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Brewing yeast Brewer’s yeast scepticism! The method was merely regarded as a means of reducing infection by wild yeasts and bacteria. In 1959 it was reported that of 39 ale yeast cultures in use commercially in Britain, 12 contained a single strain, 16 had two major strains and the rest contained three or more yeast strains. Selection of the production strain(s) varies depending on the beer type. Overall, the properties that define good brewing yeast cultures are: • A rapid fermentation rate without excessive yeast growth; • An ability to withstand high alcohol and osmotic stresses imposed during brewing; • Reproducible flavour production; • Ideal flocculation behaviour; • Efficient use of maltose and maltotriose; • Good handling characteristics, including genetic stability. A brief overview There is an old German saying, “Hopfen und malz, Gott erhalt’s”, which translates to “God save hops and malt”. So perhaps if you provide the hops and the malt, God will take care of the rest. Archaeologists have uncovered evidence of breweries and bakeries that used yeast over 4,000 years ago. However it was not until the 17th century that the globular structures in beer were first observed using simple microscopes. It took a further 120 years before the true role of yeast in fermentation was established. by Anne E Hill and Graham G Stewart ICBD, Edinburgh, Scotland I n the interim, many brewers referred to the foam used to pitch sweet wort as ‘godisgoode. By the mid 1800s the academic mainstream had been convinced that yeast was a living organism, rather than a chemical catalyst and later in the century pure culture techniques were developed at the Carlsberg Laboratory in Copenhagen. Brewing research on yeast has fluctuated since that time, with periods of intense activity and times when war or the economy has reduced the number of active researchers. Here we briefly review our knowledge of brewing yeast and discuss yeast for 21st century beer production. Outline of yeast Around 1,500 species of yeast may currently be described. These are predominantly singlecelled fungal micro-organisms (Figure 1) able to grow in both the presence and absence of oxygen. They are chemoorganotrophs, meaning that they use organic compounds as a source of energy and do not require sunlight for growth. Cell sizes vary, depending on the strain and the stage in the life cycle, from 3 to 14µ and have a mass of approximately 40ρg when dry (40g of dried yeast typically contains 1012 cells). Most yeast reproduce asexually by budding but some yeast species divide by binary fission (the parent divides into two daughter cells). The budding yeasts, or ‘true yeasts’, are classified in the order Saccharomycetales, Figure 1. Electron micrograph of a yeast cell showing multiple bud scars. the most common members belonging to the species Saccharomyces cerevisiae. Brewer’s yeast Strains of Saccharomyces cerevisiae have been used for thousands of years in both baking and fermentation. The yeast cell is also an important research model for genetics and cell biology (also called an experimental eucaryote), in addition to being useful in energy generation through microbial fuel cells and biofuel production. What, though, distinguishes brewer’s yeast from the hundreds of other yeast strains? There are basically two major kinds of yeast used in brewing: Saccharomyces cerevisiae (ale) and Saccharomyces pastorianus, also still called Saccharomyces uvarum (carlsbergensis) by some, (lager). Ale yeast operates at around room temperature (1822°C), ferments quickly, and produces the ‘fruitiness’ characteristic of many ales. They are typically top fermenters and produce a higher alcohol concentration than lager yeasts. However, with the advent of cylindroconical fermentation vessels, bottom cropping ale yeast strains are not uncommon. The use of centrifuges to crop yeast at the end of fermentation has promoted the selection of both ale and lager non-flocculent cultures. Traditionally lager yeasts are bottom fermenters. They work at colder temperatures (8–15°C), ferment slowly and utilise more wort sugars more efficiently, leaving a clean, crisp taste. Around the world, ale and lager yeasts predominate but there is an increase in the number of breweries employing Brettanomyces yeast, traditionally used in Lambic beer production. As a result of work by Emil Hansen at the Carlsberg Laboratory in the late 1880s, the practice of employing pure strains in lager production spread around the world. Aleproducing regions however, met this ‘radical innovation’ with some opposition and Living conditions Compared to other media employed for the production of fermentation alcohol (both industrial and potable) wort is by far the most complex. It is an intricate environment because it consists of a number of simple sugars, dextrins, amino acids, peptides, proteins, vitamins, ions such as zinc, magnesium, manganese, calcium, sodium and potassium, nucleic acids and other constituents far too numerous to mention. The wort sugars include sucrose, glucose, fructose, maltose and maltotriose together with dextrin material. The individual amino acids, ammonium ions and small peptides (diand tripeptides) found in wort are known collectively as free amino nitrogen (FAN). FAN is believed to be a good index for potential yeast growth and fermentation efficiency as adequate levels of FAN in wort ensure efficient yeast cell growth and hence desirable fermentation performance. High-gravity brewing employs wort at higher than normal concentration and consequently requires dilution with deoxygenated water at a later stage in processing. High-gravity worts can influence yeast performance with effects apparent upon fermentation and flocculation. The increased osmotic pressure, elevated alcohol concentration and modified nutrient balance, all have a profound influence on yeast performance during their fermentation. For example, when yeast is first pitched into high gravity wort, passive diffusion of water out of the cell occurs and this results in decreased cell viability within the first 24 hours of fermentation. The decrease in viability is exacerbated with 20°Plato compared to 12°Plato wort. However, with both lager and ale yeast the viability usually recovers later in the fermentation. Another negative effect of high gravity worts on yeast performance concerns the number of generations (yeast cycles) that can be fermented by a single yeast culture. The number of cycles that can be employed is reduced with increasing wort gravity. Brewer & Distiller International • June 2009 • www.ibd.org.uk 13 Brewing yeast Figure 2. Intracellular concentrations of glycogen and lipids in a lager yeast strain during fermentation of a 15°Plato wort. Figure 3. Order of uptake of wort sugars by yeast. How does yeast work? important in brewing, Scotch whisky production and baking, since maltose is the major component of brewing wort, spirit mash in Scotland and wheat dough. A major limiting factor in the fermentation of wort is the repressing influence of glucose (and possibly fructose) upon maltose and maltotriose uptake. Only when approximately 50% (this is yeast strain and wort composition dependent) of the wort glucose has been taken up by the yeast cells will the uptake of maltose commence. In a similar manner, the presence of glucose will repress the production of glucoamylase by S. diastaticus thereby inhibiting the hydrolysis of wort dextrins and starch. Repression of this nature has a negative effect on overall fermentation rate. A range of ale and lager yeast strains have been employed in order to explore the mechanisms of maltose and maltotriose uptake from wort. A number of studies have found that ale strains appear to have greater difficulties completely fermenting wort (especially maltotriose and particularly in high gravity wort) than lager strains. Recent studies on the utilisation of nitrogenous wort components have confirmed the early work by Jones and Pierce that amino acid uptake can be divided into four groups (Table 1) with amino acid uptake completed, with the exception of proline, within the first 48h of fermentation. An important recent finding is that yeast fermentation activity does not cease when wort amino acids are depleted. During fermentation, oligopeptides are produced as a result of larger peptide hydrolysis due to yeast protease excretion/secretion. Both lager and ale yeast strains can simultaneously use amino acids, ammonium ions, and small peptides as sources of assimilable nitrogen. The life cycle of yeast is activated from dormancy when it is pitched into the wort. Growth follows four phases, which usually overlap during fermentation: lag, log, fermentation and sedimentation phases. To grow successfully, yeast requires an adequate supply of nutrients. For healthy fermentation a ready supply of fermentable carbohydrate, nitrogen, vitamins and minerals are needed. Inadequate nutrition can result in poor beer stability, generation of mutant yeasts, poor flocculation, off flavours and incomplete fermentations. Yeast physiology, activity and nutrition requirements vary between strains and additional nutrients can be used to ensure consistent fermentations or to avoid fermentation problems. At the beginning of fermentation, build-up of unsaturated fatty acids and sterols, at the expense of the intracellular storage carbohydrate glycogen, is essential for a normal growth pattern of the yeast population during the rest of the fermentation process (Figure 2). Yeasts are unable to synthesise unsaturated fatty acids and sterols under strictly anaerobic conditions, so in practice oxygen is supplied during pitching. Optimisation of the dissolved oxygen content of wort during fermentation is important to achieve a good fermentation and a high quality product. One of the major advances in brewing science during the past 40 years has been the elucidation of the mechanisms by which the yeast cell utilises, in an orderly manner, the plethora of wort nutrients. In the normal situation, brewing yeasts are capable of utilising sucrose, glucose, fructose, maltose and maltotriose in this approximate sequence (or priority), although some degree of overlap does occur, leaving maltotetrose and the other dextrins unfermented (Figure 3). Brewer’s yeast is also capable of utilising sugars such as galactose but not lactose unless it is hydrolysed to its constituent monosaccharides – glucose and galactose. In addition, there is a closely related species (regarded as the same species by some) to S. cerevisiae, designated as S. diastaticus. This yeast species produces an extracellular glucoamylase that is capable of utilising wort dextrins to glucose units which are metabolised. The transport, hydrolysis and fermentation of maltose are particularly 14 One of the major factors when considering important characteristics during brewing or other ethanol fermentations is flocculation. The ideal brewing strain is one which in a typical fermentation, without the use of a centrifuge, remains in suspension as fermenting single cells until close to the end of fermentation when the wort sugars and most amino acids, as well as vicinal diketones are reduced. Only then should the culture rapidly flocculate and settle out of suspension to be harvested and repitched into wort for a subsequent fermentation. Genetic manipulation of brewer’s yeast strains Over the years, considerable efforts have been devoted to a study of both the biochemistry and genetics of brewer’s yeast (and other industrial yeast strains). The objectives of these studies have been two fold: (1) to learn more about the biochemical and genetic make-up of brewing yeast strains; and: (2) to improve the overall performance of such strains, with particular emphasis being placed on broader substrate utilisation capabilities, increased ethanol production, improved stress tolerance to environmental conditions such as high osmotic pressure, ethanol, temperature, salt and physical shear and to understand the mechanism of flocculation. The behaviour, performance and quality of a yeast strain is influenced by two sets of determining factors, collectively called naturenurture effects. The nurture effects are the responses made (i.e. the phenotypes) to the environmental factors which the yeast is subjected from pitching onwards. On the other hand, the nature influence is the genetic makeup (i.e. the genotype) of a particular yeast strain. Spontaneous yeast mutations are a common occurrence throughout the growth and fermentation cycle, but they are usually recessive, due to functional loss of a single gene. The characteristics that are encountered resulting from mutations that are harmful to wort fermentation are: • The tendency of yeast strains to mutate from flocculent to non-flocculent; • The loss of ability to ferment maltotriose; • The presence of respiratory deficient mutants. Table 1. The order of wort amino acid uptake during fermentation Group A Fast absorption Group B Intermediate absorption Group C Slow absorption Glutamic acid Aspartic acid Asparagine Glutamine Serine Threonine Lysine Arginine Valine Methionine Leucine Isoleucine Histidine Glycine Phenylalanine Tyrosine Tryptophan Alanine Ammonia Brewer & Distiller International • June 2009 • www.ibd.org.uk Group D Little or no absorption Proline Brewing yeast production by fusion of a flocculent strain with sake yeasts; • Construction of strains with improved osmotolerance by fusion of S. diastaticus or S. rouxii (an osmotolerant yeast strain). • Recombinant DNA techniques can also be used to make thousands of copies of the same DNA molecule to amplify DNA, thus generating sufficient DNA for various kinds of experiments or analysis. ‘Improved’ yeast strains using recombinant techniques include those with: • Glucoamylase activity from the fungus Aspergillus niger; Figure 4. Respiratory sufficient (red colonies) and • Glucanase activity from the respiratory deficient (white colonies) mutants – triphenyl tetrachloride overlay. bacterium Bacillus subtilis, the fungus Trichoderma reesii and The respiratory deficient (RD) or ‘petite’ barley; mutation is the most frequently identified • Acetolactate decarboxylase activity from mutant found in brewing yeast strains. This the bacteria Enterobacter aerogenes and mutant usually arises spontaneously when a Acetobacter spp for diacetyl control; segment of the DNA in the mitochondria • Extracellular protease for enhancing beer becomes defective to form a flawed physical stability beer; and mitochondrial genome. The mitochondria are • Modification of yeast’s flocculation then unable to synthesise certain proteins. properties. This type of mutation is also called the ‘petite’ mutation because colonies of such a Prospects for the use of recombinant DNA mutant are usually much smaller than the with brewer’s yeast and their use in the respiratory sufficient (RS) culture (also called brewing industry are still uncertain. It is ‘grande’) (Figure 4). Deficiencies in surprising that recombinant brewer’s yeasts mitochondrial function result in diminished are not commercially in use today in both ability to function aerobically and beer brewing and distilling. Permission was produced with a yeast culture that is RD is granted over a decade ago from the likely to have flavour defects and appropriate authorities in the United fermentation problems. For example, beer Kingdom for the use of a brewing strain, produced from these mutants may contain cloned with DNA from S. diastaticus that elevated levels of diacetyl and higher alcohols secretes glucoamylase to utilise wort dextrins and residual maltose and/or maltotriose. and produce low calorie beer. However public The advent of ‘new biotechnology’ has opinion does not yet support the use of such stimulated the development of novel methods manipulated strains. of genetic manipulation, such as spheroplast (protoplast) fusion and recombinant DNA The future techniques. Examples of successful fusions The economic significance of brewer’s yeast with commercial brewing and related strains cannot be overestimated. With the importance are: of the environment and the consequent need • The construction of a brewing yeast with to improve efficiency and reduce fermentation amylolytic activity by fusion of S. losses, it seems timely to take a more cerevisiae and S. diastaticus; considered look at the yeast we employ and • A polyploid strain capable of high ethanol the environment they are placed in. In March this year, the complete genome sequence of a Saccharomyces pastorianus strain was published. Analysis of the sequence shows that lager yeast is a hybrid of the ale yeast Saccharomyces cerevisiae and another yeast, Saccharomyces bayanus. New developments in gene sequence technology, such as this, will enable advances in the way yeasts are used. The sequencing of the S. cerevisiae genome (published in 1996) in conjunction with gene expression analysis has already enabled the identification of genes that have altered gene expression patterns in response to stressful environmental conditions. A ‘stress model’ has been developed to assess yeast stress resistance and evaluate the suitability of a specific strain for use in industrial ethanol fermentations. This ‘model’ could potentially be used for screening candidate yeast strains for relative stress resistance in the fuel ethanol industry and other industries where yeast encounters similar stresses. In addition, the creation of large libraries of yeast strains combined with progress in yeast screening will enable the rapid identification of strains that match desired brewery fermentation targets (such as flavour and flocculation characteristics). Over the past century we have come a long way from early perceptions of fermentation and the tools are now available to make significant improvements. Despite the increased wealth of our knowledge of brewer’s yeast though, there is still something of the Divine in the work they do! ■ ■ The authors Annie Hill is a Lecturer in Microbiology and Graham Stewart is Emeritus Professor in Brewing and Distilling both at Heriot-Watt University. NEW IMPROVED WEBSITE... Check out our website for updated information on all IBD and Beer Academy services, activities and key dates... Institute of Brewing & Distilling www.ibd.org.uk Brewer & Distiller International • June 2009 • www.ibd.org.uk 15