Brewer’s yeast Saccharomyces cerevisiae metabolism and its contribution to beer flavor

Kelly Jay

Butler UniversityApril 8th, 2015

Introduction:

A Brief History

Beer is arguably one of the most popular and important accidental discoveries that has

happened in the last 10,000 years (Alba-Lois and Segal-Kischinevzky, 2010). The beverage has

had implications on religion, health, and everyday life since the time it was discovered and

continues to have implications in those areas as well as others. While the actual process of

fermentation by which beer is produced has remained the same, the brewing process has become

highly developed and the brewing industry has evolved into part of the global economy.

When people began enjoying beer they only knew that the savory product resulted from

fruits and grains left in covered containers for extended periods of time (Alba-Lois and Segal-

Kischinevzky, 2010). When it was observed that the product inside of the barrels bubbled, as

water does when it is boiled, the process of turning fruits and grains to wine and beer became

known as fermentation, stemming from a Latin root meaning “to boil.” People began to wonder

how fermentation occurred and what was involved in the process. It became apparent that if

containers sat too long they went bad, but if they sat for too short of a time they did not achieve

the desired flavors (Alba-Lois and Segal-Kischinevzky, 2010). From the moment people started

seeking answers to these questions, the science of alcoholic fermentation and brewing began.

Hundreds of years later, with the development of the microscope, researches were finally

able to observe microorganisms that were present in these closed containers during fermentation

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(Alba-Lois and Segal-Kischinevzky, 2010). Eventually, yeast was identified as the eukaryotic

fungus responsible for fermentation. Questions around fermentation emerged as it remained

unknown how these single celled organisms turn fruit into alcohol during fermentation (Alba-

Lois and Segal-Kischinevzky, 2010). Since the discovery of alcoholic fermentation and the

microorganisms responsible, a vast amount of time, work, and money has gone in to creating a

quality final product.

Brewing

Fermentation is only one aspect of brewing that is important to the final product of beer.

An understanding of the whole brewing process is important to recognize how all aspects affect

the way yeast can metabolize and ferment sugars into alcohol and other by-products. Sanitation

is important so that only desired microorganisms are influencing the final product (Palmer,

1999). A great deal of time and energy is put into sanitizing everything that will be involved in

brewing so that other microorganisms do not have adverse affects. The true process of brewing

starts with malting barley so that it germinates and begins converting starch into proteins, sugars,

and amino acids, all of which are essential to yeast metabolism and growth. Malted barley is

soaked in hot water to release its stored sugars, which are then boiled with hops for seasoning,

and cooled so that yeast can be added to begin fermentation. At this point in the process, the

water and malt sugars are commonly referred to as wort. As the yeast converts the sugars from

the malt into glucose it is fermented and results in CO2 and ethyl alcohol. When fermentation is

complete, the beer is bottled with the addition of more sugar to provide carbonation to the final

product since the CO2 produced from that point on is trapped inside the bottle (Palmer, 1999).

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Fermentation

The type of yeast most commonly used in brewing beer is Saccharomyces cerevisiae

(Bokulich and Bamforth, 2013). S. cerevisiae is capable of quickly converting sugars to ethanol

in anaerobic conditions and grow rapidly in aerobic conditions; however, brewing capitalizes on

the anaerobic abilities of S. cerevisiae to complete alcoholic fermentation (Dashko et al., 2014).

The ability of yeast to complete fermentation directly affects their ability to survive under

oxygen-limited conditions (Bokulich and Bamforth, 2013).

Yeast use energy to grow, eat, and reproduce, which are all processes that drive

fermentation. Yeast cells gain the energy they need to survive by converting sugar (glucose) into

ethyl alcohol and carbon dioxide with ATP as an essential by-product (Alba-Lois and Segal-

Kischinevzky, 2010). The first step in fermentation is glycolysis, which does not require oxygen.

This part of respiration occurs at the beginning of the brewing process when yeast still have

available oxygen and are rapidly growing. Glycolysis is the metabolic pathway that converts one

glucose molecule into two pyruvic acid molecules while producing two ATP in the process. The

breakdown of these pyruvic acid

molecules is when fermentation begins.

Each pyruvic acid molecule is broken

down into two carbon dioxide

molecules and two ethyl alcohol

molecules, creating NAD+ in the

process. These two NAD+ molecules

are required for yeast to continue

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glycolysis since the production of ATP requires NAD+ to be replenished by fermentation (Alba-

Lois and Segal-Kischinevzky, 2010).

While the process of fermentation is vital to the survival of yeast, it is not a process that

has evolved as a long-term survival technique (Bokulich and Bamforth, 2013). Yeast cannot

tolerate excessive levels of alcohol in their environment. This is problematic since one of the by-

products of fermentation is alcohol and it remains in the liquid that the yeast live in. When yeasts

are trapped with too much alcohol and no oxygen the yeast cells die because the alcohol destroys

enzymes needed to continue to produce ATP. Most yeast strains can only tolerate up to 5%

alcohol before their enzymatic cascades begin to fail. For this reason, we normally see beers with

an alcohol content of approximately 5% (Bokulich and Bamforth, 2013).

Yeast Metabolism

In addition to ethanol and CO2, yeast produce a number of other by-products during their

metabolism. The lipid and free amino nitrogen (FAN) content of the barley used to make the

malt are necessary nutrients for yeast survival, but must be carefully proportioned to maintain

proper balance of by-product dependent flavors (Held, 2012). In addition, the temperature of

wort, oxygen conditions, and amount of yeast present affect the ability of yeast and other (less

desired) microorganisms to grow and survive (Smogrovicova and Domeny, 1998). A less than

desirable amount of any of these essential factors could cause the yeast to go dormant, while too

much of any one factor can lead to off-flavors in beer (Palmer, 1999).

Diacetyl is a compound that is the result of oxidation in wort (Smogrovicova and

Domeny, 1998). The compound is desired in small quantities (0.1-0.14mg/l), but with too much

oxygen the buttery flavor gives beer an unfavorable taste (Smogrovicova and Domeny, 1998).

Similarly, ester flavors are desirable to a certain threshold, but high levels can result in a fruity

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flavor that is unfavorable when it is too strong (Saerens et al., 2008). Other by-products also

affect the flavor of the final product of fermentation including fusel alcohols, ketones, phenolics,

fatty acids, and other trace minerals that allow yeast to carry out metabolic processes to produce

these by-products (Palmer, 1999).

Yet another desirable aspect of a final beer that depends on metabolism is a head of foam,

which is an indication of beer quality in some cases. This again results from details in the

brewing process that are unsuspected. The movement of CO2 bubbles to the surface of the beer

helps to move smaller lipids originally present in the barley to the surface (Leisegang and Stahl,

2005). Since these lipid molecules are hydrophobic they attach to the CO2 produced from

fermentation during bottling and rise to the surface. These lipids can drastically affect the flavor

and texture of beer (Leisegang and Stahl, 2005). Even the color of the foam is a direct result of

lipids that remain unused by yeast after fermentation (Marongui et al., 2015).

It is already clear that many variables go into creating a well crafted quality beer. Many

of these variables have already been studied in-depth and their affects have been clearly stated in

scientific literature. However, it remains unclear how many of these variables work together and

how the manipulation of one aspect affects the by-products produced by a different one.

Review of Significant Developments:

Increasing Efficiency

The motivation for advancement in this field is driven by economic gain through

increased production and higher quality beer. Since fermentation is the longest, and therefore

most costly, step in the brewing process it was proposed that increasing the pitching rate can

greatly increase the production of a single fermentation (Nagodawithana and Steinkraus, 1976;

Verbelen et al., 2009a). To test this Verbelen et al. (2009) tested five different pitching rates in

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lab fermentations and measured various physiological and beer quality markers. They found that

many genes involved in stress responses were expressed in yeast used in fermentations with

higher pitching rates. This suggested that higher concentrations of yeast were limited in some

way compared to those with a lower pitching rate. The buildup of unsaturated fatty acids was

lower than normal in the high pitching rate batches. Higher pitching rate also resulted in final

products with higher diacetyl levels, trehalose concentrations, and lower viability of yeast. The

low fatty acid concentration at the beginning of fermentation is concerning because yeast require

fatty acids and sterols for normal growth during fermentation. This result was confirmed when

fluorescent dye used during fermentations confirmed that cell viability and growth were lower

throughout fermentation as pitching rate increased. The accumulation of trehalose suggested that

the yeast in the high pitching rate fermentations where not getting sufficient nutrients, since

trehalose is known be stored when yeast are entering their dormant phase. Finally, diacetyl was

not reduced to the flavor inactive compounds (acetoin and 2,3-butanediol) to which it is normally

reduced to. Its high level is responsible for a buttery off-flavor in many beers. While the total

fermentation rate did decrease with pitching rate there were clearly many adverse affects to the

final product (Verbelen et al., 2009a).

Balancing Variables

This study lead to another by an almost identical group of authors that looked to stabilize

the adverse affects that high pitching rate has on the final product of beer. The study considered

the impact of oxygen during high cell density fermentations on growth rate as well as

physiological markers and by-products (Verbelen et al., 2009b). Different oxygen conditions

were applied to high cell density (high pitching rate) fermentations in the form of wort aeration,

wort oxygenation, preoxygenation of yeast and combinations of these things. Improved oxygen

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conditions lowered the glycogen and trehalose levels that were measured at the end of

fermentation, suggesting that the yeast cells were more viable at higher pitching rates with access

to more oxygen. Some genes that are expressed under high oxidative stress became active in high

cell density fermentations (HCD) with increased oxygen. This suggests that oxygen also has a

threshold in fermentation which can be damaging to the yeast if surpassed. Cell density had a

higher affect on fermentation rate as shown by a significant decrease in time to reach total

fermentation in all HCD fermentations, but relatively no difference between differing oxygen

concentrations. Net growth, viability, and FAN consumed were all positively affected by the

addition of oxygen to HCD fermentations. Sufficient amounts of oxygen are required for yeast to

produce the fatty acids that they require to grow and with better oxygen conditions fatty acids

returned to approximately normal levels. Diacetyl levels were measured above the 80ppb

threshold, suggesting that the oxygen conditions are not sufficient to return diacetyl levels in

HCD fermentations to normal levels. From this study it is clear that oxygen content is one

limiting factor in HCD fermentations, but others still exist. The combination of wort aeration and

yeast preoxygenation appears to give the most satisfying results by yielding a final product of

HCD fermentation most similar to that of a normal fermentation. Other limiting factors in HCD

fermentations need to be identified and studied in order to create a HCD fermentation that results

in an adequate final product (Verbelen et al., 2009b).

Based on the two previous studies it is clear that pitching rate and oxygen both affect

many factors of fermentation and its by-products (Verbelen et al., 2009a). These studies also

mention the importance of lipid content on yeast growth before and during fermentation, as well

as harmful effects on the final product of beer flavor and foam (Verbelen et al., 2009b). The

oxidation of unsaturated fatty acids can directly cause a stale aroma in beer, while small

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hydrophobic lipids are responsible for a stable head of foam as they bind to CO2 as it moves

toward the surface of beer released from the bottle (Leisegang and Stahl, 2005). Whereas small

lipids are beneficial, un-metabolized longer chain fatty acids can actually cause the foam to

collapse and become unstable, leading to adverse affects on flavor (Bravie et al., 2009). Bravie et

al. (2009) again confirmed that pitching has an effect on the lipid profile of wort and thus affects

both foam and flavor. In a normal cell density pitching, increased lipid content allowed for

increased growth of yeast cells since the lipids provide essential nutrients. Since a majority of

fatty acids are found within the yeast cells, the removal of yeast biomass decreased the lipid

content of final products (Bravie et al., 2009). Since oxidation of fatty acids is what causes an

off-flavor in some fermentations, it could be beneficial to look at the removal of yeast biomass

after HCD fermentations with increased oxygen conditions.

Variables Connect

Another way that scientists have looked to speed up the brewing process is by increasing

the temperature at various points of fermentation. Smogrovicova and Domeny (1998) used gas

chromatography to determine the amount of ethanol and volatile compounds in beer. Total

nitrogen, FAN, bitterness and diacetyl concentration were all measured along with cell

concentration in order to determine growth rate of yeast under different temperature

fermentations. These measures indicated how flavor can be affected by temperature changes.

Temperatures from 5-20oC were used (15oC being a typical fermentation temperature) and yeast

were either free or immobilized in calcium pectate, ӄ-carrageenan, or on DEAE-cellulose. The

concentration of diacetyl increased with temperature for free and DEAE-cellulose immobilized

batches, but decreased as temperature increased in calcium pectate and ӄ-carrageenan

immobilized batches. Higher temperatures for entrapped yeast batches showed an increase in

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most fatty acids. This combination of fatty acid increase and diacetyl decrease with higher

temperatures for entrapped yeast batches could potentially yield important results (Smogrovicova

and Domeny, 1998). Since yeast growth in HCD fermentations was mostly affected by low

levels of fatty acids at the beginning of fermentation, immobilized strains at higher temperatures

could be utilized with HCD to compensate for fatty acid production. Additionally, the decrease

in diacetyl concentrations can be beneficial in the same way to counteract HCD effects. The

production of esters was also affected by immobilized yeast at higher temperatures

(Smogrovicova and Domeny, 1998). The concentrations of esters increased, giving rise to a

fruitlike or floral taste that comes with the aromas that they produce.

Little is known about what affects the production of ethyl esters, since such low

quantities of it are produced (Saerens et al., 2008). The factors that affect ethyl ester production

have been identified as the concentrations of unsaturated fatty acids in wort during fermentation,

the carbon/nitrogen ratio, and the temperature of fermentation. A decrease in ethyl ester

production was shown when levels of unsaturated fatty acids were increased in wort during

fermentation. This was determined by filtering wort to change the beginning lipid content and

measuring ethyl ester production in wort that contain different lipid concentrations. To measure

the nitrogen-carbon ratio, the FAN content and sugar content respectively were manipulated.

Carbon was varied while nitrogen was kept constant, and vice-versa. The results showed that

specific ethyl esters were produced in different quantities at different ratios. These results did not

seem to impact the overall flavor effect of ethyl esters. However, increases in fermentation

temperature significantly decreased the ethyl ester production, as shown in previous studies.

Together, this information shows that ester production varies with a wide range of temperatures.

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Ester production can be varied in many different temperature fermentations which can provide

insight into flavor profiling (Saerens et al., 2008).

Yeast Strain

Since the strain of yeast can affect production of final by-products, it is very important to

have strains of yeast available that are known as good starters for brewing and that produce the

desired products under a set of established conditions. For this reason it is of vital importance

that breweries properly store and maintain their strains to ensure the brewery’s future ability to

produce a quality product. Of interest are strains that can add depth to the collection of known

brewing strains. Marongiu et al. (2015) considered untraditional starter strains that can introduce

genetic variation to ensure longevity of brewing strains. The study considered 12 isolated S.

cerevisiae strains from homemade sourdough breads (Marongui et al., 2015). To produce quality

by-products, each sourdough strain had to be able to withstand the boiling of wart and convert

maltose, the sugar usually used in brewing, into ethanol in a reasonable amount of time. The

study showed that many of these 12 strains are capable of producing craft beer through

fermentation. The beers obtained from the test brews were analyzed for alcohol content,

bitterness, and other flavor markers. The sourdough starter strains were successful because they

were able to ferment maltose and trehalose with comparable levels of previously mentioned

markers. Only strains S10, S15, and S16 were not able to ferment maltose, and were nullified as

potential starter strains. The baker strains were also able to multiply and grow in the beginning of

fermentation when maltose was present, another marker of success as a brewing strain. All the

sourdough strains were also able to ferment hopped wort, sometimes at a higher rate than normal

brewing strains. Based on wort fermentation rates the strains of sourdough were classified as

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strong, mild, and weak fermentors, where mild fermentors matched the fermentation strengths of

normal craft beer starter strains.

The use of strains that are very similar but differ genetically allows brewers to select

strains that may be more durable in different environments, and that can lead to differentiating

factors in final beer products. The four strains that fell into this mild characterization (S38-F2,

S38-S38, S33-F2, and S33-38) were all tasted by a panel of test consumers using 12 assessors.

The test consumers were trained and asked to describe the “presence, color and persistency of the

foam, fruity, malt and yeast character for the odor, bitter and acid for the taste and freshness and

fullness for the mouth-feel sensations.” The sensory profile reported on this scale was equivalent

for all of the sourdough strains in addition to the normal brewing strains. This suggests bakers’

yeast strains are a viable option for growing the collection of S. cerevisiae starter strains for

brewing (Marongiu et al., 2015).

Summary of the Current State of Knowledge:

Factors that affect yeast metabolism are clearly stated throughout literature on

fermentation and beer brewing and are becoming highly understood. Many of the

aforementioned studies illustrate that altering the conditions of yeast growth and the following

fermentation can drastically change the final products that yeast produce. These changes are so

important because they affect the flavor of beer. Since many of the aspects affecting by-products

such as lipids, oxygen, temperature and pitching rate are now known, scientists are now

considering ways to optimize the brewing process. It is known that temperature and pitching rate

increase the fermentation rate, but scientists are now considering how to balance the adverse

affects on other compounds to maintain stable by-products during these faster processes (Bravie

et al., 2009; Smogrovicova and Domeny, 1998; Verbelen et al., 2009b).

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Some factors negatively affect yeast growth, viability, and desired by-products from

yeast during fermentation (Nagodawithana and Steinkraus, 1976). Some are similar to the

compounds mentioned above that are necessary for a balanced fermentation. However, if they

surpass a certain threshold they become detrimental to the flavor profile of beer. For example,

the normally desired diacetyl compound produces an unfavorable buttery flavor in high

concentrations. High cell density fermentations are desirable because they are fast, but they

result in high diacetyl concentrations. As mentioned before this can be balanced by the addition

of oxygen, but only in moderation since oxygen can too cause adverse affects such as the

production of too many esters, a fruity flavor that again is undesirable past a threshold (Bravie et

al., 2009; Leisegang and Stahl, 2005; Verbelen et al., 2009b).

Other scientists have even begun to understand that the manipulation of the yeast strains

themselves is an effective way to change metabolism and the by-products produced during

alcoholic fermentation. Oxygenation during fermentation is no longer limited to wort aeration

and oxygenation, but preoxygenation can increase oxygen availability by directly oxidizing yeast

cells (Krogerus et al., 2015). Additionally, initial studies have been done on yeast hybridization.

Specifically, two lager yeasts were crossed in a hope to maintain favored properties from each

parent strain, S. cerevisiae and Saccharomyces ubayanus. The hybridization’s success was

illustrated by the presence of favorable markers considered in beer profiles, but some

inconsistencies in aroma did persist. It is also essential to further study DNA inheritance, and the

viability of future strains before this information can be fully utilized (Krogerus et al., 2015;

Styger et al., 2011).

Newer flavors can also come from isolating yeast strains from other fermented origins

such as bread. These new strains can provide slight flavor enhancements, but are still able to

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successfully ferment sugars into ethanol. New strains not only provide the opportunity for slight

differentiation in flavor, but provide the brewing industry with viable starter strains if there were

to be damage to existing strains (Marongui et al., 2015).

Suggestions for Future Research:

It is evident that scientists already know which buttons they can push to manipulate the

metabolism of S. cerevisiae. Further research can be done on balancing the effects of these

manipulations to create the highest quality and most optimized fermentation. We already know

that a higher pitching rate can be slightly counteracted by the benefits of oxygenation of wort and

preoxygenation of yeast (Verbelen et al., 2009b). Since the oxygenation is only able to

reintroduce some of the fatty acids required for adequate yeast growth before it starts to oxidize

diacetyl and other undesired targets it would be reasonable to consider if filtration for lipid

content could be used to compensate for the rest of the fatty acid required for yeast growth. We

know that lipid content can be altered so that wort starts with a higher content, so manipulating

the lipid content and oxygen conditions in response to higher pitching rate could potentially help

high density fermentations flourish with normal flavors (Bravie et al., 2009).

The genetics involved in aroma and flavor profiles also provide an interesting outlet for

new information. It is clear through the hybridization done in Krogerus et al., (2015) that

genetics have the ability to change metabolism. In another study, specific genes involved in

precise aroma profiles have been identified as well (Styger et al., 2011). Since we know that

genes involved in aroma profiles can be isolated, it is reasonable to expect that this information

can be used to determine which yeast strains would be good hybrids. For example, one strain that

has high expression of certain genes that create a desirable flavor can be crossed with another

that has high expression of other desirable genes. The question would remain if all the beneficial

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highly expressed genes would be passed from the parent to the hybrid, but the discovery of how

to cross yeast strains in this way could provide new starter strains that are highly desired by

brewers.

Finally, it would be economically beneficial to compare all of the known approaches and

any newly discovered ones for efficiency in time, resources, and cost. Analyzing which

approaches are most realistic with technologies available in a brewery and the level of training

for those who work in breweries are important to putting these discoveries into practice. This

analysis will help to find the most cost effective ways to implement laboratory science into

actual breweries to provide tangible economic savings. The potential for breweries to make beer

more effectively and more precisely control the flavor would lead to even more innovation in the

industry.

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References

Alba-Lois, L., C. and Segal-Kischinevzky. 2010. Yeast fermentation and the making of beer and wine. Nature Education. 3(9): 17.

Bokulich, N.A., and C.W. Bamforth. 2013. The microbiology of malting and brewing. Microbiology and Molecular Biology Reviews. 77(2): 157-172.

Bravie, E., G. Perretti, P. Buzzini, R.D. Sera, and P. Fantozzi. 2009. Technological steps and yeast biomass as factors affecting the lipid content of beer during the brewing process. J. Agric. Food Chem. 57: 6279-6284.

Dashko, S., N. Zhou, C. Compagno, and J. Piskur. 2014. Why, when and how did yeast evolve alcoholic fermentation. FEMS Yeast Res. 14(6): 826-832.

Held, Paul. 2012. Chemical and biochemical means to detect alcohol- determination of ethanol concentration in fermented beer samples and distilled products. Biofuel Research. 12: 29-35.

Krogerus, K., F. Magalhaes, V. Vidgren, and B. Gibson. 2015. New lager yeast strains generated by interspecific hybridization. J Ind Microbiol Biotechnol. DOI 10.1007/s10295-015-1597-6.

Leisegang, R. and U. Stahl. 2005. Degradation of a foam-promoting barley protein by a proteinase from brewing yeast. J. Inst. Brew. 111(2): 112-117.

Marongui, A., G. Zara, J.L. Legras, A. Del Caro, I. Mascia, C. Fadda, and M. Budroni. 2015. Novel starters for old processes: use of Saccharomyces cerevisiae strains isolated from artisanal sourdough for craft beer production at a brewery scale. J Ind Microbiol Biotechnol. 42:85-92.

Nagodawithana, T. and K. Steinkraus. 1976. Influence of the rate of ethanol production and accumulation on the viability of Saccharomyces cerevisiae in “rapid fermentation.” Applied and Environmental Microbiology. 31(2): 158-162.

Palmer, John. “How to Brew.” N.p., 1999. Web. 15 Mar. 2015.

Saerens, S.M.G., F. Delvaux, K.J. Verstrepen, P. Van Dijck, J.M. Thevelein, and F.R. Delvaux. 2008. Parameters affecting ethyl ester production by Saccharomyces cerevisia during fermentation. Appl. Environ. Microbiol. 74(2): 454-461.

Smogrovicova, D. and Z. Domeny. 1998. Beer volatile by-product formation at different fermentation temperature using immobilized yeasts. Process Biochemistry 34: 785-794.

Styger, G., D. Jacobson, and F.F. Bauer. 2011. Identifying genes that impact on aroma profiles produced by Saccharomyces cerevisiae and the production of higher alcohols. Appl Microbiol Biotechnol. 91: 713-730.

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Verbelen, P.J., T.M.L. Dekoninck, S.M.G. Saerens, S.E. Van Mulders, J.M. Thevelein, and F.R.Delvaux. 2009a. Impact of pitching rate on yeast fermentation performance and beer flavor. Appl Microbiol Biotechnol. 82: 155-167.

Verbelen, P.J., S.M.G. Saerens, S.E. Van Mulders, F. Delvaux, and F.R. Delvaux. 2009b. The role of oxygen in yeast metabolism during high cell density brewery fermentations. Appl Microbiol Biotechnol. 82: 1143-1156.

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