This chapter describes the importance of yeast in beer fermentation. Initially, the differences between Saccharomyces cerevisiae and Saccharomyces pastorianus in the production of “ale” and “lager” beers are analyzed. Then, the relationships between beer nutrients and yeast growth are discussed, with special emphasis on the production of the flavor compounds. The impact of the wort composition on flocculation is also discussed. Furthermore, conventional approaches to starter yeast selection and the development of genetically modified microorganisms are analyzed. Recent discoveries relating to the use of S. cerevisiae strains isolated from different food matrices (i.e., bread and wine) and the potential for the use of non-Saccharomyces starter strains in beer production are discussed. A detailed review of the selection of starter strains for the production of specialty beers then follows, such as for gluten-free beers and biologically aged beers. Yeast recovery from top-cropping and bottom-cropping systems and the methodologies and issues in yeast propagation in the laboratory and brewery (i.e., re-pitching) are also analyzed. Finally, the available commercial preparations of starter yeast and the methods to evaluate yeast viability prior to inoculation of the must are analyzed.
Beer is a very old biotechnology, with its origins dating back to around 10,000 years ago . Due to this ancient history, this alcoholic beverage has undergone three particularly important revolutions: (i) the practice of the
All brewers know that during the fermentation of the wort, the yeast have to complete two major tasks. The primary task is, as indicated, to convert the sugar into ethanol, CO2, and the aromas for the production of quality beer, while they also need growth to produce biomass that will be re-pitchable into new brews. Thus, it is imperative to satisfy the physical and nutritional requirements of the yeast. The growth rate of yeast can be modulated by manipulation of some of the fermentation parameters, such as the supply of nutrients, the dissolved oxygen, and the temperature. Furthermore, to end up with a high quality beer, there must be a balance between the nutrients absorbed and the products released.
Beer production is essentially a two-phase process: primary fermentation and secondary fermentation (or maturation). Primary fermentation is a short and vigorous step, during which almost all of the sugars are fermented, accompanied by the production of the secondary compounds that result from the yeast metabolism, most of which are associated with the final beer aroma. At the end of this stage, most of the yeast biomass is collected and separated from the “green” beer, which then undergoes secondary fermentation. During this second phase, the yeast completes the fermentation of the residual sugar, with undesirable compounds removed, and the final taste of beer defined. Among the fermentative yeast,
2. Yeast in beer
2.1. Physical and nutritional requirements of brewing yeast
Brewing yeast are mainly classified as the top-fermenting or ale yeast of
The uptake of nutrients by yeast depends on the nutrient type, the yeast species, and the fermentation conditions. Generally, glucose is transported through the cell membrane into the cell by facilitated diffusion, and maltose by active transport. A high glucose concentration in the wort can suppress the assimilation of maltose and other sugars (i.e., sugar catabolite repression). As a source of nitrogen, brewing yeast require assimilable organic (e.g., amino acids) and inorganic (e.g., ammonium salts) nitrogen for growth and fermentation. Again, high levels of ammonium ions in the wort can suppress the uptake of amino acids (i.e., nitrogen catabolite repression). Amino acid uptake occurs through two transport systems: general amino acid permease (GAP) and specific transporters for the different amino acids. The dissimilation of amino acids (i.e., decarboxylation, transamination, and fermentation) produces ammonium, glutamate, and higher alcohols (i.e., the fuel oils).
2.2. Genomic features of
S. cerevisiaeand S. pastorianusbrewing strains
Over time, there was gradual domestication and selection of yeasts . Moreover, there are two counteracting forces that act on yeast selection: yeast research needs to homogenize biological systems and to refer different yeast strains to species, while brewers need to differentiate and select yeast strains based on their fermentation characteristics.
Ale strains of
Brewing strains belonging to
Recently, it was shown that on the basis of the sequence of different isolates of lager beer strains,
Other species can contribute to wort fermentation and beer quality, including wild strains and species in open and uncontrolled fermentations.
3. Yeast management for aroma production
Generally, at the end of the boiling, the wort contains all of the nutrients that are required for yeast growth and fermentation. Thus, from a microbiological perspective, the main aspects to consider are not whether the wort is suitable for yeast growth, but rather what is the balance of the flavor compounds that will be produced by the yeast. Mathematical models have been developed to predict the final concentrations of some of these volatile compounds from the (known) quantities of their precursor(s) in the wort . However, the application of these methods requires particularly deep knowledge of the wort composition, while brewers usually conduct very basic measurements in their evaluation of wort quality. For example, there are indications that small changes in the spectrum of the wort amino acid composition can result in dramatic changes in the final beer aroma. The most important metabolites synthesized by yeast and related to beer quality are sulfur compounds, organic and fatty acids, carbonyl compounds, higher alcohols, and esters.
3.1. Sulfur compounds
Sulfur compounds, such as hydrogen sulfide, methional, and dimethyl sulfide (DMS), are active flavor components of the beer that is generated during mashing and fermentation. During fermentation, through the metabolizing of amino acids and vitamins, and through the use of the inorganic components of the wort (e.g., sulfates),
3.2. Organic and fatty acids
The organic acids in beer are derived mainly from the yeast, as they are produced during the tricarboxylic acid, or Kreb’s cycle (e.g., succinate and malate), from the catabolism of amino acids, and from redox reactions. Other organic acids, such as citrate and pyroglutamate, derive directly from the wort, and the yeast do not affect their concentrations in the beer. Overall, more than 100 organic acids have been identified in beers. These contribute to the reduction in pH during fermentation, and to the “sour” or “salty” taste of the beers. Fatty acids are of particular interest here, because of their involvement in the synthesis of esters. Yeast can incorporate saturated fatty acids and unsaturated fatty acids (UFAs) from the wort, or they can synthesize these from acetyl-CoA. However, the lack of sufficient oxygen in the later phases of fermentation makes the synthesis of UFAs impossible, and as a consequence, medium-chain fatty acids (MCFAs) are released into the medium . These MCFAs are powerful detergents, and they can influence yeast vitality, beer taste, and foam stability. In particular, the typical flavor that is characteristic of MCFAs is defined as a “rancid goaty” flavor, and hence is often described as a “caprylic” flavor.
3.3. Carbonyl compounds
The presence of aldehydes and vicinal diketones is considered undesirable for beer quality. Acetaldehydes have unpleasant “grassy” flavors that are reminiscent of green apples and dry cider. In some circumstances, such as when there is excessive wort oxygenation and high pitching rates, aldehydes can accumulate in concentrations above the flavor threshold . The vicinal diketones are important off-flavors of lager beers, which include diacetyl. During fermentation, yeast cells excrete an intermediate of valine biosynthesis, α-acetolactate, that is, spontaneously decarboxylated to diacetyl. Diacetyl has a strong aroma of toffee and butterscotch, and at concentrations above 0.05 ppm, it is considered as undesirable in lager beers. During conditioning, diacetyl is assimilated by yeast, and thus reduced to acetoin and 2-3 butandiol, which have much lower impact on beer quality. Traditionally, the rate-determining step of diacetyl accumulation in beer has been considered as the spontaneous decarboxylation of acetolactate, with yeast assimilation left with a marginal role. However, the physiological conditions of yeast are essential for diacetyl production and the time necessary for its reduction. High concentrations of valine and isoleucine in the wort inhibit vicinal diketone production by yeast. High assimilation rates have been observed at higher fermentation temperatures and when yeast is grown under aerobic de-repressed conditions. On the contrary, at higher pitching rates, the elevated concentrations of vicinal diketones produced by yeast require longer standing times .
3.4. Higher alcohols
The higher alcohols are also known as “fusel alcohols”, and these are the most abundant organoleptic compounds in beer. Isoamyl alcohol, n-propanol, isobutanol, 2-phenyl-ethanol, and triptothol are important flavor and aroma components in terms of their concentrations. Below 300 mg/L, these compounds add complexity to the beer, by conferring refreshing, flower, and pleasant notes, and imparting a desirable warming character. On the contrary, above these concentrations, these compounds can have unpleasant heavy solvent-like odors. The formation of higher alcohols by brewing yeast involves different complex pathways, and a lot of progress has been made in the determination of the roles of the key genes involved in their biosynthesis . The predominant idea for many years was that the higher alcohols are produced
Esters are chemical compounds derived from a carboxylic acid and an alcohol, and they are of major industrial interest because they have very low thresholds and define the fruity aroma of the beer. Two main classes of esters are of particular interest for brewers: acetate esters and MCFA esters. Acetate esters have concentrations above threshold levels in most lager beers (e.g., isoamyl acetate is responsible for the banana-like aroma) and ale beers (e.g., ethyl acetate gives a solvent-like aroma). It is generally believed that the acetyl-CoA that is necessary for formation of acetate esters derives from oxidation of acetaldehyde. The acyl-CoAs required for the synthesis of MCFA esters originate from intermediates in the synthesis of fatty acids. Among the MCFA esters, ethyl hexanoate (i.e., an apple-like aroma) is an important flavoring compound, with levels above threshold in ale beers. The biosynthesis of esters requires acetyl-CoA or acyl-CoAs esterification with ethanol or higher alcohols by the specific alcohol acetyltransferase enzymes ATF1 and ATF2. Different studies have focused on the manipulation of fermentation conditions and the wort composition in ways that favor the availability of these factors and that lead to increased production of higher alcohols and esters.
4. Fermentation conditions
It is generally accepted that any condition that stimulates yeast growth will increase the production of higher alcohols and their acetate esters during fermentation. In this respect, increasing fermentation temperatures leads to an accumulation of acetate esters, with no significant differences in the levels of the MCFA esters. In particular, increasing the temperature from 10 to 12°C, increases ester production by up to 75% . This phenomenon is dependent on increased alcohol acetyltransferase activity and stimulation of higher alcohol synthesis, which results from greater amino acid turnover. In addition, it has been suggested that higher fermentation temperatures increase the synthesis of a specific permease, Bap2p, that is, involved in import of the branched-chain amino acids valine, leucine, and isoleucine, which are known precursors of the higher alcohols .
Oxygen has an ambiguous role here. Indeed, oxygenation of the wort provides for better yeast growth, and consequently increased higher alcohol production. However, it is well-known that oxygenation leads to lower levels of esters in the beer. Oxygen acts in two different ways. First, availability of oxygen allows the biosynthesis of UFAs that is required to sustain yeast growth during fermentation. UFAs are esterified to glycerol to form membrane lipids, and thus, less acyl-CoA is available for synthesis of MCFA esters. Second, oxygen inhibits transcription of the alcohol-acetyltransferase-encoding gene
Another fermentation parameter that can affect ester synthesis is the hydrostatic pressure on the yeast cells. While in small craft breweries this is not a problem, the use of big cylindro-conical reactors in industrial beer production substantially increases the concentration of carbon dioxide dissolved in the beer. This has a double effect on ester synthesis. First, it inhibits yeast growth by lowering intracellular pH, and second, it directly reduces decarboxylation reactions, such as acetyl-CoA synthesis from pyruvate. As a consequence, large fermenters have been successfully used for the reduction of esters in beer, in particular during the fermentation of high gravity wort.
Stirring of the medium modulates the effects of the oxygen and carbon dioxide that are dissolved in the beer, to provide better oxygen distribution and decrease carbon dioxide super-saturation. Consequently, the synthesis of higher alcohols is stimulated, while that of MCFA esters is reduced, and the beer will have a less fruity aroma.
5. Wort composition
The amounts and types of sugars in the wort can influence the aromatic profile of the beer. Beers obtained from worts with higher percentages of glucose and fructose have higher ester levels than those obtained from maltose-rich worts. It has been suggested that glucose and fructose stimulate the glycolytic pathway and eventually lead to high levels of cytoplasmic acetyl-CoA, while maltose-rich wort only weakly induces acetyl-CoA formation . Moreover, glucose induces
It has been suggested that high nitrogen wort induces the transcription of
The effects of free UFAs on beer aroma have been well documented. Similar to oxygen, low UFA levels in the wort increase ester synthesis, by recovering optimal yeast growth. However, at higher concentrations, UFAs relieve the need for the yeast to produce acetyl-CoA for lipid biosynthesis. This in turn induces lower MCFAs production, and lower production of their respective esters. Moreover, UFAs can directly repress
Finally, zinc stimulates the breakdown of α-keto acids to higher alcohols, thus, increasing their concentrations, and those of their corresponding esters.
At the end of the primary fermentation, yeast cells must be removed from the “green”, or immature, beer. Flocculation is the process by which yeast cells aggregate and form “flocs” consisting of thousands of cells.
Thus, on this basis, the nutritional and physiochemical conditions of sweet wort inhibit yeast flocculation. Indeed, the
As well as sugars, lipids, metal ions, and nitrogenous compounds, other minor wort components can influence the flocculation of yeast. In particular, complex polysaccharides that can induce premature flocculation have been identified. These polysaccharides can act as a bridge between cells by interacting with yeast lectin-like proteins. It has been suggested that the binding between these polysaccharides and yeast cells is mediated by cationic antimicrobial peptides . These compounds are produced by barley to protect against microbial attack, or in response to fungal contamination during the malting process. Premature flocculation leads to incomplete attenuation of the wort, as aggregated cells cannot ferment the residual sugars of the wort.
Another major industrial problem is that flocculent strains can gradually lose their ability to flocculate, and thus eventually they will not form aggregates. It has been suggested that this phenomenon can be ascribed to genetic alterations during yeast multiplication and subsequent re-pitching . In the case of insufficient flocculation, the yeast cells need to be removed at the end of fermentation by centrifugation or other separation techniques. However, yeast cells exposed to the stress associated with centrifugation have lower viability and vitality. This, in turn, can negatively affect beer quality and stability.
6. Novel starters for novel beer
The use of
Over time, there has been gradual domestication and selection of yeasts . Selected
6.1. Genetic improvement of brewing strains
High quality sequencing,
The positive role of
In the last few years, the selection of starter strains has also been carried out within non-
7. Spontaneous fermentation
It is already known that some of the above mentioned yeast take part in the spontaneous fermentation processes of specialty beers. A typical example of this spontaneous process is the lambic beers. The fermentation of these beers is driven by brewery-resident microorganisms that are self-inoculated by exposing the wort in open tanks during the overnight cooling, before transferring it to wooden barrels for fermentation and aging. The fermentation of such Belgian lambic beers involves
8. Nonconventional yeast
Recently, growing attention has been given to the possible contributions of nonconventional yeast to beer production. The success of craft beers has induced brewers to look for new alternatives for fermentation, such as nonconventional yeast, to impact on the aroma and flavor, and thus to generate differentiated products. The production and increase in the aroma compounds through biological methods exploits the metabolic pathways of the yeast for the promotion of the so-called bioflavor. This approach includes microbial bioconversion of the flavor precursors, use of strains that produce the required compounds, and genetic modification of the yeast . In this regard, although still poorly investigated in the brewing sector, the use of nonconventional yeast might enhance the analytical and aromatic profiles of the final product and reduce the alcohol content .
Among the nonconventional yeast that can potentially be used in brewing,
Moreover, Michel and colleagues  screened 10
8.1. Specialty beers
In response to increased consumer demand, the brewing industry has devoted much research effort to the development of new technologies and innovations for expansion of the assortment of specialty beers. Five types of specialty beers of particular interest have been described: low calorie beer, low alcohol or nonalcohol beer, novel-flavored beer, gluten-free beer, and functional beer . Beers with a low calorie content have achieved great interest due to the problem of obesity, especially in Western populations, which accounts for a growing market segment.
This type of beer can be made by special mashing and collection of the wort with large amounts of fermentable sugars, or by inoculating microorganisms that can hydrolyze the more complex sugars, to reduce the concentrations of residual sugars in the final product. This is the case for
9. Functional beers
Functional beers are defined as beers with health benefits for those who consume them moderately. These are based on the use of nonconventional yeast that can produce or transform some beneficial compounds. This is the case for melatonin, which is a sleep-regulating hormone in mammals that has antioxidant properties and that can be produced in beer during alcoholic fermentation by the appropriate yeast . Within functional beers, there are gluten-free beers for consumers with the condition known as coeliac disease, which is a gluten-sensitive and immune-mediated enteropathy. Recently, there has been increased demand and consumer interest to develop gluten-free beers from alternative cereals, such as sorghum and maize. In addition to those mentioned above, other yeast are promising candidates for the production of specialty beers. Indeed, yeast from the genera
10. Yeast handling in the brewery
One of the common and efficient cost-reduction measures in beer production is serial re-pitching of the yeast at the end of the fermentation. The type of fermenter and yeast used define the procedures needed for the recovery of the yeast biomass. Generally, by the time a crop has formed, the yeast should be removed as soon as possible, as it has no further positive role in the fermentation. The optimum time for yeast removal is usually decided by the brewers, by considering the different parameters, such as full attenuation of the wort, or the reduction of vicinal diketones to optimal levels.
Top-fermenting (ale) yeast are removed using specific skimming systems in the fermenters. Not all of the yeast heads that are cropped are retained for re-pitching. Only a fraction of the yeast head is used, as that composed of middle to young yeast cells and relatively free from trub. When using bottom-fermenting (lager) yeast, the yeast slurry on the base of the fermenting vessel is obtained by simply removing the overlying beer. As in the case of top-fermenting yeast, for subsequent use it is necessary to retain the yeast cells that are less enriched in trub and are of a middle age. This fraction can be identified in the middle of the sediment, as the lowest layers are enriched in trub. Furthermore, some authors have suggested that the more flocculent portion of the cells settle in the middle, while the less flocculent cells are in the top portion of the cone. In the majority of breweries, pitching yeast is usually converted to a liquid slurry by adding water or by leaving enough entrained beer to facilitate yeast transport to the storage vessels via pumping. Alternatively, yeast cakes can be obtained by recovering the yeast from the entrained beer through filtering.
10.1. Yeast storage
During storage, yeast quality decreases as a function of storage conditions and procedures. This can lead to aberrant fermentation, which can be seen as slow attenuation rates, poor flocculation performance, and undesired flavor development. In particular, it is necessary to avoid yeast contamination with bacteria, wild yeast, or other starter yeast (cross-contamination). A common procedure to reduce the bacterial load is to treat the yeast slurry with a chemical disinfectant at low pH (i.e., acid washing). Usually citric and phosphoric acids are used at low temperatures (2–4°C) with continuous gentle stirring . The correct procedures during acid washing allow the removal of bacteria without affecting the yeast performances in the subsequent fermentation.
Another aspect of primary importance is the avoidance of excessive stress to the yeast cells during cropping and storage, to minimize any changes in their physiological conditions. In particular, the intracellular concentrations of storage carbohydrates (i.e., glycogen and trehalose) and sterols and other lipids are of primary importance for the duration of storage . Indeed, the storage phase is a period of starvation, and the yeast need to rely on nutritional reserves that were accumulated during fermentation. Glycogen is synthesized during mid-fermentation, and its dissimulation is directly correlated with the storage temperatures. Furthermore, supplementation of cropped yeast with linoleic acid before pitching has been suggested as a convenient way to improve the yeast physiology without affecting the yeast growth, fermentation rate, and production of volatile compounds during the subsequent beer production .
10.2. Continuous re-pitching
The influence of serial cropping and re-pitching on the consistency of fermentation performance and beer composition poses technological questions about the number of generations that can be allowed to elapse before introducing new yeast. This decision is usually made by the individual brewers, as there are no pre-determined rules. In breweries with high hygiene standards, serial re-pitching can continue for 15–20 generations, while in microbreweries, even 5–10 generations is considered excessive. Continued serial re-pitching of yeast can be associated with gradual deterioration in the yeast conditions, which can result in decline in fermentation performance. Indeed, the aging process in yeast is associated with gradual disruption of many of their metabolic processes. On the other hand, there is an economic cost to propagation, and if a re-pitched yeast is performing satisfactorily, there is less need to introduce a newly propagated yeast. Indeed, the first generation fermentation using new yeast lines is atypical, as the yeast cells are less adapted to the wort and fermentation conditions, particularly for high gravity brewing. To assist brewers in the decision of when to introduce a new yeast line, several methodologies have been developed. The most widely used assay to evaluate yeast viability involves microscopic observation of the yeast cells stained with methylene blue. While this method is economic and simple to perform, it is also subject to operator error and known to overestimate viability . Recently, flow-cytometric methods have been developed to assist brewers in the evaluation of their yeast viability and vitality. In particular, analysis of the yeast cells stained with the fluorescent dye oxonol allows automatic detection of yeast viability without interference from the wort trub . Moreover, flow cytometry assays are not limited to viability tests, but can also be used for vitality tests that are related to the yeast fermentation performance, which can be implemented using specific fluorophores.
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