Yeast Metabolism

The usual starting inoculum gives an initial yeast population of abut 5 X 106 cells per ml of wort. Depending on the activity of the yeast inoculum, a lag period of six to eighteen hours may occur. Although no increase in cell number is observed during the lag phase, metabolic activity is well under way. The yeasts are synthesizing sugar and amino acid transport systems, as well as enzymes necessary for their metabolism. As noted earlier, the wort is sparged with oxygen prior to pitching, thus the wort medium is initially highly aerobic. This oxygen-rich environment stimulates synthesis of membrane-associated sterols and un-saturated fatty acids. Sterols, and in particular, ergosterol, are essential for formation of yeast cell membranes; likewise, the unsaturated fatty acids provide membrane fluidity. Because of these requirements, yeasts may grow poorly in oxygen-deprived worts.

Following the lag phase, cells enter into the logarithmic growth phase, and a primary eth-anolic fermentation period begins. Despite the aerobic conditions initially established by sparging, sugar metabolism by yeast occurs not by aerobic respiration (i.e., the tricarboxylic acid or Kreb's cycle), but rather via the Emb-den-Meyerhoff-Parnas (EMP) glycolytic pathway. Although Saccharomyces are facultative anaerobes, enzymes of the Kreb's cycle are not even expressed during growth in wort.The repression of these genes under aerobic conditions occurs only for as long as the sugar concentration is high. This phenomenon, called the glucose effect, is due to catabolite repression by glucose. When the availability of glucose and other fermentable sugars is nearly diminished (i.e., < 1 g/L), however, metabolism in an aerobic environment shifts from fermentation to respiration (Box 9-6).

Catabolite repression by glucose also affects the specific biochemical processes that occur during the fermentation. Although brewing strains of S. cerevisiae and S. pastorium can use all of the major sugars normally present in wort (glucose, fructose, maltose, and mal-totriose), metabolism of these sugars does not occur concurrently. In fact, wort contains more maltose and maltotriose than glucose, yet glucose is still metabolized first.This preferential use of glucose occurs because: (1) at least one of the glucose transporters is constitutively expressed; and (2) the gene coding for the a-glucoside (maltose and maltotriose) transporter (AGT1) is repressed by glucose. Only when the glucose is fermented (after about one to two days), therefore, will expression of

Box 9-6. Physiology of Brewing Yeasts

Given the importance of yeast to the brewing industry, it is not surprising that so much attention has been devoted to understanding the physiological properties of Saccharomyces cere-visiae. Most brewing strains, as noted elsewhere, are very different from ordinary laboratory strains.This is because the beer and the brewing environment in which these strains have been grown for hundreds of years is quite unlike the docile conditions in the laboratory. Thus, most brewing strains have been screened and selected on the basis of their ability to grow well in wort and on their overall beer-making performance. Strains also have been modified by classical breeding programs such that specific traits are (or are not) expressed. More recently, the application of molecular techniques have made it possible to directly and specifically alter yeast physiology by introducing genes that encode for specific properties.

Although the primary function of the yeast during brewing is to ferment sugars to ethanol and CO2, it is also responsible for performing many other important duties.The yeast must not only produce adequate amounts of end products, but it must also metabolize all of the available fermentable carbohydrate to fully attenuate the beer. The main end products produced by brewing yeasts are ethanol and CO2; however, small amounts of higher alcohols, esters, aldehydes, phenolics, and other organic compounds also are produced and may affect beer flavor and quality. Some of these compounds have a negative impact, and their production is discouraged. Of course, the ability of brewing yeast to perform specific metabolic functions depends on how well it tolerates the high osmotic pressure, high ethanol concentrations, and other physiological stresses found in wort and beer. Thus, there are considerable physiological demands on yeasts during the brewing operation.

When the brewer first adds yeast to the wort, the environment is highly aerobic, due to the oxygen-sparging step that occurs just after the wort is boiled and cooled.Thus, metabolism by the facultative yeast might be expected to occur via a respiratory route (i.e., the tricarboxylic acid or TCA cycle). In fact, Pasteur demonstrated more than a century ago that when facultative organisms, such as brewers' yeast, are placed in an aerobic environment, aerobic metabolism is preferred (the so-called Pasteur effect).Aerobic metabolism, after all, is a more efficient process, yielding considerably more ATP than anaerobic metabolism (36 moles versus 2 moles ATP per glucose).

However, in the wort situation just described, yeast growth occurs via fermentative metabo-lism.Why would glycolysis occur in this environment? In part, because when the concentration of available sugar (i.e., glucose) is high (>1% or 50 mM), the reverse of the Pasteur effect occurs. That is, even under aerobic conditions, provided there is excess glucose, expediency, rather than efficiency, dictates how metabolism will occur. In fact, the enzymes of the TCA cycle are actually repressed, a situation referred to as the "glucose effect."Another reason, perhaps, for why the fermentation route proceeds under aerobic conditions is that brewing yeast strains are, in general, highly accustomed to fermentative metabolism.

this gene, as well as the a-glucosidase gene coding for maltose and maltotriose hydrolysis, occur.

Sucrose, in contrast, is hydrolyzed by an extracellular invertase, yielding its component monosaccharides, glucose and fructose. The regulation of sugar metabolism becomes especially relevant during the beer fermentation when high glucose adjuncts are used. Under these circumstances, repression of maltose and maltotriose metabolism may exist throughout the entire fermentation, and the beer will be poorly attenuated.

Following transport, intracellular metabolism of wort sugars via the EMP pathway yields mostly pyruvic acid and reduced NADH. Pyruvate is subsequently decarboxy-lated by pyruvate decarboxylase, forming CO2 and acetaldehyde. The latter is then reduced by NADH-dependent alcohol dehydrogenase, forming ethanol and re-oxidizing NAD. Because a small amount of the glucose carbon must be used to support cell growth, some of the pyruvate is diverted, via pyruvate dehydrogenase, to the Kreb's cycle, where biosyn-thetic precursors are formed. Re-oxidation of NAD then requires an alternative source of electron acceptors, leading to the formation of glycerol, higher alcohols, and other minor end-products found in beer.

The logarithmic growth phase period continues for two to three days for ales, and up to six or seven days for lagers, at which point all of the mono- and disaccharides, and most of the maltotriose, will have been fermented. Cell mass usually increases by less than two logs.

Importantly, the fermentation is exothermic, meaning that heat is generated, so the fermentation tanks must be cooled and maintained. Either internal cooling coils or external jackets are used to maintain temperatures of 8°C to 15°C for lagers and 15°C to 22°C for ales.

For ale fermentations, yeast cells, as noted earlier, rise to the surface, along with the evolved CO2.The yeast can then be skimmed off for re-use later. For lager fermentations, cells remain suspended, but there is enough CO2 evolved during the early stages of the fermenta tion to form cauliflower-shaped clumps. This thick foam layer is called krausen, and as growth and CO2 formation become more rapid, corresponding to the maximum growth rate of the cells, a period known as "high krausen" is reached. A portion of this material can be collected and re-used to initiate a secondary fermentation, as described below.

Although it is possible to monitor the progress of the fermentation simply by observing CO2 formation, the preferred means is to measure the specific gravity.As the fermentable sugars are consumed, the specific gravity, expressed as °Plato, decreases. When the specific gravity no longer is decreasing, most of the fermentable sugars will have been depleted, and the fermentation is complete. The beer is then considered to be "fully attenuated."At this point, usually four to seven days after the beginning of the primary fermentation period, the fermentation vessel is quickly cooled to 4°C or less. However, some brewers, prior to cooling, maintain a high temperature to promote use of diacetyl and related vicinal diketones that are themselves produced by yeast and which are responsible for off-flavors in beer (discussed later).This so-called "diacetyl rest" may reduce the necessary duration of the post-fermentation conditioning phase, when diacetyl reduction would ordinarily occur.

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