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BEERS | Chemistry of Brewing
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cylindroconical fermentation vessels and centrifuges (which are often, but not always, employed in tandem). There is no doubt that differences in the flocculation characteristics of various yeast cultures are primarily a manifestation of the culture’s cell wall structure. Several mechanisms for flocculation have been proposed. One hypothesis is that anionic groups of cell wall components are linked by Ca2þ ions. In all likelihood, these anionic groups are proteins. Another hypothesis implicates mannoproteins specific to flocculent cultures acting in a lectin-like manner to crosslink cells; here, Ca2þ ions act as ligands to promote flocculence by conformational changes. Most people working in the field agree that the latter hypothesis is the most credible. In addition to flocculation, there is the phenomenon of coflocculation. Coflocculation is defined as the phenomenon where two strains are nonflocculent alone, but flocculent when mixed together. To date, coflocculation has only been observed with ale strains, and there are no reports of coflocculation between two lager strains of yeast. There is a third flocculation reaction that has been described where the yeast strain has the ability to aggregate and cosediment with contaminating bacteria in the culture. Again, this phenomenon appears to be confined to ale yeast strains, and cosedimentation of lager yeast with bacteria has not been observed. As described above, flocculation requires the presence of surface protein and mannan receptors. If these are not available or are masked, blocked, inhibited, or denatured, flocculation cannot occur. The onset of flocculation is an aspect of the subject where there is great commercial interest but about which relatively little is known. As previously discussed, the ideal brewing strain remains in suspension as fermenting single cells until the end of fermentation when the sugars in the wort are depleted, and only then does it rapidly flocculate out of suspension. What signals the onset of activation or relief from inhibition? This is still an unanswered question that is currently being studied by a number of research laboratories. Yeast flocculation is genetically controlled, and research on this aspect of the phenomenon dates from the early 1950s. However, because of the polyploid/aneuploid nature of brewing yeast strains, most, but not all, of the research on flocculation genetics has been conducted on haploid/diploid genetically defined laboratory strains. Numerous genes have been reported to directly influence the flocculent phenotype in Saccharomyces spp. Four dominant flocculation genes have been identified: FLO1 (whose alleles are FLO2, FLO4, FLO8), FLO5, FLO9, and FLO10, as well as semidominant gene, flo3, and two recessive genes, flo6 and flo7. In addition, mutations in several genes, including the regulatory genes,
TUP1 and SSN6, have been found to cause flocculation or ‘flaky’ growth in nonflocculent strains. In total, at least 33 genes have been reported to be involved in flocculation or cell aggregation. Although the role of many of these genes is far from understood, FLO1 and other FLO genes have been successfully cloned into brewing strains and the flocculation phenotype expressed. See also: Alcohol: Properties and Determination; Beers: History and Types; Raw Materials; Chemistry of Brewing; Yeasts
Further Reading Reed G and Nagodawithana TW (1991) Yeast Technology.New York: Van Nostrand Reinhold. Smart K and Boulton C (2000) Brewing Yeast Fermentation Performance. New York: Blackwell Science Inc. Stewart G G and Russell L (1986) One hundred years of yeast research and development in the brewing industry. Journal of the Institute of Brewing 92: 537–558.
Chemistry of Brewing C W Bamforth, University of California, Davis, CA, USA Copyright 2003, Elsevier Science Ltd. All Rights Reserved.
Introduction The word ‘brewing’ is used in at least two contexts in beer making. Strictly speaking, the most accurate usage of the term is to describe the process by which is produced the feedstock, wort, that will be fed to the yeast. Thus it is customary for those within the industry to talk of ‘brewing and fermentation.’ Frequently, however, the word brewing is used to describe the entirety of the operation by which malted barley (and other sources of sugar) and hops are converted to beer. For the purposes of a description of the chemistry of brewing I am taking the latter definition, but we must not ignore the prior process of malting (the controlled germination of barley) because the conversion of barley into wort involves this stage also. It is very difficult to divorce a discussion of the mashing stage of brewing from malting, for in reality they are but successive stages in the enzymic conversion of barley into wort. Figure 1 summarizes the structure of barley and the distribution of the key polymers within the starchy endosperm, whilst Table 1 summarizes the key process stages of malting and brewing. I will approach this somewhat complex
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C B
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Figure 1 The structure of barley and of one of the cells of the starchy endosperm. A, embryo; B, starchy endosperm; C, aleurone; D, hull; E, micropyle; F, cell wall (75% b-glucan; 20% arabinoxylan; 5% protein; traces ferulic and acetic acids); G, protein; H, large starch granule; I, small starch granule.
issue by focusing on the chemistry of the finished product and highlighting how the balance of components in beer is determined by the various stages occurring in the maltings and brewery.
Ethanol
The equation at the heart of the production of all alcoholic beverages is:
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Yeast
C6 H12 O6 ! 2C2 H5 OH þ 2CO2
The Chemistry of Beer 0002
The properties of beer result from the presence of a diversity of chemical compounds, some large (macromolecules) and some small. For the majority of beers these materials are derived in their entirety from the raw materials or are generated through the metabolism of brewing yeast, Saccharomyces cerevisiae. Most brewers are reluctant to introduce additives at any stage of the process and will also place stringent specifications on the water, grist, and hop materials in respect of what may or may not be used in the processes of their suppliers. Some brewers will use propylene glycol alginate to protect foam on the product from the deleterious impact of lipophilic materials entering the beer at point of sale, for example, fats from food or detergent in inadequately rinsed glasses. Sulfur dioxide or ascorbic acid is sometimes (but increasingly sparingly) employed to protect beer from staling. (See Yeasts.)
In the case of the majority of beers the sugar is derived during mashing of malted barley. In fact, most of the sugar derived in conventional mashing operations is maltose, with lesser quantities of glucose, maltotriose, sucrose, fructose, and dextrins. The majority of beers worldwide contain between 3.5 and 5.5% alcohol by volume (ABV). Ethanol impacts on the quality of beer in several ways. It contributes directly to flavor, with a recognizable warming characteristic absent from nonalcoholic or low-alcohol products. It also affects the contribution to aroma of other volatiles by influencing their partitioning between the beer and its head space. Ethanol lowers surface tension, thereby promoting bubble formation. Conversely, it competes with other surface-active molecules for sites in the bubble wall, leading to a lessening of foam stability. (See Alcohol: Properties and Determination.)
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Table 1 The key events in malting and brewing Process stage
Treatments
Events
Water added, separated by air rests, to raise water Water enters through micropyle. Distribution through embryo, aleurone, and starchy content of embryo and endosperm (target in endosperm critical to enable modification. whole grain is usually 44–46%); takes up to 48 h Synthesis of hormones (including at 14–18 C gibberellins) by embryo, hydration of substrate (starchy endosperm) Germination of barley Controlled sprouting (modification) of grain – Synthesis of enzymes by aleurone (triggered by typically 4–5 days at 16–20 C hormones) and migration into starchy endosperm; sequential degradation of cell walls, some protein, small starch granules, and pitting of large granules Kilning Heating of grain via increasing temperature regime Enzyme survival greater with low-temperature (50–220 C) for desired properties: enzyme start to kilning and lower final curing survival, removal of moisture for stabilization, temperature. Increased heating of malts of removal of raw flavors, development of malty increased modification (i.e., higher sugar and flavors and color amino acid levels) gives increasingly complex flavors and colors via Maillard reactions Malt storage 3–4 weeks’ ambient storage, otherwise wort Unknown separation problems occur later Mashing Extraction of milled malt at temperatures between Enzymolysis continued, especially of starch 40 and 75 C; typically < 1–2 h. Separation of wort after gelatinization at > 62 C from spent grains by lautering or mash filtration with sparging to recover extract fully. Spent grains go to cattle feed Use of adjuncts Solid adjuncts used in brewhouse, taking Cereals with higher starch gelatinization advantage of malt enzymes; rice and corn need temperatures than for barley need ‘cooking’ at up to 100 C, < 1 h (liquid sugars are precooking before combining with main mash products of acid and enzyme action in sugar factory and added at boiling stage) Boiling 1–2 h at 100 C, before clarification (< 1 h) and To sterilize, extract hops, concentrate, and kill cooling all residual enzymes. Precipitation of insoluble materials as trub, enhancing final product stability Fermentation Wort pitched with yeast and fermented for 3–14 Fermentation of glucose, maltose, sucrose, days at 6–25 C maltotriose to alcohol; enzymic production of various flavorsome compounds (alcohols, esters, fatty acids, sulfur-containing compounds, etc.) Synthesis and removal of diacetyl as an offshoot of amino acid production Cold conditioning and filtration 1 C for 3 days; possibly stabilization (silica Precipitation, sedimentation, and removal of hydrogels, tannic acid or papain to remove solids protein; PVPP to bind polyphenol); then filtration Steeping of barley
PVPP, polyvinylpolypyrrolidone.
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A typical brewery fermentation will produce some 150 g CO2 per hectoliter for every degree Plato (1 degree Plato is basically equivalent to a 1% sugar solution). Most of this CO2 sweeps out and is lost during fermentation. Many packaged beers will contain 500–550 g CO2 hl1 and extra gas needs to be introduced to the product prior to packaging. At 1 atm pressure (as CO2) and 0 C, a beer will dissolve no more than 200 g hl1 CO2. Achievement of the very high levels of CO2 demands the pressurizing of beer. None the less, when a beer container is opened,
the gas usually stays in solution: the beer is ‘supersaturated’ with CO2. When fobbing occurs spontaneously when a can or bottle is dispensed, it is called ‘gushing.’ In the absence of agitation as a cause, the most likely reason for the phenomenon is the presence of low-molecular-weight, highly hydrophobic peptides contributed by infection of barley, notably from Fusarium. CO2 affords the ‘sparkle’ to beer, through its reaction with the pain receptor mechanism of the trigeminal nerve. Apart from this influence on mouth feel, CO2 establishes the extent of foam formation during
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dispense and of course impacts heavily the delivery of volatiles into the head space of beers and therefore the aroma.
reacts with phosphate to lower the pH to the appropriate level for mashing:
Other Gases in Beer
By precipitating out malt-derived oxalic acid in the brewhouse, calcium eliminates a material that, if surviving into beer, causes problems such as haze, gushing, and the blocking of dispense pipes.
Oxygen has major negative effects on beer via its oxidation of various components, leading to cardboard and other stale notes and to the formation of haze. Brewers therefore strive to minimize the oxygen level in beer (preferably to 0.1 mg l1 or ideally even less) by avoiding ingress into the product downstream of the fermenter (yeast is a powerful oxygen scavenger and many brewers feel that it is only after the removal of yeast that oxygen is a concern). The stale papery notes are due to a series of unsaturated carbonyl compounds, which are usually held to include trans-2-nonenal, though this has been no means fully substantiated. Several components of beer can degrade to such carbonyl substances during oxidation, including the ‘higher’ alcohols, the iso-a-acids, and the unsaturated fatty acids. Nitrogen has long been introduced to beer to promote foam stability. The foams produced on beers containing N2 tend to have populations of much smaller bubbles. These foams are more resistant to collapse and the beer displays a much smoother mouth feel. Typical levels of N2 are 20–50 mg l1 which are three orders of magnitude lower than those of CO2. Water
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The vast majority of beers contain between 90 and 95% water: apart from ethanol and carbon dioxide, the remaining constituents usually amount to less than 1% of the product. The water used for brewing must have no taints or hazardous components and water is often treated coming into the brewery to adsorb any contaminants by charcoal filtration and ultrafiltration. The ionic composition of water is also significant (although the grist also contributes salts). High levels of calcium, e.g., 200–350 mg l1, are claimed to be desirable in the production of ales. Much lower levels of calcium are traditionally associated with the brewing of lagers, for example, the water in pilsen (the home of the classic lager-style) contains less than 10 mg l1 calcium. A scientific justification for this is not entirely proven, but may relate to the role of calcium in promoting the surface behavior of the top-fermenting ale yeasts (calcium promotes the flocculation of yeast). Brewers may add salts or remove them (e.g., by reverse osmosis) to adjust the ionic composition of the product. Two key impacts of calcium are on pH and the levels of oxalic acid surviving into beer. Calcium
þ 3Ca2þ þ 2HPO2 4 ! Ca3 ðPO4 Þ2 þ 2H
Carbohydrates
Although most of the sugars found in wort are converted to ethanol by yeast, those containing four or more glucoses (i.e., maltotetraose and bigger, and known as dextrins) are not fermented. In order to sweeten the finished beer, some brewers may contrive to leave a proportion of fermentable sugar unconverted, or may add ‘priming’ sugar. Most of the starch survives malting, because it is relatively resistant to enzymatic hydrolysis when in the form of large granules. When starch is gelatinized by heating, however, its constituents (amylose and amylopectin) become accessible. Mashing, therefore, incorporates a stage, typically at around 65 C, to allow for the gelatinization of malt starch. The starch in some other cereals has higher gelatinization temperatures, e.g., rice and corn starches gelatinize over the range 70–80 C and so are cooked separately from the main mash and then mixed with the malt mash to be degraded by the amylases from malt. (See Carbohydrates: Classification and Properties.) Starch hydrolysis starts with a-amylase, a highly heat-resistant enzyme abundantly present in malt. It is endo-acting, catalyzing the hydrolysis of a-1!4 bonds within the starch, releasing dextrins. From amylose the enzyme release linear dextrins, and from amylopectin it produces dextrins with side chains. (See Starch: Structure, Properties, and Determination.) b-Amylase is an exoenzyme, which removes maltose units from the nonreducing ends of starch and dextrin molecules. This enzyme has sufficient stability at 65 C to achieve most of the conversion of starch and dextrins of which it is capable but if mashing is carried out at somewhat higher temperatures, e.g., 72 C, then b-amylase is rapidly inactivated and the ensuing wort contains a high dextrin level and low content of fermentable sugars. Such high-temperature mashing can be used in the production of low-fermentability worts which will give low-alcohol beers. b-Amylase cannot pass the a1!6 branch points in the dextrins formed from amylopectin. Limit dextrinase carries out this function but is generally present in fairly low concentrations because it is developed
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very late during barley germination and because it is inactivated by association with another protein. Furthermore, limit dextrinase is relatively heat-labile. It is because of the limited action of limit dextrinase that 20–25% of the starch remains as dextrins in most conventionally mashed beers. In so-called ‘light’ or ‘lite’ beers all of the starch is converted to ethanol. One way of achieving this is to add heat-stable glucoamylase or pullulanase from microbial sources to the mash or to the fermenter, although those brewers seeking to avoid any additions may modify their brewhouse and fermenter operations so as to take maximum opportunity of endogenous enzymes. The major component of the barley cell walls is a linear b-glucan comprising approximately 67% b1!4 links with 33% b1!3 bonds that are for the most part every third or fourth linkage. The key enzyme hydrolyzing this molecule is an extremely heat-sensitive endo-b-glucanase, which is destroyed within 5 min of mashing at 65 C. Accordingly, one of the main purposes of malting is to remove the cell walls, in large part through the action of this enzyme. In practice some cell wall material (at the distal end of the barley) will survive and if it is not properly degraded it can afford increased viscosity to the wort and cause sluggish wort separation in the brewhouse and retarded beer filtration. Some brewers mash-in at low temperatures such as 40–50 C to allow b-glucanase to act, before raising the temperature to that needed for starch gelatinization. Alternatively, heat-stable b-glucanases from bacteria (e.g., Bacillus subtilis) or fungi (e.g., Trichoderma, Penicillium) may be used. Proteins, Polypeptides, and Amino Acids
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Amphipathic polypeptides in beer stabilize the bubbles in foam. Their hydrophobic regions ‘drive’ them into the surfaces produced during foaming and are largely responsible for their interaction with other hydrophobic molecules, notably the iso-a-acids. The
matrix formed counters the force of surface tension that seeks to minimize increased surface area generated in foaming. Some of the protein in beer can react with polyphenols to form hazes. There is no advantage in having significant levels of residual amino acids in beer; rather they are a risk in so far as they represent assimilable nitrogen sources for spoilage microorganisms. However, it is important that wort contains the correct balance of amino acids to support yeast growth and fermentation. Sufficient proteolysis must occur during malting and mashing to yield these amino acids and to remove haze-potentiating proteins, whilst leaving ample foam-positive polypeptide. (See Protein: Chemistry.) The native proteins of barley undergo considerable degradation and denaturation in malting and brewing. During germination of barley, endoproteinases develop and attack the heart of substrate proteins to release polypeptides and peptides. An exoacting enzyme, carboxypeptidase, acts during malting and mashing by splitting off amino acids successively from the carboxyl-terminus of the peptides produced by the endoproteinases.
Barley comprises some 3% by weight lipid, most of it in the embryo and aleurone. Very little survives into beer, because it is removed by adsorption on insoluble matrices (e.g., spent grain, trub) during the process. Lipids are severely detrimental to beer foam, disrupting the network of proteins and iso-a-acids in the bubble wall. As observed earlier, the unsaturated fatty acid component of lipids is suspected to be at least one precursor of the stale flavors (e.g., ‘cardboard’) that develop in beer. (See Fats: Classification.)
O
Hops afford the bitterness (from the hop resins) and aroma (from the essential oils) to beer. The most important resins are the a-acids (Figure 2), accounting for 2–15% of the dry weight of the hop,
H Isomerization
HO O
trans fig0002
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Flavors from Hops
Iso-α-acids
R HO HO
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α-Acids OH O
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O
O
H R
OH
HO O
O
O F OH
cis
Figure 2 The isomerization of a-acids. R ¼ –CH2CH(CH3)2 in humulone and isohumulone. R ¼ –CH(CH3)2 in cohumulone and isocohumulone. R ¼ –CH(CH3)CH2CH3 in adhumulone and isoadhumulone.
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depending on variety and environment. The more a-acids, the greater the bitterness potential. There are three different a-acids in hops, differing in side-chain structure. The a-acids are isomerized to form the more soluble and bitter iso-a-acids during wort boiling (Figure 2). Each iso-a-acid has two isomers, cis and trans, with differing orientation of the side chains. The six iso-a-acids have a range of bitterness intensities and it is generally held that the hops should have a relatively low level of cohumulone. Apart from their impact on bitterness and foam, the iso-a-acids have strong antimicrobial properties and inhibit many Gram-positive bacteria. Hops comprise 0.03–3% w/w essential oil, comprising a complex mixture of more than 300 compounds (Figure 3). The components are very volatile and tend to be lost during wort boiling. To ensure hoppiness in beer it is necessary to add a proportion of the hops late in the boil (late hopping, associated with lager-style products) or to the finished beer (dry hopping, associated with some ales). Hops are either used in a traditional way as whole cones, though increasingly after hammer milling and extrusion into pellets, in which form they are more stable and more efficiently utilized, or after extraction by liquid carbon dioxide and isomerization by weak alkalis. The latter products can be added to beer downstream, greatly increasing the efficiency of bitterness utilization and flexibility of product formulation. The oils in such extracts may be separated from the resins and also added to beer to provide hoppy aroma. In some preparations the iso-a-acids are reduced, using hydrogen gas in the presence of a palladium catalyst. Such reduction prevents the formation of the skunk-flavored 3-methyl-2-butene-1-thiol from the side chains when the bitter compounds are exposed to light. The reduced preparations can be used in the production of beers for packaging into green
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and clear glass bottles, which are particularly prone to light damage. Phenolic Materials
The simplest phenolic acid in beer is ferulic acid, a molecule found associated with the arabinoxylan component of the starchy endosperm cell walls. It may have antioxidant properties both for the beer and for the drinker’s body. When decarboxylated, by an enzyme present in the yeast strains used to make wheat-based beers though not barley-based ones, 4-vinylguaiacol is produced, which has a distinct clove-like character. Certain flavanoids, including catechin and quercetin, derive from the outer layers of malt and from hops. They, too, may have antioxidant properties, but when oxidized they polymerize, to produce tannoids that cross-link through the proline groups in certain beer polypeptides to form the insoluble complexes responsible for haze. Haze formation is lessened by reducing oxygen ingress and by reducing the levels of haze-forming polypeptides and phenolics. Silica hydrogels, tannic acid, and the proteinase papain have been used to attend to the former, while polyphenols can be removed using polyvinylpolypyrollidone (PVPP). Vigorous wort boiling and chilling of beer to as low a temperature as possible without freezing (1 C) are both important stages in the colloidal stabilization of beer. The level of tannic materials in beer is probably too low to have any significant impact on astringency. (See Tannins and Polyphenols.)
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Other Flavor Components of Beer
The four main flavor features detected by the tongue are bitterness, sweetness, sourness, and saltiness. The nose detects the other characteristic aromas of beer and this is a balance between positive and negative notes, each of which may be due to more than a single compound from different chemical classes. Although some of these substances originate in the malt and hops, many are products of yeast metabolism.
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OH H O
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O
Linalool
Humulene epoxide
Hop ether
Myrcene
Humulene
Farnesene
Caryophyllene
Figure 3 Some components of the essential oil fraction in hops.
Esters Esters afford a fruity character to beer: two of the most important components are ethyl acetate and iso-amyl acetate. Esters are formed from their equivalent alcohols when the acetate group is available by not being needed for the synthesis of key components (lipids) of the yeast membranes. Therefore, factors that promote cell production lower ester production, and vice versa. Ester levels in beer are impacted, inter alia, by the ratio of carbon to nitrogen in the wort and by the amount of oxygen available to the yeast. The yeast strain is very
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Alcohols The alcohols are the immediate precursors of the esters. As the esters are substantially more flavor-active, it is important to regulate the levels of the higher alcohols if ester levels are also to be controlled. The higher alcohols (i.e., those larger than ethanol) are produced by transformation of amino acids, so the levels of amino acids in wort have a major impact: the more amino acids available to the yeast, the greater the production of higher alcohols. However, Saccharomyces will also produce higher alcohols as an offshoot of the metabolic pathways responsible for amino acid elaboration, pathways that are particularly significant when the amount of assailable nitrogen in the wort is low. Hence higher alcohol production is increased when both too much and too little amino nitrogen is available to the yeast. Ale strains produce more higher alcohols than do lager strains. Conditions favoring increased yeast growth (e.g., excessive availability of oxygen) promote higher alcohol formation. Sourness This is due to the organic acids, such as acetic, lactic, and succinic, produced by yeast during fermentation. It is the Hþ ion produced by their dissociation that causes sourness. Higher levels of the acids are produced in vigorous fermentations. Vicinal diketones The vicinal diketones (VDKs), diacetyl and pentanedione, afford highly undesirable butterscotch and honey characters respectively to beer. These substances are offshoots of the pathways by which yeast produces certain amino acids. Precursors leak from the yeast and decompose spontaneously to form VDKs. Yeast, however, can reassimilate the VDK, provided the cells are healthy and remain in contact with the beer. Brewers may allow a temperature rise of 2–3 C at the end of fermentation to speed up the removal of VDK. Additionally, a proportion of freshly fermenting wort is introduced to the beer as an inoculum of healthy yeast (a practice called krausening). Alternatively a bacterial enzyme (acetolactate decarboxylase) can be added to a fermentation; this enzyme converts acetolactate to acetoin, thereby avoiding the much more flavoractive diacetyl. Persistently high VDK levels may be a symptom of infection by Pediococcus or Lactobacillus bacteria. Sulfur compounds In the right levels and proportions, the sulfur compounds play a substantial role in determining the character of diverse beers. Some
ales display a distinct hydrogen sulfide character when first dispensed, albeit one which subsides to reveal the dry hop character. Lagers often have a more complex sulfury character, which for many includes dimethyl sulfide (DMS), with its cooked corn/parsnip note. DMS is the best understood of all the sulfur compounds in terms of its production route. All of the DMS ultimately originates from a precursor, S-methylmethionine (SMM), which develops in the barley embryo during germination. SMM is heatlabile, breaking down rapidly at temperatures above about 80 C in malting and brewing. Accordingly, SMM is at a lower level in the more highly kilned ale malts and, therefore, there tends to be less DMS in ales. SMM is extracted into wort during mashing and is broken down during boiling and in the whirlpool. In a vigorous boil most of the SMM is converted to DMS and lost by volatilization. In the whirlpool the temperature is still hot enough to degrade SMM, but conditions are nonturbulent and the DMS tends to linger. Brewers aiming for a finite level of DMS in their beer specify a target level of SMM in the malt and will adjust the boil and whirlpool stages so as to deliver a certain level of DMS in the pitching wort. In fermentation a great deal of DMS is swept away with the CO2, hence the level of DMS targeted in the wort is higher than that specified for the beer. Some of the DMS produced during malt kilning is oxidized to dimethyl sulfoxide (DMSO). This is not volatile but is water-soluble and enters wort, to be reduced by yeast to DMS. Hence the level of DMS in the finished beer is a function of how much DMS is present in pitching wort, how much is volatilized, and how much is replenished via the reduction of DMSO. There is no simple relationship between the level of DMS in beer and the perception of its flavor. This is because phenylethanol and phenylethylacetate interfere with the perception of DMS. There must be many other antagonisms of this type contributing to the complexity of beer flavor, but they have not been studied. Hydrogen sulfide (H2S) is produced by yeast via the breakdown of cysteine or glutathione, or by the reduction of sulfate and sulfite. A vigorous fermentation purges H2S and factors that hinder fermentation (such as a lack of zinc or vitamins) will increase H2S levels in beer. Malty notes As well as being the source of the DMS character in beer, malt contributes in other ways to flavor. Malty character is in part due to isovaleraldehyde, produced by the reaction of leucine with reductones in the malt. The toffee and caramel
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Table 2 Order-of-magnitude composition of a typical pilsner beer Component
Typicallevel (mgl1)
Original wort gravity Ethanol Total carbohydrates Glucose Fructose Sucrose Maltose Maltotriose Dextrins derived from starch b-Glucan Pentosan Protein Amino acids Thiamin Riboflavin Vitamin B6 Pantothenic acid Niacin Biotin Vitamin B12 Folic acid Potassium Sodium Sulfate Chloride Organic acids (acetic, pyruvic, citric, succinic, malic, lactic, etc.) Polyphenols Iso-a-acids Sulfur dioxide Carbon dioxide Nucleotides and nucleosides Glycerol Higher alcohols Ethyl acetate Iso-amyl acetate Acetaldehyde Essential oils from hops Dimethyl sulfide Total organic sulfur compounds
120 103 40 103 30 103 150 30 5 1500 2000 24 103 350 50 5000 1100 0.3 0.4 0.6 1.5 8.0 0.01 0.0001 0.2 500 30 200 200 700 150 30 4 5 103 300 1500 100 15 1 5 <1 0.06 <1
character from crystal malts and the roasted, coffeelike notes in darker malts are due to complex components derived from amino acids and sugars when they cross-react during kilning. Of equal importance during kilning is the disappearance of grassy and beany notes, due inter alia to cis-3-hexen-1-ol, trans-2-hexenal, trans-2-cis-6-nonadienal, and 1hexanol. The cross-linking of sugars and amino acids induced by heating in kilning and wort boiling leads to the formation of melanoidins via the Maillard reaction. The melanoidins are responsible for imparting color to beer: darker beers are produced from grists incorporating malts and other adjuncts
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that have been kilned to more intense regimes. Polyphenol oxidation, occurring during wort production, can make a significant contribution to color in some of the paler beers. Miscellaneous Acetaldehyde, which is converted to ethanol in actively fermenting yeast, imparts an undesirable ‘green apples’ character to beer. High levels of acetaldehyde are due to premature separation of yeast before fermentation is complete, poor yeast quality, or infection by the bacterium Zymomonas. The short-chain fatty acids, with their rancid notes, are offshoots in the synthesis of membrane lipids by yeast. When yeast needs fewer lipids (when it needs to grow less), these compounds accumulate. Table 2 offers a summary of the approximate chemical composition of a pilsner-type beer. See also: Alcohol: Metabolism, Beneficial Effects, and Toxicology; Antioxidants: Natural Antioxidants; Barrels: Beer Making; Barley; Beers: Raw Materials; Wort Production; Biochemistry of Fermentation; Microbreweries; Carbohydrates: Classification and Properties; Malt: Malt Types and Products; Chemistry of Malting; Packaging: Packaging of Liquids; Phenolic Compounds; Protein: Chemistry; Starch: Structure, Properties, and Determination; Tannins and Polyphenols
Further Reading Bamforth C (1998) Beer: Tap into the Art and Science of Brewing. New York: Insight. Boulton C and Quain D (2001) Brewing Yeast and Fermentation. Oxford: Blackwell. Briggs DE, Hough JS, Stevens R and Young TW (1981) Malting and Brewing Science. Volume 1: Malt and Sweet Wort. London: Chapman and Hall. Hornsey IS (1999) Brewing. London: Royal Society of Chemistry. Hough JS, Briggs DE, Stevens R and Young TW (1981) Malting and Brewing Science. Volume 2: Hopped Wort and Beer. London: Chapman and Hall. Hughes PS and Baxter ED (2001) Beer: Quality, Safety and Nutritional Aspects. London: Royal Society of Chemistry. Kunze W (1996) Technology: Malting and Brewing. Berlin: VLB. Lewis MJ and Young TW (1995) Brewing. London: Chapman and Hall. MacDonald J, Reeve PTV, Ruddlesden JD and White FH (1984) Current approaches to brewery fermentations. In: Bushell ME (ed.) Progress in Industrial Microbiology, Vol. 19: Modern Applications of Traditional Biotechnologies. Amsterdam: Elsevier. Moll M (1991) Beers and Coolers. Andover: Intercept.
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cylindroconical fermentation vessels and centrifuges (which are often, but not always, employed in tandem). There is no doubt that differences in the flocculation characteristics of various yeast cultures are primarily a manifestation of the culture’s cell wall structure. Several mechanisms for flocculation have been proposed. One hypothesis is that anionic groups of cell wall components are linked by Ca2þ ions. In all likelihood, these anionic groups are proteins. Another hypothesis implicates mannoproteins specific to flocculent cultures acting in a lectin-like manner to crosslink cells; here, Ca2þ ions act as ligands to promote flocculence by conformational changes. Most people working in the field agree that the latter hypothesis is the most credible. In addition to flocculation, there is the phenomenon of coflocculation. Coflocculation is defined as the phenomenon where two strains are nonflocculent alone, but flocculent when mixed together. To date, coflocculation has only been observed with ale strains, and there are no reports of coflocculation between two lager strains of yeast. There is a third flocculation reaction that has been described where the yeast strain has the ability to aggregate and cosediment with contaminating bacteria in the culture. Again, this phenomenon appears to be confined to ale yeast strains, and cosedimentation of lager yeast with bacteria has not been observed. As described above, flocculation requires the presence of surface protein and mannan receptors. If these are not available or are masked, blocked, inhibited, or denatured, flocculation cannot occur. The onset of flocculation is an aspect of the subject where there is great commercial interest but about which relatively little is known. As previously discussed, the ideal brewing strain remains in suspension as fermenting single cells until the end of fermentation when the sugars in the wort are depleted, and only then does it rapidly flocculate out of suspension. What signals the onset of activation or relief from inhibition? This is still an unanswered question that is currently being studied by a number of research laboratories. Yeast flocculation is genetically controlled, and research on this aspect of the phenomenon dates from the early 1950s. However, because of the polyploid/aneuploid nature of brewing yeast strains, most, but not all, of the research on flocculation genetics has been conducted on haploid/diploid genetically defined laboratory strains. Numerous genes have been reported to directly influence the flocculent phenotype in Saccharomyces spp. Four dominant flocculation genes have been identified: FLO1 (whose alleles are FLO2, FLO4, FLO8), FLO5, FLO9, and FLO10, as well as semidominant gene, flo3, and two recessive genes, flo6 and flo7. In addition, mutations in several genes, including the regulatory genes,
TUP1 and SSN6, have been found to cause flocculation or ‘flaky’ growth in nonflocculent strains. In total, at least 33 genes have been reported to be involved in flocculation or cell aggregation. Although the role of many of these genes is far from understood, FLO1 and other FLO genes have been successfully cloned into brewing strains and the flocculation phenotype expressed. See also: Alcohol: Properties and Determination; Beers: History and Types; Raw Materials; Chemistry of Brewing; Yeasts
Further Reading Reed G and Nagodawithana TW (1991) Yeast Technology.New York: Van Nostrand Reinhold. Smart K and Boulton C (2000) Brewing Yeast Fermentation Performance. New York: Blackwell Science Inc. Stewart G G and Russell L (1986) One hundred years of yeast research and development in the brewing industry. Journal of the Institute of Brewing 92: 537–558.
Chemistry of Brewing C W Bamforth, University of California, Davis, CA, USA Copyright 2003, Elsevier Science Ltd. All Rights Reserved.
Introduction The word ‘brewing’ is used in at least two contexts in beer making. Strictly speaking, the most accurate usage of the term is to describe the process by which is produced the feedstock, wort, that will be fed to the yeast. Thus it is customary for those within the industry to talk of ‘brewing and fermentation.’ Frequently, however, the word brewing is used to describe the entirety of the operation by which malted barley (and other sources of sugar) and hops are converted to beer. For the purposes of a description of the chemistry of brewing I am taking the latter definition, but we must not ignore the prior process of malting (the controlled germination of barley) because the conversion of barley into wort involves this stage also. It is very difficult to divorce a discussion of the mashing stage of brewing from malting, for in reality they are but successive stages in the enzymic conversion of barley into wort. Figure 1 summarizes the structure of barley and the distribution of the key polymers within the starchy endosperm, whilst Table 1 summarizes the key process stages of malting and brewing. I will approach this somewhat complex
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C B
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Figure 1 The structure of barley and of one of the cells of the starchy endosperm. A, embryo; B, starchy endosperm; C, aleurone; D, hull; E, micropyle; F, cell wall (75% b-glucan; 20% arabinoxylan; 5% protein; traces ferulic and acetic acids); G, protein; H, large starch granule; I, small starch granule.
issue by focusing on the chemistry of the finished product and highlighting how the balance of components in beer is determined by the various stages occurring in the maltings and brewery.
Ethanol
The equation at the heart of the production of all alcoholic beverages is:
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Yeast
C6 H12 O6 ! 2C2 H5 OH þ 2CO2
The Chemistry of Beer 0002
The properties of beer result from the presence of a diversity of chemical compounds, some large (macromolecules) and some small. For the majority of beers these materials are derived in their entirety from the raw materials or are generated through the metabolism of brewing yeast, Saccharomyces cerevisiae. Most brewers are reluctant to introduce additives at any stage of the process and will also place stringent specifications on the water, grist, and hop materials in respect of what may or may not be used in the processes of their suppliers. Some brewers will use propylene glycol alginate to protect foam on the product from the deleterious impact of lipophilic materials entering the beer at point of sale, for example, fats from food or detergent in inadequately rinsed glasses. Sulfur dioxide or ascorbic acid is sometimes (but increasingly sparingly) employed to protect beer from staling. (See Yeasts.)
In the case of the majority of beers the sugar is derived during mashing of malted barley. In fact, most of the sugar derived in conventional mashing operations is maltose, with lesser quantities of glucose, maltotriose, sucrose, fructose, and dextrins. The majority of beers worldwide contain between 3.5 and 5.5% alcohol by volume (ABV). Ethanol impacts on the quality of beer in several ways. It contributes directly to flavor, with a recognizable warming characteristic absent from nonalcoholic or low-alcohol products. It also affects the contribution to aroma of other volatiles by influencing their partitioning between the beer and its head space. Ethanol lowers surface tension, thereby promoting bubble formation. Conversely, it competes with other surface-active molecules for sites in the bubble wall, leading to a lessening of foam stability. (See Alcohol: Properties and Determination.)
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Table 1 The key events in malting and brewing Process stage
Treatments
Events
Water added, separated by air rests, to raise water Water enters through micropyle. Distribution through embryo, aleurone, and starchy content of embryo and endosperm (target in endosperm critical to enable modification. whole grain is usually 44–46%); takes up to 48 h Synthesis of hormones (including at 14–18 C gibberellins) by embryo, hydration of substrate (starchy endosperm) Germination of barley Controlled sprouting (modification) of grain – Synthesis of enzymes by aleurone (triggered by typically 4–5 days at 16–20 C hormones) and migration into starchy endosperm; sequential degradation of cell walls, some protein, small starch granules, and pitting of large granules Kilning Heating of grain via increasing temperature regime Enzyme survival greater with low-temperature (50–220 C) for desired properties: enzyme start to kilning and lower final curing survival, removal of moisture for stabilization, temperature. Increased heating of malts of removal of raw flavors, development of malty increased modification (i.e., higher sugar and flavors and color amino acid levels) gives increasingly complex flavors and colors via Maillard reactions Malt storage 3–4 weeks’ ambient storage, otherwise wort Unknown separation problems occur later Mashing Extraction of milled malt at temperatures between Enzymolysis continued, especially of starch 40 and 75 C; typically < 1–2 h. Separation of wort after gelatinization at > 62 C from spent grains by lautering or mash filtration with sparging to recover extract fully. Spent grains go to cattle feed Use of adjuncts Solid adjuncts used in brewhouse, taking Cereals with higher starch gelatinization advantage of malt enzymes; rice and corn need temperatures than for barley need ‘cooking’ at up to 100 C, < 1 h (liquid sugars are precooking before combining with main mash products of acid and enzyme action in sugar factory and added at boiling stage) Boiling 1–2 h at 100 C, before clarification (< 1 h) and To sterilize, extract hops, concentrate, and kill cooling all residual enzymes. Precipitation of insoluble materials as trub, enhancing final product stability Fermentation Wort pitched with yeast and fermented for 3–14 Fermentation of glucose, maltose, sucrose, days at 6–25 C maltotriose to alcohol; enzymic production of various flavorsome compounds (alcohols, esters, fatty acids, sulfur-containing compounds, etc.) Synthesis and removal of diacetyl as an offshoot of amino acid production Cold conditioning and filtration 1 C for 3 days; possibly stabilization (silica Precipitation, sedimentation, and removal of hydrogels, tannic acid or papain to remove solids protein; PVPP to bind polyphenol); then filtration Steeping of barley
PVPP, polyvinylpolypyrrolidone.
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A typical brewery fermentation will produce some 150 g CO2 per hectoliter for every degree Plato (1 degree Plato is basically equivalent to a 1% sugar solution). Most of this CO2 sweeps out and is lost during fermentation. Many packaged beers will contain 500–550 g CO2 hl1 and extra gas needs to be introduced to the product prior to packaging. At 1 atm pressure (as CO2) and 0 C, a beer will dissolve no more than 200 g hl1 CO2. Achievement of the very high levels of CO2 demands the pressurizing of beer. None the less, when a beer container is opened,
the gas usually stays in solution: the beer is ‘supersaturated’ with CO2. When fobbing occurs spontaneously when a can or bottle is dispensed, it is called ‘gushing.’ In the absence of agitation as a cause, the most likely reason for the phenomenon is the presence of low-molecular-weight, highly hydrophobic peptides contributed by infection of barley, notably from Fusarium. CO2 affords the ‘sparkle’ to beer, through its reaction with the pain receptor mechanism of the trigeminal nerve. Apart from this influence on mouth feel, CO2 establishes the extent of foam formation during
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dispense and of course impacts heavily the delivery of volatiles into the head space of beers and therefore the aroma.
reacts with phosphate to lower the pH to the appropriate level for mashing:
Other Gases in Beer
By precipitating out malt-derived oxalic acid in the brewhouse, calcium eliminates a material that, if surviving into beer, causes problems such as haze, gushing, and the blocking of dispense pipes.
Oxygen has major negative effects on beer via its oxidation of various components, leading to cardboard and other stale notes and to the formation of haze. Brewers therefore strive to minimize the oxygen level in beer (preferably to 0.1 mg l1 or ideally even less) by avoiding ingress into the product downstream of the fermenter (yeast is a powerful oxygen scavenger and many brewers feel that it is only after the removal of yeast that oxygen is a concern). The stale papery notes are due to a series of unsaturated carbonyl compounds, which are usually held to include trans-2-nonenal, though this has been no means fully substantiated. Several components of beer can degrade to such carbonyl substances during oxidation, including the ‘higher’ alcohols, the iso-a-acids, and the unsaturated fatty acids. Nitrogen has long been introduced to beer to promote foam stability. The foams produced on beers containing N2 tend to have populations of much smaller bubbles. These foams are more resistant to collapse and the beer displays a much smoother mouth feel. Typical levels of N2 are 20–50 mg l1 which are three orders of magnitude lower than those of CO2. Water
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The vast majority of beers contain between 90 and 95% water: apart from ethanol and carbon dioxide, the remaining constituents usually amount to less than 1% of the product. The water used for brewing must have no taints or hazardous components and water is often treated coming into the brewery to adsorb any contaminants by charcoal filtration and ultrafiltration. The ionic composition of water is also significant (although the grist also contributes salts). High levels of calcium, e.g., 200–350 mg l1, are claimed to be desirable in the production of ales. Much lower levels of calcium are traditionally associated with the brewing of lagers, for example, the water in pilsen (the home of the classic lager-style) contains less than 10 mg l1 calcium. A scientific justification for this is not entirely proven, but may relate to the role of calcium in promoting the surface behavior of the top-fermenting ale yeasts (calcium promotes the flocculation of yeast). Brewers may add salts or remove them (e.g., by reverse osmosis) to adjust the ionic composition of the product. Two key impacts of calcium are on pH and the levels of oxalic acid surviving into beer. Calcium
þ 3Ca2þ þ 2HPO2 4 ! Ca3 ðPO4 Þ2 þ 2H
Carbohydrates
Although most of the sugars found in wort are converted to ethanol by yeast, those containing four or more glucoses (i.e., maltotetraose and bigger, and known as dextrins) are not fermented. In order to sweeten the finished beer, some brewers may contrive to leave a proportion of fermentable sugar unconverted, or may add ‘priming’ sugar. Most of the starch survives malting, because it is relatively resistant to enzymatic hydrolysis when in the form of large granules. When starch is gelatinized by heating, however, its constituents (amylose and amylopectin) become accessible. Mashing, therefore, incorporates a stage, typically at around 65 C, to allow for the gelatinization of malt starch. The starch in some other cereals has higher gelatinization temperatures, e.g., rice and corn starches gelatinize over the range 70–80 C and so are cooked separately from the main mash and then mixed with the malt mash to be degraded by the amylases from malt. (See Carbohydrates: Classification and Properties.) Starch hydrolysis starts with a-amylase, a highly heat-resistant enzyme abundantly present in malt. It is endo-acting, catalyzing the hydrolysis of a-1!4 bonds within the starch, releasing dextrins. From amylose the enzyme release linear dextrins, and from amylopectin it produces dextrins with side chains. (See Starch: Structure, Properties, and Determination.) b-Amylase is an exoenzyme, which removes maltose units from the nonreducing ends of starch and dextrin molecules. This enzyme has sufficient stability at 65 C to achieve most of the conversion of starch and dextrins of which it is capable but if mashing is carried out at somewhat higher temperatures, e.g., 72 C, then b-amylase is rapidly inactivated and the ensuing wort contains a high dextrin level and low content of fermentable sugars. Such high-temperature mashing can be used in the production of low-fermentability worts which will give low-alcohol beers. b-Amylase cannot pass the a1!6 branch points in the dextrins formed from amylopectin. Limit dextrinase carries out this function but is generally present in fairly low concentrations because it is developed
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very late during barley germination and because it is inactivated by association with another protein. Furthermore, limit dextrinase is relatively heat-labile. It is because of the limited action of limit dextrinase that 20–25% of the starch remains as dextrins in most conventionally mashed beers. In so-called ‘light’ or ‘lite’ beers all of the starch is converted to ethanol. One way of achieving this is to add heat-stable glucoamylase or pullulanase from microbial sources to the mash or to the fermenter, although those brewers seeking to avoid any additions may modify their brewhouse and fermenter operations so as to take maximum opportunity of endogenous enzymes. The major component of the barley cell walls is a linear b-glucan comprising approximately 67% b1!4 links with 33% b1!3 bonds that are for the most part every third or fourth linkage. The key enzyme hydrolyzing this molecule is an extremely heat-sensitive endo-b-glucanase, which is destroyed within 5 min of mashing at 65 C. Accordingly, one of the main purposes of malting is to remove the cell walls, in large part through the action of this enzyme. In practice some cell wall material (at the distal end of the barley) will survive and if it is not properly degraded it can afford increased viscosity to the wort and cause sluggish wort separation in the brewhouse and retarded beer filtration. Some brewers mash-in at low temperatures such as 40–50 C to allow b-glucanase to act, before raising the temperature to that needed for starch gelatinization. Alternatively, heat-stable b-glucanases from bacteria (e.g., Bacillus subtilis) or fungi (e.g., Trichoderma, Penicillium) may be used. Proteins, Polypeptides, and Amino Acids
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Amphipathic polypeptides in beer stabilize the bubbles in foam. Their hydrophobic regions ‘drive’ them into the surfaces produced during foaming and are largely responsible for their interaction with other hydrophobic molecules, notably the iso-a-acids. The
matrix formed counters the force of surface tension that seeks to minimize increased surface area generated in foaming. Some of the protein in beer can react with polyphenols to form hazes. There is no advantage in having significant levels of residual amino acids in beer; rather they are a risk in so far as they represent assimilable nitrogen sources for spoilage microorganisms. However, it is important that wort contains the correct balance of amino acids to support yeast growth and fermentation. Sufficient proteolysis must occur during malting and mashing to yield these amino acids and to remove haze-potentiating proteins, whilst leaving ample foam-positive polypeptide. (See Protein: Chemistry.) The native proteins of barley undergo considerable degradation and denaturation in malting and brewing. During germination of barley, endoproteinases develop and attack the heart of substrate proteins to release polypeptides and peptides. An exoacting enzyme, carboxypeptidase, acts during malting and mashing by splitting off amino acids successively from the carboxyl-terminus of the peptides produced by the endoproteinases.
Barley comprises some 3% by weight lipid, most of it in the embryo and aleurone. Very little survives into beer, because it is removed by adsorption on insoluble matrices (e.g., spent grain, trub) during the process. Lipids are severely detrimental to beer foam, disrupting the network of proteins and iso-a-acids in the bubble wall. As observed earlier, the unsaturated fatty acid component of lipids is suspected to be at least one precursor of the stale flavors (e.g., ‘cardboard’) that develop in beer. (See Fats: Classification.)
O
Hops afford the bitterness (from the hop resins) and aroma (from the essential oils) to beer. The most important resins are the a-acids (Figure 2), accounting for 2–15% of the dry weight of the hop,
H Isomerization
HO O
trans fig0002
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Flavors from Hops
Iso-α-acids
R HO HO
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α-Acids OH O
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O
O
H R
OH
HO O
O
O F OH
cis
Figure 2 The isomerization of a-acids. R ¼ –CH2CH(CH3)2 in humulone and isohumulone. R ¼ –CH(CH3)2 in cohumulone and isocohumulone. R ¼ –CH(CH3)CH2CH3 in adhumulone and isoadhumulone.
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depending on variety and environment. The more a-acids, the greater the bitterness potential. There are three different a-acids in hops, differing in side-chain structure. The a-acids are isomerized to form the more soluble and bitter iso-a-acids during wort boiling (Figure 2). Each iso-a-acid has two isomers, cis and trans, with differing orientation of the side chains. The six iso-a-acids have a range of bitterness intensities and it is generally held that the hops should have a relatively low level of cohumulone. Apart from their impact on bitterness and foam, the iso-a-acids have strong antimicrobial properties and inhibit many Gram-positive bacteria. Hops comprise 0.03–3% w/w essential oil, comprising a complex mixture of more than 300 compounds (Figure 3). The components are very volatile and tend to be lost during wort boiling. To ensure hoppiness in beer it is necessary to add a proportion of the hops late in the boil (late hopping, associated with lager-style products) or to the finished beer (dry hopping, associated with some ales). Hops are either used in a traditional way as whole cones, though increasingly after hammer milling and extrusion into pellets, in which form they are more stable and more efficiently utilized, or after extraction by liquid carbon dioxide and isomerization by weak alkalis. The latter products can be added to beer downstream, greatly increasing the efficiency of bitterness utilization and flexibility of product formulation. The oils in such extracts may be separated from the resins and also added to beer to provide hoppy aroma. In some preparations the iso-a-acids are reduced, using hydrogen gas in the presence of a palladium catalyst. Such reduction prevents the formation of the skunk-flavored 3-methyl-2-butene-1-thiol from the side chains when the bitter compounds are exposed to light. The reduced preparations can be used in the production of beers for packaging into green
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and clear glass bottles, which are particularly prone to light damage. Phenolic Materials
The simplest phenolic acid in beer is ferulic acid, a molecule found associated with the arabinoxylan component of the starchy endosperm cell walls. It may have antioxidant properties both for the beer and for the drinker’s body. When decarboxylated, by an enzyme present in the yeast strains used to make wheat-based beers though not barley-based ones, 4-vinylguaiacol is produced, which has a distinct clove-like character. Certain flavanoids, including catechin and quercetin, derive from the outer layers of malt and from hops. They, too, may have antioxidant properties, but when oxidized they polymerize, to produce tannoids that cross-link through the proline groups in certain beer polypeptides to form the insoluble complexes responsible for haze. Haze formation is lessened by reducing oxygen ingress and by reducing the levels of haze-forming polypeptides and phenolics. Silica hydrogels, tannic acid, and the proteinase papain have been used to attend to the former, while polyphenols can be removed using polyvinylpolypyrollidone (PVPP). Vigorous wort boiling and chilling of beer to as low a temperature as possible without freezing (1 C) are both important stages in the colloidal stabilization of beer. The level of tannic materials in beer is probably too low to have any significant impact on astringency. (See Tannins and Polyphenols.)
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Other Flavor Components of Beer
The four main flavor features detected by the tongue are bitterness, sweetness, sourness, and saltiness. The nose detects the other characteristic aromas of beer and this is a balance between positive and negative notes, each of which may be due to more than a single compound from different chemical classes. Although some of these substances originate in the malt and hops, many are products of yeast metabolism.
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OH H O
fig0003
O
Linalool
Humulene epoxide
Hop ether
Myrcene
Humulene
Farnesene
Caryophyllene
Figure 3 Some components of the essential oil fraction in hops.
Esters Esters afford a fruity character to beer: two of the most important components are ethyl acetate and iso-amyl acetate. Esters are formed from their equivalent alcohols when the acetate group is available by not being needed for the synthesis of key components (lipids) of the yeast membranes. Therefore, factors that promote cell production lower ester production, and vice versa. Ester levels in beer are impacted, inter alia, by the ratio of carbon to nitrogen in the wort and by the amount of oxygen available to the yeast. The yeast strain is very
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important; some strains inherently produce much higher ester levels. 0037
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Alcohols The alcohols are the immediate precursors of the esters. As the esters are substantially more flavor-active, it is important to regulate the levels of the higher alcohols if ester levels are also to be controlled. The higher alcohols (i.e., those larger than ethanol) are produced by transformation of amino acids, so the levels of amino acids in wort have a major impact: the more amino acids available to the yeast, the greater the production of higher alcohols. However, Saccharomyces will also produce higher alcohols as an offshoot of the metabolic pathways responsible for amino acid elaboration, pathways that are particularly significant when the amount of assailable nitrogen in the wort is low. Hence higher alcohol production is increased when both too much and too little amino nitrogen is available to the yeast. Ale strains produce more higher alcohols than do lager strains. Conditions favoring increased yeast growth (e.g., excessive availability of oxygen) promote higher alcohol formation. Sourness This is due to the organic acids, such as acetic, lactic, and succinic, produced by yeast during fermentation. It is the Hþ ion produced by their dissociation that causes sourness. Higher levels of the acids are produced in vigorous fermentations. Vicinal diketones The vicinal diketones (VDKs), diacetyl and pentanedione, afford highly undesirable butterscotch and honey characters respectively to beer. These substances are offshoots of the pathways by which yeast produces certain amino acids. Precursors leak from the yeast and decompose spontaneously to form VDKs. Yeast, however, can reassimilate the VDK, provided the cells are healthy and remain in contact with the beer. Brewers may allow a temperature rise of 2–3 C at the end of fermentation to speed up the removal of VDK. Additionally, a proportion of freshly fermenting wort is introduced to the beer as an inoculum of healthy yeast (a practice called krausening). Alternatively a bacterial enzyme (acetolactate decarboxylase) can be added to a fermentation; this enzyme converts acetolactate to acetoin, thereby avoiding the much more flavoractive diacetyl. Persistently high VDK levels may be a symptom of infection by Pediococcus or Lactobacillus bacteria. Sulfur compounds In the right levels and proportions, the sulfur compounds play a substantial role in determining the character of diverse beers. Some
ales display a distinct hydrogen sulfide character when first dispensed, albeit one which subsides to reveal the dry hop character. Lagers often have a more complex sulfury character, which for many includes dimethyl sulfide (DMS), with its cooked corn/parsnip note. DMS is the best understood of all the sulfur compounds in terms of its production route. All of the DMS ultimately originates from a precursor, S-methylmethionine (SMM), which develops in the barley embryo during germination. SMM is heatlabile, breaking down rapidly at temperatures above about 80 C in malting and brewing. Accordingly, SMM is at a lower level in the more highly kilned ale malts and, therefore, there tends to be less DMS in ales. SMM is extracted into wort during mashing and is broken down during boiling and in the whirlpool. In a vigorous boil most of the SMM is converted to DMS and lost by volatilization. In the whirlpool the temperature is still hot enough to degrade SMM, but conditions are nonturbulent and the DMS tends to linger. Brewers aiming for a finite level of DMS in their beer specify a target level of SMM in the malt and will adjust the boil and whirlpool stages so as to deliver a certain level of DMS in the pitching wort. In fermentation a great deal of DMS is swept away with the CO2, hence the level of DMS targeted in the wort is higher than that specified for the beer. Some of the DMS produced during malt kilning is oxidized to dimethyl sulfoxide (DMSO). This is not volatile but is water-soluble and enters wort, to be reduced by yeast to DMS. Hence the level of DMS in the finished beer is a function of how much DMS is present in pitching wort, how much is volatilized, and how much is replenished via the reduction of DMSO. There is no simple relationship between the level of DMS in beer and the perception of its flavor. This is because phenylethanol and phenylethylacetate interfere with the perception of DMS. There must be many other antagonisms of this type contributing to the complexity of beer flavor, but they have not been studied. Hydrogen sulfide (H2S) is produced by yeast via the breakdown of cysteine or glutathione, or by the reduction of sulfate and sulfite. A vigorous fermentation purges H2S and factors that hinder fermentation (such as a lack of zinc or vitamins) will increase H2S levels in beer. Malty notes As well as being the source of the DMS character in beer, malt contributes in other ways to flavor. Malty character is in part due to isovaleraldehyde, produced by the reaction of leucine with reductones in the malt. The toffee and caramel
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Table 2 Order-of-magnitude composition of a typical pilsner beer Component
Typicallevel (mgl1)
Original wort gravity Ethanol Total carbohydrates Glucose Fructose Sucrose Maltose Maltotriose Dextrins derived from starch b-Glucan Pentosan Protein Amino acids Thiamin Riboflavin Vitamin B6 Pantothenic acid Niacin Biotin Vitamin B12 Folic acid Potassium Sodium Sulfate Chloride Organic acids (acetic, pyruvic, citric, succinic, malic, lactic, etc.) Polyphenols Iso-a-acids Sulfur dioxide Carbon dioxide Nucleotides and nucleosides Glycerol Higher alcohols Ethyl acetate Iso-amyl acetate Acetaldehyde Essential oils from hops Dimethyl sulfide Total organic sulfur compounds
120 103 40 103 30 103 150 30 5 1500 2000 24 103 350 50 5000 1100 0.3 0.4 0.6 1.5 8.0 0.01 0.0001 0.2 500 30 200 200 700 150 30 4 5 103 300 1500 100 15 1 5 <1 0.06 <1
character from crystal malts and the roasted, coffeelike notes in darker malts are due to complex components derived from amino acids and sugars when they cross-react during kilning. Of equal importance during kilning is the disappearance of grassy and beany notes, due inter alia to cis-3-hexen-1-ol, trans-2-hexenal, trans-2-cis-6-nonadienal, and 1hexanol. The cross-linking of sugars and amino acids induced by heating in kilning and wort boiling leads to the formation of melanoidins via the Maillard reaction. The melanoidins are responsible for imparting color to beer: darker beers are produced from grists incorporating malts and other adjuncts
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that have been kilned to more intense regimes. Polyphenol oxidation, occurring during wort production, can make a significant contribution to color in some of the paler beers. Miscellaneous Acetaldehyde, which is converted to ethanol in actively fermenting yeast, imparts an undesirable ‘green apples’ character to beer. High levels of acetaldehyde are due to premature separation of yeast before fermentation is complete, poor yeast quality, or infection by the bacterium Zymomonas. The short-chain fatty acids, with their rancid notes, are offshoots in the synthesis of membrane lipids by yeast. When yeast needs fewer lipids (when it needs to grow less), these compounds accumulate. Table 2 offers a summary of the approximate chemical composition of a pilsner-type beer. See also: Alcohol: Metabolism, Beneficial Effects, and Toxicology; Antioxidants: Natural Antioxidants; Barrels: Beer Making; Barley; Beers: Raw Materials; Wort Production; Biochemistry of Fermentation; Microbreweries; Carbohydrates: Classification and Properties; Malt: Malt Types and Products; Chemistry of Malting; Packaging: Packaging of Liquids; Phenolic Compounds; Protein: Chemistry; Starch: Structure, Properties, and Determination; Tannins and Polyphenols
Further Reading Bamforth C (1998) Beer: Tap into the Art and Science of Brewing. New York: Insight. Boulton C and Quain D (2001) Brewing Yeast and Fermentation. Oxford: Blackwell. Briggs DE, Hough JS, Stevens R and Young TW (1981) Malting and Brewing Science. Volume 1: Malt and Sweet Wort. London: Chapman and Hall. Hornsey IS (1999) Brewing. London: Royal Society of Chemistry. Hough JS, Briggs DE, Stevens R and Young TW (1981) Malting and Brewing Science. Volume 2: Hopped Wort and Beer. London: Chapman and Hall. Hughes PS and Baxter ED (2001) Beer: Quality, Safety and Nutritional Aspects. London: Royal Society of Chemistry. Kunze W (1996) Technology: Malting and Brewing. Berlin: VLB. Lewis MJ and Young TW (1995) Brewing. London: Chapman and Hall. MacDonald J, Reeve PTV, Ruddlesden JD and White FH (1984) Current approaches to brewery fermentations. In: Bushell ME (ed.) Progress in Industrial Microbiology, Vol. 19: Modern Applications of Traditional Biotechnologies. Amsterdam: Elsevier. Moll M (1991) Beers and Coolers. Andover: Intercept.
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