edited bv: MICHAELR. SLAEIAU~H HELENJ. JAMES
chem Impplement
Ogden, Utah 84408
The Biochemistry of Brewing Charles L. Bering SUNY College of Technology. Utica, NY 13504 Recent dramatic developments in the fields of hiochemistry, molecular biology, and molecular genetics have led to a burgeoning new industry called hiotechnology. Although the term hiotechnology conjures up images of recombinant DNA and exotic methods of producing rare and fine chemicals, the use of living organisms to produce useful chemical products is nearly as old as civilization itself. Evidence exists that nearly 6,000 years ago, man was producing a potent heverage from rotting grains and fruits. In its various forms, this biologically produced heverage has appeared as heer, wine, sake, mead, and other potions (1-4). The nroduction of beer todav is a worldwide industrv worth billions of dollars annual$, with breweries large and small throughout the globe. We know much more about the biochemical processes that occur in heer formation, hut hrewing is still as much an art as a quantitative science. In many industries, products must fall within very strict guidelines of quality control, and modern instrumentation allows a thorough analysis prior to packaging and shipment. The brewing industry also relies on quality control, hut the final arbiter-of the product's worth is the organoleptic or taste test. There is no single organoleptic quality that all beers achieve, and heer drinkers around the world have avariety of flavors from which to choose. The "best" heer is usually a matter of ~ e r s o n achoice. l The hio>hemical transformations that take place in hrewina involve several s t e ~ shut , the central Drocess is the fermentation of glucose to ethanol, catalyzed-by yeast. One can produce ethanol by simply adding yeast to a solution of glucose, hut this would not be heer. Beer requires several ingredients, combined at specific steps in the process. The main ingredients of beer are (1) malted harley seeds as a source of fermentahle carbohydrates and nitrogen, (2) water, a highly regional ingredient that greatly affects the final product, (3) hops, which are flavor enhancers and preservatives. (4) various adiuncts to increase the amount of fermentable sugar, and,"of course, (5) the yeast. The steps along the wav include maltine. mashine and wort formation. the fermentation, and various finishkg processes. This article will examine these steps with an emphasis on the hiochemical events taking place. Malting Barley supplies not only the carbohydrates that act as a carbon and energy source, hut also the nitrogen source for the subsequent fermentation. The harley kernels are rich in starch, which is made up of linear and hranched polymers of a-D-glucose (Fig. 1). The linear polymers are called amylose, while the hranched nolvmers . . are known as amvlo~ectin. During germination, several key enzymes are synthesized in the barley kernel. In malting, the kernels are first wetted a t
..
12-14 O C to initiate germination, then spread onto a malting floor or rotated in a drum at 15-20 O C for anywhere from a few days to two weeks. Moist air is blown through the grain at all times. During this time, green shoots begin to appear. On a biochemical level, some of the starch is hydrolyzed to glucose, maltose (a disaccharide of a-D-glucose), maltotriose, and other fermentahle sugars. This breakdown is due to two enzymes, a - and 8-amylase. The 8-amylase is present in the seeds initially and cleaves maltose units from the linear portion of amylose or amylopectin. Experimental evidence indicates that the amylose is preferentially attacked. a-Amylase is synthesized by the seedling during germination and cleaves glucose units a t random from the linear segments of starch. Other fermentahle sugars also appear in the malted harley, particularly sucrose and fructose. Only about 5-10% of the starch is broken down to fermentahle sugars in malting, hut the increase in amylase concentration in the seed sets the stage for further hydrolysis during mashing. CHzOH
CH20H
4
s e c t i o n of awlopecrin showing 1-6 branch
b)
Repeating vnir of amyloae
Figure 1. (a) Section of amylopectin showing 1-6 branch: (b) repeating unit of amylase.
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The other important enzymes appearing during malting are the nroteases. which cleave rotei ins and s u ~ n l vamino acids add peptides. These are "sed as a nitriied source during the fermentation. In addition, the proteases play an important role in the final clarification of the beer by removing proteins that may cause an undesirable haze. Malting is considered successful if around 95%of the seeds germinate. At the end of the malting period, the seeds are dried and heated (a process known as kilning) to lower the water content, halting many metabolic processes. Kilning also burns off the ereen shoots and rootlets that have s ~ r o u t ed from the seed.?oday malting is rarely done by thebrewerv. but the malt is sunnlied to the brewer bv malt houses. T& remaining steps ai~bccurat the brewery.. Mashlng The malted barley is next milled to produce a grist. The grist is carefully controlled and is important for improving the extraction of the products of malting during mashing. In mashing, the grist, or milled barley, is mixed with water and stirred in a large mash tun. The purpose of mashing is to nut the enzvmes that were made in the maltine staae to work. kiash:ng is a carefully controlled process h a t kikes advantnae of the fact that amylases and Droteases have different o&imum temperatures: (~nzyme-catalyzedreactions generally increase with increasing temperature as do many uucatalyzed reactions; hut the specific three-dimensional folding of the enzyme begins to unravel into a random configuration at temperatures above its optimum, and the reaction is greatly slowed.) Mashing involves either holding the malt a i d water at a single temperature, or at several temperatures for predetermined time intervals. Usually the temnerature is held for a suitable neriod a t 40 OC. where the proteases are optimal. The optimum temperature for combined a-and 0-amylase activity is about 67 "C. The result of mashing is to produce sufficient sugars for fermentation by the action of the amylases and to produce amino acids and peptides by the action of the proteases. In mashine, not all of the starch is hvdrolvzed to fermentable sugars. T h e amylases can cleavLthe i-4 bonds in the linear portions of amylase and amylopectin, but are incapable of cleaving the 1-6 branches in amylopectin. Thus a starch residue called a limit dextrin remains. Likewise the proteases do not hydrolyze all the protein in the barley to amino acids and small peptides. This is not wholly undesirable as the dextrin from starch and the intact nroteins are actually important for foam stabilization and flavor of the final nroduct. Many manufacturers increase the sugar content at this noint h v the addition of cereal adiuncts. Adiuncts are inexbensive"sources of starch that can readily 6e converted to fermentable sugars. Amone the adiuncts used are corn and rice. These ad&ncts are treated s&arately in a cooker to produce a aelatin rich in mono- and disaccharides. However, adjuncts are lacking in proteolytic enzymes, and cannot supply the nitrogen needed for fermentation. A practical maximum of around 50%adjuncts by weight of starting materials can he added. In the United States beers range from 40% adjuncts to the specialized all-barley-malt beers. The use of adjuncts in the United States is not only cost effective hut also results in a heer more in line with North American tastes. The other maior ineredient added in the mashine Drocess is the water. T K ~sugars, amino acids, peptides, &d other nutrients nroduced durine mashine leach out into the water. In addition, calcium sal&, eithernatural or added a t this stage, play an important role. Calcium sulfate (240-400 ppm) gives the final product a dry, clean flavor, while calcium carbonate (up to 50 ppm) imparts a darker color and a more bitter flavor. ("Dark beer" is actually the result of using roasted barley.) The pH of this solution is around 5.5.
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The Wort At the end of the mashing period, the solids (barley husks, etc.) are removed by filtration, and the enriched filtrate, called the wort. is collected. The solids mav then he washed with 80 ' C water to extract additional nutrients. Next, the wort is boiled for 1.5-2 hoursdurine which time the hons are added. This boiling step serves several purposes. ~ i r s thoil; ing concentrates the solutes (sugars, peptides, and other nutrients) for the subsequent fermentation. Second, it acts to sterilize the wort. Third, proteins in the wort are denatured and precipitate out of solution. This important removal of proteins is called the "hot break". Excess protein in the final product may result in a hazy beer, but some protein is necessary to give the heer body and a foam head. Thus, not all the proteins are removed in this step, and tbis precipitation can be regulated. Fourth, during the boiling period, the hops are added. Hops are the female cones of the plant Humulus lupulus. Originally, hops were added to beer as a preservative as they contain many antibacterial compounds. Today hops are one of the major flavor enhancers. Hops consist mainly of complex a- and 8-acids (lupulone and humulone), tannins, and essential oils (myrcene, geranoil, and others). Many of the volatile oils may he boiled off at this stage, but the boiling primarily serves to convert the uacids into iso-acids, which impart a desired bitterness to the heer. The tannins extracted from the hops complex with proteins to aid in the removal of proteins. Afifth result of the boiling stage is the development of color due in part to caramelization and oxidation of phenolic compounds hut more importantly due to the Maillard reaction (Fig. 2), which occurs between sugars and amines a t high temperature. This is the same reaction that browns the crust of a loaf of bread.
I
H-C-OH
I
HO--c-H + R-NHZ I
H-C-OH I
-
I I HO-f-H
H-C--OH
H-C-OH I
Figure 2. The Maillard reaction
The Fermentation The hot wort is then pumped . - to a receiving vessel where it is cooled a few degrees, and protein is pecipsated (the "cold break"). The wort is then cooled to 50-70 OC and aerated to saturation. Spent hops and precipitated proteins may be further removed by filtration or centrifugation either before or after cooling. The wort is now ready to receive the catalyst, the yeast. Two species of yeast are almost universally used by brewers-Saccharornyces cereuisiae and S. carlsbergensis (also known as S. uuarurn). The wort is pitched or seeded with about 1-2 nounds of veast slurrv ner barrel of wort (31 gallons), and the fermentation begins.. The initial staee of the fermentation is aerobic and the yeast can grow by respiration. In respiration glucose is broken down to carbon dioxide and water in the presence of oxygen. This biochemical process is highly energy efficient and allows for rapid increase in biomass or number of yeast cells. No alcohol is produced in tbis aerobic stage. In the second stage, the oxygen becomes depleted and the yeast shift to anaerobic fermentation. In this process, known as the Embden-Meyerhof pathway (51, glucose is broken down
to pyruvate, with production of energy. This involves an oxidation reaction, and electrons from glucose are passed on to a niacin-derived cofactor called nicotinamide adenine dinucleotide (NAD+;reduced form, NADH) in lieu of oxygen. This nicotinamide redox component is present in the cells in a low concentration and must he reoxidized somehow in order for glycolysis to proceed. A pathway does exist in yeast in which the nvruvate is decarboxvlated and reduced to ethanol, using'ihe enzyme alcohol dehydrogenase. The reducing agent is NADH, which becomes reoxidized and returns to its role in glycolysis. The Embden-Meyerhof pathway is exothermic, and heat is generated a t this stage. (Aerobic respiration is even more exothermic per mole of glucose oxidized, but less heat is generated in the fermentation as there is less biomass present.) T o prevent premature shutdown of the anaerobic fermentation-due to the increase in temperature above 37 "C, the fermenter must usually be cooled by coils in or around the vessel. Ethanol is a waste nroduct and can be toxic to the yeast at around 12%. The concentration never reaches this level in beer nroduction and is controlled bv the brewer. In addition to the formation of ethanol, other acids, alcohols, and their esters (called fusel oils) are formed in small amounts in the fermentation. Another product appearing during alcoholic fermentation is carbon dioxide. The Con produced is often sufficient to agitate the contents of the fermenter, so mechanical impellers may not he needed. The COs may be collected, sterilized and used in subsequent finishing processes. In the final stage of the fermentation, the yeast cells flocculate and rise to the top or sink to the bottom. This process is thought to be a function of the cell walls, which form a complex that is unable to form in the presence of high concentration of suears. Thus when the suears are denleted. flocculation occuk (3).This is fortunate ?or the hrewer be: cause this is a self-cleanine nrocess. In addition, the flocculated yeast can be skimmid-off, washed with p1;osphoric or tartaric acid to kill bacteria, and reused to seed a future fermentation.
Aging and Finishing
The product of the fermentation, called green beer, is usually somewhat rough and harsh in taste and requires aging to produce the final beer. This involves chilling or cbillproofing, during which more proteins precipitate out and are removed. The heer must also be carbonated because insufficient COz remains in the beer after the fermentation. Two wavs of carbonatine the beer are either to allow a slow secondary fermentation catalyzed by residual yeast to occur in a closed veisel or tu iniect CO, directlv into the holdine tank (for instance, the carbon dioxide that was collectei during the fermentation).to a final concentration of around 2.6% vlv. After aging for anywhere from a few weeks to several months, the beer is ready to market. Long-term problems include bacterial contamination and light damage. The effects of light on heer were discussed in a previous article in this Journal (6). Bacterial contamination is more of an organoleptic than a health problem, and is controlled either by filter sterilization or by pasteurization. The latter method is more widely used but does cause some flavor damage. Filter sterilization allows one to maintain the full flavor of the heer even with long-term room-temperature storage. True afficionados will opt, however, for draught heer, which is packaged in barrels right from the finishing tanks without pasteurization or filter sterilization and kept cool at all times. Acknowledgment
The author wishes to thank Norman Grisewood of the
F.X. Matt Brewing Company, Utica, New York, for carefully reviewing this manuscript. Literature Cited 1. Caaida, L. E., Jr. Industrial Microbiology; Wilcy: New York, 1966. 2. Peppler. H. J.; Perlman. D..Eds. Mirrobiol Techmlogy, 2nd cd.:Aeademie: New York, 1979; Vol. 11. 3. Rose, A. H. Industrial Microbiology: Bufterworths: London,1961. I . Rivlere,J. Industrial AppGcolions ofMicrobiology; Wiley: New York, 1977. 5. Bodner, G.M. J. Chem. Edue. 198% 63,566. 6. Vogler,A.;Kunkely,H. J. Cham.Educ. 1982.59, 25.
Iota Sigma Pi Awards Announced Iota Sigma Pi, a national honorary society for women in chemistry announces the 1988 recipients of the following awards. The Anna Louise Hoffman Award for Outstanding Graduate Achievement went to Athanasia Makri of the Department of Chemistry at the University of California at Berkeley far her work in evaluating Feynman path integrals by th$ Monte Carlo procedures. She has published six papers that have "made a major impact in theoretical chemical dynamics and statistical mechanics." The Undergraduate Award for Excellence in Chemistry was given to Carmen Diane Wheelock, Department of Biochemistry, Louisiana State University, who has a 4.0 in chemistry and a 3.97 overall GPA. In addition she has done summer research with the USDA and worked during the academic year with a faculty member on recombinant DNA approaches to defining the amino acids important in protein transport through chloroplast membranes. She plans to do graduate work in biochemistry next year. The Gladys Anderson Emerson Scholarship was awarded to ChristineWeise, Department of Biochemistry,University of Kansas, who is a third-year student with senior standing. She plans to complete a double major (in Biochemistryand OrganismalBiology) in May 1989 and is considering an MDIPhD program for her future work. She has already done research on the elucidation of the p26 promotor of AeNPV.
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