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).
Many yeast propagators are based on the pioneer work of Hansen and Kuehle and operate either semicontinuously or on a batch basis (88). Sterile wort is run into a vessel that has been presterilized by steam and cooled. Sterile air or oxygen is perfused through the wort, and the culture of yeast from the laboratory is inoculated. Aeration or oxygenation may be supplied continuously but, because of foam formation, it is more usually intermittent (43). In a modern example, the propagator is charged with 23 hl of wort of specific gravity 1.040 and is pitched with 91 g of pressed yeast; aeration is provided for 1 min in every 5 at 0-11 m(3)/minute. At approximately 18C exponential growth occurs for 48 hr when some 54 kg of pressed yeast is available in partly fermented beer of specific gravity 1.016. when the entire contents of the propagator are discharged into 250 hl of fresh wort there is no lag phase (5,222). With some strains of yeast, the pH levels of beers produced in the propagator are low and the cells are elongated, but these effects are lost when the yeast is used normally (43). Modern cylindro-conical fermenter may be used as yeast propagators and stirred-tank continuous fermenters are particularly good (62).
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.
In the average brewery, a large inoculum of cells is used (ca 5-15 million cells/ml of wort). In each fermentation the number of cells increases three- to four fold. Therefore, one-third to one-fourth of the yeast crop of each fermentation is used for inoculation of the next batch. If the brewery is of sufficient size (ca 1 million bbls of beer or more annually in the United States), drying of the remaining yeast for use as an animal food supplement becomes economically feasible. Alternatively, the yeast is used for manufacture of yeast extract or for fermentation in grain distilleries.
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)
Esters other than ethyl esters utilize fusel alcohols which arise from either carbohydrate or amino acid metabolism, giving a range of oxo-acids (6,106,111,115). Oxo-acids in excess of the requirements of the yeast may be enzymically decarboxylated to the corresponding aldehyde which is then reduced to yield the fusel alcohols. Thus, the uptake of isoleucine, leucine, valine, and phenylalanine from wort results in production by the yeast of 2- and 3-methyl butanol, iso-butanol and phenethyl alcohol. The choice of yeast strain, conditions of fermentation, and wort composition each affect fusel alcohol formation, thereby modifying beer flavor and aroma and providing material for ester synthesis.
Acetoin, diacetyl, and 2,3-pentanedione are normal beer constituents but in excess they spoil the beer by their musty, buttery, and honey flavors, respectively. The threshold of tolerance for vicinal diketones is in the order of 0.2-0.5 microgram/mg (53,240). Acetoin is produced from "active acetaldehyde" (hydroxyethyl-2-thiamine pyrophosphate) and free acetaldehyde in the presence of a carboligase. Yeast does not oxidize acetoin to diacetyl but in stead tend to reduce diacetyl: thus, yeast is often added to filtered beer if the level of vicinal diketones is too high. Active acetaldehyde will react with pyruvic and oxo-butyric acids to yield acetolactic and acetohydroxy butyric acids, respectively, and it is believed that these acids (which may be precursors of valine and isoleucine) diffuse to some extent from the yeast cells into the beer. By decarboxylation and oxidation within the beer, the vicinal diketones are produced (218). Strains of Pediococcus and respiratory-deficient mutants of brewers' yeasts are sometimes responsible for high levels of vicinal diketones (44).
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).