Chemistry for Everyone
The Chemistry of Beer Instability Graham G. Stewart International Centre for Brewing and Distilling, Heriot–Watt University, Riccarton, Edinburgh, Scotland, EH14 4AS;
[email protected] Biotechnology has been defined as creating products from raw materials using living organisms. By this definition, the brewing of beer is one of the oldest biotechnology industries. Brewing was one of the earliest processes to be undertaken on a commercial scale and, of necessity, it became one of the processes to be developed from a craft into a technology. A thorough review of brewing chemistry in the British Isles and highlights of the history of international brewing science have been written by Anderson (1, 2). These articles are enjoyable and enlightening and highly recommended for those interested in brewing history. Although Anderson is not a chemist, a student of chemical reactions will find the brewing process and beer stability instructive in a number of ways. Brewing requires a variety of natural ingredients. These are processed by malting, enzyme reactions, heat, and filtration. This is followed by fermentation with yeast that is collected for reuse. The immature beer is then cooled and filtered. The finished beer is always unstable as a result of a number of ongoing chemical reactions. Beer production is divided into five distinct processes: (i) malting,1 (ii) mashing and wort2 separation,3 (iii) wort boiling, (iv) fermentation, maturation, and filtration, and (v) packaging.4 The production of beer is a relatively simple process. Yeast cells are added to the nutrient medium (the wort) and the cells take up the nutrients and utilize them to increase the yeast population. The cells excrete ethanol and carbon dioxide into the medium along with a host of minor metabolites, many of which contribute to the beer flavor. The fermented medium, often after the yeast is removed, is called “green” beer.5 The beer is then aged, clarified, carbonated, and packaged. Compared to most other alcoholic beverages, beer is unique because it is unstable when in the final package. This instability can be divided into biological and nonbiological instability. Biological instability involves contamination by bacteria, yeast, or mycelia fungi. There is always a risk in brewing that beer can become contaminated by microorganisms. Fortunately, beer has an inhospitable environment for microbial growth: it has a low pH (less than 4.4), ethanol is present in a range of concentrations, there is a limited range of nutrients, there are hop acids that are bacteriostatic, the environment is anaerobic, and the liquid is carbonated. Most potential contaminants originate from the raw materials or unclean brewing equipment. Barley can contain Fusarium fungus that can release mycotoxins or cause gushing.6 It can also carry bacteria that contribute nitrosamines and cause filtration problems. Contaminants can cause flavor deterioration, turbidity, and health problems. It is important to exclude these contaminants from the brewing process. A modern plant and good hygiene will help. Many breweries pasteurize their beer to ensure biological stability, but with good hygiene and efficient filtration, the use of this expensive and potentially beer damaging process can be reduced.
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Nonbiological instability of beer involves a wide range of chemical processes and can be divided into a number of categories: physical, flavor, foam, gushing, and light. These will be discussed in detail below. Physical Instability With a few notable exceptions, consumers prefer their beer to be bright and free of particles. When beer is stored it has the potential to produce haze and the brightness is compromised. Beer’s physical stability, also called colloidal stability or simply haze formation, cannot be ensured by treating beer with one “super-product” that will solve everything. Stability will be affected by the whole brewing process; consequently, care must be taken at every stage. However, raw materials are typically the source of haze precursors. Although there are a number of types of beer haze the primary reaction is the polymerization of polyphenols and their binding with specific (sensitive) proteins. When beer is cooled below 0 C, chill haze will form that consists of a reversible association of small-polymerized polyphenols and proteins. When the beer is restored to room temperature, this haze redissolves and the beer becomes bright again. If beer is chilled and warmed a number of times, or if beer is stored at room temperature for an extended time period (six months or longer) permanent haze will form. This haze does not redissolve even when the beer is warmed to 30 C or higher. The balance between flavonoid polyphenols (tannoids) (Figure 1) and sensitive proteins largely dictates physical or colloidal stability. Beers can differ widely in the content of these species, the relative levels of which depend upon raw materials and the process conditions employed. Haze will not form, or its formation will be slowed, when either of these components is removed or the factors promoting the interaction are largely excluded.
OH OH HO
O
OH OH (+)-catechin Figure 1. A typical beer polyphenol.
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day is not as common as it once was. The enzymes employed include papain (from papaya), bromelain (from pineapple), and ficin (from figs). These enzyme preparations are not very specific and, as well as hydrolyzing haze-specific proteins, they often hydrolyze the hydrophobic foam-specific polypeptides. Consequently, the use of these enzymes often requires the addition of a foam-enhancing agent such as propylene glycol alginate. Flavor Instability The flavor stability of a beer primarily depends on the oxygen content of the packaged beer. However, it is now clear that flavor stability is influenced by all stages of the brewing process (6): preservation of reducing substances by minimizing oxygen pickup during mashing, lautering,9 and wort boiling; elimination of substances that are prone to react with flavo-active compounds like carbonyls by good mashing and wort separation procedures; prevention of ion accumulation, such as iron and copper (7); and controlled exposure of the wort to heat to limit the formation of Maillard-reaction products10 and related substances. The role of such reaction products in beer flavor-staling reactions is ambiguous and there are reports (8) of their positive and negative influences.
ribes aroma
bitter taste
sweet aroma
Intensity
Haze formation is increased by a number of factors (3) but storage temperature has the greatest influence on haze formation because an increase in temperature raises the rate of the reactions. For example, pasteurization accelerates colloidal haze formation. Oxidation (the presence of oxygen) has a great effect on beer haze formation. Extensive oxidation can increase the rate of haze appearance many fold. Heavy metal ions (particularly iron) can promote the formation of colloidal haze. Movement of beer accelerates haze formation because of rapid interaction of colloids. Light encourages oxidation and consequently haze formation. Beer chill haze consists of a loose bonding of high molecular weight proteins with highly condensed polyphenols (predominantly anthocyanogens). In these loosely bound aggregates, small quantities of carbohydrates and inorganic materials are included. This loose bonding is broken on warming. Haze formation occurs as a result of dissolved colloidal particles colliding and increasing hydrogen bonds between them. In the course of time, increasingly large aggregates come together until they are visible as haze. Haze formation correlates with the presence of sensitive proteins7 and tannoids.8 The driving force for haze formation is the interaction of hydrophilic groups on these sensitive proteins with polyphenols. There are also hydrophobic proteins in beer. These surface-active species are important for foam stability. There are a number of procedures that can be employed to retard or prevent haze formation: preventing the formation of large quantities of complex protein degradation products during beer production; enzymatically hydrolyzing the complex “sensitive” protein degradation products; removing some of the polyphenols during brewing; removing polyphenols or sensitive proteins from the beer; storing maturing beer in cold temperatures to precipitate haze precursors, and storing packaged beer in cold temperatures to retard haze formation. Employing stabilizers can produce beers with a longer shelf life. The main stabilizing agents, which can be used singly or together, are: silica gel preparations, polyvinylpolypyrolidone (PVPP), and proteolytic enzymes. Silica gel preparations are important stabilizing agents that bind hydrophilic polypeptides but have little effect on foam-promoting hydrophobic polypeptides. They are employed in quantities of 50 to 150 g兾hL and are usually dosed into the beer before filtration. There are two types of silica gel preparations used in brewing: hydrogels that have a moisture content of more than 30% and xerogels (dry gels) with a 5% water content. PVPP selectively removes phenol-containing substances. PVPP binds to polyphenols as it has a very similar structure to the amino acid proline (4). Both have fivemembered, saturated, nitrogen-containing rings with amide bonds and no other functional groups. It is not certain whether PVPP binds to the same part of the polyphenol molecule to which polypeptides bind. This selection depends on the pH-sensitive formation of hydrogen bonds that are broken again in alkaline solution with the release of the adsorbed phenol compounds. Regeneration of PVPP with hot caustic is very effective. PVPP and silica gel preparations have been used together with good results because both polyphenol and sensitive protein components are removed (5). Proteolytic enzymes are also employed as stabilizing agents, but because of the advent of silica gel preparations, their use to-
sweet taste, toffee-like aroma & flavor cardboard flavor
Time Figure 2. Sensory changes in beer flavor during aging.
Table 1. Typical Staling Flavors Taint
Flavor
Cause
Oxidized
Papery, cardboard, “dull“, toffee
Storage, oxygen
Catty/Ribes
Tomcats, black current leaves, tomato plants
High in package oxygen
Aldehyde
Rotting apples
Storage, high oxygen
“Cooked”
Over pasteurized, grainy, “dull“, toffee
Storage, high oxygen, high temperature
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In many foods, such as milk, butter, vegetables, vegetable oils, and beverages, staling is caused by the appearance of various unwanted unsaturated carbonyl compounds. It is now becoming increasingly clear that the same is true of beer staling. As already discussed, packaged beer has a limited shelf life. The phenomenon of beer aging or staling has been intensively investigated by the brewing industry with the objective to understand and control it. Despite intensive studies over the past 30 years, the mechanism of staling is not fully understood. The actual compounds responsible for stale flavor vary during prolonged storage as evidenced by changes in the flavor profile of beer (Figure 2). Typical beer staling flavors are summarized in Table 1 (9). The compounds causing the sweetish, leathery character of very old beers have not been identified. However, there is evidence that the papery cardboard character of 2–4 monthold beer is due to unsaturated aldehydes (9). The most flavor-active aldehyde that has been conclusively proven to rise beyond flavor-threshold levels is trans-2-nonenal (9). Other aldehydes such as nonadienal, decadienal, and undecadienal may also exceed threshold levels. Although there are many factors that will influence the flavor stability of beer, the oxygen level in the final package is of paramount importance. It is critical that this level in beer, immediately prior to packaging, is as low as possible (less than 100 mg兾L) and that oxygen accumulation during filling is minimal.
H R
R
O
OH
H
ⴚ
HSO3
C
S ⴚ
O
flavor active
O
O
flavor inactive
Hydrophobic Polypeptide / (mg/L)
Figure 3. Binding of bisulfite to carbonyls.
300
250
high-gravity
200
low-gravity
150
100
50
0
kettle full
kettle start post end strike ferment ferment filter
final beer
Figure 4. Change in the levels of hydrophobic peptides during the brewing process (final high-gravity beer diluted to 4.5% alcohol by volume).
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The adverse effects of oxidation on the flavor of finished beer have been known for a long time and some brewers add bisulfites or other antioxidants, such as ascorbic acid, to beer prior to packaging to provide protection against oxygen. This can improve flavor stability (9). The effectiveness of bisulfite, besides its antioxidant properties, is also its ability to bind carbonyl compounds into flavor-neutral complexes (10, 11). The reaction is reversible and excess bisulfite will increase yields of the adduct (Figure 3). Bisulfite addition to fresh beer minimizes the increase of free aldehyde concentration during aging. In addition, when added to stale beer, bisulfite lowers the concentration of free aldehydes and effects the removal of the cardboard flavor. However, over time, the bisulfite will be oxidized to sulfate, thus increasing the concentration of free aldehydes again (12). Foam Instability When beer is sold, the stability of the foam in a glass of beer is considered by some consumers to reflect the quality of the product. The increasing use of adjuncts (unmalted sources of carbohydrate) and the associated decrease in malt being used today, together with the employment of high-gravity brewing techniques, have had a negative effect on foam values in many beers. There are many foam-promoting compounds in beer, such as iso-α-acids from hops, protein, metal ions, and polysaccharides, and all have an important role to play in foam formation and stability. However, the backbone of foam is protein. Many methods have been tried and extolled for their virtues in the isolation and characterization of foam-positive beer polypeptides, for example, separation of foaming proteins by hydrophobic interaction chromatography has long been a standard technique (3). Once separated, the proteins are investigated to discover whether they are related to beer foam potential or stability. On the basis of such experiments, it has been proposed that certain sizes of proteins are important in the formation and stabilization of foam; for example, 40, 10, and 8 kD proteins have been postulated as major foam stabilizing molecules (13). However, it is now widely accepted that the polypeptides of greatest hydrophobic character produce the most stable foam and it is the hydrophobic property that is more important than size (14). The use of high-specific-gravity brewing techniques is essential for the future economic viability of the brewing industry (15). High-gravity brewing is a procedure that employs wort at higher-than-normal concentrations and therefore requires dilution with water later in the process. This process, by reducing water employed in the brewhouse, increases production capacity without adding to the existing brewing plant. Therefore, most major brewing companies worldwide have revised their production processes to accommodate high-gravity brewing procedures as a means to reduce capital expenditure. Although this process has many advantages (16), one of the problems that still exists is that beers brewed at higher gravities exhibit poor foam stability. The effect of high-gravity brewing on head retention with respect to hydrophobic polypeptide levels has been examined (using a phenyl sepharose column; ref 14) throughout the brewing and fermentation of high-gravity and low-gravity worts (Figure 4) (17).
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Three notable features of the data are highlighted. At the kettle full 11 stage the level of hydrophobic polypeptides was similar in the high-gravity and low-gravity worts.12 This is in spite of the use of twice the quantity of malt grist to produce the high-gravity wort. This implies there was a major failure to extract hydrophobic polypeptides during the high-gravity mash. There was much greater loss of hydrophobic polypeptides during fermentation of the high-gravity wort so that by the end of fermentation, the hydrophobic polypeptide content of the high-gravity fermented wort was just over 50 mg兾L, markedly lower than that of the low-gravity fermented wort. When the high-gravity beer was diluted to 4.5% alcohol by volume, equivalent to the low-gravity beer, it contained a level of hydrophobic polypeptide less than 50% of the low-gravity brewed beer (17). The head retention of the high-gravity brewed beer was less than that of the low-gravity brewed beer. This contrasts with the low-gravity brewed beer where the hydrophobic polypeptide in this foam accounted for over 40% of the total polypeptide. Therefore, not only is the polypeptide of high-gravity brewed beer reduced, but so is the hydrophobic content of its foam that would adversely influence its stability (18). The amino acid profiles of hydrophobic polypeptides recovered from beer foam, unlike polypeptides involved in haze formation (where glutamic acid and proline account for 40–50% of the total amino acid composition) have no amino acid present in a distinctive quantity (19). It has already been discussed that the fermentation stage is a key step where hydrophobic polypeptides are lost during the brewing process (Figure 4). Two factors could account for the loss of hydrophobic polypeptide during fermentation. First, fermentation is known to be responsible for the loss of a large quantity of foam-active substances and this problem is exacerbated during the fermentation of high-gravity worts. Secondly, yeast secretes proteolytic enzymes into the fermenting wort and these enzymes have a negative effect on the foam stability of finished beer through protein degradation (hydrolysis) that occurs during fermentation and storage.
9
Proteinase A Activity
8
high-gravity low-gravity
7 6 5 4 3
Gushing Excess foam in a beer is regarded as deleterious and is known as gushing or “wild beer”. Gushing is the violent, uncontrolled ejection of beer from the package at the time it is opened and involves the loss of a significant portion of the contents. There may be two different classes of gushing, namely, sporadic and epidemic. Sporadic gushing may occur as a result of minor production deviations that are generally difficult to pinpoint. Epidemic or long-term serious gushing may be caused by several factors. Perhaps the most widely discussed cause is the use of weathered (damp) barley. If barley is harvested when wet, a Fusarium or other fungus infection can develop during the malting process, resulting in beer susceptible to serious gushing. The formation of mycotoxins such as deoxynivalenal (DON) has been paralleled with the development of gushing potential. The screening of barley and malt for these metabolites may offer a means of reducing beer gushing problems (21). Other factors, such as increased levels of carbonation or a carbonating system operating without proper controls, can produce beers that have the potential to gush. Calcium oxalate micro crystals in beer are another cause of gushing. These minor crystals are thought to form nuclei for carbon dioxide gas emissions, but excess treatment and filtration will overcome this cause of gushing. Excessive levels of iron and other nuclei forming particles such as sediments will also contribute to gushing problems. Light Instability
2 1 0 0
1
2
3
4
5
6
7
8
9
10
11
12
Fermentation Time / day Figure 5. Yeast proteinase A activity released into wort during fermentation of high-gravity (20º Plato) and low-gravity (10º Plato) wort.
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Analysis of proteinase A activity (using the fluorimetric method described by Kondo and coworkers in ref 20) in wort and beer during the brewing process (Figure 5) showed, as would be expected, that freshly boiled wort contained no enzyme activity. However, during fermentation proteinase A was secreted into wort by yeast cells. Proteinase A increased throughout the fermentation with the highest enzyme activity occurring at the end. Considerably larger quantities of proteinase A were released during the 20 Plato fermentations compared to the 10 Plato fermentations. During highgravity brewing, increased stress on the yeast, in the form of elevated osmotic pressure and ethanol concentrations, stimulated the secretion of proteinase A into the wort during fermentation. Preliminary in vitro studies in this laboratory have shown that both ethanol and increased osmotic pressure (simulated using sorbitol that is not metabolized by brewer’s yeast) stimulate the secretion of proteinase A by brewer’s yeast strains. These studies are continuing in this laboratory, together with an investigation of specific hydrophobic polypeptides that occur in wort and beer and their fate during highand low-gravity brewing.
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Beer is sensitive to light, especially in the 350–500 nm range. Light of this wavelength can penetrate clear and green glass and cause nauseous off-flavors in beers bottled in such glass containers and drinking glasses. The beer is said to be “sunstruck” and the aroma and taste referred to as “skunky”. Light instability in beer results from hop components. Hops in brewing have a number of roles: they impart bitterness to beer; provide characteristic hop aromas; suppress growth of
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O
far from a complete comprehension of beer instability reaction systems.
O R
O
Notes
UV light
OH
OH
HS
iso-␣-acid HS
mercaptan
Scheme I. Mercaptan formation in light-sensitive iso-α-acid.
certain microorganisms, particularly gram-positive bacteria; assist in beer foam stability; and contribute polyphenols to the protein–polyphenol complex during wort boiling. When beer is exposed to light one of the side chains on the iso-α-acid (a component of hops) is cleaved and the highly reactive radical that is liberated combines with sulfurcontaining compounds (Scheme I) to produce 3-methyl-2butene-1-thiol (MBT). MBT has a skunky-like aroma. MBT has a flavor threshold in the order of parts per trillion, making it one of the most flavor-active substances in beer (22). Specialized hop extracts (produced using liquid CO2 or ethanol as a solvent) have been developed to combat this sensitivity to light (23). In essence, pairs of hydrogen atoms are catalytically added to the isomerized iso-α-acid. There are three principal types of such extracts (called reduced extracts) currently available on the market: RHO iso-α-acid, tetrahydro-iso-α-acid, and hexahydro-iso-α-acid. All of these materials are bitter to varying degrees, some improve beer-foam cling and stability and protect beer against light-struck-sun-induced skunky flavors. Normally all of these materials are used as a post-fermentation addition to achieve maximum benefit and optimum utilization. To achieve complete light-strike protection, no iso-α-acid can be used in any other part of the process. Even repitched yeast13 with iso-αacid absorbed onto its surface will provide sufficient material for photolytic cleavage to occur and the resultant production of MBT. Conclusions
1. Malting is the germination of the barley or other cereal and drying (or kilning) of the germinated cereal. 2. Wort is unfermented beer. 3. Wort separation is the extraction of the ground malted barley with water and separation from the insoluble material. 4. Packaging is used generically to mean kegging, bottling, and canning. 5. Green beer receives its name as it often has the aroma of green apples. 6. Gushing is the spontaneous ejection of beer from its container, either can or bottle. 7. Sensitive proteins are defined as substances precipitated with tannic acid. 8. Tannoids are defined as polyphenols adsorbed by polyvinylpolypyrolidone or PVPP. 9. Lautering is the separation of unboiled wort from solid grain material (spent grains). 10. Maillard-reaction products are produced as a result of heating sugars with amino acids. 11. The kettle full stage is when “run-off ” from the lauer tun or mash filter is complete and wort boiling begins. 12. High-gravity wort used here was measured at 20 °Plato and low-gravity wort used here was measured at 10° Plato. 1 °Plato is equivalent to 1 g of sucrose dissolved in 100 mL of distilled water at 20 °C. 13. Repitched yeast is a brewer’s yeast culture recycled through a number of wort fermentations. Only brewing recycles its yeast.
Literature Cited 1. 2. 3. 4. 5. 6. 7. 8.
9.
10.
11.
The chemistry of beer instability involves a number of complex reactions involving proteins, carbohydrates, polyphenols, metal ions, thiols, and carbonyls. There are many diverse types of beer instability involving a number of different chemical species and reactions. Our understanding of these reactions has progressed over the past 25 years, but we are www.JCE.DivCHED.org
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12.
13.
Anderson, R. G. J. Inst. Brew. 1992, 98, 85–92. Anderson, R. G. Ferment. 1993, 6, 191–195. Bamforth, C. W. J. Amer. Soc. Brew. Chem. 1999, 57, 81–90. Siebert, K. J.; Lynn, P. Y. J. Amer. Soc. Brew. Chem. 1997, 55, 73–78. McMurrough, I.; O’Rourke, T. Tech. Quart. Master Assoc. Amer. 1997, 34, 271–277. Narziss, L. J. Inst. Brew. 1986, 92, 346–353. Irwin, A. J.; Barker, R. L.; Pipasts, P. J. Amer. Soc. Brew. Chem. 1991, 49, 140–149. Bright, D. R. A Study of the Antioxidant Potential of Speciality Malt. Ph. D. Thesis, Heriot–Watt University, Edinburgh, Scotland, 2001. Dalgliesh, C. G. Proceedings of the European Brewing Convention, 16th Congress, Amsterdam, The Netherlands, 1977; pp 623–659. Collin, S.; Montesinos, M.; Meersinan, E.; Swinkels, W.; Dufour, J.-P. Proceedings of the European Brewing Convention, 23rd Congress, Lisbon, Spain, 1991; pp 409–416. Barker, R. L.; Gracey, D. E. F.; Irwin, A. J.; Pipasts, P.; Leiska, E. J. Inst. Brew. 1983, 89, 411–415. Gacey, D. E. F.; Barker, R. L.; Irwin, A. J.; Pipasts, P.; Leiska, E. Proceedings of the 18th Convention, Inst of Brewing, Aust/ NZ Section, 1984; pp 50–58, Lusk, L. T.; Goldstein, H.; Ryder, D. J. Amer. Soc. Brew. Chem. 1995, 53, 93–103.
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Chemistry for Everyone 14. Bamforth, C. W. J. Inst. Brew. 1985, 91, 370–383. 15. Murray, C. R.; Stewart, G. G. Birra Malto 1991, 44, 52– 64. 16. Stewart, G. G. Brewers’ Guardian 1999, 128, 31–37. 17. Cooper, D. J.; Stewart, G. G.; Bryce, J. H. J. Inst. Brew. 1998, 104, 283–287. 18. Cooper, D. J.; Stewart, G. G.; Bryce, J. H. J. Inst. Brew. 1998, 104, 83–87. 19. Leiper, K. A. Beer Polypeptides and Their Selective Removal with Silica Gels. Ph.D. Thesis. Heriot–Watt University,
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Edinburgh, Scotland, 2002. 20. Kondo, H.; Yomo, H.; Furukubo, S.; Fukiu, N.; Nakatani, K.; Kawasaki, Y. J. Inst. Brew. 1999, 105, 293–300. 21. Haikara, A. Proceedings of the 6th European Brewing Convention Symposia, 1980; pp 167–177 22. Irwin, A. J.; Bordeleau, L.; Barker, R. L. J. Amer. Soc. Brew. Chem. 1991, 49, 140–149. 23. Guzinski, J. A.; Stegink, L. J. Stabilized Aqueous Solutions of Tetrahydro- and Hexahydro Iso-Alpha Acids. U.S. Patent 5,200,227, 1993.
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