction
of the different cellulase components (Beguin et al., 1992; Wood, 1992b; Gilbert
and Hazlewood, 1993). In contrast, the anaerobic thermophilic bacterium
C. thermocellum degrades crystalline cellulose very effectively by means of a high
molecular mass multienzyme complex called the cellulosome (Lamed and Bayer,
1988; Beguin and Lemaire, 1996). The cellulosome of C. thermocellum has been
extensively studied and used as a model system for understanding how anaerobic
bacteria, including rumen bacteria, degrade cellulose (Wood, 1992a). The
production of cellulosomes is not restricted to C. thermocellum alone. Various other
anaerobic bacteria, such as R. albus, R. flavefaciens, F. succinogenes, A. cellulolyticus and
C. cellulovorans also produce cellulosomes on their cell surface which possess similar
properties to the cellulosome from C. thermocellum (Groleau and Forsberg, 1981;
Lamed et al., 1987; Beguin and Lemaire, 1996).
Cellulosomes of C. thermocellum degrade crystalline cellulose extensively in
the presence of Ca2+ and DTT (Lamed and Bayer, 1988). According to current
understanding, the cellulosome contains a large molecular mass, non-catalytic
subunit termed scaffoldin (CipA, S1 or SL) which possesses a cellulose-binding
domain (CBD) and numerous duplicated attachment sites called cohesins (Bayer
et al., 1994; Beguin and Lemaire, 1996; Beguin and Alzari, 1998; Bhat and Bhat,
1998). In addition to catalytic domains, the enzymatic subunits of the cellulosome
each contain a highly conserved duplicated docking domain termed a dockerin,
which interacts with the cohesins of the scaffoldin polypeptide (Bayer et al., 1994).
Some of the enzymatic subunits also contain a CBD. The exact mechanism by which
the cellulosome achieves cellulolysis is unclear, but it is evident that the efficiency of
the multienzyme complex is a function of its quaternary structure, and is dependent
on both endoglucanase/CBH synergism and clustering of the complex on to the
substrate surface (Beguin and Lemaire, 1996).
Fungal and bacterial endoxylanases

The mode of action of endoxylanases has been extensively studied (Coughlan, 1992;
Coughlan and Hazlewood, 1993a,b; Biely et al., 1997). In general, these enzymes
cleave the internal β-1,4 linkages of the xylan backbone and release mainly xylobiose,
xylotriose and substituted oligomers having two to four residues (Table 2.2). It has
been reported that most endoxylanases cleave the xylan backbone leaving the
substituent at the non-reducing end of the xylosyl chain or the oligosaccharide
(Dekker, 1985). Moreover, the presence of glucopyranosyl uronic acid sterically
hinders the hydrolysis of the second and third xylosidic linkages to the right of the
branch points, by the xylanases from S. dimorphosporum and Trametes hirsuta
(Kubackova et al., 1978; Comtat and Joseleau, 1981). Nevertheless, the xylanases
from A. niger and C. sacchari were found to cleave the substituent on the reducing
end and in the middle of the oligosaccharide chain, respectively (Dekker, 1985).
34
17 November 2000 14:58:15

By analysing the structures of the substituted products released during the

extensive hydrolysis of different xylans by purified endoxylanases, it was possible to
draw the following conclusions about their modes of action.
1. An endoxylanase from Streptomyces sp. yielded arabinoxylotrioside (IV, Fig. 2.2) as
the smallest product from wheat arabinoxylan. This revealed that the minimal spatial
requirement for the attack of β-1,4 linkage is two adjacent unsubstituted xylose units.
2. An endoxylanase from M. verrucaria liberated arabinoxylobiose (I, Fig. 2.2) from
wheat straw xylan, indicating that this enzyme attacks the glycosidic linkage adjacent
to the substitution.
3. Hydrolysis of spear grass hemicellulose by an endoxylanase from Ceratocystis
paradoxa yielded products I and III (Fig. 2.2), while a xylanase from A. niger liberated
products II and V (Fig. 2.2).
4. An extensive hydrolysis of larchwood glucuronoxylan by an endoxylanase from
T. aurantiacus yielded xylose, xylobiose and aldotetrauronic acid (VII, Fig. 2.2). This
enzyme did not cleave main-chain β-1,4 linkages between the residues carrying the
substituent and the adjacent xylose on either side.
5. Action of endoxylanase from S. dimorphosporum on redwood arabinoglucuro-
noxylan released products I, III, VI and VIII (Fig. 2.2). This endoxylanase appeared to
require at least a xylotriose group (IX) with an unsubstituted (at O-2 position) xylosyl
residue at the non-reducing end and an unsubstituted (at O-2 and O-3 positions)
central xylosyl residue, while the reducing end could be substituted or unsubstituted
(Fig. 2.2).
Based on amino acid sequence similarities and hydrophobic cluster analysis,
Henrissat and Bairoch (1993, 1996) grouped endoxylanases in glycosyl hydrolase
families 10 and 11. Family 10 includes mainly acidic and high molecular mass
endoxylanases, while basic and low molecular mass endoxylanases belong to family
11. Studies on the mode of action of endoxylanases from these two families revealed
that: (i) endoxylanases of family 10 hydrolyse heteroxylans (substituted xylan) and
homoxylans (rhodymenan with β-1,4 and β-1,3 linkages) to a higher degree than
those of family 11; (ii) only endoxylanases of family 10 are capable of cleaving
the glycosidic linkages of xylan either closer or adjacent to substituents, such as
methylglucuronic acid (MeGlcA) and acetic acid; and (iii) family 10 endoxylanases
release smaller oligosaccharides from glucuronoxylan, acetylxylan and rhodymenan
than family 11 (Biely et al., 1997). In addition, it was reported that the endoxylanases
of family 10 require two unsubstituted xylose residues between the branch
points, whereas family 11 endoxylanases require at least three unsubstituted
xylose residues in a sequence (Fig. 2.3a–c) (Biely et al. 1997). The predicted mode
of action of endoxylanases from families 10 and 11 on rhodymenan is shown in
Fig. 2.3b.
Studies of the mode of action and biochemical characteristics of endoxylanases I
and III from Aspergillus awamori indicated that they belong to families 10 and 11,
respectively (Kormelink et al., 1993). Thus, endoxylanase I (family 10) cleaves β-1,4
linkages adjacent to arabinose substitution as well as internal β-1,4 linkages, whereas
endoxylanase III (family 11) cleaves only the internal β-1,4 linkages of cereal
35
17 November 2000 14:58:15

Fig. 2.2. Chemical structures of substituted products released by different

endoxylanases. Reprinted from Xylans and Xylanases, Visser, J., Beldman, G.,
Kusters-van Someren, M.A. and Voragen, A.G.J. (eds), Coughlan, M.P., Towards
an understanding of the mechanism of action of main chain-hydrolysing xylanases,
pp. 111–139, 1992, with permission from Elsevier Science.
arabinoxylan (Fig. 2.3c) (Kormelink et al., 1993). The isolation and identification
of methylglucuronic acid and arabinose-linked oligosaccharides released from
methylglucuronoxylan and arabinoxylan by endoxylanases of family 10 revealed that
the substitution at position 3 could cause greater steric hindrance of enzyme attack
36
17 November 2000 14:59:10

Fig. 2.3. Mode of action of endoxylanases from families 10 and 11 on (a) 4-O-
methyl-D-glucurono-D-xylan, (b) rhodymenan, and (c) cereal L-arabino-D-xylan.
The arrows indicate the sites of attack on different substrates. Reprinted from
Journal of Biotechnology, Vol. 57, Biely, P., Vrsanska, M., Tenkanen, M. and
Kluepfel, D., Endo-β-1,4-xylanase families: differences in catalytic properties,
pp. 151–166, 1997, with permission from Elsevier Science.
than at position 2. Furthermore, only endoxylanases of family 10 released

monoacetylated xylobiose from acetylxylan, with the exception of a family 11
endoxylanase from T. reesei (Biely et al., 1997).
Endoxylanases from families 10 and 11 can be distinguished further, based on
their mode of action on substituted and unsubstituted oligosaccharides. In general,
the family 10 endoxylanases hydrolyse xylotriose and xylotetraose more rapidly than
those from family 11. Also, endoxylanases of family 10 hydrolyse pNPC, pNPXyl2,
pNPXylGlc, MeUmbXyl2 and MeUmbXylGlc, while endoxylanases of family 11
hydrolyse only pNPXyl2 and MeUmbXyl2. Thus, the ability to cleave β-1,4 linkages
between xylose and glucose residues is one of the major differences between
endoxylanases from families 10 and 11. Based on this information, Biely et al. (1997)
concluded that if a model β-1,4-glucoxylan is available, it is most likely that the
endoxylanases of family 10 will cleave certain β-1,4-glucopyranosyl linkages, but
those from family 11 will not.
37
17 November 2000 14:59:45

Synergism
Between cellulases
Synergism is an enhanced effect of two or more enzymes when acting cooperatively,
compared with their additive effect. Giligan and Reese (1954) first demonstrated
synergism between cellulase components during the hydrolysis of cellulose.
Subsequently several research groups have demonstrated synergism between endo-
and exoglucanases during the solubilization of crystalline cellulose (Wood et al.,
1988, 1989; Klyosov, 1990; Bhat et al., 1994). Five different types of synergism have
been reported between fungal cellulase components: (i) a non-hydrolytic protein
called C1 and endoglucanase (Reese et al., 1950); (ii) β-glucosidase and either
endoglucanase or CBH (Eriksson and Wood, 1985); (iii) two immunologically
related and distinct CBHs (Wood and McCrae, 1986); (iv) endoglucanase and CBH
from either the same or different microorganisms (Wood et al., 1989); and (v)
between two endoglucanases (Klyosov, 1990). In addition, synergism between
bacterial and fungal cellulases, as well as between the subunits of the C. thermocellum
cellulosome, has been reported (Bhat et al., 1994; Wood et al., 1994).
Synergism between fungal cellulase components has been studied most
extensively (Coughlan and Ljungdahl, 1988; Wood et al., 1988, 1989; Klyosov,
1990). The most interesting types of synergism are between: (i) endoglucanase/
exoglucanase (CBH); (ii) exoglucanase (CBH)/exoglucanase (CBH); and (iii)
endoglucanase/endoglucanase. An early model postulated by Wood and McCrae
(1972) suggested that cellulose chains cleaved by endoglucanase become the substrate
for exoglucanase, and that these two enzymes cooperatively degrade the cellulose.
However, this model did not explain the synergism seen between two different
CBHs, or the inability of CBH to synergize with endoglucanases from different
microorganisms. Subsequently, using highly purified endoglucanases and CBHs
from P. pinophilum, it was demonstrated that only two endoglucanases (EGIII and
EGV), which were strongly adsorbed on to cellulose, best synergized with CBHs I
and II (Fig. 2.4; Wood et al., 1989). The authors explained the observed synergism
between CBHs I and II and endoglucanases in terms of the different stereo-
specificities of these two enzymes.
The attack of a cellulose chain by a stereospecific endoglucanase will generate
only one of two possible types of non-reducing ends, which will be hydrolysed by a
stereospecific CBH. The successive removal of cellobiose by CBH will expose
another chain-end of a different configuration, which will be attacked by the other
stereospecific CBH. Hydrolysis of the two chain-ends by two CBHs acting
randomly, together with attack of the cellulose chains by another stereospecific
endoglucanase to generate a reducing end of a different configuration, would
facilitate the synergism observed between these enzymes. Nevertheless, it was argued
that the above synergism could be due to the strong adsorption of CBHs and
endoglucanases on to cellulose (Klyosov, 1990). Furthermore, Klyosov (1990)
reported that two endoglucanases which adsorbed strongly on to cellulose degraded
the cellulose synergistically. Although the adsorption appeared to be important, it is
difficult to explain the contribution of adsorption towards synergistic interaction
38
17 November 2000 14:59:46

Fig. 2.4. Synergism between P. pinophilum CBHs (I and II) and endoglucanases
(EI to EV) in solubilizing cotton fibre. Reproduced from Biochemical Journal,
Vol. 260, Wood, T.M., McCrae, S.I. and Bhat, K.M., The mechanism of fungal
cellulase action. Synergism between enzyme components of P. pinophilum
cellulase in solubilizing hydrogen bond-ordered cellulose, pp. 37–43, 1989,
with permission from Portland Press, London.
between endoglucanases and CBHs. Recent studies (Barr et al., 1996; Teeri, 1997)
revealed that there are two classes of CBHs (exoglucanases) which attack cellulose
chains from both reducing and non-reducing ends. Based on these results, it was
speculated that the synergism observed between CBH/CBH (exo/exo) was due to
their ability to expose new hydrolysis sites to each other, as well as their ability to act
from reducing and non-reducing ends (Barr et al., 1996). Thus, two CBHs acting
from reducing and non-reducing ends and an endoglucanase appeared to be essential
for the effective hydrolysis of crystalline cellulose. The presence of another
endoglucanase with different substrate specificity would increase the synergistic
efficiency further, as was observed in the case of the P. pinophilum cellulase system
(Wood et al., 1989).
Recent studies of the C. thermocellum cellulase system revealed that two
cellulosomal exoglucanases (S5 and S8 subunits) and an endoglucanase (S11 subunit),
together with the S1 (scaffoldin) subunit, are essential for maximum synergism
during the hydrolysis of crystalline cellulose (Bhat et al., 1994). Furthermore, the S1
subunit mediated the formation of an enzyme complex with the S5, S8 and S11
39
24 November 2000 15:11:02

subunits (Bhat et al., 1994). Using recombinant scaffoldin (CipA) polypeptides and
the endoglucanase CelD, it was demonstrated that a truncated scaffoldin polypeptide
with a CBD and a single cohesin domain was adequate for the maximum activity of
CelD towards Avicel (Kataeva et al., 1997; Beguin et al., 1998). It was also reported
that linkage of the CelD–CipA complex with the CBD was critical for maximum
synergism during Avicel solubilization, rather than the clustering of catalytic
domains (Kataeva et al., 1997; Beguin et al., 1998). These results strongly indicate
that, in the case of the aggregated C. thermocellum cellulase system, assembly of an
enzyme complex is crucial for the maximum synergistic interaction of subunits
during the solubilization of crystalline cellulose. Other studies using recombinant
cellulosomal components have confirmed the important role of the CipA CBD in
efficient cellulolysis (Ciruela et al., 1998).
Between xylan-degrading enzymes

Efficient and complete hydrolysis of xylan requires the synergistic action of main-
and side-chain-cleaving enzymes with different specificities (Coughlan et al., 1993;
Coughlan and Hazlewood, 1993a). For xylan-degrading enzymes, it is difficult to
demonstrate the synergism by measuring only the reducing sugars produced, because
of the heterogeneous nature of the substrate. Hence, the hydrolysis products must
be separated, identified and quantified in order to obtain a detailed picture of the
synergistic action of xylan-degrading enzymes. In fact, three types of synergy between
such enzymes have been described: (i) homeosynergy; (ii) heterosynergy; and (iii)
antisynergy (Coughlan et al., 1993). Homeo- and heterosynergies could be either
uni- or biproduct. Homeosynergy is the synergistic interaction between two or more
different types of side-chain-cleaving enzymes or between two or more types of
main-chain-cleaving enzymes (Coughlan et al., 1993). Homeosynergy is best demon-
strated for Neurospora crassa (Deshpande et al., 1986), Talaromyces byssochlamydoides
(Yoshioka et al., 1981) and Trichoderma harzianum (Wong et al., 1986), where endo-
xylanases of different specificities, or mixtures of endoxylanases and β-xylosidases,
cooperate in the hydrolysis of xylan. Another example of homeosynergy is reported
between ferulic acid esterase from A. oryzae and α-L-arabinofuranosidase from P.
capsulatum, where the former enzyme facilitates the release of arabinose from
feruloylated arabinoxylan by the latter (Coughlan et al., 1993).
Heterosynergy is the synergisitc interaction between side-chain- and
main-chain-cleaving enzymes (Coughlan et al., 1993). Heterosynergy is uniproduct
if the action of the main-chain-cleaving enzyme facilitates the release of a substituent
by the side-chain-cleaving enzyme, or vice versa. Heterosynergy is biproduct if the
extent of liberation of substituent, and the hydrolysis of the main chain, by the action
of combined enzymes exceeds the sum of the actions of the individual enzymes.
Heterosynergy has been reported between ferulic acid esterases and endoxylanases
(Faulds and Williamson, 1991; Tenkanen et al., 1991), α-L-arabinofuranosidases
and endoxylanases (Tuohy et al., 1992), acetyl xylan esterases and endoxylanases
(Biely et al., 1986; Lee et al., 1987), as well as between α-glucuronidases and
endoxylanases (Puls et al., 1987). A good example of heterosynergy is shown in
Fig. 2.5.
40
17 November 2000 15:00:17

Fig. 2.5. Heterosynergistic interactions in the hydrolysis of (a) feruloylxylan and

(b) arabinoxylan by fungal enzymes. Abbreviations: Ara, α-L-arabinofuranosidase;
Fae, ferulic acid esterase; Xyl, xylanase; exp, expected; obs, observed. Reprinted
from Hemicellulose and Hemicellulase, Coughlan, M.P. and Hazlewood,
G.P. (eds), Coughlan, M.P., Tuohy, M.G., Filho, E.X.F., Puls, J., Claeyssens, M.,
Vrsanska, M. and Hughes, M.M., Enzymological aspects of microbial hemi-
cellulases with emphasis on fungal systems, pp. 53–84, 1993, with permission
from Portland Press, London.
Antisynergy occurs when the action of one type of enzyme prevents the action of
a second enzyme (Coughlan et al., 1993). This can be observed in the case of either
arabinoxylan xylanohydrolase (Frederick et al., 1985) or glucuronoxylan
glucuronohydrolase (Nishitani and Nevins, 1991), an enzyme that cleaves the main
chain linkages only in the vicinity of a particular substituent. For example, in the case
of glucuronoxylan glucuronohydrolase, the presence of substituents is essential for its
action on the main-chain and the removal of the substituent by the relevant
side-chain-cleaving enzyme would preclude its action on the main chain. Although
antisynergy is unlikely to occur in vivo, it may be observed in in vitro experiments
where only one or two enzymes are used in a specific application.
Catalytic mechanism
Cellulases and xylanases catalyse stereoselective hydrolysis of the glycosidic bond with
either retention or inversion of configuration around the anomeric centre (Sinnott,
1990; Davies and Henrissat, 1995). Inversion occurs by way of a single displacement
reaction, while retention is via double displacement (Sinnott, 1990). Both reaction
mechanisms are assisted by general acid/base catalysis (Fig. 2.6).
During inversion, the protonation of the glycosidic oxygen and aglycone
departure are accompanied by a concomitant attack of a water molecule that is
activated by the catalytic base. This single nucleophilic substitution yields a product
with opposite stereochemistry to the substrate. In contrast, the retention mechanism
involves two steps: glycosylation and deglycosylation. In the glycosylation step, the
catalytic residue, acting as a general acid, protonates the glycosidic oxygen with
41
17 November 2000 15:00:34

Fig. 2.6. (a) Retention and (b) inversion mechanisms of glycosyl hydrolases.
Reprinted from Structure, Vol. 3, Davies, G. and Henrissat, B, Structures and
mechanisms of glycosyl hydrolases, pp. 853–859, 1995, with permission from
Current Biology Ltd, London.
concomitant C-O breaking of the scissile glycosidic bond. A deprotonated

carboxylate, functioning as a nucleophile, attacks the anomeric centre to give a
covalent glycosyl–enzyme intermediate. The deglycosylation step involves the attack
of a water molecule, assisted by the conjugate base of the general acid, to release the
free sugar with overall retention of configuration. Both steps proceed via transition
states with considerable oxocarbonium ion character. In both retention and inversion
mechanisms, the position of the proton donor is within hydrogen-bonding distance
of the glycosidic oxygen. In retaining enzymes, the nucleophilic catalytic base is in
close vicinity to the sugar anomeric carbon, whereas in inverting enzymes this base is
more distant, since it must accommodate a water molecule between the base and the
sugar. This difference results in an average distance between the two catalytic residues
of 5.5 Å in retaining enzymes, as opposed to 10 Å in inverting enzymes (Davies and
Henrissat, 1995).
42
17 November 2000 15:01:07

The stereochemical course of hydrolysis has been determined for glycosyl

hydrolases belonging to 42 of the 64 known families, of which families 1, 3, 5–9, 12,
26, 43–45, 48, 60 and 61 represent cellulases, and 10 and 11 represent xylanases
(Henrissat and Davies, 1997; Davies, 1998; Henrissat, 1998). Enzymes of families 1,
3, 5, 7, 10–12 and 26 proceed with retention of configuration, while enzymes from
families 6, 8, 9, 43, 45 and 48 proceed with inversion of configuration (Table 2.3).
Interestingly, all representatives of a particular family follow the same catalytic
mechanism (Gebler et al., 1992). Thus, it is possible to predict the catalytic
mechanism of a given cellulase or xylanase accurately once its family is known.
Other Characteristics of Cellulases and Xylanases

Induction and regulation
Induction and regulation of microbial cellulases and xylanases have been the subject
of detailed investigation (Ryu and Mandels, 1980; Kubicek, 1992; Biely, 1993;
Kubicek et al., 1993; Saloheimo et al., 1998). Both cellulases and xylanases are
inducible enzymes. It has been reported that cellulose or cellulose-rich material and
xylan or xylan-rich material are the best carbon sources for the production of high
levels of cellulases and xylanases by many microorganisms (Ryu and Mandels, 1980;
Biely, 1993). Since these polysaccharides cannot enter the microbial cell, it is
generally accepted that the low levels of cellulases and xylanases constitutively
produced by the microorganisms hydrolyse the corresponding polysaccharides to
soluble sugars, which enter the microbial cell and either act as, or get converted into,
true inducers and stimulate the transcription of cellulase and xylanase genes (Biely,
1993; Kubicek et al., 1993). In fact, it has been suggested that the conidial-bound
CBH of T. reesei hydrolyses cellulose to cellobiose and cellobiono-δ-1,5-lactone
(CBL), which promotes cellulase synthesis (Kubicek et al., 1988). Although cello-
biose and xylobiose are assumed to be the natural inducers of cellulases and xylanases,
the rapid utilization of these disaccharides suggested that the transglycosylated
products of these disaccharides or their positional isomers could in fact be the true
inducers (Ryu and Mandels, 1980; Biely, 1993). Sophorose (β-1,2-glucobiose), a
positional isomer of cellobiose, is reported to be an effective inducer of cellulases in
T. reesei and other species of Trichoderma, even at low concentrations (Sternberg and
Mandels, 1979). However, the inability of sophorose to induce cellulase synthesis
in other fungi (Moloney et al., 1983), and the ability of cellobiose to induce the
production of cellulase by T. reesei, in the presence of either a β-glucosidase inhibitor
or in a β-glucosidase defective mutant (Fritscher et al., 1990), strongly indicates that
cellobiose could be the true inducer of the cellulase system. Also, the positional
isomers of xylobiose (Xylβ,1-2Xyl; Xylβ,1-3Xyl) were found to be poor inducers of
xylanase in C. albidus compared with xylobiose (Xylβ,1-4Xyl), which suggests that
the latter is probably the true inducer of xylanase (Biely, 1993).
Initial studies using protein synthesis inhibitors suggested that the production
of cellulase is regulated at the translational level (Nisizawa et al., 1972). However, the
43
17 November 2000 15:01:08

34
17 November 2000 15:01:09

Table 2.3. List of cellulases and xylanases belonging to different families of glycosyl hydrolases and their catalytic mechanism.
Family Enzyme Organism EC number Mechanism Reference
1 β-Glucosidase Trifolium repens 3.2.1.21 Retention Barrett et al., 1995

3 β-Glucosidase Fungi and bacteria 3.2.1.21 Retention Warren, 1998
5 Endo A C. cellulolyticum 3.2.1.4 Retention Gebler et al., 1992
Cel B C. thermocellum 3.2.1.4 Retention Gebler et al., 1992
Cel C C. thermocellum 3.2.1.4 Retention Gebler et al., 1992
Cel H C. thermocellum 3.2.1.4 Retention Gebler et al., 1992
EG III T. reesei 3.2.1.4 Retention Gebler et al., 1992
44
Endo 5 T. fusca 3.2.1.4 Retention Gebler et al., 1992
EG Z E. chrysanthemi 3.2.1.4 Retention Barras et al., 1992
6 CBH II T. reesei 3.2.1.91 Inversion Rouvinen et al., 1990
Exoglucanase – E3 T. fusca 3.2.1.91 Inversion Barr et al., 1996

Endoglucanase T. fusca 3.2.1.4 Inversion Spezio et al., 1993
Cbh A C. fimi 3.2.1.91 Inversion Meinke et al., 1994
Cen A C. fimi 3.2.1.4 Invertion Withers et al., 1988
CHAP-2
7 CBH I T. reesei 3.2.1.91 Retention Divne et al., 1994
Endoglucanase I H. insolens 3.2.1.4 Retention Davies and Henrissat, 1995
Endoglucanase I T. reesei 3.2.1.4 Retention Claeyssens et al., 1990b
Endoglucanase I F. oxysporum 3.2.1.4 Retention Sulzenbacher et al., 1996; Mackenzie et al., 1997
8 Cel A C. thermocellum 3.2.1.4 Inversion Alzari et al., 1996
Cel C C. cellulolyticum 3.2.1.4 Inversion Fierobe et al., 1993

9 Endo D C. thermocellum 3.2.1.4 Inversion Juy et al., 1992
Cen B C. fimi 3.2.1.4 Inversion Gebler et al., 1992
Endo I T. fusca 3.2.1.4 Inversion Gebler et al., 1992
17 November 2000 15:01:10

Endo 4 T. fusca 3.2.1.4 Inversion Sakon et al., 1997

10 Xylanase A S. lividans 3.2.1.8 Retention Derewenda et al., 1994
CeX C. fimi 3.2.1.91 Retention Withers et al., 1988
Xyn Z C. thermocellum 3.2.1.8 Retention Gebler et al., 1992
Xylanase A P. fluorescens 3.2.1.8 Retention Harris et al., 1994
11 Xylanase B. circulans 3.2.1.8 Retention Wakarchuk et al., 1994
Xylanase B. subtilis 3.2.1.8 Retention Gebler et al., 1992
Xylanase S. commune 3.2.1.8 Retention Gebler et al., 1992
Xylanase T. reesei 3.2.1.8 Retention Havukainen et al., 1996
12 Endoglucanase (Cel B) S. lividans 3.2.1.4 Retention Sulzenbacher et al., 1997
45
26 Endoglucanase Bacteria 3.2.1.4 Retention Warren, 1998

43 Xylanases Fungi and bacteria 3.2.1.8 Inversion Warren, 1998

45 Endo V H. insolens 3.2.1.4 Inversion Davies et al., 1995
48 CBH b C. fimi 3.2.1.91 Inversion Shen et al., 1995
Endoglucanase – E6 T. fusca 3.2.1.4 Inversion Barr et al., 1996
CHAP-2
35
presence of high levels of cellulase mRNA in T. reesei grown on solka floc and the
complete absence of cellulase mRNA in T. reesei grown on glucose revealed that
the cellulase expression is regulated at transcriptional level (El-Gogary et al., 1989;
Saloheimo et al., 1998). The addition of low levels (1–2 mM) of sophorose resulted
in the accumulation of very high levels of mRNA in T. reesei (Ilmen et al., 1997).
Nevertheless, no cellulase mRNA was detected when sophorose was added to
glucose-grown culture or glucose was added to cellulose-grown culture of T. reesei
(Ilmen et al., 1997). These results clearly indicated that both induction and glucose
repression are operating at the transcriptional level during the regulation of cellulase
synthesis. Furthermore, all cellulase inducers promoted the synthesis of a constant
ratio of three major cellulases such as CBH I, CBH II and endoglucanase I in T.
reesei. This indicates that the expression of cellulase genes is coordinatedly regulated,
at least in T. reesei (Kubicek, 1992; Saloheimo et al., 1998).
In contrast, the induction pattern of hemicellulase genes appeared to be variable
even in T. reesei. For example, the expression pattern of xyn I and II genes was
somewhat different (Saloheimo et al., 1998). Also, genes coding for debranching
enzymes were efficiently induced by xylan having the appropriate substitutions.
Thus, regulation of hemicellulase genes appears to be more complex than cellulase
genes, even though both sets of genes are regulated through induction and catabolite
repression. Interestingly, two activator genes corresponding to proteins called ACE
I and II, responsible for the activation of cellulase expression, were isolated from
T. reesei (Saloheimo et al., 1998).
Recent studies on induction of cellulases and xylanases in Schizophilum
commune revealed that cellulose or cellulose-rich substrates greatly stimulated the
formation of both cellulases and xylanases by this fungus. Also, other carbon sources
such as cellobiose, lactose, sophorose and L-sorbose induced production of both
cellulase and xylanase (Haltrich et al., 1995). Although the production of xylanases
along with cellulases on media containing cellulose has been reported in the
case of Aspergillus sp., S. rolfsii, T. aurantiacus, P. pinophilum, etc., there are distinct
differences between these strains and S. commune (Brown et al., 1987; Yu et al.,
1987; Lachke and Deshpande, 1988; Bailey and Poutanen, 1989). Thus, these
fungi produced both cellulase and xylanase when xylan was present as a contaminant
with cellulose, and its removal from cellulose reduced their ability to produce
xylanase. In contrast, the removal of xylan from Avicel induced higher levels of
both cellulase and xylanase production by S. commune. In addition, cellulose
from Acetobacter pasteurianus, free of xylan, strongly induced the production of
both cellulase and xylanase in S. commune. Furthermore, cellobiose, the repeating
unit of cellulose, induced the production of both cellulase and xylanase by
S. commune, which strongly suggests that this fungus appears to possess a
common regulatory mechanism for the synthesis of cellulases and hemicellulases
(Haltrich et al., 1995). A similar regulatory system appears to operate in another
wood-rotting fungus, Polyporus adustus (Eriksson and Goodell, 1974). Thus, the
mechanism governing induction and regulation of cellulases and xylanases appears
to be quite complicated, and markedly different from one microorganism to
another.
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Physicochemical properties
Most fungal endoglucanases and CBHs (exoglucanases) have molecular masses rang-
ing from 20 to 100 kDa, whereas the molecular mass of β-glucosidase ranges from 50
to 300 kDa (Coughlan and Ljungdahl, 1988; Bhat et al., 1993). In general, fungal
cellulases are optimally active between pH 4.0–6.0, whereas bacterial enzymes show a
pH optimum of 6.0–7.0 (Wood, 1985). Many fungal endoglucanases and CBHs are
highly glycosylated while bacterial enzymes are less glycosylated (Wood et al., 1988;
Bhat et al., 1989). Endoglucanases, CBHs and β-glucosidases from mesophilic fungi
and bacteria are optimally active between 40 and 55°C (Wood et al., 1988; Bhat
et al., 1989; Christakopoulos et al., 1994a). Cellulases from thermophilic and
hyperthermophilic microorganisms are optimally active between 60–80 and
90–110°C, respectively (Khandke et al., 1989; Bhat et al., 1993; Antranikian, 1994).
Fungal and bacterial xylanases are almost exclusively single subunit proteins with
molecular masses ranging from 8.5 to 85 kDa, and with pI values of 4.0–10.3
(Coughlan et al., 1993). Based on their physicochemical properties, most xylanases
may be assigned to two classes: proteins with molecular mass < 30 kDa are usually
basic, whereas those with molecular mass > 30 kDa are usually acidic (Wong et al.,
1988). Also, most xylanases have acidic pH optima (Wong et al., 1988), with the
exception of xylanases from an alkalophilic thermophilic Bacillus sp., which are
optimally active at alkaline pH and are particularly suitable for paper and pulp
applications. In addition, xylanases from thermophiles are optimally active at
60–80°C, while those from mesophiles show optimal activity from 40–55°C
(Coughlan et al., 1993; Bennett et al., 1998).
Multiplicity of cellulases and xylanases
Most microorganisms capable of degrading cellulose and xylan produce multiple

forms of cellulases and xylanases with either very similar substrate specificities or
varying substrate specificities (Wong et al., 1988; Bhat et al., 1989). Based on bio-
chemical studies, it has been reported that T. reesei and P. pinophilum produce two
forms of CBHs and four to seven forms of endoglucanases (Gong et al., 1979; Wood
et al., 1988; Bhat and Wood, 1989), whereas A. niger, T. viride and T. emersonii
produce 15, 13 and 11–13 forms of xylanases, respectively (Biely, 1985; Coughlan
et al., 1993). Nevertheless, it is not certain whether the above multiple forms
of cellulases and xylanases are the products of multiple genes or the result of
post-translational modification of a single gene product by differential glycosylation
or partial proteolysis. Also, it is often difficult to distinguish the multiple forms of
cellulases and xylanases based on substrate specificity, because of the lack of defined
and structurally characterized substrates, and the preponderance of enzymes with
broad substrate specificity. It has been speculated that the apparent broad substrate
specificity of some cellulases and xylanases could be due to traces of contaminating
protein(s) present in purified samples. However, it has been clearly shown that
P. pinophilum produces seven to eight forms of endoglucanase, which differ with
47
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respect to their pI values, during the early logarithmic growth phase, and further
incubation only increases their concentration with minor variations in relative
proportions (Bhat and Wood, 1989).
The above results suggest that multiple endoglucanases must be the result of
expression of different genes, rather than post-translational modification. This fact
has been confirmed by the isolation and characterization of many cellulase and
xylanase genes from a number of bacteria and fungi (Gilbert and Hazlewood, 1993).
It has been shown conclusively that the isomeric forms of cellulases and xylanases,
observed in several microorganisms, are the consequence of large multigene families
and are not the result of processing of a single gene product. For example, at least 21
endoglucanase, three exoglucanase, six xylanase, and two β-glucosidase genes, and
one xylan esterase gene, have been isolated from C. thermocellum (Hazlewood et al.,
1988, Sakka et al., 1989; Hayashi et al., 1997). Likewise, multiple cellulase and
xylanase genes have been isolated and characterized from strains of Ruminococcus,
Pseudomonas, Cellulomonas, Bacillus, Fibrobacter and Butyrivibrio, etc. (Gilkes et al.,
1991a; Flint and Zhang, 1992; Gilbert and Hazlewood, 1993; Hazlewood and
Gilbert, 1998b–d). Furthermore, multiple cellulase and xylanase gene products
have been characterized and their structural and functional differences have been
demonstrated. Even though it is clear that multiple forms of cellulases and xylanases
are the products of different genes, a significant role for post-translational
modification in the production of some cellulase and xylanase isoforms cannot be
completely ruled out.
Adsorption
Adsorption of cellulases on to cellulose not only facilitates physical contact between

enzyme and substrate, but also plays an important role in the efficiency of the
enzymatic hydrolysis of crystalline cellulose (Klyosov, 1990). Cellulases that
adsorbed to a greater extent showed an increase in the rate and extent of crystalline
cellulose hydrolysis when compared with cellulases that adsorbed less strongly
(Klyosov, 1990). Moreover, the ability of endoglucanases to adsorb on to crystalline
cellulose varies significantly. For example, an endoglucanase from Aspergillus foetidus
showed weak adsorption on to cellulose, whereas that from C. thermocellum adsorbed
very strongly on to cellulose (Klyosov, 1990). The discovery of CBDs in some
cellulolytic enzymes explains in part the differences in adsorption ability of cellulases
(Gilkes et al., 1991b). However, a correlation between the hydrophobicity of
endoglucanases and their ability to adsorb on to cellulose has also been reported
(Klyosov, 1990). A study of the adsorption of 12 cellulase preparations and their
ability to hydrolyse amorphous and crystalline cellulose revealed that a 20-fold
increase in the adsorption capability of endoglucanases does not change the rate of
hydrolysis of CM-cellulose, but increases the rate of hydrolysis of amorphous and
crystalline celluloses by twofold and 50-fold, respectively (Klyosov, 1990). More
convincingly, Wood and co-workers (1989) demonstrated that a mixture of CBHs I
and II from P. pinophilum significantly synergized and effected extensive hydrolysis
48
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of crystalline cellulose when mixed with strongly adsorbed endoglucanases III and V
from the same source, but not in the presence of weakly adsorbed endoglucanases I,
II and IV.
Architecture and classification
The presence of separate catalytic and cellulose-binding domains in cellulases was

first demonstrated by proteolytic cleavage of cellulases from C. fimi and T. reesei
(Tomme et al., 1988; Gilkes et al., 1991b). Typically the two domains are linked by a
highly glycosylated amino acid sequence (six to 59 residues), rich in proline and
hydroxyamino acids. Some linkers are relatively rich in either Asp or Glu, or both.
The occurrence of this type of cellulase architecture has been extensively reviewed by
Tomme et al. (1995b). It was subsequently shown that many xylan-degrading
enzymes are also modular in structure. For example, in Pseudomonas fluorescens subsp.
cellulosa, four xylanases, an arabinofuranosidase and an acetylxylan esterase all have
modular architecture and, like the cellulases from this organism, all contain both
catalytic and non-catalytic CBDs, joined together by serine-rich linkers or hinges
(Hazlewood and Gilbert, 1998a,c,d). Moreover, a similar pattern of architecture
has been reported for xylanolytic enzymes from R. flavefaciens, C. fimi and Cellvibrio
mixtus (Flint et al., 1993; Millward-Sadler et al., 1994, 1995). Although the majority
of polysaccharide-binding domains mediate specific binding to cellulose, binding
domains with specificity for xylan have been described in xylanases from C. fimi
(Black et al., 1995) and T. fusca (Irwin et al., 1994). Other interesting non-catalytic
domains described in modular xylanases include thermostabilizing domains and
the so-called Nod B domains. Thermostabilizing domains have been found in
xylanases from thermophilic (Fontes et al., 1995) and mesophilic (Clarke et al.,
1996) bacteria and appear to confer stability in a broad sense on the enzymes that
contain them. Nod B domains occur in xylanases from P. fluorescens, C. mixtus and
C. fimi and have the capacity to release acetyl groups from acetylated xylan
(Hazlewood and Gilbert, 1998a).
Cellulose-binding domains
The CBDs of cellulases and xylanases from a wide range of microorganisms have
been extensively characterized (Tomme et al., 1995b; Linder and Teeri, 1997). Based
on their amino acid sequences, CBDs have been classified into more than 10 families
(Tomme et al., 1995b). Most of the CBDs characterized so far belong to families I, II
and III, while the remaining families have a few or only one member in each family.
Family I contains fungal CBDs of 30–40 amino acid residues; the best characterized
are from CBH I, CBH II, and endoglucanases II and III from T. reesei. All other
families contain bacterial CBDs, which are much larger in size than family I CBDs.
Family II CBDs are found almost exclusively at either the N or C terminus of
the enzyme and consist of typically 95–108 amino acids. The well-characterized
CBDs of this family are from Cex, CenA and CenB from C. fimi, CelA, CelB, CelC,
XylA, XylB, XylC and XylD from P. fluorescens subsp. cellulosa (Tomme et al.,
49
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1995c). Family III CBDs are found in cellulosome-producing bacteria, namely C.

thermocellum and C. cellulovorans, and contain 132–172 amino acids (Tomme et al.,
1995b). Based on amino acid sequence differences and other properties of family
III CBDs, Bayer et al. (1998) proposed three subfamilies called IIIa, b and c. Family
IIIa includes scaffoldin CBDs from C. thermocellum, C. cellulovorans and C.
cellulolyticum; family IIIb includes the CBDs of free cellulases listed in family III,
while family IIIc includes CBDs that lack cellulose-binding and anchoring residues.
Family IV CBDs are found in enzymes from C. fimi, C. cellulolyticum and other
bacteria, and contain 125–168 amino acids. CBDs of families V (E. chrysanthemi,
EgZ, 63 amino acids), VII (C. thermocellum, CelE, 240 amino acids) and VIII
(D. discoidum, CelA, 152 amino acids) are the sole members of their families, while
families VI, IX and X contain three to seven members with 85–90, 170–189 and
51–55 amino acids, respectively (Tomme et al., 1995b).
The three-dimensional structures of CBDs of families I (CBD of T. reesei CBH;
Kraulis et al., 1989), II (CBD of C. fimi Cex; Xu et al., 1995) and IV (CBD of C. fimi
β-1,4-glucanase; Johnson et al., 1996) have been determined by nuclear magnetic
resonance (NMR), while those of families IIIa (CBD of C. thermocellum scaffoldin;
Tormo et al., 1996) and IIIc (CBD of T. fusca cellulase E4; Sakon et al., 1997) were
determined by X-ray crystallography.
Although there is an overall functional similarity between CBDs from different
families, it is clear that they differ with respect to their affinity for different types of
cellulose (Linder et al., 1995, 1996; Tomme et al., 1995c, 1996). Also, the presence
of two successive CBDs in an enzyme is believed to result in synergistic binding
and an increase in affinity for the ligand (Linder et al., 1996; Tomme et al., 1996).
A number of approaches has been used to investigate the role of CBDs in cellulose
and xylan hydrolysis, including truncation of enzymes, domain swapping
and site-directed mutagenesis (Tomme et al., 1988, 1995b; Reinikainen et al.,
1992; Black et al., 1997; Srisodsuk et al., 1997), but a unifying role for CBDs has
yet to be established and their biological function continues to be a matter for
conjecture. Although the removal of CBDs from endoglucanases and CBHs does
not affect the activity of the respective enzymes against soluble substrates, there is
evidence that CBDs play a major role in potentiating the activity of cellulases against
the more resistant forms of insoluble cellulose (Tomme et al., 1988; Coutinho et al.,
1993; Hall et al., 1995). Indeed, an isolated family II CBD from C. fimi CenA was
shown to open the structure of crystalline cellulose, making it more accessible
to enzyme attack (Din et al., 1991). The fact that CBDs occur with high frequency
in xylan-degrading enzymes which have no activity against cellulose suggests
that these domains potentiate catalytic activity of hemicellulases by promoting
close contact between the enzymes and their native substrates in plant cell walls.
Support for this view has come from a number of recent studies (Hazlewood and
Gilbert, 1998b,c,d), including one which showed that the activities of a xylanase
and an arabinofuranosidase from P. fluorescens subsp. cellulosa, against plant
cell walls, were significantly enhanced by the presence of their CBDs (Black et al.,
1997).
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Catalytic domains
The catalytic domains of cellulases and xylanases are relatively large and represent
more than 70% of the total protein. Sequence analysis of these domains from various
cellulases and xylanases revealed extensive conservation of sequence and, using
hydrophobic cluster analysis, the catalytic domains of cellulases and xylanases
were initially grouped into 11 structurally related families (Henrissat et al., 1989).
According to this classification, the members of a single family possess the same
protein fold and display the same stereoselectivity (Gebler et al., 1992). This
suggested that the enzymes of a given family would follow either retaining or
inverting mechanisms (Table 2.3). Subsequently, the catalytic domains of some 1000
glycosyl hydrolases have been classified into 64 families (Henrissat and Bairoch,
1993, 1996; Henrissat, 1998). The cellulases and xylanases are contained in families
1, 3, 5–12, 26, 43–45, 60 and 61 (Henrissat and Davies, 1997; Davies, 1998;
Henrissat, 1998). This sequence-based classification has been enormously beneficial
in reflecting common structural features and evolutionary relationships, and as a tool
for deducing mechanistic information.
Structure/function relationships
Crystal structures have been determined for more than 20 glycosyl hydrolases,
including β-glucosidases, endoglucanases, exoglucanases and xylanases from families
1, 5–12 and 45. The main structural and functional properties of cellulases and
xylanases studied so far are summarized in Table 2.4. Interestingly, members of
families 1, 5 and 10 show a typical eightfold α/β barrel structure, commonly known
as a TIM barrel, but these families include β-glucosidase, endoglucanase and
xylanase, respectively (Table 2.4). Families 5, 8, 9, 12, 26 and 45 contain mainly
endoglucanases, families 10 and 11 include only xylanases, while families 6 and 7
contain both endoglucanases and CBHs.
The catalytic domains of T. reesei CBH II and endoglucanase (E2) from T. fusca
belong to family 6 and show a distorted α/β barrel structure (Rouvinen et al., 1990;
Spezio et al., 1993). Although both these enzymes belong to the same family and
possess a similar three-dimensional structure, there is a clear difference in their active
site architecture, with an enclosed tunnel consisting of four glucose-binding sites
in the case of CBH II, and an open active site in the case of the endoglucanase. The
catalytic domains of CBH I from T. reesei and endoglucanase I from H. insolens
belong to family 7 and show a similar β-jelly roll structure (Divne et al., 1994;
Sulzenbacher et al., 1996, 1997). However, CBH I has an extended active site tunnel
with seven glycosidic binding sites, while endoglucanase I from H. insolens shows a
more open structure suitable for endo-action.
CelA from C. thermocellum belongs to family 8 and folds into an α6/α6 barrel
consisting of six internal and six external helices (Alzari et al., 1996). A strictly
conserved residue, Glu-95, at the centre of the substrate-binding cleft, serves as a
proton donor. Three glucose-binding sites are identified on either side of the scissile
glycosidic linkage. Furthermore, the subsites do not lie in a straight line, which forces
51
17 November 2000 15:01:14

42
24 November 2000 15:16:59

Table 2.4. Structure and function relationships of cellulases and xylanases.
Enzyme Family Organism Structure Function Reference
β-Glucosidase 1 T. repens Eightfold α/β-barrel with pocket type Cleaves disaccharides from the Barrett et al., 1995
active site non-reducing end
Endoglucanase A 5 C. cellulolyticum Eightfold α/β-barrel with groove type Cleaves cellulose chain randomly Ducros et al., 1995
active site
Cel C 5 C. thermocellum As above with an additional segment As above Dominguez et al., 1995
CBH II 6 T. reesei Distorted (α/β) barrel with tunnel shape Releases cellobiose from the non- Rouvinen et al., 1990
active site reducing end and facilitates threading
52
of cellulose chain through it
Endoglucanase-E2 6 T. fusca As above with an open active site Randomly acting enzyme Spezio et al., 1993
CBH I 7 T. reesei β-Jelly roll with a long active site tunnel Releases cellobiose from the Divne et al., 1994
reducing end of cellulose chain

Endoglucanase I 7 H. insolens β-Jelly roll with a long substrate binding Randomly acting enzyme Sulzenbacher et al., 1996,
groove 1997
Cel A 8 C. thermocellum α/α Barrel – six internal and six external Randomly acting enzyme with Alzari et al., 1996
CHAP-2
helices with groove type active site six glycosyl binding sites

Cel D 9 C. thermocellum α/α Barrel with two distinct domains, Randomly acting enzyme with Juy et al., 1992
and extended active site six glycosyl binding sites
9 T. fusca Possess both family 9 catalytic Shows both endo- and Sakon et al., 1997
domain with an (α/α)6 barrel fold, exoglucanase activities with
and a family III CBD, having an six glycosyl binding sites
anti-parallel β-sandwich fold

Exoglucanase – 10 C. fimi Eight-parallel stranded α/β protein Releases cellobiose from the White et al., 1994
CeX with an open cleft active site, and non-reducing end of cellulose
belongs to 4/7 superfamily chain and hydrolyses xylosidic
24 November 2000 15:17:46

and glucosidic linkages

Xylanase A 10 S. lividans (α/β)8 Barrel with pocket type active Hydrolyses xylan and pNPC Derewenda et al., 1994
site and belongs to 4/7 superfamily preferentially, and possesses six
glycosyl binding sites
Xylanase A 10 P. fluorescens (α/β)8 Barrel, belongs to 4/7 superfamily Randomly cleaves xylan chain Harris et al., 1994
and with an unexpected Ca2+ binding site and possesses five glycosyl
which stabilizes the catalytic core binding sites
Xylanase 11 B. circulans β-Jelly roll with an overall structure Randomly acting enzyme with Wakarchuck et al., 1994
of partially closed right hand five glycosyl binding sites
Xylanase 11 T. harzianum As above As above Campbell et al., 1993
Xylanase I 11 T. reesei As above As above Torronen and Rouvinen, 1997
Xylanase II 11 T. reesei As above As above but with four glycosyl Torronen et al., 1994;
53
binding sites Havukainen et al., 1996

Xylanase 11 T. lanuginosus As above As above but with seven glycosyl Gruber et al., 1998
binding sites
Endoglucanase 12 S. lividans β-Jelly roll with anti-parallel β-sheets Randomly acting enzyme with five Sulzenbacher et al., 1997

(Cel B) arranged in a sandwich fashion, and glycosyl binding sites
open active site
Endoglucanase 45 H. insolens Six-stranded β-barrel with an open Releases cellobiose from the Davies et al., 1993
CHAP-2
cleft active site reducing end and possesses six

glycosyl binding sites
43
the substrate to bend at the active site and thus requires lower energy for cleavage of
the glycosidic bond. CelD of C. thermocellum belongs to family 9 and has a globular
and slightly elongated shape with a small N-terminal β-barrel closely packed against a
large, mostly α-helical domain (Juy et al., 1992). An extended active site with
six glycosyl-binding sites has been suggested since it was possible to fit cellohexaose
into the active site. Recently, the three-dimensional structure of E4 from T. fusca,
belonging to the same family, has been determined (Sakon et al., 1997). This enzyme
contains both a family 9 catalytic domain with an α/α6 barrel fold, and a family III
CBD, having an antiparallel β-sandwich fold. In addition, this enzyme functions as
both endo- and exoglucanase and possesses an extended glycosyl-binding site, similar
to that of C. thermocellum CelD.
Xylanases from families 10 and 11 follow the retaining mechanism and possess
essential glutamates in their active sites. Family 10 xylanases show a typical eightfold
α/β barrel structure (Harris et al., 1994), while family 11 xylanases possess a β-jelly
roll architecture with two parallel β-sheets and an α-helix (Torronen and Rouvinen,
1997; Pickersgill et al., 1998). Family 12 endoglucanase from Streptomyces lividans
shows a β-jelly roll topology with antiparallel β-sheets arranged in a sandwich
manner, whereas the family 45 endoglucanase from H. insolens has a six-stranded
β-barrel structure (Davies et al., 1993; Sulzenbacher et al., 1997). The family 12
enzyme possesses five glycosyl-binding sites and cleaves the glucan chain randomly,
while the family 45 enzyme has six glycosyl-binding sites and attacks between the −1
and +1 subsites at the reducing end of the cellulose chain.
Applications
In addition to their established use as animal feed additives, cellulases and xylanases
have a wide range of applications in food, paper and pulp, textile, fuel and chemical
industries (Table 2.5) (Mandels, 1985; Gilbert and Hazlewood, 1993; Beguin and
Aubert, 1994; Bhat and Bhat, 1997). In a wider context, these enzymes have
potential uses in waste management, pollution treatment, protoplast production,
genetic and protein engineering as well as other research and development
programmes (Table 2.5) (Beguin and Aubert, 1994; Bayer et al., 1995; Bhat and
Bhat, 1997). Thus, cellulases and xylanases have a wide range of potential
applications, but further research is necessary to exploit the commercial potential
of these enzymes to the fullest extent.
Conclusions
Basic and applied research carried out during the past decade has shown that
cellulases and xylanases are of great intrinsic interest, and have a range of novel
and valuable uses in biotechnology. Early biochemical studies of purified enzymes,
coupled with more recent molecular studies utilizing recombinant DNA tech-
nologies, have provided detailed information on the biochemistry, architecture,
54
17 November 2000 15:01:18

Table 2.5. Applications of cellulases and xylanases.
24 November 2000 15:20:37

Industry and research Enzyme Applications
1. Food, brewery and wine Cellulases Extraction and clarification of fruit juices and oil from seeds; increasing the soaking efficiency and homogeneous
water adsorption of cereals; improving the digestion of ball-milled lignocellulose and the nutritive quality of
fermented foods; production of cello-oligosaccharides and sugar derivatives; releasing flavours, enzymes and
proteins; improving the filtration of beer and increasing the aroma of wines (Coughlan, 1985; Mandels, 1985;
Beguin and Aubert, 1994; Caldini et al., 1994)
Xylanases Clarification of fruit juices and wines; production of food thickeners; improving the texture and quality of bakery
products, production of xylo-oligosaccharides, xylitol (artificial sweetener) and sugar derivatives (Maat et al.,
1992; Viikari et al., 1993)
2. Animal feed Cellulases and As a feed supplement for poultry, pigs and ruminants; pretreatment of forages; dehulling of cereal grains; treatment
55
xylanases of silage (Bedford and Classen, 1992; Selmer-Olsen et al., 1993; Viikari et al., 1993; Lewis et al., 1996)
3. Paper and pulp Xylanases Pre-bleaching of kraft pulps and debarking; refining pulp fibres and preparation of dissolving pulps (Wong and
Saddler, 1992, 1993; Viikari et al., 1993; Ryan et al., 1998)

4. Textile Cellulases Biostoning (removing excess dye from the denim fabric in pre-faded jeans), use in washing powders for retaining
the originality of cotton fabrics after several washing cycles by removing the cellulose microfibrils as well as
restoring the softness and colour brightness of cotton fabrics (Mandels, 1985; Beguin and Aubert, 1994)
CHAP-2
5. Waste management Cellulases and Treatment of agricultural and municipal wastes; composting and enzymatic processing of industrial pollutants
xylanases (Rivard et al., 1994; Philippidis and Smith, 1995; Scott et al., 1995; Bhat and Bhat, 1997)
6. Research and Cellulases and Production of protoplasts and hybrid strain; genetic engineering; affinity purification; understanding the plant cell
development xylanases wall structure and chemistry (Beguin and Aubert, 1994; Bayer et al., 1995; Bhat and Bhat, 1997)
45
catalytic mechanisms and structure–function relationships of these important

enzymes. Two clear models have emerged for native cellulase–hemicellulase systems.
In the first model, exemplified by aerobic fungi and bacteria, individual cellulases and
hemicellulases are secreted freely by the host, without aggregating, and interact
synergistically to degrade their target substrates in plant cell walls. Anaerobic bacteria,
and apparently also anaerobic fungi, exhibit an alternative mechanism for cellulase/
hemicellulase hydrolysis in which the individual enzymes involved form an ordered
multienzyme complex or cellulosome. In both models, individual enzymes are
typically modular in structure and, in addition to one or more catalytic domains,
contain non-catalytic functional domains, the most common of which is a
cellulose-binding domain that enables the enzyme to cluster on the surface of
cellulosic substrates. Structural studies of single enzymes have resulted in high
resolution crystal structures for representatives of more than 20 glycosyl hydrolases
families, and the identification of key catalytic residues.
Notwithstanding the progress of recent research, there are still many
unanswered questions regarding: (i) the biological significance and prevalence of
multiple domains in cellulases and xylanases; (ii) the relative efficiencies of the
multienzyme and disaggregated cellulase/hemicellulase systems; and (iii) the roles of
individual enzymes during the solubilization of complex lignocellulosic substrates.
Having the answers to such questions will not only enhance understanding of how
cellulases and xylanases function, but should also enable these enzymes to be used
more effectively as animal feed additives.
Acknowledgements
Financial support of the BBSRC is gratefully acknowledged.
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Enzymatic Characteristics of
Phytases as they Relate to Their
Use in Animal Feeds
3
D.D. MAENZ
Prairie Feed Resource Centre (Canada) Inc., Department of Animal and
Poultry Science, University of Saskatchewan, Saskatoon,
Saskatchewan, Canada S7N 0W0
D.D. Maenz of Phytases

Characteristics
3
Introduction
Substantial quantities of phytic acid occur in plant-based diets fed to production
animals throughout the world. Phytic acid is poorly digested by monogastric animals.
Disposing of manure containing excess phytate-phosphorus can damage freshwater
and other ecosystems. The poor digestibility of phytate-P increases the cost of
production since additional sources of available P are needed in ration formulation to
meet the nutritional requirements of the animal. Further, undigested phytate is
known to have a negative impact on mineral and protein digestibility. In recent years
considerable research has been directed toward understanding the process of phytate
digestion and developing methods to improve phytate-P utilization by animals. The
development of enzyme technology based on supplementing diets with sources of
microbial phytase has proven to be a practical and effective method of improving
phytate digestibility in animal diets.
Several excellent reviews have appeared covering the environmental and
nutritional consequences of phytic acid (Cheryan, 1980; Torre et al., 1991;
Ravindran et al., 1995; Rickard and Thompson, 1997), and the application,
structure and kinetic properties of phytase enzymes (Dvorakova, 1998; Liu et al.,
1998). The purpose of this chapter will not be to revisit material that has been
extensively covered in other publications. Rather, the intentions are to provide a
general overview of the subject area in combination with a specific focus on factors
that affect the efficacy of phytate hydrolysis and methods to reduce further or
eliminate indigestible phytate in plant-based diets fed to production animals.

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62 D.D. Maenz
Phytic Acid in Animal Diets

Structure of phytic acid
Structurally, phytic acid consists of a fully phosphorylated myo-inositol ring (Fig.

3.1) that exists in a chair conformation in dilute solution (Johnson and Tete, 1969).
The molecule contains a total of 12 proton dissociation sites. Six of these sites are
strongly acidic, with pKa values approximating 1.5; three sites are weakly acidic, with
pKa values of 5.7, 6.8 and 7.6; and the remaining three sites are very weakly acidic,
with pKa values > 10 (Costello et al., 1976). The structure of the molecule is consis-
tent with a high chelation capacity for multivalent cations. In solution, the stability
of phytate–mineral complexes can be measured by the pH-drop method whereby
metals displace H+ for binding to phosphate groups on the phytate molecule
(Maddaiah et al., 1964; Vohra and Kratzer, 1965). In summarizing the results of
several studies using the pH-drop method, Cheryan (1980) concluded that phytic
acid readily forms complexes with multivalent cations, with Zn2+ forming the most
stable complexes followed by Cu2+, Ni2+, Co2+, Mn2+, Ca2+ and Fe2+ in decreasing
order of stability. The phytate–mineral complex can exist as either soluble chelates or
as insoluble complexes that precipitate out of solution, depending on the concentra-
tions of phytic acid and mineral and the pH of the solution (Cheryan, 1980).
Some information is available on the structure of soluble mineral–phytate
complexes. Champagne et al. (1990) and Champagne and Fisher (1990) studied
shifts in 31P NMR spectra of phytic acid in solution at pH 7 with a relatively
low molar ratio of minerals to phytate. These workers proposed that, under the
conditions used in their experiment, a single Zn2+ ion binds to the P5 phosphate
groups and thereby forms a bridge between two molecules of phytate. In comparison,
Ca2+ and Cu2+ showed more of a tendency to associate with the P4 and P6 phosphate
groups and thus was likely to form soluble complexes by electrostatic binding within
a single phytate molecule. Mineral–phytate precipitants formed as the molar ratio of
mineral to phytate increased at neutral pH (Champagne et al., 1990).
Fig. 3.1. Structure of the fully deprotonated form of phytic acid (myo-inositol
hexakis phosphate).
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Characteristics of Phytases 63
Insoluble phytate–mineral complexes tend to form at neutral and basic pH

when the concentration of mineral exceeds the concentration of phytate. At medium
pH < 5, phytate does not precipitate out of solution in the presence of Ca2+
(Grynspan and Cheryan, 1983) or Mg2+ (Cheryan et al., 1983) even at
mineral : phytate ratios of 12 : 1. These results imply that one or more of the weak
acid phosphate groups on the molecule has a higher affinity for protons than Ca2+
or Mg2+. In both of these studies, as the pH of the medium was increased, the
mineral : phytate molar ratio required for phytate precipitation decreased. Under
conditions of excess mineral at neutral pH, it was suggested that precipitates con-
sisted of pentacalcium (Grynspan and Cheryan, 1983) or pentamagnesium (Cheryan
et al., 1983) salts of phytate. At acidic pH, partial protonation of phytic acid will
decrease net association of minerals to the molecule and thus prevent the formation
of insoluble precipitates. As described later, the structure of phytic acid as affected by
minerals and pH has profound effects on the process of phytate hydrolysis.
Occurrence of phytic acid in plants
Phytic acid serves as the storage form of P in plant seeds. Ravindran et al. (1995)
provided an extensive review of the occurrence of phytic acid in a wide variety of
plants and plant materials. Cereals (maize, barley, wheat, oats) and grain legumes
(field peas, chickpeas) that are commonly used as feed ingredients all have similar
phytate levels, approximating 0.25% of dry matter. In general, oilseed meals tend
to have higher levels of phytate-P as indicated by the percentage of dry matter
composition for soybean (0.39%), rapeseed meal (0.70%), cottonseed meal (0.84%)
and sunflower meal (0.89%). On average, 70% of the total P in the feed ingredient is
found as phytate-P.
In plants, phytic acid complexes K+ and Mg2+, and to a lesser extent Ca2+, to
form phytin. Phytin is deposited within the proteinaceous matrix of membrane-
bound protein vacuoles or protein bodies. Under the electron microscope, phytin is
often visible within the protein bodies as insoluble globoid crystal structures. In rice
grains, globoid crystals are composed of 67% phytic acid, 19% K, 11% Mg and 1%
Ca on a dry weight basis (Ogawa et al., 1975). The size of the globoid crystals is
dependent upon the ratio of divalent cations/K in the protein bodies. Ratios that
favour divalent cations result in large insoluble crystals (Lott et al., 1985). Legumes
such as soybeans and peas have higher levels of K and thus smaller globoid crystal
structures and a proportionally greater amount of phytin as predominantly K-bound
soluble forms of phytic acid (Lott and Ockenden, 1986). This soluble phytin tends
to exist as phytin–protein complexes that are evenly dispersed within the protein
bodies.
The location of phytin-containing protein bodies in the seed varies considerably
between plants. In wheat and rice phytin is found primarily in the aleurone layer and
the bran, while in maize phytin is concentrated within the endosperm (O’Dell et al.,
1972; de Boland et al., 1975). Phytin tends to be distributed evenly throughout the
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64 D.D. Maenz
seed in oilseeds and legume grains (Erdman, 1979). The form and location of phytin
within the seed are likely to be important variables that impact on the efficiency of
phytate hydrolysis. One could hypothesize that phytin as insoluble globoid crystals in
the fibrous layers of the seed coat would be relatively indigestible in comparison with
phytin that exists predominantly as soluble forms in the germ portion of the seed.
Effects of phytic acid in animal diets
Environmental effects
In the United States, agriculture is the leading source of contaminants that have a
negative impact on water quality (Parry, 1998). An estimated 60% of river miles and
50% of lakes that are classified as impaired are negatively affected by agriculture
(Daniel et al., 1998). Animal wastes are the primary source of P pollution from
agricultural practices (Daniel et al., 1998). In areas of intensive livestock production,
P added to land from animal waste can readily exceed the capacity of the crop to
incorporate the mineral. This, in turn, can lead to excess P accumulation in the
soil and the potential for runoff into lakes and rivers. Problems associated with
agriculture and P pollution are greatest in areas of intensive livestock production
with close proximity to freshwater systems. In The Netherlands there is a N, P and K
imbalance and livestock production contributes over 80% of the excess minerals
accumulating in the environment (de Boer et al., 1997). The manure problem in The
Netherlands has led to legislation that limits mineral application to soils from animal
wastes (de Boer et al., 1997).
Undigested phytate-P is the primary source of P in the manure. Any measures
that decrease unavailable phytate-P in animal diets will be effective in decreasing the
P load on the environment arising from animal production. Further, for mixed
farming operations, decreasing the P content of swine manure may benefit net
economic returns in areas that are generally not classified as environmentally
sensitive. Bosch et al. (1998) determined that reducing manure P decreases the land
area required for application based on crop P requirements which, in turn, decreases
crop production costs.
Effects on mineral utilization

Undigested phytic acid is known to decrease mineral bioavailability. The reader is
referred to the reviews of Cheryan (1980) and Torre et al. (1991) on the formation of
mineral–phytate complexes and the effects of phytate on mineral digestibility. In
general an inverse relationship exists between the phytate content of the diet and
digestibility of multivalent cations. As previously described, phytate can complex
with multivalent cations, forming insoluble complexes at neutral pH. These
complexes appear to be resistant to the digestive–absorptive process and the net result
is that a portion of the dietary mineral pool is unavailable to the animal.
In rats, Zn bioavailability is markedly affected by the phytate content of the diet
(Forbes et al., 1984; Zhou et al., 1992; Saha et al., 1994). These results are consistent
with in vitro work indicating a high stability of Zn–phytate complexes (Cheryan,
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1980). Other minerals, such as Ca, when added at increasing levels in the presence of
a constant level of phytate, further reduced Zn bioavailability in rats (Ellis et al.,
1982; Forbes et al., 1984). The mechanism of this effect is likely to involve increased
formation of insoluble phytate–mineral complexes containing both Ca and Zn that
are resistant to the digestive–absorptive process.
In humans, dephytinization of preparations of soybean protein isolates doubled
the mean fractional Fe incorporation into red cells (Davidsson et al., 1994) and
resulted in 2.3-fold increase in Mn absorption (Davidsson et al., 1995). Fe solubility
under simulated physiological conditions in vitro was increased from 3 to 53% with
dephytinization of wheat bran and from 5 to 21% with dephytinization of rye flour
(Sandberg and Svanberg, 1991).
Han et al. (1994) compared the effects of IP6 (phytic acid), IP5, IP4 and IP3 on
Fe and Zn uptake by the human adenocarcinoma cell line Caco-2 and found that
mineral solubility and uptake rates decreased with the degree of phosphorylation of
the inositol ring. Lower forms of inositol phosphates that are formed during the
hydrolysis of the parent phytate have fewer mineral-binding sites, a diminished
chelation capacity, and thus less of an effect on mineral bioavailability than is the case
for phytic acid (Tao et al., 1986).
Interestingly, certain compounds with chelation capacity have been shown to
increase mineral availability when included in plant-based diets fed to animals.
Supplementation of Zn-deficient diets with various chelators such as EDTA
promoted growth and prevented symptoms of Zn deficiency in chicks (Nielsen et al.,
1966) and turkey poults (Vohra and Kratzer, 1964). Pectins characterized by a
high degree of esterification and a low molecular weight are known to complex with
minerals and have been shown to improve Fe solubility and absorption in rats
(Kim and Atallah, 1993). Ascorbic acid and citric acid were shown to enhance Fe
bioavailability in vegetarian diets (Anand and Seshadri, 1995) and ascorbic acid
increased Fe bioavailability in soy-based infant formulas (Davidsson et al., 1994). A
mechanism of competitive chelation could account for the effect of chelators on
improving mineral bioavailability. In this model the chelator binds minerals and
thereby decreases the pool of minerals forming insoluble phytate complexes. The
mineral–chelator complex exists in a soluble form and thus could be absorbed intact
or could release minerals to binding sites on the brush border membrane of the
intestinal epithelium. The concept of competitive chelation as a mechanism for
improved phytate hydrolysis will be developed in a later section of this review.
Effects on protein digestibility

The charged phosphate groups on phytic acid can form electrostatic associations with
the terminal amino group on proteins or with the free amino group on lysine and
arginine residues within protein molecules (Cheryan, 1980). In addition, phytate–
mineral–protein complexes can form with multivalent cations acting as a bridge
between phosphate groups on the phytate molecule and the terminal carboxyl group
of proteins or free carboxyl group on aspartate and glutamate residues within protein
molecules (Cheryan, 1980). In theory, protein in the phytate-bound form may be
less susceptible to protease activity during intestinal passage. In addition, phytate
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66 D.D. Maenz
binding to proteins and minerals in the digesta has the potential to impair the activity
of digestive enzymes. Phytate has been shown to inhibit trypsin activity (Singh and
Krikorian, 1982; Caldwell, 1992) and in vitro starch digestion with human saliva
(Yoon et al., 1983). The mechanism may involve mineral chelation and thus removal
of cofactors required for optimum enzyme activity and/or the formation of less
reactive phytate–enzyme complexes.
With a few notable exceptions, a review of the literature generally supports the
concept that phytic acid does impair in vivo protein digestibility. Thompson and
Serraino (1986) reported that phytic acid did not affect rapeseed protein digestibility
and amino acid absorption. Chitra et al. (1995) found a negative correlation between
phytic acid content and in vitro protein digestibility in genotypes of mung bean,
urd bean and soybean. Phytic acid–protein interactions have been reported to
decrease the availability of protein in legumes (Camovale et al., 1988). Complete
dephytinization of a diet based on maize and soybean meal increased dialysable
protein by 12–29% (Zyla et al., 1995). Dephytinization of soy-protein concentrate
improved protein digestibility in fish (Storebakken et al., 1998). One could
hypothesize that the effect of phytate on protein and amino acid digestibility might
vary considerably between diets based on plant ingredients and the mineral status of
the diet. Phytate that is largely in the form of insoluble mineral-bound complexes
may be less active as an inhibitor of protein digestion. Clearly, interactions between
phytate, minerals and proteins and effects of these interactions on protein
digestibility are a complex issue that deserves further research.
Digestion and Utilization of Phytate

In diet formulation, plant P is often classified as available and unavailable and a figure
of 30% is routinely assigned as an estimate of available plant P. This exercise is useful
in that it permits a simple calculation of supplemental P that must be added to the
diet to meet the nutritional requirements of the animal. However, an assumption
that 30% of total plant P is available and thus 70% is unavailable is simplistic and, in
many circumstances, may be grossly inaccurate. As described in detail by Ravindran
et al. (1995), a review of the literature shows considerable variation in the digestibility
of dietary P of plant origin. As an example, Nelson (1976) reported 0% phytate-P
digestibility in chicks fed maize-based diets and 8% phytate-P digestibility with
wheat-based diets. In comparison, other studies have shown that chicks can retain up
to 60% of dietary phytate-P (Temperton and Cassidy, 1964a,b). Ballam et al. (1984)
found that phytate hydrolysis ranged from 3 to 42% in the chick and was dependent
upon the level of dietary Ca. Phytate-P retention values of 37–56% were reported for
chicks fed suboptimal levels of non-phytate-P (Edwards, 1983).
Interestingly, phytate-P when added to the diets in acidic form or as a sodium
salt is readily available to chicks and will support growth and bone mineralization to
the same extent as P originating from dicalcium phosphate (Harms et al., 1962;
Waldroup et al., 1964a). P originating from supplemental calcium phytate could
support growth and bone mineralization at levels up to 0.2% dietary P and at a
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Ca : P ratio of 0.8 : 1 (Waldroup et al., 1964b). In this study, increasing the dietary
Ca : P ratio to 1.4 : 1 or 2 : 1 decreased the apparent utilization of P from calcium
phytate (Waldroup et al., 1964). These studies provide strong support for the
concept that the form and location of phytate within the plant material is an
important factor defining phytate-P utilization.
Phytate digestibility is ultimately dependent upon the efficacy of hydrolysis of
phytate prior to feeding and during digestive passage. Hydrolysis is dependent upon
phytase activity originating from plant, animal or microbial sources. The efficiency of
this hydrolytic process is dependent upon the total phytase activity in combination
with other factors such as the form and location of phytin within the plant ingredient
and the physical conditions of the reaction.
Sources of phytase activity
Dvorakova (1998) and Liu et al. (1998) provided extensive reviews of the structure
and kinetic characteristics of phytase from microbial, plant and animal sources.
There will be no attempt to elaborate further or to assemble an extensive review of
this area here. The discussion in this chapter will be restricted to key points on what is
known of phytase structure and kinetic function.
Phytases can be broadly categorized into 6-phytase and 3-phytase classes. This
designation refers to the site of initial hydrolysis on the phytate molecule. The
6-phytases are commonly found in plants, while 3-phytases are produced by fungi
(Dvorakova, 1998). The full sequence of hydrolysis of phytic acid down to
myo-inositol has yet to be determined. However, it would appear that no single
enzyme is capable of fully dephosphorylating phytic acid and thus a combination
of phytase and non-specific phosphatase activities are involved in the process. No
information is available as to the mechanism of phytate hydrolysis by the small
intestinal phytase activity in animals.
Animal phytase
Often it is assumed, and frequently stated, that animals do not possess the capacity to
hydrolyse phytic acid. However, a specific phytase activity has been demonstrated in
the brush border membrane of the chick small intestine (Maenz and Classen, 1998).
Earlier studies demonstrated phytate hydrolysis with small intestinal mucosal
preparations from chick (Davies et al., 1970; Bitar and Reinhold, 1972; Davies and
Motzok, 1972), human (Bitar and Reinhold, 1972; Iqbal et al., 1994), calf (Bitar and
Reinhold, 1972) and rat (Bitar and Reinhold, 1972; Yang et al., 1991; Iqbal et al.,
1994). The contribution of mucosal phytase to phytate-P utilization by animals is
not known, but animals are known to utilize a portion of the total phytate-P with no
supplemental phytase in the diet. Further, a substantial body of literature indicates
that dietary supplementation with 1,25-dihydroxycholecalciferol improves phytate-P
digestibility in poultry (Ravindran et al., 1995). Rats (Yang et al., 1991) and chickens
(Davies et al., 1970) fed phosphorus-deficient diets show an increase in intestinal
phytase activity and in the case of the rat this correlated with an increase in a 90 kDa
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68 D.D. Maenz
protein isolated from the intestinal mucosa (Yang et al., 1991). Taken together, these
studies indicate that an intestinal phytase probably does contribute to the utilization
of phytate-P and the activity of the enzyme may well be regulated by the mineral and
vitamin status of the animal.
Plant phytase
Almost all plants contain some phytase activity, though the level of phytase and the
significance of the enzyme in hydrolysing phytate within the seed varies considerably
between plants. Eeckhout and De Paepe (1994) evaluated phytase levels in 51
feedstuffs used in Belgian feed mills and concluded that substantial phytase
activity occurs in cereal grains such as rye, triticale, wheat and barley while all other
feedstuffs, including soybean meal, contained marginal levels of phytase. Phosphorus
availability in wheat fed to poultry ranged from 45 to 70% (Barrier-Guillot et al.,
1996b). Barrier-Guillot et al. (1996a) measured phytase activity in 56 wheat samples
grown in France in 1992 and found a substantial variation that ranged from 206 to
775 mU g−1. In a follow-up study (Barrier-Guillot et al., 1996b), the same group
found a linear correlation between wheat phytase activity and P retention in broilers.
Pointillart (1994) pooled results on plant P availability in pigs fed cereal-based diets.
Availability ranged from less than 20% for diets with little phytase up to 60% for
diets rich in plant phytase activity and a clear linear relationship was found between
the plant phytase activity in the diet and P digestibility. Kemme et al. (1998)
measured gastric degradation of phytic acid in pigs as 3% when fed a maize-based
diet (91 phytase units kg−1), 31% when fed a maize–wheat-based diet (342 phytase
units kg−1) and 47% when fed a wheat–barley-based diet (1005 phytase units kg−1).
Clearly, the high phytase activity found in wheat and barley is significant and does
contribute to phytate digestibility. These studies argue against a simple value for
available P based solely on the total and phytate-P content of the diet. As such, the
potential exists to refine diet formulation to meet the P requirements of the animal
based on a realistic estimate of available P in a given plant-based diet.
Microbial phytase
Enzymes with hydrolytic activity directed toward phytic acid are produced by a
variety of microorganisms. Dvorakova (1998) listed 29 fungal, bacterial and yeast
species known to produce a phytase activity. Of the 29 species listed, 21 produced
an extracellular phytase. Strains of the filamentous fungi Aspergillus niger produce
a high-activity extracellular phytase (Volfova et al., 1994). Currently, commercial
phytase products are based on the phytase encoding gene originating from A. niger.
The enzymes produced by A. niger var. ficuum are by far the most extensively
studied phytase. Originally a single activity was described with an optimum pH
of 5.0 plus a secondary optimum pH at 2.5 (Van Hartingsveldt et al., 1993).
Subsequently, a second distinct phytase from A. niger var. ficuum, with a pH
optimum of 2.5 and no activity at pH 5.0, was described (Ullah and Phillippy,
1994). The pH 5 optimum enzyme has been termed PhyA and the pH 2.5 optimum
enzyme has been termed PhyB. The amino acid composition of the unglycosylated
A. niger var. ficuum PhyA indicates a molecular weight of 62 kDa (Ullah, 1988).
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The protein is 27.3% glycosylated with a molecular weight of 84 kDa in the native
form (Ullah and Gibson, 1987). PhyA has a high affinity for phytic acid with a Km
that approximates 40 µM (Ullah and Gibson, 1987). Variations in temperature and
pH were found to have little effect on the affinity of PhyA for phytic acid (Ullah,
1988). PhyA has a very low turnover number of 216 mol P formed per mol phytase
sec−1 at optimal conditions of pH 5.0 and 58°C (Ullah and Gibson, 1987). A
comparison of amino acid sequences for the protein products of expression of cloned
phyA (Van Hartingsveldt et al., 1993) and phyB (Ehrlich et al., 1993) genes revealed a
23.5% homology with four proposed catalytic sites on phyA and a single proposed
catalytic site on phyB. These differences are likely to account for the multiple pH
optimum reported for phyA and the single pH optimum of phyB.
As reviewed by Dvorakova (1998), the industrial importance of phytase from
A. niger has fostered intensive investigation and numerous patent applications to
develop cost-effective microbial expression systems for production of the enzyme.
With simple gene amplification, overexpression is repressed by inorganic P (Van
Hartingsveldt et al., 1993) and significant overexpression requires strong P-resistant
promoters (Piddington et al., 1993). Effective expression systems for phytase
cDNA originating from A. niger var. ficuum are currently described in a variety of
microorganisms (Brocades, 1991, 1993; Berka et al., 1995a,b). Efficient hydrolysis of
phytic acid down to myo-inositol and P requires phytase plus non-specific
phosphatase activities. Expression systems for DNA sequences encoding for both
phytase and acid phosphatase have been described (Alko, 1994; Panlabs, Alko, 1994).
Extracellular phytase enzymes from microbial sources are moderately heat
stable. A step-wise increase in the temperature of the reaction medium from room
temperature to 58°C resulted in a corresponding increase in the rate of phytate
hydrolysis by A. ficuum phytase (Ullah and Gibson, 1987). Further increases in
medium temperature resulted in a dramatic drop in activity, with no detectable
phytate hydrolysis at 68°C (Ullah and Gibson, 1987). The temperature sensitivity of
the enzyme must be considered in any processing situation that involves heat after
enzyme application to the feed ingredient.
Thermostable phytase activities have been described from Bacillus sp. DS11
(Kim et al., 1998) and Aspergillus fumigatus (Pasamontes et al., 1997). The
A. fumigatus phytase has a broad pH activity range and can withstand extreme
temperatures of 100°C for 20 min or 90°C for 120 min (Pasamontes et al., 1997).
The gene for the enzyme has been cloned and overexpressed in A. niger. Phytase from
A. fumigatus has commercial potential in that it can withstand conditions of feed
pelleting without significant loss of activity.
Factors affecting phytate hydrolysis during intestinal passage
Minerals
Several studies have shown that phytate-P digestibility varies with the mineral
content of the diet. Increasing the level of dietary calcium results in a decreased
phytate-P digestibility in rats (Nelson and Kirby, 1979), poultry (Scheideler and Sell,
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70 D.D. Maenz
1987) and pigs (Sandberg et al., 1993). Presumably, increasing the concentration of
a multivalent cation such as calcium will increase the formation of insoluble
mineral-bound phytin crystals, which may be resistant to hydrolysis by phytase
activities. Mohammed et al. (1991) found that decreasing the calcium level in diets
low in P from 1% down to 0.5% resulted in a 15% increase in phytate-P digestibility
in chicks. Ballam et al. (1984) found similar results in that phytate-P digestibility in
chicks was increased when dietary calcium levels were reduced from 1.0% to 0.85%
of the diet.
Mineral binding may well have negative effects on phytate hydrolysis by
endogenous phytase of animal or plant origin in the diet. In general, Ca : phytate
ratios of greater than 2 : 1 are thought to reduce intrinsic phytate digestibility (Wise,
1983). In pigs, supplementing a diet based on barley, rapeseed meal and peas with
1.25% calcium carbonate decreased phytate digestibility (Skoglund et al., 1997). In a
separate study with pigs, total gastrointestinal hydrolysis of phytate decreased with
increasing levels of dietary calcium carbonate (Sandberg et al., 1993).
In vitro studies have demonstrated that minerals impair the efficiency of phytate
hydrolysis by phytase of microbial origin. At neutral pH, Maenz et al. (1999) ranked
Zn2+ >> Fe2+ > Mn2+ > Fe3+ > Ca2+ > Mg2+ in terms of potency as inhibitors of
phytate hydrolysis by microbial phytase. Decreasing the pH resulted in a substantial
drop in the inhibitory effects of minerals. These results are consistent with the model
of pH-dependent mineral–phytate interaction whereby protonation of weak
acid phosphate groups displaces minerals and thereby converts phytate from
phytase-resistant mineral-bound forms to phytase-susceptible forms.
Other in vitro studies have demonstrated that minerals have varying effects on
the rate of phytic acid hydrolysis by animal phytase. Maenz and Classen (1998)
found that inclusion of 25 mM MgCl2 doubled the rate of phytate hydrolysis by
chick small intestinal brush border phytase. Rat mucosal phytase activity is increased
40% by low concentrations of Zn (Bitar and Reinhold, 1972). At Zn concentrations
greater than 1 mM, hydrolysis of phytic acid by chick small intestinal brush border
phytase was markedly decreased (Maenz and Classen, 1998). Animal brush border
phytase is likely to have a requirement for minerals as cofactors for optimal activity.
The activation of activity may be confounded by mineral binding to phytate and a
decrease in the susceptibility of the substrate to hydrolysis.
pH
Very little information exists describing phytate hydrolysis during intestinal passage.
In comparing pH conditions down the length of the gut one could speculate that the
majority of phytate hydrolysis by dietary plant or microbial phytase will occur during
gastric retention. The low pH in the glandular stomach of monogastric animals will
favour protonation of the weak-acids phytate groups. As such, after consumption of a
meal, one would anticipate substantial formation of partially protonated forms of
phytate that are susceptible to hydrolysis during residence in the stomach or crop.
The optimum pH value for phytase activity approximates 5, which is below the
effective pH for the formation of phytase-resistant mineral-bound complexes. In the
upper regions of the small intestine the pH of the digesta will increase and thus
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favour the reformation of mineral-bound phytase-resistant forms of phytate. In vitro,

0.5 M Ca was ineffective as an inhibitor of microbial phytase at pH 5, while 0.005 M
Ca caused complete inhibition when the pH value of the medium was increased to
7.5 (Maenz et al., 1999). The extent of hydrolysis during gastric residence may well
be limited by the amount of enzyme and the physical conditions of retention time,
pH, mineral concentration, temperature, moisture, mixing, etc., that influence reac-
tion rates. Kemme et al. (1998) determined average gastric phytate-P degradability in
pigs as 47% for wheat–barley-based diets (1005 plant phytase units kg−1), 3% for
maize-based diets (91 plant phytase units kg−1) and 47% for microbial phytase-
supplemented maize-based diets (1565 microbial phytase units kg−1). The authors
evaluated the effect of diurnal variations in gastric retention time and pH of the
stomach. At optimum conditions of prolonged retention time, phytate degradation
approached 90% in the stomach of pigs fed diets with high levels of plant phytase.
They concluded that the efficacy of phytase degradation by plant and microbial
sources of phytase is to a large extent determined by conditions of pH and retention
time in the stomach.
A model of phytate hydrolysis by microbial and plant phytase occurring
primarily in the stomach is largely hypothetical. Research quantifying phytate
hydrolysis down the full length of the gut with various dietary constituents and levels
of dietary phytase is needed for a better understanding of the physiology of lumenal
phytate hydrolysis. In the caecum, bacterial populations may provide an additional
source of phytase activity, but the significance of gut bacteria in the process of
phytate hydrolysis is unknown.
The process of phytate hydrolysis by mucosal phytase found on the brush border
membrane of the small intestine of the animal is distinct from lumenal hydrolysis by
plant and microbial phytases. The mucosal enzyme is fixed on the membrane and
the microenvironment in the unstirred water layer immediately adjacent to the
brush border is maintained at a moderately acidic pH of 6 (Lucas, 1983) down the
length of the gut. The pH of the unstirred water layer approximates the pH optimum
of intestinal phytase activity and will be less than optimal for the formation of
phytase-resistant mineral complexes. As such, brush border phytate hydrolysis will
depend upon a combination of the degree of enzyme expression and substrate access
to the binding site on the enzyme within the unstirred water layer. In broiler chicks
and laying hens the specific and total brush border phytase activity was highest in the
duodenum and decreased progressively down the length of the small intestine
(Maenz and Classen, 1998). The contribution of brush border phytase to the process
of phytate hydrolysis may well be limited by poor mixing of insoluble mineral–
phytate complexes relative to soluble nutrients in the digesta.
Temperature, duration, moisture and mixing

The rate of any enzymatic reaction is affected by the physical conditions of the reac-
tion mixture. In vitro, the activity of microbial phytase near the body temperature of
the animal is less than half of that obtained under optimal temperatures conditions of
55°C (Ullah and Gibson, 1987). Insoluble mineral-bound phytin associated with the
fibrous portions of the seed is likely to require prolonged extensive mixing in a slurry
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72 D.D. Maenz
with excess water to optimize enzyme access to the substrate. Physical conditions
of the digesta, especially in the small and large intestine, may well limit the efficacy of
lumenal phytate hydrolysis.
Improving Phytate Digestibility

Decreasing plant phytate levels
Selecting for low-phytate plants

A viable low-phytate mutant of maize (lpa1-1) has been described by Ertl et al.
(1998). When compared with conventional maize, this mutant is characterized by a
similar total P content, a 60% reduction in phytate, and a corresponding molar
increase in the inorganic P content of the seed. The inorganic P in the lpa1-1 mutant
maize supports growth and bone mineralization in broiler chicks. The lpa1-1 mutant
has the potential to provide genetic stock for production of low-phytate maize with
acceptable agronomic traits. Given the success obtained with low-phytate maize,
development of other low-phytate plants with higher levels of available P for animal
feeding can be envisaged.
Germination of plants to induce phytase activity

The function of plant phytase is to convert phosphorus from the storage form as
phytate-P to readily available inorganic phosphate during development and growth
of the plant. Germination is associated with increased phytase activity, decreased
phytate and increased inorganic phosphate in canola (Lu et al., 1987), rapeseed
(Mahajan and Dua, 1997), triticale (Niziolek, 1995) and soybean (Bau et al., 1997).
For some feed ingredients a short period of controlled germination prior to further
processing has potential as a practical and effective method of improving plant P
availability.
Maugenest et al. (1997) cloned the DNA encoding for the phytase expressed
in germinating maize seedlings. Further isolation and characterization of associated
regulatory genes may eventually allow for controlled expression of phytase in plants.
Steeping to reduce phytate levels

Steeping (water soaking) a barley–rapeseed cake–pea-based diet for 9 h caused a 50%
reduction in phytate levels and a corresponding increase in lower-level inositol
phosphates and free inorganic phosphate (Skoglund et al., 1997). The authors
speculate that prolonged soaking of the meal provided conditions whereby a
substantial portion of the phytate was susceptible to hydrolysis by plant phytase.
Enzyme pretreatment of plant meals

Pretreatment of plant meals with microbial phytase under controlled conditions has
potential as an alternative method of achieving efficient and measurable hydrolysis
of phytase. Thus far, published work on controlled dephytinization during meal
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pretreatment has been restricted to the laboratory investigation of solid-state

fermentation of plant meals with phytase-producing organisms and enzyme cocktail
application under controlled conditions in vitro.
Solid-state fermentation involves inoculation of the plant meal with an
extracellular phytase-producing organism, followed by batch incubation under
controlled conditions to maximize growth of the organism, production of phytase
and subsequent hydrolysis of phytic acid. Inoculation of canola meal with A. ficuum
resulted in a massive increase in phytase, a linear decrease in phytic acid and complete
elimination of phytate after 144 h of incubation (Nair et al., 1991). The same
research group further characterized the effects of phosphate, glucose and surfactants
on the efficacy of solid-state fermentation of canola meal with Aspergillus carbonarius
(Al-Ashed and Duvnjak, 1994a,b, 1995a,b) and A. ficuum (Ebune et al., 1995a,b).
From this work, predictive models that relate biomass, enzyme production and
reduction of phytate content to reaction conditions have been developed.
Refinement of fermentation conditions has increased the efficiency of enzyme
production such that under some conditions complete dephytinization of canola
meal occurs within 48 h (Al-Ashed and Duvnjak, 1994a, 1995a; Ebune et al.,
1995a,b). The economics and practicality of solid-state fermentation as a method for
the commercial production of dephytinized plant meals has yet to be determined.
Batch dephytinization of plant meals and mixed feed using phytase-containing
enzyme cocktails has the advantage over in-feed enzyme application in that the
physical conditions of the reaction can be set to optimize the process of phytate
hydrolysis. Zhu et al. (1990) achieved 81% phytate hydrolysis of a maize–soybean
meal–wheat bran mixture using a two-step procedure that involved a 2 h initial
conditioning of the maize–soybean meal with citric acid at pH 3.1 and a 2 h
treatment with wheat bran at pH 5.1. Presumably citric acid acted as a competitive
chelator to increase phytate susceptibility to hydrolysis by the wheat phytase during
the second stage. Zyla et al. (1995a,b) developed a cocktail of commercial fungal
phytase, acid phosphatase, citric acid and pectinase which, under simulated intestinal
conditions of the turkey, resulted in complete dephytinization of a maize–soybean
meal-based feed. Citric acid and cofactor enzymes probably functioned to improve
substrate susceptibility to hydrolysis by the phytase and non-specific phosphatases in
the cocktail. Dephytinization resulted in a 12–29% increase in dialysable protein and
utilization of plant P for growth and bone mineralization in turkey poults (Zyla et al.,
1995a, 1996). Zyla and Koreleski (1993) found that rapeseed meal could be
dephytinized during a 4 h incubation at 40°C with fungal phytase and acid
phosphatase in a 3 : 1 water : meal slurry at acidic pH. The practicalities of
commercial batch dephytinization of plant feed ingredients are not known at present.
The cost of drying the meal after dephytinization of a high-moisture slurry presents a
considerable obstacle to the application of the technology. However, if practical and
cost-effective, batch dephytinization of plant meals has a distinct advantage over
conventional in-feed enzyme application in that, by controlling reaction conditions
and measuring inorganic P, it should be possible to produce a consistent, highly
dephytinized feed ingredient.
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74 D.D. Maenz
Transgenic plants expressing foreign phytase activity

Transgenic tobacco (Verwoerd et al., 1995) and soybeans (Li et al., 1997) expressing
phytase originating from A. niger have been developed. At present, transgenic plants
expressing phytase are viewed not as a feed ingredient but as another potential
method for manufacturing the enzyme. However, the potential does exist for
developing transgenic plants with controlled expression of a foreign phytase that
could be fed directly to animals. Cereal grains that do not undergo extensive heat
treatment prior to diet formulation are the more likely targets for developing such
transgenic plants.
Increasing phytate digestibility through in-feed supplementation
Vitamin D
Numerous studies have indicated that vitamin D (Mohammed et al., 1991), the
active vitamin D metabolite 1,25-dihydroxycholecalciferol (Edwards, 1993; Mitchell
and Edwards, 1996) and various active analogues of vitamin D (Biehl and Baker,
1997; Biehl et al., 1998) are effective in increasing phytate-P availability when added
to diets fed to poultry. The magnitude of the effect of vitamin D on phytate-P
digestibility varies considerably between studies and appears to depend upon the
mineral content of the diet. Mohammed et al. (1991) measured phytate-P availability
in broiler chicks as 50.1% for control diets, 58.5% for normal Ca, high vitamin D
diets, and 76.5% for low Ca, high vitamin D diets.
The mechanism of phytate-P digestibility enhanced by vitamin D is poorly
resolved. One of the physiological functions of the vitamin is to enhance Ca and P
absorption. Increased Ca absorption could decrease Ca–phytate formation in the
digesta, which could increase the efficacy of phytate hydrolysis (Ravindran et al.,
1995). Vitamin D has been shown to induce chick intestinal mucosal phytase activity
(Davies et al., 1970). In the same study, gut phytase activity was threefold greater in
birds on P-deficient diets (Davies et al., 1970). As such, a part of the mechanism of
vitamin D action in promoting absorption of dietary P may well involve activation
of intestinal mucosal phytase. Mitchell and Edwards (1996) found that
supplementation with vitamin D3 and microbial phytase had an additive effect on
increasing phytate utilization in broiler chicks and they suggested a separate
mechanism of action. With both microbial phytase and vitamin D3 in the diet, one
could hypothesize increased lumenal and brush border hydrolysis of the substrate.
Supplementation with microbial phytase

Dietary supplementation with sources of microbial phytase is well established as an
effective and practical method of improving phytate digestibility in production
animals. Ravindran et al. (1995) summarized a large body of literature in stating
that microbial phytase supplementation generally results in a 20–45% improvement
in phytate-P utilization in poultry. A similar range for the effect of phytase
supplementation on P availability has been reported for plant-based diets fed to
pigs. Phytase supplementation of a barley–soybean meal diet resulted in a 40%
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improvement in P absorption in comparison with a control diet and a 23% improve-

ment in P absorption in comparison with a diet supplemented with dicalcium
phosphate (Nasi and Helander, 1994). Harper et al. (1997) found a linear improve-
ment in P digestibility with increasing phytase in maize–soybean meal-based diets
with low dicalcium phosphate fed to growing pigs. In the same study, faecal P excre-
tion was decreased by 21.5% with optimal phytase and low dicalcium phosphate
(Harper et al., 1997). In weanling pigs, phytase supplementation is associated with a
50% increase in P retention (Lei et al., 1993; Pallauf et al., 1994) and a 42% decrease
in faecal P (Lei et al., 1993). Similar improvements in P digestibility were reported
for sows (Kemme et al., 1997a) and growing–finishing pigs (Kemme et al., 1997b)
fed phytase supplemented tapioca–soybean-based diets. Bruce and Sundstol (1995)
found that ileal P digestibility increased from 19.4% to 26.8% with phytase
supplementation of an oat-based diet fed to growing pigs. In piglets, Mroz et al.
(1994) reported that apparent ileal phytate digestibility increased from 31% to 79%
with 800 units phytase kg−1 of a maize–tapioca–soybean meal-based diet.
The efficacy of lumenal hydrolysis of phytate is affected by the mineral content
of the diet. In turkeys, an increase in the dietary Ca : P ratio from 1.1 : 1 to 2.0 : 1
decreased the efficacy of microbial phytase as an enhancer of P digestibility (Qian
et al., 1996). Increasing Ca levels from low (0.4%) to normal (0.8%) levels in low P
maize–soybean meal-based diets markedly reduced the efficacy of microbial phytase
in improving the performance of weanling pigs (Lei et al., 1994).
Not surprisingly, the negative effect of phytic acid on mineral digestibility
is ameliorated by dietary supplementation with microbial phytase. Dietary
supplementation with microbial phytase improved Zn utilization in rats (Rimbach
and Pallauf, 1993) and broiler chicks (Sebastian et al., 1996a). In pigs, phytase
supplementation increased the apparent absorption of Mg, Zn, Cu and Fe (Adeola,
1995). Microbial phytase supplementation of maize–soybean meal-based diets
improved Ca availability in poultry (Sebastian et al., 1996a,b). Increasing the Ca : P
ratio while maintaining a constant level of phytase supplementation in maize–
soybean meal-based diets correlated with a decrease in Ca retention (Yi et al., 1996a).
This result is consistent with the formation of phytase-resistant insoluble
mineral–phytate complexes as the mineral content the diet increases.
Overall, studies on phytase supplementation of diets fed to production animals
tend to indicate improvements in protein and amino acid digestibility (Ravindran
et al., 1995). Microbial phytase supplementation of maize–tapioca–soybean
meal-based diets increased the ileal digestibility of crude protein, and most amino
acids in pigs (Mroz et al., 1994). In turkey poults fed maize–soybean meal-based
diets, microbial phytase tended to increase the ileal digestibility of N and most amino
acids (Yi et al., 1996b). Phytase supplementation of maize–soybean-based diets had
little effect on amino acid digestibilities in male broiler chicks but improved amino
acid digestibility in females, with an optimum effect on low P/low Ca diets
(Sebastian et al., 1997).
The improvement of plant P and Ca digestibility with microbial phytase
supplementation of diets fed to production animals has led to the concept of defining
microbial phytase as a dietary ingredient that contributes available P and Ca. Qian
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76 D.D. Maenz
et al. (1996) determined that 652 units of phytase kg−1 of maize–soybean meal-based
diet was equivalent to 1 g of non-phytate P to turkey poults when the diet contained
0.27% non-phytate P, while 963 units where required to supply 1 g of non-phytate P
when the diet contained 0.36% non-phytate P. With pigs, Yi et al. (1996a) found
that 1146 units kg−1 of phytase provided 1 g of available P when added to soybean
meal-based semipurified diets, while 785 units kg−1 provided 1 g of available P in
maize–soybean meal-based diets. Harper et al. (1997) estimated that 500 units of
phytase kg−1 of maize–soybean-based diets provided 0.87–0.96 g of available P to
grower–finisher pigs.
Based on the efficacy of the enzyme in improving plant P and Ca availability in
production animals, suppliers of commercially available sources of microbial phytase
have developed a set of recommendations for the P and Ca feed equivalence of the
enzyme. These recommendations are listed as levels of phytase required for a given
age and species of animal that allow for a 0.1% reduction in available P and Ca levels
in the diet. Recommendation for starter diets are in the order of 500 units kg−1 and
considerably lower for grower and finisher diets. These guidelines are useful and
provide some general indication of the sparing effect of phytase on the need for
dietary supplementation with P and Ca. However, caution should be exercised in
adapting these recommendations to a wide range of diets. As indicated in this
chapter, the efficacy of microbial phytase will vary considerably with the ingredient
composition of the diet. Maximum efficacy of phytase would be expected for a diet
characterized by a high level of plant phytate in combination with low intrinsic
(plant) phytase activity and a low multivalent mineral content. One could
hypothesize that supplemental phytase is likely to be largely ineffective in a meat
meal-based diet and of lower efficacy in a wheat-based diet with high intrinsic
phytase. Clearly, research is needed to establish accurate recommendations for
effective levels of phytase for specific types of diets.
Supplementation with mineral chelators

Chelating agents such as EDTA and EDTA-like compounds are known to increase
Zn availability to chicks (Nielsen et al., 1966) and turkey poults (Vohra and Kratzer,
1964). Citrate and ascorbate improve Fe availability in vegetarian meals (Anand and
Seshadri, 1995) and certain pectins improve Fe absorption in rats (Kim and Atallah,
1993). The mechanism of the effect of chelators on mineral absorption may involve
competitive chelation whereby soluble mineral–chelates form in the digesta and
thereby decrease mineral binding to phytic acid. In this model, minerals bound
to chelators are readily absorbed either as intact complexes or after dissociation
and rebinding to mineral-binding transport sites on the intestinal brush border
membrane. In theory, chelators may well be effective in converting phytate from
mineral-bound phytase-resistant forms to phytase-susceptible forms in the digesta.
Indeed Zyla et al. (1995) found that citrate, when added to a phytase-containing
cocktail, increased phytate hydrolysis of maize–soybean meal under simulated
intestinal conditions. With pigs fed a maize–soybean meal-based diet, citrate tended
to enhance the efficacy of phytase in promoting P and Ca digestibility (Li et al.,
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1998). Further investigation is required to determine the potential for chelators as

dietary supplements to promote phytate digestion.
Summary and Conclusions

Application of commercial products containing microbial phytase activity to
feed ingredients has proved to be a practical and effective method for improving
phytate-P utilization by monogastric animals and thereby reducing P output in the
manure. However, the process of phytate-P digestion is a complex issue and varies
considerably with the ingredient composition of the diet and the physical conditions
in the digesta. Feed ingredients such as wheat with high levels of intrinsic phytase
are characterized by relatively high utilization of phytate-P in comparison with feed
ingredients such as oilseed meals that contain low levels of intrinsic phytase.
Multivalent minerals can complex with phytate at neutral pH and form phytase-
resistant complexes. At present, oversimplistic guidelines are in place defining the
available P content for a broad spectrum of feed ingredients. The opportunity
exists to measure actual phytate-P availability for specific feed ingredients and diet
formulations and to measure the true efficacy of microbial phytase with different
feeding scenarios. This information can then be used for more precise diet
formulation based on a true reflection of available P requirements. More precise
diet formulation will improve the overall efficiency of dietary P utilization
and thereby reduce P levels in the manure. Finally, methods of reducing the phytate
content of the feed ingredient, such as developing low-phytate plants, controlled
germination, or enzyme pretreatment of feed ingredients under controlled
conditions in vitro, have the potential to produce feed ingredients characterized
by low levels of phytate and high levels of readily available inorganic P.
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Zyla, K., Ledoux, D.R., Kujawski, M. and Veum, T.L. (1996) The efficacy of an enzymic
cocktail and a fungal mycelium in dephosphorylating corn–soybean meal-based feeds fed
to growing turkeys. Poultry Science 75, 381–387.
94
17 November 2000 15:02:01

Analysis of Feed Enzymes

B.V. McCLEARY
Megazyme International Ireland Limited, Bray Business
4
Park, Bray, County Wicklow, Ireland
B.V. McCleary
Analysis
4 of Feed Enzymes
Introduction
Enzymes are added to animal feed to increase its digestibility, to remove anti-
nutritional factors, to improve the availability of components, and for environmental
reasons (Campbell and Bedford, 1992; Walsh et al., 1993). A wide variety of
carbohydrase, protease, phytase and lipase enzymes find use in animal feeds. In
monogastric diets, enzyme activity must be sufficiently high to allow for the relatively
short transit time. Also, the enzyme employed must be able to resist unfavourable
conditions that may be experienced in feed preparation (e.g. extrusion and pelleting)
and that exist in the gastrointestinal tract. Measurement of trace levels of enzymes in
animal feed mixtures is difficult. Independent of the enzyme studied, many of the
problems experienced are similar; namely, low levels of activity, extraction problems,
inactivation during feed preparation, non-specific binding to other feed components
and inhibition by specific feed-derived inhibitors, e.g. specific xylanase inhibitors in
wheat flour (Debyser et al., 1999).
In this chapter, some of the procedures used to assay for β-glucanase, β-xylanase,
α-amylase, α-galactosidase, phytase and endo-protease enzymes will be described. In
particular, some of the problems and limitations of current assay procedures will be
discussed.
b-Glucanase
The anti-nutritional properties of β-glucans (1,3 : 1,4-β-D-glucans, mixed-linkage
β-glucans) have been known for many years (Aastrup, 1979). In animal feeds the
major source of β-glucan is barley grain. Although levels are also high in oats, these
are rarely fed to chickens and pigs. The anti-nutritional properties of β-glucan are
attributed to their viscosity-inducing properties, which significantly affect the rate of
movement of barley-based diets through the digestive tract of chickens, and reduce
the rate of nutrient absorption. This problem is effectively ‘dissolved’ by the
judicious use of β-glucan-degrading enzymes.
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86 B.V. McCleary
A wide range of enzymes are active on β-glucan and these include the endo-
acting fungal cellulases [endo-1,4-β-D-glucanase (EC 3.2.1.4)] and the bacterial
β-glucanase [lichenase; endo-1,3 : 1,4-β-D-glucanase (EC 3.2.1.73)]. Both groups of
enzymes cleave within the main chain of mixed-linkage β-glucan, although their
point of action is different (Fig. 4.1). Fungal cellulases also hydrolyse cellulose
(1,4-β-D-glucan) in an endo-hydrolytic pattern, but the bacterial 1,3 : 1,4-β-
glucanases have no action on cellulose. This point of difference is important in
designing specific substrates for the assay of these groups of enzymes.
A range of substrates and assay procedures is available for the measurement of
bacterial β-glucanase and cellulase enzymes, some of which are listed in Table 4.1.
Bacterial 1,3 : 1,4-β-D-glucanase is assayed using substrates based on a 1,3 : 1,4-β-D-
glucan polysaccharide, i.e. oat or barley β-glucan or lichenan. Procedures for the
purification of barley and oat β-glucan have been developed and are reported in the
literature (McCleary, 1988), and high purity polysaccharides are commercially
available. However, the purification of lichenan is much more difficult. Lichenan is
purified from Icelandic moss, but it occurs together with isolichenan (Chandra et al.,
1957), from which it is hard to separate. Other glucose-containing polysaccharides
(starch and cellulose) are absent from purified lichenan.
Fig. 4.1. Schematic representation of the mode of action of cellulase and

1,3 : 1,4-β-glucanase on mixed-linkage β-glucan.
Table 4.1. Substrates for the assay of cellulase and β-glucanase.
Substrate Nature Assay procedure
Cellulase
Barley β-glucan Soluble Reducing-sugar or viscometric
Lichenan Soluble Reducing-sugar or gel plate
CMC-7M Soluble Reducing-sugar or viscometric
CMC-4M Soluble/gel Reducing-sugar
Azo-CMC Soluble Chromogenic substrate
Azo-barley glucan Soluble Chromogenic substrate
Cellazyme C tablets Gel particles Chromogenic substrate
Cellazyme T tablets Gel particles Chromogenic substrate
(tamarind xyloglucan)
Beta-Glucazyme tablets Gel particles Chromogenic substrate
1,3 : 1,4-b-Glucanase
Barley β-glucan Soluble Reducing-sugar or viscometric
Azo-barley glucan Soluble Chromogenic substrate
Beta-Glucazyme tablets Gel particles Chromogenic substrate
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24 November 2000 15:24:33

Analysis of Feed Enzymes 87
Reducing-sugar methods
Pure β-glucan (barley or oat) can be used directly to assay cellulase or β-glucanase
employing either a reducing-sugar or viscometric assay procedure. Of the reducing-
sugar procedures available, the only commonly used method that gives a
stoichiometric colour response with homologous oligosaccharides of varying
degrees of polymerization (DP) is the Nelson/Somogyi procedure (Somogyi, 1960).
Consequently, it is the only method that gives a true measure of glycosidic bonds
cleaved, and thus of enzyme activity. For this reason, this is the method of choice for
use as a reference method against which all other assay procedures can be compared
and standardized. Details of a suggested format for the assay of β-glucanase using
barley β-glucan and the Nelson/Somogyi reducing-sugar procedure are given in
Appendix 4.1.
Several cellulose-based substrates are available for the assay of endo-1,
4-β-glucanase (cellulase). These substrates range from highly crystalline cotton cellu-
lose to amorphous cellulose and lightly or heavily substituted chemical derivatives
such as carboxymethyl-cellulose 4M (CMC-4M) and CMC-7M. CMC-4M is a
medium viscosity (300–600 cP at 2%, 25oC) cellulose with a degree of substitution
with carboxymethyl groups of 0.4 (i.e. over ten sugar residues, only four of the 30
available hydroxyl groups are substituted by carboxymethyl groups). CMC-7M is
completely soluble, whereas CMC-4M at 1% concentration in water is somewhere
between a solution and a colloidal suspension. Many cellulase enzymes hydrolyse
CMC-7M at the same rate as CMC-4M, but for some the rate is substantially lower.
Consequentially, CMC-4M is the substrate of choice. Cellulase enzymes act on
other polysaccharide substrates, such as mixed-linkage β-glucan and tamarind-seed
xyloglucan (which is composed of a 1,4-β-D-glucan backbone, to which D-xylose
is attached in a relatively regular manner, some of which is further substituted
by D-galactose). Both of these polysaccharides serve as the base for excellent dyed
substrates for the assay of cellulases (Table 4.1).
Traditionally, cellulase activity has been standardized using a reducing-sugar
assay with either CMC-7M or barley β-glucan as substrate. In most cases the
dinitrosalicyclic acid (DNSA) reducing-sugar method (Bailey, 1988) was used with
cellobiose as the standard. The DNSA method does not give a stoichiometric colour
response with homologous oligosaccharides of varying degrees of polymerization;
thus it is not ideal as a reference method. The Nelson/Somogyi reducing-sugar
method (Somogyi, 1960) is preferable. Also, since some cellulase enzymes hydrolyse
CMC-4M more rapidly than CMC-7M, then CMC-4M or barley β-glucan are the
substrates of choice.
Viscometric methods
The Nelson/Somogyi reducing-sugar procedure (Somogyi, 1960) is an excellent

method for the assay and standardization of relatively pure β-glucanase preparations,
i.e. preparations essentially devoid of other enzyme activities active on the substrate,
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24 November 2000 15:25:06

88 B.V. McCleary
and for preparations containing low levels of reducing-sugars (which interfere

with the assay). However, the method cannot be applied to the measurement of
β-glucanase in animal feed mixtures. For such materials, other procedures need to be
employed, such as viscometric methods and methods based on the use of dyed
polysaccharide substrates. Both procedures specifically measure endo-hydrolase
activity and can be used in the presence of high levels of reducing sugars.
In industry, the most commonly used viscometric assay for polysaccharide
endo-hydrolase activity is the Institute of Brewing procedure for the measurement
of β-glucanase in malt (Bathgate, 1979). For reproducibility, accuracy and reliability,
a β-glucan preparation of high purity and a defined viscosity range is essential. In
principle, the enzyme preparation is mixed with buffered β-glucan solution
(10 mg ml−1) under controlled temperature conditions. The viscosity (as the time to
flow between two marked points on a standardized type C U-tube viscometer) is
measured at various time intervals after the addition of the enzyme to the substrate.
The inverse reciprocal viscosity is calculated and plotted against time of incubation
(see Appendix 4.2). The slope of the curve is a direct measure of enzyme activity. An
alternative substrate is CMC-7M. This is completely soluble in water and is rapidly
hydrolysed by some endo-cellulases. A procedure for the measurement of β-glucanase
or xylanase in animal feeds, based on the Institute of Brewing IRVU method, is given
in Appendix 4.2.
Agar plate diffusion procedures
Several semi-quantitative procedures have been developed for the assay of enzyme
activity based on the rate and degree of diffusion of the enzyme through agar plates.
The enzyme is applied in a central well and diffuses through agar containing a
specific substrate. The level of activity is then monitored by staining the plate with a
dye specific for the particular polysaccharide substrate. In one such procedure, Walsh
et al. (1995) detected β-glucanase using an agar plate impregnated with lichenan, and
activity was detected by staining with Congo Red. These procedures do work, but at
best are semi-quantitative.
Soluble, dye-labelled substrates
The two general types of dye-labelled substrates available for the assay of cellulase and
β-glucanase are soluble dye-labelled substrates and insoluble dyed substrates. The
soluble dyed substrates include azo-CM-cellulose (azo-CMC) and azo-barley glucan
(McCleary and Shameer, 1987). These substrates find widespread application in the
assay of β-glucanase in feeds (Rotter et al., 1990; Cosson et al., 1999). In Fig. 4.2, a
standard curve relating enzyme activity to colour released on hydrolysis of azo-CMC
(CMC-4M dyed with Remazolbrilliant Blue R dye) is shown. The curves obtained
for the two unfractionated commercial enzyme preparations and for a purified
cellulase from a Trichoderma sp. are relatively similar. Differences can be attributed
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24 November 2000 15:25:34

Fig. 4.2. Standard curves for the action of cellulase preparations on

azo-CMC (lot 60601): (A) a crude Trichoderma viride preparation; (B) a crude
T. longibrachiatium preparation; (C) a pure cellulase from a T. longibrachiatium
preparation.
to the range of activities in the crude preparations, as well as to subtle differences

in the action patterns of the individual endo-cellulase enzyme components. This
procedure has been adopted by the UK silage industry for the standardization
of cellulase activity in silage additive preparations. The major limitation in the
application of this procedure to measurement of cellulase activity in animal feed
preparations is the lack of sensitivity. A second concern is the fact that the base
substrate is CM-cellulose rather than β-glucan, which is the target substrate in animal
feeds. The action of pure Trichoderma sp. cellulase on azo-barley glucan (barley
glucan dyed with Remazolbrilliant Blue R dye) is shown in Fig. 4.3. The sensitivity
with azo-barley glucan is about three times that with azo-CMC, but unfortunately is
still not adequate for the measurement of trace levels of cellulase in animal feeds.
However, it has been found that action on azo-barley glucan is a good predictor of
in vivo response to cellulase supplementation of barley-based diets in young chickens
(Rotter et al., 1990).
Insoluble dye-labelled substrates
Insoluble dyed substrates are prepared by dyeing and cross-linking soluble polysac-
charides to give dyed cross-linked polysaccharide gel matrices. Such substrates are
readily hydrolysed and include AZCL-HE-cellulose (Cellazyme C tablets), AZCL-β-
glucan (Beta-Glucazyme tablets) and AZCL-xyloglucan (Cellazyme T tablets).
Assays based on the use of these substrates are three to ten times more sensitive
than assays using azo-barley glucan or azo-CMC. In Fig. 4.4, standard curves for
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90 B.V. McCleary
Fig. 4.3. Standard curve relating the activity of a pure T. longibrachiatium

cellulase on CMC-4M (Nelson/Somogyi assay) to action on: (A) azo-barley glucan
(lot 60602) (using EGME/zinc acetate-based precipitant) and (B) azo-CMC (lot
60601) (using ethanol/zinc acetate-based precipitant).
A
1.2
1.1 B
1.0
0.9
Absorbance, 590 nm
0.8 C
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
0 10 20 30 40 50 60 70 80 90 100
Cellulase, milliUnits/assay (i.e. per 0.5 ml)
on barley β-glucan
Fig. 4.4. Standard curves relating the activity of a pure T. longibrachiatium
cellulase on barley β-glucan (Nelson/Somogyi assay) to activity on: (A) Cellazyme
T tablets (lot 70801); (B) Beta-Glucazyme tablets (lot 50901); and (C) Cellazyme C
tablets (lot 80201).
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Trichoderma sp. cellulase on dyed cross-linked HE-cellulose (Cellazyme C), dyed

cross-linked β-glucan (Beta-Glucazyme) and dyed cross-linked tamarind xyloglucan
(Cellazyme T tablets) are compared. It is evident that, of the substrates compared, the
Cellazyme T tablets give the greatest sensitivity, and thus are the substrate of choice
for the detection and measurement of trace levels of cellulase in animal feed. The
reason for this greater sensitivity must be due to the regular distribution of xylose
residues along the cellulose backbone of the polysaccharide, compared with the
irregular to random distribution of hydroxypropyl residues in HE-cellulose.
Measurement of cellulase and β-glucanase enzymes in feed is complicated by
several factors, including inactivation of the enzyme during feed pelleting, adsorption
of the enzymes to feed components, competition with endogenous substrate and
possible enzyme inhibitors. These same problems are experienced in the assay of
xylanase and protease enzymes in feed.
Endo-1,4-b-D-Xylanase (Xylanase)
The major endosperm cell-wall polysaccharide in wheat and rye grain is
arabinoxylan, which represents about 2–5% of the grain weight. About two-thirds of
wheat-flour arabinoxylan is water insoluble (Amado and Neukom, 1985). However,
both the soluble and the insoluble components have high water-absorbing properties.
Both can absorb about ten times their weight of water (Kulp, 1968). Consequently,
arabinoxylans in feed cause problems similar to those experienced with β-glucans
(Annison and Choct, 1991). The development and application of a range of xylanase
enzymes have resolved these problems. Measurement of xylanase in feeds is
complicated by the fact that the levels added are very low, and this enzyme can
be lost through inactivation during pelleting, adsorption to feed components or
inactivation by specific xylanase inhibitors (Debyser et al., 1999).
Several assay procedures have been developed for the assay of xylanase, and these
include reducing-sugar assays, viscometric assays, and assays based on the use of
chromogenic substrates (Table 4.2).
Reducing-sugar and viscometric assays
Pure wheat arabinoxylan forms the basis of both reducing-sugar and viscometric
assays for xylanase. Reducing sugar assays are not specific and cannot be used for the
measurement of xylanase in trace levels in feed mixtures. However, the Nelson/
Somogyi reducing-sugar procedure (Somogyi, 1960) is the method of choice for the
standardization of pure enzyme preparations. These enzyme preparations can then be
used to standardize other assay procedures that use less defined substrates (e.g. the
dyed xylan and arabinoxylan substrates) and assay procedures that are expressed
in non-conventional units (e.g. viscometric assays). Viscometric assays are used
in some laboratories, and the substrate generally employed is wheat arabinoxylan
(20–30 cSt). Other xylan substrates (from oat and birchwood) are available, but the
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92 B.V. McCleary
Table 4.2. Substrates for the assay of β-xylanase.
Substrate Nature Assay procedure
Wheat arabinoxylan Soluble Reducing-sugar

Wheat arabinoxylan Soluble Viscometric
Xylazyme AX tablets Gel particles Chromogenic substrate
Azo-wheat arabinoxylan Soluble Chromogenic substrate
Azo-xylan (oat spelts) Soluble Chromogenic substrate
Azo-xylan (birchwood) Soluble Chromogenic substrate
viscosity of commercially available preparations is so low that they are of little use as
substrates in viscometric assays. While viscometric assays allow specific measurement
of endo-xylanase, the assay procedure is very tedious. With manual viscometric
equipment (type C U-tube viscometer) a single operator can assay just five samples in
1 day. This compares with about five to ten samples by a single operator in 2 h with
the newer chromogenic substrates.
Soluble chromogenic substrates
Various dye-labelled substrates have been prepared for the assay of endo-1,
4-β-D-xylanase (xylanase), including dyed oat, birchwood and beechwood xylans and
dyed wheat arabinoxylan (Table 4.2). In most cases, the dye used is Remazolbrilliant
Blue. The reason for this is that it is possible to get good colour intensity with this
dye; and because the dye is hydrophobic, it is easy to separate reaction products
from unhydrolysed substrate, by alcohol precipitation. In the preparation of
soluble dyed substrates for the measurement of xylanase there are several important
considerations, including: (i) the final substrate must be readily hydrolysed by the
enzyme, with good sensitivity and a good standard curve covering approximately one
absorbance unit (and preferably linear); (ii) the substrate must be homogeneous and
preferably completely soluble; (iii) the substrate must be specific for the measurement
of xylanase, i.e. the base polysaccharide should have a xylan backbone and not be
contaminated by other polysaccharides; and (iv) the substrate must form the base of a
simple assay procedure.
The relative sensitivities of three soluble, chromogenic xylan substrates are
compared in Fig. 4.5. Of the three, azo-wheat arabinoxylan is the most sensitive, and
as such is the substrate of choice in the assay of trace levels of xylanase in animal feeds.
This substrate is completely soluble and gives a good (essentially linear) standard
curve over one absorbance unit.
In the measurement of xylanase in feeds, a further set of problems is experienced.
The levels of enzyme added to the feed are usually low (just enough to depolymerize
the endogenous arabinoxylan), which causes analytical problems, especially since
some of this activity is lost due to pelleting or adsorption to feed components and
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Fig. 4.5. Standard curves relating the activity of a pure A. niger xylanase on
wheat arabinoxylan (Nelson/Somogyi assay) to action on: (A) azo-wheat
arabinoxylan (lot 50201); (B) azo-xylan oat (lot 60701); and (C) azo-xylan
birchwood (lot 30702).
some is inhibited by endogenous xylanase inhibitors. Assays are also complicated

by the fact that the feed material contains arabinoxylan that acts as a competing
substrate. Consequently, the analyst is left with a choice of either determining the
directly measurable activity (i.e. a simple assay on feed extracts) or trying to estimate
the level of active enzyme in the feed. In the latter case, it is essential to get some
estimate of the degree of adsorption and inhibition of the enzyme. This can be
achieved in one of two ways. In the first and preferable approach, if a sample of
essentially identical non-enzyme-treated feed is available, a standard curve can be
constructed by adding increasing quantities of enzyme to feed slurries to give final
analytical values that cover the range of the activity extracted from the feed. Then,
by reference to this standard curve, it is possible to calculate the actual amount
of enzyme in the feed. Alternatively, and perhaps more realistically, recovery
experiments can be performed. In this case, samples of non-enzyme-treated feed
are not available. Instead, a sample of the feed is extracted with buffer, or with
buffer to which set amounts of enzyme have been added. From the recovery of the
added enzyme, it is possible to calculate the actual amount of enzyme in the original
feed. Since this format can be used to estimate any enzyme in a feed mixture, it is
described in detail in Appendix 4.3.
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94 B.V. McCleary
Insoluble chromogenic substrates
In an attempt to improve the sensitivity of chromogenic substrates for the assay of

endo-xylanase, insoluble dyed substrates have been prepared by dyeing and
cross-linking soluble xylan substrates, such as oat and birchwood xylans and wheat
arabinoxylan (McCleary, 1995). Of these, the preferred starting material is wheat
arabinoxylan, because it has a high molecular weight (which is preferable for
cross-linking) and stable gel formation. Dyed, cross-linked arabinoxylan (AZCL-
arabinoxylan) is available in tablet form (Xylazyme AX) (McCleary, 1992).
Assays for the measurement of xylanase using Xylazyme AX tablets are
approximately three to four times more sensitive than assays employing azo-
wheat arabinoxylan (Fig. 4.6). Thus, where enzyme activity is present in very low
concentrations, Xylazyme AX tablets are the preferred substrate. However, in the use
of these tablets, some problems are experienced that are not experienced in the use of
azo-wheat arabinoxylan. The tablet substrate (AZCL-arabinoxylan) is quite sensitive
to salt concentration. High salt concentrations affect the swelling of the gel particles
which results in the substrate being less susceptible to hydrolysis. The effect of salt
concentration on the rate of release of coloured products from Xylazyme AX tablets
is shown in Fig. 4.7. To minimize this effect and maximize the sensitivity of the
substrate, feed samples can be extracted with 0.1 M acetic acid (instead of 0.1 M
sodium acetate buffer). The final pH in the extract must be in line with the pH
activity profile of the enzyme under evaluation. The major limitation with this
Fig. 4.6. Standard curve relating the activity of pure A. niger xylanase on wheat
arabinoxylan (Nelson/Somogyi assay) to action on Xylazyme AX tablets (lot 40602).
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Fig. 4.7. Effect of buffer salt concentration on the activity of A. niger xylanase on
Xylazyme AX tablets (lot 40602).
approach is that it is not possible to assay the pure enzyme in the extracting solution
(0.1 M acetic acid), as this will inactivate the enzyme.
On the basis of the above findings, the preferred substrate for the measurement
of xylanase in animal feeds is azo-wheat arabinoxylan. However, if extra sensitivity is
essential, then Xylazyme AX tablets should be used.
Enzyme-linked sorbent assays
An enzyme-linked sorbent assay (ELSA) has been developed for the assay of
β-glucanase and β-xylanase in animal feeds (Wong et al., 1999). This assay offers the
advantage of greater sensitivity than any of the procedures previously described.
Adoption of the method by industry will depend on numerous factors, including
assay reproducibility, accuracy and reliability, which remain to be ascertained.
a-Amylase
Several methods are available for the measurement of α-amylase in pure enzyme
preparations and in cereal flours or animal feeds. Procedures for assaying pure
enzyme preparations usually employ soluble starch as substrate and measure the
increase in reducing-sugar levels by either the DNSA (Bailey, 1988) or the Nelson/
Somogyi (Somogyi, 1960) method. Traditional methods for the assay of α-amylase
in cereal flours are based on the reaction of iodine with starch, or particularly the
β-limit dextrin of starch (Sandsted et al., 1939; Farrand, 1964; European Brewing
Convention, 1987). Hydrolysis of the starch results in a decrease in the purple-blue
colour formed on reaction with iodine.
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96 B.V. McCleary
Several newer and simpler procedures for the assay of α-amylase are now
available. Of these, the preferred method employs ‘end-blocked’ p-nitrophenyl
maltoheptaoside as substrate (in the presence of a thermostable α-glucosidase). The
principle of this assay is shown in Fig. 4.8 (McCleary and Sheehan, 1987). The
substrate is specific for endo-acting α-amylase. On hydrolysis of the oligosaccharide
chain by α-amylase, the thermostable α-glucosidase immediately hydrolyses the
nitrophenyl-oligosaccharide fragment to glucose and p-nitrophenol. The thermo-
stable α-glucosidase (in place of an amyloglucosidase/yeast maltase mixture used in
previous formulations) allows the reagent to be used in the pH range of 5.2–7.5, and
at temperatures up to 60°C. The major advantage of this method over other methods
is that the substrate is a defined oligosaccharide. With all other methods, the
substrate is starch or modified starch, and thus is ill defined.
In situations where the levels of α-amylase are very low, it may be necessary to
employ a more sensitive substrate such as Amylazyme tablets (McCleary, 1991). This
substrate is prepared by dyeing and cross-linking amylose to give gel particles that are
dehydrated and incorporated into tablets. On hydrolysis by α-amylase, soluble dyed
amylose fragments are released into solution and the non-hydrolysed material is
removed by filtration. Assays based on this substrate can be performed directly on
feed slurries, allowing an increase in sensitivity of at least tenfold. These assays are
Fig. 4.8. Schematic representation of the assay of α-amylase with Amylase HR

reagent (Ceralpha method).
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standardized in international units (U) of activity against either the Nelson/Somogyi

reducing-sugar assay or the Ceralpha assay.
a-Galactosidase
α-Galactosidase enzymes act on galactosyl-sucrose oligosaccharides (raffinose,
stachyose and verbascose) producing galactose and sucrose, which removes the
anti-nutritional properties of these oligosaccharides (Gitzelmann and Auricchio,
1965; Liebmann, 1991). To be effective, these enzymes must rapidly hydrolyse
galactosyl-sucrose oligosaccharides, and must be able to work under conditions of
limited water availability.
α-Galactosidase is readily assayed with p-nitrophenyl α-D-galactoside (10 mM)
as substrate. However, for a particular α-galactosidase, the relative rates of hydrolysis
of p-nitrophenyl α-D-galactoside, raffinose, stachyose and verbascose should be
determined to ensure that the enzyme being used is actually active on the component
of interest. Activity of α-galactosidase on galactosyl-sucrose oligosaccharides can be
determined using the particular oligosaccharide (10 mg ml−1) and the Nelson/
Somogyi reducing-sugar assay (Somogyi, 1960).
Phytase
Phytase is successfully used as a feed additive to improve phosphorus availability
in feed. This enzyme is found in plant material and is produced by many micro-
organisms, especially moulds of the Aspergillus type. Commercially available
microbial phytases are non-specific phosphomonoesterases (EC 3.1.3.8) belonging
to the group of acid phosphatases (Jongbloed et al., 1993). These enzymes catalyse
dephosphorylation of myo-inositol hexakisphosphates in a step-wise manner,
producing five classes of intermediate products (myo-inositol pentakis-, tetrakis-,
tris-, bis- and monophosphates) of variable stereochemistry (Frolich et al., 1986). A
schematic illustration of dephosphorylation of phytate is shown in Fig. 4.9.
Phytase activity in industrial enzyme preparations and animal feeds is usually
assayed by measuring the amount of orthophosphates released from phytic acid
Fig. 4.9. Schematic representation of the hydrolysis of phytic acid by phytase.
107

98 B.V. McCleary
within a period of linear release with time. One unit of phytase activity is defined as
the amount of enzyme required to release 1 µmol of orthophosphate from phytic acid
under standard assay conditions (pH 5.5, 37°C) in 1 min. However, for general
acceptance of phytase enzyme as a feed additive, there is a need for validation of this
type of assay. To satisfy this need, a major international evaluation of an assay format
has been performed under the auspices of AOAC International. The results of these
studies are currently under evaluation (P. Randsdorp, personal communication,
1999).
endo-Protease
Several substrates and assay procedures are available for the measurement of protease.
Many of those based on the use of native proteins such as casein, albumin and
haemoglobin do not distinguish between endo- and exo-protease (peptidase) activity.
Assays based on trichloroacetic acid (TCA) precipitation of non-hydrolysed substrate
and measurement of the absorption (235 nm) of the supernatant solution on
centrifugation (Kunitz, 1947; AACC, 1985) are likely to be more specific than
procedures that measure the release of free amino groups (Lin et al., 1969). However,
even TCA precipitation methods are not specific. A greater specificity can be
obtained with dye-labelled proteins (Charney and Tomarelli, 1947), or dye-labelled
and cross-linked proteins. Action of exo-acting peptidases on these substrates will be
hindered or stopped by the dye molecule, or at the point of cross-linking.
Several dyed protein substrates are commercially available, including albumin,
casein and collagen dyed with sulphanilic acid (azo-albumin, azo-casein and azo-
collagen), and collagen dyed with Remazolbrilliant Blue (Hide powder azure). These
are useful substrates and form the basis of simple assay procedures, but the quality of
commercially available substrates varies significantly, limiting their value as analytical
reagents. Standard curves for Subtilisin A on two commercially available azo-casein
substrates are shown in Fig. 4.10 and regression equations for several proteases on
one of the substrates is shown in Table 4.3 (McCleary and Monaghan, 1999). It is
evident that one of the preparations (ex. Megazyme) is more useful than the other,
with better sensitivity and linearity of the standard curve. This preparation is useful
in the assay of industrial enzyme preparations, but problems are experienced in the
measurement of enzymes in feed. Whether this is due to binding of the enzyme to the
feed, inhibition by endogenous protease inhibitors or competition by other protein
substrates is not clear.
An alternative endo-protease substrate, and one that finds considerable use, is
cowhide azure (basically collagen dyed with Remazolbrilliant Blue). This substrate
is very fibrous and difficult to weigh accurately. Consequently, an alternative
collagen-based substrate was prepared by dyeing and cross-linking collagen from the
swim bladders of fish. The structure of this collagen is very similar to that in cowhide.
The major difference in the final product is that the new product from fish collagen
(AZCL-collagen) can be milled to a fine powder and incorporated into tablets
(Protazyme OL). The suitability of this material as a general or selective substrate was
108
17 November 2000 15:04:50

Fig. 4.10. Standard curve relating the activity of Subtilisin A on casein to action
on two commercially available azo-casein preparations: (A) Megazyme (lot 81001);
(B) Sigma Chemical Co. (lot 74H7165).
Table 4.3. Action of protease enzymes on azo-casein (lot 81001).
Protease (mU ml−1)a,b Linear

Enzyme (R = 0.99) absorbance range
Papain (from Papaya latex) 270 × Abs. (440 nm) + 7 0.1–1.0

Bromelain (from pineapple stem) 460 × Abs. (440 nm) − 13 0.1–0.9
Ficin (from figs) 190 × Abs. (440 nm) + 3 0.1–1.1
Subtilisin A (from Bacillus licheniformis) 129 × Abs. (440 nm) + 4 0.1–1.0
Bacterial protease (from Bacillus subtilis) 250 × Abs. (440 nm) − 8 0.1–1.0
Proteinase K (from Tritirachium album) 140 × Abs. (440 nm) − 4 0.1–1.0
Fungal protease (A. niger; from Sigma Chemical Co.) 146 × Abs. (440 nm) − 4 0.1–1.0
aOne protease unit is defined as the amount of enzyme that will produce the equivalant of 1 µmol
tyrosine min−1 from soluble casein at pH 7.0 and at 40°C.
bAbs. = absorbance.
determined by comparing the activity of a wide range of proteases on this substrate

to a similar material prepared from casein (AZCL-casein; Protazyme AK). The
conclusion derived from this study was that a similar relative rate of hydrolysis of the
two substrates was obtained for every protease tested. Consequently, AZCL-collagen
had no particular advantage over AZCL-casein, and since the standard curves
obtained with AZCL-casein (Protazyme AK tablets) were consistently more linear,
we consider that this is the substrate of choice.
Assays with Protazyme AK substrate are four to five times more sensitive
than those with Azo-casein, and the standard curves obtained for all of the proteases
studied are linear (Table 4.4). However, the substrate tablets do not hydrate as
rapidly as do tablets containing dyed and cross-linked polysaccharide substrates.
Thus, for good reproducibility, the tube contents should be stirred during the
109
17 November 2000 15:05:05

100 B.V. McCleary
Table 4.4. Action of protease enzymes on Protazyme AK tablets (regression equations).
Enzyme Protease (mU ml−1)a,b (R = 0.99)
Papain (from Papaya latex) 76.2 × Abs. (590 nm) + 7.6

Bromelain (from pineapple stem) 304 × Abs. (590 nm) + 10
Ficin (from figs) 132 × Abs. (590 nm) + 3.5
Subtilisin A (from Bacillus licheniformis) 34.2 × Abs. (590 nm) + 0.6
Fungal protease (A. niger ; from Quest International) 42.0 × Abs. (590 nm) + 2.9
aOne protease unit is defined as the amount of enzyme that will produce the equivalant of 1 µmol
tyrosine min−1 from soluble casein at pH 7.0 and at 40°C.
bAbs. = absorbance.
reaction period. This can easily be achieved but it does make this assay a bit more
complicated than that using Azo-casein.
Even with this greater sensitivity, it is still difficult to assay protease activity in
feed mixtures accurately. Obviously, this area of analyses requires further input. The
required sensitivity may be obtained with fluorimetric substrates but, even then, the
apparent problems of adsorption of the enzyme to the feed and/or specific protease
inhibitors will still need to be resolved.
Appendix 4.1 Modified Nelson/Somogyi (Somogyi, 1960)

Reducing-sugar Method for Measurement of b-Glucanase
and Xylanase Activity
Nelson/Somogyi reagents
1. Reagent A. Dissolve 25 g of anhydrous sodium carbonate, 25 g of sodium

potassium tartrate and 200 g of sodium sulphate in 800 ml of water and dilute to 1 l.
Filter if necessary.
2. Reagent B. Dissolve 30 g of copper sulphate pentahydrate in 200 ml of water
containing four drops of concentrated sulphuric acid.
3. Solution C. Dissolve 50 g of ammonium molybdate in 900 ml of water and
carefully add 42 ml of concentrated sulphuric acid; separately, dissolve 6 g of sodium
arsenate heptahydrate in 50 ml of water and add this to the above solution. Dilute the
whole to 1 l. Warm to 55°C to dissolve completely (if necessary).
4. Solution D. Add 1 ml of reagent B to 25 ml of reagent A.
5. Solution E. Dilute solution C fivefold (50 ml to 250 ml) with distilled water
before use (stable at 4°C for about 1 week).
110
17 November 2000 15:05:07

Substrates
Dissolve 1 g of pure barley β-glucan (20–30 cSt viscosity), CM-cellulose 4 M or

wheat arabinoxylan (20–30 cSt) in 90 ml of hot deionized water. Cool to room
temperature and add 5 ml of 2 M sodium acetate buffer (pH 4.6). Adjust the pH to
4.6 with 1 M sodium hydroxide or hydrochloric acid, and adjust the volume to
100 ml. Store in a well-sealed bottle at room temperature. Add two drops of toluene
to prevent microbial contamination. If pH values other than pH 4.6 are required,
use an appropriate buffer to obtain the correct pH at a final concentration in the
substrate of 100 mM.
Procedure
1. Enzyme solution (0.2 ml) is incubated with pre-equilibrated substrate solution

(0.5 ml) at 40°C for 0, 3, 6, 9 and 12 min. The reaction is terminated by adding 0.5 ml
of solution D with vigorous stirring.
2. These tubes, along with reaction blank solutions (to which solution D is added
before the enzyme) and glucose or xylose standard solutions (which contain substrate
and glucose or xylose at 0–50 µg per tube) are incubated in a boiling-water bath for
20 min.
3. The tubes are cooled to room temperature and 3.0 ml of solution E is added. The
tubes are stirred well (10 s on vortex mixer) and allowed to stand for 10 min before
mixing again. Centrifuge (3000 rpm for 10 min) if necessary.
4. The absorbance of all solutions is measured at 520 nm against the reaction blank.
A standard curve is prepared using appropriate amounts of glucose or xylose
(0–50 µg).
5. Enzyme activity is calculated as µmol of glucose (or xylose) reducing-sugar
equivalents released per minute ml−1 or g−1 of the original enzyme preparation.
Appendix 4.2 Measurement of b-Glucanase and Xylanase

Activity by Viscometry
This method describes a viscometric assay for the measurement of endo-xylanase or
β-glucanase in microbial preparations, animal feeds and bread improver mixtures.
Principle
The activity is measured by incubating suitably diluted enzyme extract with a

solution of β-glucan or wheat-flour arabinoxylan (1% w/v, 20–30 cSt) in appropriate
buffer (sodium acetate or sodium phosphate, 100 mM) and measuring the rate of
decrease in viscosity at 40°C.
111
17 November 2000 15:05:08

102 B.V. McCleary
Unit definition
One inverse reciprocal viscosity unit (IRVU) is the increase in reciprocal viscosity
h−1 ml−1 (or g−1) of enzyme (or plant material), under standard assay conditions.
Preparation of b-glucan or arabinoxylan substrate
1. Barley β-glucan (1 g, pure, 20–30 cSt) or wheat arabinoxylan (1 g, pure,

20–30 cSt) is accurately weighed into a 120 ml dry pyrex beaker.
2. The sample is wetted with 6 ml of 95% ethanol and then with 80 ml of cold
water.
3. A magnetic stirrer bar is added to the beaker and the beaker is placed on a
magnetic stirrer-hotplate and heated at a setting of 120°C with vigorous stirring. The
beaker is loosely covered with aluminium foil and stirred and heated for about 15 min.
4. The polysaccharide should completely dissolve. If dissolution is not complete,
continue stirring for a further 30 min with the heating turned off.
5. Add 10 ml of acetate buffer (1 M, pH 4.6) or 20 ml of phosphate buffer (0.5 M,
pH 6.0) and adjust the pH to the desired value.
6. Adjust the volume to 100 ml and store in a well-sealed glass bottle. Prevent
microbial contamination by adding a few drops of toluene. The solution can be stored
at room temperature for several weeks.
Enzyme extraction and dilution
1. Plant samples are milled to pass through a 0.5 mm sieve on a Retsch centrifugal
mill (or similar).
2. Accurately weigh 1.0 g of flour or powdered enzyme preparation into a 250 ml
Erlemeyer flask and add 100 ml of appropriate extraction buffer; stir and extract over
15 min at room temperature.
3. Filter an aliquot of the slurry through a Whatman No. 1 filter circle, or centrifuge
at 3000 rpm for 10 min. Store the filtrate in an ice bath.
4. Dilute the filtrate (or supernatant) with the appropriate buffer (by sequential
dilution of 1 ml to 10 ml with dilution buffer) to give an enzyme concentration
suitable for assay (i.e. a slope value ‘A’ of 0.06 to 0.60).
5. Prepare liquid enzyme samples by dilution of 1.0 ml to 100 ml with appropriate
buffer. This solution is further diluted as for extracts of dry samples.
Viscometric assay of activity
1. Pre-equilibrate the enzyme preparation at 40°C for 5 min and then pipette 1 ml
of this solution into 12 ml of pre-equilibrated, buffered substrate solution (wheat
112
17 November 2000 15:05:09

arabinoxylan or β-glucan; 1% w/v) in a type C U-tube viscometer (in a water bath at

40°C), and mix the contents by blowing air into the viscometer tube. Immediately
start a stop clock and leave this running throughout the entire assay to record
incubation time (in minutes).
2. Using a second stop clock, take five falling time readings (in seconds) over a period
of approximately 30 min. Take the time for each reading as the elapsed time from
mixing the enzyme/substrate solutions to the mean of the falling time.
3. The viscosity (n) of the reaction digest is proportional to the falling number
according to the following equation:
ndigest = (tdigest − tsolvent)/tsolvent
where ndigest = specific viscosity of the digest; tdigest = falling time in seconds of the
digest; and tsolvent = falling time in seconds of the buffer (100 mM sodium acetate).
Calculations
The reciprocal viscosity (1/n) (Table 4.5) is calculated and plotted against incubation
time (in minutes) (Fig. 4.11). The slope (A) is determined from the linear graph in
terms of increase in reciprocal viscosity per hour.
IRV units = A × 100 × Dilution
where A = slope from graph in terms of increase in reciprocal viscosity per hour;
100 = 1 g of original enzyme preparation extracted with 100 ml of buffer (100 mM),
or 1 ml of liquid enzyme preparation diluted to 100 ml with extraction/dilution
buffer; and Dilution = further dilution of the extract or diluted liquid enzyme
concentrate required to get an appropriate activity for assay.
Appendix 4.3 Extraction and Assay of Xylanase Enzymes in

Animal Feeds
Example: Trichoderma sp. xylanase
Table 4.5. Determination of reciprocal viscosity values.
Incubation time (t) (min) Time to flow (s) Specific viscosity n = (t − to)/to 1/n
0 302.75 11.75 0.085

5 247.12 9.41 0.106
10 211.34 7.90 0.127
14 190.76 7.04 0.142
17 174.47 6.35 0.157
21 162.45 5.84 0.171
113
17 November 2000 15:05:10

104 B.V. McCleary
Fig. 4.11. Plot of reciprocal viscosity (1/n) against incubation time.
Extraction
1. Mill a feed sample (approximately 100 g) to pass a 0.5 mm screen and mix
thoroughly.
2. Weigh samples of the above feed (0.5 ± 0.01 g in quadruplicate) into glass test
tubes (16 × 120 mm).
3. Treat each sample with 5 ml of 0.1 M sodium acetate buffer (pH 4.7) and stir on a
vortex mixer. To two of these tubes, add water (0.2 ml) with stirring, and to the other
two tubes add control Trichoderma sp. xylanase (0.2 ml, 1360 mU) with vigorous and
immediate stirring on a vortex mixer.
4. Leave the slurries at room temperature with occasional stirring on a vortex mixer
over the following 20 min.
5. Centrifuge tubes (3000 rpm, 10 min) in a bench centrifuge and use the
supernatant directly. Assays should be initiated within 30 min of obtaining these
extracts to minimize loss of enzyme activity.
Assay
1. Accurately transfer 0.5 ml aliquots of supernatant solutions (in duplicate) to glass

test tubes (16 × 100 mm) at room temperature.
2. Add 0.5 ml of Azo-WAX to each tube and stir the tube vigorously. Immediately
place the tube in a water bath set at 50°C ± 0.1°C and incubate for 30 min.
3. After exactly 15 or 30 min (depending on the level of enzyme activity), add 2.5 ml
of industrial methylated spirits (IMS) and stir the tube vigorously on a vortex mixer.
Store the tube at room temperature for 5 min. This treatment terminates the reaction
and precipitates non-depolymerized dyed substrate.
4. Centrifuge the tubes at 3000 rpm for 10 min and measure the absorbance of the
supernatant solutions at 590 nm against a reaction blank.
114

The reaction blank is prepared by adding 2.5 ml of IMS to a mixture of 0.5 ml of

azo-wheat arabinoxylan and 0.5 ml of 0.1 M sodium acetate buffer (pH 4.6). The
slurry is stirred and stored at room temperature for 5 min before centrifugation
(3000 rpm, 10 min). A single reaction blank is required for each feed sample.
Calculation of activity
The level of xylanase in the flour sample is calculated as follows.

Activity in feed sample (0.5 g) = Added activity × SA / (TA − SA)
where Added activity is the amount of xylanase added to the feed slurry at the time of
assay (in the control xylanase solution; 0.2 ml) (e.g. 1360 mU); SA is the reaction
absorbance obtained for extracts of the feed to which no control xylanase was added;
TA is the total absorbance (i.e. the absorbance of extracts of the sample to which the
control xylanase was added).
Example calculation
Absorbance (590 nm)/

Sample 30 min incubation
1. Feed A 0.000
2. Feed A containing Trichoderma sp. xylanase (SA) 0.502
3. SA + 1360 mU xylanase (in the assay) (TA) 0.908
Activity in 0.5 g of feed A = Added activity × SA / (TA − SA)

where SA = absorbance of extract of sample A assayed by the standard format (e.g.
Abs = 0.502); TA = the total absorbance, i.e. the absorbance of extracts of sample A
to which the additional xylanase (0.2 ml; 1360 mU) was added (e.g. Abs = 0.908).
Thus:
Activity in the feed = (1360 / 1000) U × [0.502 / (0.908 − 0.502)]
(U per 0.5 g)
= 1.682 U.
U g−1 (or kU ton−1) = 1.682 × 2000 = 3363.
References
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24 November 2000 15:43:54

106 B.V. McCleary
Amado, R. and Neukom, H. (1985) Minor constituents of wheat flour: the pentosans. In:
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Maize: Factors Affecting its

Digestibility and Variability in
its Feeding Value
5
J.D. SUMMERS
Poultry Industry Centre, RR#2, Guelph, Ontario, Canada N1G 6H8
J.D. Summersand Feed Value of Maize

Digestibility
5
Introduction
On a world basis, maize is the most important cereal in livestock feeding. Having a
high available energy content and a relatively low protein content, it has usually been
looked on mainly as an energy source in a diet. However, since the levels used in
poultry and swine diets can approach 70%, many diets have in excess of 20% of their
protein content contributed by maize. Maize has always been considered to be a
very uniform product with slight variations in protein and energy. Thus, feed
compounders have spent little time monitoring its nutrient composition. There is
growing evidence today that the feeding value of maize samples can vary significantly,
based on animal performance. As there is little data available to suggest the reasons
for these differences in nutritive value, it is of interest to review briefly some of the
work reported on the composition of maize by the milling and food industries.
Composition and Physical Characteristics of Maize

Starch is a unique and omnipresent polysaccharide found in many foodstuffs.
Most of the energy in maize is derived from the starch fraction that is found in the
endosperm of the kernel. While many consider starch a homogeneous product it can
in fact vary considerably in composition. Regular or normal maize (NM) has most of
its starch present as amylopectin, while waxy maize (which is used mainly in the food
industry) has little amylose present as starch sources. Normal maize, often referred to
as dent maize, and waxy maize are the two most important maize varieties. Flint
maize has similar composition to NM, but it has a hard, starchy layer entirely
surrounding the outer part of the kernel. Other types of maize are important in small
specialized markets: sweet maize (sweetcorn), for example, has a high level of sugars
in its endosperm; pop maize (popcorn), used in the confectionery industry, has a
high proportion of hard starch which, on heating, expands rapidly, resulting in a
explosive rupture of the epidermis and starch granules. While these varieties differ in
119
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110 J.D. Summers
their composition compared with NM, there is little information available as to their
exact make-up.
Starch granules can differ significantly in size and composition, depending on
type and variety of cereal (South et al., 1991). Granule size and composition are also
dependent on the age of the cells in the developing endosperm. Three distinct types
of starch granules have been reported which appear to be developmentally regulated.
These vary in their content of associated protein (Wasilick et al., 1994).
Not all starch is readily digested. Some starch and products of starch digestion
have been shown not to be readily absorbed in the small intestine of healthy humans.
Indeed, large amounts of starch compounds have been found in the large bowel
contents of people who have succumbed to sudden death syndrome (Brown, 1996).
Starch has been classified as three types: (i) starch gelatinized by cooking or heating,
which is rapidly digested; (ii) native starch granules from many cereals that may be
slowly but completely digested; and (iii) starch that is resistant to digestion. Three
subcategories of resistant starch have been identified: (i) granules that are physically
inaccessible, e.g. trapped in food matrix or in partially milled or whole grain, so that
the size or composition of food particles prevents or delays the action of digestive
enzymes; (ii) native starch granules where resistance is related to structure and
conformation of granules, e.g. susceptibility to gelatinization – the gelatinization
temperature of some starches is 70°C, while for high amylose maize the complete
gelatinization temperature has to be around 170°C; and (iii) the formation of
retrograde starch material during processing. Starch composed of amylose and
amylopectin can be dispersed in water by heating beyond the gelatinization
temperature. Upon cooling, the dispersed molecules of amylose and amylopectin
spontaneously reassociate and can form crystallites that resist enzymatic hydrolysis.
Retrograde and resistant starch can vary significantly with regard to enzyme
susceptibility (Eerlingen et al., 1994). This type of starch usually passes undigested to
the lower bowel (Brown, 1996).
Amylose content increases with age and size of granule. Some of the maize
mutants differ in amylose concentration and it is this characteristic that is mainly
responsible for the wide variations seen in some of the responses with these mutants
(e.g. susceptibility to enzyme degradation, gelatinization, etc.). Maize varieties very
high in amylose are high in resistant starch and fibre and are used in a number of
novel and innovative foods.
While it is beyond the scope of this article to delve into the physical and
biochemical properties of maize and its constituents, the above short review gives
Table 5.1. Energy availability for poultry from cereal grains.
Grain Gross energy (kcal g−1) Metabolizable energy (kcal g−1) Percentage available
Maize 4.54 3.94 86.8

Wheat 4.46 3.52 78.9
Barley 4.39 3.04 69.2
Oats 4.64 2.74 59.1
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Digestibility and Feed Value of Maize 111
some insight as to how maize quality, and thus its nutritive value, can be altered with
different growing conditions, which could alter starch cell structure, and variations in
drying conditions, especially time and temperature, which could affect the physical
properties of the starch granules.
Nutrient availability of maize
Values of apparent metabolizable energy (AME) and true metabolizable energy

(TME), determined for the most part by the faecal excretion method in adult roost-
ers, have been used as a measure of the available energy in maize. The ratio of avail-
able energy to gross energy (AME : GE) is the highest for maize compared with other
commonly used feedstuffs (Table 5.1). This could well be the reason that little effort
has been expended in trying to improve the digestibility of the nutrients in maize.
A report from Indiana (Maier, 1995) shows the type of information usually
available to the purchaser of maize. A number of samples were taken from different
parts of the state and analysed for various nutrients. A wide range in these nutrients
can be noted (Table 5.2). There is little in the nutrient composition data, except
perhaps kernel density, to give an estimate of the available energy in the samples.
Several reported studies have looked at the composition of various samples of
maize. Most of these studies have investigated maize samples that, due to the type of
growing season, had a significant range in density or weight per bushel. Leeson and
Summers (1976) compared maize samples of different moisture and bushel weights
that were harvested during a wet, cold autumn and thus stage of maturity would be
expected to differ. There was a marked range in bushel weights and also composition
values. While bushel weights varied by approximately 40%, ME as determined with
adult roosters varied by only 12%. Composition data for three of the highest, lowest
and middle range bushel weight samples are shown in Table 5.3. The lower bushel
weight samples had a higher level of protein than did the more normal heavier weight
samples. Lilburn and Dale (1989) also reported higher levels of protein with lower
bushel weight maize; however, levels of lysine and methionine were not increased in
the same proportion as was total protein.
Whitacre (1987) had reported that as protein concentration in different maize
samples increased, the relative increase in non-essential amino acids was greater than
that of the essential amino acids (EAAs). A paper by Lilburn et al. (1991), in which
they studied variation in the composition of maize harvested after a season of severe
Table 5.2. Average composition of maize from nine areas of Indiana (values based on 15%
moisture). (Selected data from Maier, 1995.)
Protein Oil Starch Density
% Range % Range % Range % Range
7.7 5.7–9.7 3.3 2.6–4.9 61.7 59.9–64.8 1.26 1.20–1.31
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112 J.D. Summers
drought, again demonstrated the lower EAA values in the higher protein maize
samples. In a further paper Leeson et al. (1993) studied 26 different maize samples
produced in Ontario after a wet, cold growing season that resulted in a significant
percentage of low bushel weight maize. A selected summary of their results is shown
in Table 5.4. The composition values were quite variable and for the most part did
not correlate with bushel weight of the maize. Indeed, little difference in energy or
protein content could be traced to the bushel weight of the maize. These workers
concluded, as did Dale (1994) with a similar study, that bushel weight is a poor
estimator of the feeding value of maize. It is of interest that the low bushel weight
maize of Dale (1994) had lower protein levels as compared with the higher protein
levels reported for low bushel weight maize from a crop exposed to drought
conditions or to a wet harvesting season. Hsu and Sell (1995) studied low and high
bushel weight maize in feeding trials with young poults. They found little or no
difference in the energy value of the different maize samples. The low bushel weight
Table 5.3. Metabolizable energy and composition of maize samples (values expressed on an
85% moisture level). (Selected data from Leeson and Summers, 1976.)
Bushel weight Metabolizable energy Crude protein Fat Fibre Starch Sugar
(lb) (kcal kg−1) (%) (%) (%) (%) (%)
57 3300 7.6 3.4 2.7 55.3 5.1

55 3232 8.9 3.3 3.2 58.1 4.3
54 3410 8.9 4.0 3.1 57.6 4.3
49 3395 8.5 4.3 3.1 56.9 4.8
49 3247 9.1 3.7 2.2 57.9 6.8
48 3388 8.8 3.5 2.9 58.2 4.3
44 3054 9.1 3.2 3.8 56.3 6.2
42 3084 10.9 4.3 3.2 55.3 7.7
41 3051 10.0 4.3 2.5 56.6 6.6
Table 5.4. Bushel weight, metabolizable energy and proximate components of maize. (Selected
data from Leeson et al., 1993.)
Bushel weight Metabolizable energy Crude protein Crude fat Soluble fibre Insoluble fibre
(lb) (kcal kg−1) (%) (%) (%) (%)
58.5 3270 9.0 3.2 1.2 9.1

55.3 3306 8.9 2.7 1.4 10.6
58.5 3275 8.8 3.3 0.6 9.2
53.1 3163 7.5 2.5 1.1 8.3
53.1 2926 7.1 2.7 0.4 8.4
53.4 3363 7.7 2.9 0.7 8.7
50.2 3471 8.1 3.3 1.2 10.3
50.8 3065 7.5 2.6 1.2 8.7
45.2 2944 7.9 2.3 0.9 8.9
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maize in their study also had a lower protein content at 6.63% versus 8.53% for
maize of normal bushel weight.
Since maize quality can be reduced by the amount of broken kernels or presence
of foreign matter, Dale and Jackson (1994) took samples from various shipments of
maize and separated them into whole kernels, broken kernels and foreign matter
(which ranged from 2 to 7% in the samples). The broken kernels averaged about
2.5% and the foreign matter contained 115 kcal kg−1 less nitrogen corrected true
metabolizable energy (TMEn) than did the whole grain maize.
Potential for Improving Maize Nutritive Value

It is estimated that 60–70% of the maize produced in the world ends up in livestock
feed. Until very recently, seed companies were essentially interested in producing
varieties that increase the yield per hectare. The last number of years have seen a
shift in the goals of the plant breeders as they now are considering altering the
composition of the maize in order to improve its nutritional level.
With gene transfer technology firmly established, the stage is set for a rapid
advancement in nutrient quality improvements of cereal grains. While maize compo-
sition and quality, on a dry matter (DM) basis, has always been assumed to be fairly
constant, marked differences in composition (as shown in previous tables) have been
known for years. Because maize makes up such a large portion of swine and poultry
diets, minor changes in composition can reflect on animal performance or carcass quality.
With maize being a major contributor of energy to diets, increasing the available
energy in maize is an avenue being actively pursued. There are two strategies available
to increase energy density of maize. One is to increase the gross energy value of
the grain and, assuming that the ratio of GE : ME remains constant, an increase in
available energy will take place. A second way is to increase the percentage of GE that
can be utilized by the animal.
Oil
The approach that has received the most attention to date is to increase the GE of
maize by increasing its oil content. This has proved to be successful and there are now
maize types on the market with oil levels in excess of 8% on a dry matter basis, in
contrast to around 3–4% for NM. The oil is found almost exclusively in the germ
portion of the kernel and thus an increase in germ size brings about a corresponding
decrease in endosperm and starch content. For every 1% increase in oil content,
starch decreases by 1.3%. Since the germ is relatively high in protein, it also increases
and will be higher by approximately 0.3% with a 1% increase in oil content. For
every 0.1% increase in oil content, the available energy content of the maize is
estimated to increase by approximately 4.5 kcal kg−1. An increase in the ratio of
ME : GE from 85 to 89% would have to see an increase in the oil content of the
maize by three percentage points to equal the increased available energy produced.
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114 J.D. Summers
A survey by Maier and Briggs (1997) also showed that high oil maize (HOM)
can vary significantly in composition (Table 5.5). Han et al. (1987) investigated the
feeding value of HOM for poultry in low protein diets in order to achieve diets
containing high levels of the maize samples being tested. There was no difference
in protein quality of the HOM or NM as measured by a net protein ratio test. In a
test with broilers to 22 days of age, weight gain was equal while feed efficiency was
significantly improved when the HOM replaced NM in the basal diet (Table 5.6).
They concluded that the magnitude of the response was highly correlated with the oil
content of the maize. In a 15-week test with laying hens, feed to egg mass output was
significantly better when HOM replaced regular maize in a 17% protein diet when
the maize was substituted on an isonitrogenous basis. There were no differences for
other parameters measured between the two maize samples (Table 5.6).
Adams et al. (1994) evaluated HOM in a broiler study simulating commercial
conditions, where male birds were reared to 42 days of age. The experiment was
designed to demonstrate the ability of the HOM to increase the energy level in
broiler diets without going to high levels of added dietary fat. A range of diets con-
taining up to a level of 6% added poultry oil were compared with similar maize/soya
diets where the ratio of energy to protein was kept constant. Thus the HOM diets
contained a higher level of energy and protein. Broilers fed the HOM diets were
Table 5.5. Average composition of high oil maize from 44 samples produced in Indiana (values
based on 15% moisture). (Selected data from Maier and Briggs, 1997.)
Protein Oil Starch Density
% Range % Range % Range % Range
8.7 6.6–10.2 6.9 6.1–7.5 55.6 53.3–57.4 1.26 1.23–1.28
Table 5.6. Comparison of regular maize and high oil maize for broilers and laying hens.
(Selected data from Han et al., 1987.)
(a) Broilers (8–22 days)
Maize type Gain (g) Gain : Feed
Regular 471.5 0.672

High oila 478.3 0.687
aHOM added at same level as regular maize.
(b) Layers (23–38 weeks)
Production Egg weight Feed : egg Body weight

Treatment (%) (g) (g g−1) gain (g)
1. Regular maize (17% CP) 83.7 54.2 2.21 123.2

2. As (1), with HOM (18% CP) 84.4 54.1 2.14 183.9
3. As (2), diet kept isonitrogenous with (1) 86.8 54.7 2.08 180.0
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significantly heavier and had an improved feed : gain ratio than birds fed the NM
diets. They also had less abdominal fat than the NM-fed birds (Table 5.7). These
results show the possibility of formulating higher density diets with the use of HOM.
In a further publication from Arkansas (Saleh et al., 1997) a HOM sample was com-
pared with a NM variety. The HOM contained 2.32% more oil, 156 kcal kg−1 more
TMEn and 0.38% more crude protein than the regular maize sample. These were fed
in commercial-type diets, with equal levels of protein and energy, under commercial
conditions, to 42 days of age. There were no differences in body weight, feed conver-
sion or dressing percentage between the various diets. The HOM-fed birds had sig-
nificantly less abdominal fat. The authors again pointed out the advantage of being
able to use higher energy diets without reverting to higher levels of added dietary fat.
Bartov and Bar-Zur (1995) compared a HOM variety produced in Israel with
local NM. The composition of the maize types, HOM versus regular, was: oil
6.7–2.8%; protein 9.8–7.2%; AMEn 3658–3437 kcal kg−1, respectively. One would
expect to find improved performance of the HOM type based on its composition.
However, a broiler feeding trial from 7 to 28 days resulted in no difference in bird
performance when the HOM replaced an equal proportion of NM. Supplementing
the NM diet with protein and fat to equal levels of protein and energy in the HOM
diet resulted in a significant improvement in performance of the birds. The authors
had no ready explanation for the failure of the HOM diets to give superior
Table 5.7. Effect of high oil (HOM) vs regular yellow dent maize (YDM) in diets with various
levels of supplemental oil (male broilers to 42 days). (Selected data from Adams et al., 1994.)
(a) Body weight (g)
% Supplemental oil
Maize type 0 2 4 6 Mean
HOM 2124 2122 2257 2236 2185

YDM 2020 2056 2125 2158 2090
(b) Feed : gain

% Supplemental oil
HOM 1.71 1.69 1.61 1.57 1.65

YDM 1.80 1.75 1.72 1.66 1.73
(c) Abdominal fat (% of body weight)

% Supplemental oil
HOM 2.08 1.96 2.09 2.16 2.07

YDM 2.26 2.48 2.35 2.24 2.33
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116 J.D. Summers
performance, but they did point out that some of the reported responses to HOM
have been variable.
Vieira et al. (1997) reported on a feeding trial with a HOM variety produced
and grown in Brazil. Their HOM contained 5.7% oil compared with 3.8% in their
NM sample. However interesting, unlike most other HOM samples reported, their
sample contained slightly less protein than their NM (9.17% versus 9.39%, DM
basis). Similar to the report from Bartov and Bar-Zur (1995), their data showed that
the NM sample gave superior performance when supplemental oil was added to
make the diets equal in energy. Rand et al. (1998) reported that under commercial
conditions HOM was particularily advantageous in enhancing broiler performance
during the hot summer months. This was probably due to the more efficient use of
the higher oil diet.
Parsons et al. (1998) studied the availability of the amino acids in HOM as
compared with an NM sample. Digestibility studies with adult cockerels suggested
that digestibility for the amino acids in both samples of maize were the same. Using a
chick feeding study they demonstrated that the bioavailability of the lysine in HOM
was equal to or slightly better than that in NM.
One would have to conclude that most of the HOM varieties on the market
today are superior in feeding value to NM samples. Their greater nutrient density
and superior nutritive value, along with their advantage in producing higher density
diets without increased dietary fat inclusion, make them a potential competitor for
the maize market of the future.
Protein
Several maize varieties developed in the 1960s showed promise due to their improved
level of protein and the essential amino acids lysine and methionine. Opaque-2 maize
had a higher level of protein and significantly more lysine than NM. When compared
with NM, growth response of chicks was not as good until the opaque-2 maize diet
was supplemented with methionine to balance the higher level of lysine. Floury-2
maize had increased levels of protein as well as lysine and methionine. This maize
gave an improvement in performance when compared with NM without EAA
supplementation. Cromwell et al. (1968) compared the feeding value of a sample of
opaque-2 and floury-2 maize with that of NM. These were tested in a 21-day feeding
trial with chicks fed 15% protein diets where equal amounts of maize protein were
maintained by varying the amount of maize added. The results, shown in Table 5.8,
demonstrate the superiority of the higher protein maize samples with proper EAA
supplementation. These maize types never did gain favour with producers, due to
poor storage problems as well as low yields.
An increase in the proportion of glutelin as compared with zein protein in the
kernel is obviously the reason for the changes noted for the altered composition of the
high protein maize varieties.
Bond et al. (1991) tested two new high protein maize varieties that were
reported to be free of the problems that plagued the earlier samples. These varieties
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contained approximately 11% protein as against 9% for the NM that they used for
comparison. In a 6-month test with layers, where the diets were formulated to be
isocaloric but not isonitrogenous, production and egg size did not differ; however,
feed per kilogram of egg mass produced was significantly lower with one of the
new maize varieties (Table 5.9). In a test with broilers to 6 weeks of age, diets were
formulated with the HPM-1 sample. The diets were formulated to be isonitrogenous
Table 5.8. Effect of adding methionine to normal, opaque-2 and floury-2 maize, and to normal
maize plus lysine, in diets containing 15% protein. (From Cromwell et al., 1968.)
(a) 20 day weight (g)
Sulphur amino acids (%)
Maize type 0.45 0.60 0.80 Average
Normal 377 332 330 333

Opaque-2 299 389 382 357
Floury-2 397 380 384 387
Normal + lysine 367 368 377 371
Average 351 368 367
(b) Feed : gain

Sulphur amino acids (%)
Maize type 0.45 0.60 0.80 Average
Normal 1.74 1.77 1.74 1.75

Opaque-2 1.81 1.63 1.62 1.69
Floury-2 1.65 1.66 1.65 1.65
Normal + lysine 1.70 1.65 1.66 1.67
Average 1.72 1.68 1.67
Table 5.9. Comparison of normal maize and high-protein maize. (Selected data from Bond et al.,
1991.)
(a) Layers
NM HPM-1 HPM-2
Production (%) 69.1 70.7 68.5

Egg weight (g) 64.1 64.7 63.9
Feed intake (g per bird day−1) 109.57 106.57 109.57
Feed : egg mass 2.56 2.35 2.57
(b) Broilers
NM HPM-1
Body-weight gain (g) 1633.00 1749.00

Feed : gain 2.00 1.96
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118 J.D. Summers
as well as isocaloric. An improvement in weight gain and feed : gain ratio was noted
with the higher protein maize sample. The authors suggested that higher levels of
methionine, as well as several other EAA present, could account for the better
performance of this particular sample.
Waxy maize, while not used to any extent in commercial feeding operations, at
times does find its way into diets for poultry. Waxy maize contains amylopectin as its
only source of starch, while NM contains approximately 73% amylopectin and 27%
amylose as starch sources. Dinn et al. (1982) reported that the feeding value of the
waxy maize was similar to that of NM when fed to broilers. Ertl and Dale (1997)
compared the available energy content of normal and waxy maize of similar genetic
backgrounds, and grown under similar conditions, using adult roosters. Their results
showed that the TMEn values of the two maize varieties were quite similar, thus
suggesting that they could be substituted in diets without any fear of reduced
performance.
Increasing the Available Energy of Maize by Improving

Nutrient Digestibility
The digestibility and availability of amino acids, starch and oil and the presence of
poorly digested components of a grain determine the fraction of GE which shows up
as energy available to an animal. Imbalances of EAA in relation to an animal’s needs
obviously also reduce the nutritive value of a cereal. Most of the improved maize
varieties in the past have resulted in increases or decreases in some of the major com-
ponents such as protein, starch and oil with little or no change in the composition of
these components.
Little headway has been achieved in increasing the nutritive value of maize by
improving the digestibility or utilization of its various components. Recent work
questions whether the AME and TMEn values used today accurately predict the
energy derived by the bird, especially the young bird, from maize. Mahagna et al.
(1995) reported that the AME of maize differed from the first week post-hatch to the
third week by around 200–250 kcal kg−1. Collins et al. (1998) also reported that age
of bird was an important consideration when assigning an available energy value to
maize. It has been shown that measuring available energy with ileal rather than faecal
contents indicates that starch digestion is not complete in the small intestine. Noy
and Sklan (1995) reported low ileal digestibility of both starch and fat in young
broiler chicks fed a maize/soybean-meal diet. It was shown that digestibility for starch
in chicks from 4 to 21 days of age, up to the end of the small intestine, was as low
as 82%, with no evidence of any increase as the birds got older. This suggests that a
portion of the maize starch is reaching the hind gut, where it undergoes fermentation
with poor energetic utilization. Microscopic examination of the ileal digesta supports
this conclusion as large maize endosperm particles have been found to be undigested.
Persia and Lilburn (1998), on studying the AME and starch digestibility in the small
intestine of turkey poults, reported that there was a significant difference in AME for
wheat and maize when measured with small intestine versus colon contents. Their
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data showed 5.21% of undigested starch entering the colon from maize versus 2.67%
from wheat. Pack et al. (1998) reviewed much of the recent work in the area of maize
starch availability and reported that not only is starch not completely digested in
the small intestine but also there can be a significant amount of protein – and in
particular amino acids – entering the hind gut, depending on age of bird and maize
sample. It has been reported that amino acid digestibility is significantly lower when
measured with ileal as compared with faecal determinations. The reason for these
differences is the contribution by the hind gut in digesting starch, which is not
readily available to the animal, and also the error involved in determining amino acid
digestibility values when a correction is made for endogenous losses. There is also a
significant amount of data to indicate that overall digestion is low in the chick
to around 10 days and then increases to a plateau around 4 weeks of age. Thus
the values used for available energy for the young chick show significant room for
improvement by way of altering maize composition or the use of specific enzymes.
Noy and Sklan (1995) have shown that amylase, trypsin and lipase are low at 4 days
of age and that by 21 days of age they increase by 100-, 50- and 20-fold, respectively.
They suggest that only 82–89% of fatty acids and starch are digested in the small
intestine. While nitrogen digestion is also not complete in the small intestine, it
increased from around 78% at 4 days to 92% at 21 days of age.
Enzyme Supplementation
The action of enzymes on various starches and factors influencing starch degradation
has been well documented (Knutson et al., 1982; Planchot et al., 1995). There is
also a wealth of data reported on enhancing the feeding value of the coarse cereals by
supplementing diets with various enzymes. These enzymes degrade the non-starch
polysaccharides present in cereals such as wheat, barley, rye and oats, thus
significantly improving their available energy content. Maize with a very low NSP
level does not respond to such enzymes. However, in recent years it has been shown
that a mixture of enzymes containing amylase, xylanase and protease is effective in
enhancing the nutritive value of a maize/soybean-meal diet. Much of the recent work
in this area is covered in the reports of Pack and Bedford (1997) and Pack et al.
(1998). These reports, as well as others, suggests a 2–5% improvement in available
energy with enzyme supplementation of a maize/soybean-meal diet. More uniform
broiler weights at marketing and reduced mortality have also been noted with the
enzyme-supplemented diets. These have been attributed to the possible removal of
anti-nutritional factors present in the feed ingredients, along with more effective
nutrient digestibility in the small intestine and hence an influence on gut microbial
activity. It is of interest that Yan et al. (1998) also reported reduced mortality as one
of their consistent observations when phytase was added to a maize/soybean diet for
broilers.
Maize/soybean-meal diets have also been improved in nutritive value by the
addition of phytase enzyme. While there is some information to indicate that the
overall improvement of the diet has been enhanced with phytase supplementation, it
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120 J.D. Summers
is mainly an improvement in phytate-phosphorus availability that has been noted.

Simons et al. (1990) showed an improvement in dietary phosphorus availability for
poultry of approximately 65% with microbial phytase supplementation of diets. Van
Der Klis et al. (1997) reported a marked improvement in phytate degradation with
laying hens, from 8 to 50%, with the addition of phytase to the laying diet of the hen
(Table 5.10).
Recently, low phytic acid maize varieties have been identified and they hold
promise in markedly enhancing the utilization of the phosphorus from maize as well
as reducing faecal phosphorus excretion. Two papers from Arkansas at a recent
poultry science meeting showed that the total phosphorus in the maize was similar to
NM and that the non-phytic acid phosphorus was equal to the availability of
inorganic phosphorus supplements (Kersey et al., 1998; Li et al., 1998; Yan et al.,
1998).
Physical Properties of Maize

Another factor to consider in looking at the response to diets containing maize is
the type of grinding to which it is subjected. One of the major ways to improve the
nutritive value of cereals for livestock feeding is to grind the material. Grinding
increases the surface area and thus allows for better enzyme action in the gut. There
has been renewed interest in looking at type and degree of grinding in order to
enhance feeding value as well as to reduce energy costs for grinding. Deaton et al.
(1995) reported that hammer-milling maize through a 9.53 mm screen gave as good
performance with broilers as did maize processed through a 6.35 and a 3.18 mm
screen (Table 5.11). Work reported by Wasburn (1991) suggested that birds under
heat stress increased the rate of food passage through the gut, thus altering digestion
and subsequent feed utilization, but this was not confirmed in the study by Deaton
et al. (1995). There are a number of papers looking at particle size and diet
utilization. Nir et al. (1994) looked at particle size of maize and its influence on
diet utilization (Table 5.12) and their work suggests that degree of grinding and
particle size affect diet utilization.
Table 5.10. Effect of dietary P and microbial phytase on the ileal absorption of P in laying hens.
(Selected data from Van Der Klis et al., 1997.)
Inorganic phosphorus Phytase Ileal P absorption Phytate degradation

(g kg−1) (FTU kg−1) (g kg−1 diet) (%)
0.5 0 0.72 21.7

0.5 0 1.25 21.6
1.0 0 1.41 20.9
0.5 250 1.72 54.2
0.5 250 1.64 59.0
0.5 500 1.71 71.7
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Table 5.11. Effect of hammer-mill screen size of maize grind on male broiler weights to 49 days
of age. (Selected and rearranged data from Deaton et al., 1995.)
Screen size Body weight Mortality

(mm) (g) Feed : gain (%)
3.18 2442 1.94 3.2

6.35 2424 1.94 3.0
9.53 2434 1.93 3.5
Average values for birds reared at 21 and 31°C.
Table 5.12.
(a) Particle size of maize ground to various degrees of fineness.
Particle sizea
Parameter Coarse Medium Originalb Fine
Weight : volume 0.642 0.545 0.630 0.482

Geometric mean diameter (mm) 2.01 0.897 1.102 0.525
Calculated specific surface (cm2 g−1) 24.102 54.102 54.102 102.102
aCoarse = 1.41 mm; fine = 0.64 mm; medium = mix of fine and coarse particles.
bRegular maize ground with an 8 mm screen size.
(b) Performance of chicks fed the above fractions in maize/soybean meal diets. (Selected data
from Nir et al., 1994.)
Particle size
Original + Original +
Parameter Coarse Medium Original 18% fines 30% fines
Experiment 1 (1–7 days)

Weight gain (g) 86.102 100.102 96.102 100.102 94.102
Gain : feed 0.806 0.893 0.847 0.870 0.855
Experiment 2 (7–21 days)
Weight gain (g) 473.102 522.102 463.102 477.102 474.102
Gain : feed 0.662 0.725 0.649 0.667 0.662
An area that has received passing attention from time to time is the influence of
fertilizer on composition of maize. Bird and Olsen (1972) reported that fertilizing
with N, P and K resulted in increased crude protein content of the maize and an
increase in some of the EAA. Diets formulated taking into account the higher level of
protein in the maize, and without EAA supplementation, gave performance results
for broilers equal to the control normal maize diet. However, use of the higher
protein maize resulted in a 12% saving in soybean meal (Table 5.13).
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122 J.D. Summers
Table 5.13. Fertilizer level and % crude protein in maize (dry matter basis) for various plots.
(From Bird and Olson, 1972.)
Plot number
Fertilizer level S2 S3 S6 S9 S10
O 7.96 6.95 7.55 8.10 8.36

X 9.42 8.45 9.05 9.36 7.54
2X 10.21 8.76 8.98 11.20 9.80
X = 50 kg each of N, P2O5 and K2O per hectare.
Potential Immune Response

An interesting paper was published by Williams (1992), who investigated why birds
fed on maize diets were much less susceptible to coccidiosis than birds fed on wheat
diets. While no explanation could be given for the apparent immune response of the
maize diet, differences in some of the vitamins in wheat versus maize were discussed
as possible contributing factors.
In recent years the biotechnologists have produced several new plant varieties
that offer protection against pests. The first commercial planting of insect-resistant
maize hybrids, commonly referred to as ‘Bt’ maize, took place in 1996. These hybrids
possess a gene that enables the plants to produce an insecticidal protein that is toxic
to the larvae of the pests. Brake and Vlachos (1998) compared this type of maize with
NM in a broiler feeding test. Their results suggest that the maize is equal to NM in its
nutritive value.
There are several potential avenues for the plant breeders to investigate in their
search for novel ways of improving the nutritive value of various cereals. With maize
being such an important cereal for human as well as animal consumption, it will
receive increased attention in the years ahead as energy becomes more of a limiting
factor in producing food for an expanding world population.
References
Adams, M.H., Watkins, S.E., Waldroup, A.L., Waldroup, P.W. and Fletcher, D.L. (1994)
Utilization of high-oil corn in diets for broilers. Journal of Applied Poultry Research 3,
146–156.
Bartov, I. and Bar-Zur, A. (1995) The nutritional value of high-oil corn for broiler chicks.
Bird, H.R. and Olsen, D.W. (1972) Effect of fertilizer on the protein and amino acid content
of yellow corn and implications for feed formulation. Poultry Science 51, 1353–1358.
Bond, P.L., Sullivan, T.W., Douglas, J.H., Robsen, L.G. and Baier, J.G. (1991) Composition
and nutritive value of an experimental high-protein corn in the diets of broilers and
laying hens. Poultry Science 70, 1578–1584.
Brake, J. and Vlachos, D. (1998) Evaluation of transgenic event 176 ‘Bt’ corn in broiler
chicken. Poultry Science 77, 648–653.
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Brown, I. (1996) Complex carbohydrates and resistant starch. Nutrition Reviews 54(11),
S115–S119.
Collins, N.E., Moran, E.T. Jr and Stilborn, H.L. (1998) Corn hybrid and bird maturity affect
apparent metabolizable energy values. Poultry Science 77 (Suppl. 1), 42.
Cromwell, G.L., Rogler, J.C., Featherston, W.R. and Cline, T.R. (1968) A comparison of the
nutritive value of Opaque-2, Floury-2 and normal corn for the chick. Poultry Science 47,
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Vegetable Protein Meals and

the Effects of Enzymes
J. THORPE1 AND J.D. BEAL2
6
1University of Bristol, Department of Clinical Veterinary Science,
Langford House, Langford, Bristol BS40 5DU, UK;
2University of Plymouth, Seale-Hayne Faculty,
Newton Abbot, Devon TQ12 6NQ, UK
J. Thorpe and
Vegetable
6 Protein
J.D. Meals
Beal
Introduction
The cereal grains that constitute the bulk of animal feedstuffs also provide 30–60%
of dietary amino acids (National Research Council (NRC), 1998). However, this
protein is inadequate, not only in sufficiency but also in amino acid balance. To
obtain optimum animal performance, whether it be growth, reproduction or
lactation, protein must be added to the diet to provide both an adequate amount of
total protein and a balance of amino acids that approaches the ideal amino acid
pattern for the species and developmental stage of the animal. This chapter will
concentrate on the use of vegetable protein meals (VPM) and exogenous enzymes in
monogastric farm animals, as exogenous enzyme treatments are not in general use
in ruminant diets.
On a global scale the majority of protein ingredients incorporated into animal
feeds are supplied by vegetable proteins, with the oilseeds and legumes being the
main providers. In the UK the demand for high quality VPMs has increased due to
the ban on the use of meat and bone meal in animal feeds in the wake of the BSE
crisis. The vegetable sources of protein vary both in crude protein concentration
and in the amino acid composition of the protein. The proximate analyses of some of
the protein supplements used in animal feeds are outlined in Table 6.1. Amino acid
profiles for these meals can be obtained from various sources, for example Ewing
(1997) and NRC (1998).
Anti-nutritional Factors Found in VPMs

The availability of nutrients in feedstuffs is often limited by the presence of
anti-nutritional factors (ANFs), which may limit the use of these feedstuffs in
animal diets. ANFs can be classified in different ways. Huisman and Tolman (1992)
classified them on the basis of their effects on the nutritional value of feedstuffs, and
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126 J. Thorpe and J.D. Beal
Table 6.1. Proximate analysis of some VPMs used in animal feeds. (From Ewing, 1997.)
DE MJ ME MJ Starch and
(kg−1) (kg−1) DM CP Oil (s.e.) CF Ash sugars
Feedstuff pigs poultry (g kg−1) (g kg−1) (g kg−1) (g kg−1) (g kg−1) (g kg−1)
Cottonseed meal (s.e.) 11.1 9.1 900 410 17 159 60 90

Faba beans 15.8 13.5 860 290 18 90 34 455
Lupin meal 13.2 11.0 860 320 53 135 37 130
Peas 15.4 13.0 860 260 16 70 35 495
Phaseolus beans* – – 870 238 16 35 39 541
Soybean meal (s.e.) 14.8 10.7 880 470 20 82 72 145
Soybeans (FF) 19.3 16.9 890 400 205 60 55 120
Sunflower meal (s.e.) 10.2 7.1 880 360 20 230 70 75
Rapeseed meal (s.e.) 12.0 10.5 880 385 32 11 70 145
*Adapted from van der Poel (1991).
s.e. = Solvent extracted.
FF = Full fat.
the biological response in the animal as: (i) factors with a depressive effect on protein
digestion and on the utilization of protein (protease inhibitors, lectins, phenolic
compounds, saponins); (ii) factors with a negative effect on the digestion of carbo-
hydrates (amylase inhibitors, phenolic compounds, flatulence factors); (iii) factors
with a negative effect on the utilization of minerals (glucosinolates, phytic acid, oxalic
acid, gossypol); (iv) factors that inactivate vitamins, or cause an increase in the
animal’s vitamin requirement; (v) factors stimulating the immune system that may
cause a damaging hypersensitivity reaction (antigenic proteins); and (vi) factors in
feed that have a toxic effect (e.g. lectins, cyanide-containing compounds).
Table 6.2 shows the distribution and physiological effects of the various ANFs
found in VPMs. The toxicity and effects of the different ANFs may vary considerably
between VPMs and these toxic effects may also vary in potency between animal
species. ANFs appear to play a role in the protection of plants against predation from
moulds, bacteria, insects and birds by disturbing the digestive processes of these
organisms, and presumably act in a similar manner in domestic animals (Birk, 1989).
Reduction and Elimination of ANFs in VPMs

The ANFs in VPMs can be reduced or eliminated by processing procedures.
Maximum destruction of ANFs may require different processing treatments, due to
differences in ANF structure and their biological effects. Processing can be supplied
by physical, chemical, thermal or bacterial means. Currently, various methods can be
applied in the reduction and elimination of ANFs in legume seeds (Table 6.3).
Processing technology is conventionally applied to soybean, the most common
being some form of heat treatment, which has proved most effective at reducing
levels of trypsin inhibitors and soybean lectin. Commercial heat processing is
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Vegetable Protein Meals 127
Table 6.2. Distribution and physiological effects of ANFs found in VPMs. (Adapted from Huisman
and Tolman, 1992; Liener, 1994.)
Anti-nutritional factor Distribution Physiological effect
Proteins
Protease inhibitors Most legumes Reduction of (chymo)trypsin activity, impaired
growth, pancreatic hypertrophy, pancreas
carcinogen
Lectins Most legumes Gut wall damage, immune response, increased
endogenous nitrogen loss
Amylase inhibitors Kidney beans Impaired digestion of starch
‘Antigenic’ proteins Soybean, kidney Immune response, interference with gut wall
beans integrity
Polyphenols
Tannins Most legumes Interference with protein and carbohydrate
digestibility by formation of protein–carbohydrate
complexes
Glycosides
Vicin / convicin Faba beans Haemolytic anaemia, adverse effect on egg
production
Saponins Soybean Haemolysis, effects on intestinal permeability
Glucosides
Glucosinolates Rapeseed Impaired iodine utilization (goitogrenicity),
reduced palatability and growth
Cyanogens Linseed, traces in Respiratory failure
kidney beans and peas
Alkaloids
Quinolizidine Lupin Neural disturbances, depressed growth, reduced
palatability
Other ANFs
Phytate Most legumes Formation of complexes with minerals and
proteins, depresses mineral absorption
Gossypol Cottonseed Anaemia due to formation of iron complexes,
reduced egg weight
Sinapins Rapeseed ‘Fish’ taint in eggs
Oligosaccharides Soybeans, peas, faba Flatulence, diarrhoea, discomfort
(NSP) beans, Phaseolus beans
carefully controlled: underheating can result in inadequate inactivation of ANFs

whilst overheating may reduce availability through the occurrence of Maillard
reactions (reviewed by van der Poel, 1989, and Huisman and Tolman, 1992).
Little attention has been paid to the heat processing of other legumes and
oilseeds (post oil extraction). The insufficiencies of some processing techniques have
led to the search for new methods for the elimination of residual ANFs in VPMs.
For example, new breeding technologies have led to the development of low
glucosinolate varieties of rapeseed, and although this has made rapeseed a more
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Table 6.3. Processing techniques for reduction and elimination of ANFs in legume seeds (van
der Poel, 1989).
No. Technology
1 Breeding and genetic manipulation

2 Feed formulation: selection of ingredients, supplementation with amino acids, other
supplements, e.g. probiotics, enzymes, etc.
3 Primary processing: chemical treatments, enzymatic treatment, physical treatments
(fractionation, heat treatments)
Secondary processing: conditioning/pelleting
attractive ingredient in animal feed it has not totally solved the ANF problem. Recent
research has focused on the development of biotechnological approaches to this
problem, mainly by the application of exogenous enzymes to feedstuffs containing
ANFs.
The Use of Exogenous Enzymes

Until recently, the majority of feed enzyme research has been performed on cereal-
based diets, for example barley and wheat, particularly for poultry diets (Dierick,
1989). The type of feed enzymes currently available are outlined in Table 6.4.
There are many factors evolving in the animal feed industry which suggest that
the use of exogenous enzymes will become more important in the future (reviewed by
Johnson et al., 1993). These include: (i) an increasing shift in the use of ‘alternative’
feedstuffs in formulating diets; (ii) the use of enzymes known to be effective against
particular dietary components; (iii) the production of novel by-products (for
example, linseed meal derived after linseed oil production) that have a depressing
effect on growth; (iv) the increased availability of free amino acids which may reduce
the requirement for high quality protein supplements; (v) novel feed systems, such as
liquid feed systems and those that take advantage of pretreatment, which support the
use of in-feed enzymes; (vi) the introduction of pollution control, for example of
nitrogen and phosphorus (enzymes that reduce excretion of these substances would
be advantageous); (vii) the possibility of indirect physiological actions on problems of
commercial importance, for example the reduction of sticky litter in poultry and
post-weaning diarrhoea in pigs (Inborr and Ogle, 1988); and (viii) the possible
reduction in animal performance due to the restricted use of growth-promoting
antibiotics.
Presentation of Exogenous Enzymes to the Animal

The most common method of feeding enzymes is with dry feed, the enzymes usually
being added to the feed during blending, often prior to processing treatments such as
pelleting. However, most of the enzymes currently of interest to the animal feed
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Table 6.4. Feed enzymes in use today. (From Ogden, 1995, modified from Rotter et al., 1989;
Cowan, 1992.)
Enzyme Action Target substrate Type of feed Expected benefits
β-Glucanases β-Glucans to Barley, oats Poultry and Reduction of sticky

oligosaccharides and rye based pig diets droppings, improved
and glucose diets feed utilization
Amylases Degrade cereal High starch Early pig/calf Increased availability
starch to dextrins cereal diets diets of cereals in weaner
and sugars feeds
Cellulases Cellulose to low High fibre Poor-grade Improved energy
molecular weight diets forages availability
products and glucose
Pentosanases Arabinoxylans to low Rye, barley Pig and Improved litter
(Xylanases) molecular weight and wheat poultry diets quality, improved
products and sugars feed utilization
α-Galactosidase Degrades Soybean and Pig diets Improved energy
oligosaccharides other legumes availability, reduced
and ANFs scours in piglets
Phytases Increases availability Many different Pig and Reduces need
of phosphorus from diets poultry diets for inorganic
phytic acid phosphorus
Proteases Protein to peptides Wheat Milk replacer Higher protein
and amino acids by-products, using soybean or digestibility, lower
legume proteins soybean protein nitrogen excretion
Lipases Fats to fatty acids Animal and Pet diets/broiler Improved digestibility
vegetable fats diets of fat and enhanced
energy retention as
a result
industry, such as β-glucanase, amylases, proteases and phytase, suffer temperature-

related reduction in activity due to heat treatments (Inborr, 1990; Cowan, 1992;
Graham and Inborr, 1993; Bedford and Pack, 1998). This problem can be reduced
by the application of the enzyme in a liquid base, post-pelleting, for example spraying
or by use in liquid feed.
Action of Feed Enzymes in the Animal

The application of exogenous enzymes to dry diets presupposes that the enzyme will
be active in the digestive tract of the animal, and it must therefore fulfil a number of
criteria. The enzyme must be active under the physiological conditions prevailing
in the animal’s digestive tract; it must be able to resist proteolysis by the animal’s
endogenous proteases and supplement rather than antagonize the animal’s digestive
enzymes. Species differences in the anatomy and physiology of the digestive tract are
likely to affect exogenous enzyme activity in this respect. For example, between pigs
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and poultry these differences include the following (Partridge, 1993; Dierick and
Decuypere, 1994).
1. Anatomical: in poultry, feed passes into the crop, where any added enzymes
can act for several hours at a pH of approximately 6.0 before passing into the acid
environment of the gizzard, whereas in the pig, feed passes directly into the acid
environment of the stomach immediately after ingestion.
2. Digestive capacity: poultry have a shorter small intestine and thus reduced
possibilities for enzyme inactivation by the microflora, a shorter mean retention time
in the small intestine (1–2 h in poultry versus 4–5 h in the pig) and a lower water
content in the upper part of the gastrointestinal tract.
3. Bacterial activity: the importance of the microflora in the gut of poultry is much
less than in the pig.
4. Fibre fermentation: lower in poultry than in pigs, due to their widely different
hind gut capacities.
Survivability of enzymes in the digestive tract varies widely; for example,
Thacker and Baas (1996) and Baas and Thacker (1996) demonstrated that 84% of
pentosanase and 26% of β-glucanase activity was recovered in the duodenal digesta
of pigs 4 h after feeding diets supplemented with these enzymes. Approximately
75% of exogenous protease has been detected in the ileal digesta of young pigs fed
protease-supplemented diets.
An alternative to the use of exogenous enzymes in dry diets is the pretreatment
of the diet or its components prior to secondary processing. Adding enzymes to
complete compound feeds may be appropriate when the enzyme is targeted at cereals,
which make up the bulk of the diet. However, VPMs only constitute a small
proportion of the total diet and therefore target substrates are considerably diluted.
The dilution effect may be overcome by pretreating the VPM prior to inclusion in
the diet. This approach has been adopted in much of the recent research into the
effect of enzyme application to VPMs, much of which has focused on the effects of
proteases on soybean meals.
Pretreatment of Diets Containing VPMs

Pretreatment of soybean with proteases
The prime target of proteases in soybean are the anti-nutritional factors, such as
residual trypsin inhibitors, lectins and ‘antigenic’ protein. Pretreatment of specific
feed components allows the effect of any enzyme addition to be ascertained prior to
feeding the animal. This is a pertinent point as far as the inclusion of proteases
in animal feeds is concerned, as it is impossible to assess exogenous protease activity
in ileal digesta. Immunochemical techniques are being developed that will detect the
presence of an exogenous protease in ileal digesta; however, these give no indication
of functionality (Thorpe, 1999).
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Pretreatment of soybeans with enzymes has been used, albeit inadvertently

through the processes of fermentation, to improve the nutritional quality of legumes
in human foods for many years (Campbell-Platt and Cook, 1991). It is not the inten-
tion of this chapter to review fermentation processes extensively as little work has
been undertaken on the effect of fermenting protein meals for inclusion in animal
feeds. However, Zamora and Veum (1979) fed heated soybeans fermented with
Aspergillus oryzae or Rhizopus oligosporus to growing pigs. Table 6.5 summarizes their
results. Fermentation significantly improved feed conversion ratio (FCR) with both
organisms and average daily gain with A. oryzae, and showed an improvement
(although not significant) in nitrogen retention with both organisms. This work
indicated that fermentation improves the performance of growing pigs, possibly by
the action of fungal enzymes on the soybean present in the feed.
In the 1950s, work by Lewis et al. (1955) and Baker et al. (1956) examined the
effects of the addition of pepsin, pancreatin, a fungal protease, a diastatic protease
and papain to various soybean diets in a number of trials using pigs from 6 to 67 days
old. Pepsin and pancreatin supplementation showed some beneficial effects on aver-
age daily gain (ADG) and FCR, particularly in younger pigs. Papain and the diastatic
protease were as effective as pepsin and pancreatin, but the fungal protease showed
no beneficial effects. This work was aimed at supplementing the insufficiencies of
proteolytic and amylolytic digestive enzymes in the baby pig, as opposed to the
current work, which targets ANFs in protein meals.
More recent work has concentrated on the use of isolated enzymes produced
from large-scale microbial fermentations. This research can be divided into either
in vitro or in vivo studies. In vitro studies have been used by a number of workers in
this field to determine the effect of exogenous enzymes on VPMs prior to expensive
in vivo studies. Assessment of enzyme activity in vitro may indicate any potential
benefits the enzyme may have in vivo. A number of studies have been undertaken
utilizing proteases on soybean meals in vitro, analysing different parameters.
Huo et al. (1993) showed that one fungal and four bacterial protease enzymes
could inactivate trypsin inhibitors and lectin in raw soybean (RSB) and low-
temperature extruded soybean (LTES) in vitro, to various extents. The most effective
of these reduced trypsin inhibitor levels in RSB and LTES by 96% after 12 h
incubation at an inclusion level of 1%. Apart from being described as fungal or
bacterial proteases, there were no other details provided about the enzymes. The
bacterial proteases appeared to be more effective at breaking down trypsin inhibitors
than the fungal protease. Meijer and Spekking (1993) isolated microorganisms
Table 6.5. Summary of results of Zamora and Veum (1979).
Soybean treatment ADG (kg) FCR N retention (%)
Unfermented 0.52 2.33a 50.6

a0.60a 2.04a
Fermented with A. oryzae 55.7
Fermented with R. oligosporus 0.56 1.96a 55.0
aP > 0.05.
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producing enzymes that could utilize purified sources of Kunitz soybean trypsin
inhibitor (KSTI) and Bowman-Birk inhibitor (BBI) as sole carbon, nitrogen and
sulphur sources, and inactivate them over a time course of several days. Therefore
these microorganisms must all possess enzymes capable of hydrolysing the trypsin
inhibitors in soybeans. This does not necessarily mean that trypsin inhibitors would
be utilized in preference to the other sources of C, N or S in whole soybeans. These
enzymes may have potential activity against trypsin inhibitors in other VPMs.
Rooke et al. (1996) aimed to assess whether protease treatment of soybean meal
(SBM) could reduce its in vitro antigenicity and improve its nutritional value when
fed to newly weaned piglets. Table 6.6 (Ref. 1) describes the protease treatment of
SBM in this study. Antigenic soybean proteins were measured by a competitive
enzyme-linked immunosorbent assay (ELISA) technique. All diets, including the
Table 6.6. Summary of recent studies utilizing protease pretreatment of SBM.
Ref. Vegetable
no. protein Enzyme Treatment Reference
1 SBM Acid protease 0.1% protease added to SBM (800 g kg−1 Rooke et al.
(P2) moisture) pH 4.5. Incubated for 3 h at 50°C, (1996)
neutralized, dried at 65°C
2 SBM Acid protease 0.1% protease added to SBM (800 g kg−1 Hessing
(P2) moisture) pH 4.5. Incubated for 3 h at 50°C, et al. (1996)
dried at 55°C
3 Alkaline 0.1% protease added to SBM (800 g kg−1
protease (P1) moisture) pH 8.5. Incubated for 3 h at 50°C,
dried at 55°C
4 SBM Acid protease 0.1% protease added to SBM (800 g kg−1 Hessing
(P2) moisture) pH 4.5. Incubated for 2 h at 50°C, et al. (1996)
fed as a wet mash Rooke et al.
5 Alkaline 0.1% protease added to SBM (800 g kg−1 (1998)
protease (P1) moisture) pH 8.5. Incubated for 2 h at 50°C,
fed as a wet mash
6 SBM Protease 0.1% protease added to SBM (1 : 2 wt : vol water) Caine et al.
(B.subtilis: pH 4.5. Incubated for 16 h at 50°C, freeze dried (1997)
7 subtilisin) 50 ml of enzyme solution (pH 4.5) to give final
enzyme concentration of 0.1% sprayed on
SBM, air dried at ambient temperature for 24 h
8 FFSBM Protease (P4) 0.25% added to soybean meal (1 : 3 wt : vol Beal et al.
(autoclaved) water) incubated for 24 h at 20°C, fed as liquid (1998b)
9 Raw Protease (P4) 0.25% added to soybean meal (1 : 3 wt : vol
soybean water) incubated for 24 h at 20°C, fed as liquid
10 Micronized Protease (P3) 0.5% added to soybean meal (1 : 3 wt : vol Beal et al.
FFSBM water) incubated for 24 h at 20°C, fed as liquid (1999)
11 Raw Protease (P3) 0.5% added to soybean meal (1 : 3 wt : vol
soybean water) incubated for 24 h at 20°C, fed as liquid
SBM = soybean meal – method of processing unstated. FFSBM = full fat soybean meal. Proteases
(refs 1–11) supplied by Finnfeeds International Ltd (Marlborough, UK).
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skim milk diet, contained antigenic soybean proteins, indicating a possible problem
with the technique. The SBM treated with protease contained fewer antigenic
proteins than the other soybean-containing diets. It is assumed here, as in many
studies, that reduction in levels of antigenic proteins as measured by ELISA indicates
protein denaturation by exogenous protease treatment. This is not necessarily an
accurate assumption as, for example, antiserum from animals immunized with
soybean protein may not recognize any protein components of an enzyme-treated
soybean product, but the treated product may still contain antigenic epitopes.
Hessing et al. (1996) examined the ability of two microbial proteases (P1 and
P2) to degrade ANFs, and to determine whether enzymatically hydrolysed SBM
could improve the productive performance of newly weaned piglets or broiler chicks.
The SBM was pretreated with the protease before feeding. Table 6.6 (Refs 2–5)
describes the protease pretreatments. SDS-PAGE and Western blotting analysis
demonstrated that P1 could significantly hydrolyse the storage proteins glycinin and
β-conglycinin, and to a certain extent KSTI at inclusion levels of 1000–10,000 U g−1
material, but there were no effects on soybean lectin. It was concluded that the
potential of proteases to improve the nutritional value of soybean meal was
promising. It was cautioned that in vitro immunochemical analysis of ANFs must
be interpreted with care, as the hydrolysis of proteins such as trypsin inhibitors
may expose more antigenic epitopes, but the inhibitor itself may not be functional,
resulting in misleading results.
Caine et al. (1997) determined the optimum conditions for Bacillus subtilis
subtilisin pretreatment of SBM by measuring increases in protein solubility. They
found the optimum conditions to be incubation at 50°C and pH 4.5.
Beal et al. (1998a) used an in vitro technique (Boisen and Fernandez, 1997) as
an initial evaluation of the potential of three proteases to improve the in vitro
nitrogen digestibility of RSB and four processed SBMs. They found significant
increases (P < 0.05) of approximately 5–12% over control with the three proteases.
These results indicate the possibility of increases of protein digestibility of these meals
in vivo. Beal et al. (1998c) also examined the effects of one of these proteases (P4)
using SDS-PAGE, and found a reduction in the number and density of protein
bands with apparent molecular weights greater than 66 kDa, indicating the
hydrolysis of storage proteins.
Rooke et al. (1998) also used SDS-PAGE to examine the effects of proteases P1
and P2 (Table 6.6, Refs 4–5) on SBM. Pretreatment changed the composition of
SBM, and soluble α-amino nitrogen concentrations were increased by treatment
with P1 and P2. P1 reduced antigenic protein concentration (as determined by
SDS-PAGE) to a greater extent than P2.
Table 6.6 summarizes exogenous protease pretreatments of soybean meals
in recent studies described above. The in vivo results obtained from these trials are
summarized in Table 6.7. The results of these trials are variable. In some studies,
enzyme pretreatment produced positive effects on some of the parameters measured
whilst in others there were no significant effects. As with all such studies, it is difficult
to draw direct comparisons, due to differences in age, weight and genotype of pig and
in diet formulation.
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Pretreatment of other VPMs with exogenous enzymes
Castanon and Marquardt (1989) investigated the effects of Celluclast (cellulase),

SP249 (polygalactouronase), Alcalase (protease) and BAN (α-amylase), in various
combinations, on raw, autoclaved, or steeped faba beans for young chickens. The
Table 6.7. Summary of the results of recent trials utilizing protease pretreatment of SBM.
Cross Animal age/weight

ref. and trial duration Summary of results, enzyme treatment (T) compared with control (C)
1 Newly weaned pigs Significant (P > 0.01) increase in ADG for 7 days post weaning
21 days 155 g day−1(T) vs. 95 g day−1 (C). Significant increase in ADFI for 21
days post weaning 392 g day−1(T) vs. 347 g day−1(C). No significant
increase in FCR
2 7 day old chicks Significant increase (P < 0.01) in ADG; 41 g day−1(T) vs. 30 g
141 g, 27 days day−1(C), ADFI; 79 g day−1(T) vs. 64 g day−1(C), apparent ileal N
digestibility 0.85(T) vs. 0.76(C)
3 7 day old chicks No significant increases in ADG, ADFI or apparent ileal N
141 g, 27 days digestibility
4 Newly weaned pigs Significant increases (P < 0.01) in ADG over first 7 days
7.5 kg, 14 days postweaning; 120 g day−1(T) vs. 64 g day−1(C) and (P < 0.05) in
ADG over 14 days postweaning; 181 g day−1(T) vs.128 g day−1(C).
No significant difference in FCR
5 Newly weaned pigs Significant decrease (P < 0.01) in ADG over first 7 days
7.7 kg, 14 days postweaning; 78 g day−1(T) vs. 143 g day−1(C) and (P < 0.05) in
ADG over 14 days postweaning; 153 g day−1(T) vs. 199 g day−1(C).
No significant difference in FCR
6 Newly weaned pigs No significant differences in ADFI, ADG, FCR or apparent ileal
6.4 kg, 2 × 9 day amino acid digestibilities
changeover
7 Newly weaned pigs No significant differences in ADFI, ADG, FCR or apparent ileal
6.4 kg, 2 × 9 day amino acid digestibilities
changeover
8 Growing pigs No significant differences in ADG, ADFI or FCR
33.5 kg, 6 weeks
9 Growing pigs Significant (P < 0.05) increases in ADFI; 1.37 kg day−1(T) vs. 1.24 kg
33.5 kg, 6 weeks day−1(C), ADG 0.606 kg day−1(T) vs. 0.529 kg day−1(C). No
significant difference in FCR
10 Growing pigs No significant difference in ADG, ADFI, FCR or lean : fat ratio.
28.7 kg to slaughter Significant reduction of 4 ± 1.86 days to reach slaughter weight due
(90 kg) to protease addition
11 Growing pigs Significant (P < 0.05) improvement in ADG; 0.606 kg day−1(T) vs.
28.7 kg to slaughter 0.515 kg day−1(C) and FCR; 3.042(T) vs. 3.512(C) in finisher period.
(60 kg) Significant (P < 0.05) increase of 0.423 ± 0.158 in lean : fat ratio due
to protease addition
CP = crude protein; ADG = average daily liveweight gain; ADFI = average daily feed intake;
FCR = feed conversion ratio (feed/gain).
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authors found that cellulase and protease, and a combination of the two, significantly
increased weight gain and FCR (P < 0.05) by approximately 5–10% in both raw and
autoclaved beans, and the combination significantly improved the weight gain
(P < 0.05) and FCR (P < 0.01) of steeped beans. The other enzymes had no effect.
Näsi (1991) investigated the effects of processing, enzyme pretreatment
and multi-enzyme supplementation (cellulase, protease and β-glucanase) of SBM
and rapeseed meal (RSM) diets on the nutrient digestibility, protein utilization and
performance in growing pigs. Extrusion and the addition of enzyme premix
significantly improved the organic matter and protein digestibilities of SBM
(P < 0.05). No significant differences in growth rate, FCR or carcass quality were
seen between pigs fed diets containing differently treated SBM, relative to an
untreated control. No significant effects of enzyme treatment on any parameters were
observed with RSM.
Schulze et al. (1997) examined the effects of germination or pancreatin
pretreatment of Phaseolus vulgaris (white kidney bean) on the ANF activities and
apparent ileal digestibility of nitrogen in young pigs. The authors found germination
significantly decreased (P < 0.05) the flow of amino acids at the terminal ileum by
approximately 50% compared with pancreatin or control. Trypsin inhibitor and
lectin activity were reduced by germination, although the significance of these
reductions was not reported.
Direct Addition of Enzymes to Complete Diets Containing

VPMs
A number of pig and poultry trials have been performed using the addition of
exogenous enzyme premixes to dry diets. Pluske and Lindemann (1998) have
extensively reviewed the use of Vegpro, a mixture of protease, cellulase, pentosanase,
α-galactosidase and amylase, in pig and poultry trials using various VPMs. Cowan
et al. (1996) reviewed the use of Biofeed Plus CT (main activity protease) and
Energex MG (main activities β-glucanase and pectinase). Some other work using the
same enzymes is reviewed below.
Enzymes targeted to SBM dietary components
De Koning and van der Wel (1996) supplemented starter broiler maize/SBM diets
with Vegpro at 0.1 and 0.05%. Significant increases (P < 0.05) in weight gain after
21 days were observed with both enzyme inclusion levels, but no significant
differences in FCR were observed. Similar results were obtained by Sefton and
Perdok (1996). Swift et al. (1996) examined the effects of Vegpro on expanded
and pelleted maize/SBM diets for broilers. Enzyme treatment significantly
improved (P < 0.05) FCR in the expanded diet over the 35-day feeding period.
Vegpro significantly improved (P < 0.05) nitrogen and energy digestibility in grower
diets but not starter diets. Schang et al. (1997) evaluated the effects of Vegpro in
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maize/SBM and maize/full-fat SBM (FFSBM) diets for broilers, using high and
low nutrient density formulations. There were no significant effects of adding
Vegpro to high density diets. Significant improvements (P < 0.05) in weight gain
were seen with maize/FFSBM at low nutrient digestibilities. Spring et al. (1998)
examined the effect of Vegpro on a grain/SBM diet with reduced protein con-
centration, in weaned piglets. Significant improvements (P < 0.05) in FCR due to
Vegpro treatment were observed in diets in which the lysine concentration was
reduced to 0.97%.
Enzymes targeted to other VPM dietary components
Viveros et al. (1993) examined the effect of addition of a combination of Biofeed

Pro, a mixture of protease, amylase, hemicellulase, pentosanase and xylanase, and
BAN, a mixture of amylase and β-glucanase to diets containing 10 and 15% faba
bean hulls in diets for chickens. Addition of the enzyme mixtures significantly
improved (P < 0.05) weight gain in 10 and 15% hull diets, and FCR in 15% hull
diets. However, enzyme addition did not improve the feed efficiency of the hull diets
to the level of a maize/SBM diet. The same workers investigated the use of tannase in
a 10% hull diet. Tannase addition significantly improved (P < 0.05) weight gain, but
not FCR.
Pfirter et al. (1993) investigated the effects of exogenous protease supple-
mentation on broiler feeds containing crude or extracted lupins. No significant
improvements over control, crude lupin diets were observed.
Hadron et al. (1993) evaluated the effects of exogenous carbohydrases and
proteases on growing-pig diets containing peas. No effects of enzymes were observed
on nitrogen or energy digestibility, or on production characteristics.
Bedford and Morgan (1995) examined the effects of the addition of xylanase,
xylanase plus alkaline protease, xylanase plus neutral protease, and xylanase plus
acid protease on canola meal (CM) diets for broilers. Addition of xylanase alone
significantly improved (P < 0.05) weight gain over the CM control diet. Addition
of xylanase and alkaline protease significantly improved (P < 0.05) FCR over a
SBM-based control diet. The addition of acid protease also significantly (P < 0.05)
improved FCR, but the neutral protease had no effect.
Annison et al. (1996) examined the effects of two commercial enzyme
preparations on the nutritive value of dehulled lupins for broiler diets. Enzyme A
contained xylanase, pentosanase, and hemicellulase; Enzyme B contained
β-glucanase, hemicellulase and pectinase. Enzyme A increased the AME of lupins
(P < 0.05); Enzyme B had no significant effects.
Stanley et al. (1996) examined the effect of Vegpro addition on various levels
of cottonseed meal (CSM) in diets for broiler chicks. The inclusion of Vegpro
significantly improved (P < 0.05) FCR in diets containing 7.5, 15 and 30% levels of
CSM. Schang and Azcona (1998) evaluated the effects of Vegpro on maize/sunflower
diets as a replacement for maize/SBM diets. Sunflower meal significantly (P < 0.05)
decreased egg production, and the addition of Vegpro did not compensate for this.
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Discussion
Trials conducted to date on the effects of exogenous enzyme supplementation of
VPMs show variable results, with some showing significant changes with enzyme
inclusion, and others showing trends towards improvement, which are not actually
significant. There are a number of possible explanations for these observations. In
many cases it is impossible to draw direct comparisons between individual studies
due to differences in enzyme type, activity and inclusion level. Where enzyme
activities are actually stated, there are often differences in units and the method of
determining activity is not always stated.
The majority of trials have been performed under experimental conditions, with
high health status animals, which may not necessarily reflect commercial situations.
Basal diets can vary widely between different trials depending on the availability of
dietary components. It must be remembered that modern diets are formulated to
achieve maximum performance from an animal. Any increase in nutrient availability
due to enzyme supplementation may not elicit a response from the animal unless the
dietary factor in question is reduced to suboptimal levels. This is not necessarily
a consideration when substituting proportions of a diet with inferior feedstuffs,
provided nutrient specifications do not exceed recommended levels for that species,
age and genotype. For example, if the effect of protease inclusion in the diet is being
investigated, the protein : energy ratio needs to be reduced to suboptimal levels in
order for the animal to respond to any increase in amino acid availability due to
enzyme treatment. In the studies outlined in Tables 6.6 and 6.7, Rooke et al.
(1996, 1998), Hessing et al. (1996) and Beal et al. (1998b, 1999) indicated that
lysine : energy ratios were reduced below recommended levels, whereas Caine et al.
(1997) stated that the diets used were formulated to exceed NRC recommendations.
This could explain the lack of response to enzyme inclusion in their study. Other
studies corroborate this, such as Schang et al. (1997) and Spring et al. (1998), who
showed significant differences with diets formulated with nutrient densities below
recommendations, but not with diets formulated to meet or exceed nutrient
recommendations.
It is difficult to assess the efficacy of enzyme preparations on target substrates
when applied to complete diets, as all plant feedstuffs in a diet are likely to contain
substrates on which enzymes can act. This does not present a major problem with
enzymes designed to target substrates such as NSPs, which can present problems
in other major dietary constituents, such as cereals. However, when targeting
proteinaceous ANFs in VPMs with exogenous proteases, a number of factors need to
be considered. The VPM in a compound feed represents only a fraction of the total
feed, typically 20–30%. The target substrate for the enzyme will be even less than
this. For example, if full-fat soybean meal with a crude protein content of 40%
(see Table 6.1) is included in a diet at a rate of 25%, then the actual soybean protein
content of that compound feed will be 10%, and the ANF content even less, at 6–7%
of the total protein (Nielsen, 1983). This represents a considerable dilution of the
substrate by other feed components; the competition from other protein substrates in
a complete diet may result in little or no ANF degradation. Pretreatment of VPMs
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prior to inclusion in a diet has two advantages. Firstly, the dilution effect of other
dietary components is reduced. Secondly, pretreatment allows the effects of enzyme
activity on the specific substrate to be evaluated. Analysis of in vitro results of
pretreatment have to be interpreted with caution; for example, ELISA techniques
may demonstrate changes in protein structure, but not necessarily functionality.
However, the development of immunochemical assays which measure KSTI, BBI
and soybean lectin (SBL), both by aspects of protein structure and by biological
activity, in feed and digesta samples may allow this problem to be resolved (Thorpe
et al., 1997; Miller et al., 1998). Techniques used to measure in vitro digestibility
cannot necessarily be extrapolated to in vivo situations; however, they are useful tools
in the initial screening of the suitability of enzyme preparations.
In designing exogenous enzyme studies, the predicted application of the enzyme
needs to be considered to allow the transfer of technologies to commercial situations.
This is of particular importance with pretreatment strategies. Some of the studies
previously described use pretreatments with relatively energy-intensive processes,
such as high incubation temperatures followed by drying (Hessing et al., 1996;
Rooke et al., 1996; Caine et al., 1997). This may not be economically viable when
extrapolated to a commercial situation.
New developments in feeding systems, particularly of those for pigs, have
increased scope for effective commercial enzymatic applications. One such develop-
ment is the increased use of liquid feeding systems, which provide excellent
conditions for feed enzymes. On many UK farms, liquid feed systems for pigs are
used to enable farmers to take advantage of liquid by-products from food processors;
thus diets are often mixed on-farm (Geary, 1997). Enzyme preparations could
be added directly into the feeding system, thereby eliminating the risk of enzyme
denaturation during processing procedures such as pelleting. These systems provide
an ideal opportunity for pretreatment of individual raw materials, such as VPMs,
in situ, in a commercial environment, before addition of other dietary components.
Pretreatment of VPMs prior to incorporation into poultry diets does not appear to
have attracted so much attention. Pretreatment may not be so relevant to the poultry
industry, as liquid feed systems are generally not used and anatomical features of the
fowl digestive system effectively provide a pretreatment chamber in the crop. The
activity in the crop may explain the greater response of poultry to exogenous enzyme
supplementation in general.
Pretreatment of SBM with exogenous enzymes appears to have the greatest
effect in young animals, particularly noted in early weaned pigs (Hessing et al.,
1996; Rooke et al., 1996, 1998; Caine et al., 1997). This effect is usually greatest
in the 7 days immediately post-weaning, possibly helping to overcome the transient
deleterious effect of feeding SBM observed by workers such as Li et al. (1991a,b) and
Dreau et al. (1994). At this time the animal is often performing suboptimally, giving
a window of opportunity for the use of exogenous enzyme supplementation. Effects
of enzyme supplementation do not appear to be as great in grower/finisher pigs.
Possible reasons for this include the maturity of the digestive and immune system
and the formulation of diets to high nutrient specifications. The major VPM in use
in grower/finisher pig diets at present is SBM. This is often highly processed to
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maximize digestibility (van der Poel, 1989). Processes used are often expensive and it
is possible that the use of enzyme supplementation of less digestible SBMs or other
VPMs may allow higher inclusion rates of less expensive ingredients in diets without
affecting performance.
Future Research Requirements

To date, the majority of research has been aimed towards the improvement of
SBMs. SBM is considered to be a high quality protein source and other, possibly
lower quality, VPMs may offer more scope for improvement by exogenous enzyme
supplementation. This could allow the use of VPMs from indigenous rather than
imported sources.
The pretreatment of individual dietary components with exogenous enzymes
has not been fully explored. In particular, the application of exogenous enzyme
pretreatments in liquid feed systems in the pig industry provides a potential
opportunity for the improvement of the nutritional status of VPMs.
There is little published data on the optimum inclusion levels of exogenous
enzymes in animal diets. Inclusion levels are often extrapolated between species
and, considering the differences in anatomy and physiology of the digestive tract,
these levels may be incorrect. Maximum improvements of VPMs may not be
achieved unless optimum enzyme inclusion levels are used. These levels need to be
determined.
The implications of the recent European ban of antibiotic growth promoters
in animal diets on performance and health status have yet to be fully explored. It is
possible that this ban may result in decreased animal performance and exogenous
enzymes could be utilized to help to alleviate this.
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Enzyme Supplementation of
Poultry Diets Based on
Viscous Cereals
7
M. CHOCT
School of Rural Science and Natural Resources,
Department of Animal Science, The University of New England,
Armidale, NSW 2351, Australia
M. ChoctSupplementation of Poultry Diets

Enzyme
7
Introduction
Feed enzymes have come to be regarded by many nutritionists as being a necessary
‘ingredient’ for formulating poultry rations. This has occurred mainly since the
1990s, but the concept of enhancing animal performance using enzymes is by no
means new. For example, in the mid 20th century, various preparations of amylase
were used in an attempt to overcome poor performance of chicks fed barley diets
by increasing the availability of starch (Hastings, 1946; Fry et al., 1957). The early
work focused on the hydrolysis of specific substrates to their simple constituents
for absorption (Fry et al., 1957; Moran et al., 1968), but this approach was not
successful. Appreciable advances have since been achieved in the use of enzymes in
poultry diets with a clear understanding of the target substrates and the development
of microbiological technology. The prime example of this is the use of β-glucanases
in barley diets and xylanases (pentosanases) in rye or wheat diets.
Enhancing Bird Performance by Enzyme Supplementation

Barley
The macro-nutrient contents of barley and maize are very similar, but their nutritive
value for poultry is vastly different. This led scientists to use various treatments,
including enzyme supplementation (Hastings, 1946; Fry et al., 1957). These workers
used an α-amylase, which significantly increased the liveweight gain and feed
conversion efficiency (FCE) of chickens fed barley diets. It is now well established
that the starch in barley is totally digestible by the amylase secreted by chickens.
Therefore the reported improvements with amylase supplementation were probably
due to the impurities in the enzymes used, i.e. the crude enzyme preparation
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146 M. Choct
contained β-glucanase activity. β-Glucans are glucose polymers containing a mixture

of β1,3 and β1,4 linkages that make their physicochemical properties totally different
from cellulose that is a straight-chain glucose polymer with only β1,4 linkages. Barley
contains a high level of mixed-linked β-glucan (3–4%), which is responsible for
its poor nutritive value in chickens (Burnett, 1966). Since this significant finding,
there have been numerous studies on the use of enzymes, in particular β-glucanases
in barley-based poultry diets (Hesselman and Åman, 1986; Campbell et al., 1989),
with outstanding success. The enzymes increase growth performance and feed
conversion efficiency. Increases of up to 17% in liveweight gain (Broz and Frigg,
1986) and 19% in feed conversion efficiency (Newman and Newman, 1987) have
been reported for broiler chickens fed barley diets supplemented with β-glucanases.
Barley also contains an appreciable amount of soluble NSP other than β-glucans
and thus the majority of enzymes for barley diets have both β-glucanase and
arabinoxylanase activities.
Rye
The poor feeding value of rye was reported more than 60 years ago (Halpin et al.,
1936). In the search for an answer to the problem, Fernandez et al. (1973) extracted
rye grain with water and freeze-dried the extract. When this extract was added to a
maize-based diet, it depressed the growth of the birds and caused sticky droppings,
whereas the water-extracted rye was markedly better than normal rye. This
water-extractable factor is now known to be the soluble arabinoxylan (Fengler and
Marquardt, 1988a,b). Thus, addition of arabinoxylanases to rye-based broiler diets
significantly improves the growth performance and feed conversion efficiency
(Bedford et al., 1991; Bedford and Classen, 1992). Supplementation with increasing
levels (0.11, 0.22, 0.44 and 0.88 g kg−1) of an enzyme preparation having
arabinoxylanase and β-glucanase activities to a rye–wheat-based diet improved the
weight gain of broilers up to 27% and FCE up to 10% (Pettersson and Åman, 1988,
1989). Although enzymes always substantially improve the performance of birds fed
rye diets, they do not seem to have as large an effect on the litter quality.
‘Low-ME’ wheat
Although the large variability (up to 4 MJ kg−1 dry matter) of the nutritive quality of
wheat was reported by numerous researchers (Sibbald and Slinger, 1962; Schumaier
and McGinnis, 1967), the significance of the problem had not been widely
appreciated until recently. Connor et al. (1976) noticed that the AME value obtained
for wheats was 7–25% lower than that of sorghum, and Payne (1976) postulated that
some wheats may contain a ‘slightly toxic inhibitor’. Subsequently, two major studies
found that approximately 25% of the Australian wheats had AME values below
13 MJ kg−1 dry matter (Mollah et al., 1983; Rogel et al., 1987). When these
‘low-ME’ wheats are included at above 50% in the ration, chickens have sticky and
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Enzyme Supplementation of Poultry Diets 147
watery droppings accompanied by poor growth and feed efficiency. At very high
levels of inclusion (up to 90% of the diet), young broilers can only utilize a very
small portion of the energy (2.90 MJ kg−1) (Wiseman and McNab, 1998). It is now
generally conceded that the occurrence of low-ME wheats is due to an increased level
of NSP, in particular the arabinoxylan (Choct and Annison, 1990; Annison, 1991).
Choct et al. (1995) demonstrated that supplementation of a low-ME wheat diet with
a commercial glycanase preparation increased the AME by 24% and the FCE by
25% in 3–4 week old broiler chickens.
Mechanisms of Action
The term non-starch polysaccharide (NSP) represents a diversity of compounds
possessing different physicochemical properties; thus their nutritional effects in
poultry are also diverse and, in some cases, extreme. It is, however, generally
conceded that the detrimental effect of NSP in viscous grains is associated with
the viscous nature of these polysaccharides, their physiological and morphological
effects on the digestive tract and the interaction with the microflora of the gut. The
mechanisms include altered intestinal transit time and modification of the intestinal
mucosa, as well as changes in hormonal regulation due to a varied rate of nutrient
absorption. To elucidate the action of NSP in poultry diets, Choct and Annison
(1990, 1992a) isolated the cell wall polysaccharides from wheat and added them to
a semi-purified diet. Two fractions were prepared. Fraction 1 was extracted using
water only and hence named water-extractable arabinoxylans (WEP). Fraction 2 was
obtained by extraction with NaOH (0.2 mol l−1) and called alkali-extractable
arabinoxylans (AEP), which were also soluble in water. Addition of these soluble
arabinoxylan preparations at an equivalent of 30 g pure arabinoxylans kg−1 diet
depressed weight gain by 24.6%, feed conversion ratio by 11.2%, DM digestibility
by 8.4% and AME of sorghum by 9.9%. The anti-nutritive effects of wheat
arabinoxylans were also demonstrated in a practical type of broiler diet (Choct
and Annison, 1992a). The arabinoxylans severely depressed bird performance and
nutrient digestibilities when added at above 2% to the diet. Part of the data is shown
in Tables 7.1 and 7.2.
The wheat arabinoxylans caused a general inhibition of nutrient digestion
affecting starch, fat and protein, which indicated that they acted in a similar manner
as the anti-nutritive NSP of rye and barley. The arabinoxylans of rye have been
reported to depress fat, protein and DM retention (Ward and Marquardt, 1987;
Fengler and Marquardt, 1988b). Depressed ileal digestibilities of starch and protein
have been observed in chickens fed barley diets which had high levels of extractable
viscous β-glucans (Hesselman and Åman, 1986). Variability in the experimental data
increased as greater amounts of arabinoxylans were added to the diets. This mimics
the low-AME wheat phenomenon. When broilers were fed low-AME wheats, greater
between-bird variation was seen than when normal wheats were fed (Rogel et al.,
1987). Classen et al. (1988) demonstrated that supplementation of barley diets with
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148 M. Choct
Table 7.1. Effects of soluble arabinoxylans from wheat on AME, nitrogen retention (NR), feed
conversion ratio (FCR), weight gain (WG) and feed intake (FI) in broilers (n = 8). (Data from Choct
and Annison, 1992a.)
Arabinoxylan2 AME3 NR FCR WG FI

Diet1 (g kg−1 DM) (MJ kg−1 DM) (g d−1) (g : g) (g 6d−1) (g 6d−1)
Control 25.9 15.05a 3.05a 1.91ab 349ab 661a

WEP(20) 43.8 13.90b 2.79a 2.08ab 312ab 637a
AEP(5) 30.8 15.00a 2.89a 1.94ab 348ab 675a
AEP(10) 34.9 14.70a 2.86a 1.95ab 352ab 686a
AEP(25) 48.0 13.34b 2.42b 2.49bc 268bc 640a
AEP(40) 65.7 12.48c 1.96c 2.70cb 216cb 552b
SE (pooled) 0.22 0.13b 0.12bb 15b 19
1Valuesin parentheses are added arabinoxylans (g kg−1 DM).
2Determined values for total pentosans in diets.
3Columns sharing the same superscripts are not significantly different (P > 0.05).
WEP = water extractable arabinoxylans; AEP = alkali extractable arabinoxylans.
Table 7.2. Effects of wheat arabinoxylans on ileal digestibility coefficients of starch, protein and
lipid (n = 3). (Data from Choct and Annison, 1992a.)
Diet1 Arabinoxylan2 Starch3 Protein Lipid
Control 25.9 0.96ab 0.75ab 0.93ab

WEP(20) 43.8 0.91bb 0.70ab 0.87ab
AEP(5) 30.8 0.96ab 0.75ab 0.93ab
AEP(10) 34.9 0.95ab 0.73ab 0.92ab
AEP(25) 48.0 0.92ab 0.69ab 0.76ab
AEP(40) 65.7 0.82cb 0.61bb 0.69bb
SE (pooled) 0.02bb 0.03bb 0.06bb
1Valuesin parentheses are added arabinoxylans (g kg−1 DM).
2Determined values for total pentosans in diets.
WEP = water extractable arabinoxylans; AEP = alkali extractable arabinoxylans.
β-glucanase reduced the variability from 11.9 to 3.3% for weight gain and from 5.2
to 2.7% for FCR in broilers.
Viscous nature of soluble NSP
Increased bulk and viscosity of the intestinal contents decrease the rate of diffusion of
substrates and digestive enzymes and hinder their effective interaction at the mucosal
surface (Edwards et al., 1988; Ikegami et al., 1990). Studies in vitro with guar gum
showed that soluble, indigestible polysaccharides interact with the glycocalyx of the
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intestinal brush border, producing a thickening of the rate-limiting unstirred water

layer, which results in decreased nutrient absorption (Johnson and Gee, 1981).
Germination of barleys can lower the viscosity of their aqueous extracts and such
barleys are of better nutritive value in poultry compared with normal barleys (Fengler
et al., 1990). Barleys with high extract viscosity are more detrimental to chickens
than those with low extract viscosity (Campbell et al., 1989). These results lend
support to the hypothesis that viscosity is involved in the anti-nutritive effect of
NSP in poultry diets. Choct and Annison (1992b) compared the effects of a high
molecular weight NSP isolate (Intact-NSP) (854 g arabinoxylans and 42 g glucan
kg−1 DM; molecular weight = 758,000) and a partially depolymerized NSP isolate
(Depol-NSP; 194,000) (Annison et al., 1992), and pentoses on broiler performance
and nutrient digestibility (Table 7.3). When included at a level of 30 g kg−1 diet, the
partially depolymerized NSP increased digesta viscosity significantly compared with
pentose sugars added at the same level, but it was lower than that in birds fed intact
NSP. Bird performance was not significantly affected by the depolymerized NSP,
indicating that birds can tolerate small increases in digesta viscosity without a
detrimental effect on performance.
The anti-nutritive effects of the arabinoxylans appear largely dependent on the
polymeric nature of the polysaccharides and their viscous property. This is supported
by studies demonstrating that supplementation with enzymes capable of degrading
β-glucans and arabinoxylans increases the nutritive value of barley and rye in poultry
diets and this coincides with a significant decrease in the digesta viscosity of the
chickens (White et al., 1981; Bedford et al., 1991).
Watery and sticky droppings alone may not be a sufficient indicator of the
anti-nutritive effect of the arabinoxylans in poultry, as excreta from birds fed the diet
containing the depolymerized arabinoxylans were watery and sticky, and those from
birds given the diet containing pentose sugars also appeared more moist compared
with controls. In the rat, dietary components that are not completely digested or
absorbed in the small intestine give rise to an increased amount of osmotically active
materials in the gut content. If these materials are utilized by the hindgut bacteria,
Table 7.3. Effects of high molecular weight NSP, partially depolymerized NSP and pentoses on
weight gain (WG), feed conversion ratio (FCR), feed intake (FI) and AME in broilers (n = 8). (From
Choct and Annison, 1992b.)
Level added Digesta WG FCR FI AME

Diet (g kg−1)2 viscosity (g wk−1)3 (g : g) (g wk−1) (MJ kg−1 DM)
Control 0 1.2a 430ac 1.589ac 681 16.13a

Intact-NSP 30 3.0c 325bc 1.960bc 622 14.53b
Pentoses 30 1.2a 394ac 1.695ac 658 16.23a
Depol-NSP1 30 2.2b 404ac 1.649ac 661 15.74a
1Partially
depolymerized NSP.
2Levelsadded at equivalent to pure NSP.
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150 M. Choct
production of low molecular weight metabolites that are not readily absorbed may
result. This can lead to further accumulation of osmotically active substances which
attract a large amount of water. This may well relate to the inefficient utilization of
five-carbon sugars by chickens (Wagh and Waibel, 1966). The significant improve-
ment in performance of birds fed barley or rye supplemented with β-glucanase or
arabinoxylanase is not due to a complete hydrolysis of the polysaccharides and a
subsequent absorption of the released sugars (White et al., 1981), but is due to the
depolymerization of the polysaccharides into smaller polymers which do not greatly
elevate the viscosity of digesta (De Silva et al., 1983).
Modification of secretory response of the gut
Viscous polysaccharides cause physiological and morphological changes to the

digestive system of rats, pigs and humans (Brown et al., 1979; Cassidy et al., 1981;
Morgan et al., 1985; Ide et al., 1989; Low, 1989). The endogenous secretion of
water, proteins, electrolytes and lipids can be increased markedly by NSP
supplementation of the diet (Low, 1989). The metabolic cost of such processes can
be considerable. Prolonged consumption of diets containing viscous polysaccharides
is associated with significant adaptive changes in the digestive system in rats (Ikegami
et al., 1990). The changes in the gastrointestinal tract are characterized by enlarge-
ment of digestive organs and increased secretion of digestive juices, accompanied by
decreases in nutrient digestion. The depressed apparent ileal protein digestibility
caused by the wheat arabinoxylans, therefore, may be due to an inhibition of protein
breakdown and/or a reduction in amino acid absorption. It may also result from an
increase in the secretion of endogenous proteins, which can be derived from gut
secretions and losses of intestinal cells. In the following study (Angkanaporn et al.,
1994), the effect of soluble wheat arabinoxylans on endogenous protein (sum of
amino acids) secretions, in comparison to that of pure cellulose and an inert nutrient
diluent (polythene powder), was investigated. Endogenous and exogenous amino
acids were distinguished using the homoarginine marker technique (Hagemeister
and Erbersdobler, 1985). The apparent protein digestibility was depressed to a
similar extent by addition of wheat arabinoxylans at levels of 15 and 30 g kg−1, but
the true protein digestibility was significantly inhibited only at the higher level of
inclusion. This indicates that the anti-nutritive effect of wheat arabinoxylans on the
apparent protein digestibility is mainly due to increased endogenous secretions
of amino acids at low levels of inclusion, while at high levels a direct inhibition of
protein breakdown and/or absorption occurs. Addition of cellulose and polythene
did not have an effect although the levels of inclusion were much higher than that of
wheat arabinoxylans (Table 7.4).
The biological effects of NSP differ considerably due to their diverse physio-
chemical properties. Southon et al. (1985) reported that a diet containing 75 g
non-cellulosic polysaccharides kg−1 and 24 g cellulose kg−1 fed to rats induced higher
rates of protein synthesis in the jejunum and ileum, and more rapid mucosal cell
division than rats given a semi-purified diet containing cellulose as the only source of
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Table 7.4. Effects of wheat pentosans, cellulose and polythene on apparent (AP) and true
protein (TP) digestibility coefficients and endogenous protein secretions (EP); g kg−1 DM intake
(n = 2). (From Angkanaporn et al., 1994.)
Pentosan
Cellulose Polythene
Diet Control 15 g kg−1 30 g kg−1 92 g kg−1 92 g kg−1
AP 0.770a 0.595b 0.600b 0.701a 0.695a

TP 0.969a 0.946a 0.885b 0.955a 0.966a
EP 30.2a 53.9b 40.2a 37.4a 34.9a
Rows sharing the same superscripts are not significantly different (P > 0.05).
NSP. The lack of action of cellulose on the endogenous amino acid secretions was
further demonstrated in chickens by Parsons (1984), who found that when a
nitrogen-free diet containing 400 g raw potato starch kg−1 and 100 g pectin kg−1
was fed to laying hens, the total endogenous amino acid secretion was increased by
40%; whereas the diet containing 500 g cellulose kg−1 elicited little effect. These
observations indicate that the action of NSP in modification of the physiological
functions of the gastrointestinal tract is not a mere mechanical stimulation of the
mucosa. It is possible that the soluble NSPs interact with the gut wall in a way that
modifies the endocrine regulation.
Encapsulated nutrients
Although the insoluble NSPs have mainly been regarded as a nutrient diluent in the
diet, they can also affect digesta transit time and gut motility. Another facet of the
role of insoluble NSP in poultry diets that is worthy of reiteration is the possibility
of them acting as a physical barrier to digestive enzymes, such as amylase and
proteases, thus reducing their efficient digestion in the upper part of the gut. This
is probably particularly important in non-viscous grains, such as sorghum. There is
evidence that enzymes with affinity for insoluble NSP can also elicit a positive
response in growth performance of broilers (Cowan, 1995; Choct, 1998). This
indicates that breakdown of cell wall matrix, especially the insoluble components,
may facilitate easier access of digestive enzymes to their substrates within the short
feed transit time in birds. Wiseman and McNab (1998) also showed that the rate of
in vitro starch digestion correlates closely with the AME values in wheat, indicating
that the accessibility of amylotytic enzymes to starch granules is vastly different.
NSP and gut microflora
The gut harbours a highly evolved and complex microbial ecosystem containing a
vast number of diverse populations. For example, microbes make up approximately
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152 M. Choct
600 g kg−1 of the wet weight of poultry excreta. The proper feeding of poultry should
therefore consider the provision of ‘correct’ substrates for the microflora to keep
it stable. The consequences of altered rate of nutrient digestion in the gut may
be manifested in the number and type of microorganisms present in the gut
(Vahjen et al., 1998). An increased amount of NSP in the digesta has been
demonstrated to influence the gut microflora in a negative manner to birds (Choct
et al., 1996). The overall digestibilities of protein and long-chain fatty acids,
especially the saturated ones, were more severely depressed in intact birds than in
caecectomized birds when wheat arabinoxylans were added to the diets (Choct et al.,
1992). Bile salts are essential for emulsification of fats and activation of lipase (Tso,
1985), especially for saturated fatty acids (Johnston, 1977) as opposed to short-chain
and unsaturated fatty acids, which are more easily absorbed in the absence of bile
acids (Garrett and Young, 1975). Excessive deconjugation of bile salts is a possible
deleterious effect of gut microflora (Feighner and Dashkevicz, 1988) and it may
be exacerbated by the presence of large amounts of undigested carbohydrates
in the hindgut. This is supported in studies by Campbell et al. (1983) where
supplementation of the conjugated bile salt, sodium taurocholate, to chickens fed rye
diets significantly improved the fat retention. Furthermore, utilization of nutrients
through microbial conversion of digestible carbohydrates, such as starch, to VFAs is
not efficient compared with a direct absorption of glucose released from enzymatic
digestion (Müller et al., 1989; Carré et al., 1995). In a recent study, Choct et al.
(1996) demonstrated that addition of soluble NSP in broiler chicken diets
significantly elevated fermentation in the small intestine. Subsequent in vivo
depolymerization of the soluble NSP using glycanases almost totally overcame
this problem.
Another factor that may contribute to the negative effect of a high microbial
load in the small intestine is increased turnover of intestinal cells. According to
LeBond and Walker (1956), a 100 g rat gaining 5 g a day synthesizes 1 g mucosal
cells daily, which represents a 20% additional tissue synthesis without showing
up as weight gain. Extrapolating this to a 2 kg bird gaining 60 g daily, the bird
would synthesize 12 g of mucosal tissue to maintain the integrity of its small
intestine. Increased microbial load can exacerbate this loss (Abrams et al., 1963;
Lesher et al., 1964), since some of their fermentation products, e.g. putrescine,
have been shown to enhance small intestinal and colonic mucosal growth rates
significantly (Seidel et al., 1985; Osborne and Seidel, 1989). Furthermore,
antibiotics can significantly improve performance of birds fed high-NSP diets
(MacAuliffe and McGinnis, 1971; Misir and Marquardt, 1978a,b). Whatever the
reason, excess fermentation in the small intestine of the chicken may be detrimental
to nutrient digestion and absorption and enzyme supplementation seemed to
alleviate this problem. Perhaps reducing the viscosity of the digesta in the small
intestine hastens digesta passage and nutrient digestion rate (through removal of
the diffusional constraint of viscous gums), thereby giving less substrate and time
for the fermentative organisms to proliferate. This may in turn restore the
normal and efficient digestion (enzymatically) of starch and protein in the small
intestine.
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Predicting Enzyme Response

The concept of predicting the effect of enzymes in a particular feed is attractive
because the producer could then adjust the amount of enzymes to be added or adjust
the nutrient specifications in diet formulations. Although it is extremely difficult to
predict the specific effects of enzymes accurately in practice, some approaches have
proved to provide useful estimates of enzyme responses in viscous grains such as rye,
barley and wheat.
The NSP content of the diet
The negative correlation between NSP content of the diet and its nutritive value has
been clearly demonstrated in poultry (Choct and Annison, 1990; Annison, 1991), in
pigs (King and Taverner, 1975) and in pet dogs and cats (Earle et al., 1998). This
indicates that by measuring the NSP content of a diet, it is possible to predict the
amount of enzymes to add to the diet. However, the practicality of such an approach
is questionable. Firstly, a least-cost diet contains a number of plant ingredients,
which contain different forms of NSP. It is no way to know the amounts and types of
different NSP substrates present in a diet by a quantitative measurement of the NSP
level. Thus the responses to various types of enzyme activities added to the diet are
difficult to predict. Secondly, the NSP assay is tedious and complicated and, hence, is
not ideal for use in quality control laboratories. Thirdly, the anti-nutritive effect of
soluble NSP is related, to a large extent, to the viscous nature of the polymers, which
in turn depends on their molecular sizes and structures. Again a qualitative or a
combination of qualitative and quantitative measurements is required.
Gut viscosity
The relationship between gut viscosity and the nutritive value of barley in poultry
was first described by Burnett (1966). Only during the past 10 years has the impor-
tance of his work been recognized and a wealth of information on viscosity of the gut
contents and its effect on nutrient digestion and absorption has since emerged (Bed-
ford et al., 1991; Bedford and Classen, 1992; Choct and Annison, 1992a; Steenfeldt
et al., 1998). Whether gut viscosity can be used to predict enzyme response depends
on a number of factors, including the section of the intestine where the digesta is
sampled, the age of the bird, and antibiotic growth promoters in the diet. Firstly,
different sections of the gut will influence the viscosity value, because nutrients are
digested and absorbed as digesta moves down the gut, thus leaving the indigestible
portion (mainly NSP) to accumulate. On a relative basis, the soluble NSP content
per ml of digesta supernatant therefore increases; secondly, digesta viscosity decreases
as the bird gets older (Dusel et al., 1998; Steenfeldt et al., 1998). This also relates to
the finding that older birds cope better with low-ME wheats (Rogel et al., 1987) or
with barley (Salih et al., 1991). In addition, the relationship between gut viscosity
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154 M. Choct
and bird performance is not always apparent (Wiseman and McNab, 1998). For
example, Hughes and Zviedrans (1999) reported that two groups of birds fed wheat
had ileal viscosity value (mPa s) of 12.5 and 49, but the AME contents (MJ kg−1
DM) for the wheat were 10.6 and 12.0. This does not necessarily mean that the
viscosity has no effect on nutrient digestion and absorption; rather, it highlights the
complex nature of the interaction between digesta viscosity and the gut microflora. It
is hypothesized that as the bird ages, its gut microflora becomes more established and
it can adapt to the environment – for example, to cope with the viscous digesta better
by producing small amounts of NSP-degrading enzymes (Choct and Kocher, 2000).
This highlights the third point: that addition of antibiotic growth promoters in the
diet can have a major effect on gut viscosity and hence enzyme response. Fourthly, as
discussed earlier, removal of cell wall barriers using enzymes with an affinity for the
insoluble NSP can also benefit the bird by releasing encapsulated nutrients. The
response of such an action cannot be predicted from gut viscosity measurements.
Generally, gut viscosity values in birds fed viscous grains can be used to predict
the response to enzyme supplementation with strong viscosity-reducing property
within a grain type under set conditions. However, measuring gut viscosity is neither
rapid nor cheap.
Extract viscosity
A ground sample of ingredients or compound feed can be extracted in water or in a

buffer to measure extract viscosity. The viscosity of the extract comes predominantly
from soluble NSP in the feed (Izydorczyk et al., 1991; Saulnier et al., 1995). Since
the soluble NSP content of a diet is related to its nutritive value, it is possible
that extract viscosity can be used to predict enzyme response. Rotter et al. (1989)
demonstrated that the growth responses of chickens fed barley diets were accurately
predicted by an extract viscosity method. Choct et al. (1993) developed an extract
viscosity assay to distinguish wheats with very low AME from normal samples. More
systematic studies with promising outcomes have since been reported on the use of
extract viscosity as a predictor of nutritive value for poultry (Carré and Melcion,
1995; Choct and Hughes, 1997; Dusel et al., 1997, 1998; Wiseman and McNab,
1998). The extract viscosity method is simple and rapid and, more importantly, it
gives qualitative information on the characteristics of the NSP. However, since the
extract viscosity assay is based on the amount of soluble NSP content of the ingredi-
ent, it can only be used for predicting the response of viscosity-reducing enzymes. In
addition, the reliability of the assay may be influenced by the extraction method and
the endogenous enzyme activities in the diet or the individual ingredient.
Rate of starch digestion
Wiseman and McNab (1998) used an in vitro method to measure the rate of starch
digestion by amylase. They demonstrated a close correlation between in vitro and
164
17 November 2000 15:06:12

in vivo starch digestion for wheat (R2 = 0.65) to distinguish between wheats with
high and low metabolizable energy values in poultry. With validation, the assay may
be used to characterize wheat for poultry diets; thus it offers potential as a means of
predicting enzyme response.
Conclusion
The use of xylanase for wheat and β-glucanase for barley in poultry is a well accepted
practice today. Generally, most of the enzymes effectively depolymerize the soluble
NSP into smaller polymers, though some products with affinity for both soluble and
insoluble NSP are also used. The soluble NSPs elicit anti-nutritive activities in
poultry diets, which are closely related to their polymeric nature and ability to
increase digesta viscosity. Extract viscosity has great potential to be used as a simple
and rapid method to predict enzyme response in poultry diets. Other assays such as
the in vitro starch digestibility measurement may prove useful. Further studies on
NSPs need to address their effect on nutrient utilization in terms of relative efficiency
of enzymatic digestion vs. fermentation, and muscle growth vs. gut cell turnover.
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The Role and Efficacy of

Carbohydrase Enzymes in
Pig Nutrition
8
G.G. PARTRIDGE
Finnfeeds, PO Box 777, Marlborough, Wiltshire SN8 1XN, UK
G.G. PartridgeEnzymes in Pig Nutrition

Carbohydrase
8
Introduction
The history of use of carbohydrase enzymes into pig diets based on ‘viscous’ grains
such as wheat and barley is virtually as long as that of poultry but, until recent years,
far less convincing. There are many reasons for this, and certainly not all are related
to issues of cost effectiveness, which is always the ultimate arbitrator in the adoption
of any new feed technology. The basic structure of the pig and poultry industries
in many countries (i.e. ‘highly integrated’ versus ‘semi-extensive’, respectively) has
tended to work against the rapid adoption of novel feed technologies into the pig
sector. This situation is changing rapidly in swine markets in some parts of the world
and, in terms of the potential rapid uptake of new ideas, will undoubtedly be a move
for the better.
This chapter examines the role and efficacy of carbohydrase enzymes in
grain-based diets. Phytases and enzymes directed against vegetable protein meals are
assessed elsewhere in this book. It is recognized that, at the time of writing, the
penetration of feed enzymes into the pig sector still lags behind that of poultry but
that knowledge has accrued rapidly, particularly in the past 5 years, bringing both
more effective enzymes for pig applications and considerably more applications
expertise. Due to this rapid change the emphasis in this chapter is placed on the last
10 years of published information.
Enzyme Responses: Pigs vs. Poultry

The physiological reasons why differing responses to exogenous carbohydrases might
be expected in pigs and poultry have been covered by a number of authors on a
number of occasions, and for this reason will only be summarized here. Readers are
referred to a number of excellent papers that have been systematically published over
the past 10 years on these and other aspects of enzyme use into pig diets. A number
of these compared and contrasted responses in poultry and pigs (e.g. Dierick, 1989;
171
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162 G.G. Partridge
Chesson, 1993; Dierick and Decuypere, 1994, 1996; Graham and Balnave, 1995;
Bedford and Schulze, 1998; Danicke et al., 1999).
The main factors that may influence the potential for enzyme response in pigs
compared with poultry include gastrointestinal anatomy, digestive capacity and
digesta characteristics.
The presence of the crop in the broiler may, depending on the pH profile of
the exogenous enzyme, allow for some enzyme activation at a relatively high pH
(approximately 6) prior to passing into the acid environment of the gizzard.
Generally in the pig a much lower pH (< 3) will be more rapidly experienced by the
enzyme and its substrates in the stomach, though this will be highly dependent upon
pig age, the degree of ingredient buffering, and the extent of microbial fermentation
in the stomach leading to organic acid production (e.g. lactic acid). The pig is noted
(Moran, 1982) for its relatively gentle gastric motility, leading to regional variations
across the stomach in terms of pH and endogenous enzyme activity. All of these
factors can potentially impinge on the activity of an exogenous enzyme, depending
on its pH and temperature characteristics and its inherent rate of reaction (Km).
Mean retention times (MRTs) of solid and liquid digesta in poultry also tend
to be reduced relative to that of the pig (Moran, 1982). Solute markers in the upper
part of the gastrointestinal tract of broilers showed mean retention times in the crop,
gizzard and small intestine of 2.8, 0.3 and 1.0 h, respectively (i.e. about 4 h total to
the end of the small intestine: Dierick and Decuypere, 1994). In contrast, Dierick
and Decuypere (1994) cite a total MRT of 4–5 h through the stomach and small
intestine of the pig and Mahan (1982) quotes studies showing approximately 7.5 h
through this same part of the gastrointestinal tract. The absolute figure will clearly
vary in both species depending on diet composition, soluble/insoluble fibre level,
plane of feeding, age, etc., but the fact remains that the broiler, having less time than
the pig for effective digestion and nutrient absorption, will be potentially disrupted
more by certain anti-nutritional factors that interfere with this process (e.g. viscous,
high molecular weight soluble fibres). This may in turn have implications for enzyme
action and the relative magnitude of its effects in the two species, but it should
equally be remembered that both species lack the appropriate enzymes to break down
these anti-nutrients, irrespective of the length of the digestive tract and residence
time. Both species, therefore, will potentially suffer the same consequences of
disturbed digestion – not only less nutrients absorbed but also a rise in bacterial
numbers in the small intestine, resulting in increased competition for nutrients
between the host and its microflora.
The greater bacterial proliferation in the gut of the pig, compared with the
broiler, is often quoted as a reason why the magnitude of exogenous enzyme response
might be expected to be lower in the pig. Certainly in the pig’s hindgut the long
residence time and voluminous caecum and colon offer an ideal opportunity for
bacterial fermentation of fibrous residues to yield substantial quantities of volatile
fatty acids, which will contribute to the animal’s maintenance requirement. In the
small intestine, however, as mentioned above, such proliferation will always be a
potentially negative factor. Caution should also be observed in interpretation of some
studies reporting substantial breakdown of fibre fractions in the small intestine of
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Carbohydrase Enzymes in Pig Nutrition 163
the pig in the absence of exogenous enzymes. Some of these studies used cannulated
pigs with T-pieces inserted at the terminal ileum and, occasionally, the duodenum
(Graham et al., 1986, 1988). The opportunities for an unnatural microbial
colonization in the region of the cannula could influence the results seen.
It is well known that soluble, high molecular weight, non-starch polysaccharides
(NSPs) from viscous cereal grains interfere with nutrient digestion and absorption in
poultry, with consequences for microbial proliferation in the gut (Apajalahti and
Bedford, 1998; Bedford and Schulze, 1998). The naturally higher water content of
pig digesta (about 10 percentage units higher than poultry on similar diets) leads to
dilution effects that will negate, to some degree, this viscosity problem in the pig’s gut
(Fig. 8.1) (Danicke et al., 1999). This is not to say that, in pigs, viscosity effects are
totally irrelevant to potential carbohydrase responses in viscous grain diets, but rather
that they are likely to be of a lower order of importance to the responses seen in
broilers. Reductions in digesta viscosity have been seen in many trials in pigs offered
viscous cereal-based diets (Fig. 8.2) but it is clear that starting viscosity levels are
always considerably lower than equivalent trials in poultry. It is of interest, however,
that soluble fibre levels per se seem to be the key factor involved in negative bacterial
changes in the gut of pigs under disease challenge and fed specific types of diet, as
reviewed later in this chapter, so viscosity should certainly not be underestimated
in the pig as an influence on productive performance. It should be recognized,
therefore, that its influence may be more subtle than direct effects on nutrient
digestibility and, rather, mediated indirectly through its effects on the gut microflora.
Digesta dry matter flow rates from the stomach of young and grower pigs fed
wheat or barley-based diets have been found to be increased by certain exogenous
enzymes (Sudendey and Kamphues, 1995) (Fig. 8.3). In these trials, viscosity
reduction was apparent immediately in the stomach of the animals offered enzyme
supplemented diets. Ellis et al. (1996) noted that an increase in gastric viscosity in
8.7 36.3
5.7
2.7 2.4 11.0

1.8 1.7 6.0
3.8 3.0
1.8
Wheat var. 1 Wheat var. 2 Wheat var. 1 Wheat var. 2

Jejunum Ileum*
Fig. 8.1. Effect of wheat variety on intestinal viscosity (mPa s) in broilers, turkeys
and piglets (adapted form Danicke et al., 1999). % Broilers (3 weeks) wheat
inclusion 73%; $ turkey (4 weeks) wheat inclusion 73%; ( pig (8 weeks) wheat
inclusion 86%. * = small intestine, lower two-thirds for the pig. Wheat variety
1 = ‘Ibis’ 11 g kg−1 DM soluble arabinoxylan; wheat variety 2 = ‘Alidos’
17 g kg−1 DM soluble arabinoxylan. DM = dry matter.
173
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164 G.G. Partridge
6.4
4.2
3.3
3.1*
2.2* 2.2 1.8(*)
1.7 1.5* 1.7*
Ref. 1 Ref. 2 Ref. 2 Ref. 3 Ref. 4
Control +Enzyme complex *P < 0.05 (*)P < 0.10

Fig. 8.2. Effects of enzymes on viscosity in the small intestine of piglets.
Ref. 1. Inborr (1994); ref. 2. Sudendey and Kamphues (1995) wheat/piglet and
barley/grower; ref. 3. Dusel et al. (1997); ref. 4. Partridge et al. (1998a).
(*) 3.36 *
3.22
2.62 2.26
2.27
1.70
Wheat, piglet Barley, piglet Barley, grower

Fig. 8.3. Effect of enzyme addition to wheat or barley-based diets on dry matter
outflow rate from the stomach (Sudendey and Kamphues, 1995). Values are g dry
matter leaving the stomach h−1 kg−1 body weight. ( Control; % +Enzyme complex
(amylase, xylanase, β glucanase). *P < 0.05, (*)P < 0.10.
the pig can lead to a failure of the sieving mechanism whereby large digesta particles
normally fall to the bottom of the stomach, a feature seemingly unique to the pig. In
a viscous environment these large particles stay suspended and are thereby more
likely to exit the pylorus before being exposed to gastric secretions or particle size
reduction, which may have implications for subsequent digestion. Further down the
gastrointestinal tract, viscosity is also known to disturb peristalsis, creating laminar
rather than disturbed flow, and also to lead to disrupted pancreatic secretion via its
effects on gastric inhibitory polypeptide. Viscous non-starch polysaccharides may
also physically coat starch granules, further reducing the rate of digestion (Ellis et al.,
1996).
In successful pig trials using enzyme supplementation, a stimulation of volun-
tary food intake is frequently an important contributor to the observed improve-
ments in daily liveweight gain (Haberer et al., 1998). The fact that gastric emptying
rate and distension of the stomach are two of a number of factors involved in satiety
signals in the pig (Forbes, 1995) is unlikely to be coincidental and it is proposed that
some of the positive responses to exogenous enzymes on voluntary food intake could
be due to various influences on digesta flow rate, as well as improvements in nutrient
availability and digestibility in the small intestine. Feedback loops involving
gut hormones could also be contributors to carbohydrase effects, as outlined by
Bedford and Schulze (1998), who proposed that caecal fermentation, enhanced
174
17 November 2000 15:07:33

Fig. 8.4. The effect of water-holding capacity of the feed on voluntary food intake
in pigs (12–25 kg liveweight). Scaled feed intake = g feed kg−1 liveweight day−1
(Kyriazakis and Emmans, 1995).
by breakdown products from xylanase addition (fermentable oligomers), could

influence enteroglucagon concentrations, which in turn influence stomach motility
by depressing gastrin concentrations. These many possibilities suggest exogenous
enzymes could be potent influencers of gastrointestinal physiology through a variety
of different mechanisms, but these aspects have been little studied to date.
The relatively slow passage rate of digesta through the gut of the pig, when
compared with the broiler, may offer some advantages to exogenous enzymes when it
comes to the opportunity for insoluble cell wall breakdown, or thinning, coupled
with physicochemical changes to fibre (e.g. reduced water-holding capacity) as
digesta makes its way down the small intestine. Kyriazakis and Emmans (1995)
found that the water-holding capacity of feedstuffs for young pigs (12–25 kg) was a
major influence on voluntary food intake (Fig. 8.4). As carbohydrase enzymes are
known to reduce the water-holding capacity of feedstuffs in vitro (Fig. 8.5), it follows
that some of the potential benefits of exogenous enzyme addition in the pig may
be related to disruption of water and soluble nutrient entrapment, by both soluble
and insoluble fibre residues in the gut. Whilst the importance of soluble, viscous
polysaccharides to digestive disruption in the broiler seems to be a major mode of
action in wheat and barley-based diets (Bedford and Schulze, 1998), in the pig its
importance, as discussed, is clearly less profound. The conclusion must be that a
broader range of mechanisms is likely to be relevant to the pig, not least the ability of
an exogenous enzyme to influence both soluble and, more particularly, insoluble
fibre in the gut. This has implications for product design, as enzymes will differ in
their affinities and rates of reaction against various fibre structures and it follows that
175
17 November 2000 15:07:55

166 G.G. Partridge
−12% −29% −31% −19% −12% −17% −52% −8% −14% −5% −75%
10
0
ze
ur
rm
er
s
ea
ea
rle
at
ke
ea
SB
flo
w
ai
ge
O
fla
Ba
lo
Br
M
at
nf
s
ze
to
s
ee
he
Su
ra
ai
ta
es
W
G
M
Po
ap
R
Fig. 8.5. Effect of an enzyme complex on water-holding capacity (g g−1) of raw
materials. ( No enzyme; % +Enzyme complex (xylanase; β-glucanase; protease).
(Finnfeeds, personal communication, 1995.)
enzymes designed for maximum efficacy in the pig may involve both different levels
and/or different sources than those ideal for poultry. Xylanases, for example, even
derived from the same source organism, can vary widely in their catalytic activities on
various xylan substrates (Bedford and Schulze, 1998) and the active concentration of
xylanase will also be an important determinant of the extent of cell wall hydrolysis
(Tervila-Wilo et al., 1996).
Such issues become particularly important as commercial emphasis switches
towards carbohydrase products designed for non-viscous grains such as maize
and sorghum, which are the predominant cereals globally. In these grains, factors
associated with insoluble fibre such as the packaging of nutrients inside cell wall
material, together with starch structure and composition, seem to be more relevant
and may require a new range of enzyme solutions.
The rest of this chapter will systematically consider the major grain and
grain by-product sources available to pigs globally, examine their feeding value, its
variability and reasons for it, and then review recent work on their potential for
nutritional upgrading with appropriate exogenous enzymes.
Barley as a Grain Source for Pigs

The feeding value of conventional (hulled) barley for pigs was reviewed by
van Barneveld (1999). Estimates of DE in 16 cited studies ranged from 11.7 MJ kg−1
dry matter (DM) to 16.0 MJ kg−1 DM. Energy digestibility, rather than gross
energy content (a range of about 2 MJ kg−1 DM), had the greatest influence on this
value. In the recent studies of Fairbairn et al. (1999) the DE content of 20 barley
samples, from three locations and representing five varieties, ranged from 11.2 to
13.1 MJ kg−1 (90% DM), with less variation between varieties than within varieties
176
24 November 2000 15:53:48

(5.8%, 0.7 MJ kg−1 versus 8.4%, 1.0 MJ kg−1). This re-emphasizes the importance
of growing conditions to proximate analysis, carbohydrate composition and,
ultimately, feeding value for both swine and poultry (Aman et al., 1985; Aman
and Newman, 1986; Bach Knudsen et al., 1987).
The last decade has seen an increasing interest in the potential of hull-less barley
as a feed ingredient for monogastrics. Hull-less barley differs from conventional
barley grain in that the hull is less firmly attached to the kernel and consequently
becomes detached during threshing. Despite, consequently, having lower insoluble
fibre levels and higher levels of protein, its feeding value for pigs and poultry has
often failed to live up to its theoretical potential (Baidoo et al., 1998b; Thacker,
1999). The reasons for this are most likely associated with the soluble fibre fractions
(particularly β-glucans), which are concentrated in the endosperm of the barley
grain. Removing the hull fraction effectively concentrates these soluble components
in hull-less barley, with comparative levels of β-glucans in the two grains being
3.5–4.5% (hulled) versus 4.5–7% (hull-less) (Baidoo and Liu, 1998). The negative
effects on growth seen after feeding micronized, hull-less barley to growing/finishing
pigs are almost certainly due to further solubilization of this fibre during thermal
processing of the grain, effectively negating the potentially positive effects of the
process on starch gelatinization (Thacker, 1999). The latter study re-emphasized
the limitations of measuring total tract digestibility in the pig in isolation from
performance studies, whereby ‘apparent’ improvements in digestibility of nutrients
can be more than offset by reduced voluntary food intake coupled with implications
for microbial proliferation/competition for nutrients in the gut. This aspect will be
examined in more detail later in this chapter.
Differences in feeding value for hulled and hull-less barley varieties will also
undoubtedly be influenced by other components of the grain apart from fibre,
notably the amylose : amylopectin ratio of the starch fraction. Pettersson and
Lindberg (1997) investigated ileal and faecal digestibility of hull-less versus hulled
barleys, with varying amylose : amylopectin ratios, in growing/finishing pigs. They
found that the poorest energy and starch digestibility was in hulled/high amylose
grain and the best in hull-less/high amylopectin, at both the ileal and faecal levels.
Hull-less barley tended to give higher nutrient digestibilities overall and showed
increased hindgut digestion compared with hulled material. This would presumably
have implications for rate and pattern of microbial fermentation, but these were not
investigated in this study. Equally, productive performance was not measured on the
different barley types to see if the digestibility differences seen were actually reflected
in growth, feed intake and feed utilization. Miller et al. (1994) also described
digestibility experiments looking at the interaction between fibre (ADF – acid
detergent fibre) level in barley, β-glucan content and amylose : amylopectin ratio.
They concluded that the energy content of barley for both poultry and swine was
decreased greatly by ADF content and to a significant but lesser extent by higher total
β-glucans. They also found that β-glucans had a larger negative effect on energy
digestibility in poultry than in swine and that high amylopectin (‘waxy’) barley starch
offset some of the reductions in energy digestibility caused by a high ADF level. This
illustrates the complex interrelationships between various fractions in the grain
177
17 November 2000 15:08:32

168 G.G. Partridge
which can all have potential implications for feeding value and, importantly to this
discussion, subsequent response to exogenous enzymes.
Response of Hulled and Hull-less Barley to Exogenous

Enzymes in Pigs
The surface of the aleurone cell walls of barley contains high levels of xylan,
surrounding a core of mixed-linked β-glucans (Autio et al., 1996). For this reason,
optimal responses in rations based on barley seem to be found when both xylanase
and β-glucanase activities are supplied. This is illustrated in the studies of van
Lunen and Schulze (1996a) and Ramaswamy et al. (1996) where clear responses,
particularly in hulled barley, are only achieved in the presence of significant xylanase
activity. Some studies using exogenous enzymes in hulled barley-based rations
(Graham et al., 1989; Thacker et al., 1992b) have tended to emphasize the
β-glucanase component for reducing levels of soluble β-glucans, which are well
known to exacerbate digesta viscosity problems in broilers. In the pig these effects are
believed to be of a much lower magnitude, due to pig digesta having a much lower
dry matter content than the broiler (Bedford and Schulze, 1998), effectively diluting
the effects of these viscous fibres and partially negating their detrimental effects on
nutrient digestion and absorption. However, viscous fibre can still be relevant for its
impact on the microbial population of the gut and thereby indirectly influence
enzyme response, an aspect that will be examined in more detail later in this chapter.
Table 8.1 shows a summary of a number of published trials over the past
10 years into both hulled and hull-less barley-based diets, using a range of enzyme
additions. Although an overview is made more difficult by potential influences
of a number of factors (e.g. growth versus digestibility trials; faecal versus ileal
digestibility measurements; pig age/weight used; choice of enzyme activities;
pelleting versus meal/mash, i.e. potential for denaturation with some products;
hulled versus hull-less barley), some trends do emerge.
Responses to these various products in young pigs (< 25 kg) are strong and
quite consistent, particularly in the hull-less barley-based diets that have tended to
dominate recent publications. Baidoo et al. (1998a) describe a declining response in
pigs over the weight range 40–60 kg but an increasing numerical response to the
enzyme used as animals moved from the 9–20 kg weight range (daily gain +12%;
feed : gain −3%) to the 20–40 kg weight range (daily gain +17%; feed : gain −10%).
It should be noted that even in this trial the animals in the heaviest weight category
(40–60 kg) showed improvements in both growth and feed : gain (+4%; −12%,
respectively) after addition of the enzyme but that the large standard errors associated
with the data meant they failed to achieve statistical significance. Data from studies
with grower/finisher pigs (> 25 kg) range from no response or small responses
(Graham et al., 1989; Thacker et al., 1992a,b – but see comment above) to those
where clear, significant effects on digestibility and/or growth rate were seen (van
Lunen and Schulze, 1996a; McCracken et al., 1996; Ramaswamy et al., 1996;
Baidoo et al., 1998a,b). Even in some apparent ‘no-response’ trials (Thacker et al.
178
17 November 2000 15:08:33

1992a) significant, positive effects on faecal digestibility of protein and numerical

effects on energy were seen after enzyme addition. This is a reminder, particularly in
trials with grower/finisher pigs, that products potentially liberating more nutrients
must be tested in conditions that allow this nutrient release to be expressed in terms
of improved energy utilization, lean gain, etc. In trials where the products are added
on top of an existing formulation, which may already be in excess of an animal’s daily
nutrient needs for its lean protein deposition potential, carcass information is a
fundamental requirement to aid trial interpretation.
The studies of Baidoo et al. (1998a), McCracken et al. (1996) and Han and
Froseth (1993) illustrate that interpretation of productive and economic responses to
added feed enzymes in barley-based rations is highly dependent on an appraisal of
grain ‘quality’ in terms of energy and amino acid availability. Emphasis thus switches
to simple predictors of feeding value in barley and, in particular, their rapid estima-
tion. Fairbairn et al. (1999) described a series of predictive equations for hulled barley
for pigs in which ADF content of the barley described DE, with an R2 value of 0.85:
DE (kcal kg−1, 90% dry matter) = 3526 − (92.8 × ADF%).
More complex equations, incorporating further factors, increased the R2 value to
0.90; for example:
DE (kcal kg−1, = 3964 − (81.3 × ADF%) − (273.9 × acid
90% dry matter) detergent lignin (%)) − (56.4 × total β-glucans (%)).
Near-infrared spectroscopy (NIRS) appears currently to offer the best potential
for rapid estimation of barley DE (Zijlstra et al., 1998) and amino acid acid content
(Jaikaran et al., 1998) with sufficient accuracy to allow for its subsequent use as a tool
to maximize the cost efficacy of enzyme addition. Emphasis on DE estimation alone
as a predictor of potential enzyme response must, however, be tempered with some
caution. Experiences with wheat in pigs (Cadogan et al., 1999) have indicated that
the factors controlling voluntary food intake of different samples do not appear to be
closely related to measured DE, hence any screening method must ultimately take
account of this anomaly and understand the key factors responsible.
Wheat, Triticale and Rye as Grain Sources For Pigs

A review of the literature on wheat feeding value for pigs (van Barneveld, 1999)
found a range of 3.7 MJ DE kg−1 DM across all cultivars studied. The lowest
reported DE was 13.3 MJ kg−1 DM and the highest 17.0 MJ kg−1 DM. As for other
grains, the variability seen is a function of methodology, cultivars, growing regions,
sites and years. Most studies saw differences between ‘poor’ and ‘good’ wheats in the
range 0.8–1.4 MJ kg−1 DM.
The importance of cultivar to feeding value was illustrated in a study in the UK
(Schulze et al., 1997) where six varieties of wheat were grown on one site, to negate
environmental influences, and then used as the sole grain source in diets for piglets
from 9 kg body weight (Table 8.2). Variations in growth rate and feed intake of
179
17 November 2000 15:08:34

170
Table 8.1. Some published studies on the effects of enzyme preparations on barley-based diets. (Note that where both ileal and faecal digestibility measurements
were made, data is presented preferentially at the ileal level.)
24 November 2000 17:25:35

Basal diet – main ingredients; age/weight Enzyme(s) added, levels and inclusion Responses seen
Reference of pig; pelleted or meal/mash (where stated) rates (where stated) (C = control; +E = plus enzyme; *P < 0.05)
Graham et al. (1988) Barley; wheat pollard; meal/mash β-glucanase (16 U g−1) Ileal digestibility of protein (%): Ileal digestibility of energy (%):
19–25 kg ileal cannulated pigs xylanase (1300 U g−1) C: 64.5 C: 55.9
2 kg t−1 +E: 70.1* +E: 58.2
(P < 0.10)
Graham et al. (1989) Hull-less barley; soybean meal; meal/mash β-glucanase (20 U g−1) Ileal digestibility of protein (%): Ileal digestibility of energy (%):
versus pelleted (93°C) 5 kg t−1 C (meal): 52.2 C (meal): 60.3
80 kg ileal cannulated pigs +E (meal): 53.3 +E (meal): 61.9
C (pellet): 54.9 C (pellet): 63.3
+E (pellet): 52.3 +E (pellet): 63.9
180
Bohme (1990) Barley; wheat; soybean meal; pelleted Cellulase, β-glucanase, Growth performance:
11–25 kg α-amylase; glucoamylase C: 381 g day−1 Feed : gain 1.88
1 kg t−1 +E: 440 g day−1 Feed : gain 1.70*

Thacker et al. (1992a) Barley; soybean meal; pelleted (< 60°C) β-glucanase (750 U g−1) Growth performance:
C1 = no growth promoter pentosanase (650 U g−1) C1: 840 g day−1 Feed : gain 2.53
C2 = + salinomycin 2.5 kg t−1 +E: 860 g day−1 Feed : gain 2.59
CHAP-8
22–86 kg C2: 870 g day−1 Feed : gain 2.53

+E: 840 g day−1 Feed : gain 2.56
Faecal digestibility of protein (%):
C1: 72.2 +E: 73.7
C2: 75.3 +E: 81.3*
Faecal digestibility of energy (%):
C1: 74.2 +E: 75.6
G.G. Partridge
C2: 73.5 +E: 78.5

Bedford et al. (1992) Hull-less barley; soybean meal; meal/mash β-glucanase (1086 U g−1) Weight gain:
12–18 kg 2 kg t−1 C: 5.24 kg +E: 6.14 kg*
Feed : gain
C: 1.75 +E: 1.55 (P = 0.10)
24 November 2000 17:27:30

−1
Thacker et al. (1992b) Barley; soybean meal; pelleted β-glucanase (750 U g ) Growth performance:
(< 60°C) 2.5 kg t−1 C1: 830 g day−1 Feed : gain 3.09
C1 = no acid +E: 840 g day−1 Feed : gain 3.04
C2 = + proprionic acid C2: 860 g day−1 Feed : gain 2.89
25–98 kg +E: 790 g day−1 Feed : gain 3.00
C1: 78.6 +E: 75.3
C2: 74.3 +E: 79.2
C1: 76.3 +E: 79.3
C2: 78.7 +E: 78.1
Carbohydrase Enzymes in Pig Nutrition
Thacker et al. (1992b) Hull-less barley; soybean meal; pelleted Growth performance:
181
β-glucanase (750 U g−1)
(< 60°C) 2.5 kg t−1 C1: 254 g day−1 Feed : gain 2.20
C1 = no acid +E: 274 g day−1 Feed : gain 2.03
C2 = + fumaric acid C2: 275 g day−1 Feed : gain 1.99

C1: 78.4 +E: 77.8
C2: 77.6 +E: 81.7
CHAP-8
C1: 82.6 +E: 81.9
C2: 81.0 +E: 82.3
171
172
24 November 2000 17:41:18

Table 8.1. Continued.

Han and Froseth Nine barley cultivars of ‘high’ and ‘low’ quality β-glucanase, cellulase; xylanase; Faecal DE (kcal kg−1 dry matter):
(1993) ‘high’: < 7.6% ADF; < 4.7% β-glucans (n = 4) pectinase ‘high’ control: 3455
182
‘low’: > 9.8% ADF; > 5.4% β-glucans (n = 5) 1 kg t−1 ‘high’ +E: 3532* (+2.2%)
70 kg duodenal cannulae – mobile nylon bag ‘low’ control: 3296
technique ‘low’ +E: 3414* (+3.6%)

Inborr (1994) Hulled or hull-less barley; soybean meal; β-glucanase Growth performance:
meal/mash 2.5 kg t−1 to give 89–95 mU g−1 feed C (hull-less): 223 g day−1 Feed : gain 1.60
9.5–14 kg +E (hull-less): 229 g day−1 Feed : gain 1.52
C (hulled): 200 g day−1 Feed : gain 1.71
CHAP-8
+E (hulled): 208 g day−1 Feed : gain 1.65
Z:\Customer\CABI\A3882 - Bedford + Partridge - Enzymes\A3957 - Bedford - Enzymes #Q.vp

Effect of enzyme on growth (P = 0.07)
Effect of enzyme on feed : gain (P = 0.06)
van Lunen and Barley; rapeseed meal; pelleted E1: β-glucanase Faecal digestibility of protein (%): Faecal digestibility of energy (%):
Schulze (1996a) 35 kg E2: xylanase; β-glucanase C: 84.6 C: 80.9
+E1: 85.3 +E1: 81.8
G.G. Partridge
+E2: 86.7 +E2: 83.4*

Ramaswamy et al. Hull-less or hulled barley; soybean meal; E1: xylanase Faecal digestibility of protein (%): Measured DE (MJ kg−1):
(1996) canola meal E2: xylanase; β-glucanase C (hull-less): 82.0 C (hull-less): 14.40
47 kg +E1: 81.0 +E1: 14.73
+E2: 81.0 +E2: 14.94
24 November 2000 17:41:19

C (hulled): 76.8 C (hulled): 13.5

+E1: 81.5 +E1: 15.3
+E2: 77.3 +E2: 13.5
Enzyme P < 0.05 Enzyme P < 0.001
Barley × enzyme P = 0.06 Barley × enzyme P = 0.008
Power et al. (1996) Hulled barley; wheat; wheat middlings; β-glucanase Growth performance and digestibility at optimum inclusion level
pelleted (~70°C) (0, 500, 1000, 2000 p.p.m.) (2000 p.p.m.):
6–15 kg C: 201 g day−1 Feed : gain 2.11
+E: 227 g day−1* Feed : gain 1.95*
C: 77.4 +E: 80.3
Li et al. (1996b) Hull-less barley; soybean meal; meal/mash Faecal digestibility of protein (%): Faecal digestibility of energy (%):
183
6–11 kg A dose-response study (note the C: 81.6 C: 85.2
maximum response was shown at the +E: 88.5* +E: 89.5*
highest level of inclusion, 2 kg t−1, so only (significant linear effects of dose (significant linear effects of dose
this data is presented) rate were seen) rate were seen)

CHAP-8
173
174
24 November 2000 17:41:20


Li et al. (1996a) Hull-less barley; soybean meal; meal/mash β-glucanase (1000 U g−1) Ileal digestibility of protein (%): Ileal digestibility of energy (%):
7–11 kg 2 kg t−1 C: 65.2 C: 64.9
+E: 73.5* +E: 71.1*
Baidoo et al. (1998b) Hull-less barley (varieties ‘Condor’ and ‘Buck’); Cellulase; β-glucanase; xylanase Ileal digestibility of protein (%): Ileal digestibility of energy (%):
canola meal; meal/mash (11 U g−1; 27 U g−1; 43 U g−1) C: 57.6 C: 57.1
184
14 kg ileal-cannulated pigs 100 g t−1 +E: 61.8* +E: 63.3*
(effects were seen on both (effects were seen on both
varieties, hence these are varieties, hence these are
overall values) overall values)

Baidoo et al. (1998b) Hull-less barley; soybean meal; canola meal; Cellulase; β-glucanase; xylanase Growth performance (9–60 kg):
meal/mash or pellet (80°C) (11 U g−1; 27 U g−1; 43 U g−1) C: 721 g day−1 Feed : gain 2.01
9–60 kg 100 g t−1 +E: 782 g day−1* Feed : gain 1.84*
CHAP-8
Positive effects to enzyme addition were seen in both mash and

pelleted feed, hence overall values are shown
Baidoo et al. (1998a) Hull-less barley (’Falcon’); meal/mash E1: xylanase Ileal digestibility of protein (%): Ileal digestibility of energy (%):
10–14 kg ileal-cannulated pigs E2: β-glucanase C: 59.6 C: 62.1
E3: xylanase; β-glucanase; protease +E1: 60.1 +E1: 64.6
+E2: 66.7* +E2: 69.8*
G.G. Partridge
+E3: 67.2* +E3: 71.2*

Baidoo et al. (1998a) Hull-less barley (’Buck’, ‘Condor’ or ‘Falcon’); Xylanase; β-glucanase; protease Ileal digestibility of protein (%): Ileal digestibility of energy (%):
canola meal; meal/mash C (buck): 60.3 C (buck): 57.7
14 kg ileal-cannulated pigs +E: 64.5 +E: 67.0
C (condor): 54.8 C (condor): 56.2
24 November 2000 17:41:21

+ E: 60.7 + E: 66.3
C (falcon): 67.6 C (falcon): 60.6
+E: 67.2 + E: 61.3
Yin et al. (2000) Hulled barley (high or normal bushel Xylanase; β-glucanase Ileal digestibility of protein (%): Ileal digestibility of energy (%):
weight – HBW or NBW); pelleted (400; 400 U g−1) C (HBW): 74.8 C (HBW): 67.8
HBW = 70 kg hl−1 1 kg t−1 +E: 76.1 +E: 68.5
NBW = 64 kg hl−1 C (NBW): 72.6 C (NBW): 65.8
23 kg ileal-cannulated pigs +E: 76.8* +E: 70.2*
Gill et al. (2000) Hulled barley; soybean meal; pelleted (75°C) β-glucanase; xylanase; amylase Growth performance:
8–18 kg 1 kg t−1 C: 329 g day−1 Feed : gain 1.60
185
DE (MJ kg−1)
C: 14.86
+E: 15.08
C: 76.8

+E: 75.1
CHAP-8
175
176 G.G. Partridge
Table 8.2. Effect of variety on the feeding value of wheat (Schulze et al., 1997).
Water holding
Test wt capacity Daily gain Daily feed DEb DE intake
Varietya (lb bu−1) (filter method) (g) (g) Feed : gain (kcal kg−1) (kcal day−1)
Hussar 62.4 1.92 488 636 1.31 3365.5 2140

Hunter 61.6 1.85 470 608 1.30 3363.5 2045
Brigadier 60.9 2.04 457 609 1.34 3246.5 1977
Beaver 60.6 2.22 435 558 1.29 3308.5 1846
Dynamo 60.6 1.92 419 614 1.49 3298.5 2025
Riband 60.5 2.07 415 554 1.38 3384.5 1875
SEM – – 20 22 0.063 21.5 –
aAll varieties grown in one location.
bMeasured digestible energy.
70–80 g day−1 were seen in this trial over a 3-week period. Voluntary feed intake and
apparent DE intake (feed intake × measured faecal DE) appeared to be influenced by
the water-holding capacity (WHC) of the finished feed after pelleting and was
described by the equation:
DEI (kcal day−1) = 3325.1 − 663.64 * WHC (g g−1); (P = 0.015, r 2 = 0.81),
which in turn suggests that the fibre content of the diet (soluble plus insoluble) was a
contributory factor. This agrees with the observations of Kyriazakis and Emmans
(1995), who found that varying fibre types influenced voluntary food intake in the
young pig (12–25 kg) by increasing the WHC of the final feed (Fig. 8.4).
Cadogan et al. (1999) described similar weaner growth studies (7.5–15.6 kg) in
which ten Australian cultivars of wheat were compared immediately after harvest,
and again after 10 months storage. Growth performance across the ten cultivars
varied from 233 to 447 g day−1 (mean 388, SD 62.9) and was heavily influenced
by voluntary food intake (mean 438 g day−1, SD 70.0, range 271–514 g day−1).
Apparent DM digestibility of the ten wheats showed no relationship to pig growth
performance, illustrating the limitations of faecal digestibility in predicting pig
performance, without further knowledge of the factors influencing feed intake. After
10 months of storage the marked differences between the varieties remained,
although feed intake and daily gain were, on average, 11.3% and 9.8% higher than in
the same samples immediately post-harvest.
Variation in triticale and rye feeding value has been studied relatively little in
pigs. Van Barneveld (1999) cited data from Charmley and Greenhalgh (1987), who
found triticale feeding value was lower than that of wheat (15.1 versus 16.0 MJ kg−1
DM), but that differences between the three triticale cultivars studied were relatively
small. It is likely that the feeding value of both triticale and rye will be at least as
variable as that of wheat, and likely more so, allowing for the fact that both raw
materials will have higher total levels of non-starch polysaccharides and, particularly,
soluble arabinoxylans (Cyran et al., 1995). Rye-based diets offered to poultry are a
considerable challenge to the bird, due to their negative effects on digesta viscosity,
186
17 November 2000 15:08:42

leading to poorer nutrient digestibility and bird growth. In pigs this may be less of a
physiological problem than to the bird, but the feeding value of rye is still recognized
to be well below that of equivalent wheat-based diets and its fibre content seems to be
the main contributory factor (Bayzlo, 1990).
Response of Wheat, Triticale and Rye to Exogenous Enzymes

in Pigs
The predominant non-starch polysaccharides present in the endosperm and aleurone
layers of wheat, triticale and rye are arabinoxylans, which comprise 50–60% of the
total NSPs (Dierick and Decuypere, 1994; Evers et al., 1999). In contrast to barley
and oats, β-glucan is a minor component in these grains. The arabinoxylans in the
thin cell walls within the endosperm are water-soluble to varying degrees (depending
on variety, growing conditions, etc.) whereas those in the thick cell walls of the
aleurone layer are predominantly insoluble. Xylanases are therefore the main type of
exogenous enzyme used for these grain types, as well as for grain by-products derived
from them (e.g. wheat bran, middlings, etc.) Table 8.3 shows a summary of a
number of published trials over the past 10 years into wheat, rye and triticale-based
diets, using a range of enzyme additions.
As with the barley data in Table 8.1, there is a greater emphasis on trials with
young pigs (< 25 kg) than in grower/finishers. This is based on the assumption that
potential responses to enzymes will be greater in the younger animal where daily nutri-
ent intake, in terms of both energy and amino acids, is often the limitation to achieving
the animal’s lean gain potential. The success rate in showing positive effects of enzymes
on pig performance, measured as either growth rate, feed : gain or digestibility, seems
to be higher in the younger animal but this trend is influenced by the fact that very
few studies have made an attempt to predetermine wheat/triticale/ rye feeding value,
before adding an appropriate enzyme. In the few recent studies where this has been
attempted (Choct et al., 1999; Partridge et al., 1999) the ability of a xylanase to raise
significantly the productive performance of a ‘poor’ wheat to the level of that of ‘good’
wheat has been clearly demonstrated, irrespective of the age of the pig (Table 8.4).
The relative importance of soluble pentosans to the response to xylanase in the
pig is thrown into question by a number of studies where rye was the predominant
grain (Thacker et al., 1991, 1992a; Bedford et al., 1992; Thacker and Baas,
1996). Dusel et al. (1997b) showed that the use of a mixed-grain diet based on a
combination of wheat (high extract viscosity), rye and barley gave responses to
enzyme addition that went beyond the apparent effects on viscosity reduction (Table
8.5). As in the studies of Choct et al. (1999) and Partridge et al. (1999), addition of
xylanase or the xylanase/protease combination released an apparent limitation on
voluntary food intake by the animal in these diets such that the growth response seen
was a cumulative effect of both increased nutrient availability and intake. Other,
more subtle, changes in the microbial flora may also have influenced the response to
the enzyme. Pigs in the xylanase-supplemented group appeared to show decreases
in bacterial proliferation in the small intestine (Fig. 8.6) when compared with both
187
17 November 2000 15:08:43

Table 8.3. Some published studies on the effects of enzyme preparations on wheat, rye or triticale-based diets. (Note that where both ileal and faecal digestibility
178
measurements were made, data are presented preferentially at the ileal level.)
17 November 2000 15:08:44

Bohme (1990) Wheat; barley; maize; soybean meal; Cellulase, β-glucanase, α-amylase; Growth performance:
pelleted glucoamylase C: 411 g day−1 Feed : gain 1.81
11–25 kg 1 kg t−1 +E: 458 g day−1 Feed : gain 1.56*
Thacker et al. (1991) Rye; soybean meal; meal Pentosanase (650 U g−1) Growth performance:
(expt 1) or pellets (55°C, expt 2) 2.5 kg t−1 C (meal): 730 g day−1 Feed : gain 3.48
20–98 kg +E: 760 g day−1 Feed : gain 3.12*
C (pellet): 760 g day−1 Feed : gain 2.64
C (meal): 79.2 +E: 80.0
188
C (pellet): 77.0 +E: 77.3
C (meal): 80.8 +E: 80.1
C (pellet): 81.2 +E: 80.1

Thacker et al. (1992a) Rye; soybean meal; pelleted β-glucanase (750 U g−1) Growth performance:
(< 60°C) pentosanase (650 U g−1) C1: 740 g day−1 Feed : gain 2.40
C1 = no growth promoter 2.5 kg t−1 +E: 710 g day−1 Feed : gain 2.48
CHAP-8
C2 = + salinomycin C2: 720 g day−1 Feed : gain 2.38

C1: 72.5 +E: 76.5
C2: 78.9 +E: 79.8
C1: 78.9 +E: 80.6
G.G. Partridge
C2: 82.7 +E: 82.5

Bedford et al. (1992) Rye; soybean meal; mash/meal Xylanase (850 U g−1) Weight gain:
11–15 kg 2 kg t−1 C: 4.24 kg +E: 4.24 kg
Feed : gain:
C: 1.64 +E: 1.57
17 November 2000 15:08:46

McLean et al. (1992) Wheat; soybean meal E1: amylase; xylanase; cellulase; Ileal digestibility of protein (%): Ileal digestibility of energy (%):
Weaner pigs pectinase (700 g t−1) C: 74.0 C: 70.8
E2: β-glucanase; pectinase; cellulase; +E1: 76.6 +E1: 73.5*
hemicellulase + E2: 73.1 +E2: 72.9
(1 kg t−1)
Inborr et al. (1993) Wheat : barley (50 : 50); soybean meal; E1: β-glucanase #1; xylanase; amylase Growth performance:
meal/mash (35; 590; 3300 U g−1) C: 204 g day−1 Feed : gain 1.38
~6–10 kg 1 kg t−1 +E1: 223 g day−1 Feed : gain 1.38
E2: β-glucanase #2; xylanase; amylase +E2: 205 g day−1 Feed : gain 1.39
(41; 740; 3300 U g−1) Ileal digestibility of protein (%):
950 g t−1 C: 76.8 +E1: 78.4 +E2: 77.9

Ileal digestibility of starch (%):
189
C: 94.9 +E1: 97.7* +E2: 96.8
Officer (1995) Wheat; fishmeal; meat meal; soybean E1: protease; lipase; β-glucanase; Growth performance (after weaning):
meal; meal/mash amylase; cellulase (2 kg t−1) C: 309 g day−1 Feed : gain 1.54

~6.5–14 kg E2: protease; lipase; β-glucanase; +E: 319 g day−1 Feed : gain 1.40
amylase; cellulase; hemicellulase; (P < 0.10)
pentosanase (2 kg t−1) C: 299 g day−1 Feed : gain 1.76
E3: amylase; xylanase; β-glucanase; +E2: 282 g day−1 Feed : gain 1.82
CHAP-8
pectinase (2 kg t−1) C: 361 g day−1 Feed : gain 1.56

+E3: 332 g day−1 Feed : gain 1.49
(P < 0.10)
179
180
24 November 2000 17:26:27


Campbell et al. Wheat; lupin (W/L) or wheat; soybean Xylanases; pentosanases; Growth performance:
(1995) meal (W/S); pelleted (70°C) β-glucanases C (W/L): 355 g day−1 Feed : gain 1.21
6–13 kg 400 g t−1 +E: 388 g day−1 Feed : gain 1.15
C (W/S): 291 g day−1 Feed : gain 1.29
190
Overall effects of enzyme on growth rate (P < 0.05) and
feed : gain (P = 0.08)
Thacker and Baas Rye; soybean meal ‘Multi-enzyme preparations’ E1–E4: Growth performance (Trial 1):

(1996) Pelleted (< 65°C) E1 1 kg t−1 C: 750 g day−1 Feed : gain 2.52
23–83 kg (Trial 1) E2 200 g t−1 +E1: 780 g day−1 Feed : gain 2.42
28–92 kg (Trial 2) E3 500 g t−1 +E2: 820 g day−1 Feed : gain 2.39
E4 700 g t−1 +E3: 800 g day−1 Feed : gain 2.37
CHAP-8
Inclusion rates supplied the same +E4: 800 g day−1 Feed : gain 2.44

pentosanase activity, according to the Growth performance (Trial 2):
authors’ assay method C: 880 g day−1 Feed : gain 2.42
+E1: 940 g day−1 Feed : gain 2.33
+E2: 930 g day−1 Feed : gain 2.37
+E3: 920 g day−1 Feed : gain 2.37
+E4: 950 g day−1 Feed : gain 2.37
G.G. Partridge
Schulze et al. (1996b) Wheat; soybean meal; pelleted E1: xylanase #1 (5000 U g−1) 1 kg t−1 Growth performance:
8–22 kg E2: xylanase #1/protease (5000/500 U g−1) C: 304 g day−1 Feed : gain 1.69
1 kg t−1 +E1: 394 g day−1* Feed : gain 1.49*
24 November 2000 17:49:22

E3: xylanase #2 (5000 U g−1) 1 kg t−1 +E2: 385 g day−1* Feed : gain 1.49*
+E3: 402 g day−1* Feed : gain 1.45*
Gill and Schulze Wheat; soybean meal; meal/mash (M) or Xylanase (4000 U g−1) Growth performance:
(1996) pelleted (P) 1 kg t−1 C (M): 859 g day−1 Feed : gain 2.46
C (P): 836 g day−1 Feed : gain 2.43
Pen fouling ‘greatly reduced’ in pelleted diets + enzyme
van Lunen and Wheat : corn (60 : 0);(40 : 20);(20 : 40); Xylanase (4000 U g−1) Overall growth performance:
Schulze (1996b) wheat middlings; soybean meal; pelleted 1 kg t−1 C: 980 g day−1 Feed : gain 2.78
22–80 kg +E: 1070 g day−1* Feed : gain 2.63*
Li et al. (1996a) Wheat; soybean meal; meal/mash Ileal digestibility of protein (%): Ileal digestibility of energy (%):
191
7–11 kg 2 kg t−1 C: 68.8 C: 66.9
+E: 75.9 +E: 71.2
Li et al. (1996b) Wheat; soybean meal (W) β-glucanase, 1000 U g−1 Faecal digestibility of protein (%): Faecal digestibility of energy (%):

Rye; soybean meal (R) (a dose-response study – the C (W): 85.1 C (W): 86.8
meal/mash numerically max. responses are shown) +E: 89.1 +E: 88.4
6–11 kg 2 kg t−1 (W) C (R): 87.0 C (R): 87.2
1 kg t−1 (R) +E: 89.3 +E: 88.1
CHAP-8
181
182

24 November 2000 17:49:24

Haberer et al. (1997) Rye : barley (50 : 50); wheat bran; Xylanase (800FXU); β-glucanase Faecal digestibility of protein (%): Measured DE
soybean meal; meal/mash, wet fed (75FBG) (MJ kg−1 dry matter):
26–40 kg 750 g t−1 C: 74.9 C: 14.33
+E: 76.3 +E: 14.83*
Yin et al. (1997) Wheat; wheat/wheat middlings; Xylanase (4000 U g−1) Ileal digestibility of protein (%): Ileal digestibility of energy (%):
wheat/wheat bran 1 kg t−1 C: 76.5 C: 70.4
~20 kg ileal-cannulated pigs +E: 77.8* +E: 71.4
Dusel et al. Wheat (high extract viscosity); rye; barley; E1: xylanase (5000 U g−1) 1 kg t−1 Growth performance:
(1997b) soybean meal; pelleted or C: 354 g day−1 Feed : gain 1.92
192
and 10–25 kg E2: xylanase (5000 U g−1) +E1: 462 g day−1* Feed : gain 1.66*
Jeroch et al. Protease (500 U g−1) +E2: 480 g day−1* Feed : gain 1.65*
(1999) 1 kg t−1 DE (MJ kg−1 dry matter):
C: 13.87 +E1: 15.08*

−1
Rattay et al. (1998) Barley; wheat; wheat bran; soybean meal Xylanase (4000 U g ) Faecal digestibility of protein (%): Faecal digestibility of energy (%):
meal/mash 1 kg t−1 C: 77.9 C: 80.5
−/+ Avilamycin (A; 40 mg kg−1) +A: 82.3* +A: 83.3*
CHAP-8
23–45 kg +E: 81.6* +E: 83.1*

+A+E: 82.4* +A+E: 83.5*
Partridge et al. Wheat : barley (50 : 50); soybean meal – C1 Xylanase; β-glucanase; amylase; Growth performance:
(1998a) Wheat : maize (50 : 50); wheat pollard; pectinase Cl: 575 g day−1 Feed : gain 1.67
soybean meal – C2 (4000; 150; 1000; 25 U g−1) +E: 581 g day−1 Feed : gain 1.59*
Both diets pelleted 1 kg t−1 C2: 512 g day−1 Feed : gain 1.69
G.G. Partridge
8–30 kg +E: 557 g day−1* Feed : gain 1.62*

Callesen (1998) Wheat; barley; wheat bran; soybean meal Xylanase (4000 U g−1) Growth performance:
pelleted (81°C min.) 1 kg t−1 C: 824 g day−1 FU kg−1 gain 2.66
30–97 kg +E: 836 g day−1 FU kg−1 gain 2.59
24 November 2000 17:49:25

Protein efficiency value (protein consumption/kg lean meat gain):

C: 966 g +E: 929 g*
Gross margin/pen place/year:
C: 658 DKK +E: 706 DKK*
Partridge et al. Wheat – high (H) versus medium (M) versus Xylanase (4000 U g−1) Growth performance:
(1999) low (L) performance*; pelleted 1 kg t−1 H: 960 g day−1
28–60 kg M: 918 g day−1 +E: 945 g day−1
*Determined in weaner growth trials L: 878 g day−1* +E: 952 g day−1
Feed : gain:
H: 1.84
M: 1.80 +E: 1.81
L: 1.76 +E: 1.81
193
Dreschel et al. Triticale; rye; barley; wheat Xylanase; β-glucanase, 300 and Growth performance:
(1999) 25–110 kg 200 g t−1 in grower and finisher phases, C: 865 g day−1 +E: 893 g day−1
respectively Energy efficiency:
C: 44.0 MJ ME kg−1 gain
+E: 42.4 MJ ME kg−1 gain

Jeroch et al. (1999) Wheat (86% of diet), low (L) or high (H) Xylanase (5000 U g−1) Growth performance:
extract viscosity; pelleted 1 kg t−1 C (L): 376 g day−1 Feed : gain 1.96
10–25 kg +E (L): 383 g day−1 Feed : gain 1.93
CHAP-8
C (H): 398 g day−1 Feed : gain 1.90
+E (H): 400 g day−1 Feed : gain 1.80
DE (measured, MJ kg−1 dry matter):
C (L): 16.23 +E: 16.29
C (H): 15.91 +E: 16.07
183
184
24 November 2000 17:49:26


Choct et al. (1999) High (H), medium (M) and low (L) feed Xylanase; β-glucanase; cellulase Growth performance:
intake wheats – predetermined 120 g t−1 C (L): 230 g day−1 Feed : gain 1.38
7–16 kg +E (L): 466 g day−1* Feed : gain 1.23
C (M): 425 g day−1 Feed : gain 1.27
+E (M): 445 g day−1 Feed : gain 1.20
C (H): 460 g day−1 Feed : gain 1.14
+E (H): 479 g day−1 Feed : gain 1.20
194
Gill et al. (2000) Wheat/soybean meal (W); Xylanase; amylase; pectinase (W) Growth performance:
Wheat/beetpulp/soybean meal (WSBP); β-glucanase; amylase; pectinase (WSBP) C (W): 338 g day−1 Feed : gain 1.53
pelleted (75°C) 1 kg t−1 +E (W): 360 g day−1 Feed : gain 1.48
8–18 kg C (WSBP): 345 g day−1 Feed : gain 1.56

+E (WSBP): 363 g day−1 Feed : gain 1.48
C (WSBP) 72.1
+E (WSBP) 73.9
CHAP-8
DE (MJ kg−1)

C (WSBP) 14.10
+E (WSBP) 14.33
G.G. Partridge
Table 8.4. Effect of xylanase addition to wheats preselected for productive performance
(determined in prior growth studies with weaner pigs), 28–60 kg (Partridge et al., 1999).
Wheat −/+ Finish weight Daily gain Daily feed

performance Xylanase (kg) (g) intake (g) Feed : gain
High − 61.9 960ab 1772ab 1.84

Medium − 60.2 918ab 1622ab 1.80
Medium + 61.2 945ab 1710ab 1.81
Low − 58.3 878bb 1576bb 1.76
Low + 61.5 952ab 1728ab 1.81
SEM (NS)0.651(NS) 12.4* 33* (NS)0.021(NS)
abValues within columns with different superscripts are significantly different (P < 0.05).
*P < 0.05. NS = not significant.
Table 8.5. Effect of enzyme addition to a wheat-based diet with a high extract viscosity, piglets
10–25 kg. Dusel et al. (1997b) and personal communication (viscosity values).
Daily gain Daily feed Viscosity in the small

Wheat extract viscosity* (g) (g) Feed : gain intestine (mPa s)
Low (positive control) 388ab 717 1.85b 3.3a

High 354bb 727 1.92b 6.4b
High + xylanase 462ab 766 1.66a 3.9a
High + xylanase + protease 480ab 794 1.65a 3.1a
abValues within columns with different superscripts are significantly different (P < 0.05).
*Low = 1.3 mPa s; high = 3.3 mPa s.
control rations, suggesting that some degree of nutrient ‘sparing’ may also have
contributed to the productive response seen. These studies illustrate the difficulty in
interpreting responses, or apparent lack of responses, to exogenous enzymes in the
pig unless many parameters are examined synchronously.
Maize and Sorghum as Grain Sources for Pigs

Systematic studies on variability in the feeding value of maize for pigs are relatively
difficult to find in the scientific literature. Considering the global importance of
maize as the key feed raw material in pig and poultry diets, this apparent dearth of
information is surprising but has been remarked upon by others (e.g. Dale, 1994).
The oft-held perception that maize, assuming it is mycotoxin-free, is a grain with
consistent feeding value is clearly erroneous and this has been demonstrated in a
number of poultry trials (Leeson et al., 1993; Dale, 1994) and alluded to in the pig
(Leigh, 1994) (Table 8.6). It is equally clear that this observed variability in animal
performance on maize-based diets is a function of many interacting factors, including
bushel weight, drying conditions, particle size after grinding, variety (e.g. waxy versus
dent) and amino acid and oil content. Research in recent years has concentrated
195
24 November 2000 16:19:27

186 G.G. Partridge
0
Stomach Duodenum Jejunum Ileum Caecum
High viscous + xylanase/protease High viscous Low viscous
Fig. 8.6. Effect on microbial populations in digesta when piglets are fed diets
based on high viscous wheat −/+ xylanase/protease, or low viscous wheat.
Microscopical analysis of digesta (VTT Biotechnology and Food Research, Finland):
0 = few microbes present, 4 = high levels of microbes present. Samples from the
studies of Dusel et al. (1997a).
Table 8.6. Effect of corn bushel weight on performance of growing pigs, 63–200 lb (Iowa State
University, 1975, cited by Leigh, 1994).
Bushel weight Daily gain (lb) Daily feed (lb) Feed : gain
Low (41 lb, 9.7% CP) 1.66 5.89 3.56

Medium (48 lb, 8.6% CP) 1.69 6.44 3.82
Heavy (54 lb, 8.7% CP) 1.74 6.21 3.56
less on maize variability as an issue and more on the so-called ‘designer’ versions of
the maize crop – e.g. high-lysine, high-oil, low-phytate – all with potentially high
agronomic value for the future.
For sorghum, van Barneveld (1999) reviewed a number of digestibility studies
with pigs and found that DE estimates ranged from 15.8 to 17.4 MJ kg−1 DM, with
energy digestibility coefficients ranging from 0.72 to 0.92. Kopinski (1997) found
that the largest variation between cultivars at a single study site was 0.71 MJ kg−1
DM and within a cultivar at different sites it was 0.5 MJ kg−1 DM. Kemm and Brand
(1996), summarizing trials with samples of low- and high-tannin sorghum, found
that high-tannin varieties had reduced DE content (10–16%) compared with
low-tannin varieties. Feed conversion was correspondingly poorer on high-tannin
sorghum when compared with low-tannin sorghum and maize meal.
Response of Maize and Sorghum to Exogenous Enzymes in

Pigs
The NSP levels in maize and sorghum are similar (9–10%; Dierick and Decuypere,
1994) with insoluble arabinoxylans comprising over 40% of this total. It is known
196
24 November 2000 16:47:38

that standard milling procedures (e.g. roller milling or hammer milling through
screens of varying diameter) still result in intact packages of cell wall material,
with enclosed nutrients, entering the stomach and small intestine. As the cell wall
structure in these particular grains is predominantly insoluble fibre, and unresponsive
to endogenous enzymes, varying quantities of packaged material have been observed
at the end of the small intestine, entering the hindgut (M.R. Bedford, personal
communication, 2000). Exogenous enzyme activities of particular relevance to these
fibre components in maize and sorghum are xylanase and, to a lesser extent, cellulase
designed to break down this cell wall ‘packaging’ and disrupt its associated negative
fibre properties, e.g. water-holding capacity.
Maize starch digestibility per se will be most influenced by factors such as
the amylose : amylopectin ratio, which will vary particularly when comparing waxy
(high amylopectin) versus dent maize. The recent interest in high amylose maize
starch as an ingredient in human foods, to stimulate potentially beneficial butyrate
fermentation in the hindgut, is a reminder of the importance of this factor. Topping
et al. (1997) fed pigs on diets containing either standard maize starch or a high
amylose material and observed large differences in the amounts of starch reaching the
caecum (Fig. 8.7). This situation has implications, not only for nutrient digestibility
and availability in the small intestine, but also with respect to substrate-related
changes in the microflora in the hindgut. The relevance to exogenous enzymes is that
various amylase sources, having different properties to endogenous amylase, could
have a potential role to play in improving starch digestibility in the small intestine
and thereby indirectly influence the microbial population in the gut.
Unfortunately, relatively few studies have been reported on the effects of
exogenous enzymes on ‘simple’ maize/soybean or sorghum/soybean diets in
pigs (Table 8.7). Lindemann et al. (1997) found positive responses in the grower
phase (26–64 kg) after addition of a complex enzyme blend (protease, cellulase,
pentosanase, galactosidase and amylase) to a maize/soybean meal-based ration. The
same enzyme combination gave no response when 20% wheat mill by-product was
58.0
39.1
8.4
3.9
3.5 5.5
Time after feeding (h)
Fig. 8.7. Concentration of starch (mg g−1 dry matter) in caecal digesta of pigs
fed diets containing maize starch or high amylose starch; mean of two pigs/
treatment. (, maize starch; %, high amylose starch. (Topping et al., 1997.)
197
17 November 2000 15:09:49

188
17 November 2000 15:09:50

Table 8.7. Some published studies on the effects of enzyme preparations on maize or sorghum-based diets −/+ milling by-products (NB: where both ileal and
faecal digestibility measurements were made then data are presented preferentially at the ileal level).
Basal diet – main ingredients; age/weight of Enzyme(s) added; levels and inclusion Responses seen
Reference pig; pelleted or meal/mash (where stated) rates (where stated) (C = control; +E = plus enzyme; * P < 0.05)
Li et al. (1996b) Maize; soybean meal; meal/mash β-glucanase, 1000 U g−1 (a dose Faecal digestibility of protein (%): Faecal digestibility of energy (%):
6–11 kg response study – the numerically C: 84.4 C: 85.8
maximum responses are shown) +E: 82.7 +E: 85.7
2 kg t−1
Schulze et al. Maize; soybean meal; rice bran; meal/mash E1: xylanase #1 Growth performance:
198
(1996a) 46–93 kg E2: xylanase #1 + amylase C: 700 g day−1 Feed : gain 3.10
E3: xylanase #2 +E1: 741 g day−1 Feed : gain 2.82*
+E2: 725 g day−1 Feed : gain 2.93
+E3: 763 g day−1* Feed : gain 2.97

Lindemann et al. CH: maize; soybean meal (‘high’ energy) Protease; cellulase; pentosanase; Growth performance:
(1997) CL: maize; soybean meal; wheat α-galactosidase; amylase CH: 801 g day−1 Feed : gain 2.97
by-products (‘low’ energy) 1 kg t−1 +E: 860 g day−1 Feed : gain 2.75
CHAP-8
26–109 kg CL: 771 g day−1 Feed : gain 3.48

Enzyme × energy level P < 0.05
Schulze and Maize; soybean meal; wheat middlings; E1: xylanase #1 Growth performance:
Campbell (1998) pelleted E2: xylanase #2 C: 706 g day−1 Feed : gain 2.30
42–74 kg +E1: 781 g day−1 Feed : gain 1.83*
G.G. Partridge
+E2: 765 g day−1 Feed : gain 2.40

Partridge et al. Maize; wheat pollard; soybean meal; Xylanase (4000 U g−1 ) Growth performance at two sites:
(1998b) meal/mash 1 kg t−1 C: 659 g day−1 Feed : gain 3.07
37–78 kg +E: 694 g day−1 Feed : gain 2.76*
C: 704 g day−1 Feed : gain 3.25
17 November 2000 15:09:51

+E: 751 g day−1 * Feed : gain 3.11

Kim et al. (1998) Sorghum, soybean meal; meal/mash Cellulase Growth performance:
51–118 kg 500 g t−1 C: 930 g day−1 Feed : gain 3.51
C: 65.1
+E: 66.8
C: 80.6
+E: 81.3
Evangelista et al. Maize; soybean meal; meal/mash Amylase; β-glucanase; xylanase; Growth performance: Feed : gain:
(1998) 28–85 kg pectinase C: 792 g day−1 2.83
199
(1000; 150; 4000; 25 U g−1) 1 kg t−1 +E: 853 g day−1* 2.82
Li et al. (1999) C: Maize; soybean meal Amylases; β-glucanase; pectinase; Growth performance:
CA: Maize; soybean meal + acidifier; proteases; cellulase C: 380 g day−1 Feed : gain 1.78

meal/mash 700 g t−1 +E: 412 g day−1 Feed : gain 1.62
10–21 kg CA: 399 g day−1 Feed : gain 1.72
CHAP-8
Partridge et al. Maize; wheat by-product; pelleted Xylanase (4000 U g−1) Growth performance:

(1999) 28–60 kg 1 kg t−1 C: 886 g day−1 Feed : gain 1.88
189
190 G.G. Partridge
added to a lower energy-dense version of the same ration. Li et al. (1999) found no
significant effects of adding an enzyme blend (amylases, β-glucanase, pectinase,
proteases, cellulase) to maize-based diets for weaner pigs, with or without an acidifier
in the ration. Similarly Kim et al. (1998) failed to show benefits of adding cellulase to
a sorghum/soybean diet fed to grower/finishers. In contrast, Evangelista et al. (1998)
described significant responses in growth rate and feed intake to an enzyme blend
(amylase, β-glucanase, xylanase, pectinase) offered to grower/finisher pigs.
Van Lunen and Schulze (1996b) reported significant responses to xylanase
independent of the maize : wheat ratio in diets for grower/finishers. However, the
diet containing the highest level of maize (400 g kg−1) still contained 20% wheat and
10% wheat by-products, making interpretation of the response in relation to specific
substrates more difficult.
When maize/soybean-based rations are supplemented with various milling
by-products (e.g. wheat middlings/pollard/bran; extracted and full-fat rice bran)
a number of trials have shown a beneficial effect of exogenous enzyme supple-
mentation. Schulze et al. (1996a), Schulze and Campbell (1998) and Partridge et al.
(1998b) all showed significant benefits to xylanase supplementation on growth rate
and/or feed conversion ratio in growing/finishing pigs.
Overall, it is clear that the reported responses to enzymes in pure maize/soybean
or sorghum/soybean diets are, so far, limited and more work is needed to understand
the underlying reasons for variability in these raw materials before consistently
cost-effective carbohydrase enzyme solutions can be offered for pigs. At the same
time it is clear that when such diets are supplemented with certain fibrous
by-products, containing significant quantities of insoluble cell wall material, then
the potential for a response to an effective enzyme source (particularly xylanase) is
considerably magnified. In commercial practice such by-products are frequently
added to maize-based diets for grower/finisher pigs to offer some savings in costs
of production, so the economic opportunities for cost-effective enzyme addition
become a realistic opportunity in these situations.
Implications for Enzyme Use in Diet-induced Disease

Syndromes
Recent studies have highlighted the important interactions between diet composition
and microflora changes in the pig gut under both chronic and acute disease
challenge. In Australia, Pluske and co-workers have elegantly illustrated the role
that diet composition plays in the development of swine dysentery, provoked by
the anaerobic spirochaetal bacterium Serpulina hyodysenteriae. Both soluble,
fermentable fibre and resistant starch from various feed raw materials have been
found to be particularly provocative to this disease (Pluske et al., 1996, 1998)
(Fig. 8.8). To date, attempts to influence this particular condition by the strategic use
of carbohydrase enzymes have given equivocal results (Durmic et al., 1997) but the
concept is receiving increased research attention with the continued emphasis on
prophylaxis, rather than medication therapy, in modern pig production systems.
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Undigested soluble fibre has already been identified as one of the key influences
on the non-specific colitis syndrome affecting, particularly, pigs in the 15–40 kg
weight range offered pelleted, wheat-based rations (Taylor, 1989). Adding an
appropriate xylanase has been shown to have a positive influence on this syndrome
(Hazzledine and Partridge, 1996), negating the need for meal feeding or potentially
costly diet reformulation on affected units.
Fig. 8.8. Incidence of swine dysentery, in relation to soluble non-starch

polysaccharide concentration and resistant starch concentration in the diet
(Pluske et al., 1996).
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192 G.G. Partridge
Other studies have similarly reported positive interactions between the use of
enzymes and diet-induced diarrhoea, particularly in the post-weaned pig (Inborr
and Ogle, 1988; Florou-Paneri et al., 1998; Kantas et al., 1998). These, together
with reports on the potential synergies between enzymes and therapeutic and
subtherapeutic antibiotic addition (Florou-Paneri et al., 1998; Kantas et al., 1998;
Gollnisch et al., 1999), offer interesting possibilities for future production methods
where strategic rather than routine antibiotic use will become the norm.
Future Trends in Enzyme Research for Pig Applications?

The review of many trials published in the last 10 years, across a range of dietary
substrates (Tables 8.1, 8.3 and 8.7), suggests that the question to be asked in the next
few years is not so much: ‘Do enzymes work in pig diets?’ but far more: ‘When do
proven enzymes for pig application give their most cost-effective responses?’ Enzyme
technology has moved apace over the last decade to bring, amongst others, a range
of carbohydrase activities that have the potential to have a major impact on the
economics of pig production. However, some key challenges remain.
1. To identify quickly, cheaply and easily, the key parameters in grains and their
by-products that appear to limit both voluntary food intake and nutrient availability in
the growing pig (e.g. Schulze et al., 1997; Cadogan et al., 1999; Partridge et al., 1999).
Then to be able to relate these to responses to specific enzyme additions, given that
different sources of carbohydrases (e.g. xylanases, β-glucanases, amylases) will have
differing characteristics and relative efficacies both in vitro and in vivo.
2. To ensure, contrary to a number of published studies, that scientific trials
involving enzymes are set up in the most appropriate manner to have the opportunity
to show an effect in a marginally nutrient-limited animal. This will almost invariably
involve some prior response modelling in animals whose lean deposition rates
have been predetermined. At the same time in such trials, to superimpose some
predetermined aspects of raw material feeding value, determined in vitro (e.g. Chen
et al., 1997; Cadogan et al., 1999; Moughan et al., 1999). More attention to detail in
describing diet characteristics, particularly fibre analysis and properties, as well as
enzyme sources and levels, will also aid interpretation across a range of studies.
3. To understand better the role that enzymes have to play in gut physiology,
particularly aspects such as digesta passage rate and patterns of fermentation in
different parts of the digestive tract. The effects of these on gut hormone secretion
and the physiological control of voluntary food intake would also merit further study.
4. To understand better the potential role for feed enzymes in manipulating the
gut microflora with a view to their future use with synergistic additives in production
systems where there is far less reliance on prophylactic and therapeutic medication.
Fundamental work on how enzymes can potentially influence fermentation (e.g.
volatile fatty acid production) in the fore- and hindgut will be particularly valuable in
the pig and offers good opportunities to manipulate the gut microflora in a positive
way.
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5. To understand more clearly the interactions between different enzymes,

particularly carbohydrases, phytases and proteases, in order to maximize their cost
effectiveness across a range of dietary substrates and situations.
6. To maximize the use of appropriate enzymes in novel pig feeding applications
involving preprocessing of raw materials, with or without liquid feeding technology.
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method and alternative indigestible markers and the effects of food enzyme supple-
mentation in barley-based diets on ileal and overall apparent digestibility in growing pigs.
Zijlstra, R.T., Scott, T.A., Edney, M.J., Swift, M.L. and Patience, J.F. (1998) Measurements
to predict swine DE of barley. In: 1998 Feed Grain Quality Conference. 8th–10th
November, Edmonton, Alberta, Canada, pp. 78–81.
208
17 November 2000 15:10:54

Interaction between Cereal

Identity and Fat Quality
and Content in Response to
9
Feed Enzymes in Broilers
S. DÄNICKE
Institute of Animal Nutrition, Federal Agricultural Research Centre,
Braunschweig (FAL), Bundesallee 50, D-38116 Braunschweig,
Germany
S. Dänicke
Use
9 of Feed Enzymes in Broilers
Introduction
The use of dietary fat of plant or animal origin as a feed ingredient is a common
practice in broiler feeding, especially in intensive production systems where dietary
energy concentrations of 13 MJ ME kg−1 and greater are required. Under such
conditions, its proportion might reach 5–10% of the diet, which corresponds to
approximately 40–85% of total dietary fat. The actual proportion of a supplemented
fat in a broiler diet depends on several factors, such as local availability, relative price
and the effect on the feed manufacturing process, in addition to physiologically
derived constraints for a particular fat. The last point in particular requires
consideration as several factors modify or interfere with an effective use of a particular
type of dietary fat. For example, fat digestibility in a broiler diet depends not only on
fat type but also on the particular cereal with which it is used. Antoniou et al. (1980)
and Ward and Marquardt (1983) showed that the combination of beef tallow with
rye depressed fat digestibility much more than when wheat was the dominant cereal.
In contrast, when both cereals were tested with a vegetable oil only slight differences
in fat digestibility were found.
The interaction between cereal identity and dietary fat type became even more
interesting with the widespread use of exogenous feed enzymes, especially xylanases
and β-glucanases. These endo-enzymes are capable of partial hydrolysis of anti-
nutritive pentosans (which are more concentrated in rye than in wheat) and mixed
linked β-glucans (most concentrated in barley), respectively, thereby reducing their
viscous properties in vivo. This in turn allows the use of higher dietary proportions of
such ‘critical’ cereals. The fact that greater enzyme effects were reported for fat digest-
ibility in rye- than in wheat-containing diets (Friesen et al., 1992) further confirms the
fat by cereal identity interaction, and moreover suggests that the viscous properties of
209
17 November 2000 15:10:55

200 S. Dänicke
these cereals is the reason for their interaction with fat digestibility. In addition, the
observed enzyme effect was greater with increasing proportion of dietary rye.
In the following sections an attempt will be made to contribute to the under-
standing of the underlying mechanisms and to draw some conclusions for practical
purposes.
Principles of Fat Digestion in Broilers

Digestion and absorption of fat in poultry was extensively reviewed by Krogdahl
(1985). Briefly, the initiation of fat digestion seems to start with a possible onset of fat
emulsification in the upper digestive tract but mainly with the entrance of digesta into
the duodenum, where it is mixed with the secretions of the exocrine pancreas and gall
bladder, which contain lipase, co-lipase and conjugated bile salts, respectively. Bile salts
and phospholipids facilitate progressive fat emulsification. Binding of co-lipase at the
surface of these relatively large aggregates is the precondition for the action of the lipase
that hydrolyses the triglycerides at the 1 and 3 positions of the glycerol molecule.
Released fatty acids, the remaining 2-monoglycerides, phospholipids and bile salts
form spontaneously into mixed micelles, which are much smaller in diameter than the
emulsified fat droplets. Micelles are able to solubilize non-polar lipid compounds such
as fat-soluble vitamins or long-chain saturated fatty acids (LCSFA) within their hydro-
phobic core. The role of the micelle is to act as a shuttle between the bulk intestinal
contents and the aqueous–microvillus interface by overcoming unstirred water-layer
resistance (Westergaard and Dietschy, 1976). At this point, micelles release their
contents to the luminal membranes, from where they passively diffuse into the cell.
Under the conditions of a high fat digestibility these processes take place quantitatively
in the proximal regions of the small intestine. Bile salts released from the micelles
diffuse back to the lumen, and from this point might be used again for micelle
formation in a lower part of the small intestine. Ultimately the majority are reabsorbed
by the distal small intestine. They are transported back to the liver, where they are again
made available for bile production, thus conserving the metabolic bile salt pool.
A long-chain fatty acid that diffuses through the luminal membrane will only
enter the intracellular pool if it is bound to the intracellular fatty acid binding protein
(FABP) (Ockner et al., 1972a; Ockner and Manning, 1974; Katongole and March,
1979, 1980; for review, see Hohoff and Spener, 1998). The FABP–fatty acid complex
translocates the fatty acid to other intracellular compartments where it is normally re-
esterified to a triacylglycerol and further processed. It has been shown for broilers that
the greatest concentration of this inducible intestinal FABP is found at the main site
of fatty acid absorption, i.e. the proximal small intestine (Katongole and March, 1980).
Factors Affecting Fat Digestibility

From the above section it becomes clear that a variety of events have to take place in a
coordinated manner within a relatively short time (see below) if high quantities of
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17 November 2000 15:10:56

Use of Feed Enzymes in Broilers 201
ingested fat are to be absorbed. Hence, it is useful to take note of several factors that
influence fat digestibility in broilers before dealing with the effects of intestinal
viscosity caused by ingestion of cereal grain soluble non-starch polysaccharides
(NSPs) – and consequently the effects of the intestinal viscosity reducing
carbohydrases – in modification of fat digestibility. The first and probably the most
important factor is the chemical nature of the dietary fat itself. For example, beef
tallow is characterized by a lower fat digestibility and a lower ME content than soya
oil. The reasons are the differences in fatty acid composition of both dietary fat
sources. From their fatty acid profile (Table 9.1) it can be deduced that beef tallow
is mainly composed of saturated fatty acids (palmitic and stearic acid) and of
mono-unsaturated oleic acid, whereas linoleic acid, linolenic acid and oleic acid
are the principal fatty acids in soya oil. Differences in fat digestibility arise from
differences in absorbability of saturated and unsaturated fatty acids and can be
summarized from the literature as follows.
Degree of unsaturation and polarity of fatty acids
LCSFAs (C16 : 0, C18 : 0) are more non-polar than unsaturated long-chain fatty
acids of the C18 family and among these fatty acids the polarity increases as the
number of double bonds increases. Among the saturated fatty acids (C8 : 0 . . .
C18 : 0), the polarity increases as the chain length decreases. The more non-polar a
fatty acid is, the more it relies on an adequate presence of bile salts and phospholipids
for adequate emulsification. It has been shown that medium-chain saturated fatty
acids (MCSFA) and long-chain unsaturated fatty acids (LCUSFA) can be absorbed
in large quantities even in the absence of bile salts (Garret and Young, 1975;
Westergaard and Dietschy, 1976). The differences in polarity of fatty acids are
reflected by differences in their absorbability. In general figures, absorbability
increases in the following order: C18 : 0 < C16 : 0 < C14 : 0 < C18 : 1, C18 : 2,
C18 : 3 (Renner and Hill, 1961; Young, 1961; Garret and Young, 1975; Ketels et al.,
1987; Blanch et al., 1995; Vila and Esteve-Garcia, 1996a,b; Dänicke et al., 1997b).
This order is generally at a higher level for fatty acids derived from soybean oil than
for beef tallow, and differences in absorbability between particular fatty acids are
more pronounced for the latter. Palmitic and stearic acid from soya oil are found to
be better absorbable than those originating from beef tallow. It is thought that this is
due to the synergistic effects of LCUSFAs, which support the micellar solubilization
of LCSFAs. In this respect, monoglycerides or glycerol seem to be necessary for opti-
mized micellar dissolution (Garret and Young, 1975; Sklan, 1979). The synergistic
effects of the ratio of unsaturated to saturated fatty acids in relation to fatty acid
absorption and ME values of the fats have been described by several regression
approaches (e.g. Wiseman and Lessire, 1987; Ketels and De Groote, 1987, 1989;
Wiseman and Salvador, 1991). It should be noted, however, that the order of
digestibility of fats in addition to the absolute digestibility value might be modified
by other factors, such as dietary fat inclusion level (Ketels et al., 1987; Wiseman
and Lessire, 1987), age of the birds (Wiseman and Lessire, 1987) and free fatty
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202
17 November 2000 15:10:58

Table 9.1. Fatty acid composition of some dietary fat types (%).
Lard Soybean oil Beef tallow Linseed Palm Maize oil Coconut oil
Fatty acid
(C-atoms : double bonds) Min Max Min Max Min Max Min Max Min Max Min Max Min Max
8:0 2.6 7.5

10 : 0 3.0 7.0
12 : 0 0.1 0.1 0.0 0.2 28.4 49.5
14 : 0 1.3 1.7 0.1 0.1 2.0 4.9 0.8 1.0 12.1 19.5
16 : 0 21.2 26.6 0.7 13.5 23.7 35.4 5.0 10.0 40.7 48.8 9.4 13.6 8.5 12.8
212
16 : 1 2.1 5.3 0.0 0.1 0.1 6.1 0.1 0.1 0.1 0.3 0.1 0.1
18 : 0 11.1 17.7 1.8 7.4 13.5 36.5 3.1 7.8 3.9 5.2 1.7 6.7 2.0 8.1
18 : 1 40.3 51.8 19.1 27.3 24.5 46.4 18.2 21.4 36.7 41.6 25.8 30.1 6.0 13.7

18 : 2 8.0 10.4 44.0 59.4 0.9 5.4 13.9 17.9 7.9 12.7 42.0 58.6 1.5 23.1
18 : 3 0.1 2.1 3.3 8.2 0.1 1.1 49.4 66.2 0.5 0.5 1.0 1.0
No. of references 7.1 9.1 11.1 4.1 3.1 3.1 5.1
CHAP-9
References used: Young (1961), Veen et al. (1974), Sickinger (1975), Sibbald and Kramer (1978), Wiseman et al. (1986), Ketels et al. (1987), Huyghebaert et al.

(1988), Fritsche et al. (1991), Satchithanandam et al. (1993), Blanch et al. (1995, 1996), Halle (1996), Vila and Esteve-Garcia (1996a,b), Dänicke et al. (1999a).
S. Dänicke
acid content (Young, 1961; Wiseman et al., 1991; Blanch et al., 1995; Vila and
Esteve-Garcia, 1996c).
Bile salt availability
Bile salt availability in young broilers plays a central role in determining the extent
to which fats differ in their digestibility. It has been suggested frequently that bile
availability in the very young chick is insufficient, particularly if high proportions of
tallow are provided as dietary fat. It was demonstrated that addition of bile or several
bile salts to broiler diets containing beef tallow resulted in an improvement in fat
digestibility (Fedde et al., 1960; Gomez and Polin, 1976; Katongole and March,
1980; Polin et al., 1980; Polin and Hussein, 1982). It might be concluded that
the addition of bile salts would be beneficial in the presence of large amounts of
undigested or non-absorbed fatty acids within the small intestine. The reason for
this bile salt deficiency in tallow-fed birds might be due to an increase in bile salt
excretion whereby bile salts escape the enterohepatic bile salt cycle. This in turn
changes hepatic cholesterol metabolism and the profile of synthesized bile salts and
ultimately decreases the intestinal bile acid pool, as shown by Monsma et al. (1996)
in an experiment with rats fed different dietary fats. These authors demonstrated a
strong linear relationship between dietary intake of stearic acid and muricholic
acid-derived bile salt excretion. Furthermore, intestinal microflora might also
contribute to bile salt metabolism, as shown by Kussaibati et al. (1982) using
conventional and germ-free chickens. They found that the addition of bile salt to the
diet markedly improved the digestibility of LCSFAs in conventional birds, whereas
only slight effects were observed in germ-free chickens. The benefit of adding bile
salts to either bird type was marginal for digestibility of LCUSFAs. The authors
concluded that intestinal microbes in conventional birds deconjugate bile salts,
which are poorly absorbed and consequently escape the enterohepatic bile salt cycle.
Kritchevsky and Story (1974) demonstrated that non-nutritive fibres from dif-
ferent feedstuffs are able to bind bile salts to a different degree in vitro. Consequently,
such binding could contribute to changes in the intestinal bile salt pool.
Triglyceride structure – fatty acid positional effects
Another important point that adversely influences the digestibility of dietary fats is
the positional distribution of the fatty acids at the glycerol molecule. Generally, high
proportions of the LCSFAs at the 2-position are associated with an increased micellar
solubility of these fatty acids in the form of the respective monoglycerides compared
with their relative solubility when released as free fatty acids from the 1- and
3-positions by the action of the lipase. It has been shown that a relatively high
proportion of the LCUSFAs in beef tallow (mainly oleic acid) are bound to the
2-position, and the majority of palmitic and stearic acid are esterified at the 1- and
3-positions (Sibbald and Kramer, 1977; Ketels et al., 1987; Bracco, 1994). Such
213
17 November 2000 15:10:58

204 S. Dänicke
positional effects are presumed to be detrimental to fat digestion and were

demonstrated to influence TME values of fats in broilers (Sibbald and Kramer,
1977), energy absorption in rats (Brink et al., 1995), and fat absorption in rats
(Mattson et al., 1979) and human infants (Filer et al., 1969).
Lipase availability
Lipase activity was reported to be low in the very young turkey (Krogdahl and
Sell, 1989) and chick (Nir et al., 1993) and lipase addition to a tallow-containing diet
was shown to improve fat digestibility (Polin et al., 1980). Noy and Sklan (1995) did
not detect any increase in ileal fat digestibility in chicks between 4 and 21 days of
age (6% unsaturated fat in the diet) despite an age-related increase in lipase secretion
into the duodenum. At present, no final conclusion can be drawn under which
circumstances lipase secretion might be a limiting factor in fat digestion.
Mineral soaps
Finally, it is evident from the literature that calcium or magnesium might reduce fat
absorption by forming indigestible soaps. This is of special importance if either fat
absorption is low, i.e. high intestinal amounts of fat as in tallow digestion, or higher
levels of those minerals are present in the diet (Fedde et al., 1960; Mattson et al.,
1979; Antoniou et al., 1980; Brink et al., 1995).
Although the above sections focus mainly on beef tallow and soybean oil, it should be
noted that the effects of the other dietary fats listed in Table 9.1 might be deduced
from their fatty acid profile and other physical and chemical characteristics in a
similar manner.
Role of Intestinal Viscosity in Interference of Fat Digestion

General remarks
Increases in intestinal viscosity of broilers are mainly caused by feeding higher

proportions of soluble NSPs such as the soluble pentosans in rye and wheat and
soluble mixed linked β-glucans in barley. Although both rye and barley are
comparable in soluble NSP content, the relationship between water extract viscosity
profile and soluble NSP concentration is quite different (Fig. 9.1). It has been
emphasized that, in addition to chemical analysis of polysaccharides, their physical
properties have to be considered in evaluation of their physiological effects (Morris,
1992). Molecular weight and degree of branching of the polysaccharide chain mainly
determine the so-called intrinsic viscosity, i.e. the fractional increase in viscosity per
unit concentration of polymer (Morris, 1992). Bedford and Classen (1992) reported
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24 November 2000 16:22:28

Fig. 9.1. Water extract viscosity of rye (v) and barley (r) as influenced by
incubation volume (Dänicke et al., 1999a).
a positive correlation between high molecular weight carbohydrate complexes

(molecular weight > 500,000) and viscosity in the intestine of chickens fed diets
varying in pentosan content. Hence, the distinct structural properties might account
for differences between rye and barley with respect to in vitro and in vivo viscosity.
Annison et al. (1995) compared soluble isolates from wheat and rice bran with
respect to chemical and physical properties and found that, although both isolates
were composed mainly of arabinoxylans, they produced extract viscosities of 64 and
1.6 mPa s, respectively. This was associated with arabinose : xylose ratios of 0.58
and 1.23, respectively. These authors suggested that rice bran xylan backbone carries
more arabinose side-chains, whereas the unsubstituted sections of the wheat xylan
main chain facilitated the formation of inter-chain hydrogen bonds. Consequently,
the degree of branching within wheat varieties could be a contributing factor to
differences in extract viscosity.
It has been suggested that a viscosity of a polysaccharide solution of
approximately 10 mPa s indicates a critical polysaccharide concentration above
which viscosity responds more steeply to increasing polysaccharide concentrations.
This is thought to be the result of the onset of formation of an entangled network
from individual polysaccharide coils (Morris et al., 1981). In spite of the use of
complete cereal matrices for in vitro viscosity measurements as shown in Fig. 9.1, it
can be clearly seen that rye solution viscosity started to rise more steeply in the range
between 1 : 6 and 1 : 5 dilution, which corresponded to viscosities of approximately
5–10 mPa s. From the same graph it might be concluded that such an entanglement
was not initiated in the case of barley. The corresponding ileal viscosities in broilers
fed diets containing rye and barley (69% dietary inclusion) were measured as 480.4
and 10.2 mPa s, respectively (Dänicke et al., 1999a).
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24 November 2000 16:46:36

206 S. Dänicke
Feed passage through the digestive tract, gut motility and unstirred
water-layer resistance
Carbohydrase supplementation of diets based on barley, rye or wheat has been shown
to accelerate the total gastrointestinal tract transit time in chickens (Jeroch et al.,
1990; Salih et al., 1991; Almirall and Esteve-Garcia, 1994; Dänicke et al., 1997a)
and in turkeys (Großer and Jeroch, 1997), which in turn suggests that an increased
intestinal viscosity would result in the opposite effect and would consequently reduce
feed intake and performance. Table 9.2 summarizes some results on measurement of
feed transit time, which is expressed as the time required for 50% of marker excretion
(T50). From the results of Van der Klis and Van Voorst (1993) it would appear that
dietary soluble NSP concentration is closely related to feed transit time. The effects of
Table 9.2. Mean total gastrointestinal tract transit time of feed, expressed as time required for
50% of excretion of a marker.
1. Bird type T50

Reference 2. Age Remarks Carbohydrase (h)
Salih et al. (1991) 1. Broiler

2. 2 weeks Without 7.59
With 6.31
2..4 weeks Without 6.8
High-viscosity barley based diets With 6.45
With 6.7
With 7.21
Van der Klis and 1. Broiler Without carboxy methyl cellulose 4.58
Van Voorst (1993) 2. 5 weeks 10 g carboxy methyl cellulose 5.35
30 g carboxy methyl cellulose 7.2
Almirall and 1. Broiler High-viscosity barley Without 8.89
Esteve-Garcia 2. 3 weeks With 5.49
(1994) 1. Cocks High-viscosity barley Without 3.39
2. 1 year With 4.81
Dänicke et al. 1. Broiler Rye-based diet, 10% soybean oil Without 8.38
(1997a) 2. 4 weeks Rye-based diet, 10% soybean oil With 6.68
Rye-based diet, 10% beef tallow Without 7.96
Rye-based diet, 10% beef tallow With 6.88
Großer and Jeroch 1. Turkey Wheat-based diet, 8% soybean oil Without 7.9
(1997) 2. 3 weeks Wheat-based diet, 8% soybean oil With 8.36
Wheat-based diet, 8% beef tallow Without 14.52
Wheat-based diet, 8% beef tallow With 9.4
Wheat-based diet, 8% coconut oil Without 5.58
Wheat-based diet, 8% coconut oil With 6.07
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17 November 2000 15:11:24

different fat types and xylanase supplementation on transit times were investigated by
Dänicke et al. (1997a,b) in broiler chickens and by Großer and Jeroch (1997) in
young turkeys. Xylanase addition resulted in an accelerated transit time for broilers
fed on soya oil and beef tallow. A fat effect was not obvious in broilers. In contrast,
different fat and enzyme effects were observed in turkeys. Xylanase addition to diets
containing soya oil or coconut oil changed the transit time only slightly, whereas the
effect was dramatic (reduced transit by 5 h) in the diet containing beef tallow. Pas-
sage rate was fastest with the coconut oil diet followed by the diets containing soya oil
and finally beef tallow. A comparison between species makes it clear that the young
turkey seems to be more responsive to different fat types than the growing chick.
In a more detailed study, Dänicke et al. (1999b) examined the mean retention
time (MRT) of digesta in different segments of the digestive tract of broilers fed
rye-based diets with differing levels of added dietary fat type and xylanase (Fig. 9.2).
Again, no significant fat effect was observed. The majority of the reduction in MRT
on addition of xylanase was observed in the jejunum and ileum. Experiments with
humans and pigs revealed that an increased digesta viscosity delayed gastric emptying
(Furuya et al., 1978; Blackburn et al., 1984), thereby reducing the overall feed transit
time through the entire digestive tract. Sudendey and Kamphues (1995) observed
an accelerated rate of gastric emptying when diets based on wheat or barley fed to
piglets were supplemented with a carbohydrase. Furthermore, Meyer et al. (1986)
concluded from their experiment with dogs that meal viscosity affects gastric sieving,
which in turn could influence both gastric and intestinal digestion of nutrients. Such
effects of viscosity on gastric emptying rate have not been observed with broilers and
Fig. 9.2. Effect of fat type and xylanase supplementation on mean retention time
in successive segments of the gastrointestinal tract of male chickens (day 24 of
age, average of four birds per treatment ± standard error) (Dänicke et al., 1999b).
Crop, ]; proventriculus plus gizzard, &; duodenum, $; jejunum, *; ileum, \;
rectum, *.
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21 November 2000 15:04:06

208 S. Dänicke
the effect of different fat types on the contribution of gastric emptying to overall feed
transit changes (Fig. 9.2) has proved inconclusive. In this regard the chicken appears
to differ substantially from other monogastrics.
However, a general effect of fat as such on the MRT of digesta in the small
intestine cannot be excluded completely since its presence modifies gut motility via
stimulation of gastrointestinal neurotransmitters or hormones. Neurotensin has been
shown to decrease the frequency and strength of contractions of the gizzard and
partially of the duodenum and ileum in broilers (Degolier et al., 1997). The presence
of lipids in the duodenum was found to inhibit gastric motility in turkeys (Duke
and Evanson, 1972). Degolier et al. (1997) reported that neurotensin was released
into the hepatic-portal circulation in response to the presence of oleic acid in the
duodenum. It was suggested that, by a parallel decrease in gut motility, the efficiency
of digestion could be enhanced by a longer MRT of digesta in the intestine.
Moreover, fats differing in their contents of unsaturated and saturated fatty acids
are not equivalent in their stimulation of release of neurotensin and other gastro-
intestinal peptides. According to the results of Sagher et al. (1991) obtained with rats,
maize oil and olive oil increased neurotensin and substance P, whereas butterfat did
not, when compared with the low fat control group.
It might be concluded from these findings that both intestinal viscosity and
intestinal fatty acid composition contribute to differences in the MRT of digesta in
intestinal segments and consequently the overall gastrointestinal tract transit time,
albeit by different mechanisms. Hence, an interaction between both factors cannot
be excluded in interpreting the results shown in Fig. 9.2.
A reduction in gut motility, caused by increased viscosity, decreases the mixing
of digesta with pancreatic and biliary secretions, which is of particular importance
for emulsification of saturated fatty acids and formation of micelles. It has been
suggested by Smulikowska (1998) that gut motility-mediated shuttling of digesta
between the duodenum and gizzard increases the time of feed exposed to digestive
enzymes and favours enhanced digestion and absorption in the upper parts of the
small intestine. At the same time as intestinal viscosity is increased and mixing is
decreased, the thickness of the unstirred water layer (UWL), an extracellular barrier
covering the intestinal microvilli, is proportionally increased (Johnson and Gee,
1981, 1982; Flourie et al., 1984; Lund et al., 1989). Westergaard and Dietschy
(1976) proposed that the principal role of the micelle is to overcome the resistance of
that UWL. Consequently, an increased thickness of the UWL would increase this
resistance and decrease the transfer rate between intestinal bulk and the absorptive
site, which is, again, of particular importance for saturated fatty acids. In addition,
Smulikowska (1998) suggested that, especially in the young chicken, an increase in
the UWL would decrease the effective intraluminal diameter and as a result the rate
of digesta transit and mixing would be further impeded. However, this concept of the
UWL is open to debate. Edwards et al. (1988) and Read and Eastwood (1992)
pointed out that a reduction in mixing and an increase in the UWL are two ways of
describing the same thing. Nevertheless, strong relationships between the thickness
of the UWL and the malabsorption of sugars and of cholesterol have been reported
(e.g. Lund et al., 1989). Cholesterol absorption by everted intestinal sacs was
218
17 November 2000 15:11:49

significantly reduced even at low concentrations of oat gum and continued to fall as
the concentration increased, but proportionately less with each increment (Lund
et al., 1989). The authors concluded that this initial decrease could indicate a sieving
effect of dispersed β-glucan polymer on the micelles, which have relatively high
molecular volume (Phillips, 1986). Interestingly, this decrease coincided with a
relatively low in vitro viscosity of approximately 5–20 mPa s, which is the range
where polysaccharides in solution start to form an entangled network from individual
polysaccharide coils (Morris et al., 1981; see above section), associated with a steep
incremental increase in viscosity. Moreover, higher intestinal lipid concentrations are
paralleled by a corresponding increase in the size of the formed micelles (Ockner
et al., 1972b). This would explain why tallow feeding is more dramatically sensitive
to small increases in intestinal viscosity when compared with soybean oil feeding.
Availability of endogenous lipase and bile salts
Experiments dealing with the effects of viscous carbohydrates on lipase activity in the
intestinal contents and pancreas in relation to fat absorption are often inconclusive
and the underlying mechanisms are not fully understood. Isaksson et al. (1983a)
found an increased lipase output in rats fed a pectin-containing diet, whereas lipase
concentration per gram wet material was not changed. Total fat excretion in the
faeces was increased at the same time. These results show that pectin obviously
stimulated pancreatic lipase output but do not suggest that specific lipase activity
was responsible for the increased fat excretion. In contrast, feeding of neither low
methylated or high methylated pectin nor of wheat bran changed specific lipase
activity (units per mg) or total lipase output in rats (Isaksson et al., 1983b), whereas
protein concentration and protein content of the pancreas were altered. These results
were interpreted as adaptive changes in pancreatic enzyme production. In another
experiment, Isaksson et al. (1984) found a decrease in specific lipase activity (units
per ml) after consumption of a pectin-containing test meal in pancreatectomized
patients treated with a granulated pancreatic enzyme preparation. This decrease was
accompanied by a decrease in 14CO2 excretion after 14C triolein ingestion, which
would indicate that lipase activity indeed became a limiting factor. However, the
effect of pancreatic regulation due to pectin ingestion could not be investigated with
such an experimental design.
The decrease of in vitro lipase activity and activity of other pancreatic enzymes in
human duodenal juice was shown to be directly related to an increased viscosity
(Isaksson et al., 1982). It was also suggested that adsorption of enzymes by specific
protein enzyme inhibitors could play a role in activity depression in the case of wheat
bran. Moreover, it was shown that inhibition in lipase activity due to increased
viscosity occurred in a relatively short period of time and it remained at these
depressed levels thereafter. Therefore, it was suggested that these inhibitory effects
would be relatively independent of intestinal passage time. Finally, lipase activity
decreased as the pH was lowered from 5.7 to 5. Dänicke et al. (1997a) reported a
significant decrease in pH in the small intestine of broilers fed tallow-containing
219
17 November 2000 15:11:50

210 S. Dänicke
diets. Whether such a decrease could be a contributing factor in lower tallow

digestibility remains to be clarified.
Almirall et al. (1995) reported the specific lipase activity to be decreased in
broiler chickens and cocks after feeding barley, compared with maize. Activity was
partially restored after β-glucanase supplementation of the barley-based diets. In
addition, chickens had higher specific lipase activities and seemed more susceptible
to dietary treatments than cocks.
Dänicke et al. (1999c) demonstrated that the provision of a xylanase decreased
specific lipase activity (units per gram wet weight) in the jejunal contents and in the
pancreas of broilers fed rye-based diets supplemented with beef tallow, whereas no
effect was noted in diets supplemented with soybean oil. Total pancreatic lipase
activity remained unchanged but was significantly increased on body weight basis in
birds fed the unsupplemented tallow-containing diet. In addition, relative pancreatic
lipase activity was linearly dependent on relative pancreas weight. It would appear,
therefore, that birds fed the unsupplemented tallow-containing diet had attempted
to increase lipase activity to compensate for such poor fat digestibility and that this
was reflected in low body weights and the highest specific intraluminal lipase activity.
A decreased pancreatic lipase activity and an increased intestinal activity in rats fed
guar gum was reported by Poksay and Schneeman (1983), which suggests a similar
response. It was concluded that the observed reduction in fat digestibility following
guar gum addition to the diet could not be the result of an insufficient presence
of lipase in the intestine. Furthermore, Silva et al. (1997) found intestinal lipase
activity (corrected for body weight and weight of small intestine and contents) to be
significantly decreased in broilers fed xylanase-added rye-based diets. Interestingly,
when intestinal contents where examined in vitro, lipase activity was slightly
increased after the addition of the xylanase when compared with measured activities
before xylanase administration. It was suggested that the xylanase released some
lipase activity, which had been adsorbed by dietary fibre in the intestines of birds fed
the unsupplemented diets. Adsorption of lipase and bile salts to different fibre types
was examined by Lairon et al. (1985). It was found that binding capacity of cellulose,
purified xylan and pectin was negligible, whereas wheat bran was shown to have a
moderate capacity. Furthermore, fine wheat bran bound more lipase and bile salts
than coarse wheat bran. Pasquier et al. (1996) demonstrated, by in vitro experiments
using reconstituted duodenal medium, that the amount of emulsified lipids was
reduced and the size of emulsified droplets was increased as the concentration of
viscous fibre was increased. In the range of 0–20 mPa s the correlations to both
parameters were r = −0.79 and r = 0.88, respectively. In addition, triglyceride
hydrolysis was reduced by approximately 30% over a similar range of viscosity.
Intestinal viscosity also influences bile salt metabolism in rats. Inclusion of
psyllium, a dietary fibre rich in soluble components, was shown to increase the
excretion of bile salts and total steroids in rats significantly when compared with
inclusion of cellulose at a similar level (Buhman et al., 1998). The authors suggested
intestinal viscosity itself to be responsible for a decreased reabsorption of bile-salts
rather than bile-salt binding per se, since psyllium does not bind bile salts in vitro.
Furthermore, the hepatic cholesterol metabolism was up-regulated, as suggested by
220
17 November 2000 15:11:51

elevated activities of cholesterol 7α-hydroxylase and cholesterol 7α-hydroxylase

mRNA as a result of psyllium feeding. Ikegami et al. (1990) reported a linear
relationship between intestinal viscosity and the secretion of pancreatic enzymes and
total bile salts in rats which was accompanied by an increased pancreatic weight. It
was suggested that the animal compensates for the inefficiency of nutrient digestion
with an enlargement of digestive organs and increased secretion of digestive juices.
Gut morphology and microbial implications
Feeding soluble NSP was shown to increase the length and the absolute and relative
weight of the small intestine in rats (Johnson et al., 1984; Johnson and Gee, 1986)
and broilers (Simon, 1998) (Table 9.3). It was suggested by Johnson et al. (1984)
that the presence of large amounts of unabsorbed material in the intestine exerts
trophic effects on the intestinal mucosa. Feeding of guar gum and carboxy methyl
cellulose was shown to increase the cell division rate in the small intestine, colon and
caecum of rats, which was accompanied by reduced activity of some mucosal
enzymes (Johnson et al., 1984; Johnson and Gee, 1986). It was hypothesized that
increased cellular proliferation gives rise to a reduction in lifespan of mucosal cells
as a result of an increased rate of shedding of immature (with respect to enzyme
expression) cells at the apex of the villi. Guar gum and carboxy methyl cellulose
differed, however, in that the latter induced a greater rate of cell division and in the
more distal regions of the small intestine. The authors concluded that factors others
than intestinal viscosity per se could be involved, such as microbial activity in the
small intestine. Rakowska et al. (1993) found severe damage of intestinal villi and
mucous membranes of the duodenum and small intestine in broilers after feeding a
rye-based diet. Microbial involvement was concluded from this experiment since the
antibiotic Nisine protected the intestinal villi. An impact of intestinal viscosity on
gut morphology was also shown by Drakley et al. (1997) in that feeding of a highly
viscous wheat resulted in a significantly increased villus height and width when
compared with a low viscosity wheat. Crypt cell proliferation rate is also increased
with increasing intestinal viscosity in broilers, as demonstrated by a decrease after
feeding of xylanase-supplemented rye-based diets (Silva and Smithard, 1997).
Dietary fat type might also contribute to changes in intestinal morphology,
as shown by Sagher et al. (1991), who found that feeding of maize oil and olive oil
significantly increased villus height compared with a low-fat control diet, whereas
feeding of butterfat significantly decreased villus height. The question of whether
changes in intestinal viscosity interact with such a response remains unanswered.
Dänicke et al. (1999b) examined the effect of xylanase supplementation of rye-
based diets containing either soybean oil or beef tallow offered to broilers on tissue-
associated bacterial groups in several segments of the digestive tract (Table 9.4).
Increased intestinal viscosity is largely influenced by feeding of such diets as shown in
related experiments (Dänicke et al., 1997a,c) (Table 9.5; Fig. 9.3) and might have
caused the described intestinal morphological changes, which lead to reduced coloni-
zation surface, altered adhesion structures and an increased release of epithelial cells
221
17 November 2000 15:11:51

212
24 November 2000 16:23:55

Table 9.3. Effects of dietary fat type (S, 10% soybean oil; T, 10% beef tallow) and enzyme supplementation (−, without; +, with 1 g Avizyme 1300 kg−1 diet) in a
rye-based diet (56% dietary inclusion) on protein metabolism in male broilers (Dänicke et al., 2000a,b).
Duodenum Jejunum Ileum Pancreas

Endogenous digestive
g 100 g−1 kS g 100 g−1 kS g 100 g−1 kS g 100 g−1 kS N-losses
Fat type LW (% day−1)* LW (% day−1)* LW (% day−1)* LW (% day−1)* mg day−1 per LW (kg)0.67
222
S− 1.5a 56a 2.1a 51a 1.3a 53ab 0.4ab 52ab 87
S+ 1.4a 64a 2.0a 52a 1.2a 56ab 0.4ab 45ab 69
T− 1.9b 84b 2.6b 75b 1.6b 68bb 0.6bb 118bb 244
T+ 1.4a 61a 1.9a 58a 1.2a 50ab 0.5ab 39ab 81

* Fractional rates of protein synthesis, i.e. daily protein synthesis as percentage of tissue protein.
abValues with different superscripts within the columns are significantly different (P < 0.05).
CHAP-9
S. Dänicke
Table 9.4. Tissue-associated bacterial counts in different segments of digestive tract of chickens
(day 16 of age) as influenced by dietary fat type and xylanase supplementation (CFU, log10)
(Dänicke et al., 1999b).
Intestinal Entero- Gram-positive Entero- Total

Fat Xylanase segment bacteria cocci cocci anaerobes
Soybean oil Without Crop 6.58ba 5.04aa 4.18ab 5.89ab

Soybean oil 3000 IU kg−1 diet Crop 5.38aa 5.59ab 4.46ab 6.02bb
Tallow Without Crop 5.53aa 6.22ba 4.00ab 6.76bb
Tallow 3000 IU kg−1 diet Crop 5.94ab 5.14aa 4.28ab 4.82ab
Soybean oil Without Duodenum 6.26ba 3.96ab 3.16aa 5.74ab
Soybean oil 3000 IU kg−1 diet Duodenum 3.54aa 4.26ab 3.88ba 4.29ab
Tallow Without Duodenum 4.96ca 5.01ab 4.09ba 5.48ab
Tallow 3000 IU kg−1 diet Duodenum 5.19ca 4.72ab 3.08aa 4.66ab
Soybean oil Without Jejunum 6.35ba 4.62ba 3.43ab 6.20ab
Soybean oil 3000 IU kg−1 diet Jejunum 4.42aa 4.06aa 2.95ab 4.72ab
Tallow Without Jejunum 5.73ba 5.26ca 4.01ab 5.35ab
Tallow 3000 IU kg−1 diet Jejunum 5.71ba 4.82ba 3.88ab 5.30ab
Soybean oil Without Ileum 6.13ba 4.25aa 3.80aa 6.50ab
Soybean oil 3000 IU kg−1 diet Ileum 6.13ba 4.85ab 4.29ab 5.97ab
Tallow Without Ileum 6.49ba 6.35ca 5.90ba 6.49ab
Tallow 3000 IU kg−1 diet Ileum 5.03aa 5.06ba 3.58aa 5.52ab
Probability:
Fat 0.7967 < 0.001 0.0555 0.3749
Enzyme < 0.001 0.0147 0.1083 < 0.001
Intestinal segment < 0.001 < 0.001 < 0.001 0.0169
Fat × enzyme < 0.001 < 0.001 0.0080 0.6742
Fat × intestinal segment 0.1243 0.0849 0.0603 0.9765
Enzyme × intestinal segment 0.0869 0.3808 0.0410 0.9328
Fat × enzyme × intestinal segment < 0.001 0.0067 0.0012 0.0805
Pooled standard error of means 0.29 0.25 0.38 0.52
a–cValues with no common superscript within columns differ significantly (P < 0.05).
(and adhering bacteria) into the lumen as a result of stimulation of the proliferation
rate of mucosal cells. The observed effects on microbial colonization were most
pronounced in duodenal and jejunal samples, which exhibited short digesta retention
times and thus low steady-state concentrations of soluble arabinoxylans.
The response of cocci bacteria to enzyme supplementation seemed less drastic,
which indicates that this group of bacteria is less affected by morphological changes
or nutrient composition. On the other hand, supplementation with tallow resulted
in an increased number of cocci in the jejunum and ileum, but seemed to depress
growth of total anaerobic bacteria and enterobacteria. Significant higher pH values
were measured in most intestinal segments when tallow was used as dietary fat
223
17 November 2000 15:11:54

214
Table 9.5. Effect of dietary fat type and carbohydrase supplementation of broiler and turkey diets on body-weight gain (BWG, g), feed-to-gain ratio (FCR, g g−1),
intestinal viscosity (mPa s), apparent fat digestibility (FD%) and apparent protein digestibility (PD%).
17 November 2000 15:11:55

1. Bird type
2. Age (balance exp.) Dietary cereal inclusion (%) Dietary fat inclusion (%)
3. Age (growth exp.)
Ref no. 4. ANOVA (P) R T W M B S T L AB C E BWG FCR Vis1 FD2 PD2
[1] 1. Broiler 61 10.0 − 82.3 90.9

2. Days 7–11 61 10.0 + 87.3 91.3
61 10 − 34.0 88.8
61 10 + 51.0 89.9
1. Days 21–25 61 10.0 − 74.1 91.5
61 10.0 + 83.1 91.1
61 10 − 23.1 87.0
224
61 10 + 50.2 93.0
1. Days 32–36 61 10.0 − 62.7 89.3
61 10.0 + 79.8 89.2
61 10 39.2 79.9

−
61 10 + 73.6 88.9
3. Days 1–28 61 10.0 − 1130 1.464 250.0
61 10.0 + 1275 1.331 150.0
CHAP-9
61 10 − 138 4.254 865.0

61 10 + 1064 1.573 189.0
4. Fat *** *** *** ***
1. Enzyme *** *** *** ***
1. Age * ***
1. Fat × enzyme *** *** *** ***
S. Dänicke
1. Fat × age *** *

1. Enzyme × age * *
1. Fat × enzyme × age NS *
1. Broiler 56 10.0 − 749 1.348 17.0 91.7 87.8
17 November 2000 15:11:56

2. 21 days 56 10.0 + 761 1.352 9.3 93.0 87.9

3. Days 1–35 56 10 − 673 1.480 21.9 45.3 86.2
56 10 + 714 1.397 12.4 62.8 88.4
4. Fat *** *** ** *** NS
4. Enzyme NS ** *** ** NS
4. Fat × enzyme NS ** NS ** NS
[2] 1. Broiler 56 10.0 − 826 1.262 8.8 87.2 85.2
Use of Feed Enzymes in Broilers
2. 21 days 56 10.0 + 846 1.240 4.7 90.0 88.7

3. Days 1–35 56 10 − 778 1.366 10.2 67.4 88.3
56 10 + 824 1.291 4.6 66.7 89.2
4. Fat ** *** NS *** NS
225
4. Enzyme ** * *** NS *
4. Fat × enzyme NS NS NS NS NS
[3] 1. Turkeys 48 8.0 − ac1798ac ac1.53ac a87a.0 a87a.0
c b

2. Days 15–20 48 8.0 + 1847a 1.39b a86a.0 a91c.0
3. Days 15–42 48 8 − c1651b b1.64c a54b.0 ab88ab.0
48 8 + ac1801ac bb1.52ac a62c.0 a91c.0
48 8 − c1745c bb1.56ac a82a.0 a91c.0
CHAP-9
48 8 + c1867a bb1.48ab a79a.0 ab90bc.0
4. Fat ** * *** NS
4. Enzyme *** *** NS NS
4. Fat × enzyme NS NS NS NS
215
216
1. Bird type
2. Age (balance exp.) Dietary cereal inclusion (%) Dietary fat inclusion (%)
3. Age (growth exp.)
Ref no. 4. ANOVA (P) R T W M B S T L AB C E BWG FCR Vis1 FD2 PD2
226
[2] 1. Broiler 56 10.0 − 748 1.253 3.9 92.3 91.3
2. 21 days 56 10.0 + 742 1.236 2.9 93.1 90.9
3. Days 1–35 56 10 − 685 1.353 3.2 65.2 92.0
56 10 + 705 1.307 2.4 63.4 90.6

4. Fat *** *** *** *** NS
4. Enzyme NS ** *** 0.05 NS
4. Fat × enzyme NS NS NS NS NS
CHAP-9
[2] 1. Broiler 56 10.0 − 760 1.276 13.8 86.3 83.0
2. 21 days 56 10.0 + 797 1.257 5.0 90.2 88.3
3. Days 1–35 56 10 − 641 1.466 23.8 51.3 83.8
56 10 + 755 1.344 7.6 59.4 88.7
4. Fat *** *** ** *** NS
4. Enzyme *** *** *** NS ***
S. Dänicke
4. Fat × enzyme * ** NS NS NS
[4] 1. Broiler 30 29 6.0 − 730 2.06 49.8 75.9 83.8
2. Days 21–24 30 29 6.0 + 783 1.76 16.5 84.8 85.7
3. 30 29 3.0 3 − 712 2.12 41.2 61.7 86.3
30 29 3.0 3 + 765 1.81 16.4 73.9 86.5
30 29 6 − 681 2.33 30.7 45.5 85.2
30 29 6 + 777 1.93 20.5 63.1 86.1
30 29 6 − 690 2.07 46.2 66.4 85.6
30 29 6 + 804 1.83 17.9 77.7 85.7
4. Fat NS *** NS *** **
4. Enzyme *** *** *** *** NS
4. Fat × enzyme NS NS *** NS NS
Use of Feed Enzymes in Broilers
[5] 1. Broiler 10 50 6.5 − b636b bb1.610ab a78.3c
2. Days 24–28 10 50 6.5 + b638b b1.556ab a80.2c
3. Days 1–21 10 50 0.5 6.0 − b567a b1.748cb a60.5a
227
4. 10 50 0.5 6.0 + b621b b1.644bb a69.9b
[1] Dänicke et al. (1997b), [2] Dänicke et al. (1999g), [3] Großer and Jeroch (1997), [4] Smulikowska and Mieczkowska (1996), [5] Langhout et al. (1997).
1 Intestinal viscosity measured in digesta of jejunum plus ileum (reference [4]) or in digesta of ileum (references [1] to [3]).
2 Digestibility was measured at the faecal level (references [1], [3], [4], [5]) or at the terminal ileum (reference [2]).

Abbreviations: R, rye; W, wheat; T, triticale; M, maize; B, barley; S, soya oil; T, beef tallow; L, lard; AB, animal fat blend; C, coconut oil; E, enzyme preparation.
a–cValues with no common superscript are significantly different within columns (P < 0.05).
Probability: *P < 0.05; **P < 0.01; ***P < 0.001; NS, not significant.
CHAP-9
217
218 S. Dänicke
Fig. 9.3. Effects of dietary proportion of rye, dietary fat type and xylanase
supplementation on jejunal and ileal supernatant viscosity of broilers (Dänicke
et al., 1999c). (Without xylanase, –––; with xylanase, -----; jejunum, q; ileum, r.)
instead of soybean oil (Dänicke et al., 1997a). Thus, fat type clearly influences the
microbial composition in the intestine independently of viscosity. It is not known
if cocci growth was actively stimulated by tallow addition or if it was merely a result
of inhibition of other bacteria, possibly due to increased secretion of bile acids by
the host. Bile acids have a bacteriostatic effect, but this effect varies with each species
of interest. Cocci can be distinguished by their ability to grow in the presence of
relatively high concentrations of bile salts; thus the most resistant cocci, such as
Enterococci, may become dominant under higher bile acid concentrations. Active
de-conjugation of conjugated bile acids, as carried out by some lactobacilli, also
occurs in the intestinal tract, but it is not known if the generated bile salt hydrolase
activity is sufficient to interfere quantitatively with the bile salt metabolism.
Interactions between Dietary Fat Type and Carbohydrase

Addition in Broiler Diets Containing Different Cereals
General remarks
In the previous section an attempt was made to explain why exogenously supplied
carbohydrases capable of reducing intestinal viscosity could result in different effects
depending upon, amongst other factors, the chemical composition of the dietary fats
employed.
Several experiments have demonstrated that carbohydrase supplementation of
broiler and young turkey diets results in far greater responses in nutrient digestibility
and performance when the fat source contains more LCSFAs. Additionally, the
performance and nutrient digestibility responses are more closely related to changes
228

in intestinal viscosity in animals fed such fats. Therefore, at a comparable total fat
supply, the response to carbohydrase supplementation would increase in the order
maize < wheat < wheat/rye, triticale, barley < rye/wheat < rye (Table 9.5) since this
is the general order of increasing viscosity. Whilst this order will remain regardless
of dietary fat type, the relative differences between grains will be much smaller if
fat blends (Table 9.5) or lower dietary fat proportions are used. Under practical
conditions, one can expect a fat blend in most cases rather than pure fats in broiler
diets, for several reasons. Full-fat seeds (especially soybean) are often used as fat or
energy sources for feed-manufacture reasons and to decrease the proportion of
‘free’ supplemented fat. The use of maize or oats will itself increase the dietary plant
oil proportion substantially. Thus, the scale of response seen on enzyme supplement-
ation is mediated by total dietary fat concentration, fatty acid profile and triglyceride
structure, mineral content and age of the birds (see above sections). The interactions
are considered in more detail in the following sections.
Intestinal viscosity
It is now well established that the anti-nutritive effects of soluble NSP in

broilers are mediated by an increase in intestinal viscosity. It can be seen from the
results presented in Table 9.5 that intestinal viscosity was markedly decreased by
carbohydrase supplementation. Significant interactions between dietary fat type and
carbohydrase addition were especially observed in rye-fed birds in the experiments
by Dänicke et al. (1999c,d) (Fig. 9.3) in that enzyme effects were more pronounced
in tallow-fed birds. It was concluded that tallow itself, and other unabsorbed
materials, contributed to intestinal viscosity. These results are in contrast to those
reported by Smulikowska and Mieczkowska (1996), who found the xylanase-
dependent decrease in intestinal viscosity to be more pronounced for birds fed
soybean oil than for those fed tallow (Table 9.5). For excreta, however, a substantially
higher viscosity was measured in broilers fed unsupplemented diets containing
tallow or lard, whereas xylanase supplementation reduced excreta viscosity to
comparable levels for all fat types tested that caused the significant interactions
between fat type and enzyme addition. It was suggested that more insoluble NSP
dissolved in the last part of the digestive tract, due to a decrease in MRT mediated
by undigested fat and viscosity that could have contributed to the increased excreta
viscosity. It should be borne in mind that the described steep response of viscosity
to small incremental changes in polysaccharide concentration above a viscosity of
10–20 mPa s might cause a remarkable variation in viscosity even in the same
experimental groups.
Digestibility of fat and fatty acids
Carbohydrase supplementation of broiler diets based on rye, rye/wheat, triticale or

barley resulted in larger improvements in fat digestibility if the dietary fat was of
229
17 November 2000 15:12:24

220 S. Dänicke
animal origin compared with plant. In simple terms, it could be concluded that there
is more scope for improvement in fat digestibility when it is low to begin with. This
interpretation is probably the reverse of how such data should be viewed. An increase
in intestinal viscosity depresses fat digestibility more in animal fat-based diets, due to
the mechanisms explained above. Consequently, a reduction in viscosity due to
enzyme addition in such diets should exert a more pronounced effect. Digestibility
of different dietary fat types clearly demonstrated what was expected, i.e. a relatively
high digestibility of soybean oil, which was less drastically modified over a wide range
of dietary soluble NSP concentrations, and consequently intestinal viscosity, than
beef tallow. Intermediate effects are observed for lard and animal and vegetable fat
blends. That enzyme effect on fat digestibility strongly depends on dietary pentosan
concentration was clearly demonstrated in dose–response studies by Smulikowska
and Mieczkowska (1996) where significant interactions described by greater enzyme
responses with higher rye proportions were observed (Fig. 9.4). Moreover, Dänicke
et al. (1999c) showed that this relationship is further modified by dietary fat type.
The use of beef tallow in place of of soybean oil resulted on the one hand in a greater
reduction in fat digestibility with increasing proprtions of rye in the diet, and
consequently on the other hand in greater enzyme effects in such diets (Fig. 9.5) –
hence the significant interactions between fat type and rye proportion, and between
rye proportion and enzyme supplementation. However, in evaluation of such effects
it should be stressed that the effects of enzyme on fat digestibility decrease as birds
age (Fig. 9.6). That such age-related effects were not observed in the experiments
by Dänicke et al. (1997b) (Table 9.5) might be explained by the restricted feeding
Fig. 9.4. Effect of dietary rye proportion and xylanase addition (1.5 g Avizyme
Tx per kg of each diet) on apparent fat digestibility and metabolizability of
energy in broilers (each diet contained 4.25% soybean oil and 4.25% lard;
data from Smulikowska and Mieczkowska, 1996). (Apparent fat digestibility, –––;
metabolizability of engergy, -----; without xylanase, r; with xylanase, v.)
230
17 November 2000 15:12:48

regimen applied in these tests, which might modify the relationship between feed
supply, body weight, endogenous fat loss and mean retention time.
The differences in crude fat digestibility can be explained by differences in
digestibility of the individual fatty acids (Table 9.6, Figs 9.7 and 9.8). Unsaturated
fatty acids were more efficiently absorbed than saturated. The digestibility of
saturated fatty acids decreased with increasing chain length. Within the C18 family,
digestibility increased with increasing number of double bonds. These general
Fig. 9.5. Effect of dietary proportion of rye, dietary fat type and xylanase
supplementation on apparent crude fat digestibility at the terminal ileum
(from Dänicke et al., 1999c). Without xylanase, q; with xylanase, w.
Fig. 9.6. Effect of age and β-glucanase addition on fat digestibility in broilers fed
barley-based diets (60% and 70% barley and 5% and 6% tallow inclusion in starter
and grower diets, respectively; data from Salih et al., 1991). Without β-glucanase,
r; with β-glucanase, v.
231
17 November 2000 15:13:33

222
17 November 2000 15:13:35

Table 9.6. Effect of dietary fat type and carbohydrase supplementation on faecal digestibility of fatty acids in broilers and turkeys.
1. Bird type Dietary cereal inclusion (%) Dietary fat inclusion (%)
2. Age Carbo-
Ref no. 3. ANOVA (P) Rye Wheat Soybean oil Beef tallow Coconut oil hydrase C12 : 0 C16 : 0 C18 : 0 C18 : 1 C18 : 2 C18 : 3
[1] 1. Broiler 61 10 − 74.3 54.1 81.0 88.6 92.1

2. Days 21–25 61 10 + 81.2 61.8 86.6 92.2 94.9
61 10 − 39.0 23.4 59.5 64.3 72.3
61 10 + 46.5 28.8 71.2 74.7 80.5
3. Fat *** *** *** *** ***
3. Enzyme NS NS * * NS
NS NS NS NS NS
232
3. Fat × enzyme
[2] 1. Turkeys 48 8 67.3b 80.9a 69.0a 86.2a 88.7a 91.2a
2. Days 15–20 48 8 c c
65.8b 78.2a 66.9a 86.6a 87.9ab 89.1ab
48 8 b
79.6ab 40.1c 24.4c 74.0b 76.1c 83.1c

48 8 c c c cc
90.8a 49.6bc 34.7bc 84.6a 81.8bc 87.0abc
48 8 c cc cc
94.6a 58.1b 46.3b 79.1ab 82.4abc 86.5abc
48 8 93.8a 56.9b 44.6b c78.6ab 80.9c c84.1bc
CHAP-9
[1] Dänicke et al. (1997b), [2] Großer and Jeroch (1997, personal communication).
a–cValues with no common superscript are significantly different within columns (P < 0.05).
Probability: * P < 0.05; *** P < 0.001; NS, not significant.

S. Dänicke
relationships were shown to be influenced by dietary rye proportion and dietary fat
type, as shown in Figs 9.7 and 9.8. However, the relative decrease in digestibility of
saturated fatty acids as rye replaced wheat was greater for the tallow-supplemented
rations. Replacing wheat with rye reduced digestibility of palmitic and stearic acid
by approximately 24% and 48%, respectively, in birds fed soybean oil, whilst for
Fig. 9.7. Effect of dietary proprotion of rye, dietary fat type and xylanase
supplementation on apparent fatty acid digestibility at the terminal ileum (from
Dänicke et al., 1999c). Without xylanase, –––; with xylanase, -----; stearic acid, r;
oleic acid, q; linoleic acid, v.
Fig. 9.8. Apparent fatty acid digestibility as influenced by dietary fat type and
xylanase supplementation at different measurement sites (from Dänicke et al.,
1999c). Without xylanase, –––; with xylanase, -----; stearic acid, r; oleic acid, q;
linoleic acid, v.
233
17 November 2000 15:14:21

224 S. Dänicke
tallow-fed birds this increased to 44% and 59%, respectively. In contrast to

tallow-fed birds, digestibility of unsaturated fatty acids in those fed soybean oil was
less affected by intestinal viscosity. In all cases, increased viscosity reduced saturated
fatty acid digestibility more dramatically compared with unsaturated fatty acids,
regardless of source. However, the original source of the fatty acids had a marked
influenced on the absolute response in fatty acid digestibility to increased intestinal
viscosity. Increased viscosity reduced digestibility of a given fatty acid to a greater
extent when it was derived from tallow than when it was derived from soybean oil.
Significant interactions between fat type and xylanase supplementation are
caused by higher enzyme effects in tallow-fed birds and significant interactions
between rye level and enzyme supplementation reflect increasing xylanase effects
with increasing rye proportions, especially in tallow-fed birds.
The effects of a xylanase addition to a turkey diet containing coconut oil
(Table 9.6) have to be evaluated in a different way. Whereas both soybean oil and
beef tallow are composed of fatty acids with a chain length of 16–18 C-atoms, more
than 50% of coconut oil is saturated fatty acids with fewer than 14 C-atoms. These
shorter fatty acids are less dependent on bile salts and micelle formation than their
longer-chained counterparts, which is clearly indicated by greater absorption of lauric
acid in poults fed coconut oil (Table 9.6). The digestibility of palmitic and stearic
acid from coconut oil lies between that of soybean oil and tallow fed to poults. There
were virtually no differences in the digestibility of unsaturated fatty acids from turkeys
fed tallow or coconut oil. Such results suggest a relative deficiency of bile salts in tallow-
fed poults, whereas sufficient bile salts were available in turkeys fed coconut oil to aid
in emulsification and absorption of greater amounts of palmitic and stearic acid.
With respect to a species comparison, it should be noted that the young turkey
seemed to be more sensitive to dietary treatments than the broiler. Comparable
digestibility values were observed between species but the diets differed in that the
turkeys received diets containing less pentosans and less supplemented fat.
There were also differences in main sites of absorption observed between
saturated and unsaturated fatty acids. The latter disappeared to a greater extent from
the proximal segments of the small intestine, whereas the former were digested and
absorbed in the more distal segments (Fig. 9.8).
Digestibility of protein and amino acids
The effect of intestinal viscosity on protein and amino acid digestibility is generally
less dramatic than on digestibility of fat and fatty acids (Table 9.5). Digestibility of
crude protein and that of some amino acids at the terminal ileum was decreased
as dietary pentosan content was increased and significantly improved by xylanase
addition (Fig. 9.9). No fat effect or interactions were detected at this site. In
contrast, measurements made over the whole gastrointestinal tract showed signifi-
cantly lower protein and amino acid digestibility values for tallow-fed birds, and
significant greater enzyme effects in tallow-containing diets, especially in diets
with higher pentosan concentrations (Fig. 9.9). Langhout et al. (1997) reported the
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Fig. 9.9. Praecaecal and faecal protein digestibility as influenced by rye

proportion, dietary fat type and xylanase supplementation (Dänicke et al., 1999e).
Without xylanase, –––; with xylanase, -----; praecaecal, r; faecal, q.
improvement in faecal digestibility of amino acids after xylanase addition to a wheat/

rye-containing diet to be greater in birds fed blended animal fat compared with those
fed soybean oil. The improvements for the individual amino acids varied between
−0.5% and 1.5% in the soya oil group and 0.9% and 4.3% in the blended animal
fat group. Moreover, Rotter et al. (1990) combined a highly viscous barley with
either 4% maize oil or 4 % tallow and tested these diets without or with an enzyme
addition. A significant enzyme-related improvement was found but there was no
effect of dietary fat type. Smulikowska and Mieczkowska (1996) found that protein
digestibility did not differ significantly between diets containing either 6% tallow or
6% soybean oil in the presence of 30% rye. Xylanase addition improved digestibility
only slightly. No interactions were observed in that study. Furthermore, Großer
and Jeroch (1997) found a xylanase-related increase in apparent faecal protein
digestibility but failed to demonstrate fat effects in turkeys fed either soybean oil or
beef tallow containing wheat-based diets.
Energy metabolism
Increased intestinal viscosity results in an increase in the absolute and relative weight
of the gastrointestinal tract, especially of the small intestine. Increasing the relative
proportion of gut and liver in rats results in a simultaneous decrease in utilization of
ME for energy retention and an increase in energy maintenance requirement (MEm)
(Ferrell and Koong, 1986). Furthermore, Pekas and Wray (1991) detected a high
correlation between fasting heat production and several gut measurement variables in
pigs, such as mass and length but also gastrointestinal fill. In contrast, only weak or
no correlations at all were observed between heat production and empty body mass
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226 S. Dänicke
or carcass. These results would suggest that changes in the proportions of the gut and
gut fill in relation to liveweight due to an increased intestinal viscosity should
also affect heat production or energy metabolism. Simon (1998) calculated that a
30% increase in intestines as a proportion of body weight would increase the heat
production of a broiler of 1 kg by approximately 5%. Jørgensen et al. (1996) reported
an increase in heat production as a proportion of ME in broilers after feeding of pea
fibre containing high amounts of soluble NSP, whereas feeding of wheat or oat bran
did not increase heat production. Addition of xylanase to rye-based broiler diets was
shown to increase the caloric efficiency (Table 9.7), i.e. the proportion between net
energy and ME, the partial efficiency of ME for net energy above MEm (kpf) but also
MEm itself. Caloric efficiency includes both maintenance heat and heat of energy
accretion, thus the proportion between heat production and ME was decreased by
xylanase supplementation. An additional effect of dietary fat was mostly obvious for
metabolizability of energy, which was probably caused by differences in fat
digestibility.
Generally, improved energy utilization might be expected following
carbohydrase addition as a result of an improvement in both metabolizability of
energy (fat digestion) and an increased metabolic utilization of absorbed nutrients
(increased caloric efficiency).
Nitrogen-corrected apparent metabolizable energy (AMEN) content as a measure
of the overall nutrient effect on energy metabolism decreased with increasing dietary
pentosan content. This measure was significantly improved by addition of xylanase
and was lower in tallow-fed birds (Dänicke et al., 1999e). Again, xylanase effects were
found to be greater for tallow-fed birds and at higher pentosan concentrations.
Protein metabolism
Expected effects on protein metabolism may also be expected since increases in

intestinal viscosity result in an altered pancreatic function and an increased gut
Table 9.7. Effects of dietary fat type (S, 10% soybean oil; T, 10% beef tallow) and enzyme
supplementation (−, without, +, with 1 g Avizyme 1300 kg−1 diet) in a rye-based diet (56% dietary
inclusion) on energy metabolism in male broilers (Dänicke et al., 1999f).
Metabolizability of Energy retention

gross energy (kJ day−1 W−0.75) Fat
Caloric retention MEm
Fat Protein Fat Total efficiency (% of kpf (kJ day−1
type kJ kJ−1 (NEp) (NEf) (NEpf) (kJ kJ−1) WG) (kJ kJ−1) W−0.75)
S− 0.643ab 299ab 242ab 540a 0.421ab 11.0ab 0.665ab 468

S+ 0.706bb 315ab 295bc 611a 0.449ab 12.1ab 0.729ab 521
T− 0.606ab 301ab 286ab 588a 0.426ab 12.4ab 0.662ab 482
T+ 0.656ab 354bb 340cb 694b 0.462bb 13.6bb 0.886bb 720
a–cValues with different superscripts within the columns are significantly different (P < 0.05).
236
17 November 2000 15:14:47

weight and consequently an increased contribution of gut protein mass to the overall
protein mass of the bird. It was shown by Dänicke et al. (2000a) that tissue fractional
protein synthesis, i.e. the daily protein synthesis expressed as percentage of total tissue
protein, is significantly greater in small intestinal tissues and in the pancreas of birds
fed an unsupplemented tallow-containing rye-based diet (Table 9.3) compared with
their soybean oil-fed counterparts. Xylanase supplementation reduced intestinal
and pancreatic weights and also the fractional protein synthesis rates of these tissues
to a level comparable to birds fed the soybean oil diets. Increased intestinal protein
synthesis might be related to increased intestinal endogenous N-losses, since it was
demonstrated by Dänicke et al. (2000b) that endogenous N-losses, as measured by an
isotope dilution technique, were threefold greater in birds fed the tallow-containing
rye-based diet (Table 9.3). Moreover, Langhout et al. (1999) measured an increased
number of goblet cells per 100 villus cells when high or low methylated pectin was
added to a maize-based broiler diet, which could result in an increased secretion of
mucin.
The lack of an enzyme effect on intestinal parameters in soybean oil-fed birds
would suggest that soybean oil may have exerted a protective effect on the intestinal
villi and cell proliferation rate, since intestinal viscosity is also reduced by enzyme
supplementation in these birds.
Protein synthesis in the pancreas is closely associated with synthesis of secretory
proteins, i.e. hydrolases such as lipase, protease and amylase. Therefore, it might
be concluded that increased pancreatic protein synthesis rate in birds fed the
unsupplemented tallow-containing diet might have been related to increased
digestive enzyme secretion. Further support for involvement of both intestinal and
pancreatic contributions to digestive endogenous N-losses comes from Dänicke et al.
(2000b), who reported that the [15N]-enrichment of digesta and faeces of broilers
was equally as a result of [15N]-enrichments of pancreas and intestinal tissues of
[15N]-labelled animals.
Performance and carcass characteristics
Performance is the most important criterion from a practical point of view. It corre-
lates more or less with fat digestibility but reflects also the described interactions
between fat type and enzyme supplementation. In most experiments, liveweight gain
and feed-to-gain ratio were improved to a greater extent by carbohydrase addition in
broilers fed diets containing animal-originated fats (Table 9.5, Fig. 9.10). Although
the interactions were not always significant they should not be neglected, since the
tendency is obvious in most cases. As already shown for fat digestibility, carbohydrase
effects depend on dietary concentration of soluble NSP (Fig. 9.5). Enzyme effects
can be expected to be greater in birds fed animal fat and at higher concentrations of
dietary soluble NSP. Total mortality (including losses by culling) depends also on fat
type, carbohydrase addition and rye proportion (Dänicke et al., 1999d). Mortality
increased significantly with the introduction of rye into the diets and was much
greater in groups fed the unsupplemented tallow-containing diets, where mortality
237
17 November 2000 15:14:48

228 S. Dänicke
of 23–26% was observed. The viscosity causing stickiness of excreta was further
amplified in tallow-fed birds due to large amounts of undigested fat in the litter
and around the cloaca of the birds. Their feathers appeared dirty and wet, which
undoubtedly increases pathogenic microbial stress.
Fig. 9.10. Interactions between rye percentage (pentosan level), fat type and
xylanase supplementation for cumulative feed to gain ratio of male broilers. Day
1–35 of age: without xylanase, w; with xylanase, q. (From Dänicke et al., 1999d.)
Fig. 9.11. Interactions between rye percentage (pentosan level), fat type and
xylanase supplementation for abdominal plus visceral fat of male broilers. Day 35
of age: without xylanase, w; with xylanase, q. (From Dänicke et al., 1999d.)
238
17 November 2000 15:15:37

It should be stressed that interactions between fat type and carbohydrase

supplementation were detected both in semi-purified diets and in practical diets.
These quantitative changes in performance as a result of enzyme and fat inter-
actions are paralleled by changes in carcass characteristics and liver lipid composition.
Increased fat digestibility was shown to increase broiler fatness (Fig. 9.11), which is
closely related to energy availability and energy metabolism and affects skin colour,
since the major pigments are fat soluble. It can be seen from the relationships shown
in Fig. 9.12 that increasing fatness, as a result of either xylanase supplementation or
reduced rye levels, is associated with a more yellow skin colour (increasing b-values),
more light (increasing L-values) and less red (decreasing b-values).
The liver concentrations of the fat-soluble vitamins A and E were also enhanced
after enzyme addition as a result of improved fat digestibility (Dänicke et al., 1997b,
1999c). Alterations in liver fatty acid composition were also explained by both
dietary fat type and xylanase addition (Dänicke et al., 1999c).
Concluding Remarks
There is a strong body of evidence from a number of sources which indicates that
dietary fat type is a factor that modifies the overall effect of carbohydrase supple-
mentation in broilers. Generally, greater enzyme effects can be expected if a fat of
animal origin is used rather than one of plant origin. The importance of such
interactions increases: (i) with increasing dietary proportions and absolute amounts
of an animal fat; (ii) with increasing dietary concentrations of soluble NSP; and
Fig. 9.12. Relationship between abdominal plus visceral fat and parameters of
drumstick skin colour. Minolta L, q; a, w; b, v; S, soybean oil; T, tallow; −, without
xylanase supplementation; +, with xylanase supplementation; 0, 33, 66, 100%,
respectively, rye of total dietary cereals. (From Dänicke et al., 1999d.)
239
17 November 2000 15:16:00

230 S. Dänicke
(iii) in early fattening periods. The detection of such interactions even in practical
diets leads to the conclusion that animal fat-supplemented diets that are based on rye,
triticale, barley or wheat, or on a mixture thereof, should be supplemented with an
appropriate carbohydrase preparation to avoid adverse effects on performance.
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Digestion of Phosphorus and
Other Nutrients: the Role
of Phytases and Factors
10
Influencing Their Activity
E.T. KORNEGAY*
Department of Animal and Poultry Sciences,
Virginia Polytechnic Institute and State University,
3060 Litton-Reaves Hall, Blacksburg, VA 24061, USA
E.T. Kornegay
Role
10 of Phytases in Nutrient Digestion
Introduction
Phytase may well be the miracle enzyme of the 1990s, as soybeans were described
as the miracle crop for producing high quality plant protein in the 1940s. Dietary
addition of microbial phytase or the inclusion of high phytase ingredients in pig,
poultry and fish diets is now well documented to release a large portion of the
naturally occurring phytate P, and thus greatly reduce the amount of inorganic P that
must be added to meet the animal’s P requirement. The net result is a reduction in P
excretion that can range from 20 to 50%.
Microbial phytase was initially used as a tool to reduce P excretion because
modern commercial production of pigs and poultry had led to large amounts of
manure which, when applied to the land in excess, resulted in a build-up of nutrients
in and on the soil. This had a potential for environmental pollution that continues to
lead to legislation in many countries that require nutrient management plans for
manure from these farms.
It is becoming increasingly clear that the use of adequate amounts of phytase in
most pig and poultry diets results in improved availability of Ca, Zn, protein/amino
acids (AA) and energy. This is because seeds or products from seeds, which are the
major ingredients in pig and poultry diets, contain 60–80% of the P in the form of
phytic acid or phytate. Phytate is known to complex with other nutrients. Thus, the
unavailable phytate P and nutrients complexed with it cannot be utilized and are
excreted.
This chapter will provide a brief review of phytate, phytase, and the effectiveness
of microbial phytase in pig, poultry and fish diets for enhancing the utilization of P,
*Deceased.

238 E.T. Kornegay
Ca, Zn, amino acids/protein and energy. Factors that influence phytase activity will
be discussed.
Phytate
Occurrence and structure of phytate
The major ingredients in commercial pig and poultry diets are seeds (cereal grains) or
products from seeds (oilseed meal and grain by-products). A large portion (60–80%)
of the P in these ingredients occurs in the form of phytates, the salts of phytic acid
(Table 10.1). Phytic acid, myo-inositol 1, 2, 3, 4, 5, 6 hexakis dihydrogen phosphate,
is an essential component of all seeds. Seeds rapidly accumulate phytic acid during
the ripening period (Asada et al., 1969). The location of phytate in the seed varies
(Pallauf and Rimbach, 1995). In small grains, phytate lies mainly in the bran
(aleurone layer, testa and pericarp), and in the case of maize it is found mainly in
the germ. Generally, with legume seeds, phytate accumulates in the cotyledon. In
soybeans, phytic acid is located in protein bodies distributed throughout the seed
(Baker, 1991). Rapeseed phytates are more concentrated in seed tissues than soybean
phytates, which appear to have no specific site location (Kratzer and Vohra, 1986).
Detailed information on the phytic acid content of various foods and feedstuffs can
be found in the reviews by Oberleas and Harland (1981), Ingelmann et al. (1993),
Eeckhout and DePaepe (1994) and Ravindran et al. (1994, 1995b).
Table 10.1. Phytate phosphorus content and phytase activity of some common feed ingredients.
Phytate Pa Phytate Pa Phytase activityb

Ingredient (%) (% of total P) (units kg−1)
Cereals and by-products

Maize 0.24 72 15
Wheat 0.27 69 1193
Sorghum 0.24 66 24
Barley 0.27 64 582
Oats 0.29 67 40
Wheat bran 0.92 71 2957
Oilseed meals
Soybean meal 0.39 60 8
Canola meal 0.70 59 16
Sunflower meal 0.89 77 60
Groundnut meal 0.48 80 3
Cottonseed meal 0.84 70 NA
aData adapted from Ravindran (1996) and Ravindran et al. (1994, 1995b).
bData from Eeckhout and De Paepe (1994). One unit is defined as that amount of phytase which
liberates inorganic phosphorus from a 5.1 mM Na-phytate solution at a rate of 1 µmol min−1 at pH
5.5 and 37°C (98.6°F).
Role of Phytases in Nutrient Digestion 239
Bioavailability of phytate P
The bioavailability of P in cereal grains and products from oilseeds is generally very
low for pigs and poultry, because they have limited capability to utilize phytate P
(Table 10.2). Bioavailability estimates of P in maize and soybean meal for pigs
and poultry range from 10 to 30% (Nelson, 1967; Calvert et al., 1978; Jongbloed
and Kemme, 1990; Cromwell, 1992). This low availability of phytate P poses two
problems for producers: (i) the need to add inorganic P supplements to diets; and
(ii) the excretion of large amounts of P in the manure.
Potential for binding nutrients
The phytic acid molecule has a high P content (28.2%) and chelating potential
(Fig. 10.1) to form a wide variety of insoluble salts with di- and trivalent cations at
neutral pH (Vohra et al., 1965; Oberleas, 1973; Cheryan, 1980). One mole of phytic
acid can bind an average of 3–6 mol of Ca to form insoluble phytates at the pH of
the small intestine. Formation of insoluble phytate makes both Ca and P unavailable.
Zn, Cu, Co, Mn, Fe and Mg can also be complexed, but Zn and Cu have the
strongest binding affinity (Maddaiah et al., 1964; Vohra et al., 1965). This binding
potentially renders these minerals unavailable for intestinal absorption. Zinc may be
the trace element whose bioavailability is most influenced by phytate (Pallauf and
Rimbach, 1995). Phytic acid may have a negative influence on dietary protein
and amino acids (O’Dell and de Borland, 1976; Knuckles et al., 1985) and inhibits
proteolytic enzymes such as pepsin and trypsin under gastrointestinal conditions
Table 10.2. Bioavailability of phosphorus for pigs and nonphytate phosphorus for poultry.
Bioavailability of P for pigsa,b Nonphytate-P for poultryc

Feedstuff (%) (% of total)
Cereal grains
Maize 12 28
Oats 23 33
Barley 31 36
Triticale 46 33
Wheat 50 31
Maize, high moisture 53 –
High protein meals – plant origin
Groundnut meal 12 21
Canola meal 21 26
Soybean meal, dehulled 25 35
Soybean meal, 44% protein 35 40
aAdaptedfrom Cromwell (1992).
bRelative
to the availability of phosphorus in monosodium phosphate, which is given a value of 100.
cNRC (1994) – poultry.
240 E.T. Kornegay
Fig. 10.1. Structure of phytate and possible bonds (after Thompson, 1988).
(Singh and Krikorian, 1982). Under acidic conditions, the basic phosphate groups
of phytic acid may complex with amino groups such as lysyl, histidyl and arginine
(De Rham and Jost, 1979; Fretzdorff et al., 1995). Under neutral conditions, the
carboxyl groups of some amino acids may bind to phytate through a divalent or
trivalent mineral. Phytate–protein or phytate–mineral–protein complexes may
reduce the utilization of protein.
Starch is also known to be complexed by phytate. The in vitro hydrolysis of
either wheat or bean starch incubated with human saliva was retarded when Na
phytate was included in the mixture, but digestion was restored when Ca was added
with the Na phytate (Yoon et al., 1983; Thompson, 1986; Thompson et al., 1987).
As will be reviewed later, Ravindran (1999) reported studies with broilers that clearly
show that apparent metabolizable energy is improved when high-phytate diets are
supplemented with microbial phytase.
Phytases
Phytases are known to occur widely in microorganisms, plants and certain animal
tissues (Nayini and Markakis, 1986; Nys et al., 1996). Phytase of microbial origin
(3-phytase, EC 3.1.3.8) hydrolyses the phosphate group at the C3 position first,
whereas phytase of plant origin (6-phytase, EC 3.1.3.26) acts first at the C6 position
(Pallauf and Rimbach, 1995). Phytase produced by Aspergillus has two pH optima:
one at pH 2.5 and the other at pH 5.5. Wheat phytase has only one pH optimum: at
5.2. Aspergillus phytase has been shown to be more effective per unit of activity than
wheat phytase, probably due to these differences (Eeckhout and De Paepe, 1996).
Phytase activity is generally defined as follows: one unit of phytase activity is the
amount of enzyme that liberates 1 µmol of inorganic phosphorus in 1 min
from 5.1 mmol solution of sodium phytate at 37°C and pH 5.5. At least three
abbreviations are used in the literature for phytase activity: FTU, PU and U. The
latter (U kg−1) will be used in this chapter unless otherwise noted.
Four possible sources of phytase could breakdown phytate within the digestive
tract of pigs and poultry: (i) intestinal phytase in digestive secretions; (ii) endogenous
phytase present in some feed ingredients; (iii) phytase originating from resident
bacteria; or (iv) phytase produced by exogenous microorganisms (Nys et al., 1996).
Contents of the stomach and intestine of pigs (Jongbloed et al., 1992; Yi and
Kornegay, 1996) and crop, stomach and small intestine of chickens (Liebert et al.,
1993) have negligible phytase activity. Although endogenous phytase activity of
intestinal mucosae has been reported for pigs (Pointillart, 1993) and poultry (Maenz
and Classen, 1998), its contribution toward dietary phytate P digestibility is not
known. A higher total of small intestinal brush-border phytase activity was reported
for 50-week-old hens compared with 4-week-old broilers, but the specific activity per
unit of brush-border tissue was similar for broilers and mature hens. Phytase activity
has been detected in some animal tissue (McCollum and Hart, 1908; Bitar and
Reinhold, 1972; Pointillart et al., 1985; Yang et al., 1991); however, this activity, if
present, is negligible for improving the availability of phytate P in non-ruminant
animals (Pallauf and Rimbach, 1995). Additionally, the significance of phytase pro-
duced by resident bacteria in non-ruminants has not been demonstrated and is prob-
ably negligible, considering the lack of significant absorption in the large intestines.
Plant phytase
It has been known for more than 50 years that plant phytase has the ability to
hydrolyse phytate (McCance and Widdowson, 1944; Hill and Tyler, 1954) and its
effectiveness for improving P digesitibility in pigs and poultry has been clearly shown
(Nelson, 1967; Newton et al., 1983; Bagheri and Gueguen, 1985). Phytase activity
has been reported in a wide range of seeds, such as rice, wheat, barley, maize, rye,
soybean and oil seeds (Reddy et al., 1982; Gibson and Ullah, 1990; Eeckhout and De
Paepe, 1994); however, phytase activity of seeds varies greatly among species of plants
(Table 10.1). With the exception of wheat, rye and triticale, most dormant seeds
contain very low phytase activity. Phytase activity in maize and soybean meal is so
low that it is not of practical importance. Barley may or may not contain nutrition-
ally significant levels of phytase activity (Pointillart, 1993). The majority of the
phytase activity in wheat, rye and triticale is in the bran. Diets formulated using
ingredients having high phytase activity, such as wheat bran, wheat, triticale, rye bran
and wheat middlings, promote greater absorption of phytate P (Pointillart, 1991,
242 E.T. Kornegay
1993). Eeckhout and De Paepe (1991) reported that microbial phytase was 74%
more efficient in vivo (fed to pigs) than phytase in wheat middlings when added at an
equal in vitro activity level (500 U kg−1). They showed that genetically modified
microbial phytase was active over a wider pH range than wheat middlings phytase.
They suggest that microbial phytase may be more active at pH levels present in the
stomach than wheat phytase.
Although some feed ingredients contain native phytase activity, steam-pelleting
used in the manufacture of many commercial pig and poultry feeds results in
significant losses of this intrinsic phytase activity. Because of variation of phytase
activity among and within plant species, damaging effects of pelleting during feed
manufacturing and the lack of availability of feed ingredients of high phytase activity,
the presence of residual phytase activity often may not be considered in diet
formulation when feeds are pelleted.
Microbial phytase
Microbial phytases are found in numerous bacteria, yeast and fungi (Harland
and Morris, 1995) but they have been detected most frequently in media with the
Aspergillus genus of ascomycetous fungi (Irving and Cosgrove, 1974; Nair and
Duvnjak, 1991). Hydrolysis of dietary phytate by extrinsic microbial phytase was
probably investigated first by Nelson et al. (1971) who observed improved P
utilization by chicks fed maize–soybean meal diets containing preparations of
Aspergillus. However, the costs of adding the enzyme were very high and there was
little environmental pressure to reduce the P excretion until the late 1980s.
Two major factors led to the development of commercial phytase that could
be economically used in pig and poultry diets. The first was the advance in
biotechnology that led to techniques for genetically modifying fungi; second was
mandate that Dutch livestock farmers had to reduce P excretion. Certainly, the fact
that the phytase gene had been identified and isolated made possible the genetic
modification of A. niger. Also, an improvement in fermentation technology was a
contributing factor.
Sources of phytase
Currently, there are at least four commercially available microbial phytases, two
obtained by fermentation of a genetically modified Aspergillus (Natuphos® and
Novo phytase), and two obtained by extraction of media with Aspergillus (Finase™
and Alltech phytase). Patents influence the availability of these in some countries.
Most products are available in a powder or granular form and as a liquid. The genetic
information for both Natuphos® and Novo phytase originated from A. ficuum and
was transferred to either A. niger or A. oryzae.
Research is also underway expressing the A. niger phytase gene in seeds from
tobacco and rapeseed (Beudeker and Pen, 1995). Zhang et al. (1998a,b) reported
that Phytaseed® (rapeseed genetically modified with the A. ficuum phytase gene)
and Natuphos® were equally effective in low P pig and broiler diets for improving P
utilization. Similar findings were reported for turkeys by Ledoux et al. (1998). Other
products may be under development. Van Loon et al. (1998) reported from
short-term pig trials that a heat-stable phytase enzyme, cloned from A. fumigatus and
overexpressed in A.niger (Pasamontes et al., 1997), was more effective in reducing
P excretion than a commercially available genetically modified phytase using the
A. ficuum phytase gene. The A. fumigatus phytase was reported to have retained more
than 90% of its enzymatic activity after 20 min heating at 90°C.
Safety of genetically modified phytase
Genetically modified microbial and plant phytases were fed to pigs and poultry at
levels 5–20-fold higher than recommended levels (500 U kg−1). In a piglet (5 weeks)
and a broiler (4 weeks) trial, the performance, P retention coefficients and bone
mineralization values continued to increase (at a decreasing rate) to 2500 U kg−1, the
highest level fed in these two trials (Zhang et al., 1998a,b). General necropsy and
histological examination of liver, kidney and tibial tissues revealed no adverse effects.
In trials for both a pig (29 kg initially for 6 weeks) (Harper et al., 1999) and a turkey
(4 weeks after 7 days adjustment) (Kornegay et al., 1999), there were no differences
in mortality among phosphorus or microbial phytase treatments. It was evident that
the addition of 10,000 U phytase kg−1 did not cause any negative effects based on
a general necropsy and histological examination of liver, kidney and tibial bones
but, rather, continued to provide small positive improvements in performance, bone
mineralization and digestibility of P.
Site of phytase activity
Results from several post-slaughter and cannulation experiments with pigs have
shown that dietary phytase activity, whether from plants (Gueguen et al., 1968;
Schulz and Oslage, 1972; Lantzsch et al., 1992; Kemme et al., 1998) or fungal
sources (Jongbloed et al., 1992; Mroz et al., 1994; Yi and Kornegay, 1996), was
predominantly active in the stomach. In pigs, phytase activity or breakdown of
phytate P was generally very low or was not observed in the lower small intestine.
Significant amounts of phytate P (40–50% of the total) would be passed into the
large intestine, where most of it would be hydrolysed, but little of the hydrolysed
P would be absorbed (Jongbloed et al., 1992; Lantzsch et al., 1992; Kemme et al.,
1998). The low phytase activity in the small intestine may be due to a much more
basic pH (6.5–7.6) of the jejunum and ileum (van der Meulen and Bakker, 1991)
that is less favourable for high phytase activity, or due to proteolytic enzymes in the
small intestine that may degrade phytase.
Liebert et al. (1993) reported in chickens that 69–86% of added microbial
phytase activity was detected in the crop and that 31–38% of added phytase activity
244 E.T. Kornegay
was detected in the proventriculus. No phytase activity was detected in the small
intestine. The disappearance of phytate P in the crop and proventriculus supports the
observation that the crop and proventriculus are the main sites of phytase activity in
poultry.
Effectiveness of Microbial Phytase in Poultry and Swine Diets

Supplemental microbial phytase is well known for its effectiveness in improving the
availability of P from plant ingredients containing high levels of phytate P. Some
reports have also suggested that the availabilities of Ca, Zn, amino acids and, in some
cases, energy are also improved when phytase is supplemented in the diet. However,
the use of microbial phytase in poultry and swine diets will depend upon a number of
factors, including the cost of P and phytase, the overall effectiveness of phytase to
release P and other nutrients, and the environmental need to reduce the excretion of
P and other elements which translate into disposal costs. The remainder of this
chapter will discuss the use of microbial phytase in pig and poultry diets to enhance
the utilization of P, Ca, Zn, amino acids and energy so that excretion of these
nutrients can be reduced. Factors to consider that can influence the efficiency of
microbial phytase in these diets will be discussed.
Effects of phytase on improving the bioavailability of phosphorus
Phosphorus digestibility response equations were generated using 52 pig experiments

representing 32 references in a review reported by Kornegay et al. (1998a). Also, 23
poultry experiments representing 13 references were used to generate P retention
equations (Kornegay, 1999). Studies that had very high dietary P levels (NRC or
above) for the negative control diets were excluded. Because of limited data, no
attempt was made to separate and evaluate data by commercial inorganic phosphorus
sources. Several equations were generated for the pig and poultry data sets with the
upper levels of phytase varying from 600 to 800, 1000, 1200 or 1500 U kg−1 of diet.
An evaluation was made of estimates of digestibility (or retention) of P calculated
using each of these equations. Within the range of 100–600 U kg−1 for pigs and
200–800 U kg−1 for poultry, similar values were generally obtained for the various
equations. It was decided to use the wider range of data, but equations for the upper
level at 600 or 800 U kg−1 are also given in the figures. Unless otherwise noted, all
digestibility or retention coefficients are apparent.
Phosphorus digestibility
A nonlinear response of supplemental phytase on P digestibility for pigs and P reten-
tion for poultry was observed (Fig. 10.2). The nonlinear response of phytase on P
digestibility is in agreement with a report by Beers (1992), who found an exponential
and logistic relationship between the dosage of microbial phytase and apparent P
digestibility, and other reports where multiple levels of phytase were fed (Denbow
et al., 1995; Ravindran et al., 1995a; Kornegay and Qian, 1996; Yi et al., 1996a).
Our response curve was remarkably similar to the nonlinear response curve
(corrected for the digestibility of the negative controls) of added phytase on
Fig. 10.2. Increase of P digestibility of pigs (top) and poultry (bottom) fed low P,
low phytase activity plant-based diets supplemented with microbial phytase.
246 E.T. Kornegay
digestibility of P reported by Dungelhoef and Rodehutscord (1995) for pigs

[Y = 28.1(1 − e−0.0024X ), r2 = 0.38]. For example, in their data set there was a 19.6%
unit increase in digestibility of P when 500 U phytase kg−1 was added, and a value of
19.7% was obtained from our pig data set (Table 10.3).
The magnitude of the response per unit of phytase was much greater at the lower
phytase levels. For example, in pigs with 400–500 U phytase kg−1 added, there was a
2.17% unit increase in P digestibility; whereas for 900–1000 U kg−1 there was only a
0.58% unit increase.
In poultry, the increase in P retention was 7.8% units for 500 U phytase kg−1,
less than one-half that observed for pigs (19.7%). Similar to pigs, the magnitude of
the response per unit of phytase was greater at the lower phytase levels. For example,
for 400–500 U phytase kg−1 there was a 1.18% unit increase; whereas for
900–1000 U kg−1 there was only a 0.60% unit increase. This relationship of a
decrease in the magnitude of the response to phytase as the amount of phytase added
increased was previously described in pigs (Kornegay and Qian, 1996; Yi et al.,
1996a), broilers (Denbow et al., 1995; Kornegay et al., 1996a) and turkeys
(Ravindran et al., 1995a) for growth and bone mineralization.
Digested phosphorus
Digested P resulting from microbial phytase supplementation was calculated by mul-
tiplying the total P content of the low P diet (negative control) by the increase in P
digestibility or P retention resulting from phytase supplementation. The data are
shown in Table 10.3 for pigs and Table 10.4 for poultry.
For pigs at a dietary phytase level of 500 U kg−1, 0.075% units (0.75 g) of P
were digested. In the pig data set, the average total dietary P level of the low P diet
(negative control) was 0.381 ± 0.008% (mean ± SEM) and the Ca level was
0.588 ± 0.020. The average ratio of total Ca to total P was 1.54.
For poultry, 0.037% units (0.37 g) of P were estimated to be digested when
500 U phytase kg−1 was added to the diet. The average total P level of the low P diet
(negative control) was 0.478 ± 0.010% (mean ± SEM) and the Ca level was
0.780 ± 0.036%. The average ratio of total Ca to total P was 1.63. Although total P
levels fed are higher and retention coefficients are higher for negative control diets for
poultry than for pigs, the increase of retention coefficients due to microbial
supplementation was lower for poultry than the increase of digestibility coefficients
for pigs. As will be discussed later, the percentage utilization of inorganic phosphate
appears to be less for poultry than for pigs.
Excretion of phosphorus
Based on calculations using the digestibility equation from the pig data set, P
excretion can be reduced by 33.2% when 500 U phytase kg−1 is added to a low P
diet, compared with a positive control diet (0.481% P) containing 0.1% units more
P (Table 10.3). When the P retention equation from the poultry data set was used to
make similar estimates, P excretion was reduced by 31.9% when 500 U phytase kg−1
is added to a low P diet, compared with a positive control diet (0.57% dietary P)
which would be 0.1% units higher in P (Table 10.4). Simply lowering the level of
dietary P by 0.1% will decrease P excretion by about 8.3% in pigs and 18.4% in
poultry. Unless very high levels of P (well above the minimum requirement of the
Table 10.3. Predicted P digestibility, P digested, and percentage reduction in P excretion based
on data generated from the pig data set in this study.
Supplemental Total P P digestibilityb P digested Total P Decreased P

phytase digestibilitya by phytase by phytasec excretedd excretione
(U kg−1) (%) (%) (%) (%) (%)
0 27.9 0 0 0.275 8.3

100 34.2 6.2 0.024 0.251 16.3
200 38.9 11.0 0.042 0.233 22.3
250 40.9 13.0 0.049 0.225 24.8
300 42.6 14.7 0.056 0.219 27.0
350 44.1 16.2 0.062 0.213 28.9
400 45.5 17.5 0.067 0.208 30.6
450 46.6 18.7 0.071 0.203 32.1
500 47.6 19.7 0.075 0.200 33.2
550 48.5 20.6 0.078 0.196 34.5
600 49.3 21.4 0.081 0.193 35.5
650 50.0 22.1 0.084 0.191 36.4
700 50.6 22.7 0.086 0.188 37.1
750 51.1 23.2 0.088 0.186 37.8
800 51.6 23.6 0.090 0.184 38.4
900 52.3 24.4 0.093 0.182 39.4
1000 52.9 25.0 0.095 0.179 40.1
1100 53.4 25.4 0.097 0.178 40.7
1200 53.7 25.8 0.098 0.176 41.1
1300 54.0 26.0 0.099 0.175 41.5
1400 54.2 26.2 0.100 0.175 41.7
1500 54.3 26.4 0.101 0.174 41.9
aGenerated from the equation given in Fig. 10.2 [54.86(1 − 0.4908e−0.00263X)].
bCalculated by subtracting P digestibility of basal diet without phytase from coefficients at each
phytase level.
cCalculated by multiplying the P digestibility due to phytase by the average P content (0.381% total
P) of the basal diet. The equation of this data is = 0.1026(1 − e−0.00263X), where X = phytase level.
dCalculated by subtracting total P digestibility coefficients from 100 and multiplying the product by
the average P content (0.381% total P) of the basal diet.

eBased on an inorganic P digested equation (Y = −0.167 + 0.755X, r 2 = 0.24, where X = % P from
inorganic source); 0.0245% P was excreted for the 0.1% unit of added inorganic P above the basal
diet making the total P excreted by the positive control diet (0.481% total P) equal to 0.2995% P
(0.275 + 0.0245). Decreased P excretion was calculated by subtracting the total P excreted by the
phytase supplemented diets from the P excreted by the positive control diet (0.2995%) and then
dividing by the P excreted by positive control diet and multiplying by 100. For example, at
500 U phytase kg−1, 33.2% = [(0.2995 − 0.200)/(0.2995)]*100.
248 E.T. Kornegay
pig) are fed, the amount of P excreted in pig urine will generally be minimal and was
not considered in the calculations in this chapter; this is consistent with conclusions
reached by Jongbloed (1987).
Table 10.4. Predicted P digestibility, P digested, and percentage reduction in P excretion based
on data generated from the poultry data set in this study.
Supplemental Total P P digestibilityb P digested Total P Decreased P

phytase digestibilitya by phytase by phytasec excretedd excretione
(U kg−1) (%) (%) (%) (%) (%)
0 52.1 0.0 0.010 0.229 18.4

100 54.1 2.0 0.010 0.219 21.8
200 55.9 3.8 0.018 0.211 24.8
250 56.7 4.6 0.022 0.207 26.2
300 57.4 5.3 0.025 0.203 27.5
350 58.1 6.0 0.029 0.200 28.6
400 58.8 6.7 0.032 0.197 29.7
450 59.4 7.3 0.035 0.194 30.8
500 60.0 7.8 0.037 0.191 31.9
550 60.5 8.4 0.040 0.189 32.7
600 61.0 8.9 0.042 0.186 33.5
650 61.5 9.3 0.045 0.184 34.3
700 61.9 9.8 0.047 0.182 35.0
750 62.3 10.2 0.049 0.180 35.7
800 62.7 10.6 0.050 1.178 36.4
900 63.4 11.2 0.054 0.175 37.6
1000 64.3 11.8 0.057 0.172 38.6
1100 64.7 12.4 0.059 0.170 39.5
1200 65.0 12.8 0.061 0.168 40.3
1300 65.4 13.2 0.063 0.166 41.0
1400 65.7 13.6 0.065 0.164 41.6
1500 66.0 13.9 0.066 0.162 42.1
aGenerated from the equation given in Fig. 10.1 [68.18(1 − 0.2354e−0.00134X)].
bCalculated by subtracting P digestibility of basal diet without phytase from coefficients at each
phytase level.
cCalculated by multiplying the P digestibility due to phytase by the average P content (0.478% total
P) of the basal diet. The equation of this data (%) = 0.07672(1 − e−0.00134X), where X = phytase level.
dCalculated by subtracting total P digestibility coefficients from 100 and multiplying the product by
the average P content (0.478% total P) of the basal diet.

eBased on an inorganic P digested (retained) equation (Y = −0.000936 + 0.484X, r 2 = 0.69, where
X = % P from inorganic source); 0.0516% was excreted for 0.1% added inorganic P above the
basal diet making the total P excreted by the positive control diet (0.578% total P) equal to
0.2804% P (0.2288 + 0.0516). Decreased P excretion was calculated by subtracting the total P
excreted by the phytase supplemented diets from the P excreted by the positive control diet
(0.2804%) and then dividing by the P excreted by positive control diet and multiplying by 100.
For example, at 500 U phytase kg−1, 31.9% =[(0.2804 − 0.191)/(0.2804)]*100.
Digested P vs. P equivalency value of phytase
Phosphorus equivalency value, a term used to describe the replacement or

substitution value of phytase, is defined as the amount of inorganic P that can be
removed by a given amount of added or intrinsic phytase. For direct comparison of
equivalency value of phytase for P and digested P, equivalency values must be
adjusted by the estimated digestibility of the inorganic P sources that phytase
replaces. Kornegay et al. (1998a) estimated that the digestibility of P in several
feed-grade P sources was 76.7% for pigs. The retention of P from several feed-grade
P sources was estimated to be 46.2% for broilers and turkeys (Kornegay, 1999).
Equivalency values (or equations) are usually obtained from non-linear or linear
equations generated from body-weight gain, bone mineralization, and sometimes
digested P data obtained by feeding multiple levels of P without phytase addition and
multiple levels of added phytase to a low P diet; these equations are set equal
and solved. This procedure was described in detailed by Denbow et al. (1995).
Using a small data set of four pig studies from Virginia Tech, Radcliffe et al.
(1998a) reported an average P equivalency value of 1.0 g inorganic P for 500 U
phytase kg−1 based on average daily gain, bone mineralization and P digestibility.
Using growing and finishing pig data reported by Shih and Hsu (1997), Veum
(1996), O’Quinn et al. (1997) and Harper and Kornegay (1997), non-linear and
linear equations were generated for the response to phytase, and the average P
equivalency value of 500 U phytase kg−1 was 1.0 g inorganic P.
For example, if the average equivalency value of 1.0 g P is multiplied by 76.7%
(0.767 g P digested per 1 g of inorganic P fed), the product, 0.767 g P (1 × 0.767) is
similar to a digested P value of 0.75 g (0.075%) obtained from the pig data set shown
in Table 10.3. Remember that equivalency values must be adjusted by the apparent
digestibility of the inorganic P source phytase that is being replaced. Jongbloed et al.
(1996a) estimated that 500 U phytase kg−1 was equivalent to 0.8 g digestible P,
which was equivalent to 1.0 g P from monocalcium phosphate.
Yi and Kornegay (Virginia Polytechnic Institute and State University, 1998,
unpublished data) developed and organized equivalency values from several
published broiler studies. For broilers, usually from hatch to 3 weeks of age, the
average mean P equivalency of 500 U phytase kg−1 was 0.70 g inorganic P based on
body-weight (BW) gain and toe ash percentage. In a 3-week starter turkey trial, the
average P equivalency of 500 U phytase kg−1 was 0.82 g inorganic P based on BW
gain and toe ash. If the 0.70 g P and 0.82 g P are multiplied by 46.2% (0.462 g P
retained per 1 g inorganic P), the products are 0.32 g P (0.7 × 0.462) and 0.38 g P
(0.082 × 0.462). A value of 0.37 g retained P (0.037%) was obtained in the poultry
data set for 500 U phytase kg−1 (Table 10.4).
Higher P equivalency values for BW gain and tibia ash percentage were reported
by Kornegay et al. (1998a) for growing–finishing broilers: 1.0 and 0.9 g P,
respectively, for 400 U phytase kg−1, the highest level of phytase fed in this study.
However, the calculated digested P in this study was only slightly more (0.036 vs.
0.032 g P) than the calculated digested P value obtained for 400 U phytase kg−1
from the poultry data set (Table 10.4).
250 E.T. Kornegay
Although studies have reported that the digestibility of inorganic P sources

vary, our research results (Kornegay and Radcliffe, 1996; Kornegay et al., 1996b)
with young pigs and turkeys suggest little to no difference in the bioavailability
of monocalcium/dicalcium, dicalcium/monocalcium and defluorinated phosphate
from ‘good quality’ commercial sources in the United States. Several commercial
feed phosphate sources were used in the studies included in our pig and poultry
data sets shown in Tables 10.3 and 10.4, but calculations could not be made for the
different P sources, because of inadequate data and variation observed.
Based on the similarity of digested P values calculated from P equivalency
estimates, and digested P values derived from equations generated in the pig and
poultry data sets, the estimates of P excretion calculated from these data sets should
be accurate for a range of situations. However, a larger response than observed in
these data sets is possible if careful attention is given to ingredient composition and
diet formulation (optimal Ca and P levels), and if quality processing procedures are
followed, as will be discussed later.
Response of layers to phytase supplementation
In a series of trials reported by Simons et al. (1992), Peter and Jeroch (1993), Simons
and Versteegh (1993), Vahl et al. (1993), Van der Klis et al. (1997) and Gordon and
Roland (1997), phytase supplementation of a low P diet for layers was very effective
as a replacement for inorganic P. A range of P equivalencies, 0.5–1.2 g P as mono-
calcium phosphate, have been reported for 200–300 U phytase kg−1. Van der Klis
et al. (1997) reported that the effect of phytase supplementation (250 and 500 U
kg−1) on ileal P absorption was 12% units greater when added to a low P basal diet
containing 3.0% Ca compared with a low P basal diet containing 4.0% Ca. Leske
and Coon (1998) reported that phytate P retention was 36.7, 29.0 and 14.8% units
greater with phytase supplementation (300 U kg−1 of diet), respectively, for soybean
meal, maize and rice bran, but total P retention was only 16.6, 16.1 and 7.1% units
greater. As has been reported for pigs, broilers and turkeys, the response of phytase
supplementation was greater at the lower levels of total P. Phytase supplementation is
very effective at releasing P in layer diets, which can result in reduced dietary P levels
and reduced P excretion. The efficiency appears to be greater for layers than for
broilers and turkeys. Phytase supplementation of layer diets is also simplified because
most diets are fed in a mash form and thus heat stability issues are averted.
Factors influencing phytase response
The dietary level of P influences the response to phytase (Dungelhoef and

Rodehutscord, 1995; Kornegay and Qian, 1996; Kornegay, 1996a,b). Results of
studies in pigs (Kornegay and Qian, 1996; Yi et al., 1996a), broilers (Denbow et al.,
1995; Kornegay et al., 1996a) and turkeys (Ravindran et al., 1995a) clearly
demonstrate that the response to microbial phytase is influenced by dietary levels of
available or non-phytate P, and that digested P per unit of phytase decreases as the
amount of phytase increases per unit of diet. Extremely high and low levels of dietary
P should be avoided, to optimize the response to phytase.
Vitamin D probably influences phytase activity indirectly by increasing Ca
absorption, therefore limiting the formation of insoluble Ca-phytates, which are
more resistant for phytase hydrolysis (Pointillart, 1993). High levels of Ca or wide
Ca : P ratios will reduce the response for phytase (discussed later).
Effects of phytase on calcium bioavailability
Broilers and turkeys

Schoner et al. (1991, 1993, 1994) reported improved Ca retention in broilers fed
supplemental phytase. In a broiler study designed to measure the effect of phytase on
Ca availability, Schoner et al. (1994) reported that 500 U microbial phytase were
equivalent to 0.46 g Ca based on BW gain and phalanx ash. Calcium retention and
DM digestibility were improved when phytase was added to broiler diets (Kornegay
et al., 1996a, 1998c; Yi et al., 1996b). In our study with turkey poults (Kornegay
et al., 1996c), estimates based on BW gain, gain : feed and digested Ca suggest that
500 U phytase are equivalent to 0.87 g Ca. Both P and Ca retention were sensitive
to the addition of phytase at two non-phytate (nP) levels and four Ca : tP ratios
in turkeys (Qian et al., 1996b), or two vitamin D3 levels and four Ca : tP ratios in
broilers (Qian et al., 1997). Calcium retention, as well as P retention, linearly
increased as the amount of supplemented phytase increased, and decreased as the
Ca : tP ratios became wider in both broilers and turkeys.
Pigs
Radcliffe et al. (1995) reported results of two pig trials conducted to determine the
effectiveness of microbial phytase for improving the bioavailability of Ca. Based on
this data, Kornegay et al. (1996c) estimated Ca equivalency values of 1.08 and 0.38 g
Ca per 500 U of microbial phytase for pigs in Trials 1 and 2, respectively, with an
average of 0.73 g Ca. Calcium equivalency estimates for phytase were based on daily
gain during weeks 3 and 4, digestible Ca and tenth-rib ash percentages.
The interaction between dietary Ca and microbial phytase were reported by
Jongbloed et al. (1996c) in two trials. In both trials, the apparent total tract digestibil-
ity of P linearly decreased with increasing Ca level. No significant interaction could
be demonstrated between dietary Ca level and microbial phytase. Microbial phytase
enhanced not only the apparent total tract digestibility of P, but also the apparent
total tract digestibility of Ca. They estimated that an extra 0.8 g of P and between 0.4
and 0.7 g of Ca were absorbed with 500 U supplemental phytase kg−1.
Using primarily cereal phytase, Pointillart (1993) reported that improved P
utilization was generally accompanied by improved Ca retention. Simecek et al.
(1995) also reported that P as well as Ca digestibility was improved when two forms
of microbial phytase were supplemented in a barley–soybean meal diet for growing
pigs. In other studies, the apparent absorption of Ca and N (Kornegay and Qian,
252 E.T. Kornegay
1996; Yi et al., 1996a) in pigs was improved when phytase was added to the diet.
Eeckhout and De Paepe (1991) reported a highly positive correlation between Ca
and P digestibilities and phytase supplementation to a low P pig diet. They suggested
that this relationship could be explained by the fact that phytic acid acts as a
Ca-binding agent in the proximal small intestine. Hydrolysis of phytate in the
stomach as a result of phytase activity results in increased digestibility, not only of P
but, indirectly, of Ca.
Influence of phytase on zinc bioavailability
Zinc is probably the most vulnerable mineral to phytate complexation. In early

research in chicks, swine, rats and other species it was observed that dietary Zn
requirement was affected by dietary phytate (Prasad, 1966). Oberleas and Harland
(1996) suggest that it is imperative that much of the endogenously secreted minerals
needs to be reabsorbed lest perpetual deficiencies result. Oberleas and Harland
(1996) reported results that clearly showed that phytate was a significant factor in the
development of zinc deficiency. The presence of Ca aggravated the effect of phytate
on zinc utilization, probably through an insoluble Ca–phytate–Zn complex that is
formed which prevents the absorption of Zn.

The addition of 800 U phytase kg−1 to a diet containing 27 mg Zn kg−1 increased
the retention of Zn and decreased Zn excretion of chicks (Thiel and Weigand, 1992).
Thiel et al. (1993) reported that the femoral Zn content of chicks fed a diet
containing 30 mg Zn kg−1 plus 700 U phytase kg−1 of diet was equal to that of chicks
fed a diet containing 39 mg Zn kg−1 without phytase. Using chicks fed a glucose–
soybean concentrate diet (13 mg Zn kg−1 diet) with multiple levels of added Zn,
phytase or 1,25-dihydroxycholecalciferol (di-OH-D3), Biehl et al. (1995) reported
that phytase and di-OH-D3 supplementation both increased growth rate and tibial
Zn to a similar extent. Based on Biehl et al. (1995) estimates using tibia Zn, the Zn
equivalency of 600 and 1200 U phytase kg−1 was 3.8 and 5.5 mg, respectively. In
contrast, Roberson and Edward (1994) did not consistently observe an improvement
in Zn absorption or retention in broilers when 600–750 U phytase kg−1 was added
to a maize–soybean diet containing 32 mg Zn kg−1 diet.
In our laboratory, day-old male broilers were fed a maize–soybean isolate basal
diet containing 20 mg Zn kg−1 alone and supplemented with multiple levels of Zn
and phytase for 21 days (Yi et al., 1996d). Non-linear or linear response equations of
the effects of Zn and phytase levels were generated for BW gain, feed intake, Zn
retention, Zn concentration of toes, tibia and liver and used to calculate an average
Zn equivalency of 5.4 mg kg−1 for 600 U phytase kg−1.
Pigs
Based on enhanced growth, increased plasma Zn concentration and alkaline
phosphatase activity, the bioavailability of Zn for pigs was improved when phytase
was added to a low P (0.3%) and Zn (30 mg kg−1) maize–soybean meal diet (Lei
et al., 1993). Adding microbial phytase to the diets of young pigs significantly
improved apparent absorption of Zn and Mg (Pallauf et al., 1992; Nasi and
Helander, 1994). Using a low Zn diet (23 mg kg−1) containing 0.8% Ca and 0.62%
P, Adeola et al. (1995) reported that growth rate and the retention of Zn, Cu, Ca and
P were increased when 1500 U phytase kg−1 diet was fed. A Zn equivalency for
phytase has not been established for pigs.
Influence of microbial phytase on amino acids and nitrogen

bioavailability
Phytate can bind with protein/amino acid (AA) at low and neutral pH (De Rham
and Jost, 1979; Cosgrove, 1980; Anderson, 1985; Thompson, 1986; Fretzdorff et al.,
1995). Phytate–protein/AA complexes may occur in foodstuffs in the native state,
and may be formed in the upper gastrointestinal tract. Complexing of phytate
with proteolytic enzymes may also occur in the upper gastrointestinal tract. These
potential phytate–protein complexes thus may reduce the utilization of proteins
and amino acids.
If phytate is hydrolysed, then its inhibitory effects are reduced. Most of the early
research with microbial phytase was conducted to measure the effects of phytase on
P utilization, and results were inconsistent when total tract N digestibility was
measured. Amino acid digestibility was rarely evaluated. Kemme (1998) provides a
good review in his dissertation.
The opportunity to show improvements in protein/AA utilization is influenced
by the dietary level of protein/AA. If the protein/AA retention is maximum, then the
potential to show an improvement is greatly reduced. Furthermore, the use of total
tract (faecal) digestibility may not be reliable because of the influence of the microbial
population in the large intestine. Ileal digestibility is a more appropriate method of
evaluating the influence of phytase on protein/AA utilization.
Binding of free amino acids (particularly lysine) by phytate has been demon-
strated in vitro (Rutherfurd et al., 1997). Approximately 20% of free lysine-HCl was
bound following incubation with rice pollards (rich in phytate). Half of that amount
was liberated after the addition of phytase. In broilers, Ravindran and Bryden (1997)
and Ravindran et al. (1999a) reported that dietary addition of phytic acid as rice
pollards reduced ileal digestibility of N and AA (lysine, threonine, isoleucine, leucine,
valine, phenylalanine and histidine). These adverse effects of phytic acid were
effectively overcome by supplemental phytase.

The apparent N retention of broilers was improved when phytase was added to a
23% crude protein maize–soybean diet (Kornegay et al., 1996b). Yi et al. (1996c),
using Large White turkey female poults fed a maize–soybean meal diet, reported that
apparent and true ileal digestibilities of N and AA were generally improved when
750 U phytase kg−1 was added to both 22.5 and 28.0% CP diets that contained
254 E.T. Kornegay
0.45% nP. Improvements were only observed for birds fed 22.5% CP that contained
0.60% nP. Ravindran and Bryden (1997) similarly observed that improvements in
AA digestibility were higher in low P wheat–sorghum–soybean meal diets.
Using young broilers, Kornegay (1996b) reported that ileal digestibilities of all
amino acids was linearly decreased (P < 0.001) as the CP level increased (17, 20 and
23%). Ileal digestibilities of all amino acids except methionine and proline were
linearly increased (P < 0.05 to 0.001) as phytase (250, 500 and 750 U kg−1) was
added to the 17 and 20% CP diets. In a broiler finishing study, Kornegay et al.
(1998b) reported that when dietary protein/AA levels were reduced from 95 to
85% of the NRC (1994) recommendation (1.5–2.0% units of CP), additions of
300–450 U phytase kg−1 diet prevented the decrease in performance (slightly lower),
breast meat yield, and ileal CP/AA digestibilities observed.
Supplemental phytase (1200 U kg−1) was reported by Ravindran et al. (1999b)
to improve ileal digestibilities of protein/AA of three cereals (maize, sorghum and
wheat), four oilseed meals (soybean meal, canola meal, cottonseed meal and
sunflower meal) and two cereal by-products (wheat middlings and rice polishings).
The magnitude of the response varied among feedstuffs and individual AA.
Using a low lysine (85% NRC) wheat–sorghum–soybean meal diet with
multiple levels of crystalline lysine or microbial phytase and based on BW gain
and feed : gain ratios of broilers, Ravindran and Bryden (1998) reported that
500 U phytase kg−1 diet was equivalent to 0.7 g lysine kg−1 diet. Apparent
metabolizable energy was also linearly increased as phytase was added, but not as
lysine was added. Ravindran and Bryden (1998) suggest that BW gain and feed
efficiency responses to phytase may have resulted from both lysine and energy effects.
In a similar study using broilers fed a single level (600 U kg−1) of phytase or multiple
levels of crystalline lysine added to a low lysine (0.8%) maize–soybean meal diet,
Johnston and Southern (1999) reported an average equivalency, based on BW gain
and feed efficiency, of 0.23 g lysine kg−1 diet for 600 U phytase kg−1 diet.
Pigs
Similar findings have been reported in pigs. Officer and Batterham (1992) and Khan
and Cole (1993) reported that microbial phytase improved the apparent ileal and
total tract digestibility of crude protein by 2.3–12.8% units. Using cannulated pigs
in two studies, Mroz et al. (1994) and Kemme (1998) reported that microbial
phytase (800 and 900 U kg−1, respectively) enhanced the apparent ileal digestibility
of N and most amino acids. Added microbial phytase generally enhanced the total
tract digestibility of N and the retention of N. Li et al. (1998) reported improved
DM digestibility and N retention when phytase was added to a maize–soybean meal
diet fed to weaned pigs. In a literature review, Jongbloed et al. (1996d) reported in 17
experiments using microbial phytase that the average apparent total tract digestibility
of protein was enhanced by 0.85 ± 1.70% units.
Faecal N digestibility, ileal AA and N digestibilities and N retention were
determined in a feeding trial and N-balance study, and a cannulation study using
finishing pigs fed diets containing: (i) NRC (1998) CP levels (12% CP); (ii) 7.5%
CP reduction (11% CP); (iii) 15.0% CP reduction (10% CP); (iv) diet 3 (10% CP)
plus 250 U phytase kg−1; and (v) diet 3 plus 500 U phytase kg−1 (Kornegay et al.,
1998a). Apparent digestion coefficients for N and AA increased as the dietary levels
of CP increased and they increased as microbial phytase was added to the 10% CP
diet. Coefficients were generally equal to or greater than values for pigs fed the 11%
CP diet (a 7.5% reduction in CP). Using a conservative reduction of CP of 1% unit
(7.1% reduction), and assuming a N excretion (faeces and urine) value of 40% of N
intake, N excretion was estimated to be reduced by 7.1% when phytase was added to
10–11% CP pig diets at a level of 500 U kg−1. Therefore, phytase supplementation
has the potential to reduce P and N/AA excretion when P and N/AA levels are
reduced appropriately.
Influence of microbial phytase on energy metabolism
Thompson and Yoon (1984) indicated that, in the native state, phytate could
complex with starch. Rajas and Scott (1969) reported that the apparent
metabolizable energy (AME) of cottonseed meal and soybean meal for chicks was
improved following treatment of the meals with a crude phytase preparation from
Aspergillus ficuum. Later, Miles and Nelson (1974), using chicks and a similar
product, reported improvements in the AME value of cottonseed meal and wheat
bran, but not soybean meal, when treated with the phytase preparation.
More recent studies, primarily in Australia, used a genetically modified phytase
(reviewed by Ravindran, 1999). Small but significant improvements in AME
were observed for broilers fed sorghum–soybean meal-based diets (Farrell et al.,
1993; Selle et al., 1999), and sorghum–soybean meal–canola meal–cottonseed
meal–wheat middling diets (Selle et al., 1999) when phytase was supplemented to the
diets. The AME values of a sorghum–soybean meal-based diet and in diets with 60%
rice bran fed to ducks was improved when phytase was added (Martin and Farrell,
1994).
Findings from three very recent studies (Bryden and Ravindran, 1998;
Ravindran et al., 1999a,b), which were designed to determine the influence of
microbial phytase on protein/AA and energy utilization in poultry, clearly show that
supplemental phytase improves the AME value of wheat- and sorghum-based poultry
diets (reviewed by Ravindran, 1999). In agreement, Kornegay and Denbow (Virginia
Polytechnic Institute and State University, 1998, unpublished data) observed that
phytase supplementation (600 U kg−1) improved AME coefficients in 4-week-old
broilers fed maize–soybean meal, maize–wheat middlings–soybean meal, maize–
meat meal–soybean meal or wheat–maize–soybean meal diets. It was suggested,
based on literature reported by Ravindran (1999), that phytic acid may exert its
effects on starch digestion in one or more ways: (i) by binding α-amylase or by
chelating Ca2+, which is needed for the normal activity of amylase; and (ii) by
binding starch directly through a protein linkage. Of course, hydrolysis of phytate
P would release the enzyme or free the starch.
256 E.T. Kornegay
Influence of dietary calcium and calcium : phosphorus ratio on the

effectiveness of phytase
The response to a given level of supplemental phytase will be influenced by dietary

Ca level and/or Ca : P ratio, dietary P level and dietary phytate level (Lei et al., 1994;
Dungelhoef and Rodehutscord, 1995; Kornegay, 1996b). A high molar ratio of Ca
to phytate in the diet can lead to the formation of extremely insoluble Ca–phytate
complexes under intestinal conditions, making the phytate molecule inaccessible to
phytase. The presence of such strong complexes could explain the apparent inactivity
of phytase in calcium-rich diets rather than a direct inhibition of the enzyme by Ca2+
(Wise, 1983; Bos, 1988). The importance of maintaining a narrow ratio of total
Ca to total P (or for that matter available Ca to available P) has recently been
demonstrated in broilers (Qian et al., 1997), turkeys (Qian et al., 1996b) and pigs
(Qian et al., 1996a; Liu et al., 1998).
In broilers and turkeys, a total Ca : total P ratio of 1.1 : 1 to 1.4 : 1 appeared to
be equally effective. Feeding diets with wider ratios reduces performance, P utiliza-
tion and bone mineralization. In young broilers, using data reported by Qian et al.
(1997), a 14.5, 8.5 and 8.3% decrease was calculated for BW gain, P retention and
toe ash percentage, respectively, when the Ca : total P ratio was increased from
1.1 : 1 to 2.0 : 0. In young turkeys, using data reported by Qian et al. (1996b), an
8.7, 10.8 and 6.6% decrease was calculated for BW gain, P retention and toe ash
percentage, respectively, when Ca : total P ratio was increased from 1.1 : 1 to 2.0 : 1.
A Ca : nP (weight/weight) ratio of 2 : 1 is suggested for poultry with the
exception of laying birds (NRC, 1994). A high level of dietary Ca was shown to affect
adversely the availability of phytate P; whereas a high level of vitamin D was shown to
have a positive effect (Mohammed et al., 1991; Edwards et al., 1992). Schoner et al.
(1993) reported in broilers that feeding high levels of Ca with a constant level of P
(0.35%) reduced the increase in BW gain, feed intake and P and Ca retention that
was observed when phytase was added. From their lowest (0.6%) to highest (0.9%)
levels of Ca, the Ca : total P ratio varied from 1.7 : 1 to 2.57 : 1.
In pig studies, a greater response to phytase (better utilization of P) is obtained
when the total Ca : total P ratio is maintained between 1 : 1 to 1.1 : 1 (Qian et al.,
1996a). For example, in growing–finishing pigs, Liu et al. (1998) reported a 4.5, 8.2
and 9.7% decrease in ADG, P digestibility and metacarpal breaking strength, respec-
tively, when the Ca : total P ratio was increased from 1.0 : 1 to 1.5 : 1. Lei et al.
(1994) reported for pigs fed a maize–soybean meal diet with no added inorganic P
(0.31% tP) that the ability of phytase to improve phytate–P availability was greatly
reduced at 0.92% dietary Ca (Ca : tP ratio was 3.1) compared with the 0.50%
dietary Ca (Ca : tP ratio was 1.6 : 1). Dungelhoef and Rodehutscord (1995) also
observed from this review of the literature that high Ca levels reduce the absorption
of P and the utilization of phytate. Similar findings have also been reported by
Jongbloed et al. (1996c,d).
The decrease in the effectiveness of phytase as the Ca : tP ratio becomes wider
could be explained as follows: (i) phytate P utilization in maize–soybean diets fed to
broilers is influenced by Ca and P levels in the diet (Edwards and Veltmann, 1983;
Ballam et al., 1984); (ii) the extra Ca can bind with phytate to form an insoluble
complex that is less accessible to phytase; or (iii) the extra Ca may directly repress
phytase activity by competing for the active sites of the enzyme (Wise, 1983;
Pointillart et al., 1985). The negative effect of wide Ca : tP ratios is greater at lower
levels of supplemental phytase and at lower levels of aP or nP, because less P would be
released as a result of reduced phytase activity.
Phytase and organic acids
The use of organic acids in combination with phytase to enhance the effectiveness of
phytase has produced mixed results. It is known that the inclusion of organic acids
in the diet will generally lower dietary pH (Kirchgessner and Roth, 1982; Giesting
and Easter, 1985; Risley et al., 1992) with a resultant smaller effect on lowering the
stomach pH (Risley et al., 1992; Radcliffe et al., 1998b). Also, it is well documented
that there are two optimal pH values for maximizing microbial phytase activity – 2.5
and 5.5 (Shieh et al., 1969; Simons et al., 1990) – with the activity about 50% greater
at pH 5.5. Organic acids (fumaric and citric) have been reported to promote some
improvement in the total tract apparent digestibility of minerals (Hohler and Pallauf,
1993; Ravindran and Kornegay, 1993). Complexes are known to be formed between
organic acids and various cations such as minerals, and these may result in increased
intestinal absorption of minerals (Ravindran and Kornegay, 1993).
Jongbloed (1987) pointed out that a lower intestinal pH enhanced the solubility
of P and phytate, and thus increased P absorption. Jongbloed et al. (1996e) reported
two trials (cannulation and grower feeding trial) in which a combination of phytase
and lactic acid was fed in trial 1 and a combination of phytase and lactic acid,
formic acid and propionic acid was fed in trial 2. In trial 1, lactic acid enhanced the
effectiveness of phytase on P digestibility but not in trial 2. In trial 2, only formic acid
enhanced the effectiveness of phytase. In two trials, Radcliffe et al. (1998b) reported
that the addition of citric acid to weanling pig diets lowered gastric pH and improved
pig performance. However, the lowered gastric pH did not enhance the efficacy of
microbial phytase. In fact, when high levels of phytase were supplemented to diets
containing citric acid, there was a decrease in the amount of phytase activity
recovered from the stomach after slaughter (Yi and Kornegay, 1996). This did not
affect phytase efficacy, because no citric acid interaction with phytase was observed
on growth performance, feed efficiency, dry matter, Ca and P digestibility or bone
measurements.
Valencia and Chavez (1997) reported that 1.0% acetic acid addition to a
maize–soybean meal diet enhanced the effectiveness of microbial phytase as
measured by performance and apparent mineral utilization of young pigs. In a
balance trial with young pigs, Li et al. (1998) reported that microbial phytase
supplementation of a maize–soybean meal diet improved digestibilities of P, Ca, N
retention and dry matter, and only non-significant trends for further improvements
were observed when 1.5% citric acid was added to the diet. Boling et al. (1998)
258 E.T. Kornegay
observed only additive effects of dietary addition of 6% citrate and 1450 U

phytase kg−1 using bone ash and weight gain of young chickens.
Further research is needed to understand the relationship between diet
acidification and phytase effectiveness.
Effects of age and physiological status
The effects of age and physiological status of the pig on the efficacy of phytase in
rendering phytate P available for absorption have been inconsistent. Kemme et al.
(1997a) reported that the efficacy of the phytase for improving digestible P decreased
in the order of lactating sows, growing–finishing pigs, sows at the end of pregnancy,
piglets and sows at mid-pregnancy. Efficacy of phytase was particularly low in sows
on day 60 of pregnancy and high in lactating sows, with the growing–finishing
pigs intermediate (Kemme et al., 1997b). They suggested that the differences among
the categories probably relate to differences in gastric retention time, but also the
requirement for digestible P by the specific category has to be taken into account.
Harper and Kornegay (1997) reported slightly higher apparent digestibility
coefficients for finisher pigs compared with grower pigs in one trial, but the opposite
effect was observed in a second trial. Rodehutscord et al. (1998) recently compared
the efficacy of microbial phytase fed in combination with a low P semipurified diet
in growing pigs of either 16 or 39 kg body weight (BW). Digestibility of P was very
similar for both BW groups. From these studies there is no indication that the
efficacy of microbial phytase is dependent on BW of young pigs.
Effect of soaking on phosphorus availability
As pointed out by Helander (1995) in her dissertation review, the positive effect of
soaking on P availability has been reported in many experiments involving different
feedstuffs (Irving, 1980). The P in high-moisture maize or grain sorghum is known
to be considerably more available than in dry grain (Trotter and Allee, 1979a,b; Boyd
et al., 1983; Ross et al., 1983; Jongbloed et al., 1991). Although phytase is present in
mature seeds, it appears to have little effect on phytate in dry or dormant seeds
(Maga, 1982). Irving (1980) reported that water was necessary for the hydrolysis of
phytate.
Kemme and Jongbloed (1993) reported that the efficacy of microbial phytase
was enhanced by soaking the diet in water in one experiment, but in a second
experiment, soaking had no effect on P digestibility. A pelleted diet with a different
composition was used in the second experiment and the soaking time was much
shorter; these factors, together, may explain why soaking had no effect on P utiliza-
tion. A higher incubation temperature may also have increased phytate hydrolysis
(Hill and Tyler 1954; Han, 1988). The pH of the diet is also most important.
Tossenberger et al. (1993) reported that the degradation of sodium phytate was 81%
and 93%, respectively, for 2.5 and 5.0 h of soaking in a pH 5.0 solution. The
respective values were only 38% and 47% when the pH was increased to 6.0.
Beneficial effects of soaking on P utilization with whey were observed for a
barley–rapeseed meal diet fed to growing pigs (Nasi et al., 1994), whereas no effect of
soaking a pelleted barley–soybean meal diet was observed by Nasi and Helander
(1994). Liu et al. (1997) reported improved P utilization when a soybean meal-based
diet was soaked. A soaking by phytase quadratic interaction was observed; soaking
the 250 U kg−1 diet increased P absorption to a level similar to that obtained with the
500 U kg−1 diet fed dry.
Wet feeding of growing–finishing pigs has increased, especially in Europe and
has the potential of enhancing phytase effectiveness and improving P digestibility.
Reports from the industry are that phytase is more efficient in these feeding systems.
Effectiveness of Microbial Phytase in Fish Diets

Feed ingredients of plant origin are being used in greater amounts in intensive fish
farming. Like pigs and poultry, several species of fish utilize only one-third of the
phytate P in plant feedstuffs. The addition of inorganic P is necessary to meet the P
needs of the fish for normal growth. It has also been observed that high dietary
phytate levels lead to depressed growth, feed intake and protein utilization, which
may be a result of phytate complexing with cations in the gastrointestinal tract so that
Zn, protein and energy bioavailability are reduced (Spinelli et al., 1983; Richardson
et al., 1985, 1986; McClain and Gatlin, 1988).
A number of studies with striped bass (Hughes and Soares, 1998), carp (Schafer
et al., 1995), catfish (Jackson et al., 1996; Eya and Lovell, 1997; Li and Robinson,
1997), rainbow trout (Cain and Garling, 1995; Rodehutscord and Pfeffer, 1995;
Lanari et al., 1998; Vielma et al., 1998) and Atlantic salmon (Storebakken et al.,
1998) have shown that microbial phytase is very effective in enhancing P digestibility
and utilization. Generally, only single and usually high levels of phytase have been
fed, but multiple levels have been used in a few studies (Schafer et al., 1995; Jackson
et al., 1996; Li and Robinson, 1997). Reported improvements in P absorption and
retention range from 15 to 45%, and are influenced by phytase level and sources of
ingredients. Reduction in P excretion is reported to range from 30 to 88%.
In summary, microbial phytase is very effective in improving P availability of
diets based on plant ingredients and can significantly reduce P excretion and accu-
mulation in the water when dietary P levels are reduced. Because of very limited data
where multiple levels of phytase were fed, it is not possible to derive a response curve
for any measurement so as to determine equivalency value based on good data sets.
Summary
When pig and poultry diets are formulated using significant amounts of plant-based
ingredients that are low in native phytase, microbial phytase supplementation is very
260 E.T. Kornegay
effective for improving the availability of phytate P. Because phytate is known to

complex a number of other minerals, amino acids/protein and even starch, their
bioavailability is enhanced when phytate is hydrolysed by phytase. Thus, the
excretion of P, Ca, Zn and N can be reduced significantly when diets are properly
formulated using phytase. The dose–response curve of phytase for improving P
utilization is non-linear for pigs and poultry (broilers and turkeys) and the response
has been described over a wide range of phytase levels. The dose–response curves for
the effects of phytase on Ca, Zn, amino acid/N and energy utilization are not as
clearly understood as they are for P.
The dose response of phytase will, however, depend upon: (i) the level of phytase
used; (ii) the level of total P in the diet; (iii) the level of phytate P in the diet; (iv) the
level of Ca and the Ca : P ratio; (v) the intrinsic level of phytase in foodstuffs; and (vi)
processing and pelleting methods. The effects of physiological age and the influence
of organic acids are not clearly understood, if they exist.
Microbial supplementation of diets based on plant ingredients for several species
of fish is very effective in improving P availability, which leads to reduced P excretion
and accumulation in the water.
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Enzymes in Ruminant Diets
T.A. McALLISTER,1 A.N. HRISTOV,1
K.A. BEAUCHEMIN,1 L.M. RODE1
11
AND K.-J. CHENG2
1Agriculture and Agri-Food Canada, Lethbridge Research Centre,
PO Box 3000, Lethbridge, Alberta, Canada, T1J 4B1;
2Faculty of Agricultural Sciences, University of British Columbia,
Vancouver, British Columbia, Canada, V6T 1Z4
T.A. McAllister
Enzymes
11 in Ruminant
et al. Diets
Introduction
Exogenous enzymes have been used extensively to remove anti-nutritional factors
from feeds, to increase the digestibility of existing nutrients, and to supplement the
activity of the endogenous enzymes of poultry (Classen et al., 1991; Bedford, 1993).
Researchers in the 1960s examined the use of exogenous enzymes in ruminants
(Burroughs et al., 1960; Rovics and Ely, 1962; Rust et al., 1965), but responses were
variable and no effort was made to determine the mode of action of these products.
Furthermore, production of exogenous enzymes was expensive at the time and it was
not economically feasible to apply these preparations at the concentrations necessary
to elicit a positive animal response. Recent reductions in fermentation costs, together
with more active and better defined enzyme preparations, have prompted researchers
to re-examine the role of exogenous enzymes in ruminant production (Chen et al.,
1995; Beauchemin et al., 1997; McAllister et al., 1999). Several studies have
attempted to define possible modes of action of these additives (Judkins and Stobart,
1988; Feng et al., 1996; Hristov et al., 1998a,b; Yang et al., 1999). Exogenous
enzymes could exert a number of effects, both on the gastrointestinal microflora and
on the ruminant animal itself. It is highly probable, therefore, that physiological
responses to exogenous enzymes are multifactorial in origin.
This review will summarize production responses to supplementary exogenous
enzymes obtained to date in ruminants. Possible mechanisms by which these
products may improve nutrient utilization by ruminants will be discussed and
suggestions will be made with regard to strategies that may further enhance the
efficacy of these products for ruminants.

274 T.A. McAllister et al.
Sources of Enzymes
Although enzyme products marketed for livestock number in the hundreds, they are
derived primarily from only four bacterial (Bacillus subtilis, Lactobacillus acidophilus,
L. plantarum and Streptococcus faecium) and three fungal (Aspergillus oryzae,
Trichoderma reesei and Saccharomyces cerevisiae) species (Muirhead, 1996). Other
fungal species, including Humicola insolens and Thermomyces anuginosus, are being
marketed to a lesser extent. It is unlikely that this list of source organisms will expand
substantially, given that no petition to the Food and Drug Administration to add a
new organism has been successful (Pendleton, 1996).
Enzymes are naturally occurring biocatalysts produced by living cells to bring
about specific biochemical reactions. In the context of feed additives for ruminants,
enzymes are employed to catalyse the degradative reactions by which substrates
(i.e. feedstuffs) are digested into their chemical components (e.g. simple sugars,
amino acids, fatty acids). These are in turn used for cell growth, either by ruminal
microorganisms or by the host animal.
Complete digestion of complex feeds such as hay or grain requires literally
hundreds of enzymes. Enzyme preparations for ruminants are marketed primarily
on the basis of their capacity to degrade plant cell walls and, as such, are often referred
to as cellulases or xylanases. However, none of these commercial products is a prepa-
ration of single enzymes; secondary enzyme activities such as amylases, proteases
or pectinases are invariably present. Degradation of cellulose and hemicellulose
alone requires a number of enzymes, and differences in the relative proportions and
activities of these individual enzymes impacts the efficacy of cell wall degradation by
the marketed products. Even within a single microbial species, the types and activity
of enzymes produced can vary widely, depending on the strain selected and the
growth substrate and culture conditions employed (Considine and Coughlan, 1989;
Gashe, 1992).
The diversity of enzyme activities present in commercially available enzyme
preparations is advantageous, in that a wide variety of substrates can be targeted by a
single product, but it presents problems in terms of quality control and extrapolation
of research findings among different preparations. For ruminants, enzyme products
are usually standardized by blending crude enzyme extracts to obtain specified
levels of one or two defined enzyme activities, such as xylanase and/or cellulase.
These products are not currently standardized for secondary activities. In fact, these
activities, which may well be affecting the overall effectiveness of a given product, are
seldom even measured.
Measurement of Enzyme Activity

Enzyme activity is assayed by measuring over time either the disappearance of a
defined substrate or the generation of a product from the biochemical reaction
catalysed by the enzyme. Activities of enzymes for use in the feed industry are most
commonly measured using the latter approach, and are expressed as the amount of
Enzymes in Ruminant Diets 275
product produced per unit time. These measurements must be conducted under
conditions closely defined with respect to temperature, pH, ionic strength, substrate
concentration and substrate type, as all of these factors can affect the activity of an
enzyme (Headon, 1993). For example, the relative ranking of cellulase activity of
three enzyme preparations differs depending on the test substrate (cellulose, carboxy-
methylcellulose (CMC) or β-glucan) selected for analysis (Table 11.1). Furthermore,
release of reducing-sugars from CMC or xylan is not directly proportional to enzyme
concentration. Consequently, the ratio of enzyme to substrate will affect enzyme
activity estimates (Fig. 11.1) (Hristov and McAllister, unpublished data).
Enzyme activity can also be assessed using synthetic substrates, which usually
consist of chromophores linked to molecules chemically similar to the natural
substrate. Enzyme activity is measured as the release of the dye or chromophore
(Biely et al., 1985). These synthetic substrates offer uniformity among assays, but are
subject to criticism in that they do not represent the substrate found in intact feeds
such as cereal grains or forages. Furthermore, the assays used to assess enzyme activity
are not representative of the conditions in the digestive tract, where ultimately the
level and persistence of enzyme activity may be most important. For these reasons,
measurement of enzyme activity using traditional assay techniques may have little
relevance to the potential efficacy of an enzyme as a feed additive for ruminants.
Researchers have attempted to develop biological assays that may be more
indicative of the value of a given enzyme preparation for ruminants. These methods
usually involve in vitro incubation of enzyme and feed with ruminal contents, and
measurement of the disappearance of substrates (e.g. cereal grain, straw, hay)
representative of those consumed by the animal (Forwood et al., 1990; Varel et al.,
1993; Hristov et al., 1996b, 1998a). Alternatively, the amount of gas produced by
the mixed culture can be used as an indication of digestion (Iwaasa et al., 1998),
which enables rapid screening of different enzyme products and application rates.
These procedures may provide valuable information on the extent to which
exogenous enzymes complement the digestive activity of ruminal microorganisms.
However, extrapolation of information from these procedures to whole-animal
Table 11.1. Relative activities of three commercial enzyme preparations on substrates that
reflect cellulase activity. (Adapted from Hristov et al., 1996b.)
Substrate1
Product % Protein Cellulose2 CMC3 β-Glucan
1 11.5 102.1 737.9 1016.4

2 21.3 126.1 727.1 1042.1
3 15.6 225.6 699.7 1638.3
1For all substrates, activity is expressed as nmol reducing-sugar released mg per product min−1,
after a 3 h incubation of the substrate with the enzyme product in 0.2 M sodium phosphate (pH 6.0)
buffer at 39°C.
2Microcrystalline cellulose.
3Carboxymethylcellulose.
Fig. 11.1. Release of reducing-sugars (RS) from xylan and carboxymethylcellulose

(CMC) during a 5 h incubation with three commercially available enzyme
preparations. Note that the relationship between reducing-sugar release and
enzyme concentration is not linear, indicating that concentration of enzyme
selected can affect estimates of activity. (Hristov and McAllister, unpublished.)
situations is limited by: (i) variations in microbial composition among inocula from
different donor animals; (ii) differences in growth of microbial populations in the in
vitro system versus in the rumen; and (iii) artefactual accumulation of end products
that alter enzyme activity. Additionally, these assays do not consider the possible
impact of exogenous enzymes on biological parameters such as feed intake, rate of
passage or post-ruminal digestion of nutrients.
Because viscosity of intestinal digesta is closely correlated with growth and feed
efficiency in poultry, viscosity measurements have been used as a standard for assess-
ing the biological value of exogenous carbohydrases for poultry (Sabatier and Fish,
1996). For ruminants, however, the value of enzymes can presently be assessed only
through expensive and time-consuming production experiments with beef or dairy
cattle, which makes screening large numbers of products impractical. This lack of an
adequate bioassay for assessing the value of exogenous enzymes is perhaps the greatest
impediment to the development of more efficacious enzyme products for ruminants.
Production Responses to Exogenous Enzymes

Beef cattle
Evidence that exogenous enzymes could improve average daily gain and feed
efficiency in beef cattle was first recorded in a series of ten feeding trials reported 40
years ago (Burroughs et al., 1960). When given diets of ground ear maize, oat silage,
maize silage or lucerne hay treated with an enzyme cocktail containing amylolytic,
proteolytic and cellulolytic activities (Agrozyme, Merck Sharp and Dohme
Research Laboratories), cattle gained 6.8–24.0% more weight and exhibited feed
efficiencies improved by 6.0–21.2%, relative to cattle fed untreated control diets.
In the same year, four different enzyme preparations (Agrozyme, Zymo-Pabst,
Rhozyme and Takamine; Merck and Company, Rahway, New Jersey), given in
combination with diethylstilbesterol, were shown to increase gain by cattle fed a
maize–lucerne hay diet by an average of 14.0% (Nelson and Damon, 1960).
Further studies confirmed that enzyme supplements could improve average
daily gain (ADG) and feed efficiency in cattle fed high silage diets (Rovics and
Ely, 1962), but not all responses by feedlot cattle to enzyme supplementation were
positive. Leatherwood et al. (1960) added a fungal enzyme to a grain supplement
for calves fed a lucerne hay-based diet and found no improvement in ADG or feed
efficiency. Two enzyme preparations containing primarily amylase and protease
activities also failed to increase ADG by cattle given a diet comprising 80%
concentrate and 20% chopped lucerne hay (Clark et al., 1961). In a separate study,
Agrozyme even reduced the ADG of cattle by 20.4% when it was fed with a maize
carrier to beef cattle given a maize silage diet (Perry et al., 1960). Similarly, Kercher
(1960) found that ADG was reduced when Zymo-Pabst was fed with a maize
carrier to cattle given a diet of steam-rolled barley, lucerne hay and maize silage. Perry
et al. (1966) attributed an 18.2% decline in ADG observed in cattle fed Agrozyme
with maize cob diets to a 6.8% reduction in feed intake, because the enzyme had
been shown to enhance fibre digestibility in metabolism experiments.
Although these early studies provided valuable information on the potential
benefits of enzymes for beef cattle, they did little to address the impact on animal
responses of factors such as the composition of the diet, types and levels of enzyme
activities present, or the method of enzyme application. More recent studies have
been designed specifically to address these issues. Different feed types (Beauchemin
et al., 1995, 1997), application levels (Beauchemin and Rode, 1996; Michal et al.,
1996; McAllister et al., 1999), enzyme products (Pritchard et al., 1996) and enzyme
application methods (Beauchemin et al., 1998; Hristov et al., 1998b; Yang et al.,
1999) have been compared under controlled conditions. Application of different
levels (0.25–4.0 l t−1) of a mixture of xylanase and cellulase products and cellulase
increased ADG of steers fed lucerne hay or timothy hay cubes by 30 and 36%,
respectively, but had no effect on ADG when applied to barley silage (Beauchemin
et al., 1995). When this same mixture was applied to a 95% grain diet, feed efficiency
of cattle fed barley was improved by 11% but performance of cattle fed maize was
unaffected (Beauchemin and Rode, 1996). Application of a different mix of fungal
enzyme preparations at rates up to 5.0 l t−1, however, increased the final weight
and ADG of feedlot cattle given diets based on lucerne silage (Michal et al., 1996;
Pritchard et al., 1996) or barley silage (McAllister et al., 1999). Treatment of 82.5%
maize diets with ‘multiple stabilized enzymes’ increased ADG and feed conversion by
feedlot cattle by 10 and 7.5%, respectively (Weichenthal et al., 1996), and similar
improvements in feed efficiency have been reported for cattle fed sorghum-based
diets treated with amylase (Krause et al., 1989; Boyles et al., 1992).
Dairy cattle
The effect of exogenous enzymes on milk production in dairy cows was first
examined in the mid-1990s (Chen et al., 1995; Lewis et al., 1995; Stokes and
Zheng, 1995) and recently there has been a flurry of research activity in this area
(Luchini et al., 1997; Nussio et al., 1997; Kung et al., 1998; Yang et al., 1998, 1999;
Beauchemin et al., 1999). As in studies using beef cattle, production responses by
dairy cattle to exogenous enzymes have also been variable. Application of enzyme
mixtures on to sorghum did not improve milk production by Holstein cows (Chen
et al., 1995). Similarly, milk responses to exogenous enzymes depended on the
method of application to barley-based diets (Beauchemin et al., 1999). Applying a
cellulase/xylanase mixture to diets containing 45–50% concentrate and lucerne
silage, lucerne hay or a mixture of lucerne silage, maize silage and lucerne hay also
failed to increase milk yield (Luchini et al., 1997; Nussio et al., 1997). In contrast,
spraying two similar enzyme preparations on to maize silage in a 50% concentrate
diet increased milk production by 2.5 kg day−1 without altering milk composition
(Kung, 1996). Although it was demonstrated that these enzyme preparations could
increase milk production in cows fed lucerne hay/silage-based total mixed rations
(Lewis et al., 1995; Stokes and Zheng, 1995; Sanchez et al., 1996), positive responses
in milk production were highly dependent on the level of enzyme applied (Sanchez
et al., 1996). For example, research at the Lethbridge Research Centre has shown that
increasing the amount of enzyme applied to lucerne cubes from 1 g kg−1 to 2 g kg−1
increased milk production from 23.7 kg day−1 (control) to 24.6 kg day−1 and
25.6 kg day−1, respectively (Table 11.2) (Yang et al., 1999). Responses to this
enzyme preparation were even more dramatic in early lactation, when treatment of a
diet comprising 24% maize silage, 15% lucerne hay and 61% barley-based concen-
trate increased milk production by 4 kg day−1. The efficacy of this enzyme was appar-
ently dependent upon the method of its application, as spraying the enzyme on to the
total mixed ration did not affect milk production, whereas applying it to the concen-
trate did (4 kg day−1) (Beauchemin et al., 1998).
Lambs
It was shown in the 1960s that feeding a mixture of amylolytic, cellulolytic and
proteolytic enzymes (Agrozyme; 1.5, 3 and 6 g day−1), as well as a potent
Table 11.2. Dry matter intake and yield and composition of milk from lactating cows fed
enzyme-treated diets.
Diet1
Item Control LH HH HT SE2
Dry matter intake (DMI)

kg day−1 20.4 20.7 20.7 20.8 0.7
% of BW 3.29 3.39 3.32 3.42 0.14
Body weight (kg) 621.11 623.11 626.11 619.11 3.11
Milk yield3 (kg day−1)
b23.7b ab24.6ab b25.6a ab25.3ab
Actual 0.6
b22.4b ab22.9ab b24.6a b24.2a
4% FCM 0.7
b22.2b ab23.2ab b24.4a b24.2a
SCM 0.7
Milk composition (%)
Fat 3.79 3.70 3.78 3.76 0.11
Protein 3.36 3.41 3.48 3.49 0.11
b4.56b ab4.61ab ab4.60ab b4.62a
Lactose 0.11
Milk DMI−1 (kg kg−1) 1.2 1.22 1.29 1.25 0.1
abWithin a row, means bearing unlike superscripts differ (P < 0.05).
1LH = low level of enzyme added to cubed lucerne hay; HH = high level of enzyme added to cubed
lucerne hay; HT = enzyme added to both cubed lucerne hay and concentrate.
2SE = standard error.
3FCM = fat-corrected milk; SCM = solids-corrected milk.
proteolytic enzyme (Ficin, Merck and Company; 5, 10 and 20 mg day−1) did not
alter feed conversion or the ADG of fattening lambs fed ground maize or lucerne hay
(Theurer et al., 1963). Recently, McAllister et al. (2000) have also found that
fibrolytic enzymes did not increase feed intake or ADG by lambs fed lucerne hay- or
barley-based diets.
Learning from animal experiments
The positive effects of exogenous enzymes on growth production both by beef and by
dairy cattle have been demonstrated definitively, but the information required to
improve the consistency and increase the magnitude of these responses is unfortu-
nately still lacking. Comparisons between experiments are exceedingly difficult,
because many enzyme products are poorly defined. Further, several studies have
shown that over-application of enzyme is possible, such that increased application
costs are not recovered by corresponding improvements in animal performance
(Beauchemin et al., 1996; McAllister et al., 1999). Thus, application of one enzyme
preparation at a given concentration provides little information with regard to the
potential effect on animal performance of a different application level, let alone a
different product. Method of application also influences production responses; they
have been shown to differ between dry forage, fresh forage and silage (Beauchemin
et al., 1995; Feng et al., 1996), and if the enzyme is infused directly into the rumen,
or applied to the complete diet or to the concentrate component only (Lewis et al.,
1996; Beauchemin et al., 1998; McAllister et al., 1999). It is obvious that many
factors may influence enzyme efficacy in ruminants. Therefore, an understanding of
the modes of action by which enzymes improve nutrient utilization in ruminants is
key to obtaining consistent positive responses to exogenous enzymes over a broad
range of diets and animal types.
Modes of Action
Upon initial consideration, exogenous enzymes might be expected to alter feed utili-
zation in ruminants either through their effects on the feed prior to consumption, or
through their enhancement of digestion in the rumen and/or in the post-ruminal
digestive tract (Fig. 11.2). In actuality, all of these possible modes of action are inter-
twined and enzyme-mediated alteration of the feed prior to consumption likely has
ramifications on ruminal and post-ruminal digestion of nutrients. Preconsumptive
effects of exogenous enzymes may be as simple as the release of soluble carbohydrate
or as complex as the removal of structural barriers (Fig. 11.2A) that limit the
microbial digestion of feed in the rumen. Within the rumen, exogenous enzymes
could act directly on the feed or could indirectly stimulate digestive activity through
synergistic effects on ruminal microorganisms (Fig. 11.2B). Exogenous enzymes may
remain active in the lower digestive tract, contributing to the post-ruminal digestion
of fibre, or they could indirectly improve nutrient absorption in the lower tract by
reducing viscosity of intestinal digesta (Fig. 11.2C). They may also supplement
enzyme activity in the faeces, thereby contributing by accelerating decomposition of
waste (Fig. 11.2D). Ultimately, the goal of enzyme supplementation is to improve
the efficiency of feed utilization in ruminants and reduce waste production.
Undoubtedly, the mode of action of exogenous enzymes in ruminants is exceedingly
complex and continues to be a major focus of the research presently being conducted
with these additives.
Preconsumption effects
There is ample evidence that exogenous enzymes can release reducing-sugars

from feedstuffs prior to consumption (Beauchemin and Rode, 1996; Hristov et al.,
1996a,b). The degree of sugar release depends on both the type of feed and the
type of enzyme. For example, only two of 11 enzyme preparations tested released
significant amounts of reducing-sugars from barley silage (Hristov et al., 1996a).
Moreover, the preparations most effective at releasing reducing-sugars from lucerne
hay were not those that released the most reducing-sugars from barley silage. There is
evidence that exogenous enzymes may be more effective when applied to dry forage
as opposed to wet forage (Feng et al., 1996; Beauchemin et al., 1998). At first this
seems improbable, given that the role of water in the hydrolysis of soluble sugars
Fig. 11.2. Possible modes of action of exogenous enzymes in ruminants. (A) Prior
to consumption, exogenous enzymes may partially digest feed or weaken structural
barriers that impede microbial digestion in the rumen. (B) In the rumen, exogenous
enzymes may hydrolyse feed directly or work synergistically with ruminal micro-
organisms to enhance feed digestion. (C) In the small intestine, exogenous enzymes
may improve nutrient absorption by reducing intestinal viscosity, or by hydrolysing
substrates that escape ruminal digestion. (D) In the faeces, exogenous enzymes may
increase the rate of decomposition.
from complex polymers is a fundamental biochemical principle (Lehninger, 1982).

However, feed offered to ruminants is seldom absolutely dry; even feeds that
nutritionists would describe as ‘dry’ (e.g. grain, hay) contain 6–10% moisture.
Release of soluble sugars from these ‘dry’ feeds suggests that their water content is
sufficient to enable hydrolysis.
Release of sugars from feeds arises at least partially from the solubilization of
NDF and ADF (Hristov et al., 1996a; Gwayumba and Christensen, 1997). This is
consistent with observed increases in the soluble fraction and rate of in situ digestion
(Feng et al., 1996; Hristov et al., 1996a; Dong, 1998; Hristov et al., 1998a; Yang
et al., 1999). However, most studies have not found that exogenous enzymes
improve the extent of in situ or in vitro DM digestion (Feng et al., 1996; Hristov
et al., 1996a). These results suggest that enzyme additives only degrade substrates
that would be naturally digested by the endogenous enzymes of the rumen
microflora. Using scanning electron microscopy, we have observed that high
concentrations of fibrolytic enzymes can cause digestive pits in the cell walls of bar-
ley straw (Fig. 11.3). However, no similar degradation was observed when enzymes
Fig. 11.3. Scanning electron micrographs of barley straw, (a) untreated or (b)
incubated in a 1 : 10 dilution of concentrated enzyme product containing
cellulases and xylanases. Note that this exceedingly high concentration of the
enzyme caused visible degradation of the barley straw. In other studies, enzymes
were applied at levels recommended by the manufacturer (e.g. 2.0 l t−1) and no
visible damage of the surface of the straw was observed (data not shown).
Bars = 250 µm. (McAllister, Bae and Cheng, unpublished.)
were applied at the concentration recommended by the manufacturer (McAllister

et al., unpublished data).
Although exogenous enzymes do effect release of soluble carbohydrates, the
amount liberated represents only a minute portion of the total carbohydrate present
in the diet. It is difficult to attribute observed enzyme-associated production
responses solely to the generation of soluble carbohydrates prior to consumption,
given that comparable increases in yield were not seen when up to 9% of total dietary
DM was supplied as molasses (Wing et al., 1988). Additionally, there is ample evi-
dence that through associative effects soluble carbohydrates can actually depress fibre
digestion in ruminants (Huhtanen, 1991). Some exogenous enzyme preparations
contain soluble carbohydrates, which can affect in vitro gas production (Varel et al.,
1993), but it is unlikely that they contribute significant levels of soluble carbohydrate
to the feed at practical application levels. Thus, it is most likely that at least a portion
of the positive production responses observed to accompany enzyme supplements is
due to alterations in ruminal or post-ruminal digestion.
Ruminal effects
Direct hydrolysis
Until recently it was assumed that, upon introduction into the rumen, exogenous
enzymes would be rapidly degraded by the array of proteases produced by ruminal
microorganisms (Kung, 1996). Indeed, fungal cellulases incubated with ruminal

fluid were rapidly degraded to the extent that after 6 h of incubation, less than
25% of their original activity remained (Kopency et al., 1987; Vandevoorde and
Verstraete, 1987). However, experimentation with other enzyme products showed
that CMCase activity and xylanase activity remained constant after 6 h of incubation
with ruminal fluid (Hristov et al., 1998b). Furthermore, exogenous enzymes
increased xylanase and cellulase activity in the rumen (Hristov et al., 1998a,b, 2000)
and there is evidence that declining exogenous enzyme activity in ruminal fluid is
associated both with inactivation of the enzymes and with their outflow with the
fluid phase of ruminal contents (Fig. 11.4) (Hristov et al., 1996b).
The fact that exogenous enzymes remain active in the rumen raises the
possibility that they may improve digestion through the direct hydrolysis of ingested
feed. Several researchers have shown that exogenous enzymes can enhance fibre
Fig. 11.4. Decline in endo-cellulase and β-glucanase activities in the rumen

during a 15 h period following administration of two enzyme products directly into
the rumen. Note that decline of both enzyme activities differs between the two
products, and that the declines are related to the passage of fluid from the rumen,
as measured by the decline in Cobalt-EDTA. RS, reducing-sugars. (Adapted from
Hristov et al., 1996b.)
degradation by ruminal microorganisms in vitro (Forwood et al., 1990; Varel et al.,

1993; Feng et al., 1996; Hristov et al., 1996a; Dong et al., 1999) and in situ (Lewis
et al., 1996). This effect has been confirmed in some (Beauchemin et al., 1999; Yang
et al., 1999) but not in all (Firkins et al., 1990; Varel and Kreikemeier, 1994) studies
conducted using cattle with ruminal and duodenal cannulae. Although adding
exogenous enzymes may increase the activity of xylanases and cellulases in ruminal
fluid, enzyme activity in the fluid usually represents less than 30% of the total
enzyme activity in the rumen, the remainder being associated with the feed particles
(Minato et al., 1966; Brock et al., 1982). For example, applying fibrolytic enzymes to
a grass hay diet for sheep prior to consumption increased endo-glucanase activity and
xylanase activity in ruminal fluid, but this activity accounted for only 0.5% of the
total endo-glucanase activity in the rumen (Table 11.3) (Dong, 1998). Given that
exogenous enzymes represent only a small fraction of the ruminal enzyme activity,
and that the ruminal microbiota are inherently capable of readily digesting fibre
(McAllister et al., 1994), it is difficult to envision how exogenous enzymes would
enhance ruminal fibre digestion through direct hydrolysis.
Synergism with ruminal microorganisms

Enhancement of fibre digestion in the rumen would seem more feasible if these
products are working synergistically with ruminal microbes. Logically, this concept
implies that exogenous enzyme preparations contain enzymatic activities that would
normally be limiting to digestion of plant cell walls by ruminal microorganisms.
Table 11.3. Effect of exogenous enzymes on endoglucanase and xylanase activities1 in the
rumen of sheep fed grass hay. (Adapted from Dong, 1998.)
% Total ruminal activity
CON ENZ CON ENZ
Activity in liquid phase (units ml−1 h−1)

a0.026a a0.029b
Endoglucanase – –
a0.315a a0.344b
Xylanase – –
Activity in particulate phase (units g DM−1 h−1)
Endoglucanase 3.14 3.12 – –
Xylanase 2.37 28.27 – –
Total activity in liquid phase (units × 103)
Endoglucanase 0.14 0.165 3.6 4.6
Xylanase 1.76 1.95 5.4 6.4
Total activity in particulate phase (units × 103)
Endoglucanase 3.79 3.45 96.4 95.4
Xylanase 30.5 28.54 94.6 93.6
a,bWithina row, means bearing unlike superscripts differ (P < 0.05).
1Endoglucanase activity was standardized against a commercial enzyme preparation from P.
funiculosum (EC 3.2.1.4, Sigma Chemical Co., St Louis, Missouri) and xylanase activity was
standardized against a commercial xylanase activity from A. niger (EC 3.2.18, Sigma Chemical).
Incubations were conducted in sodium phosphate buffer (pH 6.5) at 39°C for 30 min.
Limitations to plant cell wall digestion in the rumen could result from insufficient
quantities or types of enzyme production by the ruminal microbes, from an inability
of degradative enzyme(s) to interact with the target substrates, or from conditions in
the rumen not being optimal for enzyme activity (e.g. low ruminal pH). At least 21
different enzymatic activities have been identified as being involved in the hydrolysis
of the structural polysaccharides of the plant cell wall, all of which are produced by
a normally functioning ruminal microflora (White et al., 1993). Researchers have
shown that extracts from Aspergillus oryzae can increase the number of ruminal
bacteria (Newbold et al., 1992a,b) and can work synergistically with extracts from
ruminal microorganisms to enhance release of soluble sugars from hay (Newbold,
1995). The extent of cross-linking by p-coumaryl and feruloyl groups to
arabinoxylans has been identified as one factor that limits the digestion of plant
cell walls (Hatfield, 1993). A. oryzae has been shown to produce an esterase capable
of breaking the ester bridges formed between ferulic and p-coumaric acids and
arabinoxylan (Tenkanen et al., 1991); and these activities have been proposed to
function synergistically with ruminal microorganisms (Varel et al., 1993). However,
many of the ruminal fungi (e.g. Neocallimastix spp.: Borneman et al., 1990) and
some species of ruminal bacteria (e.g. Fibrobacter succinogenes: McDermid et al.,
1990; Butyrivibrio fibrisolvens: Dalrymple et al., 1996) produce esterases capable of
hydrolysing linkages with phenolic acids. The fact that exogenous enzymes usually
only increase the rate and not the extent of digestion (Varel et al., 1993; Feng et al.,
1996; Hristov et al., 1996a) suggests that the activities contributed by these additives
are not novel to the ruminal environment. Recent work in which oligonucleotide
16S rRNA probes are used to study rumen ecology suggests that there may be several
species of fibrolytic ruminal bacteria (e.g. clostridia, ruminococci) that have yet to be
cultured in the laboratory (Forster and Whitford, 1998). If this is the case, these
microorganisms may also be contributing to the diverse array of enzyme activities
required for efficient digestion of plant cell walls.
Hydrolysis of cellulose and hemicellulose is accomplished either by free enzymes
or by cellusomal structures comprising multiple enzymes bound non-covalently to
form an organized complex (Teeri, 1997). Aerobic fungi, the principal commercial
source of exogenous enzymes, hydrolyse plant cell walls by means of free enzymes,
whereas hydrolysis of plant cell walls by Clostridium spp. and the anaerobic ruminal
fungi involves cellulosomal structures (Beguin et al., 1998; Ljungdahl et al., 1998).
There is also evidence that the ruminococci may rely on a cellulosome-like
multienzyme complex for fibre degradation (Flint et al., 1998; Ohara et al., 1998).
Destruction of these multienzyme complexes during the extraction process may
explain why enzymes from mixed ruminal microorganisms failed to release as much
soluble sugar from hay and straw as extracts from A. oryzae (Newbold, 1995).
Evidence has also been presented that cellulosomes may play a role in adhesion
of microbial cells to their substrates (Pell and Schofield, 1993; Beguin et al., 1998).
The process of adhesion is absolutely essential for efficient digestion of forages
and cereal grains in the rumen (McAllister et al., 1994; McAllister and Cheng, 1996).
Cellulose-binding domains may be involved in the attachment of ruminal bacteria to
cellulose (Pell and Schofield, 1993). Presently it is not known if exogenous enzymes
block microbial adhesion sites on the surface of feeds, or expose additional ones.
Administering an aqueous solution of mixed fibrolytic enzymes (3.3% vol/vol)
directly into the rumen of cannulated sheep actually lowered DM digestion
(McAllister et al., 1999), and similar results were reported when enzymes were
infused into beef steers (Lewis et al., 1996). These studies suggest that enzymes are
not as efficacious when ruminally infused as when applied directly to the feed.
Alternatively, this response may be enzyme dependent, given that administration of
other enzymes directly into the rumen did not affect DM digestion (Hristov et al.,
2000). Treating lucerne cubes with exogenous enzymes prior to consumption
increased bacterial colonization and in situ DM disappearance of forage between 3 h
and 24 h of ruminal incubation (Yang et al., 1999). These responses were supported
by a concurrent numerical increase in digestibility of fibre in the rumen, and by
significantly increased digestibility of fibre in the total gastrointestinal tract (Yang
et al., 1999).
Considering the low fibre content of high concentrate diets, it is surprising
that fibrolytic enzymes have improved feed digestion (Krause et al., 1998) and
performance of cattle fed high cereal grain diets (Beauchemin et al., 1997; Iwaasa
et al., 1997). An explanation of this phenomenon may come from comparing the
pH optima of the fibrolytic enzymes produced by ruminal microorganisms with
the pH optima of exogenous fibrolytic enzymes produced by aerobic fungi. It is well
documented that growth of fibrolytic bacteria is inhibited (Russell and Dombrowski,
1980), and that fibre digestion is severely compromised (Hoover et al., 1984) when
pH falls below 6.2. Most of the fibrolytic enzymes produced by ruminal micro-
organisms function optimally at pH values above 6.2 (Greve et al., 1984; Matte
and Forsberg, 1992). In contrast, the pH optima of fibrolytic enzymes produced by
aerobic fungi typically range from 4.0 to 6.0 (Gashe, 1992; Muzakhar et al., 1998).
This point is illustrated by an experiment in which the extent to which Trichoderma
longibrachiatum enzymes enhanced gas production was shown to increase as the pH
declined from 6.5 to 5.5 (Table 11.4) (Morgavi et al., unpublished data). Further,
although a decline in pH from 6.5 to 5.5 reduced DM disappearance from maize
silage in mixed ruminal cultures supplemented with T. longibrachiatum enzymes, the
negative effect of low pH on DM disappearance was more pronounced in the absence
of added enzyme (Table 11.4). Ruminal pH can be below 6.0 for a significant
portion of the day in dairy cattle (Nocek, 1998; Yang et al., 1999) and in feedlot
cattle (Krause et al., 1998). Under these conditions, exogenous enzymes could make
a significant contribution to ruminal fibre digestion. The higher fibre content of
barley, as compared with maize, may explain why exogenous enzymes improved feed
conversion in finishing cattle fed barley grain but did not affect feed conversion by
finishing cattle fed maize (Beauchemin et al., 1997).
Some evidence also suggests that non-enzymatic factors in crude enzyme extracts
may work synergistically with ruminal microorganisms. In early experiments with
fungal cellulases, boiling the extract for 20 min failed to reduce its effect on cellulose
digestion in vitro (Bowden and Church, 1959). In that study, adding valine or
proline to the incubation medium enhanced cellulose digestion to the same extent
as adding fungal cellulases. A more recent study showed that autoclaved A. oryzae
Table 11.4. Effect of pH and T. longibrachiatum enzyme preparations on in vitro gas production
and dry matter disappearance from maize silage during 48 h of incubation with mixed ruminal
cultures.1
Enzyme
Item pH No enzyme Autoclaved Unautoclaved
Gas production (ml) 6.5 a 10.4a 11.9a

9.4
a7.3a 8.0a 9.8b
6.0
a6.4a 7.1a 9.0b
5.5
DM disappearance (%) 6.5 32.1a 31.5a 36.2b
a23.6a 23.5a 31.8b
6.0
a23.2a 22.8a 32.7b
5.5
a,bWithina row, means bearing unlike superscripts differ (P < 0.05).
1Consecutive batch culture techniques were used to adapt mixed ruminal microorganisms to each
respective pH prior to incubation (Morgavi et al., unpublished data).
extract (8% vol/vol) enhanced cell wall degradation in vitro (Varel et al., 1993). The
researchers attributed the effect to the presence of soluble substrate in the extract,
but concluded that it would not be relevant at the concentration of extract
(i.e. 0.067 mg ml−1) expected at recommended in vivo dosages. The effects of non-
enzymatic factors are likely expressed more in vitro than in vivo, because microbial
populations have limited adaptive capabilities in vitro, and they are exposed to higher
concentrations of the additive due to the absence of flow or dilution. Enzyme extracts
often contain preservatives to prolong their shelf life, as well as emulsifying agents
(e.g. surfactants) that maintain the enzymes in suspension and facilitate application
of the product to the feed. Work in our laboratory has shown that surfactants can
alter microbial activity and feed degradation in the rumen (McAllister et al., 2000).
Unfortunately, few enzyme manufacturers list the non-enzymatic components
of their products and the consequent difficulty in distinguishing between non-
enzymatic and enzymatic effects of enzyme products continues to hamper progress
toward defining the mode of action of these products.
Digesta flow
Enzymes have been shown to enhance (Feng et al., 1996; Dong, 1998) and to exert
no effect on (Beauchemin et al., 1999; Yang et al., 1999) the rate of passage of
particulate matter from the rumen. Increases in passage rate can be associated with a
faster rate of particle size reduction in the rumen and a corresponding increase in feed
intake (Mertens et al., 1984). Although exogenous enzymes have improved both
intake and digestion in some experiments (Stokes and Zheng, 1995; Sanchez et al.,
1996), in other srudies enhanced digestion is not associated with an increase in feed
consumption or particulate passage rate (Judkins and Stobart, 1988; Krause et al.,
1998; Kung et al., 1998; Beauchemin et al., 1999). These studies suggest that some
portion of the effects of exogenous enzymes may be post-ruminal.
Post-ruminal effects
Experiments in our laboratory have shown that exogenous enzymes not only
heighten fibrolytic activity in the rumen, but also increase fibrolytic activity in the
small intestine (Hristov et al., 1998a,b, 2000). This phenomenon was particularly
evident with xylanase activity. Supplementary enzymes increased duodenal xylanase
activity by 30% (Hristov et al., 1998a). In the same study, enzyme supplementation
increased cellulase activity at the small intestine by only 2–5%, because these
enzymes were largely inactivated by the low pH and pepsin in the abomasum. Other
researchers have reported that xylanases from both mesophilic and thermophilic
microorganisms resist proteolysis (Fontes et al., 1995), possibly due to their high
degree of glycosylation (Gorbacheva and Rodionova, 1977), but our work has shown
that acidity, not pepsin, is the primary factor responsible for the inactivation of
xylanases in the abomasum (Hristov et al., 1998a). At pH 2.0–2.6 (Sturkie, 1970)
gastric fluids in the gizzard of poultry are typically less acidic than digesta in the
stomach of swine or in the abomasum of ruminants, and they have shorter residence
times. This difference in gastrointestinal physiology may explain why positive
responses to enzyme supplementation are more consistent in poultry than in swine or
ruminants (Campbell and Bedford, 1992; Baas and Thacker, 1996).
Administering large quantities of enzymes (400 g day−1) directly into the rumen
can significantly increase cellulase activity in the small intestine (Hristov et al., 2000).
Exogenous enzymes supplemented in this manner flow mainly with the fluid
phase of ruminal contents (Hristov et al., 2000) and at these elevated feeding levels
a portion of the cellulases even escapes inactivation by the low pH and pepsin in the
abomasum. We have observed that increased xylanase activity in the small intestine is
associated with a decline in intestinal viscosity (Table 11.5) (Hristov et al., 1998b,
2000). Because viscosity of duodenal digesta increases with increasing levels of grain
in the diet (Mir et al., 1998), enzyme-mediated reductions in viscosity could improve
nutrient absorption in the small intestine of cattle fed grain diets. Reduced intestinal
viscosity was associated with 1.2% and 1.5% increases in total tract digestibility
of DM when enzymes were applied to the feed or infused into the abomasum,
respectively (Hristov et al., 1998a). However, intestinal viscosity in cattle is only
between 1 and 2 cP (Mir et al., 1998) whereas intestinal viscosity in poultry
may exceed 400 cP (Bedford, 1993). Improved growth performance in poultry
supplemented with enzymes is often associated with tenfold reductions in intestinal
viscosity (Bedford, 1993; Graham, 1996). Consequently, it is difficult to compre-
hend how the relatively modest declines in intestinal viscosity observed in ruminants
supplemented with high levels of enzymes results in a substantial improvement in
nutrient absorption in the small intestine.
In studies with dairy cows fed barley grain diets, improvements in total tract
digestion were attributed primarily to an improvement in the digestibility of fibre
and starch in the lower tract (Beauchemin et al., 1999). Hydrolysis of complex carbo-
hydrates by exogenous enzymes in the small intestine and subsequent absorption of
released sugars would offer energetic and nitrogen balance benefits to the animal that
would not be accessible if these substrates remained undigested or were fermented by
Table 11.5. Enzyme activity1 in duodenal digesta and faeces of cattle with and without enzyme
supplementation.
Experiment 12 Experiment 23
Item Control EF EA Control EFL EFM EFH P4
Ruminal contents
CMCase 42.7 51.9 41.1 43.3 48.0 48.6 52.9 ***
Xylanase 215.2 298.1 208.1 179.1 237.3 264.0 299.1 ***
a190.1a ab160.5ab b95.9b
Amylase 144.1 138.1 137.9 144.0 NS
AB3.16AB B2.99B A3.34A
Viscosity (mPa s) 3.33 3.06 2.90 2.74 ***
Duodenal contents
CMCase ND 0.75 2.20 0.18 0.79 1.89 6.32 ***
a3.6a a45.1b b190.1b
Xylanase ND 6.72 13.7 37.4 ***
Amylase 0.98 0.82 0.77 0.92 0.87 0.98 2.88 ***
Viscosity (mPa s) 1.73 1.56 1.54 1.75 1.53 1.73 1.49 ***
Faeces
CMCase NM NM NM 30.7 19.6 26.5 46.0 NS
Xylanase NM NM NM 658.5 686.0 832.1 900.0 ***
Amylase NM NM NM 447.3 479.2 512.1 450.2 NS
Total tract digestibility (%) 80.2 81.4 81.9 74.9 76.2 75.3 74.5 NS
1Expressed as nmol reducing-sugars released ml−1 min−1.
2EF, enzyme applied to feed; EA, enzyme infused into abomasum. Adapted from Hristov et al.
(1998).
3Enzyme introduced directly into the rumen at rates of 0 (Control) or 100, 200 and 400 g day−1
(EFL, EFM and EFH, respectively). Adapted from Hristov et al. (2000).
4Linear effect of enzyme supplementation on enzyme activity (P < 0.001).
abWithin a row and experiment, means bearing unlike superscripts differ (P < 0.05).
ABWithin a row and experiment, means bearing unlike superscripts differ (P < 0.10).
NS, not significant (P < 0.05).
microbial populations residing in the large intestine. It is possible that exogenous

enzymes work synergistically with the microbes even in the large intestine, given
that we have determined that xylanase activity in the faeces increases linearly with
increasing levels of enzyme supplementation (Table 11.5) (Hristov et al., 2000). This
heightened fibrolytic activity may have important ramifications with regard to the
rate at which faecal material decomposes in the environment.
Steps toward Improving Exogenous Enzymes for Ruminants

Matching the enzyme to the feed
Not all exogenous enzymes are equally effective at digesting complex substrates
such as lucerne and barley grain (Table 11.6). Feedstuffs are exceedingly complex
structurally, and lack of knowledge of the factors that limit the rate and extent of feed
Table 11.6. Release of reducing-sugars1 from lucerne hay and hull-less barley by a number of
commercially available fibrolytic enzyme products.
Substrate
Product Lucerne hay Hull-less barley
A 379.0 4980.32
B 102.8 1384.4
C 122.3 1785.5
D 106.4 1201.8
E 30.2 387.0
F 31.0 489.5
G 134.7 1808.7
H 156.1 2780.3
I 170.7 2424.9
J 62.8 2558.3
1Activity is expressed as p.p.m. of glucose released by the product (present at 0.250 mg ml−1)
under standardized test conditions (Hristov and McAllister, unpublished observations).
digestion impedes the engineering of enzyme preparations designed to overcome

constraints to feed digestion. With some feeds, specific targets can be identified. In
maize, for example, the protein matrix surrounding the starch granules, as opposed to
the properties of the starch itself, dictates the extent and rate of starch digestion in
that grain (McAllister et al., 1993). Thus, exogenous enzymes designed to improve
the utilization of maize should contain proteases capable of digesting the protein
matrix and exposing starch granules to digestion by endogenous ruminal or host
enzymes. An exogenous preparation containing amylase but not protease activity
would not be expected to substantially improve utilization of maize by ruminants. In
straw, the major barriers to microbial digestion are apparently silica, wax and cutin
(Bae et al., 1997), whereas in unprocessed grain the pericarp dictates the extent of
grain digestion (Wang et al., 1998). Enzyme preparations that digest the structural
components limiting the extent of feed digestion in the rumen would be expected to
be more efficacious than preparations that just increase the rate of feed degradation in
the rumen. Many enzyme preparations are currently in use, with no attempt being
made to define the types or activities of the enzymes they contain. Such random
employment of enzymes on feeds, without consideration for specific substrate
targets, will only discourage or delay adoption of exogenous enzymes for more
standard use in the animal production industry. Ultimately, enzyme cocktails
should be designed specifically to overcome the constraints limiting digestion of
a particular type of feed. Component enzymes in such cocktails might vary even
for a given forage, targeting particular maturity levels and structural barriers. Recent
developments in biotechnology make it feasible to engineer such enzyme cocktails
containing xylanase and β-glucanase activities, but we presently lack the technology
for specific production of many other potentially important enzymes (e.g. cutinase,
ferulic acid esterase, acetylxylan esterase, arabinofuranosidase).
Lowering enzyme cost
Once the mechanisms of action and specific targets for degradative exogenous
enzymes have been identified, steps can be taken to optimize the application of these
preparations. Application concentrations can be minimized by ensuring that the
preparations contain the enzyme activities most likely to elicit an improvement in
feed utilization. In some instances, the activity of crude enzyme preparations may be
increased by including specific enzymes most likely to overcome the constraints to
feed digestion. Recently, application of recombinant DNA technology has enabled
manufacturers to increase the volume and efficiency of enzyme production, and to
create new products (Ward and Conneely, 1993; Hodgson, 1994). Genes encoding
superior enzymes can be transferred from organisms such as anaerobic bacteria and
fungi, typically impractical for commercial production, into well-characterized
industrial microbial production hosts (e.g. Aspergillus and Bacillus spp.). Expression
of genes encoding novel enzymes in plants such as canola and potato may be a partic-
ularly effective means of lowering the cost of enzyme production (Pen et al., 1993;
van Rooijen and Moloney, 1994). At the Lethbridge Research Centre, a β-glucanase
gene from the ruminal bacterium F. succinogenes has been expressed in a number of
lines of potato, and a tenfold difference in level of expression has been observed
among lines (Table 11.7). The line exhibiting the highest expression levels is now
being evaluated in diets for poultry. It is possible that similar technologies (e.g.
grasses expressing cutinase, esterase) may have applications in ruminant production.
Conclusion
The use of feed enzymes in the monogastric animal production industry has
increased dramatically in recent years and numerous commercial products are
presently being marketed. In many instances, the mechanisms and modes of action
Table 11.7. Accumulation of glucanase in the leaf and tuber tissue of seven transgenic lines1 of
potato (Solanum tuberosum).
Glucanase (as % of total protein)
Line Leaf tissue Tuber tissue
1 0.011 0.004
2 0.016 0.005
3 0.020 0.011
4 0.031 0.023
5 0.067 –
6 0.105 0.050
7 0.047 0.021
1β-glucanase from F. succinogenes was expressed in potato using the cauliflower mosaic virus
(CAMV) 35S promoter (Armstrong et al., unpublished observations).

of these preparations have been defined. In contrast, few commercial preparations of

exogenous enzymes exist for ruminants and many of these just now are entering the
marketplace. Although positive responses in animal performance have been observed,
results have been inconsistent. Characteristics of the ruminant digestive tract
(e.g. complex microbial populations producing numerous endogenous enzymes)
complicate the elucidation of the mechanisms of exogenous enzyme action in
ruminants. There is evidence that exogenous enzymes initiate digestion of feeds prior
to consumption and that they can improve feed digestion in the rumen and lower
digestive tract. The challenge for researchers is to determine the modes of action,
singly or in combination, that enable exogenous enzymes to improve feed efficiency
and increase growth and milk production.
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Microbial Interactions in
the Response to Exogenous
Enzyme Utilization
12
M.R. BEDFORD1 AND
J. APAJALAHTI2
1Finnfeeds, PO Box 777, Marlborough, Wiltshire SN8 1XN, UK;
2Danisco-Cultor Innovation, Kantvik, FIN-02460, Finland
M.R. Bedford
Microbial
12 Interactions
and J. Apajalahti
with Exogenous Enzymes
Introduction
The intestinal microflora are an integral part of the digestive system of all animals.
The fact that they are living organisms means that they have nutritional and spatial
requirements. This implies that the ability of the digestive system to digest and
absorb nutrients is, in part, dependent upon the species distribution and total
population of resident microbes. The species distribution determines the range of
metabolic processes that can be found and the total population the degree or extent
of such processes. The microfloral population is itself very much dependent upon the
diet as the ultimate source of substrates for metabolism (Wagner and Thomas, 1987;
Savory, 1992). As a result, changes in dietary composition or nutrient density can
have dramatic effects on the intestinal microfloral populations (Gibson et al., 1996;
Hillman, 1999; Reid and Hillman, 1999), which in turn influence the ability of the
animal to digest and absorb nutrients therein. Exogenous enzymes have frequently
been shown to improve nutrient digestibility and some authors have identified
concomitant changes in the intestinal flora (Hock et al., 1997; Vahjen et al., 1998).
Whether such changes are merely an adaptation to the new environment or whether
these changes play a large role in the physiological response seen in the host animal
on use of exogenous enzymes is the focus of this chapter. Due to the relative paucity
of data in pigs, the majority of this chapter will focus on poultry.
Physicochemical Environment of the Intestinal Tract

There are various estimates of the numbers of different strains of bacteria that inhabit
the gut of the broiler, but many settle in the range of 400–500. Most strains are
unknown, many unculturable and thus knowledge of this ecosystem is at best rudi-
mentary. The conditions in the intestine range from the relatively aerobic environ-
ments of the crop and duodenum to progressively anaerobic conditions of the ileum
300 M.R. Bedford and J. Apajalahti
and ultimately the caecum and colon where the redox potential is often less than
−400 mV (J. Apajalahti, unpublished data), equivalent to that found in the rumen.
Differences exist in the pH of these environments, either as a result of the digestive
activity of the host (e.g. acid secreted in the stomach/proventriculus or neutralization
by secretion of sodium bicarbonate in the duodenum) or the microbes themselves
(production of volatile fatty acids, amines, etc.). The substrates available for fermen-
tation range from complex plant and animal polymeric compounds such as cellulose,
xylan and proteins to the simplest monomeric sugars, amino acids and fatty acids.
The conditions existing in the compartment of the intestine where such substrates
find themselves limits and dictates the metabolic processes through which they can
be utilized. Anaerobic fermentation dominates in the large intestine whilst facultative
anaerobes and aerobes are more frequent in younger animals (Naqi et al., 1970) and
in the upper reaches of the intestine. Bacterial fermentation may provide resources to
the host in terms of vitamins, essential amino acids and fatty acids, energy in the form
of volatile fatty acids (VFAs) and many growth factors, which include VFAs and
polyamines (Annison et al., 1968; Just et al., 1983; Dierick et al., 1989, 1990; Choct
et al., 1996; Noack et al., 1996). On the other hand they can produce toxins and
carcinogens, compete for nutrients directly and create disease conditions directly or
indirectly (Muramatsu et al., 1994; Choct et al., 1996; Johnston, 1999; Schutte and
Langhout, 1999; Titball et al., 1999; Uchida et al., 1999). Ultimately the microfloral
population resident in the gut is dependent upon the resources provided (principally
derived from the diet and subsequent metabolites, and from the intestine itself –
mucin, endogenous losses, etc.) and the initiating flora (which depends upon envi-
ronmental exposure and species interactions). Since diet, sex, age and housing, which
represent a fraction of all the parameters involved in determining the subsequent
microfloral ecosystem, vary with each experiment, it is not surprising to find discrep-
ant data in the literature. The microbial population permutations are potentially lim-
itless. It is therefore currently not possible to describe consistently and adequately a
given microfloral population on the basis of the diet offered to the host and it is even
more difficult to describe its contribution to the nutritional or health status of the
host. Whilst some of the effects and capabilities of individual species may be under-
stood in isolation, in a living gut the roles that each species plays and the potential
interactions with others, be it beneficial or detrimental to the host, are not clear at all.
Given these constraints, however, it is still clear that performance responses to
exogenous enzyme addition are likely mediated to be in part through changes in the
microflora species distribution and total population, and therefore it is important
that their role should be determined.
Microbial Responses to Use of Enzymes

Enzyme effects in the ileum
Much of the early work with exogenous enzymes addressed the problematic grains,
rye and barley. The response to enzymes targeting these grains was considered to be
Microbial Interactions with Exogenous Enzymes 301
due to their ability to increase nutrient digestibility and hence assimilation through
depolymerization of the viscous cell wall carbohydrates in these grains (discussed
in more detail in Chapter 7). Viscous digesta was blamed for reduced rates of
convection and diffusion, which slowed the rate of nutrient extraction from the
intestines (White et al., 1981, 1983; Annison and Choct, 1991; Bedford and
Classen, 1992; Classen et al., 1995). Enzymatic depolymerization reduced viscosity
and improved nutrient extraction rate and the result was more rapid growth.
However, even in such early work it was quickly established that the detrimental
effects of feeding rye- and barley-based rations could be partially, if not largely,
overcome by use of antibiotics (Moran and McGinnis, 1968; MacAuliffe et al., 1976;
Korelski and Rys, 1985). More recent work, in which germ-free and conventional
birds were fed highly viscous diets, demonstrated that the negative effects of viscosity
were virtually absent in the germ-free bird (Schutte and Langhout, 1999). Such
observations suggest that viscosity per se is not relevant unless taken in context with
the microbial status of the bird. Subsequent work with wheat and more recently
maize and sorghum-based diets has identified additional and alternative mechanisms
of direct enzyme action (cell wall mechanisms, starch digestion mechanisms) which,
again, may only be relevant when considered along with the microfloral status of the
host. The proposition of this chapter is that exogenous enzymes in use today (with
the notable exception of phytase) improve animal performance largely through
interaction with the intestinal microflora. It is proposed that the exogenous cell
wall-degrading enzymes have two types of activity that need consideration: removal
and provision of substrates for microbial fermentation. The former is more likely to
be an effect seen in the small intestine and the latter in both lower small and large
intestine. Each activity and its consequences are discussed in detail below.
Ileal phase – substrate removal
The small intestine of monogastrics, particularly chickens, was for a long time
thought to be largely devoid of bacteria, and any counts were assumed to be contami-
nants rather than established flora. It was later established that in fact there was a very
rapid colonization of the small intestine of both turkeys and chickens within the
first few days post hatch (Lev et al., 1957; Naqi et al., 1970; Vahjen et al., 1998).
Subsequent work has established that there is a specific progression from bacteria
staining Gram-positive to those staining Gram-negative in the jejunum of turkeys as
they age from 1–21 days old. This evolutionary pattern varies, depending upon the
source flock and farm on which the birds were raised (Morishita et al., 1992).
The direct source of substrate for much of this flora is the diet of the host, with
poorly digested diets leaving far more substrate for microbial fermentation than
highly digestible diets. Dramatically different ileal flora are evident when comparing
rye with maize-based diets (Wagner and Thomas, 1987), the rye diet being very
poorly digested and supporting much larger numbers of anaerobes, and in particular
spore-forming anaerobes, than the maize-based diet. Supplementation of the same
maize diet with a small percentage of pectin resulted in a dramatic increase in
numbers of anaerobes and in particular the spore formers. This was likely brought
about through two properties of pectin that are relevant to this chapter, namely the
rheological properties of pectin, which would slow down the rate of digestibility and
reduce oxygen tension through reduced digesta mixing, and its role as a substrate for
bacterial fermentation. Reducing oxygen tension and provision of extra substrate will
evidently favour increases in the numbers of anaerobes, and the fact that the pectin
itself is fermentable will select for those strains capable of using the constituent
uronic acids.
Such observations are supported by more recent work, where addition of
exogenous enzymes reduces viscosity of wheat- and rye-based diets and as a result
reduces populations of small-intestinal flora (Choct et al., 1996, 1999; Hock et al.,
1997). Presumably, the addition of the enzyme increases the rate at which starch and
protein are removed from the small intestine, which effectively leads to less material
being available for microbial fermentation.
In many respects, the numerous studies that have reported significant
improvements in ileal digestibility of nutrients on enzyme addition (Pettersson et al.,
1991; Bruce and Sundstol, 1994; Jondreville et al., 1995; Dänicke et al., 1997b;
Kemme et al., 1999; Ravindran et al., 1999; Wyatt et al., 1999) have underestimated
the significance of their results. Whilst extra nutrients captured by the terminal ileum
do indeed contribute to a greater extent to the productive energy of the animal
than those captured in the large intestine, the effect of such a response on the small-
intestinal microflora is ignored by most. For example, if starch digestibility is 80%
at the terminal ileum, then there is 20% remaining which has the potential to feed
the microflora in the large intestine. If addition of an enzyme accelerates the rate
of digestion of the starch such that 85% has been digested by the terminal ileum,
then the classic response of a nutritionist would be that the animal has extracted
5% more starch and as such will gain more productive energy from the diet. The
microbiologist will recognize that there is now only 75% of the fermentable starch of
the control diet entering the large intestine. Moreover, that proportion of the starch
that has been removed by enzyme addition was likely the most easily degradable of
the residual starch. With less substrate available to support starch fermenters in the
lower small intestine, populations fall (Fig. 12.1). Similar conditions will apply for
protein digestibility and protein utilizers.
Highly indigestible diets will support greater intestinal microfloral populations,
and population reductions will be greatest with the most effective enzyme applica-
tion. Figure 12.2 demonstrates that in birds fed a reasonable quality wheat-based
diet, the application of a xylanase-based enzyme results in a 60% reduction in micro-
bial numbers. The antibiotic evidently reduces populations by direct activity against
the target species, and the numbers are reduced more dramatically than that seen
with enzyme use. Whilst both reduce microfloral populations, their modes of action
are evidently quite different. The performance gain on use of an antibiotic is thought
to be due to many factors, such as reduced competition for nutrients in the small
intestine, reduced local inflammation due to control of pathogens, and reduced
intestinal thickening and length as a result of improved digestibility and pathogen
loading (Thomke and Elwinger, 1998a,b). The latter two mechanisms result in a
Fig. 12.1. Micrograph of the terminal ileum contents of broilers fed wheat-based
diets without (top panel and bottom left) and with enzyme addition. Top panel
stained for starch, bottom autofluorescing bacteria in digesta sample.
more efficient digestion and reduced maintenance energy requirement. As a result of

the total population changes and species shifts, many of these benefits apply to use of
enzymes (Brenes et al., 1992, 1993; Gollnisch et al., 1996; Hock et al., 1997; Simon,
1998; Vahjen et al., 1998). Whilst the issues discussed to date relate to the ability of
enzymes to remove fermentable substrate from the small intestine, it is apparent that
this is not the only mechanism by which they interact with the microflora.
Caecal phase – substrate provision
By their very nature, enzymes that reduce the size of carbohydrates produce smaller,
more fermentable products. Xylanases and cellulases ultimately produce xylose
and glucose, respectively, and oligomers of various chain length and degrees of
substitution (in the case of xylans). The small intestinal concentration of carbohy-
drates with an apparent molecular weight of 40,000 Da or less was increased almost
twofold on addition of high levels of a xylanase-based enzyme (Bedford and Classen,
1992). More recent work (Apajalahti and Bedford, 1999), shown in Fig. 12.3, breaks
such data into additional categories of smaller molecular weight fragments. The effect
of the enzyme is to increase dramatically the concentration of all xylo-oligomer
fractions, but it is much more pronounced for those that are less than ten xylose
sugars long (fivefold increase compared with 2.3-fold for the DP < 500). These
oligomers are not, by their very nature, either digested or absorbed by the host and
as a result remain in the aqueous phase of the intestinal digesta. Whilst inert to
host enzymes, such oligomers are broken down by bacterial enzymes that are present
in the caeca (Dänicke et al., 1999a) into their constituent sugars, which can then be
utilized by many organisms inhabiting the digesta. Xylo-oligomers fed to diabetic
rats were shown to increase caecal acetate levels more than threefold (Imaizumi et al.,
1991), indicating their potential in supplying substrate to the large intestine. As a
result there is a reduction in the number of starch and protein users (Vahjen et al.,
1998) and an increase in xylose users on incorporation of a xylanase enzyme in the
Fig. 12.2. Effect of addition of either an antibiotic (Avoparcin) or a xylanase on

the total population of bacteria inhabiting the small intestine of 21 day broilers.
Fig. 12.3. Addition of xylanase to a wheat-based diet increases the concentration

of small, soluble xylo-oligomers in the small intestinal tract.
ration (Fig. 12.4., Apajalahti and Bedford, 1999). In this work, broilers were fed a
wheat-based diet with or without supplementation with a xylanase enzyme. The
entire small intestinal digesta was removed and washed in buffer, and the extracted
bacteria were grown in a medium in which xylose provided the only carbohydrate
source. Microscopic enumeration of the total bacteria in each treatment prior to
incubation revealed that the enzyme-treated birds yielded only 40% of the number
of organisms found in the control. Despite this fact, the results demonstrated a
considerable degree of adaptation of the microflora towards utilization of xylose in
enzyme-treated animals. Clearly the continued generation of this sugar in situ by
exogenous enzymes exerts a significant selection pressure on the inhabitants of the
intestine. This is seen in alterations in total numbers of bacteria and/or species
dominance on feeding xylanase to pigs and poultry (Gollnisch et al., 1996; Hock et
al., 1997; Vahjen et al., 1998). As a result of alterations in the intestinal flora profile
in both the small and large intestines, it is likely there will be concomitant alterations
in intestinal morphology, digestive enzyme secretions, and stability of these
secretions and local and systemic immune function.
Some effects are clearly direct. Some species, particularly enterococci and
lactobacilli, numbers of which are often reduced on feeding enzymes, have
been shown to secrete hydrolases that deconjugate taurine and cysteine from
bile acids, rendering them less effective in fat digestion (Campbell et al., 1983;
Bovee-Oudenhoven et al., 1999; Schutte and Langhout, 1999; Tanaka et al., 1999).
As a result, the digestion of fat, particularly saturated fats, is compromised (Dänicke
et al., 1997a, 1999b) and with it the digestibility of many fat-soluble compounds,
Fig. 12.4. Influence of previous exposure to xylanase enzyme on ability of total

small-intestinal flora isolated from broiler intestines to utilize xylose as the only
carbon source. Incubation in media containing only xylose as source of carbon.
Total bacterial contents of digesta washed and collected and used as starter culture
in each experiment. Since 60% fewer bacteria were harvested and hence used per
incubation test for the xylanase-treated birds, the differences between treatments
for each parameter is actually much larger on a per bacterial count.
including vitamins and pigments (Roxas et al., 1996). Application of an enzyme

results in fewer of these organisms and hence improved digestibility of these nutrients
(Hock et al., 1997).
Indirect Interactions between Host and Microflora Mediated

by Enzyme Use
Some effects of enzyme-mediated changes in the microflora have a less direct effect
on host nutrition. For example, use of enzymes has been reported to alter the
concentrations of polyamines and volatile fatty acids (Choct et al., 1996, 1999;
Jansman et al., 1996; Silva and Smithard, 1996), compounds that are known to
influence many processes in intestinal growth and development (Seidel et al., 1985;
Osborne and Seidel, 1990; Shinki et al., 1991; Gee et al., 1996; Noack et al., 1996;
Raul and Schleiffer, 1996). Furthermore, interaction of the microflora with the
intestine will have far-reaching effects on digestion, due to modulations in villus
structure and turnover rate, mucin production and growth rates of the animal as
a whole (Seidel et al., 1985; Smith, 1990; Bhatt et al., 1991; Furuse et al., 1991;
Sharma and Schumacher, 1995; Gee et al., 1996; Noack et al., 1996; Silva and
Smithard, 1996; Sharma et al., 1997). The interaction of the microflora with the
intestines also has effects on the immune status of the animal which will inevitably
interact with and alter the microbial community profile in itself (Ouwehand et al.,
1999) and the susceptibility of the host to antigen damage and some nutrient
deficiencies (Fukushima et al., 1999; Mengheri et al., 1999). The effects of shifts
in fermentation in the caeca can also influence activity elsewhere in the digestive
tract. In rats, the provision of fermentable carbohydrates has been shown to have
profound effects on secretion of enteroglucagon from the colon, which in turn
influences gastric secretions and emptying rates (Gee et al., 1996). As a result the
microfloral status of the large intestine can influence the gastric phase of digestion,
the extent of which in turn will influence the provision of substrates into the small
and large intestines.
In many respects it appears that the nutrition of the animal cannot be divorced
from the process of microfloral interaction with the diet. Digestion and absorption of
nutrients by the host seem to be accomplished through a two-way negotiated process
between host and intestinal microflora that establishes a sustainable partition of the
available resources. Use of exogenous enzyme seems to move this equilibrium more
in favour of the host, hence the animal benefits.
The absence of a microflora will thus reduce much of the benefit of addition
of an enzyme. It is the very fact that the populations of intestinal flora are probably
so different between experiments that results vary so much between experiments.
The next section focuses on some of the factors that need to be considered when
interpreting data from enzyme trials.
Microbial Modulators with Potential to Interact with

Response to Enzyme Usage
The species diversity and density within the intestinal tract of an animal will vary,
depending upon the environment and diet. Environment evidently influences the
composition and density of the resident flora – ranging from the germ-free to the
deliberately challenged or seeded environment (Muramatsu et al., 1994; Droleskey
et al., 1995; Noack et al., 1996; Hofacre et al., 1998; Schutte and Langhout, 1999).
Nipple drinkers are cleaner than bell drinkers (Belyavin, 1995), and floor pens with
litter will provide for greater recycling and build-up of bacterial numbers than
wire-floor cages. Even litter material can significantly influence intestinal flora and
health (Corrier et al., 1993; Bilgili et al., 1999).
The diet is of course a major source of the variation in intestinal flora diversity
and total population. Provision of specific fermentable sugars can significantly influ-
ence composition of the flora, with some (for example, the fructo-oligosaccharides)
being put forward as carbohydrates that confer a favourable fermentation pattern
(Oyarzabal and Conner, 1995; Grizard and Barthomeuf, 1999), whilst others (such
as the raffinose, stachyose and verbascose series of oligosaccharides) have a more
questionable value (Brenes et al., 1989; Krause et al., 1994; Iji and Tivey, 1998). In
any given study, the dietary concentrations of fermentable compounds derived from
ingredients such as soybean meal, canola, peas, etc. are mostly unmeasured.
Antibiotics and coccidiostats with antimicrobial activity markedly influence the
population and composition of the resident flora (Ohya and Sato, 1983; Gollnisch et
al., 1996; Hock et al., 1997). There are also many dietary nutrients, particularly met-
als such as zinc and copper, which, when fed at elevated levels, have a pronounced
influence on the microfloral populations either directly or through interaction with
the immune system (Kidd et al., 1996; Yenigun et al., 1996; Leeson et al., 1997).
Many nutrients interact with the immune system, either positively or negatively
(Korver and Klasing, 1997; Klasing, 1998), which can significantly alter the popula-
tions of the intestines.
When reviewing the literature, it is of value to consider the microbiological
challenge of each study as determined by chick handling, caging and rearing, dietary
ingedients, antimicrobial compounds present, and the status of the immune system.
All such factors, and many more, contribute to the condition and description of the
resident flora. Since enzymes alter substrate flow into the small and large intestines,
the subsequent response to their use will vary according to the populations present
at time of administration and their reaction to such changes. It is not surprising,
therefore, that given the huge range in microfloral conditions likely to exist between
studies, the responses to enzyme use, rather than being absolute, are a continuum
or population of responses from detrimental to highly positive. Determination of
the key bacterial species involved in enzyme response will allow for more thorough
interpretation of future research where such measurements are made.
Conclusions
Many of the factors that govern the scale of response to enzyme addition are just as
relevant to those influencing the response to antibiotics. The response to antibiotics
very much depends on the conditions under which the animals are grown, with
responses being much more marked in conventional than germ-free animals
(Bywater, 1998). Indeed, in conventional birds, the general response to antibiotic
administration is very much influenced by the relative performance of the
non-supplemented controls. The greatest responses are observed in those trials where
the controls performed particularly poorly, and virtually no benefit is reported in
those trials where the controls performed very well (Rosen, 1995). The response to
exogenous enzymes is subject to the same constraints, the greatest responses being
observed with the poorest performing controls (Barrier-Guillot et al., 1995; Classen
et al., 1995; Scott et al., 1998). Scale of response to exogenous enzymes seems to
depend upon not only initial cereal quality but also the microfloral status of the bird.
In studies where both antibiotics and enzymes have been used, in general the
response to enzyme in the presence of the antibiotic is reduced in comparison with
that observed in its absence (Elwinger and Teglof, 1991; Broz et al., 1994; Vranjes
and Wenk, 1996, 1997; Hock et al., 1997). Such observations suggest that the
response to enzymes is reduced when the microflora is controlled by the presence of
an antibiotic. In those studies where the response to enzyme is just as great in the
presence or absence of antibiotic, it is interesting to note that the response to
the antibiotic alone is not particularly great (Allen et al., 1996; Esteve-Garcia et al.,
1997). Reduced intestinal microbial populations probably limit the scope for
improvements in performance due to enzyme supplementation. When digestion is
slowed through addition of carboxymethyl cellulose (CMC), which is a viscous
non-fermentable carbohydrate, the result is more apparent in conventional compared
with germ-free chicks (Smits and Annison, 1996). In essence, addition of an enzyme
to a wheat- or barley-based diet has much the same rheological effect as removal of
the CMC from the diet in the work of Smits and Annison (1996). The implications
of this work and that discussed earlier in this section are that the response to enzyme
addition probably depends on the initial microfloral status of the animals, which in
turn depends on the digestibility of the ration and microfloral challenge. Nutrition,
enzymes, antibiotics, prebiotics and probiotics are in effect all tools for negotiation
for nutrient abstraction from the intestines between the host and its residents.
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1269–1274.
Enzymes: Screening,
Expression, Design
and Production
13
K. CLARKSON, B. JONES, R. BOTT, B. BOWER,
G. CHOTANI AND T. BECKER
Genencor International Inc., 925 Page Mill Road, Palo Alto,
CA 94304-1013, USA
K. Clarkson et of
Development
13 al.New Enzyme Products
Introduction
Enzymes were discovered in the latter part of the 19th century and have been used in
industrial and food processes since the early 1900s. Examples include the use of
amylases in textiles for removal of the starch sizing, bovine rennin in cheese making,
pancreatic enzymes in production of leather and degumming of raw silk, and
proteases for beer stabilization. As a result of the advances in biotechnology over the
past 10–20 years, especially in the areas of genetics and protein engineering, there
is now widespread use of enzymes in detergent, textile, grain wet milling, food,
and pulp and paper applications. Incorporation of enzyme additives in feed rations
is more recent. The benefits of enzymes in feed applications include (Annison,
1993; Bedford and Schulze, 1998; Simon, 1998): (i) balancing deficiencies in the
endogenous enzyme system of the animal; (ii) elimination of anti-nutritionals; and
(iii) increased utilization of the feed. As the value of enzymes continues to grow and
new markets are developed, technology, which can provide methods for more rapid
identification and development of new or improved enzymes, will be a key element
for success. This chapter is a review of current and emerging technologies used in
the discovery, research and development of new enzyme products with a focus on
microbial production hosts.
Background
Enzyme classes and sources
Enzymes are extremely efficient and highly specific catalysts. They also exhibit low
toxicity and act over wide pH and temperature ranges. There are several classes of
enzymes (International Union of Biochemistry and Molecular Biology, 1992):
316 K. Clarkson et al.
1. Oxidoreductases catalyse the transfer of hydrogen from a donor to an acceptor

molecule. These enzymes are also referred to as dehydrogenases or reductases. In cases
where the hydrogen acceptor is oxygen, the enzymes are referred to as oxidases.
2. Transferases carry out the transfer of a group, such as a glucosyl, from one
compound to another.
3. Hydrolases catalyse the cleavage of specific bonds including carbon–oxygen,
carbon–nitrogen and carbon–carbon bonds, and oxygen–phosphorus bonds.
Essentially these are transfer reactions where water is the acceptor molecule. Many of
the enzymes currently used in feed applications, such as amylases, xylanases, cellulases,
proteases and phosphatases, are hydrolases.
4. Lyases can cleave similar bonds as hydrolases but do so by a mechanism of
elimination resulting in the formation of double bonds or rings. They can also carry
out the reverse reaction and add groups to double bonds.
5. Isomerases carry out structural changes within a molecule.
6. Ligases catalyse the coupling of two molecules in conjunction with the hydrolysis
of a high-energy phosphate bond such as ATP.
The majority of enzyme products to date have been derived from mesophilic
and, to a lesser extent, alkaliphilic microorganisms. While these sources continue to
serve as valuable reservoirs of commercial enzymes, other microbial systems, such
as extremophiles, are rapidly gaining attention (Kristjansson and Hreggvidsson,
1995; van der Oost et al., 1996; Vielle and Zeikus, 1996; Adams and Kelly, 1998;
Davis, 1998; Horikoshi and Grant, 1998). Extremophiles are organisms that reside
in extreme environments and they include thermophiles (> 70°C and often in excess
of 100°C), psychrophiles (< 20°C), barophiles (> 1 atm) and halophiles (high salt).
Plant-derived enzymes as well as genetic engineering of plant cells and transgenic
plants are also being explored (Kahl and Weising, 1993, Willmitzer, 1993; Flavell,
1995; Ma et al., 1995; Mazur, 1995; Moffat, 1995; Evangelista et al., 1998; Gale
and Devos, 1998; Kusnadi et al., 1998).
Enzyme properties
It is not unusual for a microorganism to produce an array of enzyme representatives

from each class, subclass and category of enzyme. Many have even evolved multi-
enzyme complexes or systems. The cellulolytic enzyme system, which is a mixture of
cellulases that act synergistically to degrade cellulose, is one example (Wood and
Kellogg, 1988). The cellulolytic system produced by each microorganism is unique
and often comprises several types of cellulases, which can differ in their mode of
action and specificity (substrate preference). Endoglucanases (endocellulases) attack
the internal glucosidic bonds of the cellulose polymer in a random fashion and
produce multiple cellulose chains of varying length as well as oligosaccharides.
Another type of cellulase, cellobiohydrolases, attacks the cellulose chain ends and
produces primarily cellobiose. The more chain ends produced by the endoglucanases,
the more substrate there is for the cellobiohydrolases. Build-up of cellobiose, a potent
Development of New Enzyme Products 317
end-product inhibitor of cellobiohydrolases, is alleviated by β-glucosidase, which

converts cellobiose to glucose. Therefore, the complete or whole cellulase system is
essential for driving the degradation of cellulose to glucose, an important energy
source for many microorganisms. Of further interest is the fact that cellulolytic
microorganisms often generate a variety of endoglucanases and/or cellobiohydrolases.
These enzymes can differ in their physical and biochemical properties such as molec-
ular weight, isoelectric point (pI), pH and temperature activity profile, stability,
and substrate specificity. Production of multiple xylanases, amylases, proteases,
phosphatases, esterases, etc., is also not uncommon. This arsenal of enzymes allows
the microorganism to access nutrients from a variety of sources and to adapt to a wide
range of environmental conditions (pH, temperature, etc.). Through sophisticated
control mechanisms, which have evolved over millions of years, the cell can react to
these types of changes and turn on genes in order to express the required enzymes and
proteins or shut down non-essential protein synthesis and metabolic pathways. In
this way, energy is conserved and directed to growth, reproduction and survival of the
organism.
Although many of the enzymes that are of value in feed applications can be
found in nature, the levels produced by wild-type organisms are relatively low,
making the economics unsuitable for most commercial applications. An added issue
is the fact that many microorganisms produce a mixture of enzymes. While these
mixtures may show a benefit in the application, only one or two of the enzymes
present may actually be responsible. Also, while certain enzymes produced by
a microorganism may be of benefit, others may exert a negative effect in the
application. An example is an enzyme mixture containing both an endo-type
xylanase, which is useful in reducing the viscosity of xylan, and a xylosidase, which
catalyses the hydrolysis of short xylooligosaccharides to xylose. Given that xylose is
known to cause cataracts, diarrhoea and anorexia in some animals, this side activity is
not desirable (Schutte, 1990; Schutte et al., 1990, 1991). Therefore, before designing
a production organism or host, it is important to know which enzyme(s) provides the
benefit in order to develop a host and process that are optimized for its production,
thus reducing undesirable side activities while providing improved economics.
Identification and Development of Novel Enzymes

Enzyme identification
The development of enzyme products often relies on screening a large number

of organisms for an enzyme activity with a specific set of biochemical and physical
characteristics that suit the targeted application. Traditionally, microbial screening
processes have involved evaluation of purified enzymes isolated from: (i) pure cul-
tures obtained from in-house or accessible culture collections throughout the world;
(ii) microorganisms isolated from environments rich in the substrate of interest; and
(iii) microorganisms that have a history of being good enzyme producers, such as
Trichoderma, Aspergillus and Bacillus. While these approaches have met with success
over the years, there are limitations in that they provide access to only a very small
portion of the immense microbial population or diversity. Indeed it is believed that,
to date, only a very small number of microbial species, possibly less than 1%, has
been cultured (Pace et al., 1986; Ward et al., 1992; Amann et al., 1996; Pace, 1997).
Part of the problem lies in the fact that the majority of microbes are not culturable
using standard techniques. While traditional sources and approaches are still valid,
several methods for handling unculturables and for speeding up their analysis have
recently become available (Olsen et al., 1986; Pace et al., 1986; Saiki et al., 1988;
Weller and Ward, 1989; Ward et al., 1992; Amann et al., 1995, 1996). Most of
these involve nucleic acid approaches in which the need to culture an organism is
eliminated. One such method is sequencing of the total genome of specific organisms
in order to screen for DNA sequences that are related to known DNA sequences,
which express the desired type of enzyme activity. Other methods include extraction
and amplification of DNA from unculturables or mixed culture populations. These
approaches and others, including use of RNA, will be discussed in more detail later
in this chapter. To complement these techniques much effort is also being invested
in development of automated, miniaturized, highly sensitive, accurate and rapid
methods for obtaining and analysing nucleic acids and enzymes from various sources.
Despite the diversity of enzymes available in nature, it is sometimes the case that
these enzymes do not possess all of the features necessary for a targeted application.
Instead of rescreening for an enzyme that possesses one additional property, it is
possible to protein-engineer a deficient enzyme by modifying its sequence such
that the lacking feature is incorporated into the enzyme without loss of the existing
desirable features.
Protein engineering of an enzyme can be carried out by site-directed and/or
random mutagenesis. To a limited extent chemical methods, such as acetylation or
amidation, can also be used. Although the chemical approach has met with some suc-
cess, overall it is less predictable. Site-directed mutation involves modification of the
enzyme properties by specific changes in the DNA code through the use of recombi-
nant technology (Carter and Wells, 1987; Bott et al., 1988; Estell, 1993; Cleland
et al., 1996). These changes can be selective amino acid substitutions, deletions or
insertions, or a selective combination of specific structural units of two enzymes to
generate hybrid or chimeric enzymes (Schneider et al., 1981; Mas et al., 1986). A
protein engineering approach can be carried out by elucidating and studying the
three-dimensional structure of the protein by X-ray diffraction, nuclear magnetic
resonance (NMR) and other methods. Molecular graphics and modelling studies can
be used to predict the effect of single or multiple amino acid substitutions. In the past
40 years or so, over 3000 protein structures have been elucidated (PDB, 1996;
Godzik, 1997). Even so, it is still difficult to predict protein structures based on
amino acid sequences. Fortunately some alternative approaches exist, such as random
mutagenesis and, more recently, directed evolution, which has emerged as a powerful
alternative to site-directed protein design. These techniques can be applied to
produce designer catalysts without much knowledge of the protein structure.
Random mutagenesis has been used for decades as a tool for increasing genetic
variability and improving the properties of microbial strains as well as plant cultivars.
Improved strains or enzymes can be generated by random modification of the nucleic

acid sequence followed by screening for the desired properties. Modification or
mutation of the DNA sequence, which encodes for the targeted enzyme, can be
achieved by physical (heat, freezing, UV, etc.) or chemical methods. With random
mutagenesis there is less control over the number and types of mutations made
compared with site-directed engineering, which tends to generate modifications in
localized regions of the amino acid sequence. This means that a huge number of
mutants must be generated and screened. These types of approaches require highly
efficient and rapid screening techniques.
Directed evolution involves multiple cycles of mutagenesis with selection or
screening. Mutants that show improved properties after each cycle are subjected
to further treatment. An accumulation of beneficial mutations is then achieved
(Kuchner and Arnold, 1997). A suitable host for expression of the gene, appropriate
methods for generating genetic variability, and suitable selection and screening
methods are all needed. Again, to ensure a high success rate, a large but manageable
number of clones need to be screened. Another approach that can be applied is
DNA shuffling. This technique involves random fragmentation of DNA sequences
followed by assembly of the fragments into genes by various recombination methods.
By combining enzyme screening with modern techniques such as protein
engineering and directed evolution, hybrid molecules with improved performance
under specific application conditions can be generated.
Enzyme development and production
Once the targeted enzyme has been identified, the gene which encodes the enzyme is
then inserted into a suitable production host. As mentioned previously, many natural
isolates, from which an enzyme can be derived, are usually not acceptable production
hosts. Although it may be possible, to some extent, to alter the type and amount
of protein produced by natural isolates or mutants through modification of the
fermentation conditions, in most cases this is not sufficient to achieve the desired
purity, process reproducibility and economics. Of equal importance are safety
concerns and the fact that production hosts are usually limited to those with a history
of safe use. Therefore, recombinant methods are often employed. Given that the gene
sequence in a recombinant host is well characterized, there should be no safety issues
beyond the normal procedures, which regulate all enzymes derived from genetically
engineered organisms.
Attractive production systems or hosts include those microorganisms that
have the ability to: (i) produce and secrete large amounts of intact protein; (ii) carry
out proper folding of the protein and post-translational modifications such as
signal sequence cleavage, proteolytic processing, glycosylation, disulphide bridge
formation, etc.; (iii) generate stable recombinant cells or transformants; and (iv)
comply with regulatory requirements such as ‘generally regarded as safe’ (GRAS)
status (van den Hombergh et al., 1997).
Regardless of the nature of the production system developed, it needs to be com-

patible with industrial-scale fermentation and recovery processes. These processes
should be reliable, meet all safety and quality assurance requirements, and provide
a reproducible source of high quality product. Ideally, the strain development
and fermentation development efforts should be highly interactive. In turn, the
fermentation development effort needs to be synchronized with the recovery efforts,
since the nature of the fermentation broth exerts a large impact on downstream
processes such as cell separation and product recovery. The fermentation and
recovery processes must together generate a liquid concentrate that can be formulated
as a stable liquid or solid product without significant cost. Successful fermentation
development requires: (i) a good understanding of the physiology of the microbial
host; (ii) development of a suitable medium; and (iii) optimization of the interactions
between the microorganism and its chemical and physical environment in the
fermentor (Arbige et al., 1993). The chemical and physical environment, once
defined, can be controlled by computers and automated processes. Given all the
factors that can exert an effect upon the productivity, statistical–mathematical
approaches are often employed to optimize the various parameters.
The type of medium employed as well as by-products produced by the cells can
have a large impact on the protein recovery yields and the cost of the downstream
processes. Even enzyme products that are sold as crude preparations require down-
stream recovery after fermentation. Once the enzyme has been isolated from the
broth, it can then be concentrated by ultrafiltration, precipitation, extraction or other
methods prior to formulation. Where purity is of high concern, further processing
involving one or several fractionation steps is needed. The yield and product purity
criteria need to be balanced with the economics associated with each step, which is
incorporated into the downstream process. Also, the nature of the ingredients used in
the fermentation and recovery processes can have a significant effect on the cost of
waste disposal.
After the enzyme has been recovered and concentrated, it is usually formulated
in order to meet the product stability specifications suited to the application and
handling practices employed by the end-user. Food-grade ingredients are required for
food and feed applications. For solid products, the enzyme concentrate or formulated
product, if suitable, can be granulated or dried in a manner that provides a solid
product that meets health and safety requirements, such as low dusting.
Screening
Screening in essence means searching for a particular gene coding for the target
enzyme. In the past this involved exclusively the screening of living microorganisms
(‘classical microbial screening’), but today, by the application of modern tools of
molecular biology, a screening may be performed without the need to culture the
organisms involved. As with all methods, there are limitations and a combination of
techniques (‘combinatorial screening’) often provides the most success.
The most rewarding starting point for the process of search and discovery is the
rich diversity of microorganisms in nature (Bull, 1992). It is seldom appreciated that
for 85% of the earth’s history, life was restricted to microbial forms. The metabolic
diversity this has created is truly immense. During more than 4 billion years of
evolution, enzymes have emerged that are perfectly adapted for maintaining cellular
processes. However, many enzymes are required to perform in a milieu that is far
removed from the natural physiological conditions and may even involve exposure to
chemicals known to degrade or denature enzymes, which leads to loss of activity.
Usually, enzyme screening and selection strategies are based on knowledge of the
application and the physical and chemical conditions under which the enzyme
must operate. Therefore, selection for enzyme performance under the application
conditions is an essential early step in the screening process. Access to a large gene
pool is a prerequisite for a successful screening programme. The approaches can
involve the indiscriminate examination of heterogeneous material (e.g. soil) gathered
from the environment, or an ecological approach with a targeted examination of
specific habitats where a particular activity is likely to be found. Strategies can be
adapted to search specifically in poorly explored environments in order to access
a greater variation in genomes from novel microbes. In the search for new genes,
exploration of extreme environments such as volcanic hot springs, hypersaline lakes
and polar soils has yielded many novel microorganisms with peculiar properties
(Horikoshi, 1995; Kristjansson and Hreggvidsson, 1995; Horikoshi and Grant,
1998). Alternatively, a taxonomic-based approach involving a systematic study of
known organisms can also be rewarding.
Natural isolate screening
The source material can be plant or animal matter, or microbes – both prokaryotes
(e.g. bacteria and archaea) and eukaryotes (e.g. yeasts and fungi). Currently, micro-
organisms are the major source for industrial enzymes in the feed sector. The classical
method of screening natural microbial isolates is a well established process having its
roots in antibiotic discovery (e.g. penicillins, streptomycin) in the decades after 1945.
It involves the examination of thousands of samples of soil, plant material, etc., and
the random isolation and screening of the resident microbial flora. Although capable
of significant automation, the throughput capacity largely determines the speed
of progress. In the past, screening strategies using soil samples as a starting point
were highly empirical but much can be done to reduce the need for extensive
experimentation. These processes involve subjecting an environmental sample (soil,
etc.) to selective pressure using the principles of culture enrichment established
early in the 20th century by microbiologists such as Beyerinck and Winogradsky.
By manipulating the growth conditions, only those microorganisms expressing a
particular enzyme are able to grow and survive. For example, by providing only
xylan as the source of carbon and energy, only microbes producing a xylanase either
constitutively or by induction will grow and the culture will become enriched in
these organisms. The system is capable of considerable refinement; for example,
should an acidic xylanase be required, then the pH of the enrichment culture can be
designed to permit growth of microbes producing the enzyme at pH 4–6. The out-
come can be improved by a judicious choice of environment for examination – for
example, acidic soils, peat bogs and forest litter.
The time between collection of the environmental sample and examination in
the laboratory can be critical since changes will inevitably occur. Even in the best-
preserved samples chemical and physical changes may occur, leading to the death
of some organisms and the reduced viability of others. The longer the time lapse
involved, the more serious these changes can be. There are many advantages to
treating the sample immediately in the field since this can lead to the isolation of a
totally different population of microbes. It can be useful to provide ‘bait’ for specific
microbes or types of microbial activity. An example of this in situ enrichment is
placing cotton linter in an environment such as lake sediment and retrieving the
sample at a later date. The sample can then be taken back to the laboratory and
screened for cellulolytic activity.
The first problem in processing of samples is often one of numbers. Composts
and soils rich in organic matter contain enormous numbers of microbes. Some form
of dilution is required, or targeting of specific groups. Selective techniques can
involve, for instance, careful air drying of the (soil) sample, which reduces the
numbers of Gram-negative bacteria, or a brief heat treatment at 80°C, which will
kill most microorganisms but leave the resistant spores of bacteria such as Bacillus
intact. Antibiotics may be incorporated into the growth medium. For example,
Gram-positive bacteria are more sensitive towards penicillin than Gram-negative
bacteria. Many techniques have been developed empirically, such as the use of water-
or chitin-agar for isolation of slow-growing actinomycetes, and such ideas are
limitless (Williams et al., 1984).
An ecological approach takes advantage of the natural selective pressure of
the environment. For example, an alkaline environment such as a soda lake at pH 9
is inhabited only by alkaliphilic microorganisms. It is likely that these organisms
have extracellular enzymes that are stable and perform optimally at a high pH,
which is indeed the case (Jones et al., 1998). For screening of feed enzymes such
considerations are often less clear-cut. What, for example, is the natural habitat for
a phytase-producing microorganism? Phytate, a storage phosphate, is often a major
component of some seeds and screening microorganisms in intimate association
with the roots of germinating seeds (rhizosphere) would be an appropriate niche to
examine.
Whatever the screening strategy adopted, all of these approaches rely on
some form of selection criteria for the organism–activity combination being sought.
The most widely applied methods involve growing the microbes on an agar plate
containing the appropriate enzyme substrate for detecting the desired enzyme
activity. Often the methods employed are proprietary and frequently constitute more
of an art than an exact science. Some methods, however, are well known. For
instance, incorporating casein as sole carbon source into an agar medium providing
an opaque surface will detect protease-secreting organisms as a clear halo of
hydrolysed casein around the growing colony. Specific protease cleavage reactions
can be detected by dye release from appropriate chemicals carrying a chromophore

that is only visible after the specific hydrolysis reaction has taken place.
The selection of microorganisms is a very critical stage in the screening process
and can be quite subjective, especially if the numbers of microorganisms involved are
very large. Even though the screening has followed some form of selection process,
it is often not easy to distinguish the different types of microorganisms growing on
an agar medium. This is particularly true of bacteria, which, unless they are clearly
coloured, all look much the same. This gives rise to one of the biggest drawbacks of
the random approach to isolation and screening of naturally occurring strains: the
problem of redundancy. The same microbe will recur time and again from different
samples collected from different locations, which may explain why so many of the
current products come from a very limited range of microbes. Until relatively
recently most screening strategies were fairly conservative. The tendency to use rich
media which favour fast-growing, nutritionally undemanding organisms has led to
an overemphasis on particular groups of microorganism. This is coupled to the fact
that certain fungal genera such as Aspergillus and Trichoderma, and Bacillus and
Pseudomonas species among the bacteria, have traditionally been a rich and proven
source of commercial enzymes.
To ensure greater diversity and as a measure of how extensive a screening has
been, some estimate is required of the biodiversity of the population screened.
A decade ago this was a daunting task involving a detailed examination of each
organism, but nowadays fast molecular methods are available. A range of techniques
will rapidly group collections of unknown strains into clusters of related organisms.
Now, techniques such as total cell protein patterns, amplified fragment length poly-
morphism, ribotyping, amplified ribosomal DNA restriction analysis, inter-
nally transcribed spacer region–polymerase chain reaction (PCR), single-strand
conformational polymorphism combined with sophisticated electrophoresis meth-
ods, image analysis for data acquisition and computation can provide information on
the diversity of a strain collection. The ease with which small subunit ribosomal RNA
(ssu rRNA) gene sequencing can be performed today, combined with a large and
growing database for comparison, means that specific organisms can often be quickly
assigned to recognized taxonomic groups, and that novel microorganisms can be
recognized when found. The clustering of organisms into related groups of strains
can be combined with screening selection data, thus permitting the identification of
specific attributes with recognized groups of organisms. This information allows
particular procedures to be devised for the isolation of a specific group and the
imposition of more stringent selection criteria. Even so, it is still too easy to overlook
potential novel isolates, especially when confronted by many hundreds of culture
dishes, each containing several hundred similar-looking microbial colonies.
Nowadays, the task can be performed by colony-picking robots and visual imaging
systems coupled to computers.
As indicated earlier, one problem often encountered early in the screening
process is that natural microbial isolates usually produce commercially important
enzymes in exceedingly low concentrations. Environmental manipulation that
optimizes the growth and production conditions is limited by the intrinsic maximum
ability of the wild-type organism. Classical genetic manipulation by selecting

mutants with improved properties can lead to an increase of potential yield. The
classical techniques of mutation and selection for microorganisms with improved
attributes have been applied for many decades. Modern methods of molecular
biology have in recent years accelerated the possibilities of creating in the laboratory
microorganisms with new or improved activities. However, there is increasing
evidence suggesting that many of the techniques associated with ‘genetic engineering’
do, in fact, take place in the natural environment between groups of quite diverse
microorganisms. It is part of the opportunistic nature of microbes that allows them
to cope with changing environmental conditions. This can lead directly to the
acquisition of new activities within a microbial community. New activities are often
due to selection pressure, especially where recalcitrant chemicals, such as a new herbi-
cide, are introduced into the environment. This can be mimicked in the laboratory
by microbial selection in a continuous culture chemostat. This should not be
confused with the idea of the creation of a new microbe. Microorganisms react in a
manner to preserve their genetic individuality, and in any case, the sudden emergence
of an entirely new organism is not an event we are ever likely to be able to witness.
Molecular screening
The last decade has witnessed spectacular progress in the technology enabling the
direct sequencing of nucleotides of a DNA strand. Already there are thousands of
gene sequences in public databases, although most relate to genes involved in the
translation of the genetic code. The complete sequencing of microbial genomes has
also accelerated quickly in recent years. At present some 35 prokaryotic (Bacteria and
Archaea) and three eukaryotic microbial genomes have been reported in their entirety
and the sequencing of some 70 other microbial genomes is currently in progress.
There are several ways this can be done. If a suitable ‘lead’ enzyme (for example,
an acid xylanase) has been identified in a particular species, then a search can be made
for homologous enzymes in related species. The process requires the application of
‘reverse genetics’. Enzymes are polymers of amino acids covalently bonded in a
defined sequence. The order in which the amino acids are arranged in the enzyme
can be analysed by a process known as N-terminal sequencing. Since the genetic code
is universal it is possible to predict the probable sequence of nucleotide bases in
the gene coding for the specific enzyme. Using this information a probe can be
constructed consisting of 15–20 nucleotides (primer set) in the correct sequence,
which because of the double helix nature of DNA will bind to the complementary
strand. By using the primer set in PCR on chromosomal DNA from related
organisms, a homologous gene can be amplified and transferred to a host organism
such as Escherichia coli for the expression of the gene product – a homologous enzyme
(Dalborge and Lange, 1998). In practice the procedure can be a little more complex
than is described here but it is possible to track down enzymes with identical
functionality but with a significantly different sequence of amino acids, where only
the crucial elements of the enzyme, such as the active site, have been conserved
through evolution. In application tests these ‘new’ enzymes may have significantly
different properties, such as improved temperature or pH stability, compared with
the ‘lead’ molecule. In this way, whole families of related organisms can be screened.
Even though this is a very powerful technique it is limited by the ability to grow and
extract suitable DNA from the target organisms. It is also confined to known micro-
organisms that have been isolated, described and deposited in culture collections. It
has been estimated that fewer than 1% of all microorganisms that exist in nature have
been isolated and even fewer have been adequately characterized and described
(Amann et al., 1995). It is thought that fewer than 25% of the microbes in a sample
taken from the environment can actually be cultured in the laboratory. There may be
many reasons for this ‘unculturable’ portion of the microbial community. Some cells
may be in a resting phase, or be damaged or moribund; some may not be true
residents and not form part of the active population; others may be opportunists
waiting for a shift in environmental conditions in their favour; while others may have
unknown requirements for culture that remain unmet in the laboratory.
Fortunately, recent developments have permitted access to at least a portion of
these uncultivated microbes (Hugenholtz and Pace, 1996). It is possible to extract
DNA directly from the microbial community present in a sample taken from the
environment. Although there are many techniques for extracting chromosomal DNA
from natural microbial communities, not all cells are equally susceptible to lysis and
the DNA recovered is never 100%. Another concern is the detection of organisms
that form only a minor proportion of the total community. Their DNA will
be under-represented unless efforts are made to normalize the sample. A recent
examination of sequences of ssu rRNA genes in environmental DNA clones derived
from habitats as diverse as polar seawater from the Southern Ocean and hot springs
in Yellowstone National Park revealed a vast biodiversity of ‘yet to be cultivated’
microorganisms unsuspected a few years ago (Hugenholtz and Pace, 1996; DeLong,
1997). It has also revealed that the cultivated microorganisms may not be the
dominant flora in these environments, which provides a further challenge to the
screening-by-enrichment culture methods. The environmental DNA can be tested
for known genes using the PCR techniques described above. This requires good
quality DNA since the PCR screening is particularly sensitive to disturbance by
contaminating substances in the environment. Or, the DNA can be probed using
DNA carrying a radioactive or fluorescent label by a technique called hybridization.
Alternatively, the environmental DNA can be cut into smaller fragments using
restriction enzymes and the fragments cloned into an expression host such as E. coli.
The clones can be screened for new enzymes’ genes by techniques similar to many of
those described for the screening of natural microbial isolates.
However, all these molecular methods based on similarity screening have one
big drawback. They rely on a comparison with data that has already been recorded.
Since more than 70% of the putative genes revealed by total genome sequencing
projects (Bacillus subtilis, for example) have no known function, these data indicate
that the resources for screening of novel enzymes are still largely untapped. It also
remains true that the greatest diversity of life forms on this planet is microscopic. It
probably makes little difference whether the exploration and screening of microbial
and gene diversity for novel enzymes for food and feed application use the techniques
of the ‘hunters and gathers’ (conventional microbial screening) or those of the
‘breeders and selectors’ (molecular screening); the resources available are truly
immense and the ultimate performance of the enzyme under application conditions
still remains the crucial factor in selection.
Protein Engineering
The challenges for feed enzymes often require modification of enzymes to function
in a new environment or to possess a new property. Through protein engineering
techniques, tailored enzymes for cleaning and starch processing have been developed
and shown to outperform the best naturally occurring enzymes. The enzymes used
in cleaning and starch processing face quite unnatural environments: detergent
formulations contain surfactants and buffers to stabilize highly alkaline conditions
and starch processing involves liquefaction of the raw starch at temperatures often
near 105°C. The challenge has been to improve enzymes taken from quite different
environments in nature. Examples include subtilisin (protease) from Bacillus lentus
(a microorganism growing on plants and in soil) for cleaning, and α-amylase from
Bacillus licheniformis for starch processing. While neither enzyme would be expected
to function under commercial conditions, both perform the best of any found
in nature thus far. Although isolated from a mesophilic organism, the α-amylase
from B. licheniformis is equal or superior to the α-amylase from the thermophile,
B. sterathermophilus, in starch processing; and B. lentus subtilisin, while normally
operating in near neutral environments, seems to excel in alkaline detergent
environments. Thus natural enzymes can often be found that are already surprisingly
robust. These enzymes have evolved over time and as a consequence possess relatively
stable structures that can accommodate the accumulation of amino acid changes
necessary for the process of evolution. What has been undertaken in the process of
‘engineering’ enzymes is nothing more than a directed evolution of the natural
enzyme to be better suited for the commercial task and conditions.
There are a number of complementary approaches to develop an enzyme with
improved characteristics. Generally, as previously noted, the first step is to survey
naturally occurring enzymes and identify the best starting enzyme. Sampling the
available natural diversity and comparing functional properties versus the amino acid
sequences may provide clues as to what properties are associated with each desirable
molecular trait. The difficulty is that, over the evolutionary process, enzyme amino
acid sequences have diverged considerably. With so much variation, the problem
of identifying which sequence changes confer the desired trait can be extremely
difficult. For example, subtilisin Carlsberg differs from subtilisin BPN′ in 87 of a
possible 275 amino acid residues (Siezen et al., 1991). These two enzymes possess
quite different kinetic parameters, yet, as will be described below, changes in as few as
one to three of the possible 87 amino acids appear to be able to shift the kinetic
parameters. The protein tertiary structure plays an important role in identifying the
likely structural elements that result in these functional differences.
Subtilisin engineering
Enzymes are synthesized as linear polypeptide chains. The amino acid sequence is
dictated by the DNA sequence of the structural gene. After being synthesized, the
enzyme folds into a stable three-dimensional structure that brings residues far
removed in the linear sequence into close juxtaposition. The juxtaposition of these
residues and the creation of a substrate binding surface confer the enzyme molecule
with its catalytic properties. As an example, the structure of subtilisin BPN′, a serine
protease, is shown in Fig. 13.1. In this figure, three residues – aspartic acid (Asp) 32,
histidine (His) 64 and serine (Ser) 221 – are brought into position to form the
‘catalytic triad’ that is common to all serine proteases, not only subtilisin type but
enzymes belonging to two other classes as well. There is also a wide depression where
a segment of the polypeptide chain, from the substrate, binds to position a peptide
bond to be attacked by the activated Ser 221 hydroxyl oxygen.
Knowledge of what residues form the substrate binding surface is invaluable
when deciphering which of the multitude of possible changes may contribute to an
Fig. 13.1. Three-dimensional structure of subtilisin BPN′ (Bott et al., 1988). The
linear sequence adopts a stable three-dimensional fold to juxtapose the catalytic
triad residues: Asp32, His64 and Ser221. The catalytic triad is situated in a
depression that forms the substrate binding region.
enzyme having a desired property. Focusing on which residues differ between

subtilisin BPN′ from B. amyloliquefaciens and subtilisin Carlsberg from B.
licheniformis allowed the identification of three of 87 possible substitutions that were
likely to contribute to the difference in catalytic activity between the enzymes (Wells
et al., 1987). Subtilisin Carlsberg has a turnover number, kcat, that is approximately
tenfold greater than subtilisin BPN′ and a different specificity (substrate preference)
as well. The three-dimensional structures of subtilisin Carlsberg and subtilisin BPN′
share a common folding pattern despite the differences in their sequences. It is possi-
ble to superpose the two enzymes and relate residues that, while chemically different,
are structurally homologous. This homology in tertiary structure along with the
relative restricted fraction of residues forming the catalytic site and substrate binding
region allowed the identification of three amino acid residues out of the 87 that were
likely to affect the kinetic parameter and specificity of the enzymes. By introducing
three substitutions – Ser for glutamic acid (Glu) 156 (E156S), alanine (Ala) for
glycine (Gly) 169 (G169A) and leucine (Leu) for tyrosine (Tyr) 217 (Y217L) – a
subtilisin BPN′ variant enzyme having the kinetic parameters and specificity corre-
sponding to subtilisin Carlsberg was generated (Wells et al., 1987). Of these changes,
a single substitution was found to exert a major effect on the difference seen for the
turnover numbers, kcat. Even at this site as shown in Fig. 13.2 for the Y217L variant,
only subtle rearrangement is necessary to accommodate the substituted residue (note
the superimposed leucine and tryosine residues at amino acid position 217).
Fig. 13.2. Comparison in three-dimensional structures of native subtilisin BPN′

and a variant having the substitution of Leu for Tyr at position 217.
Amylase engineering
A similar strategy has been successfully employed with amylase. The α-amylase from
B. licheniformis consists of 458 amino acid residues, which in three dimensions
consist of three domains as shown in Fig. 13.3. The active site and substrate binding
region are situated between domains A and B.
The engineering goal for amylase was to increase the stability of the enzyme.
Numerous variants with increased stability were found by exploiting the diversity
found in related α-amylases from different species. Several clues were initially
obtained from variants derived from a screen of randomly created variants. These led
to a strategy of exchanging key amino acids with larger structurally homologous
residues observed in different α-amylases. It was proposed that bulkier residues,
known to be accommodated in homologous locations in these other α-amylases,
would fill surface cavities and thus stabilize the enzyme. An example is presented in
Fig. 13.4, where the substitution of Ser for Ala at position 379 has been modelled.
This method has been used to create variants having increased pH and thermal
stability (Day et al., 1998).
The strategy is based on the close homology seen in the structures of different
α-amylases. A residue found in one position in α-amylase from one species can often
Fig. 13.3. Three-dimensional structure of α-amylase from B. licheniformis. The

molecule consists of three domains A, B and C. Tha catalytic and substrate binding
regions are formed between domains A and B. (Courtesy of Dr Andrew Shaw,
Genencor International.)
Fig. 13.4. Space filling representation of the substitution of Ser for Ala at position
379. At position 379 the additional hydroxyl atom fills an external crevice and
leads to a more stable variant. (Coordinates of the modelled variant provided by
Dr Anthony Day, Genencor International.)
be accommodated in other α-amylases, which are homologous. The strategy thus

relies on the diversity of nature to provide clues such as use of crevice-filling residues
in the common α-amylase fold. These crevice-filling residues were then recruited
into the B. lichenformis α-amylase. What is striking about the strategy was that the
homologous residues selected for substitution were found in the sequences of other
mesophilic α-amylases having lower stability than B. licheniformis. These residues
were presumed to be very compatible with the α-amylase fold.
The overall process of selecting substitutions resulting in the most stable variant
includes rational site-specific substitutions, recruiting changes from variants isolated
from random mutagenesis and screening, and from the strategy of homologous
recruitment described above. The common thread is that all diversity can be
exploited successfully for clues as to how to make an enzyme better suited for a
particular function and environment.
Combinatorial and other methods
The most recent advance in designing enzymes is the realization that, with advanced
recombinant DNA technology, it is possible to entertain the possibility of accelerat-
ing evolution to a point where thousands of mutations can be created in successive
generations that are subject to either direct selection or to indirect selection by way of
a rapid screening strategy. This approach has been termed ‘directed evolution’. To
date there are numerous examples in the literature when either enzyme functionality
or stability has been improved using this approach. Subtilisin E variants have been
produced to perform in radically different non-aqueous environments using
these techniques (Chen and Arnold, 1993). While these demonstrations may be
academically successful, what is required for an industrial success is the ability to
screen or select for relevant properties and conditions.
These techniques generally produce numerous incrementally beneficial variants.
A leapfrog technique that involves recombining the individual substitutions by shuf-
fling their DNA has also been exploited. All of these techniques share the common
element of exploiting the range of diversity found in homologous functioning
enzymes. We see that a multitude of different but homologous sequences produce
enzymes having a common tertiary structure and function. Different combinations
of substitutions from just a single amino acid substitution to substitutions ranging
over the entire molecule result in enzymes with detectable differences but which are
able to perform a common function.
It may well be an inherent property of natural enzyme structures to have a robust
tertiary fold that is tolerant of considerable sequence variation. Thus evolution is
allowed to sample a relatively broad range of variation, providing sufficient diversity
for enzymes better suited for specific environmental factors and functional
requirements facing a particular organism.
Engineering Microorganisms for Enzyme Production

Classical mutagenesis
Classical mutagenesis is one of the most powerful techniques used to increase enzyme
yields from microorganisms. Dramatic productivity improvements in the order
of tenfold, or even 100-fold, are common. Classical mutagenesis techniques were
originally developed by the pharmaceutical industry to improve the yields of
antibiotics. The same techniques are used to improve enzyme yields.
Classical mutagenesis techniques are not unlike the process of evolution. Both
require mutations in the DNA of an organism to bring about changes in genetic
traits. While evolution selects for traits that confer greater reproductive fitness upon
an organism, classical mutagenesis can select for traits chosen by the experimenter.
Cells or spores are treated with mutagens to increase the frequency of mutation above
the natural rate. The mutagens can include ultraviolet light, gamma ray irradiation or
chemicals reactive with DNA. After treatment with mutagens, a diverse population
of cells is obtained. A screening process is used to identify cells having mutations
conferring desirable traits. The enzyme productivity of a microorganism is a trait that
can be altered through mutagenesis. While many mutations are detrimental or lethal
to an organism, a few can lead to increased enzyme productivity.
Classical mutagenesis was used to improve the yields of α-amylase enzyme
production by the bacterium B. subtilis. The enzyme α-amylase is used for liquefying
maize starch for the production of high fructose maize syrup and for increasing the
digestibility of maize starch in animal feed. However, natural isolates of B. subtilis
produce very low levels of α-amylase. Classical mutagenesis and screening of a
wild-type strain resulted in the isolation of a more productive and thus commercially
viable strain. Figure 13.5 shows a comparison of the wild-type strain and the
improved mutant strain growing on a nutrient agar supplemented with insoluble
starch. Degradation and clearing of the starch provides an indication of the presence
of α-amylase. Large clear haloes surrounding the cells of the improved mutated strain
indicate that the cells are producing and secreting α-amylase. Practically no halo
is apparent surrounding the wild-type cells, indicating low levels of α-amylase
production.
Genetic engineering to alter enzyme expression
Genetic engineering allows the in vitro manipulation of DNA with the ability to
reintroduce the modified DNA back into host cells. The genetic elements of genes
as well as their regulating elements can be altered to achieve changes in enzyme
expression. The typical bacteria or fungi used in enzyme production can have
between two and 10,000 genes. The bacterium B. subtilis encodes its genes in 4.2
million bases of DNA (Kunst et al., 1997) on one chromosome (one continuous
length of DNA). The fungus Aspergillus niger uses approximately 35 million bases of
DNA to encode its genes (Debets et al., 1990) on eight chromosomes. Each gene
specifies the amino acid composition of a protein. One gene confers the sequence for
Fig. 13.5. Comparison of α-amylase production from Bacillus sp. strains growing
on starch agar. The plate on the left shows an industrial overproducing α-amylase
strain generated using classical mutagenesis; the plate on the right shows a wild
isolate strain. Haloes surrounding the colonies result from α-amylase hydrolysis of
the insoluble starch.
one protein. The length of a typical bacterial enzyme gene, encoding a protein of 300
amino acids, is in the order of 1000 nucleotides. In addition to segments of DNA
that encode genes, there are also segments of DNA called promoters that control the
function of genes. Only a fraction of the genes of an organism are ‘on’ at any given
time. Coordination of the expression of genes is controlled by promoter elements.
These regulatory segments of DNA can be thought of as light switches for genes, able
to turn a gene on, off or to a state in between.
A key breakthrough in the 1970s that made genetic engineering possible was the
discovery of specialized enzymes called restriction endonucleases. These enzymes are
capable of cutting DNA in precise locations, in effect acting as molecular knives.
Another group of enzymes, called DNA ligases, can join cut ends of DNA together,
in effect acting as molecular splicers. Segments of DNA can thus be excised from
chromosomes and recombined.
Genetic engineering requires the ability to transfer recombined DNA into a host
organism, a process referred to as transformation. The bacteria E. coli and B. subtilis
can be transformed fairly easily: mixing cells with calcium chloride renders the cells
competent (susceptible) to take up DNA. When genetically engineered DNA is
added to such competent cells, the DNA adheres to the cell walls and is transported
inside the cell. Though transformation of fungi is more complex, a method was
found in the early 1980s to transform the fungus Aspergillus nidulans (Balance et al.,
1983). Minor variations soon followed to allow transformation (and hence genetic
engineering) of the fungi A. niger and Trichoderma reesei, both of which are
frequently used for enzyme manufacturing.
To enable high expression levels of enzymes, DNA can be recombined into an
overexpression vector. An overexpression vector is a specialized assembly of DNA
that when placed into a host cell causes a specific enzyme to be expressed (produced)
above normal levels. Several DNA elements are essential to create a suitable vector.
An example of an overexpression vector is one that has been used for production of
ferulic acid esterase A from A. niger (the enzyme is abbreviated as FAEA and the gene
encoding this enzyme is abbreviated as faeA). FAEA (de Vries et al., 1997) can cleave
the ester link between ferulic acid and arabinose present in the cell walls of many feed
substrates. For this overexpression vector (Fig. 13.6), recombination of four elements
was required: a strong promoter glaA, the faeA gene, the glaA terminator and the
pyrG selectable marker. The glaA promoter regulates the faeA gene. The glaA
promoter is known to result in high-level expression of genes that it regulates, and
for this reason was chosen for the vector. When the overexpression vector depicted
in Fig. 13.6 was transferred into an A. niger host, production of the FAEA enzyme
increased significantly.
The selectable marker gene allows for a way to determine if a host organism has
been transformed with the vector DNA. Before transforming the vector into the A.
niger host strain, the A. niger cells are initially mutated to make the native pyrG gene
non-functional. The pyrG gene produces an enzyme required to synthesize the DNA
building block, pyrimidine. If pyrimidine cannot be made, the cell cannot synthesize
DNA, which abolishes any possibility of cell growth. After transformation with the
vector, the cells are placed on selective nutrient media that lacks any pyrimidine. In
Fig. 13.6. A. niger expression vector for ferulic acid esterase A (faeA). The
vector shows required elements to produce high level faeA expression: a strong
glucoamylase (glaA) promoter, the expressed gene, faeA and a selectable marker,
pyrG.
order to grow, cells must take up the overexpression vector DNA containing a pyrG
gene. Those cells that take up the vector can synthesize pyrimidine and DNA.
Genetic engineering to delete enzyme genes
The use of genetically engineered overexpression vectors does not prevent non-target
enzymes from also being produced by a microorganism. Ideally a production
microorganism maximizes the expression of the enzyme of interest while minimizing
production of inconsequential or deleterious enzymes such as proteases or enzymes
with interfering activities. Deletion of genes coding for undesirable enzymes can be
performed when necessary to prevent their production.
T. reesei is a filamentous fungus found in warm moist environments growing on
wood. The original isolate was put through classical mutagenesis and screening
programmes which resulted in higher-producing cellulase strains. However, T. reesei
produces at least six different cellulase enzymes, each with its own unique enzymatic
properties. Additionally, these enzymes are not produced at equal ratios. Typical
T. reesei cellulase strains produce an enzyme mixture consisting of approximately
40–60% cellobiohydrolase I (CBHI), about 5–20% each of cellobiohydrolase II
(CBHII), endoglucanase I (EGI) and endoglucanase II (EGII) and less than a few
percent of EGIII (see Fig. 13.7, lane B). While this enzymatic ratio may be useful for
Fig. 13.7. Progression of genetically engineered T. reesei strains. The SDS-PAGE

gel shows the fermentation products produced by a series of strains starting from
the parent production strain and ending with the EGIII overproducing strain. Lane
A, molecular weight markers; B, parent strain; C, double deleted strain (Dcbh1,
Dcbh2); D, quad deleted strain (Dcbh1, Dcbh2, Deg11, Deg12); E, eg13
overexpression in quad deleted strain, late fermentation sample.
T. reesei to convert the cellulose in plant material to glucose as an energy source, it is

problematic for applications where only a single cellulase enzyme is required.
In order to tailor the cellulase enzyme composition to a specific application, a
strain of T. reesei was genetically engineered to prevent the production of CBHI,
CBHII, EGI and EGII. Deletion of the genes that encode for the non-desired
cellulase enzymes resulted in an altered enzyme profile. An example of the gene
deletion method, using the cbhI gene as a model, is illustrated in Fig. 13.8. The cbhI
deletion vector DNA cannot replicate independently upon entering the cell but must
integrate itself into an existing chromosome, otherwise the vector DNA will not be
replicated. This integration can happen at random. However, since identical DNA
has an affinity for itself, the deletion vector was made with the selectable marker, the
pyr4 gene, flanked on either side by cbhI DNA. The pyr4 gene is used as a selectable
marker in the equivalent manner that the pyrG gene is used in A. niger. The cbhI
portions of the vector can then recombine with the cbhI portions of the chromosome
of the host strain, resulting in the replacement of the cbhI gene with the pyr4 gene.
Upon deletion of the cbhI gene, production of the CBHI enzyme is not possible.
Thus, presence of the selectable marker can be used to identify those cells that no
longer contain the cbhI gene.
Fig. 13.8. Deletion of the T. reesei cellobiohydrolase I gene (cbhI).
Deletion vectors were also made for the three other major cellulase genes:
cellobiohydrolase II (cbhII ), endoglucanase I (eglI ) and endoglucanase II (eglII ).
These deletion vectors were used to delete these three genes sequentially. The effect
of gene deletions upon the enzymatic profile of various T. reesei strains can be seen in
a protein gel (Fig. 13.7). The protein gel (SDS-PAGE) separates proteins on the basis
of size (molecular weight). Fermentation broth samples were taken from various
genetically engineered strains of T. reesei. When the cbhI and cbhII genes are deleted,
the enzyme bands corresponding to these proteins (CBHI, CBHII) disappear (com-
pare lanes B and C in Fig. 13.7). When the eglI and eglII genes are deleted, the EGI
and EGII enzyme bands are no longer present (compare lanes C and D, Fig. 13.7).
The resulting deleted strain was further engineered to achieve higher production
of endoglucanase III (eglIII ) using an eglIII overexpression vector. This vector
contained the strong cbhI promoter to regulate high expression of the eglIII gene. The
vector was transformed into the strain that had been deleted for cbhI, cbhII, eglI and
eglII. The resulting strain showed an enhanced production of EGIII as compared
with all the strains that preceded it (compare lane E with lanes B, C and D in
Fig. 13.7).
Classical mutagenesis and the genetic engineering techniques of gene deletion
and gene overexpression can result in dramatic changes in the enzyme production of
a microorganism. By using such techniques, the enzymatic production of a microor-
ganism can be tailored to fit specific applications needs.
Fermentation Process – Design and Development

The main goal of fermentation process development is to maximize the productivity
and minimize the cost. Also, the process needs to be reproducible and must
meet all regulatory, health and safety, and quality assurance requirements. Ideally,
fermentation and strain development should take place in an integrated manner.
Strain development must take into account the fermentation process design options
and limitations, and the fermentation development must address critical strain
requirements.
Fermentor design
Fermentors are operated in batch, fed-batch, or continuous mode (Fig. 13.9).

They are controlled by monitoring and estimating several critical parameters, such as
temperature, pH, dissolved oxygen, redox potential, respiration rate, foam level and
mass transfer coefficient (Fig. 13.10). During batch fermentation, the production
rate increases and then declines. This is often associated with the cell state: exponen-
tial growth, stationary or sporulating. In fed-batch fermentation, by careful design of
batch and feeding medium, cell growth is regulated and production is maximized.
During continuous culture, production is affected by continuous growth, repression,
low or non-producing mutants, and dilution. A continuous cell recycle system
decouples growth and production and maximizes cell density, which results in high
productivity. If extracellular enzyme production under minimal growth is sustain-
able, then an immobilized viable cell fermentor design would obviate the need for cell
separation and result in even higher productivity.
When cells are cultured on a rapidly utilizable carbon source (e.g. glucose and
rich nutrients such as amino acids) the production of many enzymes is repressed.
This is referred to as catabolite repression. Regardless of how catabolite repression is
controlled inside a cell, either production strains must be able to make products in
the presence of carbon/nitrogen in the fermentor, or fermentors must be operated
under carbon/nitrogen limitation. The maximum rate of product synthesis of many
extracellular enzymes occurs during the late exponential or early stationary phase
of growth, as the cells become committed to sporulation (Doi, 1989). The use
of sporulation-deficient strains is therefore common in the industry (Priest, 1977).
For production, the default industrial mode of operation is batch/fed-batch
fermentation, with production cycles ranging from 12 to 300 h. Production
338
K. Clarkson et al.
Fig. 13.9. Fermentor modes.
Fig. 13.10. Parameters affecting fermentors.
maximization with this type of fermentation usually entails optimization of the

strain, the medium, the carbon feed and the run conditions (temperature, pH,
aeration, agitation, pressure). What exactly determines the limited period of
production is not known. The impact of other control systems (e.g. related to
nutrient starvation, cell cycle events) is likely to be involved.
Fermentation process design is a highly interdisciplinary effort, requiring
integrated use of concepts and methodologies of both chemical engineering and
microbial physiology, to accomplish scale-up. Fermentor ‘scale’ commonly refers to
fermentor volume. Scale-up is a procedure for designing and building a large-scale
system based on small-scale models. In practice, it involves the study of the effects of
scale on physiochemical and biological phenomena in fermentation process design.
Scale-up effects are more pronounced for aerobic (carried out in aerated and agitated
vessels) than for anaerobic fermentations (Atkinson and Mavituna, 1991). Therefore,
as a ‘rule of thumb’, in an aerobic fermentor, the constant oxygen transfer rate and
dissolved oxygen concentration are generally maintained in the scale-up. While
thermodynamic and intrinsic kinetic phenomena are independent of scale,
momentum, mass and heat transfer are dependent on scale. For example, mixing,
aeration and cooling are well controlled and uniform at the 10 l scale. However, not
all transport parameters can be maintained in this manner at a greater than 10,000 l
scale. Further scale-up complications arise from cell growth, adaptation and decay.
The development of entirely new processes or the improvement of existing

processes requires the evaluation of a wide range of strains and cultivation conditions
in a short period of time. Shake-flask fermentation studies have a cost advantage
but lack the process control options (pH, nutrient addition, aeration). This often
leads to use of laboratory scale (about 10 l size) agitated-aerated fermentors, with
adequate instrumentation and control. Almost all fermentation processes can be
translated to production scale by use of laboratory-scale fermentors. However,
pilot-scale fermentors are often necessary for the downstream process scale-up.
Process development
A considerable amount of published information is available on laboratory-scale

fermentation processes but commercial-scale process information is for the most part
proprietary and is generally not published in detail. Mutation, genetic manipulation
and strain selection contribute the most to the industrially improved processes.
Once the host of choice has been decided, development of a culture, which can
be used to inoculate the seed flask, begins with the process of correctly storing
and maintaining the organism. Usual methods include use of culture stocks stored:
(i) in 10–20% glycerol or DMSO at liquid nitrogen temperature (−70°C); (ii)
as freeze-dried cells; or (iii) on agar nutrient plates/slants kept refrigerated and
transferred periodically. In the process of scaling up a fermentation from a flask to
the production stage, a proper seed for inoculating the production fermentor needs
to be generated. The requirement of the seed is to reach high cell densities in the
pre-production fermentor so as to minimize the lag phase in the production vessel,
without compromising the cells. A typical seed train for inoculation of a production
fermentor is shown in Fig. 13.11.
Seed flask inocula are prepared from frozen, freeze-dried or plated stock cultures.
Erlenmeyer flasks, containing nutrient broth designed for optimal growth, are first
Fig. 13.11. Production fermentor: typical seed train for inoculation.

inoculated with a stored culture and incubated on an orbital shaker for about 4–48 h.
Both the time of transfer and the cycle stage of the cells are critical factors. The
purpose of the seed is also to adapt the cells for growth in the production media.
Therefore the seed medium must be designed based on the physiological needs of the
organism.
All microorganisms have basic requirements, such as water, a source of energy,
carbon, nitrogen, salts, trace metals and possibly growth factors. The challenge is to
develop a medium and conditions that optimize growth, by meeting the organism’s
basic needs, while producing the desired product economically. The media used
to isolate and screen production hosts are not necessarily those that can be used
in production fermentors. Development of an industrial process requires medium
components that provide carbon, nitrogen, phosphorus, potassium, magnesium,
sulphur and trace nutrients. The carbon source also provides energy for synthesis
of cell mass and product. The objective of fermentation-medium development is
therefore to maximize productivity, minimize side-products and costs, and ensure
steady product quality. Generally, large-scale fermentation media are made up of
complex natural materials supplemented with inorganic/organic salts, alkali and
acids. Development of a balanced fermentation medium is based on knowledge of
physiology as well as empirical work. Medium development combines growth and
production data obtained with agar plate, microtitre plate, shake-flask culture
and fermentor studies. Statistical methods (Plackett–Burman, Box–Benkhen) are
used for media screening and optimization (Monaghan and Koupal, 1990). The
media and conditions used may change from shake-flask through the fermentor
stages. Although many models have been developed for providing the basic needs
of the organism (Pavlou and Fredrickson, 1989; Priest and Sharp, 1989; Arbige et al.,
1993), the investigator is usually left to rely on either traditional recipes or media
development based on stochiometric requirements (growth, maintenance and
production) and statistical design of experiments.
When choosing raw materials for a production-scale process, many inexpensive
nutrients are available that provide good sources of the basic requirements, and many
contain inducers for product gene expression. Among these are soy flours, cottonseed
meal, cornsteep liquor and yeast and protein hydrolysates for nitrogen sources; and
glucose, molasses, starch, cellulose and peat for carbohydrate sources. The macro-
elements can be added as the salts (e.g. magnesium sulphate, potassium phosphate,
sodium phosphate) while the trace elements can be added separately as salts, or may
be sufficiently available in the complex raw materials. Among metal ions, only
potassium and magnesium are required as bulk salts, since these are involved mostly
as osmo- and energy-regulators, while the other trace elements have more specific
physiological roles (Hughes and Poole, 1989). Yeast extract usually contains all the
metals required for growth, especially copper, iron, magnesium and zinc.
When designing media for product formation, many factors affecting protein
expression and secretion need to be considered. In recombinant production hosts,
vector selection plays an important part in determining the medium and the process.
Also, when choosing a carbon source, the phenomenon of catabolite repression must
be considered. Regulation of nitrogen and phosphorus metabolism is an additional
consideration. It has been observed on numerous occasions that some organic

and inorganic nitrogen substrates have negative effects on exocellular protease
production. While oligopeptides and proteins seem to be the inducers, salts and free
amino acids seem to be strong inhibitors (Chu et al., 1992). Repression by amino
acids appears to be due not to any particular one, but rather by the supply of a
number of amino acids in sufficiently high concentrations. Production can also be
affected by high levels of carbon. In amylase production, where the composition of
the medium has a profound effect on product yields, the presence of phosphates can
have a significant effect on enzyme production (Thirunavukkarasu and Priest, 1980).
It has also been shown that production is enhanced by using more slowly
metabolized carbon sources, such as starch or lactose, or by slow feeding of glucose
to maintain a very low concentration.
When developing media formulations optimized for growth and production,
one area often neglected is the elimination of unwanted by-products, either coming
from a raw ingredient or produced via metabolism. Common metabolites produced
by bacilli are acetate, propionate and the metabolites of the butanediol cycle. These
acids not only represent a waste of carbon; they can also lead to inhibition of growth
or even cell death at high levels of accumulation.
Preparation of the fermentor is the next step after designing the basic media
for growth and production. Some general texts have been written on fermentor
preparation (Rehm and Reed, 1993), but because of the advances in genetic, enzyme
and pathway engineering, the investigator may also consider special requirements of
the production host. Selection and regulation of ingredients can make the difference
between a good and bad fermentation run. Another factor is the method of
sterilization, which refers to the physical or chemical processes of inactivation or
elimination of contaminants present in liquid and gas phases before the fermentation
takes place. Besides the initial sterilization procedure, it is also important to prevent
contamination during the run. There are several conditions that affect the probability
of contamination: temperature, pH, sugar concentration, defined/complex nutrients,
oxygen, back pressure, osmotic strength, and antibiotics present in the fermentor
(Bader, 1986). Typical fermentation media can support contaminating moulds,
yeasts and bacteria but generally fermentations are more susceptible to
contamination by spore-forming bacilli and bacteriophages. The sterilization kinetics
vary dramatically between vegetative cell and bacterial spore, and between dry and
wet heat sterilization. The lower the initial contaminants and particulate matter, the
more effective the sterilization procedure. Good housekeeping helps in lowering
the bioburden and eliminating difficult-to-sterilize residues. Steam sterilization, as
opposed to dry heat, is often preferred because of more efficient heat transfer and
higher reactivity, resulting in greater cell kill, most probably by rapid denaturation of
cellular protein. A rule of thumb is that all surfaces be exposed to saturated but not
superheated steam, for time periods of 10–60 min at 121°C. These conditions are
sufficient to inactivate all organisms and heat-resistant bacterial spores. Germination
and pasteurization (heating to 63°C for 30 min, or 80°C for 15 s) can also inactivate
heat-resistant spores of the Bacillaceae family. On the other hand, phage
contaminants are difficult to contain and tend to spread rapidly throughout the
plant. The only effective way to prevent phage attack is to develop resistant cultures.
In addition to sterilization, high-solids media are sometimes pretreated by enzymes
or acid to avoid mass transfer limitations before seeding the fermentor with cells,
since the cells utilize salts and trace nutrients only if available in the right form. As the
medium components are utilized by the cells, the broth rheology changes, CO2 is
produced and foaming occurs, making the use of anti- or de-foaming agents
necessary. Besides sterilizing all liquids and surfaces in contact with fermenting cells,
it is equally important to sterilize the air used for cultivation. Air is first compressed
to increase its pressure to as high as 100 psi, resulting in temperatures up to 260°C.
Heat of compression reduces contaminants, including heat-resistant spores and
phages, but is generally not solely relied upon for sterilization. So, filtration is
considered the primary method of sterilization of dry air. The standard air ‘inlet
filter’ is membrane based. These membranes are able to eliminate spores as well as
cells, and are also able to withstand steam sterilization conditions.
Once the media handling and fermentor sterilization are completed, and the
process variables are optimized, the performance of the host production strain in its
controlled environment is studied. At this stage, the primary goal is to obtain the
maximum biomass or product per unit of substrate in the minimal amount of time.
Optimizing growth yields while maintaining cell viability is key to the success of the
process development. Avoiding nutrient and O2 depletion and understanding
by-product formation are also important for maximizing the yield and minimizing
stress responses.
Downstream Processing and Formulation of Enzymes

The overall goal of downstream processing and formulation is to recover the enzyme
of interest cost effectively at sufficiently high yield, purity and concentration, and in a
form that is stable, safe and easy to use in a target application.
The variety of separations technologies available is large and the possible order
and permutation of sequential processing steps is vast. Several general considerations,
however, limit the range of practical choices for a process design (Becker, 1995).
Intracellular and membrane-bound enzymes are more difficult to recover than those
that are secreted, requiring physical or chemical disruption of cells and the challenge
of separating the enzyme from viscous or entraining substances such as nucleic acids
and cell wall materials. Fortunately, xylanases, proteases, amylases, cellulases and
phytases – virtually all feed enzymes of commercial interest today – are extracellular,
allowing for a cleaner first-stage separation. Most microbial fermentations are batch
or fed-batch, and are typically not harvested until the cell mass, enzyme and other
solids are quite concentrated, often requiring some post-harvest dilution to avoid
entrainment losses and low fluxes.
Typically, very high purity is not required of feed and food enzymes as it would
be of, say, human pharmaceutical proteins for injection. On the other hand, raw
materials must be food grade or otherwise shown to be safe and suitable, and new
enzyme formulations must be tested for toxicological properties and shown to be safe
for the intended use. Finally, economic considerations constrain both the range
of raw materials and separation processes that can be used. While multiple
chromatographic steps are routinely included in the downstream processing of
human therapeutic proteins, few feed or food enzymes include even a single
chromatographic step. Purification, if any, is accomplished by less expensive
techniques such as extraction or crystallization, as described below.
Harvest and cell separation
In general, cells should be removed from a fermentation broth within hours after
harvest in order to prevent cell lysis. After cell separation, the clarified fermentation
broth is usually stable and can be stored refrigerated for days. Upon harvest, the broth
may need to be cooled, pH adjusted, and certain stabilizers added in order to mini-
mize enzyme degradation. For the more labile enzymes such as proteases, control of
temperature, pH, oxidants, inhibitors and activators will be important throughout
the recovery process. For example, it is important to maintain a molar excess of
calcium ions to ensure the thermal stability of certain Bacillus proteases and amylases
that contain calcium-binding sites (Lloyd et al., 1970; Letton and Yunker, 1982).
If minor proteases and other side activities cannot be deleted from the strain itself,
finding ways to remove or neutralize them early in recovery becomes imperative.
Cell separation is usually accomplished by means of either depth filtration or
centrifugation. Fungal fermentations, such as those of Trichoderma or Aspergillus,
lend themselves particularly well to cell separation by means of filtration through a
rotary drum vacuum filter (RDVF) because of the ease with which fungal mats can be
thinly shaved off across the drum’s knife, renewing the filter cake surface to maintain
high filtration flux. High-capacity filter presses can be a reasonable alternative to
RDVFs. Bacterial fermentation broths can usually be processed by either filtration or
centrifugation, but the much smaller size of bacteria generally requires the addition
of a polymeric flocculant. Cell flocculants are generally cationic, and function by
bridging the negative surface charges on neighbouring cells to increase the particle
size and facilitate either sedimentation rate or filtration flux and clarity. In addition,
the choice of flocculant and optimization of dosage is usually a delicate balance
between considerations of separation quality, yield, cost and minimization of residual
excess polymer in the product.
The main challenge in centrifugation is to find flocculant and machine-
operating conditions such that cell mass or sludge is easily conveyed or discharged
from the centrifuge bowl without break-up or carry-over in the centrate. While
centrifugation can handle a high concentration of cell solids, filtration may provide
more complete removal of the most finely suspended solids, which can interfere with
downstream concentration or purification steps. On the other hand, the presence of
high solids and sometimes slimy polysaccharide fermentation by-products can limit
filtration fluxes without the judicious use of filter aids – usually diatomaceous earth,
perlite, or other mined materials. The presence of flocculants and filter aids in cell
wastes often gives rise to added disposal costs.
Microfiltration – the use of tangential flow anisotropic membranes to permeate

enzyme while retaining suspended solids – would appear to be an attractive cell
separation technique because it does not require the use of flocculants or filter
aids. However, microfiltration yields are typically low due to progressive fouling of
membranes. Equipment and membrane replacement costs are currently higher than
capital and operating costs of filters and centrifuges of equal production capacity.
Clarified filtrates or centrates are usually too dilute for use in feed applications, so
substantial amounts of water must be removed.
Concentration
Industrial concentration methods, such as evaporation and solvent extraction, are

unsuitable for dewatering enzymes because of their potential for thermal or chemical
denaturation, and evaporation gives rise to high energy costs. Chromatography is
generally uneconomical for concentrating feed enzymes, for reasons detailed below.
The most common concentration technique in use today for industrial enzymes is
ultrafiltration (UF), using hydrophilic tangential flow membranes with molecular
weight cutoffs in the range of 10,000–100,000 Da. UF fluxes and yields are often
significantly enhanced by removal – or omission – upstream of potential membrane
foulants such as certain polysaccharides or anti-foams. Precipitation, crystallization
and extraction can also be used for concentration, but are more typically utilized as
purification techniques, and therefore will be discussed below.
Purification, decolorization and finishing
The main ‘purity’ consideration for feed enzymes is not the removal of proteinaceous
impurities, but rather ensuring that only food-grade raw materials and non-
pathogenic, non-toxigenic production organisms are used. In some cases, an
undesirable side activity must be removed through some means of purification,
particularly when it is not feasible to delete the side activity genetically. Even if
minor proteases co-secreted by the host organism do not cause significant
degradation of the product enzyme during processing, they are more likely to wreak
havoc over several months of storage in the final product formulation. In other cases,
side activities must be removed because they interfere in the ultimate application.
Although chromatography offers the greatest potential and diversity of mechanisms
for fractionation, it is usually not cost effective in feed enzyme manufacture due to
the expense and low protein-binding capacity of most chromatography resins, and
the complexity and control required for reproducible operation.
Purification can often be accomplished with surprising effectiveness by two of
the oldest known protein purification technologies: fractional precipitation and
crystallization. Fractional precipitation is most useful when the protein of interest is
only one of several enzymes or other proteins present. Chaotropic salts, such as
ammonium sulphate, or water-binding polymers, such as polyethylene glycol, are
added in increments to a concentrate containing the enzyme, and those fractional

‘cuts’ are combined that have the most favourable balance of net enzyme purity and
recovered yield. When a higher level of purity is desired than can be attained through
fractional precipitation, crystallization is an attractive purification technique. To
the surprise of those unfamiliar with it, it is also a highly scalable, reproducible
and cost-effective technique (Becker and Lawlis, 1991; Visuri, 1992). The purity
attainable is comparable to what can be achieved through multiple chromatographic
steps. The major challenge is the often time-consuming screening of precipitants, pH
and temperature optimization, and mapping of the solubility phase behaviour. Once
the development work is completed, however, the process can be linearly scaled to
multiple tonnes. It should be noted that highly pure forms of certain proteases and
other enzymes are in some cases less stable than the impure forms, which often
contain stabilizing peptides and other impurities that inhibit degradation.
In the last one to two decades, aqueous two-phase extraction has become an
attractive concentration technique. It provides the selectivity of classical solvent
extraction without its denaturing potential. Utilizing incompatible two-polymer and
polymer–salt combinations and adjustments in pH and ionic strength, this technique
separates proteins based on differences in hydrophobicity, surface charge and
molecular weight. One outstanding application of the technique has been the
purification of genetically engineered chymosin from multiple side activities
produced by the fungal host (Heinsohn et al., 1992).
Decolorization is sometimes preferred in certain applications, mostly as an aes-
thetic preference. Colour can often be minimized by choice of fermentation medium
components and control of the sterilization cycle so as to minimize the browning
reaction between nitrogen and carbon sources. Colour can also be reduced by treat-
ment with activated carbon, use of antioxidants, and diafiltration through membranes.
‘Finishing’ refers to one or more filtration steps at the end of downstream
processing which have the goal of clarifying and reducing the microbial load of
the product. Solids are most conveniently removed using a filter press loaded with
cellulosic pads, or precoated with filter aid; disposable submicron-gauge filter
cartridges can be used to remove microbes, to the point of sterility if required.
Liquid formulation
The majority of feed enzymes are sold as formulated liquid concentrates, which may
be further formulated as liquid or solid products suitable for the end-user. The major
requirements for a liquid formulation are enzymatic stability and preservation against
microbial growth. It is sometimes not appreciated that the dominant factor affecting
enzyme stability is the intrinsic stability of the enzyme itself; formulation can do
very little to correct for a structurally labile protein. It is advisable, therefore, to make
stability an important criterion of the initial screening process.
As most feed enzymes are hydrolases, they are subject to the three principal
means of deactivation: denaturing or unfolding, catalytic site inactivation, and
proteolysis (Becker et al., 1997). Denaturation is best minimized by controlling
temperature and pH and avoiding the presence of chemical denaturants. Catalytic

site inactivation is prevented by supplying sufficient levels of cofactor, typically a
metal cation, and preventing oxidation of the active site – for example, by formulat-
ing with antioxidants. Alternatively, oxidative resistance can be enzyme engineered
into the protein structure. Finally, proteolysis – or autodigestion, in the case of
proteases – can be minimized by upstream deletion of proteases, by reducing
water activity through the use of water-sequestering compounds, or by addition of
inhibitors. Useful water-sequestering compounds include sugars and other polyols,
such as glucose, glycerol, sorbitol and propylene glycol. Useful inhibitors include
substrate analogues such as peptides and acid salts.
Microbial growth is fairly easy to prevent by the addition of food-grade
antimicrobials such as sodium benzoate, methyl paraben and various commercial
preparations. The prevention of precipitation and hazes is often a highly empirical
challenge and one that is very specific to the enzyme and the specific process raw
materials used. The best advice is to use ingredients with high solubilities, and to
screen potential formulations by extended storage at high and low temperatures.
Solid formulation
Most feed enzymes are supplied as liquid formulations at the end-user level, as these
are convenient to use. It is mostly dry enzyme premixes that are being sold to the feed
manufacturer. However, solid formulations can provide some significant advantages,
such as enhanced stability, delayed or controlled release, and protection against deac-
tivation during harsh applications. One example of the latter is the use of granules to
encapsulate enzymes against deactivation in the steam pelleting process. The use of
effective stabilizers and coatings can prevent or retard exposure to moisture under
high heat, yet allow release of the enzyme in the subsequent feed application.
Dry enzymes, in most industries, are almost universally supplied as encapsulated
granules in order to meet strict industry standards for control of airborne dust. The
primary technologies for enzyme granulation include prilling, extrusion and
spheronization, high-shear granulation, and fluidized-bed spray coating. The latter
two techniques are superior in producing low dust granules. The advantage of the
fluidized-bed method is that the entire granulation process can be carried out within
a single, contained piece of equipment. In addition, the spray-coating process
involved in this method allows the sequencing of layers of different thicknesses and
compositions with almost infinite flexibility.
Regulatory and Environmental Aspects

Along with the transfer of the fermentation, recovery and formulation processes to
the manufacturing plant, validated quality control methods and assays, which are
suitable for assuring product quality, must also be provided. Various agencies
throughout the world are involved in the regulation of enzyme products for feed. In
the USA, all ingredients used in feed, including silage ingredients, need to be listed in
the official publication of the Association of American Feed Control Officials
(AAFCO Manual ), and registered with the states in which they are sold, as well as
tonnage fees paid to the states. Any enzyme not listed in the AAFCO Manual requires
an assessment of safety and efficacy by the Center of Veterinary Medicine at the Food
and Drug Administration (FDA), who will then recommend listing in the Manual.
In the EU, all enzymes require submission of a full dossier and specific approval
by trade name. Although the regulations can vary from country to country, in gen-
eral most require (Wohlleben et al., 1993; Chapman, 1996): (i) the use of well-char-
acterized non-pathogenic non-toxigenic organisms; (ii) good manufacturing prac-
tices (GMPs) involving well-documented, safe and reproducible fermentation and
recovery processes in which only approved raw materials are used; (iii) the absence of
toxins or pathogens; and (iv) low levels of heavy metals or other contaminants. Not
only are genetically engineered organisms well characterized; they have also been
engineered for poor survival outside a controlled environment. This, along with the
fact that recombinant organisms are grown under contained good industrial
large-scale practices (GILSPs), minimizes the safety issues relating to environmental
release. Biotechnology has also had a favourable environmental impact compared
with traditional techniques. For example, enzymes derived from recombinant
organisms usually have higher fermentation yields and therefore reduce the
consumption of raw materials used for their production.
Conclusions
Advances in biotechnology over the past few decades have had an immense impact
not only in the areas of therapeutics, pharmaceuticals and health care but also in
industrial, food and feed applications. Recombinant DNA technology has proved to
be an effective means for increasing the production of enzymes, including foreign or
minor enzyme components, and for generating tailored enzyme compositions for
numerous applications. Recent breakthroughs in screening methods and genomics
have allowed greater and faster access to unique enzymes from culturable as well as
unculturable organisms. The ability to genetically engineer additional features into
these naturally occurring enzymes and to accelerate the evolutionary process in a
directed manner has provided routes to the design of unique enzymes that are
tailored to a specific application. These technologies, coupled with the development
of well-characterized generic hosts and optimized and controlled fermentation,
recovery and formulation processes, have assured a reliable flow of safe, stable and
cost-effective products.
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Liquid Application Systems
for Feed Enzymes
P. STEEN
14
Finnfeeds, PO Box 777, Marlborough, Wiltshire SN8 IXN, UK
P. SteenApplication Systems for Feed Enzymes

Liquid
14
Introduction
Enzymes have found widespread application in those countries where animal diets
are based principally upon wheat, barley, oats, triticale or rye. The rapid commercial
uptake of this new technology in recent years has resulted from extensive research,
yielding a greater understanding of the anti-nutritive properties of these cereals and
of the enzyme types and dosages required to reduce their negative effects. This in turn
has stimulated the commercial availability of a number of feed enzyme products that
can offer consistent and economic benefits to the user.
All animals use enzymes in the digestion of food. These are produced either by
the animal itself or by the microbes naturally present in its digestive tract. However,
the digestive process is less than 100% efficient. Therefore, the supplementation of
animal feeds with enzymes to increase the efficiency of digestion can be viewed as an
extension of the animal’s own digestive process.
Recent developments in feed processing technology have resulted in increased
processing temperatures (e.g. expanders). This has provided benefits such as
improvements in feed hygiene, production efficiency and pellet durability. However,
the increases in processing temperature have also focused attention on thermo-
stability of some of the more heat-sensitive feed ingredients, such as vitamins, amino
acids and enzymes. This has presented an interesting dilemma for the feed industry in
striking a compromise between what these ingredients can endure and what process
conditions will allow. The situation is confounded by the fact that there are no set
standards worldwide for feed processing. In effect there are almost as many variations
in feed conditioning retention times, temperatures and pellet die sizes as there are
feed mills (Pickford, 1992).
Enzymes are proteins and can therefore be susceptible to degradation by external
factors such as pH, hydrothermal conditioning and frictional forces and by the heavy
metals that are added to certain animal feeds. Stability of granular enzymes can be
improved through the selection of activities with inherent high thermal stability,
selection of specific carriers and through novel manufacturing techniques such as
coating. Consequently, some granular feed enzymes have been shown to maintain
354 P. Steen
efficacy after exposure to conditioning temperatures of up to 90°C. However, where

processing conditions exceed this temperature it is generally advisable to apply
enzymes as liquids, post-pelleting, thereby avoiding exposure to high temperatures.
Application of Liquid Enzymes

Use of enzymes in the feed mill
As various pressures mount on the global animal feed industry, so the demand to
produce ‘hygienic food’ for farm animals has increased. This has resulted in the
adoption of new processing technologies that have elevated processing temperatures
beyond those achieved with conventional steam conditioning. The expander is one
such example of a new processing technique. Typically, expanders are capable of
processing feed at 105–130°C and at pressures of 10–40 bar (145–580 psi). Hydro-
thermal processes – expanders or extruders in particular – are often regarded as, at
best, neutral or even potentially destructive to certain feed additives (Pickford, 1992).
As feed-processing temperatures increase in areas of the world where viscous
cereals are employed, so the intestinal viscosity of the targeted animal (broiler, layer,
etc.) fed untreated diets will increase (Table 14.1). The application of enzymes
addresses this dilemma, although when feed-processing temperatures exceed 90°C
the application of heat-sensitive additives such as enzymes, vitamins, probiotics and
certain antibiotics, etc., favours liquid addition at the end of the manufacturing
process.
Addition of liquid enzymes
A major consideration for successful use of liquids is the method and accuracy
of dosing. Whilst other micro-ingredients, such as amino acids, are routinely
Table 14.1. Effect of (i) pelleting and (ii) expanding, followed by pelleting, on broiler performance
and foregut viscosity (Harker, 1996).
Pelleted Expanded and pelleted
7–28 days Control + Granular enzyme Control + Liquid enzyme
Liveweight gain (g) 1042ab.37 1060aa.37 1010b.37 1064a.37

Feed : gain1 1.37 bc
1.34ab
1.39 c
1.31a
Foregut viscosity (mPa s) 6.9ab 4.3ba 20.0c 3.8b
(CV) (22) (13) (44) (11)
a–cP< 0.05.
1Feed intake corrected to dry matter.
60% wheat, 20% soybean meal, 5% sunflower oil; pelleting = 75°C, expanding = 105°C/Amandus
Kahl; batch-type liquid dosing system.
Liquid Application Systems for Feed Enzymes 355
incorporated as liquids, they are dosed directly into the mixer as opposed to post-
pelleting. Dosing into the mixer is a relatively simple process typically involving
only a dosing pump, flow meter and spray nozzle. Some micro-ingredients, such as
flavours/spices, are sprayed on to pelleted feed via low-cost and relatively inaccurate
spray systems. However, the relative value of these products and importance of
dosing accuracy is typically low compared with feed enzymes. Feed enzymes require
a different approach due to the role they play in the feed formulation and the
nutritional value they add to the feed. Ineffective application of the enzyme to the
feed can result in an energy-deficient diet leaving the feed mill, and subsequent poor
performance of the targeted animal.
There are two recognized positions within the feed manufacturing process
for the application of enzymes: in process (post-pellet sieving) or at bulk outloading
as the finished feed is loaded into the delivery truck.
The advantage of the installation in the loading line is that only one post-
pelleting application (PPA) device is required, though it must be correspondingly
large. In the other case, several spray systems are necessary, depending on the number
of pelleting lines. Application directly before loading additionally offers the option of
manufacturing compound feed of one particular composition in large quantities and
then adjusting it to individual customer wishes directly prior to loading. Further-
more, unwanted dispersal or cross-contamination with critical components such as
liquid medication can be largely ruled out in this way. This simplifies the production
process and improves accuracy (Schwarz, 1998).
Application of Enzymes in the Feed Manufacturing Process

In any post-pelleting liquid application, we must first consider the upstream and
downstream processing equipment and how it operates. We must also consider the
maximum processing capacity, which will not necessarily be the pelleting capacity.
These two factors are essential to the successful design and operation of the
equipment.
Today, many feed mills have installed counter-flow coolers. These coolers
operate in a semi-continuous mode, retaining the product in the cooler for a
predetermined time, then discharging the product by opening the discharge gate,
allowing product to flow en masse from the cooler. This discharge rate will be
higher than the output capacity of the pellet press. Therefore, if a PPA system is
installed to match the pelleting capacity, the plant will be bottlenecked at the
PPA system, due to the reduction in capacity at this point in the process. In order to
overcome this problem, the capacity of the PPA system should be calculated against
the maximum transfer capacity (volumetric flow rate × maximum feed bulk density)
of the mechanical handling device immediately below the cooler. In feed mills where
the horizontal cooler is still employed, the feed rate from the cooler is consistent with
the pelleting capacity. It follows, therefore, that if the pelleting capacity changes then
this upstream fluctuation will affect the operation of any downstream processing
equipment unless adequate precautions are taken.
356 P. Steen
Another critical aspect in the effective application of liquid enzymes is the

location of the PPA system. If the feed is to be screened, for the removal of fines,
before being transferred to the finished product bin(s), then it is essential that the
application of the enzymes be applied to the feed post-screening. If not, it has been
demonstrated that 24–50% of the enzyme activity can be recovered from the fines,
which typically amounts to between 5 and 10% of the feed (Gunther et al., 1997; van
Laarhoven, 1998). Fines will be particularly high in enzyme activity, due to their
large surface area and absorptive capacity. If these fines are removed from the finished
product by sieving after the PPA of the enzymes, then the enzyme activity will return
to the pelleting system for reprocessing. As the thermal stability of liquid enzymes is
far less than their granulated equivalents, then most of this activity risks denaturation
during subsequent re-processing of the feed. This will have a major influence on the
overall economics of enzyme use.
From Table 14.2 it can be seen that the xylanase activity level of the fines is
more than three times higher than the xylanase activity in the pellets. The fines are
formed by attrition of the pellets and it can be concluded from these experiments
that the absorption of the enzyme into the centre of the pellet is very limited, with
the result that the enzyme activity is confined to the outer layers (Engelen, 1998).
Consequently the better the pellet quality, in terms of durability and hardness, the
higher will be the enzyme recovery during subsequent enzyme analysis.
Table 14.2. Xylanase activity in pellets and fines for different samples (Engelen, 1998).
Xylanase activity in samples Relative proportion of pellets and

(U kg−1) fines (%)* Total xylanase
activity in a
Sample ID1 Pellets2 Fines Pellets Fines sample (U kg−1)
A1 6210.7 19,580.0 89.6 10.4 7600.3

A2 6410.7 19,260.0 89.8 10.2 7717.4
A3 6380.7 19,720.0 85.5 14.4 8308.3
A4 5960.7 19,690.0 88.3 11.6 7557.3
A5 5430.7 19,550.0 93.4 6.6 6362.3
A6 7480.7 19,670.0 92.7 7.3 8369.4
A7 5480.7 19,240.0 94.3 5.7 6259.2
A8 5995.7 19,800.0 91.5 8.5 7168.1
A9 5490.7 19,470.0 91.3 8.6 6698.5
A10 5720.7 19,140.0 94.8 5.2 6414.2
Average 6056.7 19,512.0 91.1 8.8 7245.5
STD 587.5 19,216.0 – – 747.5
CV 9.7 19,261.1 – – 10.3
1Water activity of feed 0.86 at 25°C.
quality: Holmen 76.4%; Kahl 2.0 ± 0.5.
2Pellet
*Xylanase is sprayed on the feed in a batch mixer and the resultant pellets and fines are analysed
separately for enzyme activity.
Eliminating fines from the feed-manufacturing process may be virtually

impossible, but there are devices on the market, in addition to the pellet sieve,
to improve pellet quality. Although pellet quality will not directly improve the
efficiency of the PPA system, it will reduce the analytical variation of the assay by
reducing the percentage of fines in the final sample.
Post-pelleting Application of Liquid Enzymes

Measurement and control of the liquid and dry flow rates
The accuracy of any liquid application system is based upon a number of essential
factors; the most fundamental of these is the measurement and control of the liquid
and dry flow rates. To achieve high homogeneity of application (low coefficient of
variation) and precision dosing of the enzyme on the feed it is essential to control,
and not simply measure, the dry flow rate. This is because fluctuations in the dry
flow rate can result from upstream processing equipment, changes in manufacturing
capacity, variations in formulation, etc. Without the necessary control logic to
manage these immediate changes, long feedback times will render any advantages
expected from measuring the dry flow an expensive exercise.
There are principally two methods for controlling the dry and liquid flow
rates, namely by volumetric or gravimetric means (Figs 14.1 and 14.2). With the
volumetric system, both the liquid and dry elements are dosed independently of
each other. No control or adjustment is made for variations in specific gravity or bulk
density. Therefore, both elements are dosed on a theoretical capacity. Accuracy of the
volumetric system is based upon the variations in the manufacturing process for the
pellets (bulk density and product form) and repeatability of the dosing equipment for
the liquids, plus any changes in specific gravity of the liquid.
The gravimetric method of measurement is when both the liquid and dry
elements are gravimetrically weighed or measured in mass terms (mass/time). The
dry flow can be measured in a number of different ways, e.g. impact weigher, belt
weigher, batch weight, etc. The liquid flow rate has fewer options for the gravimetric
measurement of the flow, being limited to the batch weigher in the case of a batch
system or as a loss-in-weigh system for measurement of presumed use. The more
common option is the use of a mass flow meter based on the Coriolis measuring
principle.
The major benefit of the gravimetric (mass rate of flow) system has to be the
accuracy that is achievable. With the gravimetric system, both the dry flow and liquid
flow rates are gravimetrically measured, with the flow rate being equated to mass. If
the flow rate of either the dry or liquid changes, then the corresponding change is
made to the other component to offset any inaccuracies. This type of system is not
affected by changes in bulk density or specific gravity, flow rates or flow profile,
although the more constant the flow rate, the greater is the degree of accuracy.
Economically the level of automation needed for such systems makes it feasible to
incorporate a number of ‘production aids’ to the benefit of the feed mill operator
358 P. Steen
Fig. 14.1. Typical mill schematic arrangement for a gravimetric system.

and mill manager. It also makes it possible to link the application control system
with the mill formulation computer so that the required inclusion rate for a specific
formulation can be set by the nutritionist within the formulation computer.
Volumetric systems are lower in specification and therefore lower in cost. For
the same reasons the installation of the equipment is much simpler. This type of
system comprises a dosing pump without the use of a flow meter and relies solely on
the calibrated displacement and repeatability of the dosing pump to deliver a known
volume of liquid. The other component of the volumetric system is the volumetric
Fig. 14.2. Typical mill schematic showing the arrangement for a volumetric
system.
flow of the pellets. The major deficiency of the volumetric system is the non-
relationship between the two flow rates, dry or liquid, i.e. if the capacity of pellet flow
increases, the liquid flow rate does not. It is relatively easy, therefore, to appreciate
how the capacity of this type of system can change when manufacturing a different
product form or simply a change in bulk density or specific gravity of the liquid.
360 P. Steen
Application System Types

The three commonest systems for the application of liquids are the semi-continuous,
continuous and batch type.
Semi-continuous system
The semi-continuous system is the most commonly adopted system for the post-
pelleting addition of liquids. This type of system was introduced for economic
reasons, balanced by its flexibility and accuracy. Initially used as a volumetric system
for the addition of fat, in recent years the system has been further developed to
incorporate gravimetric measurement for the addition of aqueous-based liquids.
The installation of a surge hopper to accumulate the flow of dry product will
eliminate any flow fluctuations from upstream processing equipment. The hopper
has sufficient capacity to accommodate a volume of product, predetermined to
maximize the operation of the dosing system, therefore limiting the frequency with
which the complete system has to stop and start. The hopper is fitted with a discharge
device that is automatically sequenced to operate by the level of the product in the
surge hopper. This is achieved by the inclusion in the hopper of a working-level and
low-level probe. The control logic of the system is that when the low level and then
the working level are covered by product, the discharge device will operate. When the
Fig. 14.3. Sprout-Matador Micro Fluid System (Sprout Matador Ltd).

working level and then the low level are uncovered, the discharge device will stop.
The position of the low-level probe is such that a residue of product will always
remain in the feeder and the surge hopper. This mode of operation ensures that when
the discharger next operates the volumetric discharge capacity is as the previous
operation, eliminating any surging effect.
Advances in technology have seen the introduction of the gravimetric weigh
feeder. Although the system operates in the same way as detailed above, the feeder is
suspended on a fulcrum immediately under the outlet of the surge hopper. The
opposing end of the feeder is suspended on a load cell, which measures the product
flow through the feeder.
The volumetric feeder ensures a constant volumetric flow rate from the
feeder without compensating for changes in bulk density. This can be a distinct
disadvantage if a number of different products are to be manufactured. However, it is
possible to compensate for these changes, either manually or automatically, if the
changes in the bulk density fall within a range. Manually, the mill operator can adjust
a feed gate, precalibrated, at the feeder inlet, and the capacity of the system can be
altered by either increasing or decreasing the bed-depth in the feeder. The same can
be completed automatically by the use of a variable speed drive fitted to the feeder.
This degree of control can provide the flexibility to maintain a constant throughput
capacity, given the variations of feed bulk density.
Continuous system
The processing of friable products is best suited to the continuous system, due to the
ability of the system to follow the manufacturing rate. As the operating capacity of the
application system is matched to the upstream processing equipment, it is possible to
optimize the system with only a relatively small surge hopper, so preventing degrada-
tion of the product but also optimizing the dry and liquid flow rates (Fig. 14.3).
For the continuous system to operate effectively, it must be capable of adjusting
to upstream fluctuations. For this to be possible, it must be capable of determining
the upstream capacity and adjust accordingly the operating capacity of the dosing
system. This can be achieved by the installation of an ultrasonic level measurement,
positioned in the surge hopper before the dosing system. The device is calibrated
with an empty hopper and with a full hopper, the scale in between being calibrated
against the known manufacturing rates for the given products. The capacity of the
dosing system increases as the hopper fills and decreases as the level in the hopper
diminishes. The objective is to maintain a consistent capacity through the dosing
system by minimizing any large fluctuations in the capacity of the system.
The control of the dosing system must be such that it can respond to the
necessary changes in capacity. A continuous liquid dosing system requires extra
accuracy in determining the pellet flow and the liquid flow (Engelen, 1998). This
means that both the dry and liquid flow rates have to be changed proportionally. For
this to be possible, both elements have to be gravimetrically measured to ensure the
accuracy of the system.
362 P. Steen
Batch system
The batch system is a discontinuous system that operates (as the name implies) on a
batch-by-batch basis. The system provides the flexibility to apply multiple liquids
and combine multiple dry product forms. There are several different arrangements
for the batch system and these can be defined in two categories, as either a pre-
weighing system or a combined weigher–mixer.
The weigher–mixer combination is when the batch mixer is supported on
load cells and performs the dual function of both weighing and mixing. The main
disadvantage of this system is that the capacity of the mixer needs to be greater for the
same manufacturing capacity as a pre-weighing system, as the batch cycle time is
increased. This will increase the cost of the mixer, but this cost may be offset against
the cost of the weigh hopper(s) and associated control items. It may also rule out the
requirement for additional processing equipment such as conveyors and elevators, as
the height required for the installation is reduced.
The pre-weighing system shown in Fig. 14.4 comprises a twin weigh hopper
arrangement. This system is configured for the batch weighing of both the dry mate-
rial and liquid additives. The principle of the batch system is to operate using the
actual batch weight and not the theoretical batch weight. For this reason, the system
is extremely accurate, proportioning the correct quantity of liquid additives to the
corresponding weight of dry material. For this principle to work, the batch weight of
the dry material must be known before weighing the liquid additives. Therefore,
when the initial batch of dry material is weighed, the corresponding batch weight for
the liquid additives is then weighed. For the succeeding batches, the weighing of both
the dry material and liquids is completed before the finish of the preceding batch.
Batch-weighing multiple liquids can have its disadvantages. The principle for
the addition of micro-liquids to the batch mixers differs from the addition of fat and
other high-inclusion liquids. If these liquids are weighed in the same weigher, then a
compromise may have to be made regarding the addition method to the mixer, i.e.
addition time, nozzle type, nozzle capacity, etc.
The more commonly adopted method for the addition of micro-liquids to the
batch mixer is to meter each liquid individually. This provides finite control and
flexibility. Unlike other systems, the batch system does not require continuous
measurement and control of the liquid flow. The liquid addition is calibrated to dose
the theoretical quantity of liquid to the mixer in a predetermined time, the addition
time being important to the performance of the system. The dosing capacity of the
pump being constant, the enzyme is metered until the set point is achieved.
Measurement and Control of the Liquid Flow Rate

The dosing precision of liquid enzymes is critical, due to the known dose–response
relationship that exists between enzyme dose and animal performance (Fig. 14.5).
Under-dosing can result in significantly reduced efficacy and bird response,
ultimately reducing feed quality, whilst over-dosing can be costly in terms of wastage
of feed enzymes and reduced animal performance under certain circumstances.
Soon, enzymes may not be blanket-fed throughout the animal’s life but targeted
with various dose levels at different stages of growth or reproduction (Overfield,
1999a). In addition, some enzyme suppliers are developing modelling tools able to
predict the optimum dose rate based on cereal quality. For this to be of practical use
within the feed mill, the dosing system needs to be such that the application rate of
Fig. 14.4. Typical mill arrangement for a batch system.

364 P. Steen
Fig. 14.5. Non-linear (exponential) dose–response curve for five trials combined
(M. Bedford, Finnfeeds, personal communication, 1999).
the enzymes can be varied easily, without being recalibrated, and yet still maintain its
accuracy.
Flow meters
The measurement and control of the liquid flow rate can be achieved with the
incorporation of a flow meter in the liquid line. The process signal from the flow
meter is used to vary the speed of the drive motor on the dosing pump, thereby
altering the liquid flow rate. The process signal from the flow meter is proportional
to the required application rate and dry flow capacity.
There are three main types of meters used in the measurement of liquids and a
brief explanation of each of these follows.
Positive displacement or turbine meters

These are mechanically operated flow meters. The internal arrangement of the meter
contains moving parts – either oval gears or a Pelton wheel type arrangement. The
rotation of the rotors is sensed by an electronic system, using the interaction of a
ferrite material imbedded in the rotor tips. A sensing device is mounted in the top of
the meter body to pick up the rotation of the rotors. The sensor will then generate
and transmit the resulting signal. The speed of the rotor and the resulting output are
directly proportional to the linear flow rate of the liquid through the meter.
The main concern with this type of meter is the low flow rate used in the
application of enzymes and the possible build-up of material on the gears of the
meter. Either of these parameters can affect the accuracy of the meter or prevent it
from operating.
Electromagnetic meters
This type of meter has no moving parts, which eliminates the possibility of
entrapment of material in the sensing unit. The measuring principle is based
on Faraday’s law of magnetic induction. This is when a voltage is induced into a
conductor, which moves in a magnetic field, the flowing liquid being the conductor.
The induced voltage is proportionally related to the flow velocity, and through
knowledge of the cross-sectional area of the meter sensor, the volumetric flow
rate can be calculated. The application of the meter is limited to materials that are
electrically conductive above 5 µS cm−1.
Mass flow meters

The measuring principle for these meters is based on the controlled generation of
Coriolis forces. These forces are always present when both transitional (straight-line)
and rotational movement occur simultaneously. When there is no flow, the
measuring tubes both oscillate in phase. When there is flow, the tubes’ oscillation is
decelerated at the inlet and accelerated at the outlet. As the flow rate increases, the
phase difference also increases. The oscillation of the measuring tubes is determined
using electrodynamic sensors at the inlet and outlet. The measuring principle of the
mass flow meter is independent of temperature, pressure, viscosity, conductivity or
flow profile.
Dosing pumps
The other essential element in controlling the application of liquid enzymes is

the correct selection of the dosing pump. There are numerous pumps available
and capable of dispensing liquid enzymes. The most commonly adopted type
of pump for this application is the diaphragm pump, which can be mechanically or
electronically coupled. One of the benefits of the diaphragm pump is its ability
to meter volumes of liquid precisely. It is also self-priming and can handle
particulate-filled and shear-sensitive liquids.
The pump (Fig. 14.6) operates in two stages, with a positive and negative stroke.
For the pump to operate efficiently, the suction valve (1) has to allow liquid into
the head on the suction stroke and allow it through the discharge valve (2) on
the delivery stroke. This is achieved due to the motion of the diaphragm. As the
diaphragm moves backward (negative) on the suction stroke, a partial vacuum is
caused in the pump head. This lifts the suction ball valve, allowing liquid to flood
into the pump head. The vacuum created also ensures that the delivery ball valve
remains tightly seated. When the diaphragm moves forward (positive) on the delivery
stroke, the suction ball valve is forced back on to its seat. The pressure in the pump
head rises until it is greater than the system pressure. At this point, the ball in the
delivery valve lifts and the liquid is displaced. Once the diaphragm has reached its
limit of travel, the system pressure on the delivery ball valve forces the ball back on to
its seat. The cycle is then repeated.
366 P. Steen
Fig. 14.6. Cross-section of a diaphragm pump head.
Flow oscillation is common to all diaphragm pumps. To overcome this and to

achieve a consistent and even flow through the flow meter, it is necessary to install a
pulsation damper after the pump installation. The gas in the pulsation damper is
compressed until the system pressure is overcome, resulting in low-pulsation flow
from the discharge of the damper.
Enzyme Application to the Feed Pellets

Feed compounders need reassurance that the products they incorporate into their
feed are not just present, but present in the desired quantity. It is impossible for
enzymes to achieve repeatedly the perfect application criteria of 100% recovery
and a coefficient of variation of zero, given the external influencing factors, such
as assay procedure, sample collection, sample size, product quality, etc. To achieve a
homogeneous distribution, enzyme should be sprayed uniformly on to the feed, the
number of droplets per gram of feed must be sufficiently large, and the pellets
sprayed with enzyme must be thoroughly distributed (Gunther et al., 1997).
There are two accepted methods for the application of enzymes on to feed. One
involves spraying into a free-falling curtain of material, or a mechanically formed
curtain (mist-coater); the other is by spraying into a fluidized area of pellets (batch
type mixer, etc.). Fodge et al. (1997) conducted an extensive survey of the different
application points and equipment options for the PPA of enzymes. These data will be
equally applicable to any heat-labile micro-ingredients, across a range of mill systems,
and for this reason are detailed below.
Figure 14.7 shows the application of the enzyme at the mist-coater, followed by
subsequent mixing in distribution augers. The left-hand Y-axis indicates the level of
enzyme activity in millions of units per ton. The right-hand Y-axis indicates the
uncorrected coefficient of variation (CV) (%).
Of the seven systems depicted in Fig. 14.7, system G yielded an uncorrected
coefficient of variation (CV) of less than 10% and the smallest spread of average
activities. The next best uniformities were observed with systems F, C and D.
Systems A, B and E were the least uniform.
Both G and F were continuously fed systems in which an impact-style load cell
is positioned in the feed line just prior to the roto-coaters. Feed strikes the impact
plate, a proportional voltage signal from the impact plate is forwarded to a variable
Fig. 14.7. Application of enzyme at roto-coaters followed by mixing in distribu-

tion augers (Fodge et al., 1997). Lower dotted line is the uncorrected CV, obtained
from spraying Hemicell into a batch mixer (use right y-axis). Upper dotted line is
the desired amount of enzyme per ton applied to the feed (use left y-axis). Above
each letter are ten data points where each point represents the average enzyme
activity for ten feed samples collected in approximately 60 s. The bar above each
letter is the average CV obtained over a period of several weeks from ten sample
sets.
368 P. Steen
frequency drive that controls the output of the fat and enzyme pumps in synchrony
with feed flowing into the roto-coaters. Feed slides off the impact plate in a curtain-
shape, roughly 50 mm thick and 410 mm wide, directly above the roto-coater. One
spray nozzle was positioned on either side of this curtain and sprayed the enzyme on
the feed rather than applying it inside the roto-coater (Fodge et al., 1997).
A and B roto-coaters were semi-continuous fed systems that were very old and
in a feed mill designed for about 50% of the current output. Both systems often
had problems and had never performed smoothly. The feed-flow rate varied by 10%,
or more, and the mixing of the feed was poor. Systems C and D were batch-fed
roto-coaters in a well-maintained old feed mill, also producing feed at greater than
the design capacity. Preventive maintenance programmes for C and D were well
developed and effective, and the feed mill did not experience unusual problems that
slowed their performance. Roto-coater E was constructed by a feed mill maintenance
shop. It was fed semi-continuously, and although the accuracy was reasonable, the
uniformity of mixing was poor (Fodge et al., 1997).
Applying enzymes to the feed at an existing fat coater is a widely adopted and
accurate solution. The above figures help to support this, but they also indicate that,
if the correct operating practices are not adopted, then poor results can also occur,
as demonstrated by systems A, B and E. In the case of volumetric systems it is
vital that changes can be made for variations in product bulk density to compensate
for increase or decrease in the system capacity. Roto-coater type systems have been
developed primarily for the application of fat; however, testing with enzymes has
been conducted and has resulted in excellent uniform coating (Decksheimer, 1998).
Figure 14.8 shows the results of spraying enzyme on to pelleted feed in four
different augers. The accuracy of enzyme application was undesirable in three of the
four and the CV was slightly less than 20% on two systems and worse than 20% on
the third system. In system A, enzyme application was linked to feed flow rate, which
was measured, whereas in systems B–D it was not measured. The main problems
with using augers are: (i) the need for accurate measurement of feed flow rates in
them; and (ii) the difficulty of keeping liquid product from building up on either the
walls or the drive shaft in the auger (Fodge et al., 1997).
The results shown for system A are not typical for a measured flow rate, with all
samples indicating an over-application of enzyme. It may be the case here that the
enzyme and dry flow rate were not correctly calibrated. Either the dry flow rate was
low for the given enzyme set point or the enzyme set point was high for the dry flow
rate.
The application of enzyme in conveying augers is not generally recommended,
due to (i) above. However, in applications such as a fat coater complete with a blend-
ing screw, this configuration can provide good opportunities for enzyme application.
Additionally, with care and diligence regarding the nozzle selection, positioning and
air pressure, it is possible to overcome the associated problems with (ii) above.
The data collected from four feed lines where the enzyme was applied by
spraying it on to curtains of feed falling from the end of weigh-belt coaters are shown
in Fig. 14.9. Spray nozzles for spraying fat were positioned on either side of this
curtain. For adding enzymes, nozzles were positioned on both sides of the curtain of
Fig. 14.8. Enzyme application in conveying augers (Fodge et al., 1997). Lower
dotted line is the uncorrected CV, obtained from spraying Hemicell into a batch
mixer. Upper dotted line is the desired amount of enzyme per ton applied to the
feed. Above each letter are ten data points where each point represents the average
enzyme activity for ten feed samples collected in approximately 60 s. The bar
above each letter is the average CV obtained over a period of several weeks from
ten sample sets.
feed, either above or below the fat nozzles, and occasionally included the enzyme
with the fat. System A operates at a constant rate, systems B–D at varying rates
(Fodge et al., 1997).
Feed mills may not have roto-coaters, augers or horizontal weigh-belt coaters.
To develop an alternative application site the product was applied beneath the cooler
just before the feed drops into a chain drag. The transition piece between the cooler
exit and the chain drag was replaced with a redesigned transition piece that collected
the feed forming a curtain of feed 25–50 mm thick. The new transition piece
comprised two compartments, each with two internal baffles that helped to form the
feed into a curtain-like shape, and spray nozzles were inserted into the transition
behind these curtains. Positioning the nozzles behind the curtain of feed helped to
protect the spray patterns from the airflow turbulence that is prevalent at these sites
(Fodge et al., 1997).
Figure 14.10 shows the results obtained at four such transition systems installed
in poultry feed mills. Accuracy and uniformity were quite good in A and B, accept-
able in C but still problematic in D. System D had more airflow turbulence problems
than the others. It was concluded that these modified systems were an acceptable
means to apply heat-labile products and were cost effective (Fodge et al., 1997).
Spraying at transitions and spouts is a very contentious topic. From the data
above it can be seen to work, but also in other circumstances to fail. It is a very
370 P. Steen
Fig. 14.9. Enzyme application results with weigh-belt coaters using spray nozzles
(Fodge et al., 1997). Lower dotted line is the uncorrected CV, obtained from
spraying Hemicell into a batch mixer. Upper dotted line is the desired amount of
enzyme per ton applied to the feed. Above each letter are ten data points where
each point represents the average enzyme activity for ten feed samples collected
in approximately 60 s. The bar above each letter is the average CV obtained over
a period of several weeks from ten sample sets.
demanding application point, in that it needs constant inspection and continual

measurement to ensure the system stays within the predefined limits of acceptance.
Proportion of sprayed pellets
The homogeneous distribution of the additive (liquid) on the feed is critically

determined by the proportion of the sprayed pellets. The system should work in
such a way that the CV is minimized. A value of less than 20% should be aimed for.
The more pellets that are sprayed, the sooner this value is achieved (Schwarz, 1998).
The volume of liquid applied to the feed in a PPA system is relatively small (typi-
cally 500–1000 g tt−1). To disperse this quantity of liquid across the feed, the use of
air atomizing nozzles is required. Air is used to shear the liquid; this mechanism pro-
duces a broad spectrum of droplet sizes, often varying from submicron up to several
hundred micrometres in the same spray. A perspective of droplet diameters can be
gained by realizing that there are 1000 µm mm−1 and the required range of droplets
to be applied to the feed would need to have a mean of 300–400 µm. It is critical that
the liquid is not over-atomized by the use of too high an air pressure. This will result
in a micro-mist, with many of the micro-droplets condensing on the internal surfaces
of the application equipment. In addition, the smaller the droplet size, the smaller is
Fig. 14.10. Spraying in transitions beneath coolers, before augers (Fodge et al.,
1997). Lower dotted line is the uncorrected CV, obtained from spraying Hemicell
into a batch mixer. Upper dotted line is the desired amount of enzyme per ton
applied to the feed. Above each letter are ten data points where each point repre-
sents the average enzyme activity for ten feed samples collected in approximately
60 s. The bar above each letter is the average CV obtained over a period of several
weeks from ten sample sets.
the mass and, as a result, droplets can be affected by the airflow in the application
equipment, with the droplets being drawn away from the application point.
To maximize the exposure of enzyme and feed pellets, it is critical to present
both elements so that the pellets come into contact with the enzyme spray pattern for
as long as possible, and that the pellets are in suspension during this period. The
nozzle design, application or orientation should be such that, if used in a continuous
or semi-continuous system of varying capacities, the varying flow rate through the
nozzle would not in any way affect the exposure of the enzyme and pellets, reducing
the efficiency of the system.
Addition of water with enzymes
The inclusion rate of some concentrated enzymes can be as little as 50–100 g tt−1.
This does not present problems for the metering or measurement of the liquid, but it
does make it a considerable task to apply the liquid to the feed accurately. These con-
centrated enzymes need to be diluted to increase the volume of liquid to feed,
improving the exposure of the enzyme to the pellets.
Although the objective is a minimum level of water inclusion, a 1000 g tt−1
total inclusion has proved effective (Decksheimer, 1998). Figure 14.11 demonstrates
372 P. Steen
Fig. 14.11. Water/enzyme concentration at 1 kg tt−1 (Decksheimer, 1998).

Samples 1–2, pelleted hog grower, 15 t h−1, spray chamber. Samples 3–6, pelleted
17% layer, 22 t h−1, spray chamber. Samples 7–8, pelleted broiler, 45 t h−1,
roto-coater. Samples 9–12, pelleted broiler, 15 t h−1, mixing auger.
that acceptable coefficients of variation were achievable with enzymes sprayed

on to samples of feed using different production rates and application points
(Decksheimer, 1998).
Predilution of liquid enzyme products to aid application also raises potential
issues of product stability. In practice many companies advise against prolonged
storage of diluted products, in order to avoid the risk of microbial contamination
(e.g. Schwarz, 1998). Any dilution with water should take place immediately prior to
the spray nozzle and mixing should be with a static mixer, without moving parts. The
mixer, nozzle and connecting parts should also be automatically flushed after each
addition (Overfield, 1999b).
Pre- and post-fat addition of enzymes
There are conflicting opinions regarding the application of enzymes at the fat coater.
Should enzymes be applied to the feed before the fat and then be subjected to the
high fat temperature, or applied post-fat application, with concern then focusing on
the enzyme’s ability to penetrate the fat coating on the pellets?
Assay work carried out with samples from commercial feed mills has shown
there to be no clear interaction between enzyme recovery and stability and pre- or
post-application of enzymes at the fat coater. Research cited by Engelen (1998)
(Tables 14.3 and 14.4) also supports this view. The recovery of xylanase LC was
shown to be unchanged irrespective of the fat temperature, the fat level and the
spraying order.
Table 14.3. Effect of spraying fat before or after enzyme addition on measured enzyme recovery
(Engelen, 1998).
Enzymeb recovery (%)
Enzyme sprayed Enzyme sprayed immediately Enzyme sprayed 30

Dieta Control before fat spray after fat spraying min after fat spraying
Starter 0 100 90 99
Grower 0 100 104 99
a50% barley based.
b2 l xylanase LC t−1 of feed.
Table 14.4. Effect of fat level and fat temperature on the recovery of a liquid enzyme (Engelen,
1998).
Enzyme recovery (%) in feeda,b
Fat temperature (°C) Fat level: 1.5% Fat level: 3.0%
64 114 118
80 110 117
88 105 117
a50% barley based.
b2 l xylanase LC t−1 of feed.
Liquid Enzyme Stability

Liquid enzymes are inherently less storage stable than their granular counterparts. In
liquid form, water activity is sufficiently high for the component enzymes to remain
active, whereas in the granular form the low water activity renders the component
enzymes relatively inactive prior to ingestion by the animal. Commercially available
enzymes are typically less than 100% pure and often contain side enzyme activities in
addition to the desired main activity or activities. These side activities can include
trace levels of proteases which, even at low activity, can cause some degradation of the
main activities (e.g. xylanase). An aqueous environment also introduces a significant
risk of microbial contamination and proliferation, which can cause rapid decline
in enzyme activity in liquid products. The problem of storage stability is increased
further when different liquid enzymes are mixed to produce ‘multi-enzyme’ liquid
products and when liquid enzyme products are stored at ambient temperatures in
different parts of the world.
Enzyme suppliers generally guarantee the shelf-life of liquid enzyme products
for between 3 and 6 months following the date of manufacture, if stored at or below
22°C (e.g. Fig. 14.12). For shipment to tropical countries, where the ambient
conditions exceed 22°C, suppliers should ship and store enzymes in refrigerated
containers that maintain the product at a constant temperature below 5°C prior
to delivery to the feed mill. Once liquid enzymes have been applied to the feed,
374 P. Steen
Fig. 14.12. Storage stability of a Trichoderma xylanase, product stored for 18

months at 22°C (Cultor Technology Centre, Finland).
the component enzymes are rendered inactive and stable during the period prior to
consumption by the animal.
Delivery of Enzymes
Liquid enzymes can be delivered in a range of different sized containers to the feed
producer’s requirements. The more commonly adopted sizes are either a 1000 l
intermediate bulk container (IBC) or a 210 l barrel. Containers are manufactured
from high-density polyethylene with UV stabilizers.
Discharge from the 210 l container is from the 50 mm valve at the top of the
container. The discharge from the IBC is via a 50 mm valve located at the bottom of
the IBC. The IBC has a 150 mm screw cap, which incorporates a degas/regas vent for
balancing internal and external pressure. The 1000 l container is self-emptying, due
to the bottom discharge and sloped bottom of the IBC. The IBC is mounted on a
pallet for handling by forklift truck.
The IBC offers greater flexibility, ease of handling and self-emptying, and
minimizes risk of exposure to operators, etc. The 210 l barrel offers flexibility for
installations when access/handling of IBCs is not possible.
Handling of Liquids
During the period prior to dosing, liquid enzymes must be stored carefully in the feed
mill to maintain stability. Ideally the product should remain in its original container
and be stored in cool dry conditions. If transfer to another container or tank is neces-
sary, then this must be sterilized before use and at regular intervals. These containers
must also be ‘closed’ to prevent ingress of dust and other sources of contamination.
Microbial contamination of liquid enzymes may not only reduce their stability prior
to dosing, but also recontaminate the feed following heat treatment.
Appropriate health and safety precautions must be observed whilst handling
liquid enzymes. These precautions will be found in the supplier’s Material Safety
Data Sheet.
Measurement of Dosing Accuracy vs. Bird Performance

Measurement of the accuracy of dosing enzymes on to feed pellets relies upon a
sensitive assay procedure. Currently, feed enzymes stand alone from many other
high-value feed additives due to the absence of universally accepted assays for
their determination, both as products and when incorporated in-feed. The assay
procedures used will be influenced by the properties of the enzyme concerned, e.g.
pH, temperature characteristics. The assay is undertaken to measure the effectiveness
of the application of the enzymes to the feed, the main criteria for measurement
being homogeneity (coefficient of variation) and absolute recovery of enzyme activity
from the feed. It can be seen that, provided an accurate dosing system is employed,
productive performance is similar whether the product is applied post-pelleting
(liquid) or pre-pelleting (granular, Fig. 14.13).
Summary
The choices of liquid application system vary both in cost and in accuracy. The use of
the simple volumetric dosing system to apply enzymes is an economical choice in
Fig. 14.13. Effect of temperature on response in broiler performance (0–42 days)

to enzyme supplementation in granular (Az 1300) vs. liquid (Az 1310) products
(Finnfeeds Int. Technical Report 1300.UK.98.29).
376 P. Steen
terms of the equipment only. Frequently such systems do not offer the accuracy
required to exploit the economic relationship that exists between enzyme dose, target
substrate and resultant animal performance.
The installation of the dosing equipment must be at the right location in the
process (post-screening) and be correctly sized for the feed mill capacity, which may
not necessarily be the same as the pelleting capacity. The measurement and control of
the dry flow rate are as vital to accuracy of the system as the measurement and control
of the liquid flow rate.
The choice of system must include the present and future requirements of
the feed producer, provide the flexibility for the system to dose different numbers
of liquids at varied application rates, and handle the different number of product
forms manufactured by the feed mill.
References
Decksheimer, D. (1998) Accurate application of liquid ingredients. Proceedings of the Eastern
Nutrition Conference. Montreal, Canada, pp. 111–126.
Engelen, G.M.A. (1998) Technology of Liquid Additives in Post Pelleting Applications.
Wageningen Institute of Animal Science, The Netherlands, 101 pp.
Fodge, D., Smith, A., Redman, C., Humg-Yu, H. and Treidel, B. (1997) Post-pelleting
application of heat-labile products explored. Feedstuffs, 29 September, 18–26.
Gunther, C., Beudeker, R.F. and Vahl, J.L. (1997) Choosing a liquid enzyme system. Feed
Mix 5(5), 24–27.
Harker, A.J. (1996) Application of liquid enzymes in poultry feeds. Proceedings of the Carolina
Nutrition Conference. Charlotte, North Carolina, 12 pp.
Overfield, N. (1999a) New standards with liquid application system. Agricultural Supply
Industry 29(38), 8.
Overfield, N. (1999b) New liquid enzyme application system sets high standards. Feed
Compounder, June/July, 16–20.
Pickford, J.R. (1992) Effects of processing on stability of heat labile nutrients in animal feeds.
In: Garnsworthy, P.C., Haresign, W. and Cole, D.J.A. (eds) Recent Advances in Animal
Nutrition. Butterworth-Heinemann, Oxford, UK, pp. 177–192.
Schwarz, G. (1998) Liquid feed additives, an alternative in modern animal nutrition (Part 2).
Kraftfutter 11/98, 506–511.
van Laarhoven, H. (1998) Future developments in liquid applications of enzymes. Feed
Expo 98, UK. The European Feed Seminar, 6 pp.
Process Stability and

Methods of Detection
of Feed Enzymes in
15
Complete Diets
M.R. BEDFORD,1 F.G. SILVERSIDES2 AND
W.D. COWAN3
1Finnfeeds, PO Box 777, Marlborough, Wiltshire SN8 1XN, UK;
2Denman Island, British Columbia, Canada; and 3Novo Nordisk,
Krogshoevej 36, Bagsvaerd, Denmark 2880
M.R. Bedford
Stability
15 and Detection
et al. of Feed Enzymes
Introduction
Enzymes are now routinely added to monogastric diets in many parts of the world. It
is clear that their use is based on determined benefits in animal performance, but
proving their presence in manufactured animal feeds is a daunting task and has been
the focus of much research. Enzymes are proteins and as such are susceptible to the
rigours of feed manufacture, as are all other feed proteins. Whilst the structural
conformation need not be maintained for feed proteins to deliver their benefit in
terms of amino acid content, it is critical that feed enzymes are not irreversibly
denatured during feed manufacture or they will no longer function. It is clear that
detection of their activity in complete feeds is therefore important for confirmation
of survival. Whilst it is not within the remit of this chapter to discuss the methods
available for their detection (this is covered in Chapters 4 and 15), the idiosyncrasies
of each major class of enzyme used in animal feed are of interest and will be covered.
Issues addressed will include differences between source enzyme stability to thermal
denaturation in vitro, problems with analysis due to interaction with the feed matrix
(and methods of limiting this effect), data from in-feed processing trials, and future
trends.
The vast majority of mixed feed for poultry and swine undergoes some sort
of processing. For many years, feed has been pelleted, a process whereby the
feed mixture is conditioned by adding steam, then pressed through a die. Pelleting
increases nutrient density, improves the handling characteristics of the feed, and
reduces the microbial load. Pelleting commonly uses temperatures between 65 and
95°C (Gibson, 1995), which can be damaging to heat-sensitive nutrients, including
enzymes.
387
17 November 2000 15:29:43

378 M.R. Bedford et al.
In the past few years, concern for feed-borne pathogens and factors of pellet
quality have caused feed manufacturers to increase the processing temperature, time
and pressure, and feed has been submitted to double pelleting or expansion
(Pickford, 1992). The increase in the severity of the processing treatment underlines
the importance of enzyme stability. Several approaches have been taken to overcome
the difficulties, including avoidance of the treatment altogether by adding the
enzymes as a liquid after the pellets have cooled. Despite the possibility of
post-pelleting supplementation, feed enzymes are often added to the mash before
treatment. The effects of heat may be reduced by protection of the enzymes with
hydrophobic coatings or selection of heat-resistant enzymes.
Until recently, there had been limited research published on the survivability of
feed enzymes (Chesson, 1993). Yet enzyme stability is of critical importance for the
producers of feed enzymes and we must assume that in-house evaluations preceded
the marketing of enzyme preparations. Since 1993, several research trials have been
reported in the popular press or in symposia proceedings, and a few trials have
been described in the peer-reviewed scientific literature. Clearly, measuring enzyme
activity in vitro, either in solution or in the feed, is of critical importance. Recent
research suggests that measurements of in vitro activity must be tempered by the
results of tests on in vivo efficacy.
Phytase
A surprising amount of research has been reported on the temperature stability of
phytase, considering that phytase makes up less than 20% of commercial enzyme
usage (Bedford and Schulze, 1998). The interest is likely because phytic acid, which
renders phosphorus and other nutrients unavailable, is present in many plant sources
of feed ingredients (Cheryan, 1980; Eeckhout and dePaepe, 1994; Ravindran et al.,
1995) yet endogenous phytase activity in monogastric animals is minimal or absent
(Pallauf and Rimbach, 1997). To complicate the situation, many of the same plant
sources of phytic acid also contain significant amounts of phytase and the nutritional
problem of phosphorus digestion is compounded by the environmental problem of
phosphorus deposition on the land. Phosphorus pollution has become a limitation to
production in areas with intensive animal production.
Phytase enzymes are diverse in both their sources and their characteristics.
Liu et al. (1998) summarized the literature to 1998 and showed that phytases from
bacteria, fungi, yeast and plants have optimal temperatures that range from 45 to
77°C, a difference of 32°C. A phytase isolated from Aspergillus niger was described by
Dvorakova et al. (1997) as having activity at temperatures between 25 and 65°C with
an optimum at 55°C. Incubation of the enzyme for 10 min at 60°C destroyed 5% of
its original activity, while incubation at 80°C destroyed 80% of the activity. As part
of an effort to find heat-stable enzymes, Wyss et al. (1998) studied heat denaturation
of purified phytases isolated from A. fumigatus and A. niger. Both enzymes were
denatured at temperatures as low as 55°C. However, even when submitted to a tem-
perature of 90°C, the enzyme from A. fumigatus refolded into an active conformation
388
17 November 2000 15:29:44

Stability and Detection of Feed Enzymes 379
on cooling, while that from A. niger did not. Some of the heat-resistant phytases will
undoubtedly become available commercially in the near future.
Heat inactivation of enzyme in solution does not translate directly into heat
inactivation in the feed, because of an interaction of the enzyme with the feed matrix.
In effect, the feed ingredients can protect the enzyme from steam or elevated
temperatures for a short period of time (Chesson, 1993). Measuring phytase activity
in pelleted feed provides a more accurate evaluation of the commercial importance of
inactivation. Simons et al. (1990) added phytase to a ‘common pig feed’, which they
heated to either 50 or 65°C before pelleting. Heating to 50°C resulted in a pellet
temperature of 78 or 81°C but did not lower the enzyme activity. However, heating
to 65°C produced a pellet temperature of 84 or 87°C, resulting in inactivation of
17 and 54%, respectively, of the original enzyme activity. Gibson (1995) added three
phytase preparations to a wheat-based feed and pelleted the feed at temperatures
of 65–95°C. Two of the preparations suffered inactivation even at 65°C and only
a commercially available stabilized preparation retained substantial activity with
pelleting at 85°C or above. In addition to studying enzyme stability in solution,
Wyss et al. (1998) added phytase enzymes to commercial feed before pelleting at
75 or 85°C. Recovery of the activity was similar for the two enzymes after pelleting
at 75°C, but pelleting at 85°C resulted in a greater inactivation of the enzyme from
A. niger than that from A. fumigatus, supporting their results on denaturation
kinetics. Eeckhout et al. (1995) added a commercial phytase to feed and reported
that pelleting temperatures between 69 and 74°C destroyed 50–65% of the enzyme
activity.
Inactivation is not limited to microbial enzymes that may be added to the
feed, but also affects the enzymes naturally contained in feed ingredients. Gibson
(1995) found that pelleting at temperatures of 85°C or above largely inactivated
the endogenous phytase in wheat. Eeckhout and dePaepe (1994), in their survey of
phytase activity in different feedstuffs, reported that pelleted samples of wheat
bran, which is particularly rich in phytase, had only 56% of the phytase activity of
unpelleted wheat bran. In three experiments, Jongbloed and Kemme (1990) found
that pelleting at temperatures approaching 80°C decreased enzyme activity in pig
feeds that were based on feedstuffs that were rich or poor in phytase activity. They
carried their experimentation a step further by measuring the effect of pelleting on
the apparent absorption of phosphorus. In two experiments, they found that
pelleting of phytase-rich diets decreased phosphorus absorption, supporting the data
on endogenous phytase inactivation.
Research interest in phytase stability by both the research community and the
feed industry is due to the higher processing temperatures currently being used
and the increased importance of phosphorus absorption for both nutritional
and environmental reasons. Thermal protection of available exogenous enzymes by
encapsulation or granulation provides a solution at present. A more fundamental
solution may be the isolation of enzymes that are heat-resistant or that can reform
their active structure after denaturation. Unfortunately, neither of these solutions
prevents high processing temperatures from destroying endogenous enzymes
contained in feed sources.
389
17 November 2000 15:29:45

b-Glucanase
The commercial availability of enzyme preparations containing β-glucanase activity
has allowed barley to be incorporated into poultry diets without the depressed
performance and sticky droppings that are associated with high β-glucan levels
(Campbell and Bedford, 1992). The use of β-glucanase is now widespread in areas of
the world where barley is commonly grown. There are a limited number of reports on
the effect of heat treatment on β-glucanase enzyme that has been added to feed.
Eeckhout et al. (1995) measured the activity of a β-glucanase included in a
commercial piglet feed conditioned at temperatures between 50 and 95°C and
pelleted at temperatures between 72 and 91°C. Even at the lowest temperatures,
40% of the original activity was lost, and at the highest temperatures, only 7% of the
original activity remained after processing. More than two thirds of enzyme inactiva-
tion occurred in the conditioner. On the other hand, Esteve-Garcia et al. (1997) did
not find major inactivation of enzyme in broiler feed with conditioning and pelleting
temperatures approaching 80°C. The enzyme that they used was described as a
microgranulate, suggesting that it was incorporated into the feed in a stabilized form.
At least two trials have measured the performance of broiler chicks fed
enzyme-supplemented diets that were submitted to heat treatment. McCracken et al.
(1993) supplemented a barley-based diet with a stabilized commercial enzyme
mixture containing β-glucanase and xylanase activities and heated it to 85°C for
15 min before pelleting. Heat treatment of the feed without exogenous enzyme
decreased the apparent digestibility of nutrients, increased the viscosity of the
intestinal contents, and reduced faecal dry matter content. The addition of the
enzyme improved nutrient digestibility and eliminated the negative effects of
heat treatment, demonstrating clearly that the enzymes remained active at this
temperature. Vukic-Vranjes et al. (1994) tested a commercial enzyme mixture,
including β-glucanase, xylanase, amylase and pectinase activities, in two diets, one
of which contained 20% barley. The feed was conditioned at 70–75°C, and pelleted
or extruded with the temperature in the extruder barrel reaching 110–120°C.
Compared with pelleting, extrusion produced negative effects on chick performance.
Extrusion also resulted in increased in vitro viscosity of the feed, suggesting that
the higher temperature increased the solubility of non-starch polysaccharides. The
enzyme mixture improved chick performance whether the diet was pelleted or
extruded, likely indicating that there was enzyme activity even after exposure to the
extrusion process. Vukic-Vranjes et al. (1994) also showed that supplementation
with the enzyme mixture reduced the viscosity of the feed extract, demonstrating that
the enzyme was active in the feed before ingestion by the bird. They did not rule out
enzyme action in the feed even before processing.
Inborr and Bedford (1994) measured both enzyme activity in the feed and
bird performance after supplementation and heat treatment. These authors found
significant inactivation of added β-glucanase (the same product used by McCracken
et al., 1993) in a barley-based diet after conditioning at a temperature as low as 75°C
followed by pelleting. Conditioning at 95°C for 30 s and 15 min destroyed 84 and
91%, respectively, of the original β-glucanase activity. Inborr and Bedford (1994)
390
17 November 2000 15:29:46

wondered if measuring enzyme activity in vitro or in vivo was the most accurate
method of evaluating the effect of processing on enzyme activity. While recovery of
enzyme activity was highest at the lowest processing temperatures and lowest at
the highest temperatures, this was not accurately reflected in bird performance. The
highest value for weight gain and the lowest value for the feed-to-gain ratio were
obtained with the intermediate conditioning temperature of 85°C. Clearly, the
enzyme remained active at this temperature, and the positive effects of heat treatment
outweighed the negative effects of enzyme inactivation.
Pickford (1992) showed that stabilization of β-glucanase protected it from a
pelleting temperature of 75 but not 95°C. Cowan and Rasmussen (1993) reported
that a commercial β-glucanase lost significant activity with pelleting at a temperature
above 65°C, but that coating the same product provided protection to 75°C. Both
these reports are brief and there is very little published research relating specifically to
increasing the temperature stability of β-glucanase.
Xylanase
Xylanase represents by far the greatest share of worldwide enzyme sales (Bedford and
Schultz, 1998) and its use allows high-viscosity wheat to be incorporated into poultry
diets without negative effects. Perhaps because of the economic importance, there
is considerably more research reported in the scientific literature on the stability of
xylanases than there is on the stability of β-glucanases.
Xylanase enzymes are produced by many species of fungi and bacteria, and varia-
tion in the optimum conditions for activity of these is not surprising because each
organism is best adapted to a specific environment. In a review, Bedford and Schulze
(1998) show that the optimum temperature for a number of bacterial and fungal
xylanases can range from 30 to 105°C and the optimum pH can range from 2.0 to
10. Pickford (1992) compared the stability of three commercial enzyme preparations
without providing information on the type of activities that they contained. At a
pelleting temperature of 80°C, 85% of the original activity was retained by one prep-
aration, 55% by a second, and only 33% by the third. No enzyme activity was shown
for any preparation with pelleting at 95°C. Expansion, with a higher temperature but
shorter exposure, caused greater loss with similar differences between enzymes.
Pettersson and Rasmussen (1997) demonstrated differences in temperature
stability of xylanase enzymes isolated from Thermomyces, Humicola and Trichoderma.
At a conditioning temperature of 75°C, the Trichoderma enzyme lost significant
activity, while at 85°C both the Thermomyces and Humicola enzymes retained more
than 80% of their original activity. Even with conditioning at 95°C the Thermoyces
xylanase retained 70% of its activity. Gibson (1995) observed variation between nine
xylanase preparations, seven of which were commercially available at the time of the
trial. Activity remaining after treatment at 90°C was above 90% for one preparation
and lower than 10% for several of the others. It is not clear from the report how
much of this variation was due to the source of the enzyme and how much was due to
stabilization of the enzyme. Perez-Vendrell et al. (1999a) tested the in vitro stability
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of eight commercially available preparations, including coated or encapsulated

products, after feed processing at 65–70, 75–80 and 85–90°C before pelleting.
Measurement in vitro showed that, even at the lowest temperatures, most products
lost at least 30% of their activity, while at the highest temperatures, activity loss
reached 90%. As they found for β-glucanase, Esteve-Garcia et al. (1997) reported
that the microgranulated xylanase preparation that they studied was not inactivated
by conditioning and pelleting temperatures that approached 80°C.
In the preceding paragraph reference was made to several methods of thermal
protection. The most fundamental protection is that suggested by the study of
Pettersson and Rasmussen (1997) in which heat-resistant enzymes are prepared
from heat-resistant organisms. At the other extreme, the problem can be avoided
altogether by spraying the enzyme on the finished pellets (Perez-Vendrell et al.,
1999b), though this requires some additional equipment and an extra step in the
manufacturing process.
Dry enzyme preparations can also be stabilized to increase heat resistance and
allow their addition to the feed before pelleting without undue inactivation. Methods
of stabilization were the subject of a previous chapter. Much of the inactivation of
enzymes is due to the steam used for conditioning. Thus, stabilizing a preparation
relates largely to protecting the enzyme from the steam by absorbing it on to a
carrier or by coating it with a hydrophobic substance (Cowan, 1996). Pickford
(1992) provides an example whereby stabilization increased activity remaining
after pelleting at 75°C from 48 to 76%, and after pelleting at 95°C from 12 to 34%.
Clearly, even protected enzymes have a limit of temperature stability; Steen (1999)
suggests that if a feed is to be processed at a temperature above 90°C, even enzymes
that have been stabilized should be added after processing.
Tests of in vitro enzyme activity are valuable tools in determining enzyme
activity loss, and they suggest that exposure to even relatively low temperatures
results in significant destruction. However, in vitro tests are but one tool among
many. Clearly, enzyme activity in a buffer solution at the optimum pH provides only
a very limited view of activity under the conditions of feed processing and digestion.
In fact, most investigators (Vukic-Vranjes et al., 1994; Pettersson and Rasmussen,
1997; Perez-Vendrell et al., 1999a; and others) have recognized that interactions
between the enzyme and the feed are important and they have measured activity in
complete feeds. Measurement of the viscosity of feed extracts (Spring et al., 1996;
Bedford et al., 1997) can provide a measurement of the effects of enzyme action
before the feed is ingested by the bird. Preston et al. (1999) demonstrated this action
by showing that even with apparently complete enzyme inactivation in the finished
feed, supplementation resulted in improved bird performance.
To complete the story of enzyme action in relation to processing, the effects
of enzyme addition and heat treatment of the feed must be measured by bird
performance. Enzyme inactivation does not always translate directly into perfor-
mance differences (Bedford et al., 1997; Perez-Vendrell et al., 1999a; Silversides and
Bedford, 1999). With processing temperatures between 65 and 105°C, Bedford et al.
(1997) found that apparent inactivation of enzyme was linear, whereas performance
(body-weight gain, feed conversion ratio) was best when the feed was processed at
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81–83°C. Silversides and Bedford (1999) also demonstrated this effect of heat
treatment, as is shown in Fig. 15.1. As the processing temperature increases, enzyme
activity decreases in a linear fashion (R2 = 0.97) while the change in broiler body
weight with increasing processing temperatures was quadratic (R2 = 0.84). Graphing
the results for the feed conversion ratio produces very similar results (R2 = 0.98).
The highest body weight and lowest feed conversion ratio were at a processing
temperature between 80 and 85°C. When such a performance response is achieved
but is juxtaposed with apparent enzyme inactivation in an in vitro assay, it suggests
two possibilities: either the enzyme completes its function almost entirely in the
conditioner, making post-pelleting analysis largely irrelevant, or enzyme activity is
not effectively measured by the assays used.
Data from enzymes derived from coated Thermomyces species do not seem to fall
into the same category as those from uncoated Trichoderma or Aspergillus, probably
due to a lack of interaction with the feed matrix of the former. Under such conditions
the enzyme is more easily extracted and a correlation between performance and
recovered enzyme activity has been demonstrated (Cowan and Rasmussen, 1993;
Pettersson and Rasmussen, 1997; Andersen and Dalbøge, 1999). These contrasting
observations clearly indicate that there is no single method that can be applied for
determining subsequent bird performance from analysis of a feed sample when
identity of the enzyme is unknown.
Improved performance with a decline in in vitro enzyme activity underlines
the danger of relying solely on measuring activity, and it is not as contradictory
as it appears. Heat affects many things in addition to enzymes (Pickford, 1992).
Moderate heat increases starch gelatinization and cell wall destruction, increasing
the availability of nutrients and subsequently improving performance as well. This
is, after all, one of the principal reasons for pelleting feed. Higher processing
Fig. 15.1. Changes in broiler body weight (q) and xylanase activity (x) with
increasing processing temperatures. (From Silversides and Bedford, 1999.)
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17 November 2000 15:29:51

temperatures also cause increased solubilization of non-starch polysaccharides,

leading to higher intestinal viscosity and reduced performance, and actually
increasing the need for exogenous enzymes. Silversides and Bedford (1999) showed
that without exogenous xylanase in a wheat-based diet, the viscosity of the intestinal
contents of broilers increased dramatically as processing temperature increased
(Fig. 15.2). Addition of xylanase produced low digesta viscosity even with high
processing temperatures, and the actual reduction in viscosity was greatest at the
highest temperatures. The greater enzyme action with high processing temperatures
is likely because more substrate is available for enzyme action. The enzyme may be
active before or during processing, reducing the viscosity measured either in vitro or
in vivo. High processing temperatures reduce performance not only due to increased
digesta viscosity and decreased exogenous enzyme action, but also because vitamins
and other enzymes are also inactivated, and starch and protein digestibility is
decreased. With heat-resistant or stabilized enzyme preparations available, heat
damage to vitamins and nutrients other than exogenous enzymes will likely limit the
processing temperatures that will be used commercially.
Conclusions and Trends

The activity of enzymes in solution can be reduced at temperatures of 60°C or lower.
Enzymes in mixed feed are somewhat protected by the feed ingredients and most
are likely to be reasonably stable at a slightly higher temperature. However, with
Fig. 15.2. Changes in the viscosity of the intestinal contents of broilers given feed
supplemented (w) or unsupplemented (z) with xylanase and processed at different
temperatures. (From Silversides and Bedford, 1999.)
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24 November 2000 17:04:46

processing temperatures as high as 95°C there is a serious loss of enzyme activity.

Several approaches have been used or suggested to reduce the negative effects of
heat treatment, including protecting the enzymes from steam penetration, using
heat-resistant enzymes or simply adding the enzymes as a liquid after processing.
Feed enzymes may have some activity before the animal ingests the feed, so that total
loss of in vitro activity may not always mean total loss of benefit. This seems to be at
least partially dependent on the enzyme and the assay method. In some cases in vitro
analysis is misleading, whereas in others there is a clear link between enzyme content
and in vivo performance. Common processing temperatures cannot increase
indefinitely because of the increased damage to vitamins, proteins and starch, which
may be even more susceptible to heat damage than exogenous feed enzymes. The
further development of enzymes selected for heat resistance and the utilization of
enzyme activity before processing or ingestion by the animal may be promising
approaches to obtaining the maximum benefit of added enzymes for the feed
industry.
References
Andersen, L.D. and Dalbøge, H. (1999) Future trends in feed enzymes. Proceedings of the
American Soybean Association Joint Seminar, Bangkok, Thailand, pp. 1–9.
Bedford, M.R. and Schulz, H. (1998) Exogenous enzymes for pigs and poultry. Nutrition
Research Reviews 11, 91–114.
Bedford, M.R., Pack, M. and Wyatt, C.L. (1997) Relevance of in feed analysis of enzyme
activity for prediction of bird performance in wheat-based diets. Poultry Science 76
(Suppl. 1), 39.
Campbell, G.L. and Bedford, M.R. (1992) Enzyme applications for monogastric feeds: a
review. Canadian Journal of Animal Science 72, 449–466.
Cheryan, M. (1980) Phytic acid interactions in food systems. CRC Critical Reviews in Food
Cowan, W.D. (1996) Animal feed. In: Godfrey, T. and West, S. (eds) Industrial Enzymology,
2nd edn. MacMillan Press, London, pp. 71–86.
Cowan, W.D. and Rasmussen, P.B. (1993) Thermostability of microbial enzymes in expander
and pelleting process and application systems for post-pelleting addition. In: Wenk, C.
and Boessinger, M. (eds) Proceedings of the 1st Symposium on Enzymes in Animal
Nutrition. Kartause Ittingen, Switzerland, pp. 263–268.
Dvorakova, J., Volfova, O. and Kopeck, J. (1997) Characterisation of phytase produced by
Aspergillus niger. Folia Microbiology 42, 349–352.
Eeckhout, W. and dePaepe, M. (1994) Total phosphorus, phytate phosphorus and phytase
activity in plant feedstuffs. Animal Feed Science and Technology 47, 19–29.
Eeckhout, M., De Schrijver, M. and Vanderbeke, E. (1995) The influence of process
parameters on the stability of feed enzymes during steam-pelleting. In: van
Hartingsveldt, W., Hessing, M., van der Lugt, J.T. and Somers, W.A.C. (eds) Proceedings
of the 2nd European Symposium on Feed Enzymes. Noordwijkerhout, The Netherlands,
pp. 163–169.
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Esteve-Garcia, E., Brufau, J., Pérez-Vendrell, A., Miquel, A. and Duven, K. (1997)
Bioefficiency of enzyme preparations containing β-glucanase. Poultry Science 76,
1728–1737.
Gibson, K. (1995) The pelleting stability of animal feed enzymes. In: van Hartingsveldt, W.,
Hessing, M., van der Lugt, J.T. and Somers, W.A.C. (eds) Proceedings of the
2nd European Symposium on Feed Enzymes. Noordwijkerhout, The Netherlands,
pp. 157–162.
Inborr, J. and Bedford, M.R. (1994) Stability of feed enzymes to steam pelleting during feed
processing. Animal Feed Science and Technology 46, 179–196.
Jongbloed, A.W. and Kemme, P.A. (1990) Effect of pelleting mixed feeds on phytase activity
and the apparent absorbability of phosphorus and calcium in pigs. Animal Feed Science
Liu, B.-L., Rafiq, A., Tzeng, Y.-M. and Rob, A. (1998) The induction and characterization of
phytase and beyond. Enzyme and Microbial Technology 22, 415–424.
McCracken, K.J., Urquhart, R. and Bedford, M.R. (1993) Effect of heat treatment and
enzyme supplementation of barley-based diets on performance of broiler chicks. Proceed-
ings of the Nutrition Society 50A.
Pallauf, J. and Rimbach, G. (1997) Nutritional significance of phytic acid and phytase.
Archives of Animal Nutrition 50, 301–319.
Perez-Vendrell, A.M., Fish, N.M., Sabatier, A.M., Frapin, D. and Geraert, P.A. (1999a)
Thermostability of powder enzymes: in vitro recoveries and in vivo efficiencies.
Proceedings of the Australian Poultry Science Symposium. Australian Poultry Science,
Sydney, p. 172.
Perez-Vendrell, A.M., Brufau, J., Uzu, G. and Geraert, P.A. (1999b) Spraying enzyme before
or after fat coating: in vitro recoveries and in vivo efficiencies. Proceedings of the Australian
Poultry Science Symposium. Australian Poultry Science, Sydney, pp. 105–107.
Pettersson, D. and Rasmussen, P.B. (1997) Improved heat stability of xylanases. Proceedings
of the Australian Poultry Science Symposium. Australian Poultry Science, Sydney,
pp. 119–121.
Pickford, J.R. (1992) Effects of processing on the stability of heat labile nutrients in animal
feeds. In: Garnsworthy, P.C., Haresign, W. and Cole, D.J.A. (eds) Advances in Animal
Nutrition. Butterworth Heinemann, Nottingham, pp. 177–192.
Preston, C.M., Ellis, S., Bedford, M.R. and McCracken, K.J. (1999) Effects of feed enzymes
during heat processing on diet characteristics and subsequent broiler performance.
World’s Poultry Science Association in press.
Ravindran, V., Bryden, W.L. and Kornegay, E.T. (1995) Phytates: occurrence bioavailability
and implications in poultry nutrition. Poultry and Avian Biology Reviews 6, 125–143.
Silversides, F.G. and Bedford, M.R. (1999) Effect of pelleting temperature on the recovery
and efficiency of a xylanase enzyme in wheat based diets. Poultry Science 78, 1184–1190.
Simons, P.C.M., Versteegh, H.A.J., Jongbloed, A.W., Kemme, P.A., Slump, P., Bos, K.D.,
Wolters, M.G.E., Beudeker, R.F. and Verschoor, G.J. (1990) Improvement of
phosphorus availability by microbial phytase in broilers and pigs. British Journal of
Spring, P., Newman, K.E., Wenk, C., Messikommer, R. and Vukic Vranjes, M. (1996) Effect
of pelleting temperature on the activity of different enzymes. Poultry Science 75,
357–361.
Steen, P. (1999) Modern milling requires thermostabile granular enzymes. Feed Technology
3(3), 43–47.
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Vukic-Vranjes, M., Pfirter, H.P. and Wenk, C. (1994) Influence of processing treatment and
type of cereal on the effect of dietary enzymes in broiler diets. Animal Feed Science and
Wyss, M., Pasamontes, L., Rémy, R., Kohler, J., Kusznir, E., Gadient, M., Müller, F. and
vanLoon, A.P.G.M. (1998) Comparison of the thermostability properties of three acid
phosphatases from molds: Aspergillus fumigatus phytase, A. niger phytase, and A. niger pH
2.5 acid phosphatase. Applied and Environmental Microbiology 64, 4446–4451.
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Future Horizons
R.R. MARQUARDT1 AND
M.R. BEDFORD2
16
1Departmentof Animal Science, University of Manitoba,
Winnipeg, Manitoba, Canada R3T 2N2; 2Finnfeeds, PO Box 777,
Marlborough, Wiltshire SN8 1XN, UK
R.R. Marquardt
Future
16 Horizonsand M.R. Bedford
Introduction
To date, the use of exogenous enzymes in animal diets has had a great impact on the
livestock industry. They are routinely used to improve the nutritive quality of diets
containing cereals such as barley, wheat, rye and oats, especially for poultry, and as a
result they have improved the quality of the environment by reducing the output of
excreta and pollutants, such as phosphate and nitrogen, including ammonia. Since
enzymes tend to increase performance of animals fed low apparent metabolizable
energy (AME) cereals more so than those fed cereals with higher AME values, a
further benefit that results is improved animal uniformity within and between
experiments. In commercial situations this results in reduced variance between
flocks.
Enzymes have also been shown to decrease the size of the gastrointestinal (GI)
tract, which increases partitioning of nutrients into edible tissue. Their use has also
been shown to alter microbial fermentation, which again may affect the availability of
nutrients and the health status of the animal. Most of the research on enzymes as feed
additives has been with poultry but studies with pigs, especially young pigs, have also
demonstrated a positive response to enzyme supplements. The recent appearance on
the market of recombinant enzymes, especially phytase, should further accelerate the
adoption of enzymes by the feed industry. For some recent reviews on the use of
enzymes in the feed industry, see Annison and Choct (1991), Bedford (1995) and
Jeroch et al. (1995).
Although enzymes have proved to be highly beneficial, their use is still in its
infancy. Many problems need to be solved before their full potential is reached. The
purpose of this chapter is to highlight areas of further research that should enhance
not only the use but also the value of exogenous enzymes in animal nutrition.

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390 R.R. Marquardt and M.R. Bedford
Areas Requiring Additional Research

Improved enzyme assays
Of great concern to current users of enzymes is not only how to select an enzyme
from the multitude on offer, but also how to assay for the presence of the product in
complete animal feed. Currently, there is no single standard procedure for assessing
the quality or quantity of all commercial enzyme products, nor has a satisfactory assay
been developed for monitoring quantities of enzymes present in complete diets. If
xylanases are taken as an example, the current commercial products are derived from
at least four different genera of organisms. Even within one genus, some products
contain one, two, three or even more xylanases of differing characteristics (pH and
temperature optimum, substrate specificity, etc.). As a result it is not valid to assign
one assay for determination of the ‘relative activity’ of all products since any one
product can be made to appear superior by judicious choice of assay conditions.
A further problem is that, once added to a complete feed, some xylanases exhibit a
significant binding to the feed matrix, and so estimation of activity is difficult to
accomplish (Bedford, 1993).
Some of the commonly used enzyme assays include: measurements of the
reducing sugars liberated by the enzymes; the use of coloured substrates;
immunological methods, such as the enzyme-linked immunosorbent assay (ELISA);
and assays based on the ability of an enzyme to reduce the viscosity of water-soluble
non-starch polysaccharides (WSNSPs) (Cowan and Rasmussen, 1993; Hendon and
Walsh, 1993). Cowan and Rasmussen (1993) reported that, among the different
methods, the release of dye from dyed substrate is one of the easiest and most
sensitive methods but it is not sensitive enough to detect enzymes in feeds readily.
The ELISA procedure can detect enzymes in feeds, but antisera are not available
for all of the different enzymes, and ELISA sometimes shows a weak reaction to
inactivated enzymes. Measurement of the reducing-sugars liberated by enzymes in a
feed matrix is inaccurate because of the high background level of reduced sugars
in feed. A viscosity-based assay has been reported but its use is not common in
complete feeds (Vahjen et al., 1997). McCleary discusses the strengths and
weaknesses of each of these methods and others in more detail in Chapter 4. The
limitations of the current situation do point to a major goal for future research
which, if successful, will significantly improve the trust that feed manufacturers place
in enzymes. This goal is to produce a meaningful assay, or series of assays, that will
determine not only the amount of enzyme present in the complete feed, but also its
biological relevance. Clearly, there should be an association between the activity
determined in the feed and its activity in vivo.
Government regulatory bodies, enzyme manufacturers, the feed industry and
professional societies should therefore benefit from development of standardized
assays for feed enzymes. This is important, as standardized assays would: (i) allow the
manufacturers and purchasers of enzymes to compare enzymes based on their activity
values; (ii) enable livestock producers to determine the amount of active-enzyme
product that was added to a diet or the amount that survived the rigours of the
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Future Horizons 391
environment, including heat treatment during processing; (iii) provide a means of

assessing how well the enzymes survive in different sections of the GI tract, especially
in the section where they are most efficacious (the benefits of this are discussed in the
next section); and (iv) bear some relation to the physiological effects that the enzymes
are supposed to have (for example, it may be possible to run an assay at pH 8 and
60°C, but the results would be irrelevant to the feed industry).
Site of action of enzymes
Fundamental information is lacking on where in the GI tract enzymes produce most

of their beneficial effects. It is not known, for example, whether the main site of
action of enzymes in chickens is in the crop, proventriculus, gizzard, duodenum,
ileum, rectum, or all or part of the GI tract. Data presented in Chapter 15 suggest
that enzyme activity may be as much as 60–70% completed during the conditioning
process of a wheat-based diet at 85°C. Confirmation of such information may assist
in the selection of enzymes appropriate for the target substrate and the conditions at
the site where they are most efficacious. The type of enzymes selected for use in poul-
try, for example, could be very different if the major site of action turned out to be
the crop rather than the lower sections of the GI tract. The ability of the enzymes to
resist proteolysis, their optimal operating temperature (particularly if most activity
is expended during feed manufacture) and pH would be considerations for their
selection. Sites of activity may also vary considerably between animal species, since
the conditions in the GI tract vary considerably between pigs, poultry and ruminants.
As a result, whereas today most enzymes destined for use in pig diets are derived from
those used in products designed for poultry, it is likely that future ruminant, swine
and poultry products will be designed specifically for the requirements of the target
species. Indeed, differences clearly exist between young and older animals of the same
species, and within the grouping of poultry there are significant differences between
turkeys and chickens, for example. Determination of the sites and modes of action of
specific enzymes in each of these species will ultimately lead to better products that
are more cost efficient for the industry as a whole.
Production of new enzymes
Whilst the current generation of enzymes are highly beneficial, their usefulness
will undoubtedly be surpassed when new activities with greater activity are made
available. Sources other than those currently used, such as those produced by
microorganisms in the rumen of cattle, from thermophilic organisms or from other
extremophiles, will probably provide enzymes with the properties desired for
maximal activity. Such properties will include: (i) highly specific activities (substrate
turnover rates per unit of protein) under the conditions where they act; (ii) high
levels of resistance to inactivation by heat treatment, low pH and proteolytic
enzymes; (iii) low production costs; (iv) long shelf-life under ambient storage
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conditions; (v) lack of apparent binding to feed matrix to facilitate the quantitative
determination of enzyme in finished feed; and (vi) specificity to suit the purpose for
which they are designed.
Substrate and product identification
The focus so far has been on the developments in enzyme properties that are likely to
occur in the next few years. Without a doubt the advances will only be made possible
through better identification of the target substrates and the desired products of
enzyme activity. Such knowledge will permit more efficient, rapid and focused
development of enzymes that will have a biological effect, and will hopefully supplant
the current empirical approach that is largely used for product development.
Many researchers have hypothesized that the principal mode of action of most
xylanase and β-glucanase enzymes is the destruction of gel-forming polysaccharides
leached from cereal cell walls in amounts sufficient to depress performance (Annison
and Choct, 1991; Bedford, 1993; Chesson, 1993). Hesselman and Aman (1986)
proposed an alternative explanation: namely, that the β-glucans and arabinoxylans
that form the endosperm wall of cereals restrict the access of enzymes to nutrients.
They postulated that the disruption of intact walls and the release of entrapped
nutrients are the major factors in the improvement of the nutritive value of diets
with exogenous enzymes. Chesson (1993) postulated that a single enzyme should
be effective if the beneficial effects of enzymes are attributable solely to reduced
viscosity. The reason is that viscosity is partially a function of chain length and so it is
only necessary to break the chain in a few sites to reduce substantially or destroy its
gel-forming capacity. However, if the beneficial effects of added enzymes are due to
disruption of intact cell walls and release of entrapped nutrients, rather than reduced
viscosity, then many more enzymes would be required. Regardless of mechanism, it is
fair to say that the actual structure of the anti-nutritive factor in the intestinal lumen
is not known. If the structure were known, then in vitro work using such a substrate
would enable rapid screening of new enzyme candidates for use in new product
design. With modern discovery and mutagenic techniques it is possible to supply
many hundreds, if not thousands, of candidates for such screening methods.
High-throughput robotic methods allow many thousands of assays to be completed
in very short periods of time. As a result, the more faithful the screening method is
in reproducing the ‘real life’ conditions and substrate, the more likely it is that the
process will lead to provision of successful and cost-effective research.
Of further interest are the saccharides of various degrees of polymerization
released in the process of cell wall carbohydrate hydrolysis, i.e. the products of
enzymatic activity. Recent work discussed in this book is beginning to show that the
response to addition of cell wall hydrolysing enzymes is due in large part to changes
in the intestinal microfloral populations that accompany their use. The feeding of
a xylanase selects for ileal populations of bacteria that can utilize xylose and
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Future Horizons 393
concomitantly alters caecal populations dramatically (e.g. Apajalahti and Bedford,

1999). Such shifts can be seen as a response to the addition of the enzyme and
perhaps not a route through which they act on animal performance. Work with
germ-free animals, however, tends to suggest that the animal performance responses
observed on addition of enzymes may in fact be due to microfloral responses to the
alteration in the availability of a number of substrates. Some substrates, such as starch
and protein, are more rapidly digested in the presence of an effective enzyme and as a
result bacteria that rely on such substrates are less prevalent. Others, such as cell wall
oligo- and polysaccharides, increase in concentration and as a result populations that
can use such substrates thrive. The areas for future investigation must therefore
include a detailed understanding of the microflora of the gut. Desirable and
undesirable species need to be identified and their nutritional requirements
determined so that enzymes can be designed to supply the correct balance of
oligosaccharides and peptides for beneficial organisms, whilst at the same time deny
the pathogens their nutrition. Clearly such work is in its infancy but is likely to yield
significant benefits.
Raw material pretreatment
Whilst partial hydrolysis of cell wall NSPs reduces intestinal viscosity and may
accelerate digestion through permeation of endosperm cell walls, it is also likely that
in many cases there is some degree of complete hydrolysis to monosaccharides.
Glucose from cellulose and galactose from the α-galactosides can be readily utilized
by most animals, whereas the pentoses cannot be readily utilized by all classes of
livestock, especially poultry (Schutte et al., 1991, 1992). Most poultry, and even
swine to some degree, however, do not have a long enough digesta residence time to
enable complete cell wall hydrolysis. The development of raw material pretreatment
procedures for the complete hydrolysis of the hemicellulose fraction cereals, and
even less digestible feedstuffs such as straw, would be of great economic importance,
as it could provide a basis for using products that are present in vast quantities
but cannot be utilized to any appreciable extent by monogastric animals. The
pretreatment of high-phytate ingredients such as rapeseed meal and cottonseed meal
would allow a far greater degree of phytate hydrolysis than is currently achievable
in vivo. Pretreatment would yield the added benefit that the content of anti-nutrients
can be monitored throughout the process and controlled so that there is consistency
from batch to batch. Currently there is variation in efficacy of enzymes added to
complete feed, due to variations in dosage rate, survival through processing and
intestinal tract conditions. Control of enzyme addition rates, pH, temperature
and moisture during processing would likely increase uniformity of product and
hence of animal nutrition. Such procedures, in order to be economic, would need to
be at minimal cost, which inevitably means low moisture and temperature. Such
requirements set the challenge for enzyme discovery projects.
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Targeted enzymes
Knowledge of the exact conformation of the substrate to be degraded may confer

additional benefits to future enzyme discovery programmes. Often, the target may be
a protein, and as a result enzymes are selected on their ability to degrade the
anti-nutrient. Blanket administration of a such an enzyme to a complete diet has two
problems. The first is that the target protein substrate may represent only a few per
cent of the total protein in the diet. In order to be effective, large amounts of protease
need to be added in order to provide enough activity to seek out the desired target
and render it ineffective. In such a scenario, much of the total protein in the diet is
degraded to a lesser or greater extent compared with the target, the extent depending
upon its relative susceptibility to the enzyme. Whilst this may be of benefit in
speeding up digestion of protein in general, it may also produce bitter peptides in
the process, which are thought to decrease diet palatability. As a result, it would
be desirable if the added enzyme hydrolysed only the targeted protein and left other
proteins intact. Not only would the enzyme be more specific, but in all likelihood far
less would be needed. The development of such enzymes, sometimes referred to as
catalytic antibodies, is a key area of research. Once the identity of the substrate is well
characterized, this technology will lead to the production of antibodies with
enzyme-like properties designed to meet the requirements of specific feedstuffs and
classes of livestock and poultry (Lerner et al., 1992; Mayforth, 1993). Modifications
of these procedures will undoubtedly develop. Alternatively, antibodies could be
linked to specific proteases to concentrate the enzyme on its target substrate.
Alternative sources of enzymes
Enzymes are not only produced by fungi that have been improved using traditional
methods but also expressed in microorganisms, such as bacteria, and in plants,
such as in canola seed. Production in plants is seen as a far less costly method of
manufacture. An abundant supply of enzymes in canola seed, for example, would
dramatically reduce the cost of enzyme production and hence costs to the livestock
producer. Production of phytase in canola or xylanase in rye, if successful, would also
result in a higher value feedstuff. Whilst enzyme selection and discovery are still in
their infancy, however, it is likely that production techniques need to be able to react
rapidly to new discoveries. At present it is quicker to bring a new product to market
through fermentation methods than it is through production in plants. Once the
rate of improvement in next-generation enzymes slows down, and the base enzyme
activities effectively become commodities, then considerations of costs of production
will likely drive enzyme production through plant ‘biofactories’. Whilst emphasis
here is on reducing costs and economics, it must be born in mind that by reducing
costs the scope for enzyme usage increases dramatically. Ten years ago, xylanases were
too expensive to consider as viable additives in pig feed. Current prices have allowed
rapid expansion into this sector in recent years. Use of enzymes in ruminant feeds is
mostly constrained by the cost : efficacy ratio, a ratio that will improve over the next
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Future Horizons 395
few years and as a result lead to greater enzyme use in this, as yet, untapped area of
animal feed.
An alternative strategy for the hydrolysis of anti-nutritive compounds in animal
feeds is to construct transgenic monogastric animals able to digest cellulose,
β-glucans, xylans or phytic acid. Forsberg et al. (1993) concluded that it should be
feasible to use bacterial DNA constructs to express and secrete glycanases in rat
pancreatic acinar cell lines. The major challenge will be to obtain sufficiently high
levels of the expression of glucanase genes and other genes in pancreatic cells to effect
adequate hydrolysis of the glucans, xylans, etc., in the intestine. Again, such a strategy
would require that the enzymes of interest were commodities and not likely to
be improved upon by genetic manipulation. Only under these circumstances would
the gene inserted into the animal be competitive with the bacterial, fungal, or
plant-derived products freely available on the market at the time of introduction of
the animal to the marketplace. The animal breeders would have to be certain of such
a state of affairs, since the multiplication time required from germ-line breeding
stock to food-producing animals can take as long as 5 years, which is more than
sufficient time for development of superior products through fermentation methods.
Enzyme dosage and synergies
Most enzymes present on the market are simple xylanases, β-glucanases or phytases.
It is fair to say that whilst there are some data describing the dose–response
characteristics of many of these products, this relationship is for the most part poorly
described, particularly when compared with the data available for several essential
amino acids, for example. As a result the current usage of such products is far from
optimized. Dose-titration studies and linkage of the enzyme dose to the substrate
quantity present in any complete diet will certainly help to improve on the
economics of current enzyme usage. Zhang et al. (1996) recently showed that they
could predict the response of poultry to a particular feed enzyme by using a simple
linear model. The model predicts that the response to enzyme supplementation is a
function of enzyme concentration. When this is converted to a logarithmic value, a
doubling or halving of the response to enzyme treatment can be achieved only by
varying the amount of enzyme by a factor of 10, not 2 as may be expected. Clearly, as
the costs of feed and enzyme change in proportion to one another, the economically
optimum dose of enzyme will change also. The ability to match feed and enzyme
costs with substrate concentration and thus predict optimum enzyme dosage via
computer models is currently available in wheat- and barley-based rations. The intro-
duction of near-infrared techniques for prediction of substrate concentrations, hence
allowing for an ‘on-line’ enzyme dosage optimization at the level of the feed mill, is a
clear goal for optimizing the economy of enzyme usage. The advantages of titration
of the correct amount of enzyme are realized in terms of not only more cost-efficient
enzyme usage, but also greater uniformity of feed and hence animal production.
Nearly all the research done on the response of poultry to enzymes (with the
exception of phytase) has used crude fungal extracts with high levels of the desired
405
17 November 2000 15:30:02

activities, such as of β-glucanase or xylanase, but also with considerable amounts of

other side activities or contaminating enzymes, including proteases. Research carried
out using different combinations of pure enzymes (that is, devoid of contaminating
and potentially synergistic enzyme activities) has and will allow determination of
whether the principal enzymes have synergistic, antagonistic or additive effects. For
example, the ability of enzymes to reduce the viscosity of the water-soluble
arabinoxylans in wheat or rye may depend on the amount not only of endo-xylanase
but also of arabinofuranosidase, and possibly β-glucanase, acetylxylan esterase and
feruloyl esterase, in the preparation (Forsberg et al., 1993). Recent work suggests that
there may be synergies between phytases and xylanases (Ravindran et al., 1999).
Detailed descriptions of substrates will allow for more focused research into enzyme
mixtures based on likely synergistic interactions.
Studies with other animals
Although studies with chickens have clearly established the benefits of enzymes as
feed additives, only a limited number of studies have been carried out with other
species of poultry, such as turkeys, ducks, geese and ostriches. Also, very few studies
have been carried out using fish, eels, alligators, turtles, other exotic animals, pets
(such as dogs and cats) and fur-bearing animals that have simple stomachs. Enzymes
may be particularly beneficial in the diets of animals that tend to be carnivorous,
as they also tend to have GI tracts with smaller large intestines and therefore do
not house a large population of anaerobic microorganisms capable of hydrolysing
complex carbohydrates.
Conclusions
In the past several years, the use of enzymes as supplements in feeds has expanded
dramatically. The research that led to this was mostly carried out at universities and
research institutions and funded in large part by the industry, in cooperation with
government agencies. Although dramatic progress has been made in the past decade
on the use of enzymes in the diets of poultry and livestock, additional research will be
needed in many areas to exploit the full potential of this very powerful and beneficial
technology. Areas of research and development should include the following:
• Development of more sensitive, accurate and (most importantly) biologically
meaningful assays.
• Precise identification of the catalytic properties of enzymes most suited to
different classes of livestock and poultry through characterization of the
substrates and environments in which they reside.
• A better understanding of the effects that enzymes have on the physiological,
microbiological and endocrine responses of animals fed cereal-based diets.
406
17 November 2000 15:30:03

Future Horizons 397
• Development of methods to predict substrate concentrations and use of models

to predict responses to enzyme treatment and hence optimize economy and
efficacy of usage.
It is clear from the above that, in order to be successful, research into this field needs
to move from what has been a relatively simple approach to one that is more
multidisciplinary.
Whilst the roadmap for development of new products from a research viewpoint
seems clear, it is by no means clear when viewing the current climate of animal feed
production. Bovine spongiform encephalopathy, dioxins in fat sources, sewerage in
animal feed and, most importantly, the use of antibiotics in animal production have
in recent times increased consumer, regulatory and media interest in food production
processes. Genetically modified organisms are currently suffering acute attention
and rejection in some parts of the world. Such attention may well increase the time
required to introduce new products into some parts of the world, which serves as a
reminder that the identification, improvement and production of these products is
only one step in the process of their introduction into the marketplace.
References
Annison, G. and Choct, M. (1991) Antinutritive activities of cereal non-starch polysaccharides
in broiler diets and strategies for minimizing their effects. World’s Poultry Science Journal
47, 232–242.
Apajalahti, J. and Bedford, M.R. (1999) Improve bird performance by feeding its microflora.
World Poultry 15, 20–23.
Bedford, M.R. (1993) Matching enzymes to application. Feed Management 44, 14–18.
Bedford, M.R. (1995) Mechanism of action and potential environmental benefits from the use
of feed enzymes. Animal Feed Science and Technology 53, 145–155.
Cowan, W.D. and Rasmussen, P.B. (1993) Thermostability of microbial enzymes in expander
and pelleting processes and application systems for post-pelleting addition. In: Wenk, C.
and Boessinger, M. (eds) Enzymes in Animal Nutrition. Kartause Ittingen, Thurgau,
Switzerland, pp. 263–268.
Dänicke, S., Simon, O., Jeroch H. and Bedford, M.R. (1995) Effect of fat source and xylanase
supplementation on the performance and intestinal viscosity of rye-fed birds. In: van
Hartingsveldt, W., Hessing, M., van der Lugt, P. and Somers, W.A.C. (eds) Proceedings
of the Second European Symposium on Feed Enzymes, 23–27 October, Noordwijkerhout, The
Netherlands. TNO Nutrition and Food Research Institute, Zeist, The Netherlands,
pp. 102–107.
Forsberg, C.A.W., Cheng, K.-J., Knell, J. and Phillips, J.P. (1993) Establishment of rumen
microbial gene pools and their manipulation to benefit fibre digestion by domestic
animals. Proceedings, 7th World Conference on Animal Production, Edmonton, AB,
Canada. University of Alberta Press, Edmonton, Alberta, Canada, vol. 1, pp. 281–316.
Hendon, D.R. and Walsh, G.A. (1993) Activity analysis of enzymes under field conditions.
In: Wenk, C. and Boessinger, M. (eds) Enzymes in Animal Nutrition. Kartause Ittingen,
Thurgau, Switzerland, pp. 233–240.
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Hesselmann, K. and Aman, P. (1986) The effect of β-glucanase on the utilization of starch and
nitrogen by broiler chickens fed on low- or high-viscosity barley. Animal Feed Science and
Hutchison, C.A. III, Philips, S., Edgell, M.H., Gillam, S., Jahnke, P. and Smith, M. (1978)
Mutagenesis at a specific position in a DNA sequence. Journal of Biological Chemistry
253, 6551–6560.
Jeroch, H., Dänicke S. and Brufau, J. (1995) The influence of enzyme preparations on the
nutritional value of cereals for poultry: a review. Journal of Animal Feed Science 4,
263–285.
Lerner, R.A., Kang, A.S., Bain, J.D., Burton, D.R. and Barbas, C.F. III (1992) Antibodies
without immunization. Science 258, 1313–1314.
Mayforth, R.D. (1993) Designing Antibodies. Academic Press, San Diego, California, 207 pp.
Ravindran, V., Selle, P.H. and Bryden, W.L. (1999) Effects of phytase supplementation,
individually and in combination with glycanase, on the nutritive value of wheat and
barley. Poultry Science 78, 1588–1595.
Schutte, J.B., Van Leeuwen, P. and Lichtendonk, W.J. (1991) Real digestibility and urinary
excretion of D-xylose and L-arabinose in ileostomised adult roosters. Poultry Science 70,
884–891.
Schutte, J.B., de Jong, J. and Van Weerden, E.J. (1992) Nutritional implications of
L-arabinose in pigs. British Journal of Nutrition 68, 195–207.
Schutte, J.B., de Jong, J. and Langhout, D.J. (1995) Effect of endo-xylanase preparation in
wheat-based rations for broilers. Feed Compounder 15, 20–24.
Vahjen, W., Glaser, K., Froeck, M. and Simon, O. (1997) Non starch polysaccharide
hydrolyzing enzymes as feed additives – detection of enzyme activities and problems
encountered with quantitative determination in complex samples. Archives Animal
Ward, P.P. and Conneely, O.M. (1993) Using biotechnology to improve enzyme yields: from
DNA to the market place. In: Wenk, C. and Boessinger, M. (eds) Enzymes in Animal
Nutrition. Kartause Ittingen, Thurgau, Switzerland, pp. 17–21.
Zhang, Z., Marquardt, R.R., Wang, G., Guenter, W., Crow, G.H., Han, Z. and Bedford,
M.R. (1996) A simple model for predicting the response of chicks to dietary enzyme
supplementation. Journal of Animal Science 74, 394–402.
408
17 November 2000 15:30:04

Index
Index
Acetivibrio 14 insoluble, dye-labelled substrates
Alpha-galactosidase 89–91, 94–95
assay methods 97 interfering factors 91, 92–93, 94
Amylase RBB xylan 18
assay methods 95–97 reducing-sugar methods 87, 91–92,
engineering 329–330 100–101
pH and thermal stability 329–330 soluble, dye-labelled substrates 88–89,
three-dimensional structure 329–330 92–93
Amylopectin 109 Somogyi–Nelson 18
Amylose 109 viscometric methods 87–88, 91–92,
Antibiotic growth promoters 101–103
interaction with enzymes 192 see also individual enzymes, e.g.
Antibiotics 9, 301, 307, 308 β-glucanase; Xylanase
Antigenicity
effects of enzymes on, in soybean
132–133 Bacterial metabolites 300
Arabinan 12 Bacterial proliferation
Arabinofuranosidase 14 effects of enzymes 162–163, 185–186
Arabinoxylans 5, 13 Barley 5, 7
effects in poultry 147–148 amylose : amylopectin ratio 167
properties 91 effects of enzymes in pigs 166–169,
see also Xylans 170–175
Aspergillus 14 effects of enzymes in poultry 145–146
Assay methods for enzymes energy variation in pigs 166–167
agar plate diffusion 88 prediction of energy value for pigs 169
Avicel 15, 23 Beef cattle
coloured substrates 18 response to enzymes 277
Congo Red 18 Bile salts 203
DNS 18 deconjugation 203
ELSA (enzyme-linked sorbent) 95 effects of NSPs on 152
general 16, 17 Bioavailability of phosphorus 239
399
409
17 November 2000 15:30:05
A3882:Bedford:AMA:First Proof: 04/09/00 CHAP-Index

400 Index
Calcium to phosphorus ratio 70 response to enzymes 278

Caloric efficiency Diet digestibility
influence of enzyme addition 226 influence on intestinal microflora 302
Catalytic mechanisms 31–33, 34–35 Dietary fat
Cell wall influence on gut morphology 211
degradation 282, 285 Digestion
encapsulation of nutrients-effects of efficiency 4
enzymes on 151 simulation methods 275
Cellobiohydrolase 19 variability 4
assay 15 Directed evolution 319, 326, 330–331
mode of action 22–23 Disease
stereospecificity 28–29 role of non-starch polysaccharides and
Cellulase 11, 316–317 enzymes
adsorption 38 colitis in pigs 191–192
applications 45 swine dysentery 190–191
architecture 39 DNA
assay 15 expression and overexpression 333
catalytic domain 41 ligase 333
fungal vs. bacterial sources, properties promotors 333
86 restriction endonuclease 333
mode of action 22–24 transformation 333
physicochemical properties 37 Dockerin 24
regulation 33, 36
synergy with CBH 28
three-dimensional structure 41, 42, 43 Emulsification of fat 200, 209
variety of forms 37–38 Endocrine system
Cellulose 11, 12, 14 effect of enzymes on 151
structure 12 Endogenous losses
Cellulose binding domain 24, 38, 39, 40 effects of NSPs and enzymes 150–152
Cellulosomes 24, 285 Endoglucanase 19
effect on bacterial adhesion 285 stereospecificity 28–29
Cereal Energy
growing conditions 5 availability from cereal 110
variety 5 Enzyme
Chelators activity
value in phytic acid rich diets 65 during feed processing 380
Citric acid 73, 257 effect of substrate concentration
Classical mutagenesis 331–332 275–276
Clostridium 14 of exogenous in small intestine 289
Coccidiostats 307 influence of amino acid changes 326
Copper 307 adsorption 38
Corn see Maize assays 390
Cottonseed meal-effects of enzymes on 136 augmentation of ruminal activity 284
characteristics of value to feed industry
391–392
Dairy cattle classes 316
influence of enzyme activity 278–279 concentration from broth 345
influence of method of enzyme crystallization 346
application 278 dosage 395
410
17 November 2000 15:30:06

Index 401
dose–response 279 elimination of need to culture source

expression in animals 395 organism 318
expression in plants 291, 394 substrate ratio 275
factors influencing recovery from survival in rumen 283
fermentation broth 320, 340 synergy 28–31, 395
families 34–35 exogenous with endogenous 284
and fat three-dimensional structure effect on
influence on broiler performance activity 327–331
and digestion 214–217, 227, uses in industry 315
228 Enzymes (exogenous)
influence on rate of passage in survival in the gut 130
ruminants 287 Esterase 285
influence on substrate availability to Exogenous enzyme
microflora 302 influence on endogenous enzymes 284
inhibition 21 as proportion of endogenous 284
interaction with antibiotics 308 Exoglucanase 19
kinetics 20 see also Cellobiohydrolase
liquid formulation 346–347 Expression vector 334
market size 3, 4, 6, 8 Extrusion
mediated provision of substrate for effect on enzyme activity 380
microflora 303–304
methods for targeting to substrate 394
mode of action 392 Faba beans
multiple forms 317 effects of proteases on 134
non catalytic effects in ruminant 286, Faecal moisture
287 effects of enzymes 149–150, 192
post-ruminal effects 288 Faeces decomposition by enzymes 289
pre-processing of raw materials 393 Fat digestibility
processing from fermentation broth effects of NSPs on 152
343–344 influence of barley, fat source and
products 25 β-glucanase 221
and effects 392–393 influence on fat soluble vitamin storage
purification 345–346 229
response influence on pigmentation 229
factors involved 307 influence of rye, fat source and xylanase
influence of dietary ingredients 277 221, 222, 223
screening 321–325, 332 influence of wheat, fat source and
enrichment techniques 321 xylanase 222
factors limiting discovery 318 Fat source
molecular techniques 324, 325, effect on intestinal protein turnover
330–331 227
limitations 325 effect on protein and amino acid
organism identification by digestibility 225
sequencing techniques 323, 324 fatty acid composition 202
redundancy 322 interaction with cereal 199
selective environments/criteria 321 Fatty acid binding protein 200
site of action 391 Fatty acid digestibility
solid formulation 347 influence of cereal, fat source and
sources 274 enzyme 221–224
411
24 November 2000 16:45:08

402 Index
Fatty acids β-Glucanase

degree of saturation, effects on assay methods 85–91
digestibility 201 correlation of in feed activity with in
site of absorption 224 vivo performance 381
Feed passage rate effects of coating on
effects of enzymes and fat type 207, thermostability 381
208 microgranulate thermostability 380
influence of viscosity 206 recovery in feed 381
Feed processing 377, 383 thermostability 380
effect on intestinal viscosity 384 Glucomannan 12
effect on nutrient availability 384 β-Glucosidase 19
implications for enzymes 353–354 assay 15
Feed production 1, 2, 3 α-Glucuronidase 14
Fermentation Grain
bacteriophage control 342–343 by-products (rice and wheat)
common substrates 341 effects of enzymes in pigs 188–190
control 337, 338, 339 extract viscosity
inhibitors 341–342 as predictor of enzyme response in
maximizing yield 337, 338, 341 poultry 154
preparation of fermentor 342–343 Granulation 347
process 320, 337–340 Grinding of cereals 120
scale-up 340–341 Grist size 120
separation of cells from enzymes Gut
344–345 effects of NSPs on the mucosa 150
types 338 microflora
Fibre digestion effects of NSPs on 151–152
pH effect 286, 287 transit time
role of surfactants 287 effects of enzymes 152, 163–165
Fibrobacter 14
Fibrolytic activity post rumen 288
Filtration 344–345 Hammer milling see Grinding of cereals
Fines in pellet production Hemicellulase 11
implications for liquid enzyme Hemicellulose 11, 12
application 356 Hind gut fermentation 119
Formic acid 257 Humicola 14
Fractional precipitation 345–346
Fructo-oligosaccharides 307
Fungi 14 Immune system 307
Fusarium 14 Immunochemical techniques for protease
detection 130
In-feed activity of enzymes 280
Galactan 12 Intestinal
Gastric digestion damage
effects on enzyme function 162 microbial involvement 211
Gene deletion 334–337 microbial populations
Germ free chickens 301 influence of enzyme and fat type 213
β-Glucan 5 microflora
properties 85, 145–146 adaptation to diet 305
412
17 November 2000 15:30:07

Index 403
changes with age 301 effect of fertilizers on nutritive quality

effect on bile acids and fat digestion 121, 122
305 flint 109
effects on gut physiology 306 floury 116, 117
interactions between species 300 genetic modification 122
numbers 299 germ 113
response to viscosity 302 high oil varieties 113, 114, 115, 116
phytase 241 high protein varieties 116, 117
substrates 300 ileal digestible energy 118
viscosity immunity 122
ruminants 288 low phytic acid varieties 120
see also viscosity normal 109
Intestines nutrient composition 111, 112
environment 299–300 nutritive value 114, 115, 116
oil 113
opaque 116, 117
Lactic acid 257 protein 112, 116
Lambs 279 effects on starch digestion in
Lectins 5 ruminant 290
protease effects on 131 true metabolizable energy 118
Lichenase function 86 use of enzymes 119
Lipase in turkeys 204 waxy 109, 118
Liquid enzymes 9 Manure 5, 6
application systems 355–363 Marker genes 333
coefficient of variation in different Meat consumption 2
application systems 367–371 Micelles 201
desired droplet size at spraying Mineral soaps 204
370–371 Mode of action of enzymes
dilution and application rate 371–372 ruminants 280–281
dosing pumps 365 Mortality 119
flow meters 364 influence of fat source and enzyme
handling 374 addition 228
importance of dosing accuracy 375
pre- or post fat addition 372–373
stability in storage 373 Neocallimastix 14
Liquid feeding of pigs NIRS (near-infrared spectroscopy)
application of enzymes 138 prediction of raw material feeding
Lupins value 169
effects of enzymes on 136
Oligosaccharides 307
Maize 6
amino acids 116
amylose : amylopectin ratio 187 Particle size 120
breeding 113, 122 Penicillium 14
bushel weight 111, 112 Phenyl esterase 14
dent 109 Phytase
digestibility 118 animal sources 67
effects of enzymes in pigs 185–190 Aspergillus, structure 69
413
17 November 2000 15:30:07

404 Index
Phytase continued site of activity in animal 243

assay methods 97–98 sources 67, 68
brush border see Phytase, intestinal temperature sensitivity 378, 379
competitive chelation 65, 73, 76 thermostability 69
dose–response relationship with P of plant sources 379
availability 246 through coating 379
effect of Ca on activity 75 units of activity 241
effect on units equivalence for Ca and P
Ca availability 251 replacement 76
Ca and P digestibilty 76 variability in response 76
energy metabolism 255 Phytic acid
phosphorus availability 244–245 binding of minerals 239
phosphorus pollution 75, 246–248 complexes with metal ions 62, 63, 70
protein and amino acid availability content in feed ingredients 63, 238
252–255 digestion and utilization 66
protein and amino acid digestibilty effect of calcium on digestibility 70
75 effect on
zinc availability 252 digestive enzyme activity 66
factors influencing response 250–251, mineral utilization 64–65
254, 256 phosphorus pollution 64
in fish diets 259 protein digestibility 65–66
in genetically modified plants 74, germination effects on content 72
242–243 hydrolysis pattern 67
influence of location in ingredients 238
age on response 258 pKa values 62
Ca to P ratio on activity 256 selection for low phytate varieties 72
feed matrix on thermostability 379 solubility of complexes 63
interaction with structure 62, 238, 240
EDTA see Phytase, competitive Phytin 63
chelation location in feed ingredients 63–64
organic acids 257 Pig vs. poultry-comparative responses to
phosphatases 67 enzymes 129–130, 161–165
vitamin D 256 Piromyces 14
intestinal 67, 70, 71 Production organisms 319
bacteria 71 Protease 5
in laying hen rations 250 assay methods 98–100
microbial 68 bacterial vs. fungal, efficacy 131
sources 242 effects on
optimum animal performance 131, 133
conditions 71 soybean 130–134
temperature 378 Protein engineering 318–319, 326–331
pH optima 241, 259
phosphorus equivalence values
246–249 Random mutagenesis 319
plant 68, 70, 71 Rapeseed
sources 241 antinutrients 127–128
pretreatment of plant meals 72–73 effects of enzymes on 135
safety 243 Regulatory authorities 347–348
site of action in animal 69–71 Rennet 3
414
17 November 2000 15:30:08

Index 405
Rice bran see Grain, by-products energy variation in pigs 176

Ruminal effects of enzymes 282 Trypsin Inhibitor 5
Ruminal infusion 280 protease effects on 131
Ruminococcus 14
Rye
effects of enzymes in pigs 176–185 Uniformity
effects of enzymes in poultry 146 body weights 119
Unstirred water layer 208
Scaffoldin 24
Soaking of cereals Vegetable protein meals
effect on P availability 258 antinutrients and their elimination
influence on phytase response 258 125–128, 134
Sorghum-effects of enzymes in pigs effects of enzymes on animal
186–190 performance 135–136
Soybean meal see also individual raw materials, e.g.
antinutrients and their elimination Soybean meal, Rapeseed
130 Viscosity 5, 204–205
effects of proteases 130–134, 138 changing effect on fat digestibility with
Sporotrichum 14 age 220
Stabilization of enzymes 320 effect of fat type, enzyme and rye
Starch 109 concentration 218
granule 110 effect on
resistant 110 bile salt metabolism 210
Substrate fat digestibility 219–221, 224
availability lipase activity 209–210
influence on microflora 301 pancreatic lipase secretion 209
identification of relevant target for new individual bird variability 219
enzymes 392 influence of
influence of enzyme activity 290 concentration of solute 205
Substrates 16, 17 molecular weight of solute 205
Subtilisin influence on
effects of amino acid substitution on gut morphology 211
activity 328 maintenance energy 225–226
three-dimensional structure 327–328 protein and amino acid digestibility
turnover number 328 224, 225
relevance for microflora 301
Viscosity in the gut
Talaromyces 14 physiological effects in poultry and pigs
Thermomyces 14 148–151, 164
Thermostabilizing domains 39 pig vs. poultry
Thermostability 9 implications for enzymes 163–164
Thermostable phytase 378–379 as predictor of enzyme response in
Transferase reactions 21 poultry 153–154
Trichoderma 14 Vitamin D
Triglycerides influence on phytic acid digestibility
fatty acid positional effects 203–204 74
Triticale Voluntary food intake-effects of enzymes
effects of enzymes in pigs 176–185 164–165, 177
415
17 November 2000 15:30:08

406 Index
Water holding capacity of the feed-effects of family 10 25–27

enzymes 165–166, 176 family 11 25–27
Wet feeding systems and fat type
opportunity for phytase 259 influence on protein metabolism 212
Wheat 5 influence of feed matrix on
effects of enzymes in pigs 169–185 thermostability 382
energy variation influence on
in pigs 169 endogenous N losses 227
in poultry 146–147, 154–155 lipase activity 210
starch digestibility 154–155 mode of action 22–25
varietal effects on feeding value in pigs physicochemical properties 37
169, 176 post-pelleting application 382
Wheat bran see Grain, by-products recovery in feed 381
White kidney bean (Phaseolus vulgaris) regulation 33, 36
effects of enzymes on 135 stabilization 382
synergy with other activities 30–31
thermostability 381, 382
Xylanase 12, 13, 14, 19 three-dimensional structure 41, 43, 44
antisynergy 31 variety of forms 37–38
applications 45 Xylans 13
assay methods 91–95, 103–105 Xylo-oligomers 304
catalytic domain 41 β-xylosidase 14
correlation of in feed activity with
in vivo performance 382–383
effects of coating 382, 383 Zinc 307
416
17 November 2000 15:30:09

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ENZYMES IN FARM ANIMAL NUTRITION

A3882:Bedford:AMA:First Proof: 04/09/00 CHAP-

A3882:Bedford:AMA:First Proof: 04/09/00 CHAP-

ENZYMES IN FARM ANIMAL

A3882:Bedford:AMA:First Proof: 04/09/00 CHAP-

CABI Publishing is a division of CAB International

CABI Publishing CABI Publishing

Tel: +44 (0)1491 832111 Tel: +1 212 481 7018

Library of Congress Cataloging-in-Publication Data

SF98.E58 E59 2000

ISBN 0 85199 393 1

Typeset in Garamond by AMA DataSet Ltd

A3882:Bedford:AMA:First Proof: 04/09/00 CHAP-

1. The Current Feed Enzyme Market and Likely Trends 1

2. Enzymology and Other Characteristics of Cellulases and Xylanases 11

3. Enzymatic Characteristics of Phytases as they Relate to Their Use in

4. Analysis of Feed Enzymes 85

5. Maize: Factors Affecting its Digestibility and Variability in its

6. Vegetable Protein Meals and the Effects of Enzymes 125

7. Enzyme Supplementation of Poultry Diets Based on Viscous Cereals 145

8. The Role and Efficacy of Carbohydrase Enzymes in Pig Nutrition 161

A3882:Bedford:AMA:First Proof: 04/09/00 CHAP-Contents

9. Interaction between Cereal Identity and Fat Quality and Content in

10. Digestion of Phosphorus and Other Nutrients: the Role of Phytases

11. Enzymes in Ruminant Diets 273

12. Microbial Interactions in the Response to Exogenous Enzyme

13. Enzymes: Screening, Expression, Design and Production 315

14. Liquid Application Systems for Feed Enzymes 353

15. Process Stability and Methods of Detection of Feed Enzymes in

16. Future Horizons 389

A3882:Bedford:AMA:First Proof: 04/09/00 CHAP-Contents

J. Apajalahti, Danisco-Cultor Innovation, Kantvik, FIN-02460, Finland

A3882:Bedford:AMA:First Proof: 04/09/00 CHAP-Contributors

E.T. Kornegay (deceased), Department of Animal and Poultry Sciences, Virginia

A3882:Bedford:AMA:First Proof: 04/09/00 CHAP-Contributors

A3882:Bedford:AMA:First Proof: 04/09/00 CHAP-Preface

A3882:Bedford:AMA:First Proof: 04/09/00 CHAP-Preface

The Current Feed Enzyme

Global Animal Feed Production

A3882:Bedford:AMA:First Proof: 04/09/00 CHAP-1

1998 production 1999 production Percentage difference

North America 159.8 160.7 +0.6

A3882:Bedford:AMA:First Proof: 04/09/00 CHAP-1

The Current Market and Likely Trends 3

Sector 1993 (%) 1998 (%)

Background to Enzyme Technology

A3882:Bedford:AMA:First Proof: 04/09/00 CHAP-1

Why use enzymes in animal feeds?

A3882:Bedford:AMA:First Proof: 04/09/00 CHAP-1

The Current Market and Likely Trends 5

A3882:Bedford:AMA:First Proof: 04/09/00 CHAP-1

Phytic acid-degrading enzymes

The Current Feed Enzyme Market

A3882:Bedford:AMA:First Proof: 04/09/00 CHAP-1

The Current Market and Likely Trends 7

A3882:Bedford:AMA:First Proof: 04/09/00 CHAP-1