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Saccharomyces spp. role in brewing process and its serial repitching impact
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In: Beer: Production, Consumption and Health Effects ISBN: 978-1-63485 -704-8
Editor: William H. Salazar © 2016 Nova Science Publishers, Inc.
Chapter 5
SACCHAROMYCES S PP. ROLE IN BREWING PROCESS
AND ITS SERIAL REPITCHING IMPACT
Cátia Martins1,2, Tiago Brandão3,4,
Adelaide Almei da2 and Sílvia M. Rocha1,
1Departamento de Química and QOPNA,
Universidade de Aveiro, Aveiro, Portugal
2Departamento de Biologia and CESAM,
Universidade de Aveiro, Aveiro, Portugal
3Unicer Bebidas, SA, Leça do Balio, Portugal
4President of the European Brewing Convention, russels, Belgium
ABSTRACT
Beer is the second most consumed beverage in the world, accounting ca. 35%
of all recorded alcohol consumed in 2010. Brewing industry is extremely competitive,
highlighting the importance of constant innovation. The success of global beer
commercialization is its intrinsic quality that depends of a network of variables, namely
raw materials, yeast strain and the brewing process itself. The biochemical performance
of yeasts, usually Saccharomyces spp., is one of the parameters with significant
importance in brewing once it limits the alcoholic content of final beer and produces
different metabolites with crucial impact on beer aroma and flavor. It will be given focus
to the different metabolic pathways with si gnificant impact on beer’s aroma profile,
namely the carbohydrates and nitrogen compounds metabolism; and also the formation of
aldehydes, higher alcohols, vicinal diketones, esters, fatty acids, sulfur and terpenic
compounds. Environmental changes can promote stress in yeasts along brewing. The
stress factors will be systematized in this book chapter, as well as the yeasts cellular
effects and their biological response. Furthermore, brewing companies tend to reused (or
repitch) yeasts several times to minimize resources costs, while maintaining quality of the
final product. Also, different approaches will be described in order to monitor yeast
quality. Therefore, the impact of yeasts repitching along brewing will be systematized,
considering its associated advantages and drawbacks. In summary, this book chapter aims
Corresponding Author address; Email: [anonimizat].
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Cátia Martins, Tiago Brandão, Adelaide Almeida et al. 214
to elucidate about the raw materials and the brewing process, and also to give an
overview of the Saccharomyces spp. metabolism (special attention will be done on its
impact on beer aroma profile), as well as to understand the effects of serial repitching on
yeasts’ behavior.
Keywords : Saccharomyces spp., metabolism, repitching, beer, raw materials, brewing
process
BEER GENERAL CHEMICAL COMPOSITION AND CONSUMPTION
The name of beer comes from the Latin word “bibere” that means to drink. Historically,
beer was started to be produced by the summarians in Mesopotamia (now Iraq) from more
than 6000 years ago. In this region, the oldest surviving beer recipe was found in cuneiform
(clay tablets) that refers it in a hymn to Ninkasi , the patron goddess of brewing. In that period,
it is known that the beer was made by women bakers, from barley via bread. Then, a new
civilization appeared in Mesopotamia, around 1770 BC, the Babylonians, which had beer in
their diet, made from barley or emmer ( Triticum dicoccum , wheat’s ancient form), and they
developed more than twenty different beers, ruled by Hammurabi code. This brewing
tradition had spread from Mesopotamian region to Egypt, where beer was the primary
beverage for Pharaoh and all peasants (fact recorded in several pictures and sculptures), and
was also considered as a product with medical and religious purposes. Egyptians’ brewing
techniques were passed to Greeks and Romans (around 500 BC), but beer was not well
accepted by the privileged classes that preferred to drink wine. In the Middle Age, European
monasteries had an important contribution in the development of modern beer, namely in the
introduction of hops flowers to flavor and preservation of beers (rule written in 822 by abbot
Adalhard in a monastery in Corbie, northern France ) (Barth, 2013). At this time, beer was
used as commodity for commerce, payment and tax es, being disseminated in Europe. From
16th century, beer became a global beverage due to the universal expansion of the brewing
techniques. The Industrial Revolution (18th century) had a huge impact on the brewing
industry once the beer production at an artisanal manufacture was converted into an industrial
scale. Several scientific advances were recorded, such as the development of hydrometers and
thermometers, saccharometer, and artificial refrigeration, which allowed a greater knowledge
about the final product and better process control. Moreover, between 1855 and 1876, Louis
Pasteur discovered the pasteurization methods to prevent the development of undesirable
microorganisms. Also, Emil Christian Hansen had an important contribute to brewing
science, once he isolated and classified the yeasts as bottom-fermenting lager yeasts
(Saccharomyces uvarum , or also called Saccharomyces carlsbergensis ) and top-fermenting
ale yeasts ( Saccharomyces cerevisiae ). His work contributed to the beer quality, and also the
flavor standardization.
In the 21st century, the great developments of the brewing industry allow that consumers
have hundreds of different beers available on markets, being the actual trends concerning
about the quality and stability control of beer, as well as the developing microbrewing
industries (in Europe, there was an increase of 16%, from 2013 to 2014) and the development
of innovative products (e.g., non-alcoholic and flavored-mixed beers) to attract consumers.
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Saccharomyces spp. Role in Brewing Process … 215
The latest statistical results (Figure 1 ) report that the beer global consumption and,
consequently, its production, has been stagnating either considering the world, as well as the
European Union (EU) plus 3 countries (Norway, Switzerland and Turkey). This stagnation of
the beer consumption was not only induced by the actual economic context but there are other
major factors that are correlated such as: lower per capita consumption; higher taxes for beer
in bars, restaurants in some countries, which leads to the consumption of beer at consumer’s
house; and the consumers buy less beer premium brands. Only Asia and Africa markets have
a contrary trend, where an slight increase of beer consumption have occurred, for instance
representing attractive markets for Europe ’ exportation that increased 15% since 2008 until
2014.
Figure 1. Beer production and consumption in the European Union plus 4 countries (Croatia, Norway,
Switzerland and Turkey) and in world, between 2009 and 2014 (Kirin Beer University Report, 2015;
Walle, 2015).
Beer is a fermented beverage that is produced from several raw materials: water, hops or
other hop products (e.g., pellets or hop oil), malted grains (normally barley), and yeast. Some
other cereals can be combined with barley malt (e.g., wheat malt, unmalted cereal adjuncts )
contributing for other starch- and/or sugar-containing raw materials. Usually the final product
contains: ethanol, compounds from hops, high concentration of carbon dioxide (CO 2, around
0.5% p/p), low pH value (between 3.8 and 4.7) and low concentration of oxygen (< 0.1 ppm)
(Dragone et al., 2008). Figure 2 represents the average chemical composition of a lager beer ,
which main component is water ( ca. 90 – 94%). The major yeasts fermentation products are
ethanol and carbon dioxide. Yeasts, as well as other raw materials (derived from malt and
hops that survive wort boiling), are responsible for most of the volatile compounds formation
(responsible for the bouquet or aroma of beer), which then that can be found in beer, namely
higher alcohols (10 – 300 mg/L), organic acids (50 – 250 mg/L), esters (25 – 40 mg/L),
aldehydes (30 – 40 mg/L), vicinal diketones, sulfur compounds (1 – 10 mg/L), and terpenic
compounds (Briggs et al., 2004; Buiatti, 2009). Despite their lower amount ( ca. 0.5%),
volatile components have great impact on beer flavor, as well as their interaction with non-
volatile components, thus contributing for the achievement of the aroma characteristics of
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2009 2010 2011 2012 2013 2014
Worlwide data (x103kL)EU data (x103 hL)
YearEU production
EU consumption
Worldwide production
Worldwide consumption
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each beer style. There are also non-volatile components present in beer ( ca. 4%), such as
inorganic salts, sugars, amino acids, nucleotides, polyphenols, vitamins, lipids, and hop
resins. Additionally, the majority of the carbohydrates, present in wort, are metabolized by
yeasts, being only detected the residues that yeasts can not metabolize (e.g., D-ribose, L-
arabinose, or D- xylose) or those that come from ‘primings’ addiction, that are other sugar
sources without being barley (Briggs et al., 2004; Boulton, 2013). Thus, hundreds of different
compounds were already identified in beer, which belong to different classes, being their
concentration dependent of the different raw materials that are used, the different brewing
steps applied by each brewing company, as well of the yeast strain (Boulton, 2013).
Figure 2. Average chemical composition of a lager beer based on data collected from (Vanderhaegen et
al., 2006; Eßlinger, 2009; Eßlinger and Narziß, 2012).
1. BREWING PROCESS
This topic is divided in two sections, the first one will comprise the four raw materials
used in beer production, and then, the complexity of the brewing process will be explained
through the description of the different steps, being each one clarified from a scientific
point of view. The brewing process comprises several steps, represented in Figure 3, which
can be sectioned in three different and independent zones within a company: brewhouse;
fermentation, and storage cellar; and filtration and filling. Furthermore, it is also indicated in
which step of the process the different raw materials can be added.
Extract 4 %
Ethanol 3-5 %
Carbohydrates (80 –85 %)
Proteins (4.5 –5.2 %)
Minerals (3 –4 %)
Bitter substances ( 2 –3 %)
Organic acids (0.7 –1.0 %)CO20.5 %Water
90-94%Volatiles 0.5 %
Higher alcohols
Volatile organic acids
Esters
Aldehydes
Vicinal diketones
Sulfur compounds
Terpenic compounds
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Figure 3 . Brewing process workflow, in which is indicated the steps where raw materials are added.
1.1. Raw Materials
In order to produce beer, there are four main ingredients: malted grains, hops, water, and
yeast. A brief description of each raw material will be presented, as well as some key
concepts that are important to understand the brewing process.
Malted Grains
The cereal grain that is more often used is barley ( Hordeum vulgare ), due to the easier
regulation and control of the germination process of these grains comparing with others; it has
a favorable price; and allows the creation of innovative beer styles through special coloring
and flavoring (O’ Rourke, 1999) . The barley seeds are the raw material used in the production
of malted barley (starch ’ source and enzymes required for starch break down) (Barth, 2013) .
The main carbohydrates presen t in barley are the α -glucans, namely amylose and amylopectin
of starch (55 – 57%). Moreover, barley proteins (9 – 10.5%) are important for beer’ stability ,
foam and taste, and yeast nutrition; while lipids are only partially used on malting process.
Wort
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Another important constituents are phosphates ( ca. 0.3%), minerals (2.5 – 3.5%), vitamins
(ca. 0.5 × 10-3%), and phenolic substances ( ca. 0.2%) (Eßlinger and Narziß, 2012). Mixtures
of malted and unmalted barley can be used, nevertheless the portion of unmalted grains
should not exceed 50%, because it can promise the activity of the endogenous hydrolases,
present in malt, during the mashing process (Meilgaard, 1976; Eßlinger, 2009) .
Other cereal grains (e.g., wheat, maize, rice, sorghum) can be added as adjuncts, in order
to have different sources of extract (fermentable sugars). They can be unprocessed raw
cereals or semi-purified extracts . Regarding the solid cereal adjuncts (‘mash tun’ adjuncts), it
is required some prior processing (e.g., milling and mashing) for starch release and/or is
required the use of protein-rich barley malts with very high enzyme contents (amylases)
(Briggs et al., 2004; Eßlinger and Narziß, 2012). In the case of liquid extracts (soluble sugars
or syrups), they may be added during boiling wort (‘copper’ -or ‘kettle’ -adjuncts), increasing
and adjusting the wort fermentability that is required for high-gravity brewing; or they can be
added post-fermentation (called ‘primings’) for flavoring and/or coloring the finished beer
(usually that undergo a secondary conditioning process) (Briggs et al., 2004).
Partially unmalted wheat ( Triticum aestivum ) can be added to the mash, being its
composition similar to barley: contains 65% of starch and other carbohydrates, 12 – 14% of
proteins, and 1.7% of fat. Maize or corn (Zea mays L.) is used as a malt replacement (requires
prior starch purification through dry and wet milling), with extract yields of 87 – 91% (after
removing the oil-rich embryo), 8.5 – 9% of proteins, and residual fat content (< 1%)
(Eßlinger, 2009; Eßlinger and Narziß, 2012). Rice ( Oryza sativa L.) requires a pre-processing
to be used as adjunct, and it is composed by extract 93 – 95%, protein 8 – 9%, and fat 0.5 –
0.7%. Sorghum ( Sorghum vulgare ) can be used as a malt additive, however it has only
regional importance in Africa (Eßlinger and Narziß, 2012).
In the case of some cereals such as sorghum, rice, or maize, it is required highe r
temperatures (60 – 90°C) along mashing, comparing with barley (50 – 70°C), thus requiring a
separate treatment in a cereal cooker (Boulton, 2013).
Water
Another important raw material is water, which content varies between 90 – 94% in beer.
As it is the main component of beer, its chemical and biological composition requires high
quality control , namely be ‘aesthetic’ (clear, colorless, neutral in odor and taste), without
presence of pathogens and heavy metals (namely iron and manganese), and should not be
corrosive (Briggs et al., 2004; Eßlinger and Narziß, 2012). Water contributes mainly with the
inorganic constituents present in beer, having impact on the palability of the finished beer,
once the ionic composition of the water varies with the geographical area (Boulton, 2013).
The major cations are the potassium, sodium, calcium, and magnesium; while the major
anions are chloride, sulphate, nitrate, and phosphate. There are many ions that are essential
for yeast growth, even at trace amount (e.g., zinc, copper or iron); while in case of other
constituents, their larger amounts may be toxic to yeasts (e.g., nitrate), being their content
limited by law (Briggs et al., 2004).
Hops
Hops are the dried cones of the female part of the plant Humulus lupulus . They can be
added as flowers or by the form of pellets/hop extract, depending on the wanted amount of
resin and essential oils. They are widely used as beer raw material due to several reasons,
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namely they give a bitter taste and specific aroma, they improve and stabilize the beer foam,
and also increase the biological shelf-life through their antibacterial properties (especially
against Gram-negative, but also against Gram-positive bacteria) (Eßlinger, 2009). The hops
bitter components are α -acids (humulones, 3 – 17%), β -acids (lupulones, 2 – 7%) and the
oxidation products of bitter acids, namely, the soft and hard resins . The α -acids are the most
important components because of their high bitterness potential (Eßlinger, 2009; Eßlinger and
Narziß, 2012). In conventional brewing, hops are added to the sweet wort and are boiled
during one hour and half to two hours, in which the α -acids are isomerized into the iso- α-
acids, and the essential oil components (up to 3%) are vaporized. Nevertheless, a portion of
choice ‘aroma’ hops can be added later (‘kettle -hop aroma’), in order to replace the loss of
some volatile compounds, mainly in lager beers. Moreover, dry hopping technique can be
applied, which corresponds to the introduction of dry hops in beer, either in casks or
conditioning tanks , thus increasing the ‘dry -hop aroma’ (mainly applied in ale beers
production) (Briggs et al., 2004).
Hop essential oil is present in the lupulin glands, being responsible for the characteristic
pleasant aroma of hops, and composed by a complex mixture of more than 300 compounds .
Each individual variety has specific compositions and concentrations of components.
Nevertheless, hydrocarbons are the major fraction (50 ± 80%), being the major components
the monoterpene myrcene (17 – 37%) and the sesquiterpenes β -caryophyllene and
humulene (Briggs et al., 2004; Eßlinger, 2009). The ripeness of the cones can be reflected
in the myrcene percentage, while humulene/caryophyllene ratio is considered a varietal
characteristic. The hydrocarbons components are more volatile and then more easily lost
during wort boiling when comparing with the oxygenated fraction, which are more probable
to be found in beer. The oxygenated fraction have intense odors and aromas, and can
contribute with different aromas to beer (e.g., citrus, floral, spicy), being linalool the major
impact compound for hop aromatic beers (Boulton, 2013).
Hops flavor is conditioned by several parameters, such as growing temperature, soil,
moisture, and other climate issues. As small craft brewers cannot have a big hop inventory,
their product cannot be so consistent between batches (Barth, 2013).
Yeast
Brewing yeasts are unicellular fungi, that reproduce vegetatively by multilateral budding
or fission, and usually they belong to the Saccharomycetaceae family and Saccharomyces
genus (Eßlinger and Narziß, 2012). They are able to ferment either in anaerobic or semi-
anaerobic conditions, and are also able to ferment one or more sugars (e.g., glucose, fructose )
(Barnett, 1992).
There are two types of Saccharomyces yeasts involved in beer fermentation: top-
fermenting (or ale) and bottom-fermenting (or lager) yeasts, which vary according to their
fermentative ability, rate of sugar utilization, tolerance to temperature , flocculation
characteristics and volatiles’ profile (Eßlinger, 2009). While ale yeasts are usually classified
as S. cerevisiae; lager yeasts’ classification is still a controversy, but S. pastorianus, S.
carlsbergensis and S. uvarum are classified as lager yeasts. There are other yeasts species,
namely some of the genus Brettanomyces , that are commonly used in a Belgian ale style
called lambic (Barth, 2013).
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1.2. Brewing Process Steps
The first step on beer production (Figure 3) is the conversion of grains or seeds of
different plants (barley is the most commonly used) into malt through a malting process,
which is usually performed by an independent company. Malting involves three main steps:
steeping, germination, and kilning. Firstly, the grains are immersed in water (steeping), being
allowed their germination under controlled temperature and humidity. It is promoted the
activation of some enzymes (amylolytic, proteolytic, and cellulytic) present in barley, leading
to the partial degradation of the starch (present in the grains’ endosperm ). The germination is
stopped by heating (kilning), forming the green malt in its finished form (water content of
malt of ca. 4%), which then can be stored in silos (Eßlinger and Narziß, 2012). Furthermore,
Maillard reactions can also be promoted in the kilning step, leading to formation of
darker malts. These malts can be employed to contribute with different and specific
aromatic/coloring compounds to beers, although enzymes might not resist the applied high
temperatures.
The brewing process itself starts at the brewhouse, in which the wort is produced through
several main steps, namely milling, mashing, filtration, wort boiling, and wort clarification.
The malt grains and other solid adjuncts are broken down and reduced into small particles,
through a mill, obtaining thick flour called grist. The milling conditions should be adapted in
order to obtain the best yield of the obtaining extract during mashing.
Grist (in combination with other solid adjuncts or enzymes) is added to hot water (step
called mashing) at controlled time (or periods of time), temperature (or range of temperatures)
and pH, in order to promote the most efficient hydrolysis of starch and other biomolecules ,
e.g., proteins and lipids. At high temperatures (around 65șC), the starch granules start to
gelatinize, allowing an easier access to amylase enzymes ( – and -amylase), which initiates
the starch degradation (breaking of the -1,4 glycosidic bonds) into fermentable sugars.
Moreover, some protein degradation can occur due to some remaining peptidase activity
(most of these enzymes which have high concentrations in malt before kilning, are denatured
in the kilning step). Mashing can take from 2 to 4 hours, and finish at approximately 75șC.
The variation of time, pH and temperature, as well as the presence of coenzymes, cofactors,
substrate concentration or presence of activators and inhibitors, will allow the activation and
inactivation of enzymes, in a sequential manner along mashing step. Sweet wort is obtained
after mashing, being composed mainly by fermentable sugars (e.g., glucose, malto se,
maltotriose, and sucrose) and non-fermentable sugars (such as small branched dextrins), and
the sum of these sugars corresponds to the wort extract, that is usually measured in degrees
Plato (șP): sugar content expressed in grams of sugar per 100 grams of wort (Huuskonen et
al., 2010).
Very high gravity (VHG) fermentations are a current brewing trend that allows to obtain
beer with 14 – 16% (v/v) of ethanol content instead of 4 – 5% in low or normal gravity
fermentations. The main difference is related with the amount of dissolved solids in wort, in
which high amount of fermentable sugars are available (18 șP or more, instead of 11 – 12 șP).
VHG fermentations have several advantages such as: considerable reducing amount of water
need, increased productivity (more alcohol content), lower energy needs, less capital loss,
among others; thus increasing the overall brewery productivity. However, the high osmo-
stress conditions that yeasts are submitted in VHG fermentations, compromise their
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fermenting ability and viability, as recently review by Puligundla et al., 2011. This stress can
lead to an unbalanced flavor composition to beer (mainly overproduction of acetate esters )
(Anderson and Kirsop, 1974; Saerens et al., 2008; Yu et al., 2012) due to changes the
expression of several genes associated to the metabolic pathways as observed by Nam et al.,
2009 . The increase of fermentation temperature can lead to higher fermentation rate, but can
promote higher stress to yeasts. Nevertheless, the efficiency of the fermentation can be
increased if the oxygen conditions were improved, without interfering with the physiological
conditions of yeast and quality of beer, namely using preoxygenation of yeast combined with
aeration as Verbelen et al., 2009c suggests.
Sweet wort can be filtrated to separate an insoluble part (spent grains – currently sold for
animal feed) and the soluble part (wort), usually by the use of a lauter tun. This step takes 2 –
3 hours and occurs at 75 – 80șC. Furthermore, wort is vigorously boiled around one hour and
half to two hours, in a vessel called kettle, in which hops are added. The boiling process leads
to loss of most of the hop oil volatile components (Simpson, 1993; Kishimoto et al., 2005) ,
which can lead to th e addition of ‘aroma’ hops in the end of boiling, the ‘kettle -hop aroma.’
The boiling process is performed in order to allow: the solubilisation and transformation of
the hops substances, namely the extraction of -acids and their isomerization into iso- -acids
that contribute for the bitter taste (Schönberger and Kostelecky, 2011); elimination of
unpleasant volatile compounds (mainly derived from malt; e.g., decomposition of S-
methylmethionine into dimethyl sulfide, that has a cooked vegetable flavor (Barth, 2013));
sterilization of the wort; precipitation of proteins with high molecular weight (trub) and the
formation of complexes between proteins and polyphenols (important in beer haze); fixation
of the final concentration of wort (original extract); denaturation of the exogenous enzymes
and malt, for guarantee unforeseen alterations along the following brewing steps. At this step,
Maillard reactions are also promoted due to the applied high temperatures, which lead to
melanoidins formation, which contributes to wort color. Moreover, liquid adjuncts can be also
added at this phase. Subsequently, a decanter (gravity ’s action) or whirlpool (centripetal
force) is used to separate the wort from trub and hops components that did not solubilize.
Wort is then cooled until 7 – 22șC (depending on the fermentation temperature) and airy in
sterile conditions. Thus, wort is a rich and complex source of nutrients for yeasts, being
mainly composed by carbohydrates ( ca. 90 – 92% of the total solids) (Barth, 2013). The
nitrogen-compounds represent ca. 5% of the total solids, from which 85% are represented by
free amino acids, peptides, and proteins. In fact, the wort amino acids and small peptides
come from malts and adjuncts (that contain nitrogen) and their content is expressed as free
amino nitrogen (FAN), representing the main source of assimilable nitrogen by yeasts (Briggs
et al., 2004; Boulton, 2013).
Prior to fermentation, it is needed to perform yeast propagation, whereas yeasts are
transferred consequently into larger wort volumes, until is obtained large amount of yeasts
needed to pitch (pitching rate). Cold oxygenated wort is inoculated with yeasts (pitching)
during its transfer into the fermenter. The fermentation takes place in closed vessels, usually
cylindroconical vessels, being the selection of yeast strain dependent of the required beer
type: ale or lager. Typical ale fermentation occurs at 16 – 25șC, while lager fermentation is
usually performed at 7 – 14șC, requiring more days to be completed. Normally, yeasts tend to
reproduce between four- and six fold, during fermentation. Throughout this step, yeasts
uptake amino acids and sugars from the wort, which metabolize for reproduction and
production of energy and two main products, ethanol and CO 2 (Figure 4). Moreover, several
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other biochemical reactions are mediated by yeasts, which lead to the release of hundreds of
chemical compounds that not only can contribute to beer flavor, but also to other beer
desirable characteristics. The different metabolic pathways associated to yeasts fermentation
will be further explained in detail.
Figure 4. Summary of the main biochemical reactions mediated by yeasts in the brewing fermentation
(adapted from (Lewis and Young, 1995) ).
The subsequent step is the beer maturation, where green beer is transformed into
its finished form. It is enabled the adjustment of the beer carbonation, once the remaining
yeasts perform a secondary fermentation; moreover, some off-flavors components, like
vicinal diketones (VDK’s) diacetyl (2,3 -butanedione) and 2,3-pentanedione, are converted
enzymatically by yeasts, reducing their content until it is below the flavor threshold.
Furthermore, after yeasts had converted all the available sugars and other nutrients, there is a
reduction of the convention currents (promoted by CO 2 formation), the metabolism rate starts
to decrease, and yeast cells tend to adhere and form flocs – flocculation process. This inherent
characteristic of yeasts is extremely important once if it happens too late, it will interfere in
the filtration; but if it occurs too earlier, beer will have high sugars concentration, low ethanol
concentration, and presence of off-flavors (Verstrepen et al., 2003; Lodolo et al., 2008). After
flocculation process is completed, yeasts are removed from the fermenter (usually as
concentrated slurry), being transferred to a collection vessel, in which they are maintained at
4șC, until be reused again (repitching ).
Clarification is performed by the beer filtration, in order to bright it, once consumers tend
to consider bright beer as a quality parameter. There is the removal of particles down to about
0.5 μm. Nowadays, kieselguhr (light soil consisting of siliceous diatom remains) is the most
common filter. Clarification can be combined with stabilization, where beer is allowed to
stabilize at – 1 – 2șC (during 1 – 3 days), in order to promote the precipitation of proteins and
polypeptide-polyphenols complexes, thus inhibiting the haze formation and colloidal
instability. There are some stabilizing agents that can be added: to adsorb polyphenols (e.g.,
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polyvinylpolypyrrolidone – PVPP) or proteins (e.g., silica gel); to degrade proteins and
polypeptides (proteases, for instance papain); or to precipitate proteins (with tannic acid).
The last step is filling where the beer can be packed in different containers: bottles (glass
or plastic), cans, kegs, casks, or bulk tanks. Some important factors should be considered for a
suitable package, especially the barrier properties in order to prevent the CO 2 loss and
entrance of oxygen that could induce beer staling (Vanderhaegen et al., 2006). The biological
stabilization can be done at low temperatures by filtration, or high temperatures through
pasteurization (flash or tunnel).
2. SACCHAROMYCES SPP. ROLE IN THE BREWING PROCESS
This second topic is divided in four sections, the first one will comprise the
characterization of the two main yeasts types used in the brewing process (lager and ale), as
well as aspects related to their taxonomy; then the yeasts metabolic pathways associated to
the volatile compounds formation will be presented in the second sub-section; the third part
will describe the different stress factors that yeasts suffer during brewing process and their
biological response, and also the description of the different parameters (viability and vitality )
that can be used to monitor yeasts quality; and the fourth part will comprise the impact of
serial repitching on yeasts.
2.1. Saccharomyces spp. Used in the Brewing Process and Their Taxonomy
Yeasts that are used in the brewing process are mostly members of the genus
Saccharomyces , being their taxonomy classification described in Figure 5. However, some
difficulties have emerged for determination of the current taxonomy of Saccharomyces
species, once initially brewers classified yeasts according to their brewing properties (e.g.,
flocculation process). Currently, the development of molecular tools allows a more accurate
determination, and new classifications have occurred. Thus, in this sub-section, it is intended
to illustrate the progress of the yeasts taxonomy over the years, as well as to describe the
differences between the two main types of yeasts used in the brewing process: ale and lager.
Saccharomyces cerevisiae was firstly named as top (ale) fermenting brewing yeast in
1883, when Emil Hansen from Carlsberg brewery was able to characterize Saccharomyces
pure cultures, describing its unique and reproducible industrial fermentations skills.
In 1908, Hansen called the bottom (lager) brewing yeast as Saccharomyces
carlsbergensis (Barnett, 1992; Lodolo et al., 2008; Saerens et al., 2010), which sometimes is
used as a synonym for S. pastorianus . Martini and Martini, 1987 reported the existence of
three different species of Saccharomyces based on the DNA sequences : S. cerevisiae and S.
bayanus only had 22% of their genomes in common; S. pastorianus shared 52 and 72% of the
genome with S. cerevisiae and S. bayanus , respectively, which led to the possibility of S.
pastorianus to be a natural hybrid of S. cerevisiae and S. bayanus . Rainieri et al., 2006 ,
through polymerase chain reaction – restriction fragment length polymorphism (PCR-RFLP)
analysis confirmed that S. pastorianus yeasts contain components of S. cerevisiae-like
genome and non- S. cerevisiae portions of genome derived from S. bayanus and/or S. uvarum .
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Only Libkind et al., 2011 finally isolated (association with Nothofagus trees in Patagonia) and
identified S. eubayanus as the non- S. cerevisiae parent of S. pastorianus . The genome of S.
eubayanus was almost completed sequenced by Baker et al., 2015. These authors verified that
these yeasts have experienced increased rates of evolution since their hybridization with S.
cerevisiae yeasts (formation of domesticated alloploid hybrids), and also some alterations on
the expression of certain genes associated to metabolism.
Figure 5. Taxonomic classification of Saccharomyces species.
The most recent definition of the Saccharomyces sensu stricto group includes seven
different species : S. cerevisiae , S. paradoxus (encompassing S. cariocanus), S. mikatae , S.
kudriavzevii , S. arboricolus , S. eubayanus , and S. uvarum (Borneman and Pretorius, 2015). S.
cerevisiae is used to produce wine, bread, ale beer and sake; S. eubayanus that can produce
wine and cider; while S. pastorianus is involved in lager beer fermentation (Rainieri et al.,
2003).
Two distinct lineages can be associated to lager yeasts:
I. Group I: Saaz. They are taxonomically classified as S. carlsbergensis, they have
slightly greater capacity to grow at low temperatures and relatively poor fermentation
performance (lower maltose and maltotriose utilization, comparing with Frohberg
strains) (Wendland, 2014). They have retained proportionally more DNA derived
from the S. eubayanus parent (Dunn and Sherlock, 2008).
II. Group II: Frohberg. They are taxonomically classified as S. pastorianus . Their
fermentation performance is slightly better than group I, thus they are more
predominant in modern industrial-scale brewing (Gibson et al., 2013). They have
retained proportionally more DNA from the S. cerevisiae parent (Dunn and Sherlock,
2008).
Domain Eukarya
Kingdom Fungi
Phylum Ascomycota
Sub-phylum Saccharomycotina
Class Saccharomycetes
Order Saccharomycetales
Family Saccharomycetaceae
Genus Saccharomyces
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Nevertheless, both lineages are alloploid hybrids from S. cerevisiae S. eubayanus ,
which produce different esters concentrations, thus contributing with different impact on beer
aroma (Dunn and Sherlock, 2008; Gibson et al., 2013; Wendland, 2014). For instance, these
different features can be achieved through yeasts domestication. Hybridization studies hav e
been vastly explored in order to produce yeasts with different fermentation performances,
being able to be used in high-gravity brewing and serial repitching (Steensels et al., 2014a,
2014b; Baker et al., 2015; Krogerus et al., 2015).
Brewing yeasts are divided in two types: ale and lager. According to their flocculation
pattern, ale yeasts (Figure 6A) are classified as top-fermenting, while lager yeast (Figure 6B)
are bottom-fermenting. Ale yeasts show flotation, trapping the CO 2 bubbles, and going to the
top of the vessel; while lager yeasts clump together, sedimenting on the vessels ’ bottom
(Speers et al., 1992; Verstrepen et al., 2003). This purely natural property of yeast cells to
flocculate allows the brewing industry to have an cost-effective, environment-friendly, and
simple method to take apart yeast cells, at the end of fermentation, from green beer
(Verstrepen et al., 2003). Nevertheless, in modern brewery industries, the vessels type
(cylindroconical) and volume allow that ale yeasts also sediment on the bottom at the end of
fermentation (Soares, 2010; Vidgren and Londesborough, 2011). There are two possible
reasons: one is related to greater hydrostatic pressure in vessels that limits the CO 2 bubbles
size and also promote more turbulence, which leads the liberation of CO 2 bubbles from flocs;
other is related with a possible selection of ale mutants in order to have a more suitable
cropping of yeasts (Vidgren and Londesborough, 2011).
Figure 6. Flocculation pattern of ale (A) and lager (B) yeasts. Ale yeasts were initially top-fermenting
but in modern breweries they are bottom-fermenting such as lager yeasts.
Flocculation occurs in the presence of calcium ions that ensure that the lectin-like
proteins (flocculins that are only present in the flocculent strains) have the correct
conformation for the connection with the α -mannan residues (associated with mannoproteins)
of the cell wall of the neighbor cell. The flocculins synthesis seems to be associated with the
presence of FLO genes , being the yeasts’ flocculation dependent of several factors, namely
the presence of cations (e.g., calcium), pH (the optimum value is between 3.0 – 5.0),
temperature, oxygen, sugars (competition with the flocculation receptors, so their promote the
Yeasts
Yeasts
A B
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reversible dispersion of flocs), ethanol, chronological age (older cells tend to be more
flocculent), cell density (it is required a minimum threshold), and mechanical agitation
(promotes the physical proximity between yeasts cells) (Soares, 2010).
Besides the difference at the flocculation behavior, ale yeasts (e.g., S. cerevisiae) are
genetically more diverse and require high temperatures to ferment (between 16 – 25șC), while
lager yeasts (e.g., S. pastorianus ) are more conserved and need low fermentation temperatures
(between 7 – 14șC) (Saerens et al., 2010).
The phenotypic characteristic of the two yeast types can be distinguished based on
their colony morphology due to the formation of light green colonies, being ale yeasts
characterized by an undulating appearance, while lager yeasts have smooth surface (Powell
and Diacetis, 2007), when grown in a specific growth media – Wallerstein Laboratory
Nutrient Broth, WLN. The budding process is different between the yeast types, being
possible to be observed under a microscope: ale yeasts tend to be stuck together, even when a
new bud occurs; while lager yeasts tend to separate after a budding process (Eßlinger, 2009).
Yeasts can also be differentiated by fermentation characteristics, namely melibiose
(disaccharide that can be present in wort) is only metabolized by lager yeasts, once ale yeasts
do not have the enzyme α-galactosidase (melibiase) (Lodolo et al., 2008; Eßlinger, 2009) .
Moreover, the yeast strain has a huge impact on the final product, as Tosi et al., 2009 showed
through the comparison of fermentation performance of two strains ( S. cerevisiae and S.
uvarum ). They observed different patterns according to the strain used, namely the content of
compounds with direct impact on aroma, such as ethanol, ethyl acetate, and 2-phenylethanol.
Ale beers tend to be more fruity and estery, whereas lager beers do not have such a complex
aroma, which is partly sulfurous (Eßlinger, 2009).
Besides the common phenotypical approach, other attempts have been used to
distinguish the yeast strain, namely: spectroscopic methodology (pyrolysis mass
spectrometry and Fourier transform infrared spectroscopy) developed by Timmins et al.,
1998 ; genetic approach through PCR-RFLP (homologous gen es) or amplified fragment
length polymorphism (AFLP, genetic fingerprinting) used by Pope et al., 2007; metabolic
footprinting through direct injection mass spectrometry and gas chromatography time-of-
flight mass spectrometry applied by Pope et al., 2007, complemented with multivariate
analysis (PCA – Principal Components Analysis and CVA – Canonical Variants Analysis).
American Society of Brewing Chemists recommends the use of PCR of inter-delta regions of
chromosomal DNA, for genetic fingerprinting, which allows strains identification and
mutants’ detection (Boulton, 2013).
2.2. Saccharomyces spp. Metabolism
Yeasts metabolism is conditioned not only by the raw materials that are used, but also by
their initial concentration and the initial oxygen content. This section intends to explain the
importance of these parameters that can influence the yeasts metabolism. Moreover, a special
attention will be given to the metabolic pathways involved in the formation of volatile
compounds that will have further impact on beer flavor.
Saccharomyces spp. are added to oxygenated wort during its fermenter transfer, at a
certain concentration (pitching rate). Yeasts pitching rate corresponds to the amount of viable
cells per wort volume, being usual to use 10 millions of viable cells per milliliter in a 10 șP
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wort. This parameter should be monitored and revised according to the wort gravity and the
amount of viable cells (> 95%).
At the beginning, the presence of oxygen promotes the yeasts growth (cell size and
number). It is important to satisfy the yeasts requirements during fermentation through the
presence of enough dissolved oxygen in the pitched wort, in order to allow their adaptation
from aerobiosis to anaerobiosis. Moreover, oxygen is also important for yeasts survival
along repitching, once they suffer some starvation after the end of previous fermentation and
storage steps (Boulton, 2013). Indeed, new yeast cell biosynthesis needs the production of
lipids (main cell membrane component), that requires the presence of oxygen. Oxygen
promotes the formation of unsaturated molecules (like unsaturated fatty acids – UFA) and
sterols, both from acetyl coenzyme A (CoA) molecule (formed through carbohydrate
metabolism or from wort fatty acids) (Verbelen et al., 2009c).
Table 1. Major yeast volatile metabolites present in beer, their flavor threshold and
concentration in beer, as well as their aroma descriptor (Kobayashi et al., 2008; Russell
et al., 2009; Boulton, 2013)
Metabol ites Flavor threshold
(mg/L) Typical concentrations
in beers (mg/L) Aroma descriptor
Aldehydes
Acetaldehyde 25 2 – 20 Sweat, pungent
Higher alcohols
Ethanol 14000 25000 – 50000 Alcohol
n-Propanol 600 5 – 50 Alcohol
Iso-butanol 100 5 – 100 Alcohol
2-Methylbutanol 50 – 70 10 – 130 Alcohol, banana, sweet,
aromatic
3-Methylbutanol 50 – 65 25 – 180 Alcohol, banana, medicinal,
aromatic
Phenyl ethanol 40 5 – 102 Roses, sweet, fragrant
Esters
Ethyl acetate 25 – 30 5 – 35 Fruity, solvent -like
Ethyl hexanoate 0.20 0.05 – 0.20 Apple
Ethyl octanoate 0.90 0.04 – 0.50 Aniseed
Isoamyl acetate 1 0.30 – 4.00 Pear drops, banana
Isobutyl acetate 0.40 – 1.60 0.10 – 0.30 Fruity
Phenyl ethyl acetate 3.50 0.10 – 0.80 Rose or floral
Vicinal diketones
2,3-Butanedione 0.070 – 0.15 0.008 – 0.600 Butterscotch or toffee
2,3-Pentanedione 0.9 0.008 – 0.600 Honey
Sulfur compounds
Hydrogen sulfide 0.004 – 0.005 0.001 – 0.002 Rotten eggs
Dimethyl sulfide 0.050 0.010 – 0.150 Asparagus, corn, molasses
3-Methylthio -1-
propanol 1.2 0.002 – 0.050 Cauliflower, cabbage,
potatoes
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At anaerobic stage, yeasts metabolize the fermentable carbohydrates present in the wort .
Through the nutrients available (mainly carbohydrates and nitrogenous compounds), yeasts
convert them principally into ethanol and CO 2; they also produce energy (adenosine
triphosphate – ATP), and use it to generate new cell components or new biomass, as well as
the synthesis and secretion of hundreds of metabolic products into wort. If these metabolites
are volatile, they can give characteristic flavor and aromas to beer (Figure 4) (Lewis and
Young, 1995), being the major yeasts volatile metabolites presented in Table 1.
2.2.1. Metabolic Pathways
Carbohydrates Metabolism
Maltose is the main sugar present in wort, around 50 – 55% of the total amount of
carbohydrates, and its uptake depends first by its transport into yeast cell by maltose
permease; then occurs its hydrolysis, into two molecules of glucose, by maltase ( –
glucosidase) (Figure 7). Nevertheless, yeasts prefer to uptake firstly monosaccharides, such as
glucose and fructose, followed by the increase of the carbohydrates complexity, namely
disaccharides (maltose), and then trisaccharides (maltotriose) (Lodolo et al., 2008). Dextrins
are not metabolized by yeasts, so they will remain in beer, contributing for its body and
mouth feel.
Brewing yeasts have ability to grow either at aerobic and anaerobic conditions, being
capable of a fully oxidative respiratory growth. Nevertheless, relatively high amount of
fermentable carbohydrates can switch off the genes responsible for this respiratory oxidative
phosphorylation, leading to the carbohydrates catabolism via glycolysis. This phenomena is
called catabolite repression or Crabtree effect (Boulton, 2013). The high carbohydrates
concentration present in wort promote the metabolization of glucose residues by yeasts
(Figure 7), through the glycolytic pathway to produce pyruvate that, in anaerobic conditions,
is converted into ethanol and CO 2, via acetaldehyde. This produced ethanol allows yeasts
to have an advantage in natural environments due to its inhibitory effects on many
microorganisms. The CO 2 is usually recovered from the fermenters for beer carbonation
(Lewis and Young, 1995).
Aldehydes Formation
Yeasts can remove carbonyl compounds from wort (e.g., 2-methylbutanal, 3-
methylbutanal, and 3-methylpropionaldehyde), that are not desired in beer due to their
“worty” aroma. They can also lead to the production of other carbonyl compounds that are
intermediates in higher alcohols formation.
Acetaldehyde is formed from pyruvate (by pyruvate decarboxylase action, Figure 7 ),
which content can be accumulated in mid- to late fermentation, which leads to its increase
until concentrations above the flavor threshold (25 mg/L) (Table 1). Nevertheless,
acetaldehyde concentration can decrease during cool conditioning by yeasts. Some factors
contribute for high levels of acetaldehyde in beer, namely elevated: fermentation
temperatures, wort oxygenation or pitching rates. Acetaldehyde accumulation in beer is not
desired once it contributes with green apple and grassy flavors to beer (Boulton, 2013).
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Figure 7. Carbohydrates metabolism of yeasts, via aerobic and anaerobic conditions. Maltose,
maltotriose, sucrose, fructose and glucose are the main carbohydrates present in wort.
Nitrogen Compounds Metabolism
Besides the metabolism of wort carbohydrates, also nitrogen compounds (main sources in
wort: amino acids, ammonium ion, and some di- and tripeptides) are metabolized by yeasts.
Oligosaccharides and proteins are not metabolized by yeasts, so they will remain in beer, and
can contribute for haze and head formation (Boulton, 2013). Wort-free amino nitrogen (FAN)
level and composition have a great impact on the content of several yeasts metabolites,
namely: higher alcohols, esters, vicinal diketones, and hydrogen sulfide, most of them with
impact on beer flavor. The uptake of nitrogen compounds is dependent of the yeast strain,
being performed by general (GAP) or specific amino acids permeases.
Like fermentable carbohydrates, yeasts tend to metabolize amino acids according with
the following order groups: group A – arginine, asparagine, aspartate, glutamate, glutamine ,
lysine, serine and threonine; group B – histidine, isoleucine, leucine, methionine and valine ;
group C – alanine, glycine, phenylalanine, tyrosine, tryptophan, and ammonia (only absorbed
after total consumption of amino acids from group A); and group D – proline (Lodolo et al.,
2008). Moreover, Procopio et al., 2013 evidenced that the amino acid uptake by yeasts is
correlated with beer flavor profile, where leucine, isoleucine, valine, glutamine, cysteine, and
proline were the amino acids that had more impact on the synthesis of aroma-active
compounds in fermentations performed by S. pastorianus .
Higher Alcohols Formation
The amino acids from wort can be incorporated directly to protein production, or they can
be metabolized leading to formation of higher/fusel alcohols. Higher alcohols formation can
Maltose
PyruvatePyruvateGlucose
Glycolysis
AcetaldehydeEthanol
CO2
Acetate
Acetyl-CoA Acetyl-CoAEthanolGlucose
Maltose
MaltoseCO2
TCAFructoseHexose
transporter
Fructose
MaltotrioseMaltotrioseSucrose Glucose + FructoseInvertase
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occur through the amino acids catabolism , “Ehrlich pathway,” or from the amino acid
synthesis “Genevoi s pathway ” (where α -keto acids are formed in carbohydrates metabolism )
(Eßlinger, 2009). Basically, in the Ehrlich pathway (Figure 8), the amino group from the
amino acid may be transferred by an enzyme called transaminase (transamination) leading to
the formation of an oxo- acid (α-keto acid) product, that then suffers a decarboxylation by
decarboxylase enzyme, producing CO 2 and an aldehyde. This aldehyde is then reduced
(action of alcohol dehydrogenases) to a higher/fusel alcohol (Lewis and Young, 1995; Pronk
et al., 1996; Pires et al., 2014). Around 90% of the higher alcohols have been synthetized at
the end of the primary fermentation (Eßlinger, 2009).
Figure 8. Ehrlich pathway for higher alcohols formation by yeasts.
Higher aliphatic and aromatic alcohols are important flavor compounds once they are the
most abundant volatile components present in beer. Nevertheless, their specific impact on
beer aroma is not always noticeable, once these compounds are present, in some cases, at
concentrations lower than their flavor threshold (Table 1).
Higher fermentation temperatures lead to higher fermentation rates with increased
concentration of higher alcohols, as observed by Landaud et al., 2001. Moreover, higher
pitching rates and oxygen levels in wort, as well as high level of FAN in wort, contribute for
higher alcohols concentration in beer; but the main key factor is the yeast strain (ale strains
tend to produce higher alcohols concentration than lager yeasts) (Boulton, 2013).
Vicinal Diketones Formation
Vicinal diketones (VDK’s) production (Figure 9) is directly correlated with amino acid
metabolism, once their precursors, -acetolactate and -acetohydroxybutyrate, are
intermediates of the biosynthesis of valine and isoleucine, respectively (Lewis and Young,
1995; Lodolo et al., 2008). These intermediates ( -acetolactate and -acetohydroxybutyrate)
are released outside the cell, where they suffer a spontaneous oxidative decarboxylation,
leading to the formation of diacetyl and 2,3-pentanedione, respectively. Yeasts can uptake
again these compounds, reducing them into 2,3-butanediol and 2,3-pentanediol, which
threshold is higher, not contributing for beer flavor (Eßlinger, 2009).
VDK’s have low flavor thresholds, namely 0.07 – 0.15 mg/L for diacetyl and 0.9 mg/L
for 2,3-pentanedione (Table 1). Due to their intense butterscotch or toffee aromas, these
compounds are considered defect in pilsner-type lager beer; while in some ales and stouts,
they can be desirable compounds if present in concentrations above their threshold.
The amino acids concentration in wort is the main key factor for diacetyl formation,
namely valine , once it inhibits the α -acetolactate formation (Boulton, 2013). Krogerus and
Gibson, 2013 showed that modifications in the amino acid profile of wort (namely valine
and other branched-chain amino acids) can be a successful way to reduce the diacetyl
concentration along fermentation.
CO2
Fusel aldehyde α-Keto acid Amino acid Higher alcoholTransaminationDecarboxilationReduction
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Figure 9. Metabolic pathway of vicinal diketones (diacetyl and 2,3-pentanedione) formation by yeasts.
Maltose, maltotriose, sucrose, fructose and glucose are the main carbohydrates present in wort.
Esters Formation
Esters are the most important aroma compounds produced by yeasts, once they have low
thresholds (µg to mg/L, Table 1). Nevertheless, their concentration is lower comparing with
other yeast metabolites. The formation of an ester molecule involves the reaction between an
acetyl CoA molecule (derived from pyruvate or from direct reaction between CoA and
acetate, through acetyl-CoA synthase activity) with an alcohol molecule. These reactions
occur during the vigorous stage of the primary fermentation (Boulton, 2013; Pires et al.,
2014).
Beer esters can be divided in two groups: acetate esters (e.g., ethyl acetate, isoamyl
acetate, and phenyl ethyl acetate), where there is a reaction between an acyl-CoA and ethanol
or higher alcohol; and ethyl esters (e.g., ethyl hexanoate, ethyl octanoate and ethyl
decanoate), where first occurs a reaction between CoA and medium-chain-length fatty acid ,
and then with an alcohol (by action of an alcohol acyl transferase). The main key factor that
promotes higher esters concentration is the increase of temperature.
Ester compounds contribute with floral and fruity flavors in beer (Table 1), being already
identified around 100 esters in beer (Lodolo et al., 2008; Pires et al., 2014). However, higher
ester concentrations can lead to bitter taste instead of fruity taste, contributing with negative
flavors to beer, so brewers need to have special attention to the optimum conditions for a
proper balanced ester profile in beer (Pires et al., 2014). The most frequent esters present in
beer are described in Table 1 (Lewis and Young, 1995).
SpontaneousMaltosePyruvateGlucose
Glycolysis
α-AcetolactateGlucose
Maltose
MaltoseFructoseHexose
transporter
Fructose
MaltotrioseMaltotrioseα -AcetolactateCO2Diacetyl
Spontaneous Acetoin DiacetylAcetoin
2,3-Butanediol2,3-Butanediol
Threonine α-Acetohydroxybutyrate
α-Acetohydroxybutyrate
CO22,3-Pentanedione2,3-Pentanedione2,3-Pentanediol 2,3-PentanediolValine Isoleucine
ThreonineSucrose Glucose + FructoseInvertase
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Short-Chain Fatty and Organic Acids Formation
The short-chain fatty acids (e.g., butanoic, hexanoic, and octanoic acids), formed during
lipid metabolism, can be released in the beer, contributing for yeasty flavors in beer and
inhibiting the formation of beer foam; while long-chain fatty acids (e.g., palmitic, linoleic,
stearic, and oleic) are uptake from wort by yeasts (Briggs et al., 2004; Lodolo et al., 2008;
Eßlinger, 2009).
Organic acids (e.g., pyruvate, citrate, malate, acetate and succinate) can also be produced
from pyruvate or from the repressed tricarboxylic acid cycle. They are responsible for the pH
decrease during fermentation and also sour flavors (Lodolo et al., 2008).
Sulfur Compounds Formation
Yeasts can also produce sulfur compounds, being hydrogen sulfide (H2S) and sulfur
dioxide (SO 2) the main ones. Volatile sulfur compounds are usually not desired in beers, due
to their strong vegetable aromas. The formation of these compounds is related with the
metabolism of sulfur-containing amino acids (e.g., cysteine and methionine) and sulfate ions
from wort . Moreover, some dimethylsulfide (DMS) can be produced by yeasts that have
dimethyl sulphoxide (DMSO) reductase activity, contributing with vegetables or celery
aromas to beer (Eßlinger, 2009; Boulton, 2013).
During primary fermentation, firstly exogenous sulfur-containing amino acids are
used for sulfur compounds formation, while sulphate uptake is depressed and the S-
adenosylmethionine pool is small. S-adenosylmethionine is an important intracellular
metabolite that monitors the formation of S-containing metabolites: when its concentration is
low, occurs the uptake of sulphate (by specific permeases), which is reduced by a specific
NAD+ linked reductase, into sulphite and sulphide (used in the biosynthesis of S-containing
metabolites); the excess of S-adenosylmethionine leads to inhibition of genes expression
related with sulphate uptake and its utilization. Then, the formed SO 2 is used for the amino
acids synthesis. The decline of the metabolic rate in mid- to late fermentation promotes the
excretion of SO 2 into extracellular medium; an accumulation of S-adenosylmethionine pool
can occur, and consequently of sulphite; and then there is a stagnation of sulfur compounds
production. Furthermore, sulphite can bind to aldehydes and form adducts, being important
during beer staling due to its possible reaction with staling precursors, namely (E)-2-nonenal.
Thus, aldehydes may persist in beer, once they are not available for enzymatic reactions
(Boulton, 2013).
Duan et al., 2004 reported that there were increased levels of sulfur compounds when
wort had higher concentration of cysteine. Despite their biosynthesis pathway is closed
linked, Duan et al., 2004 verified that the environmental conditions can constrain differently
their rate of formation.
Terpenic Compounds Formation
Terpenic compounds mainly derive from the raw materials, particularly the hops .
Nevertheless, yeasts seem to have ability to produce these compounds that can contribute
with pleasant flavors to beer.
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Figure 10. Terpenic compounds formation through mevalonate pathway, based on KEGG (Kanehisa et
al., 2016).
Terpenic compounds can be de novo synthesized through mevalonate pathway, in yeasts
cytosol (Figure 10)(Carrau et al., 2005; Khor and Uzir, 2011; Rodriguez et al., 2014) .
Additionally, they can suffer bioconversion by yeasts (King and Dickinson, 2003; Takoi et
al., 2010; Gamero et al., 2011), in fact King and Dickinson, 2000 showed the S. cerevisiae
ability of geraniol and nerol biotransformation into linalool and α -terpineol.
Metabolic engineering of Saccharomyces spp. have also been performed so that yeasts
can produce higher concentrations of terpenic compounds (Hu and Lu, 2015). Basically,
genes of certain enzymes, namely geraniol synthase (Pardo et al., 2015), linalool synthase
(Amiri et al., 2016), or farnesyl diphosphate synthase (Erg20) (Fischer et al., 2011; Ignea et
al., 2015), are cloned from a vegetable source and introduced in a plasmid from Escherichia
coli, which is expressed in a Saccharomyces spp. This approach was successful in the
application of wine production (Gamero et al., 2011; Pardo et al., 2015).
2.2.2. Yeast Stress Factors in Brewing Process
Yeast cells are exposed to severe environmental changes along the brewing process,
namely during yeast propagation, fermentation, and yeast storage. The different stress factors
that yeasts can suffer along the brewing process are presented in Table 2, as well as the
cellular effects and respective yeast response.
At the first period of fermentation, temperature shock, hyper osmotic solution and
oxidative stress are experienced by yeasts, in aerobic conditions. Then, there is an
anaerobiosis period, in which hydrostatic pressure inside fermenters increases, as well as the
amount of acetaldehyde and ethanol. Internal acidification and starvation are the main
parameters that the yeasts are exposed (Bleoanca and Bahrim, 2013).
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Table 2. Yea st stress factors along the brewing process (Lodolo et al., 2008; Bleoanca and Bahrim, 2013)
Stress Cellular effect Yeast response Brewing steps aff ected and main effects
Oxidative Proteins, lipids and DNA are the cellular
components more affected;
Lipid peroxidation – decreased membrane
fluidity, inactivation of membrane receptors and
enzymes ;
Protein oxidative damage – formation of
hydrogen peroxide , protein aggregation or
fragme ntation, changes in electrical charges;
DNA – mitochondrial DNA is more prone; if
the damage is not repaired, can occur punctual
mutations, intrachromosomial recombination, or
crossing – over, deletions or insertions. Non- enzymatic antioxidant
mechanisms: ROS binding by
glutathione , polyamines ,
erithroascorbic acid ,
metallothioneines,
flavohemoglobines;
Enzyma tic antioxidant
mechanisms: catalase, superoxide
dismutase, glutathione – peroxidase,
glutathione -reductase,
thioredoxinperoxidase, and
peroxiredoxins. Yeast propagation (increased catalase
activity and elevated concentrations of
glycogen and trehalose);
Fermentation (activation of a oxidative
stress regulator responsible for the
activation of several genes to respond to
several stress conditions);
Yeast storage (different consumption
of trehalose according to the storage
temperature) .
Thermal Hydrogen bonds and hydrophobic interaction
are broken;
Denaturation of proteins and nucleic acid s;
Under high temperatures – death, atypical
budding , appearance of respiratory -deficient,
lower pH , increased fluidity of plasma membrane ;
Under lower temperatures – shrinkage of cells,
destruction of vacuolar membranes, membrane
integrity is compromised because of a transitory
gel-like phase (fatty acids/ sterols) . Increase synthesis of the Heat
Shock Proteins (HSP);
Accumulation of protecting
compounds (trehalose and glycerol )
or enzymes – catalase, mithocondrial
superoxide – dismutase;
Presence of poliamines
(spermine and spermidine) that
improve the membrane integrity. Yeast propagati on (higher temperatures
comparing with the rest of the process);
Yeast storage (low temperatures used;
and depending on the period of time,
different stora ge temperatures should be
applied).
Mechanical
(physical or
shear stress) Glycogen consumption and variation of the
trehalose content;
Reduction of viability and vitalit y;
pH increase in slurry;
Leakage of intracellular proteases. Release of cell wall enzymes
(invertase and melibiase) and cell
wall polysaccharides (mannan and
glucan) in the slurry superna tant;
Haze generation in beer and
impaired flocculation performance. Whenever yeasts are moved within
brewery: natural process (convection
current within the fermentation vessel) or
artificial process (e.g., pumping,
centrifugation).
Osmotic Contact with hypertonic medium leads to
plasmolisis;
Adaptation – restructuring of the actinic
cytoskeleton , temporary arrest of life cycle and
metabolisms’ reprogramming; also increase of the
amount of glycerol , elimination of toxic ions ;
Contact with hypoosmotic medium leads to
turgescence. Induction of Stress Responsive
Elements (STRE). Ferme ntation (mainly hyperosmotic
stress );
The new brewing trend of Very High
Gravity Brewing promotes this type of
stress on yeasts.
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Stress Cellular effect Yeast response Brewin g steps affected and main effects
High pressure Similar effects as thermal and oxidative stress ;
Cellular membranes could be an effect of the
lipids ’ high sensitivity to high pressure , once
affects the membrane fluidity;
Vacuoles suffer acidification due to proton
dissociations;
DNA conformation changes (from double –
helix into zig -zag-ed str ucture) . Homeoviscous adaptation –
increase of the membrane fatty acid
unsaturation (e.g., cholesterol );
Accumulation of trehalose. Fermentation – mainly gaseous
pressure due to the formation of CO 2 by
yeasts.
Ethanol Increase of membrane fluidity and
permeability ;
Deficient transport system of essential
compounds (amino acids and glucose );
Growth, cellular metabolic rate, and viability
reduction;
Repression of cellular pathways (e.g., protein
biosynthesis , cell growth , RNA metabolism );
Activation of cellular pathways, such as cell
integrity pathway and ergosterol synthesis. Appeara nce of vacuolar
acidification;
Reduction of saturated fat ty
acids (e.g., palmitic acid ), and
increase of unsaturated fatty acids
(e.g., oleic acid );
Biosynthesis of squalen and
ergosterol;
High concentration of trehalose;
Increased of the activity of
superoxidismutase. Fermentation and storage –
accumulation and persistence of ethanol
leads to danger on the yeast cell;
The new brewing trend of Very High
Gravity Brewing promotes this type of
stress on yeasts due to higher ethanol
contents produced;
Yeast storage – combination with
higher temperatures, decreases the yeast
viability.
Nutritional Starvation for “natural” nutrients (carbon ,
phosphate , nitrogen , or sulfate ) – low death rate s;
Starvation of amino acids or other metabolites
in auxotrophic mutants – rapid loss of viab ility;
Inhibition of ribosomal RNA synthesis ;
Variations on trehalose and glycogen
concentration;
Autophagy – membrane transport catabolic
phenomenon. Interruption of fermentation if
assimilable wort nitrogen is below
150 mg/L;
Zinc depletion leads to
“sluggish” fermentations;
Impaired flocculation capacities
(changes on cellular surface and
malfunction of genes responsible
for intracellular adhesion) . Yeast storage – if yeasts are stored for
longer periods, there is an induction of
delayed flocculation which le ads to
problems in yeast separation, and beer
quality is compromised.
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If yeast cells are exposed to toxic levels of intracellular reactive oxygen species (ROS),
disturbance in yeasts performance may occur, and therefore, lead to a cellular damage
response, process called oxidative stress. ROS are produced by mitochondria in the normal
aerobic metabolism of yeasts. Gibson et al., 2008 showed that aging may cause damage in
mitochondrial DNA, thus more tendency for yeast cells to become respiratory-deficient
yeasts. Indeed, respiratory-deficient yeasts are the most frequent mutations along the brewing
process, where they have a loss of the respiratory function possible due to mitochondrial
DNA damage caused by oxidative stress (Gibson et al., 2008), especially if the yeasts are
serial repitched.
Measurement of glycogen, trehalose and sterol levels may also be used to predict if
yeasts are under stress and if they are properly stored (Winkler et al., 1991; Jenkins et al.,
2003b; Cheong et al., 2007). This information allows performing an adequate pitching of
yeast when they are reused, letting to have increased fermentation performance. Glycogen is
an intracellular carbohydrate that reflects the nutritional status of the cell, and allows to
indicate if there is an efficient storage and yeast handling. Glycogen is required for sterol
synthesis (Jenkins et al., 2003b), while trehalose is produced to confer stability to plasma
membrane; both compounds are synthesized when cells are under stress. Trehalose is also
used during starvation periods, being regulated by stress-responsive elements (STRE)
(Jenkins et al., 2003b). The 2,3,5-triphenyltetrazolium chloride (TTC) overlay technique is
most used methodology to verify the existence of respiratory-deficient yeasts (Jenkins et al.,
2003b; Gibson et al., 2008).
2.3. Parameters of Yeasts Quality Control
The physiological and healthy state of yeasts influences their performance, being
extremely important to manage their quality, not only along the brewing process, but also in
serial repitching. In fact, the improvement of quality assurance procedures allows the
production of more consistent beer products, namely their quality, flavor, alcohol content, etc.
Yeasts quality can be evaluated through their viability and vitality, being in this sub-section
presented the different methodologies used and the reference values established for some
methodologies are presented in Table 4.
Viability determines the number of living cells, providing the cells ability to grow and
reproduce when they are pitched into wort (Briggs et al., 2004). The pitching rate
conventionally used in brewing fermentations is around of 5 – 20×106 viable cells/mL (Erten
et al., 2007; Verbelen et al., 2009a). A healthy culture is considered when it has 95% of
viable cells (Table 4 ), and it is inadvisable to use cultures, especially for repitching, with
viabilities lower than 85% (Lewis and Young, 1995).
One possible approach to measure yeasts viability is the cells staining with a vital dye
(e.g., methylene blue, methylene violet, or 1-anilino-8-naphthalenesulphonic acid (MgANS)).
Methylene blue is the most used stain test in brewing industry, in which the dead cells are
stained in blue once they cannot exclude the dye, while viable cells uptake the dye, they will
reduce it through cellular dehydrogenase activities, turning it colorless. The yeasts
measurement is performed in a haemocytometer counting chamber, using a simple light
microscopy. Nevertheless, it is time consuming and has user-dependent variations. Other
main problem of this procedure is correlated with the reliability for viabilities below 90%
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Saccharomyces spp. Role in Brewing Process … 237
(Briggs et al., 2004). This method is recognized as international method by the Institute of
Brewing, European Brewing Convention, and the American Society of Brewing Chemists.
Another possible staining test is with methylene violet, which action is similar with
methylene blu e (Smart et al., 1999; Maskell et al., 2003). The fluorescent dye MgANS test
consists in the penetration of the dye into the nonviable cells and binds to the cytoplasmatic
proteins to produce yellow/green fluorescence. The necessity of having a fluorescence
microscopy led to exclude this method to be implement in brewery industries (McCaig,
1990). Flow cytometry can also help brewers to measure the yeasts viability once it measures
the number of yeasts and DNA content, it also gives information about cell cycle (Novak et
al., 2007) . Novak et al., 2007 reported the use of flow cytometry to monitor the propagation
of yeasts under anaerobic and aerobic conditions, using acriflavine (ACR) and fluorescein
isothiocyanate (FITC) as graduating fluorescent dye solutions; Kobayashi et al., 2007 also
used this approach but with different fluorescent dyes: dehydrorhodamine 123 (DHR) and
bis-(1,3-dibutylbarbituric acid) trimethine oxonol (OXN). In traditional microbiology, the
viability of yeast cells is quantified through the colony counting by plating serial dilutions
(Jenkins et al., 2003a; Maskell et al., 2003). The viable cell concentration is expressed
through the number of colony forming units (CFU) (Briggs et al., 2004). This technique can
also be applied to detect contaminations by other microorganisms, by selecting the proper
culture medium and incubation temperature (Lewis and Young, 1995). Particle counter can
also be used to monitor the population growth and the cell size, as showed by Albertin et al.,
2011 . More recently, Laverty et al., 2013 developed an automated method that allows the
quantification of yeast budding percentages and yeasts viability, through the use of an image
cytometry method. This automated method employs a dual-fluorescent nucleic acid dye that
specifically penetrates into stain live cells, thus allowing the distinction of the morphological
characteristics of budding yeasts. Saldi et al., 2014 applied this image cytometry method,
compared its efficiency with several others, and it showed to be a good alternative to the
current methodologies, not only to monitoring yeast viability, as well as their vitality.
Table 4. Sum up of the reference values for some methodologies used to control yeasts
quality (viability and vitality)
Yeasts’ quality
control parameters Methodologies Reference valu e Reference
Viability Vital dye staining
95% of viable cells (healthy culture) (Lewis and
Young, 1995) Flow cytometry or
particle counter
Colony counting
Vitality Acidificatio n
power (AP) Between 2.4 -2.7 (highly active with
good fermentation potential);
Below 2 (reduced metabolic activity);
Below 1.8 (low metabolic
competence). (Gabriel et
al., 2008)
Intracellular pH
(ICP) High vitality : pH 6.2 – 7.2;
Stressed populations: pH < 5.2. (Weigert et
al., 2009)
Flocculation
capacity Industrial brewer’s yeasts: 40 – 90%;
Extremely flocculent laboratory yeasts:
90 – 100%;
Nonflocculent laboratory yeasts: 0 –
15%. (Verstrepen
et al., 2003)
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The fermentation process can be highly affected by yeasts because they may be viable,
yet they may not be actively proliferating. Indeed, yeasts vitality has been described for
several purposes such as the measurement of activity after the transfer from a nutrient-poor to
a nutrient-rich media, the fermentation performance, and the yeasts behavior in the presence
of physiological stress (Smart et al., 1999). The available tests for vitality are based in one
specific feature of yeast metabolism. These tests have different degrees of relevance for
brewing industry, so there is not only one recommended vitality method until now.
Flow cytometry has been used to monitor yeasts vitality, through the determination of the
physiological state of single cells during propagation or to set the vitality before fermentation
(the optimal time for pitching propagated yeast cultures is different for each yeast strain,
being the amount of yeast cells in phase G2/M determines the optimal conditions for pitching
(Novak et al., 2007)). With this technique is possible to establish the population distribution
within the cell cycle, once the signal obtained is proportional to the amount of yeast cells in
each phase (Hutter, 2002; Kobayashi et al., 2007; Lodolo and Cantrell, 2007; Novak et al.,
2007). It also allows to distinguish the yeast type, once the DNA profile achieved with flow
cytometry has 1/2 and 3 or more peaks for lager (diploid DNA) and ale (polyploid or
aneuploid DNA) yeasts, respectively (Hutter, 2002).
Acidification power (AP) test is a fast and simple method that allows the determination of
yeast vitality through their ability to perform a successful fermentation. This method
promotes the metabolic activity of cells through a glucose-induced medium acidification,
which leads to the excretion of acidity compounds (production of CO 2 and organic acids ,
H+/K+ exchange), lowering the pH of the medium (Sigler et al., 2006; Gabriel et al., 2008) .
Gabriel et al., 2008 established that AP values from 2.4-2.7 means that yeast was highly
active with good fermentation potential, AP values below 2 was registered when yeasts had
reduced metabolic activity, and AP values below 1.8 when yeast showed low metabolic
competence (Table 4).
The intracellular pH (ICP) is another parameter that allows the yeasts vitality monitoring.
The majority of the yeast enzymes have an optimal activity pH in a neutral range. During
fermentation or propagation, the production of CO 2 and organic acids lowers the extracellular
medium pH, which leads to the protons ’ pumping by yeasts, in order to maintain the
extracellular pH. Higher proton extrusion rate corresponds to higher yeasts metabolic activity,
thus higher ICP indicates higher yeasts vitality. ICP was recently monitored by flow
cytometry, in which Weigert et al., 2009 were able to differentiate between exponential and
stationary phases along yeasts propagations (bottom-fermenting S. cerevisiae strain W34/70),
as well as to detect less vital yeast subpopulations. ICP varied between 6.2 and 7.2 for highly
active yeasts, while stressed populations had ICP lower than 5.2 (Table 4).
Yeasts sedimentation is one of the most important measurable parameter of industrial
strains, which can be measured through yeasts flocculation capacity according to several
parameters: bond strength, morphology, extent of sedimentation and rate of sedimentation
(Verstrepen et al., 2003). The flocculation capacity, based on degree of sedimentation, counts
the free cells in a flocculating culture, and compares them to the total cell number (before
flocculation or after deflocculation). Helms sedimentation test (Jenkins et al., 2003b; Strauss
et al., 2003) is the most used method in brewing (also the method recommended by ASBC)
(Verstrepen et al., 2003) and also the modified Stratford flocculation test (Novak et al., 2007) .
It is estimated that extremely flocculent extremely laboratory strains have around 90 – 100%
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Saccharomyces spp. Role in Brewing Process … 239
flocculation, most industrial brewer’s yeasts have 40 – 90%, toward the end of fermentation ,
and nonflocculent laboratory yeast strains have 0 – 15% (Table 4) (Verstrepen et al., 2003).
2.4. Saccharomyces spp. Serial Repitching
Each brewery has its own yeast culture line, which requires the propagation before the
first fermentation. Thus, yeasts are pitched, cropped, and then repitched in consecutive
fermentations, in order to have more efficient processes. Between the reuses, yeasts are stored
with a high concentration cell suspension (slurry) or maintained in extended stationary phase.
The yeasts performance along serial repitching can be affected by several parameters, which
will be described in this sub-section (namely yeasts age and population selection). Moreover,
it is intended to list the research papers that mention the application of yeasts serial
repitching, and to understand the impact of this brewing procedure in yeasts performance.
The number of yeast reuses is dependent of some parameters, such as: microbial stability ,
yeast apparent robustness, final product quality, and the company policy. The number of
reuses can vary between 7 and 20 times, or sometimes longer, especially for top-cropping
yeasts, once they have lower contamination with trub and other non-yeast solids. However,
the number of yeasts repitching may be compromised due to the possible stress that yeasts
can suffer, thus the number of repitching should be reduced to 5 – 10 times (Powell et al.,
2003). Moreover, the application of high-gravity brewing practices affects yeasts repitching
once it is limited by yeasts’ capacity to tolerate higher ethanol levels and to attenuate very
concentrated worts. In some cases, it is possible to adapt some parameters, such as the
composition of malts and the ionic composition of wort (adjust or supplement metal ions ,
e.g., Mg2+) for less yeasts stress and consequently less problems along repitching (Boulton,
2013).
Yeasts age can be measured chronologically by the number of times that yeasts are
recycled along repitching. In order to maintain the yeasts quality, it is required to reduce their
metabolism through decrease of the storage temperature (Eßlinger, 2009). Before a new
fermentation, it is needed to determine the yeasts viability, to adjust the amount of pitching
yeast (Powell et al., 2000). Aeration and short-term storage are essential for the good
physiological conditions of yeasts (Eßlinger and Narziß, 2012).
Lager beers represent around 90% of the beer market, so most of the research has been
focused on lager yeasts (Saerens et al., 2010). After the fermentation, where the brewing
yeasts typically divide 4 – 6 times (Boulton, 1991), lager yeasts are allowed to settle on the
cylindroconical vessels. The yeast sedimentation seems to occur within an age gradient as
reported by Hodgson et al., 1997, where older yeasts are in the bottom of the cone and
younger yeasts are more frequently found on the cones’ top. These results confirmed the
hypothesis formed by Barker and Smart, 1996 that already mentioned that older cells tended
to flocculate earlier, while younger cells had the opposite behavior. The formation of bud
scars in the surface of yeast cells are correlated with the replication lifespan (range from 10 –
30 divisions) of yeasts and can be used as an indicator of cell age – budding index. This
analysis can be done by confocal microscopy as reviewed by Powell et al., 2000, where virgin
cells tend to have smooth surfaces, with practically no protruding structural components;
while older cells have rough surface, with more wrinkles, leading to more tendency to
flocculate, supporting previous reports (Barker and Smart, 1996; Hodgson et al., 1997).
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The selection of the yeast population has impact on the fermentation performance: if the
population contains higher older cells concentration, the cells will have slower division rates,
reducing the size of the inoculum, and then slowing the yeasts growth and high lag phase.
Moreover, Barker and Smart, 1996 reported an increased tendency of older “mother” cells to
retain “daughter” cells, influencing the flocculation process. If the population has mainly
newly budded virgin cells, it will need more time to reach the critical size needed for the first
division. So, it is important to compromise both types of cells in order to not have
inappropriate fermentative characteristics, which may lead to fermentation inconsistency,
poor flavor development, and reduced flocculation performance (Hodgson et al., 1997;
Powell et al., 2003). Powell et al., 2004 suggested that the yeast strain and the dimensions of
the fermentation vessel influence the localization of aged cells within the cone, that cannot
follow the previous reports; but still the bottom yeasts had lower fermentation capacities.
These authors also mentioned that yeast crop is composed by a diverse range of cells with
different phenotypes, and not by a single homogenous mass, which can promote some
variability on fermentation performance. Thus, the population of yeast to repitch should be
with young to middle aged cells, once they are the most active portion (fast growth and yeast
biomass production) (Hodgson et al., 1997; Powell et al., 2000) . Bühligen et al., 2014 also
verified an age gradient along yeasts sedimentation, in which the older and younger cells
fractions had the higher dead cells concentration, and should not be used along serial
repitching. Nowadays, in the brewing process, after the sedimentation, the first fraction of
cropped yeast is discarded as waste, because it contains older and dead cells among protein
debris (trub). The rest of the yeasts (middle-aged and virgin cells) are transferred to a yeast
collection vessel, until be used into a new fermentation (Powell et al., 2000, 2003).
Several studies (Smart and Whisker, 1996; Jenkins et al., 2003a; Lodolo et al., 2008)
reported the deterioration of yeasts along serial repitching and cropping due to: hygiene issues
(cross-contamination with others microorganisms such as wild yeast or bacteria); selection of
yeasts with certain characteristics that affect the performance such as age, cell size, increased
flocculation capacities; and selection of yeasts with certain characteristics related with
quality, for instance respiratory-deficient yeasts, which physiological changes can be caused
by stress and genetic changes.
In order to understand the impact of serial repitching on yeasts (Table 5), different
parameters have been studied, namely their: viability, vitality (through the analysis of
flocculation capacity, acidification power test, the glycogen and trehalose contents, and petite
mutations), fermentation performance (by analysis of volatile compounds, sugars and amino
acids/proteins uptake and degradation, lipids analysis), and genetic modifications.
As previously mentioned, the yeasts taxonomy suffered several modifications along time,
especially through the development of molecular tools that allowed a more consistent yeasts
classification. This problem reflects on the yeasts strain characterization along Table 5, which
shows the diversity of yeasts names and some incorrect taxonomy, for instance Miller et al.,
2013 refer that S. cerevisiae is a synonymous of S. pastorianus . Thus, it is difficult to verify
the impact of serial repitching in each yeast strain due to this duality of criteria used in yeasts
taxonomy.
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Table 5. Sum up of the different studies implied with yeasts serial repitching, regarding the following studied factors: yeast
strain, tested parameters, repitching times and the main results/conclusions
Yeast strain Parameters Repitching time and wort
conditions Main results / conclusions Ref.
Ale Viability
(methylene blue );
Flocculation
(Helm's test). 30 successive
fermentations (lab scale)
Malt wort (10-12 șP) First 10 fermentations: increase of viability and vitality until
100%, being stable until 24 fermentations;
After 24 fermentations: flocculation and viability became
highly variabl e and decreased in overall magnitude. (Smart and
Whisker, 1996)
S. cerevisiae Viability
(methylene blue );
Vitality
(acidification
power test);
Sugar analysis
(HPLC);
Ethanol content. 5
Wort
(12 and 20 șP) Shaking flasks fermentation :
– No effects on yeasts viability along serial repitching ;
– At 5th fermentation in a 20 șP, yeasts were not able to
attenuate properly, being the ethanol amount decreasing along
serial repitching ;
– Decrease of yeasts vitality along serial repitchi ng;
Static fermentation :
– Viability decreases in static fermentations;
– Decrease of yeasts vitality along serial repitching ;
– No effects on yeasts ferm entation performance;
Oxygen stimulates yeasts growth, allowing them to tolerate
stress conditions (e.g., acid treatments, high -gravity
fermentations). (Cunningham and
Stewart, 2000)
S. cerevisiae
5610 Volatile esters
and alcohols (HS-
GC-FID) . 7
(lab scale)
~12 șP Substantial increase in isoamyl acetate (up to 2.25 fold), and
ethyl acetate content (from 25 to 32 ppm) along repitching ;
Isoamyl acetate content increased steadily up until 5th
fermentation, and then began to decrease. (Quilter et al.,
2003)
S. cerevisiae 95 Acidif ication
power test. 4 AP value decreased with repitching, having higher SD along
repitching. (Gabriel et al.,
2008)
S. cerevisiae
(lager) Lipid analysis
(phospholipids and
fatty acids) . 3 Phosphatidylcholine (PtdCho) and
Phosphatidylethanolamine (PtdEtn) content altered along
repitching , but mostly in 1st generation, and then yeasts
gradually accommodated to unfavorable fermentation
conditions;
Yeasts are capable to adapt to stress conditions partially by
modifying the PtdCho/PtdEtn ratio and the unsaturation degree
of their fatty acids. (Jurešic et al.,
2009)
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Table 5. (Continued)
Yeast strain Parameters Repitching time and wort
conditions Main results / conclusions Ref.
S. cerevisiae var.
carlsbergensis
(lager) Neutral lipids. 3 Proportion of neutra l lipid classes did not vary significantly
along repitching (high intrinsic stress resistance );
Triacylglycerols together with steryl esters and squalene
constituted the major part of neutral lipids (70%);
Increased ergosterol content and an increased
unsaturated/saturated (UFAs/SFAs) ratio (major changes: shift
from 16:0 to 16:1, but less from 18:0 to 18:1 species) . (Rupčić and
Jurešič, 2010)
S. carlsbergensis
BH092 Ethanol and
isoamyl alcohol
content (GC);
Cell staining and
flow cytometry. 8
(lab scale)
Yeast extract -dextrose
medium Specific growth rate and maximum optical density
decreased, otherwise there was an increase in isoamyl alcohol
and ethanol content along repitchin g;
Gradually increase of intensities of fluorescence dyes along
repitching, indicating higher exposition to reactive oxygen
species. (Kobayashi et al.,
2007)
Lager: SCB1,
SCB2, SCB3,
SCB4 Viability
(methylene blue ,
MgANS, plate
counts);
Acidification
Power Test;
Glycogen and
trehalose content. 5 (SCB2),
8 (SCB4)
10 (SCB1 and SCB3) Viability decreased as generation number increased for all
strains (reduction not statistically significant, between 80 –
100%);
AP declined with increased generation number to a greater
extent;
Overall increase in glycogen and intracellular tre halose
levels. (Jenkins et al.,
2003a)
Lager SCB3 Petite mutation ;
Flocculation;
VDKs
determination. 8 Appearance of 2% petite mutations only in 5th fermentation ,
and then 7% in the 6th fermentation;
As the fermentation progresses, the ability of yeasts to
reduce diacetyl decreases;
Low VDK uptake of propagation slurries due to the
requirement for enzymes ’ induction as a result of acetolactate
generation and beer diacetyl challenge;
Slight decrease on flocculation, but remained unchanged in
4 to 6 fermentations (around 80%). (Jenkins et al.,
2003b)
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Yeast strain Parameters Repitching time and wort
conditions Main results / conclusions Ref.
Lager yeast
CB11 Petite mutations ;
mtDNA RFLP
analysis. 1
3
9
10 No accumulative increase in petite mutation after six
repitchings;
Majority of petites isolated from yeasts slurries exhibited
identical mtDNA restriction digest patterns;
Disruption of mitochondrial DNA may not occur randomly . (Lawrence et al.,
2012)
Diamond lager
yeast (Lallemand,
Inc.) (wet and
dried) Viability
(methylene blue );
Flocculation;
Petite mutations ;
Volatile
compounds (GC-
FID) . 5
(lab scale)
Lager -style wort (12.5
șP) Different flavor profiles (esters and higher alcohols ) were
exhibite d in beer from generation 0 and generation 4;
No statistical changes in flocculation along repitching ;
Despite the lower viability of G0 dried yeasts, repitched
cultures consistently co ntained high proportion of live cells;
No phenotypic or genetic variants accumulated over time
(no petite mutations) . (Powell and
Fischborn, 2010)
S. cerevisiae
(lager brewing
yeast, syn. S.
pastorianu s) Sugar analysis
(HPLC);
Amino acids
analysis. 2
(lab scale)
Wort 80:20 (malt: high
maltose syrup adjunct)
14.7 șP No significant differences in the rate of sugars assimilation ;
Repi tching had impact on the utilization of nitrogenous
compounds by yeasts, in wort ;
Increased intracellular storage of amino acids may occu r as
a result of repitching, and may progressively be reduced the
uptake of particular amino acids from wort on subsequent
fermentation. (Miller et al.,
2013)
S. cerevisiae
(pastor ianus )
CMBSPV09 Viability (flow
cytometry );
Volatile analysis
(GC-FID);
Glycogen and
trehalose content;
Total fatty acids
of yeasts. 8
(lab scale)
15 șP Six to eight generations do not cause a significant increase
in cell age (preoxygenated wort );
No trend was observed in glycogen concentration after
repitching , while for trehalose concentration there was a
decreased trend (ad aptation of yeasts to the fermentation and
storage related stresses);
More stress in non -preoxygenated yeasts;
Both higher alcohols and esters are not influenced by
repitching ;
(Verbelen et al.,
2009b)
Diacetyl content increased drastically after two repitchings
(possibly due to increased fermentation speed starting from 3rd
generation);
Decrease of unsaturation of fatty acids along repitching.
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Table 5. (Continued)
Yeast strain Parameters Repitching time and wort
conditions Main results / conclusions Ref.
S. pastorianus 95 Acidification
power test;
Volatile analysis
(GC-ECD and GC –
FID) . 3
(lab scale)
12, 16 and 20 șP AP decreased (being up to 10%) with increased wort
osmolarity and yeasts generation , however values were always
very high;
Acetaldehyde level in green beer was slightly above or
within the flavor threshold;
Levels of esters ethyl acetate were within the usual limits or
slightly above them, and had small variations; same trend for
higher alcohols. (Sigler et al.,
2009)
S. pastorianus
RIBM 95 Acidification
power. 2
(lab scale)
Malt wort (12 șP) Small drop in AP value was observed with repitching,
nevertheless AP remained high throughout. (Matoulková and
K. Sigler, 2011)
S. pastorianus
HEBRU RT-qPCR;
Microarrays
(expression of
genes for aging,
flocculation,
metabolism and
stress) . 20
(4 different cycles )
12 șP Signs for advanced aging could not be detected after 20
repitchings;
Expression of flocculation genes remained stable during all
serial repitching s;
Higher wort aeration led to increased expression of stress
response genes , espe cially connected to ROS defense;
Higher amounts of oxygen in the wort led to lower levels of
isoamyl acetate in beer. (Bühligen et al.,
2013)
S. pastorianus
HEBRU Bud scar
counting and size
measurement ;
Viability ;
mRNA analysis;
Expression of
specific genes. 6 and 17 (industrial
scale)
12, 12.5 and 14 șP Supplementation of oxygen in worts allow the rejuvenation
of yeasts cells and prevent cell aging;
No loss of flocculation during serial repitching ;
No significant changes were observed for genes associated
to stress ;
Gradient of age observed for yeasts sedimentation , being the
older cells the first to flocculate;
Older and younger cells fractions contained the higher dead
cells concentration, and should not be used to repitch;
No increase of replicative age cells during serial repitching. (Bühligen et al.,
2014)
S. pastorianus
308 and B4 Viability
(methylene blue );
Glycogen and
trehalose content;
Volatile ana lysis
(GC-FID) . 10 (10 șP)
8 (15 șP) No detectable changes in yeasts fermentation ability;
No changes in viability of yeasts after repitching ;
Drop in the glycogen level during fermentation of 15°P wort
was observed earlier than 10°P wort – higher osmotic stress
and ethanol concentration generated when higher gravity wort
was fermented; (Kordialik –
Bogacka and
Diowksz, 2013)
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Yeast strain Parameters Repitching time and wort
conditions Main results / conclusions Ref.
No significant var iation in beer flavor produced along
repitching; no difference in the ‘higher alcohols to esters’ ratio
was noticed either.
S. past orianus
TUM 34/70 Ethanol
determination;
Metal ions
analysis;
Fermentable
carbohydrates
analysis. 11 (lab scale) Extract consumption and ethanol production was consistent
along serial repitching ;
Serial repitching does not affect the initial and overall
uptake of zinc ions by yeasts;
After the 6th fermentation, there was a slight increase of
glucose utilization. (Deželak et al.,
2015a)
S. pastorianus
TUM 34/70 Amino acids
analysis. 11 (lab scale) Asn, Ser, Val, Ph e, Ile, Leu, and Lys assimilation profiles
were less affected by serial repitching ;
Gln, Gly+His, Arg, Ala, and Trp assimilation profiles were
more affected by serial repitching. (Deželak et al.,
2015b)
S. pastorianus
TUM 34/70 Yeast karyotype ;
Protein
profiling. 11 (lab scale) Only 3 middle -molecular weight chromosomes had
significant and consistent s ize changes;
Total protein synthesis declines along serial repitching. (Deželak et al.,
2014)
S. pastorianus
TUM 34/70 Volatile
compounds (GC-
FID) . 11 (lab scale) No obvious or little pattern that influences methanol and
acetaldehyde formation during serial repitching , respectively;
Low influe nce of serial repitching in higher alcohols
formation (2- and 3 -methylbutanol, 2 -phenylethanol, 1 –
propanol, and isobutanol);
Higher rate of esters formation (ethyl acetate, isoamyl
acetate, and 2 -phenylethyl acetate) from 1st to 4th repitching. (Deželak et al.,
2015c)
BridgePort ale
and lager strains Flocculation;
RAPD
fingerprinting (
regions);
RFLP
fingerprinting (Ty
elements). 98 generations for the
ale strain
135 generations for the
lager strain
13.5 șP Some changes in macromorphological characteristics of ale
strain, however there were no genomic variations or changes in
terms of fermentation characteristics;
No significant differences were registered for fermentation
analysis, flocculation assessment and genetic fingerprinting of
nuclear DNA, between fresh lager yeasts and after 135
generations. (Powell and
Diacetis, 2007)
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Cátia Martins, Tiago Brandão, Adelaide Almeida et al. 246
Taking into account the serial repitching of S. cerevisiae (ale yeast), only two parameters
can be compared: viability and vitality (AP test). Smart and Whisker, 1996 registered
decrease of yeasts viability after 24 fermentations at lab scale, but in most of the cases not
registered viabilities lower than 80%. Cunningham and Stewart, 2000 only verified decrease
on yeasts viability when there was no wort oxygenation . S. cerevisiae vitality was monitored
by AP test which revealed that there is a decrease of the yeasts acidification power along
serial repitching, nevertheless the values were superior to 2.2 (Cunningham and Stewart,
2000; Gabriel et al., 2008), showing that yeasts were not critically affected by the handling
during the brewing process.
Regarding lager yeasts’ (in the broadest sense) serial repitching , it is only possible to
compare yeasts vitality through the analysis of petite mutations. Jenkins et al., 2003b only
registered the presence of petite mutants after the 5th (2%) and 6th repitching (7%), while
Lawrence et al., 2012 verified the presence of petite mutants in different serial repitchings,
but concentrations always lower than 1%. These lower values of p etite mutants’ percentage
were obtained, once warm cropping techniques were used, causing less stress in yeasts, which
lead to less formation of petite mutants.
Several studies report the serial repitching effects of S. pastorianus strains, covering a
wide range of studied parameters. Regarding yeasts viability, there were registered no
significant changes along repitching, even using high-gravity worts (8 to 10 times) (Verbelen
et al., 2009b; Kordialik-Bogacka and Diowksz, 2013). Oxygen dissolved in wort proved to be
important for yeasts growth, and consequently for their viability. Yeasts use oxygen to
produce unsaturated fatty acids and sterols, which are used for the plasmatic membrane
structure and its integrity. Thus, there is an efficient biomass production that can tolerate
different stress conditions (e.g., acid treatments, high-gravity fermentations) which yeasts can
be exposed along serial repitching, and prevent cell aging (Verbelen et al., 2009b; Bühligen et
al., 2014).
As observed for S. cerevisiae strains, there is a decrease of the S. pastorianus
acidification power along serial repitching, however yeasts were not critically affected by the
handling. The flocculation capacity is another parameter that reflects yeasts vitality, and S.
pastorianus serial repitching showed no changes in the flocculation genes expression, until 20
repitchings (Bühligen et al., 2013, 2014).
Sugars uptake by yeasts seem to not have significant interference along serial repitching
(Miller et al., 2013; Deželak et al., 2015a) . Regarding amino acids uptake by S. pastorianus
strain, Deželak et al., 2015b verified different trends of assimilation of the amino acids along
11 serial repitchings. Miller et al., 2013 also observed reduced uptake of several amino acids
by S. pastorianus strain, only in 2 serial repitchings, due to an increase of amino acids
intracellular storage. The total protein synthesis in S. pastorianus strain also declined during
11 serial repitchings, as registered by Deželak et al., 2014 .
Kordialik-Bogacka and Diowksz, 2013 verified an increase of trehalose content in S.
pastorianus strain until 10 serial repitchings, while the glycogen content decreased in older
cultures. Regarding glycogen, Kordialik-Bogacka and Diowksz, 2013 showed a greater
decrease when using a high-gravity wort (15 șP) that promotes higher osmotic stress to yeasts.
However, Verbelen et al., 2009b registered no trend in glycogen concentration and a decrease
of trehalose content along 8 serial repitchings, which results suggest that yeasts adapt to
fermentation and storage related stress cycles. These results obtained by Verbelen et al.,
2009b and Kordialik-Bogacka and Diowksz, 2013, are contradictory maybe due to strain
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Saccharomyces spp. Role in Brewing Process … 247
variability, fermentation conditions (one work was performed at laboratory conditions and
other at industrial scale), and wort composition.
In terms of yeast volatile producing compounds (higher alcohols and esters), Verbelen et
al., 2009b and Kordialik-Bogacka and Diowksz, 2013 showed that their formation by S.
pastorianus strains was not influenced until 10 serial repitching, while Sigler et al., 2009
observed slight variations, but only repitched S. pastorianus strain 3 times. On the other hand,
Deželak et al., 2015c verified little influence on higher alcohols formation (2- and 3-
methylbutanol, 2-phenylethanol, 1-propanol, and isobutanol) during 11 serial repitchings of S.
pastorianus , but higher rates of esters formation (ethyl acetate, isoamyl acetate, and 2-
phenylethyl acetate) from 1st to 4th repitching.
DNA techniques such as random amplified polymorphic DNA – polymerase chain
reaction (RAPD-PCR) and RFLP were also used to understand the genetic changes during
long term serial repitching by Powell and Diacetis, 2007. Either for ale, as well as for lager
yeasts, Powell and Diacetis, 2007 did not have significant differences along the 98 and 135
generations, respectively, in terms of genetic fingerprinting. These results showed that with
absence of selective pressures, yeasts can be resistant to genetic changes, being that
phenomenon related to the yeast strain used. Bühligen et al., 2013 also verified the same
trend: with more than 20 repitchings of S. pastorianus, there was not observed an advanced
aging through mRNA (messenger ribonucleic acid) expression; as well as no significant
changes in the expression of genes associated to aging, stress, storage compounds metabolism
and cell cycle through the analysis by low-density microarrays (Bühligen et al., 2014) .
However, no studies to detect phenotypic or metabolic alterations of yeast cells were done by
the authors. Yeast karyotype was performed by Deželak et al., 2014 , and only three middle-
molecular weight chromosomes had significant changes along S. pastorianus serial
repitching.
CONCLUDING REMARKS AND FUTURE TRENDS
Beer aroma profile is dependent of a network of several effects, from which raw
materials and the yeast strain have a key role. In fact, Saccharomyces spp. are the most
commonly yeasts used in brewing, and through their complex metabolic pathwa ys, different
metabolites can be formed, namely volatiles which contribute for the beer sensorial
characteristics. Moreover, serial repitching showed to have slight impact, even when applied
in high-gravity fermentations, in different studied parameters, namely viability, vitality ,
oxidative stress, genetic modifications or volatile compounds formation. The presence of
oxygen revealed to be one important parameter in yeasts serial repitching once it is essential
for yeasts growth. Nevertheless, there is still required to have a deep knowledge about the
metabolic pathways correlated with the formation of compounds that will have impact on
beer sensorial characteristics.
The competitive environment that brewing companies are involved, promotes their
constant innovation, either for the development of new products or for the maximization
resources and reduction of costs. Indeed, the production of different beer styles is a current
brewing trend, once consumers wish to have different sensorial experiments. Furthermore, the
use of by-products from others industries, as adjuncts, is an increasingly common practice,
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Cátia Martins, Tiago Brandão, Adelaide Almeida et al. 248
either for the development of new products, but also because of their cheap price. However, it
is required to investigate more about the yeasts performance in these conditions, mainly due
to initial high carbohydrates content. High-gravity fermentations are another current brewing
trend, which require yeasts selection or genetic modifications in order to be able to tolerate
higher ethanol contents. Yeasts genetic modifications are a possibility for the achievement of
interesting beer products.
In conclusion, the metabolic complexity of yeasts reveals a great potential to be
exploited, nevertheless they do not reach the goal by themselves, being necessary the
combination with different raw materials to lead to the constant innovation in this sector, and
consequently to the development of new products.
ACKNOWLEDGMENTS
Thanks are due to FCT/MEC for the financial support to the QOPNA research Unit (FCT
UID/QUI/00062/2013) and CESAM (project PEst-C/MAR/LA0017/2013), through national
founds and where applicable co-financed by the FEDER, within the PT2020 Partnership
Agreement. C. Martins thanks the FCT/MEC for the PhD grant (SFRH/BD/77988/20112010)
through the program POPH/FSE.
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