Beverages 02 00034 v2

beverages
Review
Saccharomyces species in the Production of Beer
Graham G. Stewart
The International Centre for Brewing and Distilling, Heriot-Watt University, Riccarton, Edinburgh EH14 4AS,
Scotland, UK; Profggstewart@aol.com
Academic Editor: Edgar Chambers IV

Received: 20 October 2016; Accepted: 25 November 2016; Published: 2 December 2016
Abstract: The characteristic flavour and aroma of any beer is, in large part, determined by the
yeast strain employed and the wort composition. In addition, properties such as flocculation, wort
fermentation ability (including the uptake of wort sugars, amino acids, and peptides), ethanol and
osmotic pressure tolerance together with oxygen requirements have a critical impact on fermentation
performance. Yeast management between fermentations is also a critical brewing parameter. Brewer’s
yeasts are mostly part of the genus Saccharomyces. Ale yeasts belong to the species Saccharomyces
cerevisiae and lager yeasts to the species Saccharomyces pastorianus. The latter is an interspecies
hybrid between S. cerevisiae and Saccharomyces eubayanus. Brewer’s yeast strains are facultative
anaerobes—they are able to grow in the presence or absence of oxygen and this ability supports
their property as an important industrial microorganism. This article covers important aspects of
Saccharomyces molecular biology, physiology, and metabolism that is involved in wort fermentation
and beer production.
Keywords: amino acids; ethanol; fermentation; malt; peptides; Saccharomyces species; sugars;
wort; yeast
1. Introduction
The flavour and aroma of any beer is, in large part, determined by the yeast strain employed
together with the wort composition. In addition, yeast properties such as flocculation, fermentation
ability (including the uptake of wort sugars, amino acids, small peptides, and ammonium ions),
osmotic pressure, ethanol tolerance, and oxygen requirements have a critical impact on fermentation
performance. Proprietary strains, belonging to individual breweries, are usually jealously guarded
and conserved. However, this is not always the situation. In Germany, for example, most of the
beer is produced with only four individual yeast strains and approximately 65% of it is produced
with a single strain sourced from the Weihenstephan Brewing School, which is located in the Munich
Technical University.
No satisfactory definition of yeast exists that encompasses commonly encountered properties
such as alcoholic fermentation and growth by budding. The latter is not unusual in yeast and nearly
all brewer’s yeast strains multiply by budding (Figure 1). Brewer’s yeast cultures predominantly
come from the genus Saccharomyces—a minority of non-Saccharomyces yeast cultures employed in
brewing will be discussed in Section 8. Yeast is cultured in an acidic aqueous sugary solution called
wort prepared from barley malt and other cereals such as corn (maize), wheat, rice, sorghum, and
also cane and beet sugar. The cells absorb dissolved sugars, simple nitrogenous matter (amino
acids, ammonium ions, and small peptides), vitamins, and ions through their plasma membrane.
Subsequently, they employ a series of reactions known as metabolic pathways (glycolysis, biosynthesis
of cellular constituents, etc.) and use these nutrient materials for growth and fermentation. It is
important to emphasise that the primary products of glycolysis are: ethanol, glycerol, and carbon
dioxide (Figure 2).
Beverages 2016, 2, 34; doi:10.3390/beverages2040034 www.mdpi.com/journal/beverages

Beverages 2016, 2, 34 2 of 18
Beverages 2016, 2, 34 2 of 18
Beverages 2016, 2, 34 2 of 18
Beverages 2016, 2, 34 2 of 18

Figure 1. An electron micrograph of a budding yeast cell containing bud and birth scars.

D-Glucose
D-Glucose ATP
Glucose 6-phosphate ADP
ATP
Fructose 6-phosphate
D-Glucose ATP
Fructose 6-phosphate ATP
Fructose 1, 6-diphosphate ADP
ATP
Glyceraldehyde 3-phosphate
Fructose 6-phosphate ATP
Glyceraldehyde 3-phosphate
Dihydroxy-acetone phosphate
+
Glycerol Dihydroxy-acetone phosphate NAD
Glyceraldehyde 3-phosphate NADHNAD+ 2
Glycerol 1, 3-Diphosphoglycerate NADH
Dihydroxy-acetone phosphate ADP 2
1, 3-Diphosphoglycerate
3-Phosphoglycerate NAD+
ATP
ADP
Glycerol
3-Phosphoglycerate NADH
ATP
2-Phosphoglycerate
1, 3-Diphosphoglycerate H2 O 2
2-Phosphoglycerate ADP
H
Phosphoenolpyruvate 2O
3-Phosphoglycerate ADP
ATP
Phosphoenolpyruvate
Pyruvate ATP
ADP
2-Phosphoglycerate H2 O CO2
Pyruvate
Acetaldehyde ATP
Phosphoenolpyruvate NADH2 CO2
Acetaldehyde ADP+
NAD
Ethanol NADH
Pyruvate ATP+ 2
NAD CO2

Ethanol
Acetaldehyde
Figure 2. Formation of ethanol, glycerol, and CO2 from glucose NADH by 2 the Embden‐Meyerhof‐Parnas
NADby +
Figure 2. Formation of ethanol, glycerol, and
(EMP) Pathway. CO2 from glucose
Ethanol the Embden‐Meyerhof‐Parnas
Figure 2. Formation of ethanol, glycerol, and CO2 from glucose by the Embden-Meyerhof-Parnas
(EMP) Pathway.
(EMP) Pathway.
Figure 2. Formation of ethanol, glycerol, and CO2 from glucose by the Embden‐Meyerhof‐Parnas
Saccharomyces cerevisiae (ale yeast) has the ability to take up a wide range of sugars, for example,
(EMP) Pathway.
glucose, fructose, mannose, galactose, sucrose, maltose, maltotriose, and raffinose (in part). In
Saccharomyces
glucose, fructose, cerevisiae (alegalactose,
mannose, yeast) hassucrose,
the ability to take
maltose, up a wide and
maltotriose, range of sugars,
raffinose
addition, as will be described later, a sub‐species of S. cerevisiae, Saccharomyces diastaticus, is able to (in for example,
part). In
glucose, fructose, mannose, galactose, sucrose, maltose, maltotriose, and raffinose (in
addition, as will be described later, a sub‐species of S. cerevisiae, Saccharomyces diastaticus, is able to
utilise dextrins (partially hydrolysed starch). Also, Saccharomyces pastorianus (lager yeast) strains are part). In addition,
glucose, fructose, mannose, galactose, sucrose, maltose, maltotriose, and raffinose (in part). In
utilise dextrins (partially hydrolysed starch). Also, Saccharomyces pastorianus (lager yeast) strains are
as will be described later, a sub-species of S. cerevisiae, Saccharomyces diastaticus, is able to utilise dextrins
able to utilise the disaccharide melibiose (glucose—galactose) in addition to the sugar spectra taken
addition, as will be described later, a sub‐species of S. cerevisiae, Saccharomyces diastaticus, is able to
(partially hydrolysed starch). Also, Saccharomyces pastorianus (lager yeast) strains are able to utilise the
up by S. cerevisiae. This melibiose utilisation property can be used in diagnostic tests to distinguish
utilise dextrins (partially hydrolysed starch). Also, Saccharomyces pastorianus (lager yeast) strains are
disaccharide
between melibiose
ale (glucose—galactose)
and lager in addition
yeast strains. The enzymatic to the sugar
hydrolysis spectra
of starch, as taken
would by S.during
upoccur cerevisiae.
between
Thismashing
melibiose ale and 3),
(Figure lager
utilisation yeast
leads to
property strains.
a can The
fermentable
be usedenzymatic
medium
in hydrolysis
(wort)
diagnostic of consists
that
tests to starch, of
as a
distinguish would
number
between occur
of
ale during
simple
and lager
mashing (Figure 3), leads to a fermentable medium (wort) that consists of a number of simple
sugars—glucose, fructose, sucrose, maltose, and maltotriose together with unfermentable dextrins.
yeastbetween
strains. ale
Theand enzymatic hydrolysis
lager yeast strains. ofThe
starch, as would
enzymatic occur during
hydrolysis mashing
of starch, (Figure
as would 3), leads
occur during to a
The predominant sugars in most brewer’s worts are: glucose, maltose, and maltotriose (Table 1) [1].
fermentable
mashing medium (wort)
(Figure 3), leads that
to a consists of a number
fermentable medium of simple
(wort) sugars—glucose,
that fructose,
consists of a number sucrose,
of simple
maltose, and maltotriose together with unfermentable dextrins. The predominant sugars in most
Malt Adjunct Hot Wort Tank
brewer’s worts are: glucose, maltose, and maltotriose
(rice, corn, wheat) (Table 1) [1].
Malt Adjunct Hot Wort Tank
Mill (rice, corn, wheat)
Plate Cooler
Mill Cereal Cooker
Malt Adjunct Plate Cooler
Hot Wort Tank
Cereal Cooker
(rice, corn, wheat)
Mash Mixer Yeast + 02
Mill
Mash Mixer Yeast
Plate + 02
Cooler
Cereal Cooker
Syrup Hops Fermenter
Lauter Tun or
MashFilter
Mash Mixer Syrup Hops Fermenter
Yeast + 02
Lauter Tun or
Mash Filter Kettle Centrifuge
Spent Grain Syrup Hops Centrifuge
Fermenter
Lauter Tun or Kettle
Spent Grain Aging
Mash Filter
Aging
Packaging Bright Beer
Kettle Carbonation Centrifuge
(bottle,
Spentcan,Grain
keg) Tank Filtration
Packaging Bright Beer
(bottle, can, keg) Tank Carbonation Filtration
Aging

PackagingFigure 3. The typical brewing process.
Bright Beer
(bottle, can, keg) Tank Carbonation Filtration
Figure 3. The typical brewing process.

Beverages 2016, 2, 34 3 of 18
Beverages 2016, 2, 34 3 of 18
Table 1. Typical sugar spectrum of wort.

Table 1. Typical sugar spectrum of wort.
Percent Composition
Percent Composition
Glucose
Glucose 10–15 10–15
Fructose
Fructose 1–2 1–2
Sucrose
Sucrose 1–2 1–2
Maltose
Maltose 50–60 50–60
Maltotriose
Maltotriose 15–20 15–20
Dextrins
Dextrins 20–30 20–30
2. The Malting and Brewing Processes and Wort Composition
2. The Malting and Brewing Processes and Wort Composition
The beer production process is outlined in Figure 3. Malting, mashing, and fermentation/aging
The beer production process is outlined in Figure 3. Malting, mashing, and fermentation/aging
are essentially enzymatic
are essentially enzymatic processes.
processes. The
The major
major brewing
brewing rawraw material
material is malt
is malt usually
usually (not always)
(not always) from
from barley and it contains extract components (starch, proteins, etc.) and enzymes
barley and it contains extract components (starch, proteins, etc.) and enzymes (amylases, proteases, (amylases,
proteases,
glucanases,glucanases, etc.).
etc.). However, However,
malt malt is raw
is an expensive an expensive
material and raw material cereals
unmalted and unmalted
(adjuncts)cereals
[2] are
(adjuncts) [2] are extensively used in most brewing countries.
extensively used in most brewing countries.
The
The exception
exception to to this
this use
use of of adjuncts
adjuncts is
is Germany,
Germany, where
where the
the Purity
Purity Law
Law (also
(also called
called the
the
Reinheitsgebot),
Reinheitsgebot), was was adopted
adopted in in Bavaria in 1516.
Bavaria in 1516. As
As Germany
Germany unified,
unified, Bavaria
Bavaria campaigned
campaigned for for
adoption
adoption ofof this
this law
law nationally.
nationally. TheThe
onlyonly ingredients
ingredients that be
that could could
usedbe used in Germany
in Germany for the
for the production
production of beer were water, barley, and hops. The principal purpose of the law was to prevent
of beer were water, barley, and hops. The principal purpose of the law was to prevent price competition
price competition with bakers for wheat and rye and to ensure the availability of bread [3]. When
with bakers for wheat and rye and to ensure the availability of bread [3]. When yeast was described by
yeast was described by Antonie van Leewenhoek in 1680 and its role in fermentation was described,
Antonie van Leewenhoek in 1680 and its role in fermentation was described, it was added to modern
it was added to modern versions of the Reinheitsgebot. It is worth noting that Norway and Greece
versions of the Reinheitsgebot. It is worth noting that Norway and Greece also commonly subscribe to
also commonly subscribe to this law [2].
this law [2].
The objectives of malting are to develop a spectrum of enzymes in cereals such as barley and
The objectives of malting are to develop a spectrum of enzymes in cereals such as barley and
wheat. These enzymes are able to hydrolyse the cereal constituents in order to produce a fermentable
wheat. These enzymes are able to hydrolyse the cereal constituents in order to produce a fermentable
extract called wort (Figure 4). As already discussed, wort is a medium that will support yeast growth
extract called wort (Figure 4). As already discussed, wort is a medium that will support yeast growth
and
and fermentation
fermentation with
with beer
beer as
as the
the end
end product.
product. It It is
is important
important that
that beer
beer is
is drinkable
drinkable (beer
(beer is
is not
not
usually supped, it is drunk!) and it exhibits a number of stability properties, i.e., flavour, physical,
usually supped, it is drunk!) and it exhibits a number of stability properties, i.e., flavour, physical,
foam, and biological characteristics [4]. Malt contributes a large number of materials to wort [5]. The
foam, and biological characteristics [4]. Malt contributes a large number of materials to wort [5].
principal wort components (not the only ones) are free amino nitrogen (FAN), fermentable sugars,
The principal wort components (not the only ones) are free amino nitrogen (FAN), fermentable sugars,
and unfermentable dextrins.
and unfermentable dextrins.
Barley
Steeping
Grain hydration
Germination
Enzyme generation
Kilning
Drying and curing
Malt

Figure 4. Typical barley malting process.
Figure 4. Typical barley malting process.
Compared to other media used in the production of fermentation alcohol (both industrial and
Compared to other media used in the production of fermentation alcohol (both industrial and
potable), wort is by far the most intricate. Therefore, when yeast is pitched (inoculated) into wort it
potable), wort is by far the most intricate. Therefore, when yeast is pitched (inoculated) into wort it is
is introduced into a complex environment because it consists of simple sugars, dextrins, amino acids,
introduced into a complex environment because it consists of simple sugars, dextrins, amino acids,
peptides, proteins, vitamins, ions, nucleic acids, and other constituents too numerous to mention.
peptides, proteins, vitamins, ions, nucleic acids, and other constituents too numerous to mention.
One of the major advances in brewing science during the past 40 years has been the elucidation of
One of the major advances in brewing science during the past 40 years has been the elucidation of the
the mechanisms by which yeast cells utilise in an orderly manner, the plethora of wort nutrients [1,6].
mechanisms by which yeast cells utilise in an orderly manner, the plethora of wort nutrients [1,6].
FAN is the sum of the individual wort amino acids, ammonium ions, and small peptides (di‐
and tripeptides). FAN is an important general measure of yeast nutrients which constitute the yeast
assimilable nitrogen during brewery fermentations [6]. Wort also contains the sugars sucrose,
Beverages 2016, 2, 34 4 of 18
FAN is the sum of the individual wort amino acids, ammonium ions, and small peptides (di- and
tripeptides). FAN is an important general measure of yeast nutrients which constitute the yeast
assimilable nitrogen during brewery fermentations [6]. Wort also contains the sugars sucrose, fructose,
glucose, maltose, and maltotriose, together with dextrin material. A typical percentage sugar spectrum
of brewer’s wort is shown in Table 1.
3. Wort Fermentation
The objectives of wort fermentation are to consistently metabolise wort constituents into ethanol,
carbon dioxide, and other fermentation products in order to produce beer with satisfactory quality and
stability. Another objective is to produce yeast crops that can be confidently re-pitched into subsequent
brews [7]. It is noteworthy that brewing is the only major fermentation process that recycles its yeast
culture from one fermentation to another. Fermentation processes such as most potable and industrial
distilled alcohol production, viticulture, saké brewing, and cider production only use their yeast culture
once. As will be discussed later, the management of brewer’s yeast culture between fermentations is a
critical part of the brewing process. It is important to jealously protect the quality of cropped yeast
because it will be used to pitch a subsequent wort fermentation and will, therefore, have a profound
effect on the quality of the beer resulting from it (see Section 7).
Over the years, considerable effort has been devoted to studies on the biochemistry and
genetic/molecular biology of brewer’s yeast (together with industrial strains in general). The objectives
of these studies have been two-fold:
• To learn more about the biochemical and genetic makeup of brewing yeast strains and
• To improve the overall performance of such strains, with particular emphasis on broader substrate
utilisation capabilities, increased ethanol production, and improved tolerance to environmental
stress conditions such as temperature, elevated osmotic pressure, and ethanol, and finally to
understand the mechanism(s) of flocculation.
There are several differences in the production of ale and lager beers, one of the main ones being
the characteristics of the ale and lager yeast strains employed and the fermentation temperatures.
Considerable research by many breweries and institutions on this topic has been conducted [8–10] and
the typical differences between the ale and lager yeast strains have been established (Table 2). With the
advent of molecular biology-based methodologies, gene sequencing of ale and lager brewing strains
has shown that S. pastorianus is an interspecies hybrid between S. cerevisiae and S. eubayanus, with
homologous relationships to one another and also to Saccharomyces bayanus, a yeast species used in
wine fermentation and identified as a wild yeast in brewing fermentations.
Table 2. Differences between ale and lager yeast strains.
Ale Yeast
Saccharomyces cerevisiae (ale type).
Typical fermentation temperature—18–25 ◦ C.
Maximum growth temperature—37 ◦ C or higher.
Does not ferment melibiose.
“Top” fermenter.
Lager yeast
Saccharomyces pastorianus.
Typical fermentation temperature—8–15 ◦ C.
Maximum growth temperature—34 ◦ C.
Ferments melibiose.
“Bottom” fermenter.
Beverages 2016, 2, 34 5 of 18
Gallone et al. [8] have proposed that current industrial yeasts (including brewing and distilling)
can be divided into five sublinkages that are genetically and phenotypically separated from wild strains
and originate from only a few ancestors through complex patterns of domestic and local divergence.
Large-scale phenotyping and genome analysis further show strong industry-specific selection for stress
tolerance, sugar utilization, and flavor production—details to follow.
In 2011, a paper entitled “Microbe domestication and the identification of the wild genetic 5 of 18
Beverages 2016, 2, 34 stock
of lager-brewing yeast” was published [11]. This study confirmed that S. pastorianus is a domesticated
yeast species created by the fusion of S. cerevisiae with a previously unknown species that has now
yeast species created by the fusion of S. cerevisiae with a previously unknown species that has now
been designated
been designated Saccharomyces
Saccharomyces eubayanus
eubayanus because
because of itsof its relationship
close close relationship to S. They
to S. bayanus. bayanus. They
proposed
proposed
S. eubayanus S. exists
eubayanus exists exclusively
exclusively in the forestsin ofthe forests of
Patagonia! Patagonia!
Since Since this S.publication,
this publication, S.
eubayanus has
eubayanus has also been isolated and identified in Tibet [12] and in the USA [13]. The initial paper
also been isolated and identified in Tibet [12] and in the USA [13]. The initial paper from Argentina
from Argentina contains a draft genome sequence of S. eubayanus; it is 99.5% identical to the non‐S.
contains a draft genome sequence of S. eubayanus; it is 99.5% identical to the non-S. cerevisiae DNA
cerevisiae DNA fragment of the S. pastorianus genome sequence. This suggests specific changes in wort
fragment of the S. pastorianus genome sequence. This suggests specific changes in wort sugar and
sugar and sulphate metabolism, compared to ale strains, that are critical for determining lager beer
sulphate metabolism, compared to ale strains, that are critical for determining lager beer characteristics.
characteristics. The complete DNA sequence of S. eubayanus has recently been published [14]
The complete DNA sequence of S. eubayanus has recently been published [14].
4. Uptake and Metabolism of Wort Sugars

4. Uptake and Metabolism of Wort Sugars
It is very important that wort sugars and the FAN complex are efficiently taken up by a brewer’s
It is very important that wort sugars and the FAN complex are efficiently taken up by a brewer’s
yeast culture. In normal situations, brewing yeast strains (both ale and lager strains) are capable
yeast culture. In normal situations, brewing yeast strains (both ale and lager strains) are capable of
of utilising sucrose, glucose, fructose, maltose, and maltotriose in this approximate sequence (or
utilising sucrose, glucose, fructose, maltose, and maltotriose in this approximate sequence (or priority)
priority) (Figure 5), although some degree of overlap does occur. The majority of brewing yeast strains
(Figure 5), although some degree of overlap does occur. The majority of brewing yeast strains leave
leave the maltotetraose
the maltotetraose (G4) (G4)
and and other
other dextrins
dextrins unfermented.
unfermented. However,
However, S. S. diastaticusis isable
diastaticus able to
to utilise
utilise
dextrin material, albeit inefficiently. Wort dextrin utilisation is possible by this yeast sub-species
dextrin material, albeit inefficiently. Wort dextrin utilisation is possible by this yeast sub‐species due
due to the extracellular secretion of glucoamylase. However, this enzyme produced by S. diastaticus,
to the extracellular secretion of glucoamylase. However, this enzyme produced by S. diastaticus, is
is incapable of hydrolysing the α-1,6 bonds of dextrins whereas it is able to hydrolyse the α-1,4
incapable of hydrolysing the α‐1,6 bonds of dextrins whereas it is able to hydrolyse the α‐1,4 bonds
bonds [15,16]—details to follow.
[15,16]—details to follow.

Figure 5. Uptake order of wort sugars by brewer’s yeast cultures.
Figure 5. Uptake order of wort sugars by brewer’s yeast cultures.
The initial step in the utilisation of any wort sugar is either its passage intact across the yeast
The initial step in the utilisation of any wort sugar is either its passage intact across the yeast
plasma membrane, or its hydrolysis outside the cell membrane, followed by entry into the cells of
plasma membrane, or its hydrolysis outside the cell membrane, followed by entry into the cells of
some or all of the hydrolysis products (Figure 6). Maltose and maltotriose (Figure 7) are examples of
some or all of the hydrolysis products (Figure 6). Maltose and maltotriose (Figure 7) are examples
sugars that pass intact across the cell membrane, whereas sucrose and dextrins are hydrolysed by
of sugars that pass intact across the cell membrane, whereas sucrose and dextrins are hydrolysed by
extracellular enzymes (invertase for sucrose and glucoamylase (amyloglucosidase) for dextrins) and
extracellular enzymes (invertase for sucrose and glucoamylase (amyloglucosidase) for dextrins) and
the resultant simple sugars taken into the cell. An important metabolic difference between the uptake
the resultant simple sugars taken into the cell. An important metabolic difference between the uptake
of monosaccharides such as glucose and fructose and disaccharides such as maltose and maltotriose
of monosaccharides such as glucose and fructose and disaccharides such as maltose and maltotriose
uptake is that energy (ATP conversion to ADP) is required for maltose and maltotriose uptake (active
uptake is that energy (ATP conversion to ADP) is required for maltose and maltotriose uptake (active
transport) whereas glucose and fructose are taken up passively with no energy requirement [17]. As
transport) whereas glucose and fructose are taken up passively with no energy requirement [17].
maltose and maltotriose (Table 1) are the major sugars in brewer’s wort, the ability of a brewing yeast
As maltose and maltotriose (Table 1) are the major sugars in brewer’s wort, the ability of a brewing
strain to use these two sugars depends on the correct genetic complement. Brewer’s yeast possesses
yeast strain to use these two sugars depends on the correct genetic complement. Brewer’s yeast
independent uptake mechanisms (maltose and maltotriose permeases) to transport these two sugars
across the cell membrane into the cell. Once inside the cell, both sugars are hydrolysed to glucose
units by the α‐glucosidase system (Figure 6). The transport, hydrolysis, and maltose fermentation
systems are particularly important in brewing, distilling, and baking since maltose is the major sugar
component of brewing wort, spirit mash, and wheat dough. Maltose fermentation by brewing,
Beverages 2016, 2, 34 6 of 18
possesses independent uptake mechanisms (maltose and maltotriose permeases) to transport these
two sugars across the cell membrane into the cell. Once inside the cell, both sugars are hydrolysed
to glucose units by the α-glucosidase system (Figure 6). The transport, hydrolysis, and maltose
fermentation systems are particularly important in brewing, distilling, and baking since maltose is
the major sugar component of brewing wort, spirit mash, and wheat dough. Maltose fermentation by
brewing, distilling, and baking yeasts requires at least one of five unlinked MAL loci, each consisting
of three genes encoding:
• the structural gene for α-glucosidase (maltase) (MALS)

• maltose permease (MALT) and
Beverages 2016, 2, 34 6 of 18
Beverages 2016, 2, 34 6 of 18
• an activator whose product co-ordinately regulates the expression of the α-glucosidase and
• an activator whose product co‐ordinately regulates the expression of the α‐glucosidase and
permease genes.whose product co‐ordinately regulates the expression of the α‐glucosidase and
• an activator
permease genes.
permease genes.
Maltose Maltotriose
Maltose Active transport Maltotriose
(G-G) (G-G-G)
Active transport
(G-G) (G-G-G)
Maltose α‐glucosidase Maltotriose

Maltose α‐glucosidase
enzyme Maltotriose
enzyme
GLUCOSE (G)
GLUCOSE (G)
Yeast cell wall Metabolic Pathways
Yeast cell wall Metabolic Pathways
Glucose (G) Fructose (F) Invertase in the periplasmic space
Yeast cell membrane Glucose (G) Fructose (F) between the cell wall and membrane
Invertase in the periplasmic space
Yeast cell membrane hydrolyses sucrose to glucose and
between the cell wall and membrane
fructose which then enter the cell by
hydrolyses sucrose to glucose and
facilitated diffusion
fructose which then enter the cell by
facilitated diffusion
Glucose (G) Facilitated Fructose (F) Sucrose (G-F)

Glucose (G) Facilitated
Diffusion Fructose (F) Sucrose (G-F)
Diffusion
Figure 6. Uptake of wort sugars by a yeast cell.

Figure 7. Structure of maltose and maltotriose.
The expression of MALS and MALT is regulated by maltose induction and repression by glucose.
When glucose concentrations are high (>10 mg/L), the MAL genes are repressed, and only when 40%–
When glucose concentrations are high (>10 mg/L), the MAL genes are repressed, and only when 40%–
50% of the glucose has been taken up from the wort will maltose and maltotriose uptake commence
When glucose concentrations are high (>10 mg/L), the MAL genes are repressed, and only when
50% of the glucose has been taken up from the wort will maltose and maltotriose uptake commence
(Figure 5).
40%–50% of the glucose has been taken up from the wort will maltose and maltotriose uptake
(Figure 5).
Several ale and lager yeast strains have been used to explore maltose and maltotriose uptake
commenceSeveral ale and lager yeast strains have been used to explore maltose and maltotriose uptake
(Figure 5).
mechanisms in wort. For example, a 16°Plato all‐malt wort was fermented in a 30 liter static vessel at
mechanisms in wort. For example, a 16°Plato all‐malt wort was fermented in a 30 liter static vessel at
15 °C (Figure 8). Under these conditions, lager strains utilised maltotriose more efficiently than ale
15 °C (Figure 8). Under these conditions, lager strains utilised maltotriose more efficiently than ale
strains, whereas maltose utilisation efficiency was not dependent on the type of brewing strain [18,19].
This supports the proposal that maltotriose and maltose possess independent, but closely linked,
This supports
uptake the proposal
(permease) systems that maltotriose
[18,19]. and maltose
In addition, possess difference
this consistent independent, but closely
between linked,
ale and lager
uptake (permease) systems [18,19]. In addition, this consistent difference between ale and
strains supports the observation that ale strains appear to have greater difficulty than lager strains to lager
Beverages 2016, 2, 34 7 of 18
Several ale and lager yeast strains have been used to explore maltose and maltotriose uptake
mechanisms in wort. For example, a 16◦ Plato all-malt wort was fermented in a 30 liter static vessel at
15 ◦ C (Figure 8). Under these conditions, lager strains utilised maltotriose more efficiently than ale
This supports the proposal that maltotriose and maltose possess independent, but closely linked,
uptake (permease) systems [18,19]. In addition, this consistent difference between ale and lager
strains supports the observation that ale strains appear to have greater difficulty than lager strains to
completely ferment wort, particularly high gravity wort (>16◦ Plato) [20].
Beverages 2016, 2, 34 7 of 18
Beverages 2016, 2, 34 7 of 18

Figure 8. Maltotriose (A) and maltose (B) uptake profiles from a 16°Plato wort.
Figure 8. Maltotriose (A) and maltose (B) uptake profiles from a 16◦ Plato wort.

In order to investigate the MAL‐gene cassettes further, a strain containing two MAL2 and two
Figure 8. Maltotriose (A) and maltose (B) uptake profiles from a 16°Plato wort.
In order
MAL4 to copies
gene investigate the MAL-gene
was constructed using cassettes further,
hybridisation a strain[21].
techniques containing two
The wort MAL2 and
(16°Plato)
two MAL4 gene copies was constructed using hybridisation techniques [21]. The wort (16◦ Plato)
fermentation rate was compared to a strain that only contained one copy of MAL2. As expected, the
In order to investigate the MAL‐gene cassettes further, a strain containing two MAL2 and two
overall fermentation rate with the strain containing multiple MAL genes was considerably faster than
MAL4 gene
fermentation copies
rate was was constructed
compared using
to a strain thathybridisation
only containedtechniques [21]. of
one copy The wort As
MAL2. (16°Plato)
expected, the
overall fermentation rate was compared to a strain that only contained one copy of MAL2. As expected, the
the strain containing
fermentation the the
rate with single MAL2
strain copy (Figure
containing 9). MAL
multiple The principal
genes was reason for this faster
considerably faster than
overall fermentation rate with the strain containing multiple MAL genes was considerably faster than
fermentation rate was due to an increased rate of maltose uptake and subsequent metabolism
the strain containing the single MAL2 copy (Figure 9). The principal reason for this faster fermentation
the strain containing the single MAL2 copy (Figure 9). The principal
compared to the yeast strain containing a single MAL2 copy (Figure 9). reason for this faster
rate wasfermentation
due to an increased rateto
rate was due ofan
maltose uptake
increased and
rate of subsequent
maltose uptake metabolism compared
and subsequent to the yeast
metabolism
strain containing a single MAL220copy (Figure 9).
compared to the yeast strain containing a single MAL2 copy (Figure 9).
M AL2/M AL2;
16
20 M AL4/M AL4
MM AL2/mal2;
AL2/M AL2;
Plato
Degree Plato
16
12 Mmal4/mal4
AL4/M AL4
M AL2/mal2;
Plato
Degree Plato
12 mal4/mal4
8
Degree
8
Degree
4
0
0 24 48 72 96
0
Fermentation time (hours)
Fermentation time (hours)
0 24 48 72 96
*Shaking Fermentation Note: Shaking
fermentations atfermentations
time (hours)
Fermentation time (hours) 21°C at 21 °C

Figure 9. Fermentation *profile
Shaking of a Note: Shaking
fermentations
16°Plato atfermentations
21°Cand a atdiploid
wort 21 °C
yeast strain containing multiple
maltose (MAL) genes.
Figure 9. Fermentation profile of ◦a 16°Plato wort and a diploid yeast strain containing multiple
Figure 9. Fermentation profile of a 16 Plato wort and a diploid yeast strain containing multiple maltose
maltose (MAL) genes.
(MAL) genes.
5. Carbohydrate Metabolism
It has already been described that S. diastaticus is a subspecies of S. cerevisiae. It differs from the
It has already been described that S. diastaticus is a subspecies of S. cerevisiae. It differs from the
parental species by producing the extracellular enzyme glucoamylase (α‐1,4‐glucan glucohydrolase)
parental species by producing the extracellular enzyme glucoamylase (α‐1,4‐glucan glucohydrolase)
[22]. This enzyme removes successive glucose units from the non‐reducing ends of dextrins (partially
It has already been described that S. diastaticus is a subspecies of S. cerevisiae. It differs
[22]. This enzyme removes successive glucose units from the non‐reducing ends of dextrins (partially
fromhydrolysed starch). Using hybridisation techniques, three non‐allelic genes have been identified and
the parental species by producing the extracellular enzyme glucoamylase (α-1,4-glucan
hydrolysed starch). Using hybridisation techniques, three non‐allelic genes have been identified and
characterised [22].
glucohydrolase) from This
strains of S. removes
enzyme diastaticus [23]. Two glucose
DEX genes (DEX1 and DEX2) have been
characterised from strains of S. diastaticus successive
[23]. Two DEX genes units from
(DEX1 and the non-reducing
DEX2) have been ends of
identified that codes for the production of S. diastaticus glucoamylase, with glucose being the sole
dextrinsidentified that codes for the production of S. diastaticus glucoamylase, with glucose being the sole
(partially hydrolysed starch). Using hybridisation techniques, three non-allelic genes have
hydrolysis product. A third dextrinase gene (DEX3) was found to be allelic to STA3, a gene reported
hydrolysis product. A third dextrinase gene (DEX3) was found to be allelic to STA3, a gene reported
to control starch fermentation in Saccharomyces sp. [24,25].
to control starch fermentation in Saccharomyces sp. [24,25].
Using hybridisation
Using hybridisation techniques
techniques [21],
[21], a number of
a number of diploid
diploid strains
strains have
have been
been developed,
developed, each
each
being either heterozygous or homozygous for the individual DEX genes. Wort fermentations with
being either heterozygous or homozygous for the individual DEX genes. Wort fermentations with
the DEX containing cultures showed that in addition to the wort sugars (glucose, fructose, sucrose,
the DEX containing cultures showed that in addition to the wort sugars (glucose, fructose, sucrose,
maltose, and
maltose, maltotriose),
and maltotriose), the
the dextrins
dextrins (G4–G15) were partially
(G4–G15) were partially fermented.
fermented. There
There was
was residual
residual
Beverages 2016, 2, 34 8 of 18
been identified and characterised from strains of S. diastaticus [23]. Two DEX genes (DEX1 and DEX2)
have been identified that codes for the production of S. diastaticus glucoamylase, with glucose being
the sole hydrolysis product. A third dextrinase gene (DEX3) was found to be allelic to STA3, a gene
reported to control starch fermentation in Saccharomyces sp. [24,25].
Using hybridisation techniques [21], a number of diploid strains have been developed, each being
either heterozygous or homozygous for the individual DEX genes. Wort fermentations with the DEX
containing cultures showed that in addition to the wort sugars (glucose, fructose, sucrose, maltose,
and maltotriose), the dextrins (G4–G15) were partially fermented. There was residual dextrin in the
Beverages 2016, 2, 34
fermented worts because the glucoamylase from S. diastaticus strains was unable hydrolyse 8 of 18 the α(1-6)
branch points, only α(1-4) linkages.
dextrin in the fermented worts because the glucoamylase from S. diastaticus strains was unable
Beer produced with DEX-containing yeast strains has been studied. Glucose concentration in the
hydrolyse the α(1‐6) branch points, only α(1‐4) linkages.
pasteurized beer increased during storage for three months at room temperature—this indicates
Beer produced with DEX‐containing yeast strains has been studied. Glucose concentration in
that this glucoamylase is not heat sensitive [24]. Also, overall beer flavour produced by these
the pasteurized beer increased during storage for three months at room temperature—this indicates
DEX-containing yeast strains was unacceptable because it contained 4-vinylguaiacol (clove-like),
that this glucoamylase is not heat sensitive [24]. Also, overall beer flavour produced by these DEX‐
estery, sulphury, and musty aromas [25]. Nevertheless, the brewing industry has continued to be
containing yeast strains was unacceptable because it contained 4‐vinylguaiacol (clove‐like), estery,
sulphury, and musty aromas [25]. Nevertheless, the brewing industry has continued to be interested
interested in the glucoamylases produced by S. diastaticus strains. Consequently, recombinant DNA
in the glucoamylases produced by S. diastaticus strains. Consequently, recombinant DNA (rDNA),
(rDNA), plasmids, and transformation techniques have been employed to clone DEX genes into
plasmids, and transformation techniques have been employed to clone DEX genes into brewing
brewing strains [26]. In order to enhance glucoamylase activity, the glucoamylase gene from Aspergillus
strains [26]. In order to enhance glucoamylase activity, the glucoamylase gene from Aspergillus niger
niger was also cloned into a brewing yeast along with the STA2 gene. The A. niger glucoamylase has
was also cloned into a brewing yeast along with the STA2 gene. The A. niger glucoamylase has the
the advantage of possessing debranching activity, unlike the yeast enzyme, so that it can hydrolyse
advantage of possessing debranching activity, unlike the yeast enzyme, so that it can hydrolyse more
moreof the wort dextrins [26]. This enzyme is also sensitive to pasteurisation conditions.
of the wort dextrins [26]. This enzyme is also sensitive to pasteurisation conditions.
The The
cloned amylolytic
cloned strains
amylolytic strains have
have received approvalby
received approval bythe
theU.K.
U.K. Agriculture
Agriculture and and Health
Health
Ministries, who concluded in 1995 that there were no food safety reasons why this yeast should
Ministries, who concluded in 1995 that there were no food safety reasons why this yeast should not
not bebe used
used [27].
[27]. The
Thereduced
reduced dextrin beer
dextrin produced
beer with this
produced withcloned yeast was
this cloned called
yeast was Nutfield
called Lyte
Nutfield
(Figure 10) and was the first beer in the world to be produced from this genetically modified yeast to
Lyte (Figure 10) and was the first beer in the world to be produced from this genetically modified
yeastreceive
to receiveapproval.
approval.After considerable
After deliberation,
considerable it was
deliberation, decided
it was that that
decided specific labelling
specific was was
labelling
unnecessary. However, a back label to Nutfield Lyte bottles explained the process by which it had
unnecessary. However, a back label to Nutfield Lyte bottles explained the process by which it had been
been produced and the advantages of doing so. Reaction to this beer from the general public has been
produced and the advantages of doing so. Reaction to this beer from the general public has been neutral
neutral or positive. Media reports were largely factual and supportive. There was some criticism from
or positive. Media reports were largely factual and supportive. There was some criticism from the
the environmental lobby. This criticism has not diminished over the years and the production of
environmental lobby. This(including
modified foodstuffs criticism alcoholic
has not diminished
beverages) over the years
in Europe has and
been, theand
production of modified
still is, severely
foodstuffs (including
restricted [28]. alcoholic beverages) in Europe has been, and still is, severely restricted [28].

Figure 10. Nutfield Lyte.
Figure 10. Nutfield Lyte.
6. Uptake and Metabolism of Free Amino Nitrogen (FAN)
6. Uptake and Metabolism of Free Amino Nitrogen (FAN)
FAN is a general measure of a yeast culture’s assimilable nitrogen efficiency. It is a good index
FAN is a general
for yeast measure
growth and of a yeastfermentation
consequently culture’s assimilable
efficiency nitrogen efficiency.
and sugar It isWort
uptake [29]. a good index
FAN is for
yeastessential for the formation of new yeast amino acids, the synthesis of new structural and enzymatic
growth and consequently fermentation efficiency and sugar uptake [29]. Wort FAN is essential
for the formation of new yeast amino acids, the synthesis of new structural and enzymatic proteins,
proteins, cell proliferation, and cell viability and vitality. Lastly, FAN levels have a direct influence
on beer flavour compounds (for example, higher alcohols, carbonyls, and esters).
There are differences between lager and ale yeast strains with respect to wort assimilable
nitrogen uptake characteristics [30]. Nevertheless, with all brewing strains the amount of wort FAN
content required by yeast under normal brewery fermentation conditions is directly proportional to
yeast growth and also affects certain aspects of beer maturation. There has been considerable
Beverages 2016, 2, 34 9 of 18
cell proliferation, and cell viability and vitality. Lastly, FAN levels have a direct influence on beer
flavour compounds (for example, higher alcohols, carbonyls, and esters).
There are differences between lager and ale yeast strains with respect to wort assimilable nitrogen
uptake characteristics [30]. Nevertheless, with all brewing strains the amount of wort FAN content
required by yeast under normal brewery fermentation conditions is directly proportional to yeast
growth and also affects certain aspects of beer maturation. There has been considerable problems
regarding the minimal wort FAN required to achieve satisfactory yeast growth and fermentation
performance in conventional gravity (10–12◦ Plato) wort, considered to be 130 mg FAN/L. For rapid
attenuation of high gravity wort (>16◦ Plato), increased levels of FAN are required [31]. However,
optimum wort FAN levels differ from fermentation to fermentation and from yeast strain to yeast
Beverages 2016, 2, 34 9 of 18
strain. Furthermore, the optimum FAN values are dependent on different wort sugar levels and
type FAN/L. For rapid attenuation of high gravity wort (>16°Plato), increased levels of FAN are required
[6].
Studies on the uptake of wort nitrogen by brewer’s yeast strains began over 50 years ago. During
[31]. However, optimum wort FAN levels differ from fermentation to fermentation and from yeast
the 1960s, Margaret Jones and John Pierce, working in the Research Department of the Guinness
strain to yeast strain. Furthermore, the optimum FAN values are dependent on different wort sugar
levels and type [6].
Brewery in Park Royal, London, conducted notable studies on nitrogen metabolism during brewing,
mashing,Studies on the uptake of wort nitrogen by brewer’s yeast strains began over 50 years ago. During
and fermentation [32]. They reported that the absorption and utilisation of exogenous
the 1960s,
nitrogenous Margaret
wort Jones and
compounds andJohn
theirPierce, working
synthesis in the Research
intracellularly Department
are controlled byof three
the Guinness
main factors:
Brewery in Park Royal, London, conducted notable studies on nitrogen metabolism during brewing,
• total wortand
mashing, fermentation
concentration of[32]. They reported
assimilable that the absorption and utilisation of exogenous
nitrogen
nitrogenous wort compounds and their synthesis intracellularly are controlled by three main factors:
• the concentrations of individual nitrogenous compounds and their ratio and
• • competitive
the total wort concentration of assimilable nitrogen
inhibition of the uptake of these components (mainly, but not exclusively, amino
• the concentrations of individual nitrogenous compounds and their ratio and
acids) via various permease systems [33].
• the competitive inhibition of the uptake of these components (mainly, but not exclusively, amino
acids) via various permease systems [33].
A unique classification of amino acids according to their rates of consumption during brewing
wort fermentation has been established. There are four uptake groups of amino acids. Three groups
A unique classification of amino acids according to their rates of consumption during brewing
wort fermentation has been established. There are four uptake groups of amino acids. Three groups
of wort amino acids are taken up at different stages of fermentation and the fourth group consists of
only of wort amino acids are taken up at different stages of fermentation and the fourth group consists of
one amino acid, proline (the largest concentration amino acid in wort), which is not taken
only one amino acid, proline (the largest concentration amino acid in wort), which is not taken up
up during brewing fermentations (Figure 11). This is because of the anaerobic conditions that
during brewing fermentations (Figure 11). This is because of the anaerobic conditions that prevail
prevail late in the fermentation (oxygen is necessary for proline uptake). When this classification was
late in the fermentation (oxygen is necessary for proline uptake). When this classification was
developed, the methodology employed liquid chromatography for measuring individual amino acids
developed, the methodology employed liquid chromatography for measuring individual amino
and was iconic. Similar measurements today employ automated computerized high-performance
acids and was iconic. Similar measurements today employ automated computerized high‐
liquidperformance
chromatography liquid (HPLC), and it is(HPLC),
chromatography difficultand
to envisage and appreciate
it is difficult to envisage the challenges
and appreciate that
the were
facedchallenges that were faced and overcome 50 years ago! Indeed, advances in analytical methods have
and overcome 50 years ago! Indeed, advances in analytical methods have been a primary reason
for our increasing knowledge of wort fermentation control.
been a primary reason for our increasing knowledge of wort fermentation control.
3.5 4.5
4.0
3.0
Group A, Group B (mmol/L)
Group C, Proline (mmol/L)
3.5
2.5
3.0
2.0
2.5
1.5 2.0
1.5
1.0
1.0
0.5
0.5
0.0 0.0
0 19 24 43 48 67 72 96
Fermentation Time (hours)
Group A Group B Group C Proline

Figure 11. Amino acid absorption pattern during wort fermentation.
Figure 11. Amino acid absorption pattern during wort fermentation.
The Jones and Pierce amino acid classification [32,33] continues to be the basis of our current
The Jones and Pierce amino acid classification [32,33] continues to be the basis of our current
understanding of the relative importance of individual wort amino acids during fermentation. This
understanding of the relative importance of individual wort amino acids during fermentation. This
information has assisted manipulation of wort nitrogen levels by the addition of supplements such
as yeast extract or specific amino acids, particularly during high gravity brewing. However, this
assimilation pattern is often specific to the conditions employed. The nutritional preferences of a
particular yeast strain are paramount. The absorption and utilisation of wort amino acids has recently
been re‐examined [34] and the overall Jones and Pierce classification was confirmed using
contemporary analytical techniques. However, the order of methionine uptake has been transferred
Beverages 2016, 2, 34 10 of 18
information has assisted manipulation of wort nitrogen levels by the addition of supplements such
as yeast extract or specific amino acids, particularly during high gravity brewing. However, this
assimilation pattern is often specific to the conditions employed. The nutritional preferences of
a particular yeast strain are paramount. The absorption and utilisation of wort amino acids has
recently been re-examined [34] and the overall Jones and Pierce classification was confirmed using
contemporary analytical techniques. However, the order of methionine uptake has been transferred
from Group B to Group A (Table 3).
Table 3. Order of wort amino acids and ammonia uptake during fermentation.
Group A Group B Group C Group D

Fast Absorption Intermediate Absorption Slow Absorption Little or No Absorption
Glutamic acid Valine Glycine Proline
Aspartic acid Leucine Phenylalanine
Asparagine Isoleucine Tyrosine
Glutamine Histidine Tryptophan
Serine Alanine
Methionine Ammonia
Threonine
Lysine
Arginine
It has also been reported [35] that approximately 30% of incorporated nitrogen compounds come
from sources other than amino acids. Although the utilisation of small peptides by brewing yeasts
was confirmed over 50 years ago, an understanding of their role in yeast nitrogen requirements is still
limited. Small peptides can be used as nutritional sources of amino acids as both carbon or nitrogen
sources and precursors of cell wall peptides during yeast growth, although, growth is much slower
when they are the sole nitrogen source [36]. Polypeptides may also used as a substrate because yeasts
can generate proteolytic enzymes extracellularly to provide additional assimilable nitrogen to the
cells—see below and reference [37].
Most brewing strains transport peptides with no more than three amino acid residues but this
limit is strain dependent [35]. Nevertheless, small wort peptides are an important source of assimilable
nitrogen and 20%–40% of wort oligopeptides are used during fermentation. Similar to single amino
acids, peptides probably contribute to beer character and flavour. Wort peptides are taken up in a
specific order dependent on their amino acid composition. The determination of small (two and three
amino acids) peptides has been an impediment to this area of research. However, a method to measure
these small oligopeptides has now been developed [35]. In brief, the sample is deproteinised, the
supernatant ultrafiltered through a membrane with a 500 Da exclusion limit, the filtrate is acid and
alkaline hydrolysed, and the hydrolysate is subjected to amino acid analysis by HPLC.
It has already been briefly discussed that yeast secretes proteinase into fermenting wort (mainly
proteinases). Therefore, the hydrolysis of medium-sized peptides into smaller peptides occurs, which
can be taken up by yeast cultures, and this activity continues throughout fermentation [35]. This fact
highlights a difference between the uptake of sugars and FAN during brewing. The spectrum of wort
sugars at the beginning of fermentation is fixed because after wort boiling, all malt amylases have been
inactivated. Whereas, because of proteinase secretion by yeast, the spectrum of nitrogen compounds
is dynamic. This is not the case during malt and grain whisky fermentations where the wort is not
boiled. Consequently, the wort still contains amylase activity (along with proteinase activity) and
microbial contaminants. Consequently, the spectrum of wort carbohydrate is flexible [38]. Additionally,
stress effects on yeast can increase proteinase secretion and this occurs to a greater extent during the
fermentation of high gravity worts [37,38].
Regarding wort fermentation by brewing yeast, the metabolism of wort sugars and FAN during
fermentation has already been discussed but a plethora of other metabolic reactions also occur. As there
Beverages 2016, 2, 34 11 of 18
are many reactions, they will not be discussed in detail here, but are covered in greater detail in
reference [39]. As a consequence, only three reactions will be considered:
• yeast management, including the initial stages of fermentation, where the intracellular storage
carbohydrate glycogen (Figure 12) is used in the presence of oxygen in order to synthesize
unsaturated fatty acids (UFAs) and sterols which are incorporated into yeast membrane
structures [40].
• the formation of diacetyl during the exponential stages of fermentation (Figure 13A) followed by
• theBeverages 2016, 2, 34
later stages of fermentation overlapping into maturation (aging/lagering). This11 of 18 involves the
Beverages 2016, 2, 34 11 of 18
management (reduction) of diacetyl and other vicinal diketones (VDK—see Figures 13B and 16).
• the later stages of fermentation overlapping into maturation (aging/lagering). This involves the
Such
• compounds impart a butterscotch flavour in beer. and
the later stages of fermentation overlapping into maturation (aging/lagering). This involves the
management (reduction) of diacetyl and other vicinal diketones (VDK—see Figures 13B and 16).
• management (reduction) of diacetyl and other vicinal diketones (VDK—see Figures 13B and 16).
the harvesting (cropping) of the yeast culture for reuse either
Such compounds impart a butterscotch flavour in beer. and by flocculation or with a centrifuge,
as • Such compounds impart a butterscotch flavour in beer. and
discussed later.
the harvesting (cropping) of the yeast culture for reuse either by flocculation or with a centrifuge,
• the harvesting (cropping) of the yeast culture for reuse either by flocculation or with a centrifuge,
as discussed later.
as discussed later.

Figure 12. Structure of glycogen.

Carbohydrate
Ethanol Carbohydrate
Acetic Acid
Ethanol
Pyruvate Acetic Acid
Pyruvate
Ethanol Acetic Acid
Enzymatic
Ethanol Acetic Acid
Conversion
Enzymatic
α-Acetohydroxy- Conversion
α-Acetolactate butyrate Passive
α-Acetohydroxy-
α-Acetolactate butyrate Diffusion
Passive
Plasma
Diffusion
Membrane
Plasma Valine Iso- Non-Enzymatic
Membrane leucine leucine
Iso- Decomposition
Valine Non-Enzymatic
leucine leucine Decomposition
α-Acetohydroxy-
α-Acetolactate butyrate
α-Acetohydroxy-
α-Acetolactate butyrate
Diacetyl Pentanedione
Diacetyl A Pentanedione
Diacetyl A
Diacetyl
Plasma
Diacetyl Membrane
Plasma
Diacetyl Membrane
Enzymatic
Conversion
Acetoin Enzymatic
Conversion
Acetoin Plasma
Diffusion
Plasma
Butanediol Diffusion
Butanediol
Acetoin
AcetoinButanediol
ButanediolB
B
Figure 13. Diacetyl metabolism of a fermenting wort. (A) Formation of diacetyl and 2,3‐pentandione
Figure 13. Diacetyl metabolism of a fermenting wort. (A) Formation of diacetyl and 2,3‐pentandione
during logarithmic and early stationary growth phases; (B) Conversion of diacetyl to acetoin and 2,3‐
Figure 13. Diacetyl metabolism of a fermenting wort. (A) Formation of diacetyl and 2,3-pentandione
during logarithmic and early stationary growth phases; (B) Conversion of diacetyl to acetoin and 2,3‐
butanediol late in the fermentation.
during logarithmic and early stationary growth phases; (B) Conversion of diacetyl to acetoin and
butanediol late in the fermentation.
2,3-butanediol late in the fermentation.
7. Yeast Cropping—Flocculation and/or Centrifugation
Flocculation is one of the major factors when considering important characteristics of yeast
Flocculation is one of the major factors when considering important characteristics of yeast
strains during brewing fermentations [7]. Most brewing yeast strains are classified as either flocculent
strains during brewing fermentations [7]. Most brewing yeast strains are classified as either flocculent
or non‐flocculent (powdery) (Figure 14) [41–43]. One definition of flocculation that has been used for
or non‐flocculent (powdery) (Figure 14) [41–43]. One definition of flocculation that has been used for
many years is: “the phenomenon wherein yeast cells adhere in clumps and either sediment from the
many years is: “the phenomenon wherein yeast cells adhere in clumps and either sediment from the
Beverages 2016, 2, 34 12 of 18

Flocculation is one of the major factors when considering important characteristics of yeast strains
during brewing fermentations [7]. Most brewing yeast strains are classified as either flocculent or
non-flocculent (powdery) (Figure 14) [41–43]. One definition of flocculation that has been used for
Beverages 2016, 2, 34 12 of 18
many years is: “the phenomenon wherein yeast cells adhere in clumps and either sediment12 of 18
Beverages 2016, 2, 34 from
the medium in which they are suspended or rise to the medium’s surface” [43]. This
medium in which they are suspended or rise to the medium’s surface” [43]. This definition excludes definition
medium in which they are suspended or rise to the medium’s surface” [43]. This definition excludes
excludes forms ofgrowth”
“clumpyand
growth” and “chain formation” [44]will
which
forms of “clumpy “chain formation” [44] which not will not be discussed
be discussed further further
here
forms
here of “clumpy
(Figure 15). growth” and “chain formation” [44] which will not be discussed further here
(Figure 15).
(Figure 15).

Non‐flocculent Flocculent
Non‐flocculent Flocculent
Figure 14. Flocculation characteristics of Saccharomyces cerevisiae strains.

Figure 15. Chain‐forming yeast strain.
Figure 15. Chain‐forming yeast strain.
Figure 15. Chain-forming yeast strain.
It is important to crop a yeast culture at the end of fermentation either using a culture’s
flocculation characteristics (Figure 16) or with a centrifuge (Figure 17). Flocculation is a cell surface
It is important to crop a yeast culture at the end of fermentation either using a culture’s flocculation
phenomenon
characteristics and the cell
(Figure wall structure of a culture is critical (Figure is18). Components such as
phenomenon and the 16)
cell orwall
with a centrifuge
structure of a (Figure
culture 17). Flocculation
is critical (Figure a18).
cell surface phenomenon
Components such as
mannoprotein are found distributed throughout the entire yeast wall and therefore the wall’s layered
and the cell wall structure of a culture is critical (Figure 18). Components such as mannoprotein
arrangement exhibits zones
are found distributed of enrichment. It is wall
a matrix, not a sandwich! Nevertheless, the
arrangement exhibits throughout the entire yeast
zones of enrichment. It is a and therefore
matrix, not a the wall’s layered
sandwich! arrangement
Nevertheless, the
mannoprotein is an integral structure that determines a yeast culture’s flocculation characteristics [7].
exhibits zones of enrichment. It is a matrix, not a sandwich! Nevertheless, the mannoprotein is an
integral structure that determines a yeast culture’s flocculation characteristics [7].

phenomenon and the cell wall structure of a culture is critical (Figure 18). Components such as
arrangement
Beverages exhibits zones of enrichment. It is a matrix, not a sandwich! Nevertheless,
2016, 2, 34 13 ofthe
18

Figure 16.
Figure 16. Flocculation
Flocculation characteristics
characteristics of
of a brewer’s yeast
a brewer’s yeast strain
strain during static fermentation
during static fermentation and its
and its
influence on wort diacetyl levels.
influence on wort diacetyl levels.
Beverages 2016, 2, 34 13 of 18
Beverages 2016, 2, 34 13 of 18

Figure 17. A disc stack centrifuge (5 hL/h).
Fibrillar Layer
Fibrillar Layer
Mannoprotein
Mannoprotein
β Glucan
β Glucan
β Glucan-Chitin
β Glucan-Chitin
Mannoprotein
Mannoprotein
Plasma Membrane
Plasma Membrane
Components such as mannoprotein occur as a matrix and are
distributed throughout
Components the entire walloccur
such as mannoprotein and therefore theand are
as a matrix
layered arrangement
distributed throughout should show
the entire wallzones of enrichment.
and therefore the
layered arrangement should show zones of enrichment.
Figure 18. Schematic diagram of the yeast cell wall architecture.
Flocculation usually occurs in the absence of cell division (not always) during late logarithmic
and early stationary growth phases and only under rather circumscribed environmental conditions
involving specific yeast cell surface structures (mainly protein and carbohydrate (mannan)
involving specific
components) yeast
and also an cell surface of structures
interaction (mainly
calcium ions protein
[45]. Only and carbohydrate
a microamount (mannan)
of calcium ions is
components) and also an interaction of calcium ions [45]. Only a microamount of calcium ions
necessary for the flocculation of brewer’s yeast strains. Cells can be deflocculated as a result of the is
necessary for the flocculation of brewer’s yeast strains. Cells can be deflocculated as a result of the
Beverages 2016, 2, 34 14 of 18
involving specific yeast cell surface structures (mainly protein and carbohydrate (mannan) components)
and also an interaction of calcium ions [45]. Only a microamount of calcium ions is necessary for the
flocculation of brewer’s yeast strains. Cells can be deflocculated as a result of the addition of sugars
such as mannose and also treatment with chelating agents such as ethylenediamine tetraacetic acid
(EDTA) that complex with Ca++ ions. Cells will usually reflocculate rapidly upon the addition of Ca++
ions [45].
Although yeast separation often occurs by sedimentation, it may also be by flotation because of
cell aggregation entrapping bubbles of CO2 as is the case of hydrophobic top-cropping ale brewing
yeast strains. Co-flocculation [46] (also called mutual agglutination [47], mutual aggregation, and
mutual flocculation) is an aggregation process between two yeast strains (yeast-bacteria co-flocculation
has also been reported [48,49]). When two ale strains (one being non-flocculent and the other weakly
flocculent) are mixed together in the presence of Ca++ , flocs form and the culture rapidly settles out
of suspension or rises to the surface during static wort fermentation. Co-flocculation has not been
reported in lager yeast strains [50].
Genetic studies on yeast flocculation began over 60 years ago. Thorne [51] and Gilliland [52]
independently confirmed that this phenomenon was an inherited characteristic with the flocculation
phenotype being dominant over non-flocculence. The first flocculation gene (FLO gene) to be studied in
detail was FLO1. Using traditional gene mapping techniques (mating, sporulation, micromanipulation,
tetrad analysis, etc.) [21], it was shown that FLO1 is located on chromosome I, 33cM from the centromere
on the right-hand side of the chromosome [53].
Novel genetic techniques have been developed, the principal of which is the sequencing of the
Saccharomyces genome [54]. A sequenced laboratory yeast strain contains five FLO genes, four located
near the chromosome telomeres FLO1, FLO5, FLO9, and FLO10 and one neither at the centromere
nor at the telomeres, FLO11 [55]. At least nine genes—FLO1, FLO5, FLO9, FLO10, FLO11, FLONL,
FLONS, and LgFLO in S. cerevisiae and S. pastorianus—encode flocculin proteins. The flocculin encoded
by FLO11 differs from other FLO genes in that it is involved in filamentous growth, adhesion to solid
surfaces, and floc formation rather flocculation per se [56]. Another FLO gene, FLO8, encodes for a
transposable factor regulating the expression of other FLO genes [57]. It has already been stated that
many yeast strains usually possess several FLO genes in their genome [56].
FLO genes are very long (up to 4 Kbp) because of the number of tandem repeated DNA sequences
of about 100 nucleotides that are repeated 10–20 times in each gene [56]. Tandem repeated DNA
sequences of the FLO genes are highly dynamic components of the genome—they change more rapidly
than other parts of the genome. They enable rearrangements both between and within flocculin genes.
The longer the FLO protein, the stronger the strain’s flocculation ability [56]. FLO1 has been identified
as the longest FLO gene, with the most repeats and confers the strongest flocculation phenotype to a
yeast strain [57].
The sedimentation performance of a brewer’s yeast strain often changes during repeated
re-pitching in a brewery [39,58]. This could be due to irreversible or reversible genetic change.
Alternatively, it could be due to long lasting physiological changes—perhaps modifications in yeast
handling and fermentation environments (for example, the use of high gravity worts). When a
genetic change, conferring a non-flocculent phenotype, occurs in a yeast culture, it will gradually
become a mixture of flocculent and non-flocculent cells [39]. Often within a lager yeast population,
exhibiting moderate flocculent characteristics, a more flocculent variant culture can be isolated. An
example of this development was when a Canadian brewing company began brewing its beer, under
contract, in breweries in the United Kingdom. Most of the contracted breweries employed vertical
fermenters. However, at that time, the Canadian breweries only possessed horizontal tanks (as
both fermentation and maturation vessels). This difference in tank geometry influenced the yeast
culture’s sedimentation characters. In vertical fermenters, this yeast culture was too non-flocculent
Beverages 2016, 2, 34 15 of 18
(powdery), with too much yeast remaining in suspension at the end of fermentation (centrifuges were
not available at the time). It was thought possible that the culture contained a spectrum of isolates
that exhibited various flocculation intensities. Consequently, one of the variants from this strain, with
more intensive flocculation characteristics, was successfully isolated and employed in the vertical
fermenters. The result was less yeast in suspension at the end of fermentation. However, care had to
be taken to ensure that the flocculent variant used was not too flocculent because under-fermented
wort and residual unwanted beer flavours (for example, diacetyl and other VDKs—Figure 16) could
have been the result [39].
The alternative way to crop yeast for reuse and to clarify primary fermented wort is with the use
of a disk stack centrifuge (Figure 17). However, when a yeast culture is passed through a centrifuge it
can experience mechanical and hydrodynamic shear stresses unless the centrifuge is correctly operated
under the appropriate conditions. This shear stress can cause a decrease in cell viability/vitality, cell
wall damage, injury to the mitochondrial DNA leading to formation of respiratory deficient mutants,
increased extracellular proteinase A levels, hazier beer, and reduced foam stability [59].
8. Non-Saccharomyces Yeasts in Brewing

Many yeast species are emerging as candidates for the production of specialty beers. Innovation
not only relies on obtaining new products with more complex aromatic and flavour characters, but
also increasing the range of approaches to achieve different results in a growing market. Using
non-Saccharomyces yeasts for pitching, in sequential or co-inoculation, in bottle conditioning, or many
other strategies, has presented the possibility to unveil old and new methods to produce specialty and
original beers. Brewers are now reinterpreting the role of different yeast species—previously regarded
as spoilage microorganisms in uncontrolled or in the absence of good manufacturing practices—and
learning to find a multitude of applications and benefits with them. Yeasts from the genera Dekkera,
Hanseniaspora, Pichia, Torulaspora, Wickerhamomyces, and others, can offer a diversified enzymatic
apparatus and bioconversion abilities that are allowing brewers to work with new concepts that
include bioflavouring, beers with reduced calories and alcohol contents, and also various functional
beers. Nonetheless, the introduction of a new yeast culture must be carefully assessed, because each
microorganism is unique, and can develop a range of process adaptations and flavours in contact with
different substrates and/or conditions. Finally, it is important not to ignore the importance of the
ongoing craft brewery revolution that is occurring in many parts of the globe. This is a movement
that is paving the way to explore such traits and possibilities in order to identify a growing market
of enthusiastic and expert consumers [60]. The impact of non-Saccharomyces yeasts on the volatile
components and sensory profile of beer, wine, spirits, and other fermented beverages has recently
been reviewed [61].
9. Conclusions
The brewing process involves the use of four major raw materials: water, barley malt, hops, and
yeast. As well as focusing on brewing yeast, research has productively compared yeast’s use with
the production of other products such as distilled spirits, sake, wine, baked goods, and food extract.
Yeast is also employed as a model eukaryote and it is interesting to note that the 2016 Nobel Prize in
Physiology or Medicine has been awarded to a Japanese cell biologist for research on autophagy (the
degradation and recycling of cellular components). This research has been conducted with S. cerevisiae
by Yoshinori Õhsumi [62]. Another recent example of yeast’s contribution to our knowledge of
basic biology, that has been applied to medical science, concerns the structure and function of yeast
mitochondria [59]. There are a group of diseases caused by dysfunctional mitochondria often as a
result of mitochondrial mutation of its DNA (mtDNA). Current research removing the mtDNA from
the ovaries of a diseased patient and replacing it with unmutated mtDNA from a donor to produce
healthy zygotes is a novel technique with significant potential (the three parent concept) [63]. Without
Beverages 2016, 2, 34 16 of 18
basic mitochondrial research on brewer’s yeast and its close relations, these medical developments
would have been inhibited, impeded, and protracted [59].
Acknowledgments: The invaluable assistance and support of Anne Anstruther in developing this review paper
is gratefully acknowledged.
Conflicts of Interest: The author declares no conflict of interest.
References
1. Stewart, G.G. Studies on the uptake and metabolism of wort sugars during brewing fermentations. Tech. Q.
Master Brew. Assoc. Am. 2006, 43, 265–269. [CrossRef]
2. Stewart, G. Adjuncts. In Brewing Materials and Processes; Bamforth, C.W., Ed.; Academic Press: Boston, MA,
USA, 2016; pp. 27–46.
3. Winkelmann, L. 500 years of beer purity law. Brauwelt Int. 2016, 34, 188.
4. Davies, N. Malts. In Brewing Materials and Processes; Bamforth, C.W., Ed.; Academic Press: Boston, MA, USA,
2016; pp. 1–25.
5. Palmer, G.H. Barley and malt. In Handbook of Brewing, 2nd ed.; Priest, F.G., Stewart, G.G., Eds.; Taylor &
Francis: Baca Raton, FL, USA, 2006; pp. 139–160.
6. Lekkas, C.; Stewart, G.G.; Hill, A.; Taidi, B.; Hodgson, J. The importance of free amino and nitrogen in wort
and beer. Tech. Q. Master Brew. Assoc. Am. 2005, 42, 113–116.
7. Speers, A. Brewing fundamentals, Part 3: Yeast settling—Flocculation. Tech. Q. Master Brew. Assoc. Am. 2016,
53, 17–22.
8. Gallone, B.; Steensels, J.; Prahl, T.; Soriaga, L.; Saels, V.; Herrera-Malaver, B.; Merlevede, A.; Roncoroni, M.;
Voordeckers, K.; Miraglia, L.; et al. Domestication and divergence of Saccharomyces cerevisiae beer yeasts. Cell
2016, 166, 1397–1410. [CrossRef] [PubMed]
9. Pulvirenti, A.; Nguyen, H.-V.; Caggia, C.; Giudici, P.; Rainieri, S.; Zambonelli, C. Saccharomyces uvarum,
a proper species within Saccharomyces sensu stricto. FEMS Microbiol. Lett. 2000, 192, 191–196. [CrossRef]
[PubMed]
10. Rainieri, S.; Zambonelli, C.; Kaneko, Y. Review Saccharomyces sensu stricto systematics, genetic diversity and
evolution. J. Biosci. Bioeng. 2003, 96, 1–9. [CrossRef]
11. Libkind, D.; Hittinger, C.T.; Valério, E.; Gonçalves, C.; Dover, J.; Johnston, M.; Gonçalves, P.; Sampaio, J.P.
Microbe domestication and the identification of the wild genetic stock of lager-brewing yeast. Proc. Natl.
Acad. Sci. USA 2011, 108, 14539–14544. [CrossRef] [PubMed]
12. Bing, J.; Han, P.J.; Liu, W.Q.; Wang, Q.M.; Bai, F.Y. Evidence for a Far East Asian origin of lager beer yeast.
Curr. Biol. 2014, 24, R380–R381. [CrossRef] [PubMed]
13. Peris, D.; Sylvester, K.; Libkind, D.; Gonçalves, P.; Sampaio, J.P.; Alexander, W.G.; Hittinger, C.T. Population
structure and reticulate evolution of Saccharomyces eubayanus and its lager-brewing hybrids. Mol. Ecol. 2014,
23, 2031–2045. [CrossRef] [PubMed]
14. Baker, E.C.; Wang, B.; Bellora, N.; Peris, D.; Hulfachor, A.B.; Koshalek, J.A.; Adams, M.; Libkind, D.;
Hittinger, C.T. The genome sequence of Saccharomyces eubayanus and the domestication of lager-brewing
yeast. Mol. Biol. Evol. 2015, 32, 2818–2831. [CrossRef] [PubMed]
15. Sills, A.M.; Stewart, G.G. Production of amylolytic enzymes by several yeast species. J. Inst. Brew. 1982, 88,
313–316. [CrossRef]
16. Sills, A.M.; Sauder, M.E.; Stewart, G.G. Isolation and characterization of the amylolytic system of
Schwanniomyces castellii. J. Inst. Brew. 1984, 90, 311–314. [CrossRef]
17. Bisson, L.F.; Coors, D.M.; Frankel, A.L.; Lewis, D.A. Yeast sugar transporters. CRC Crit. Rev. Biochem.
Mol. Biol. 1993, 284, 259–308. [CrossRef] [PubMed]
18. Zheng, X.; D’Amore, T.; Russell, I.; Stewart, G.G. Factors influencing maltotriose utilisation during brewery
wort fermentations. J. Am. Soc. Brew. Chem. 1994, 52, 41–47.
19. Russell, I.; Stewart, G.G. Transformation of maltotriose uptake ability into a haploid strain of
Saccharomyces spp. J. Inst. Brew. 1980, 86, 55–59. [CrossRef]
Beverages 2016, 2, 34 17 of 18
20. Stewart, G.G.; Zheng, X.; Russell, I. Wort sugar uptake and metabolism—The influence of genetic and
environmental factors. In Proceedings of the 25th Congress of the European Brewery Convention, Brussels,
Belgium, 14–18 May 1995; pp. 403–410.
21. Sherman, F.; Fink, G.R.; Lawrence, C.W. Methods in Yeast Genetics; Cold Spring Harbour Laboratory:
Cold Spring Harbor, NY, USA, 1979.
22. Gilliland, R.B. Saccharomyces diastaticus—A starch fermenting yeast. J. Inst. Brew. 1966, 72, 271–275. [CrossRef]
23. Erratt, J.A.; Stewart, G.G. Genetic and biochemical studies on yeast strains able to utilize dextrins. J. Am. Soc.
Brew. Chem. 1978, 36, 151–161.
24. Erratt, J.A.; Stewart, G.G. Fermentation studies using Saccharomyces diastaticus yeast strains.
Dev. Ind. Microbiol. 1981, 22, 577–586.
25. Erratt, J.A.; Stewart, G.G. Genetic and biochemical studies on glucoamylase from Saccharomyces diastaticus.
In Current Developments inYeast Reserch; Stewart, G.G., Russell, I., Eds.; Pergamon Press: Toronto, ON, Canada,
1981; pp. 177–183.
26. Hammond, J.R.M. Genetically-modified brewing yeasts for the 21st century: Progress to date. Yeast 1995, 11,
1613–1627. [CrossRef] [PubMed]
27. Baxter, E.D. The application of genetics in brewing. Fermentation 1995, 8, 307–311.
28. Sofie, M.G.; Saerens, C.; Duong, T.; Nevoigt, E. Genetic improvement of brewer’s yeast: Current state,
perspectives and limits. Appl. Microbiol. Biotechnol. 2010, 86, 1195–1212.
29. Lekkas, C.; Stewart, G.G.; Hill, A.E.; Taidi, B.; Hodgson, J. Elucidation of the role of nitrogenous wort
components in yeast fermentation. J. Inst. Brew. 2007, 113, 3–8. [CrossRef]
30. O’Connor-Cox, E.S.C.; Ingledew, W.M. Wort nitrogenous sources—Their use in brewing yeasts: A review.
J. Am. Soc. Brew. Chem. 1989, 47, 102–108.
31. Casey, G.P.; Magnus, C.A.; Ingledew, W.M. High-gravity brewing: Effects of nutrition on yeast composition,
fermentative ability and alcohol production. Appl. Environ. Microbiol. 1984, 48, 639–646. [PubMed]
32. Jones, M.; Pierce, J. Absorption of amino acids from wort by yeasts. J. Inst. Brew. 1965, 70, 307–315. [CrossRef]
33. Jones, M.; Pierce, J. Nitrogen requirements in wort—Practical applications. In Proceedings of the 10th
Congress of the European Brewery Convention, Brussels, Belgium, 1965; pp. 182–194.
34. Stewart, G.G.; Hill, A.E.; Lekkas, C. Wort FAN—Its characteristics and importance during fermentation.
J. Am. Soc. Brew. Chem. 2013, 71, 179–185. [CrossRef]
35. Lekkas, C.; Hill, A.E.; Taidi, B.; Hodgson, J.; Stewart, G.G. The role of small peptides in brewing fermentations.
J. Inst. Brew. 2009, 115, 134–139. [CrossRef]
36. Ingledew, W.M.; Patterson, C.A. Effect of nitrogen source and concentration on the uptake of peptides by a
lager yeast in continuous culture. J. Am. Soc. Brew. Chem. 1999, 47, 102–108.
37. Cooper, D.J.; Stewart, G.G.; Bryce, J.H. Yeast proteolytic activity during high and low gravity wort
fermentation and its effect on head retention. J. Inst. Brew. 2000, 106, 197–201. [CrossRef]
38. Russell, I.; Stewart, G.G. Distilling yeast and fermentation. In Whisky: Technology, Production and Marketing,
2nd ed.; Russell, I., Stewart, G.G., Eds.; Elsevier: Boston, MA, USA, 2014; pp. 123–143.
39. Stewart, G.G.; Russell, I. An Introduction to Brewing Science and Technology, Series III: Brewer’s Yeast, 2nd ed.;
Institute of Brewing and Distilling: London, UK, 2009.
40. Quain, D.E.; Thurston, P.A.; Tubb, R.S. The structural and storage carbohydrates of Saccharomyces cerevisiae,
changes during fermentation of wort and a role for glycogen catabolism in lipid biosynthesis. J. Inst. Brew.
1981, 87, 108–111.
41. Soares, E.V. Flocculation in Saccharomyces cerevisiae: A review. J. Appl. Microbiol. 2011, 110, 1–18. [CrossRef]
[PubMed]
42. Speers, R.A. A review of yeast flocculation. In International Brewers Symposium: Yeast Flocculation, Vitality and
Viability; Master Brewers Association of the Americas: St. Paul, MN, USA, 2012.
43. Stewart, G.G.; Russell, I. Yeast flocculation. In Brewing Science; Pollock, J.R.A., Ed.; Academic Press: Toronto,
ON, Canada, 1981; Volume 2, pp. 61–92.
44. Guinard, J.-X.; Lewis, M.J. Study of the phenomenon by agglomeration in the yeast Saccharomyces cerevisiae.
45. Stratford, M. Yeast flocculation: Calcium specificity. Yeast 1989, 5, 487–498. [CrossRef]
46. Stewart, G.G. Co-flocculation of brewer’s yeast. Tech. Q. Master Brew. Assoc. Am. 1972, 9, 25.
Beverages 2016, 2, 34 18 of 18
47. Eddy, A.A. Composite nature of the flocculation process of top and bottom strains of Saccharomyces cerevisiae.
48. Zarattini, R.; de Wiliams, J.W.A.; Ernandes, J.R.; Stewart, G.G. Bacterial-induced flocculation in selected
brewing strains of Saccharomyces cerevisiae. Cerevisia Biotech. 1993, 18, 65–70.
49. Peng, X.; Sun, J.; Iserentant, D.; Michiels, C.; Verachtert, H. Flocculation and coflocculation of bacteria by
yeasts. Appl. Microbiol. Biotechnol. 2007, 121, 31–37. [CrossRef]
50. Stewart, G.G.; Russell, I.; Garrison, I. Further studies on flocculation and co-flocculation in brewer’s yeast
strains. Proc. Am. Soc. Brew. Chem. 1973, 31, 100–106.
51. Thorne, R.S.W. Some aspects of yeast flocculation. In Proceedings of the 3rd Congress of the European
Brewery Convention, Brussels, Belgium, 1951; pp. 21–34.
52. Gilliland, R.B. The flocculation characteristics of brewing yeasts during fermention. In Proceedings of the
3rd Congress of the European Brewery Convention, Brussels, Belgium, 1951; pp. 35–58.
53. Russell, I.; Stewart, G.G. Revised nomenclature of genes that control flocculation. J. Inst. Brew. 1980, 86,
120–121. [CrossRef]
54. Goffeau, A.; Barrell, B.G.; Bussey, H.; Davis, R.W.; Dujon, B.; Feldmann, H.; Galibert, F.; Hoheisal, J.D.;
Jacq, C.; Johnston, M.; et al. Life with 6000 genes. Science 1986, 274, 563–567. [CrossRef]
55. Querol, A.; Bond, A. The complex and dynamic genomes of industrial yeasts. FEMS Microbiol. Lett. 2009,
293, 1–10. [CrossRef] [PubMed]
56. Halme, A.; Bumgarner, S.; Styles, C.; Fink, G.R. Genetic and epigenetic regulation of the FLOI gene family
generates cell-surface variation in yeast. Cell 2004, 116, 405–415. [CrossRef]
57. Stewart, G.G.; Maskell, D.L.; Speers, A. Brewing fundamentals—Fermentation. Tech. Q. Master Brew.
Assoc. Am. 2016, 53, 2–22.
58. Stewart, G.G. Biochemistry of brewing. In Biochemistry of Foods, 3rd ed.; Eskin, N.A.M., Shahindi, F., Eds.;
Elsevier: Boston, MA, USA, 2013; pp. 291–318.
59. Stewart, G.G. Yeast mitochondria—Their influence on brewer’s yeast fermentation and medical research.
Tech. Q. Master Brew. Assoc. Am. 2014, 51, 3–11.
60. Basso, R.F.; Alcarde, A.R.; Portugal, C.B. Could non-Saccharomyces yeasts contribute on innovative brewing
fermentations? Food Res. Int. 2016, 86, 112–120. [CrossRef]
61. Varela, C. The impact of non-Saccharomyces yeasts in the production of alcoholic beverages. Appl. Microbiol.
Biotech. 2016, 100, 9861–9874. [CrossRef] [PubMed]
62. Tsukada, M.; Õhsumi, Y. Isolation and characterization of autophagy—Defective mutants of Saccharomyces
cerevisiae. FEBS Lett. 1993, 333, 169–174. [CrossRef]
63. Rheinhardt, K.; Dowling, D.K.; Morrow, E.H. Mitochondrial replacement, evolution, and the clinic. Science
2013, 341, 1345–1346. [CrossRef] [PubMed]
© 2016 by the author; licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC-BY) license (http://creativecommons.org/licenses/by/4.0/).

Beverages 02 00034 v2

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Beverages 02 00034 v2

Uploaded by

Copyright:

Available Formats

beverages

Academic Editor: Edgar Chambers IV

Beverages 2016, 2, 34; doi:10.3390/beverages2040034 www.mdpi.com/journal/beverages

Table 1. Typical sugar spectrum of wort.

Table 2. Differences between ale and lager yeast strains.

4. Uptake and Metabolism of Wort Sugars

• the structural gene for α-glucosidase (maltase) (MALS)

Maltose α‐glucosidase Maltotriose

Glucose (G) Facilitated Fructose (F) Sucrose (G-F)

Group C, Proline (mmol/L)

Fermentation Time (hours)

Group A Group B Group C Proline

Group A Group B Group C Group D

7. Yeast Cropping—Flocculation and/or Centrifugation

8. Non-Saccharomyces Yeasts in Brewing

You might also like