It is usual to select strains of yeast for brewing from yeasts already in commercial use. While the application of genetic principles to the production of new strains of bakers' yeast has been successful (65), there have been few instances of induced hybridization for commercial brewing (112). Mutation and transformation (178) have also been suggested for producing brewing strains with new properties but there has been no commercial exploitation. Desirable features in a brewing yeast include:
In batch fermentation, it is desirable that the yeast separates readily from the beer at the conclusion of fermentation although less necessary if centrifuges are used for separation (37). Selection is normally based on the results of small-scale fermentations (213). For continuous fermentation using unstirred towers, it is necessary to have a yeast which is strongly sedimentary throughout the fermentation in order to maintain a yeast plug at the base of the tower. (4, 32, 198).
Some breweries isolate, select and maintain their yeast strains but others engage specialist laboratories to provide this service. The entire yeast within a brewery may be derived from a single cell, from several isolated cells, from a single yeast colony or from several colonies (32). Again, some breweries choose to have two or more strains that may be employed in mixture or separate fermentation vessels. Proportions of strains in a mixture may, however, change because of alterations in materials or procedure, and individual strains may be eliminated (103). Nevertheless, a yeast of several strains may adapt more successfully than a single clone. Cultures may be maintained at 10C on wort-agar slopes or at 4C in carbohydrate media such as 10 per cent sucrose, wort, or Wickerham's malt extract medium (243). Subculturing is carried out at regular intervals (24), preferably at less than three-month intervals. Lyophilized cultures have not been used extensively because there is a high mortality of cells during freeze-drying, and thus mutants and variants may be selected (251).
Propagation of brewers' yeast enables a brewery to replace the entire stock of yeast on a predetermined basis. Frequently, a batch of yeast is used only about 12 times before it is discarded. there are, however, breweries claiming that their yeast has not been changed for 50 years or more (139). The changes in a yeast that persuade brewers to discard them relate either to infection with bacteria or wild yeast, poor settling near the end of fermentation if a bottom yeast, or partial loss of ability to grow, ferment, and produce the expected quality of beer.
Yeast collected for repitching is usually mixed with 2-3 volumes of chilled water and passed through a vibrating screen to help remove bitter cold trub particles (196). In a modern brewing operation, the screened yeast passes directly into a scale hopper thereby providing the required amount of yeast for repitching (Editorial 1959, Brewers Digest 24:11). One danger in washing with water is a change in metabolic activity from fermentation to respiration (31), thereby increasing susceptibility to autolysis (116). Conversely, storage under chilled water is believed to hold autolysis to a minimum (100). Yeast to be stored for a prolonged period of time is best left in the fermenter under beer (38). One danger of prolonged storage is incomplete ability to ferment upon reuse (197). A minimum 24-hr rest period is believed necessary before reusing a yeast (197), but present practice in Britain with top and bottom yeasts in cylindro-conical vessels belies this belief.
Some suggestions for reducing yeast autolysis include iron enrichment and maintenance of a high C to N ration (117), and the addition of unsaturated fatty acids to wort (223, 224)). An important index of yeast autolysis is increased Proteolytic activity (10).
Yeast contaminated with beer spoilage bacteria may either be replaced with a pure culture or washed with acids such as phosphoric acid (45), ammonium persulfate (27), or a combination thereof (7), thereby eliminating the necessity for replacement. Yeast replacement or acid washing can affect beer flavor since it usually requires several fermentations for fresh yeast to become acclimatized to the brewery (16). Related information on yeast replacement and acid washing is found in sections Selections and Propagation of Brewers' Yeast and Microbiological Control in Brewing, Fermentation, and Packaging Including Sanitation.
Brewers' wort (145) commonly has 8-14 per cent total solids, of which 90-92 per cent are carbohydrates. The major carbohydrate components of wort are glucose, fructose, maltose, sucrose, maltotriose, and a group of linear and perhaps also branched polymers of glucose containing four or more units. Brewers' yeast uses the sugars up to maltotriose but not the larger molecules (91). More fermentable worts are produced if the malts used are rich in amylolytic enzymes; unkilned malts are particularly rich. Lowering the mashing temperatures increases fermentability (86). Raising the proportion of unmalted cereal or the temperature of mashing diminishes wort fermentability (13, 110). Similarly, the concentration of nitrogenous material in the wort is influenced by the malt and other materials used in wort making and by mashing and wort boiling conditions (109, 200). Commercial worts commonly have 70-110 mg N/100 ml, and the nitrogenous constituents include ammonia, simple amines, amino acids, purines, and simple peptides to complex proteins (145). The most important source of nitrogen is the amino acids. Proline, an imino acid, is abundant but is scarcely used (113). Biotin, inositol, pantothenic acid, pyridoxine, and thiamine are present in wort and utilized by brewers' yeast. The total ash content of wort represents about 2 per cent of the wort solids; phosphates, chlorides, sulfates and other anions are present with the cations Na, K, Ca, Mg, Fe, Cu, and Zn. Phosphate content is in the range 60-120 mg/ 100 ml (64), and sulfate content in the region of 400 mg/liter (125). Dissolved oxygen content varies from about 4-14 mg/liter (154).
The growth and metabolism of brewers' yeast have recently been reviewed (191). Yeast cells readily take up monosaccharides by facilitated diffusion (120) but di- and trisaccharides enter the cell by means of a permease system (92, 93) which is inducible in some strains, constitutive in others. Maltotriose is the last fermentable carbohydrate to be taken up. There is also a sequence of uptake of amino acids (Table 2) probably because of competition at the permease sites between the various acids (113, 114). The yeast is able to synthesize certain amino acids more easily than others. Thus, lysine, histidine, arginine, and leucine yield oxo-acids which are not furnished to any extent from carbohydrate metabolism and therefore changes in their concentration may affect the general metabolism of the yeast and hence the quality of the final beer. Nitrogen nutrition is complicated, however, by the ability of yeasts to release amino acids and nucleotides especially when changing the medium, thereby causing alteration in membrane permeability (49,136).
When yeast is pitched into aerated or oxygenated wort, there is at first a lag period when the cells actively take up materials from the wort, including the dissolved oxygen. It is not certain why the oxygen is important for the growth of the yeast but it may well permit synthesis of unsaturated lipids (2,23) and influence mitochondrial function (36). The level of oxygen (about 4-14 mg/liter) is insufficient for any significant aerobic respiration and indeed the high levels of fermentable sugar ensure by the Crabtree effect (47, 211) that the metabolism is anaerobic. The major energy-yielding pathway is the glycolytic Embden-Meyerhoff-Parnas (EMP) one, but the hexose monophosphate shunt mechanism operates to a limited extent, mainly for the synthesis of pentoses (102). Pyruvic acid, the product of the EMP pathway, undergoes enzymic decarboxylation and reduction to ethanol and carbon dioxide. While this is the outstanding feature of yeast metabolism during beer production, special flavors and aromas of beers may arise from minor biochemical reactions, notably those stemming from pyruvic acid. For instance, esters arise from an intracellular reaction involving acetyl-CoA compounds, alcohols, and ATP (175). Ethyl acetate is thus produced from acetyl-CoA and ethanol, both products of pyruvic acid metabolism. The various fatty acids available with the cell compete in ester synthesis, except that propionic, isbutyric, and isovaleric acids do not furnish ethyl esters. Leakage of acetyl CoA-compounds from the synthesis of higher fatty acids may also contribute to the level of esters, for instance, ethyl caprylate (174)
Yeast requires sulfur for the production of proteins, coenzymes, vitamins, etc., and takes up organic sulfur from wort, chiefly as methionine, and inorganic sulfur in he form of sulfate (152). Hydrogen sulfide is generated during yeast metabolism and depends, in brewery fermentation, on the yeast strain used, the temperature, and the wort composition (123). The gas, unpleasant over certain threshold levels, arises either from leakage of sulfide ions during the enzymic reduction of sulfate or more likely by the action of cysteine desulfhydrase on cysteine (133). Mercaptans, sulfides, and thicarboxyls have been implicated in the flavor of beer (227). Nevertheless, growth of yeast in synthetic media and wort gives rise to no significant levels of volatile organic sulfur compounds (97,176). these compounds arise from nonenzymic reaction in the beer (170) and from the metabolism of spoilage bacteria (1).