CHAPTER 5
Yeast
Yeast are nonphotosynthetic, relatively sophisticated, living, unicellular fungi, considerably larger than bacteria. Brewers’ yeast are of the genus Saccharomyces. In an aciduric aqueous solution, they absorb dissolved vitamins, minerals, and simple nitrogenous matter (amino acids and very simple peptides) through their hemicellulose cell membranes. Then they employ a structured series of reactions known as metabolic pathways to break down these substances into nutrients, mainly amino acids to nitrogen and sugars to carbon. They obtain oxygen for metabolism from what is dissolved in the solution, or they split it off of molecular compounds.
Yeast, although living organisms, are actually highly organized enzyme collectives, each and every reaction of the yeast cell being controlled by a separate enzyme. In one reaction, simple sugars are reduced to alcohol and carbonic gas in the presence of a constitutive intracellular enzyme group called zymase and a phosphoric coenzyme. This process is known as fermentation. Yeast metabolism directly determines the degree of attenuation of any wort, and its character greatly affects the flavor of the finished beer. In fact, just as every living organism varies from every other, every yeast strain, and even every fermentation, has qualities distinctly its own. These depend on a number of factors. Particular strains of yeast produce different flavor characteristics; variations in the pH, temperature, or composition of each ferment result in slight to significant changes in the metabolic products.
Although brewing dates back to prehistory, it was not until 1841 that Mitcherlich discovered that yeast were essential to fermentation. Further research by Pasteur and Buchner revealed that yeast produced alcohol only as a by-product of carbon metabolism, and that it was in fact the nonliving zymase enzyme that was responsible for the fermentation of sugar.
Bottom fermenting began with Gabriel Sedlmayer in Munich and Anton Dreher in Vienna in 1841, using mixed strains of yeast that were not purely bottom fermenting. Emil Hanson, working at Jacob Christian’s Carlsberg brewery in Copenhagen, set the stage for modern lager brewing by isolating two distinctly different pure-culture yeasts, that is, strains propagated from a single cell and therefore all exhibiting the same characteristics. These were a top-fermenting Saccharomyces cerevisiae and the bottom-fermenting Saccharomyces carlsbergensis (S. uvarum). Pure-culture, bottom-fermenting yeast were first employed at Carlsberg in 1883; within the decade, lager culture yeast were being employed in refrigerated fermentation throughout Europe and America.
Besides their visually different flocculating characteristics, the yeast operate at different temperatures and ferment different sugars. The top-fermenting yeast strains are generally only effective at 55 to 75 degrees F (13 to 24 degrees C). They form colonies that are supported by the surface tension of the beer and create a very thick, rich yeast head; in general, they ferment glucose, fructose, mannose, galactose, maltose, sucrose, xylulose, and maltotriose, and partially ferment the trisaccharide raffinose. (S. cerevisiae splits off and ferments the fructose molecule from raffinose, leaving the disaccharide melibiose.) “Bottom-fermenting” lager yeasts, which don’t have as great an ability to chain and cling together, form smaller colonies that make a thinner, less tenuous head and that sediment out on the bottom of the fermenter more rapidly. They operate best at temperatures below 50 to 55 degrees F (10 to 13 degrees C). They ferment glucose, fructose, mannose, galactose, maltose, sucrose, melibiose, xylulose, and maltotriose, and fully ferment raffinose. Neither yeast ferments lactose, and all but the monosaccharide sugars need to be reduced by specific yeast enzymes before they can be fermented; sucrose must be split into glucose and fructose by invertase (sucrase), and maltose and maltotriose must be reduced to glucose by maltase (a-glucosidase). Maltose is able to be absorbed into the yeast cell before being hydrolyzed, but all the other disaccharides need to be reduced to monosaccharides by excreted enzymes before they can be transported into the yeast cell; this is the basis of maltose’s ready fermentability.
Top, left to right: Saccharomyces carlsbergensis, Lactobacillus, Pediococcus, Exiguus
Middle: Acetobacter, Acetomonas, Hafnia
Center: Pichia membranaefaciens (wild yeast)
Bottom: Torulopsis (wild yeast), Klebsiella, Zymomonas, Mycoderma
There are two distinctive subdivisions of the bottom-fermenting yeast S. carlsbergensis. The Frohberg type (F.U., dusty or “powdery” yeasts) ferment very strongly, and attenuation is very rapid. Because they do not clump well, they remain in suspension longer and consequently have a greater effect upon wort attenuation. They ferment isomaltose as well as maltose. The Saaz type (S.U., or “break” yeasts) settle out of the ferment more satisfactorily than do the powdery yeast strains. Consequently, they are very weak fermenters and reduce the extract very slowly. They do not ferment isomaltose.
Different yeast strains span the spectrum between these two major classifications, producing very different aspects of taste, mouthfeel, alcohol, and clarity in the finished beer; the yeasts that ferment the quickest and most completely are not often the yeasts that produce the best beer. Yeast strains are selected for the character of their fermentation, their ability to form colonies, their ability to ferment with or without forming esters, and their viability rather than their ability to attenuate the wort rapidly.
Chemically, yeasts are constituted of proteins (especially volutin, a nucleoprotein visible as small, shiny bodies in the vacuoles and cell plasma), glycogen (a starchlike reserve not usually found in older or stressed cells), minerals, enzymes, and vitamins (especially those of the beta complex).
Yeasts require various nutrients to renew these elements of their cellular structure. They absorb simple protein from hydrolytic solution, which they refine to a very high quality amino-acid group that composes roughly half of the yeast cell. Another 10 percent of the cell is calcium based and requires renewal, as do the minerals and trace elements that account for up to 5 percent of its structure. The minerals, besides calcium, are mostly the inorganic salts of phosphorus and potassium, with some magnesium, sodium, and sulfur. Yeast obtain these from mineral compounds in the ferment. The trace elements, especially zinc, boron, and manganese, are almost always available in small amounts from the malt, hops, or water. Yeast cells also require readily available oxygen for membrane synthesis; this is particularly important during the reproductive phase.
Yeasts reproduce by cell division, known as binary fission or budding. They reproduce only in a nutrient-rich environment; one daughter cell emerges and grows to the size of the mother cell in two to six hours in a suitable solution.
There are numerous strains of yeast, and each operates successfully within a very narrow pH and temperature range. It is necessary to carefully control these factors during brewing because the metabolic reactions and the reproduction rate of the yeast greatly influence the nature and flavor of the beer being brewed.
Yeast operate in suspension in a sugar solution, until they clump together and are brought to the surface by attached CO2 or are sedimented by virtue of their increased mass. They cease to have a considerable effect on attenuation once they have clumped.
As yeast cells age, their previously colorless, homogeneous plasma (protoplasm) becomes bubbly, then separates into solids and liquid substances by forming vacuoles that envelop the liquid plasma secretion; later they become granulated, and gradually the plasma turns to fat (visible as round bodies of varying sizes within the cell walls). Although they are incapable of sporulation, yeast can be sustained in an unsuitable environment for long periods by these fatty bodies.
In solutions lacking obtainable nutrients, the culture yeast will cease reproducing. When they can no longer sustain their own metabolic functions, albumin-, hemicellulose-, and vitamin-dissolving enzymes are activated, which reduce the yeast cell to amino acids and other simple substances. This autolization releases typical organic decomposition flavors into beer that is not racked off its sediment.
Because a ferment lacks nutrients needed by the culture yeast, or because the temperature or the pH of the ferment does not suit the particular yeast strain does not mean that wild yeast strains, mutations, or other microbes will not find the conditions ideal. Under normal conditions, one in a million yeast cells spontaneously mutates; under hostile conditions mutations increase dramatically. Either a wild yeast strain or one of these genetically altered mutations may become the dominant fermentation organism, to the detriment or ruin of the finished beer.
Wild yeast cause spoilage, including clove, sour, vinegar, sulphuric, phenolic/medicinal, fusely, and diacetyl flavors, and create film formation on the beer surface. Because wild yeast do not tend to cling together as well as culture yeast, and consequently remain in suspension longer, they almost invariably cloud the beer. The offending yeast may even be a wild strain of S. uvarum (S. carlsbergensis) or S. cerevisiae, but this does not make their presence any more desirable. Other common spoilage yeasts are Torulopsis, Candida, Dekkera, and Pichia species. It is essential to ferment with solely the culture yeast alone, maintaining its purity, ensuring its adequate nutrition, and carefully controlling its metabolic functions through manipulation of the nutrient spectrum, pH, and temperature of the ferment.
Culturing Pure Yeast Strains
Pure-culture yeasts are strains propagated from a single cell. Yeast from a successful primary fermentation that has exhibited good brewing characteristics are collected and mixed into a small amount of distilled water until the solution just becomes cloudy (approximately 100,000 cells/milliliter). One drop of the yeast solution is then mixed into one fluid ounce of diluted wort gelatin (beer wort diluted with sterile water at 4 to 8 °Plato [SG 1016 to 1032] mixed with 5 to 10 percent pure vegetable gelatin). The yeast is distributed by thorough agitation before the mixture is thinly spread over a clean cover glass, allowed to congeal, and placed in a sterile, moist container. The glass is then fixed to a graduated stage and microscopically examined at powers of 400 to 1,000 magnification, and the location of isolated, healthy-looking (white, hemispherical, nonreflective, uniformly sized) cells marked.
After twenty-four hours at 68 degrees F (20 degrees C), the glass is reexamined. Colonies should have formed. If they appear healthy, sample yeast cells from several isolated chains that are known to have grown from a single cell are removed with a flame-sterilized platinum or stainless-steel wire loop. Where a microscope is unavailable, the loop can be used to take a sample directly from a cloudy yeast solution, and an isolated colony can be chosen from the petri dish in the next step.
Each sample is streaked onto the surface of a sterile, staining nutrient agar (WL nutrient agar) in a petri dish. The inoculating streak is cross-hatched to isolate individual cells from it. When visible colonies have formed, an isolated clump (“rosette”) is microscopically examined, and if it is uncontaminated, it is used to inoculate an agar wort slant (eight fluid ounces of wort diluted to 4 to 8 °Plato [SG 1016 to 1032] with sterile water, mixed into five grams of prepared agar or vegetable gelatin, at room temperature, heated to boiling after fifteen minutes, and boiled [or autoclaved at 15 psi] for fifteen minutes). This is poured into sterile twenty-milliliter test tubes tilted fifteen degrees from the horizontal, that are then capped or plugged with cotton and allowed to cool.
The temperature is maintained at 50 to 68 degrees F (10 to 20 degrees C), until fermentation is apparent (usually two to four days); then the culture may be refrigerated for three months at 39 degrees F (4 degrees C) for lager yeast, or at above 50 degrees F (10 degrees C) for ale yeast. The medium can be covered with a layer of sterile mineral oil to maintain an anaerobic environment.
The slants may be recultured by adding one-half inch of wort to each of the older tubes, and after fermentation begins, using that mixture to inoculate four freshly prepared slants. All culturing must be done under strictly sanitary conditions using sterile labware.
For the brewer who does not have the laboratory equipment necessary to isolate and incubate pure cultures (basically, a microscope, wire loop, and the several items of glassware mentioned), purchase of commercially prepared vials or slants is the best source of a yeast culture. Frozen yeast is a reasonable alternative, as are properly handled liquid cultures. Granulated dry yeasts are the least-desirable alternative, as they are likely to contain many dead cells and be contaminated by bacteria during the drying process.
Slants are activated by covering the culture with one-half inch of wort. After forty-eight hours, that pure liquid-culture is used to inoculate a sterile, narrow-necked eight- or twelve-ounce vessel (or Erlenmeyer flask) filled with four fluid ounces of (sterile) aerated wort; this volume can be successfully inoculated directly from the cover glass if slant culturing must be omitted. The bottle must be capped or covered with a fermentation lock.
After twenty-four hours at 82 degrees F (28 degrees C), each four fluid ounces of wort should yield two to four grams of pure culture yeast. Cooler temperatures, however, are generally employed to retard the yeast’s reproduction rate; for lager yeasts, 50 to 68 degrees F (10 to 20 degrees C) for two to three days is usual. If capped, the lid must periodically be loosened to release pressure. When strongly fermenting, the culture may be roused into one quart of wort at a slightly warmer temperature than is usual for the brewery fermentation. It may be cooled to as low as 39 degrees F (4 degrees C) if the culture is not needed immediately. It is ready to pitch when it comes into active kraeusen. Each quart of starter should yield sixteen grams or more of pure culture yeast.
Aerate starter cultures often and well, so that the increase in available oxygen will stimulate greater yeast growth. In professional practice, a swab of yeast from the starter is cultured on a slide and microscopically examined for contamination before the parent culture is pitched.
Storing Yeast
Starters may be held at 39 degrees F (4 degrees C) for up to three weeks, or until fermentation subsides. The beer above the yeast can then be decanted, and the yeast covered with cold wort before it is refrigerated again.
For longer storage, after one week at 50 degrees F (10 degrees C), the yeast may be forced to sediment by lowering the temperature. The liquid above the yeast is decanted, and the yeast sediment pressed to remove at least all of the free liquid. The yeast mass is formed into a ball, tightly covered with plastic wrap, placed in chipped ice, and frozen.
Yeast prepared in this manner may be stored for several months. When reactivation is desired, it is crumbled into a quart of well-aerated wort.
Yeast may be collected from each brewing and successively subcultured until undesirable changes occur in the beer flavor or the strain’s fermentation profile. Most breweries repitch yeast only three to fifteen times before going back to the pure culture.
Washing Yeast
Usually yeast requires only rinsing before reuse, but periodically cultures should be washed to destroy bacterial contaminants. (This will not, however, destroy wild yeast; they can only be eliminated by reculturing.)
To wash, chill the vessel to sediment the yeast, then decant off the liquid above the yeast cake. Rinse the yeast by covering with, and then decanting off, distilled or biologically sterile water. Cover again with a solution of sodium metabisulfite, phosphoric or tartaric (winemakers) acid at pH 2.8, or a .75 percent solution of acidified ammonium persulfate (one teaspoon of tartaric acid with two teaspoons of ammonium persulfate in one quart of water, at pH 2.8) in water or sterile beer, equal in volume to the amount of yeast being washed. Agitate the yeast into temporary suspension. When the yeast have completely settled, or within two hours, decant off the liquid above the yeast, rinse several times, and cover with sterile wort. Some yeast may display abnormal characteristics in the first fermentation cycle following an acid wash; they should be cultured through at least one fermentation cycle before being pitched. The above precautions notwithstanding, many breweries wash their yeast regularly, often pitching the yeast, still in the acid solution at pH 2 to 2.5, after two hours.