The word "beer" is used for alcoholic beverages made from cereal grains. In the narrower sense beer is the carbonated beverage prepared by fermentation of mashes containing predominantly malt. In the wider sense the word is applied to sake, kaffir beer, and other alcoholic beverages. It is also used for the fermented mashes of distillers and for the fermenter contents of bakers' yeast fermentations although, with modern methods, such liquors may contain less than 0.1% alcohol. In the following pages the word "beer" will be used in the narrower sense.
In contrast to wine fermentations, which are highly seasonal, beer is brewed all year long since the principal raw material, malt, is always available. One of the main distinctions between beer fermentations and the fermentation of distillers' mashes is the sequence of operations. For the production of beer the mashes are enzymatically hydrolyzed to the desired extent, and the worts are clarified and boiled. Boiling of the wort is carried out for one or more of the following reasons: (a) to sterilize the wort; (b) to precipitate protein; (c) to solubilize and isomerize hop compounds; (d) to generate flavor through browning reactions; and (e) to concentrate the wort. The boiling also arrests enzyme action and results in the presence of dextrins in the beer. In contrast distillers' mashes are partly hydrolyzed by malt enzymes; and then fermentation proceeds simultaneously with hydrolysis of the remaining oligosaccharides.
The enzymes used in the conversion of cereal starches are obtained by malting of cereal grains. "Malt" is generally understood to be barley malt. Other cereal malts are designated as wheat malt, rye malt, etc. For the production of beer, barley malt is preferred because, in contrast to wheat and other cereal malts, the liquefied and saccharified extract can be readily separated from the husks and other insoluble material. In some countries the mash bill (or grist) is entirely composed of malt; in others, adjuncts such as rice, corn, corn syrup, sucrose or glucose are added. In the U.S. up to 40% of the mash bill consists of corn and rice, which are the preferred adjuncts for American lager beer. In Britain sugar syrups are often used alone or in conjunction with rice. The amount of adjuncts is usually limited to 35% of the mash bill. Recently the use of unmalted barley has been suggested as a suitable raw material for the production of beer. It may be used in concentrations up to 80% of the mash bill, if 20% malt and microbial enzymes are added.
The mashing procedure is of considerable importance for the subsequent fermentation. It will not be discussed in detail but some of the more significant effects must be mentioned. In the so-called infusion process, the temperature of the slurry of ground malt is slowly raised to a point that permits enzyme action. At 40°C proteinase action predominates, resulting in the production of soluble nitrogen, which plays an important part in yeast nutrition. Generally mashes are held longer at temperatures between 62 and 68°C to permit saccharification of starches by the carbohydrases of the malt. A typical mashing schedule is as follows: Hold at 52°C for 30 min; raise from 52 to 63°C for 20 min; hold at 63°C for 60 min; raise from 63 to 78°C for 40 min; hold at 78°C for 10 min, for a total mashing time of 160 min. Within the past decade new varieties of barley have yielded malts with higher enzyme activity, and mashing cycles as short as 100 min can be achieved.
The lower limit of the range (62 to 63°C) favors action of b-amylase. Mashes held at this temperature yield worts in which 80% of the total extractable carbohydrate is in the form of fermentable sugars. At the higher limit of the range (68°C) B-amylase action is limited because the enzyme is less heat-stable than a-amylase. Worts from such mashes may contain 70% or less of the soluble carbohydrates in the form of fermentable sugars. Consequently low mashing temperatures (with regard to the B-amylase optimum) result in the production of beer with a higher alcohol content and a lower residual extract. The unfermentable carbohydrates consist of oligosaccharides with 4 or more glucose molecules, loosely called dextrins.
The mashing procedure may be modified as in the "decoction" process. In this process a portion of the malt mash is boiled to gelatinize the starch completely. The boiled portion of the mash is then used to raise the temperature of the main mash to the conversion temperature of about 65°C. High yields of fermentable sugars can be obtained because gelatinized starch is more susceptible to enzyme action; but the malt used in the main mash must supply all the required enzyme, since the enzymes of the boiled portion of the mash have been inactivated. It is, of course, essential to boil rice, corn or other starchy adjuncts which do not contain amylases in order to facilitate enzymatic conversion of their starches by malt enzymes.
The sequence of brewing operations is shown in Fig.1, including the vessels used for preparing the mash and for boiling of the adjunct portion of the mash. The figure provides a scheme of the process and the sequence of brewing operations.
The effect of mashing temperature on yield of fermentable sugars has been determined by Hudson. At 62.5°C the percentage of fermentable sugars was 78%' at 65.5°C it was 76%; and at 68°C it was 72% (based on total soluble carbohydrates). The nitrogen levels of the three mashed worts were 73, 69 and 65 mg N per 100 ml of wort, respectively, the lowest mashing temperature giving the highest nitrogen value.
After the main mash cycle is complete the entire slurry of solubilized and insoluble ingredients is transferred from the mash tun to the false-bottom lauter tun. In the lauter tun the insoluble residual grain and husks settle to the bottom on a slotted floor, and the liquid portion is clarified by recycling through this heavy layer of insoluble material. Some breweries use a plate-and-frame type mash filter for this purpose. The clarified liquid is now transferred to the brewing kettle where it is hopped and boiled. After additional settling or filtration of the wort, it is cooled and is now ready for fermentation.
The amounts and proportions of mashing ingredients are chosen in such a way that worts of definite specific gravities result. A specific gravity of 1.047 to 1.050 is desired in the U.S. for premium beer and of 1.044 to 1.047 for less expensive beer. This gravity corresponds to about 11 to 12.5°. Balling or to 11 to 12.5° Plato in the terminology of the brewing trade. The soluble substances of the wort are called extract. During fermentation extract values of the beer diminish. Since ethanol is formed, determination of the specific gravity and expression as Plato indicates an extract value lower than the true value. The results of this measurement are sometimes called "apparent Plato." In order to determine the true extract value, one must distill off the ethanol and bring the residual liquid to the original volume of the beer by adding water.
The composition of worts has been reviewed by MacWilliam. Individual components which are important for fermentation or yeast nutrition will be discussed below. The following figures give an approximation of the gross composition of 100 ml of wort made with an all-malt mash: Total solids 10 to 12.5 g, total carbohydrates 9 to 11.3 g, nitrogenous substances (proteins, amino acids, amines, etc.) 0.4 to 0.5 g, lipids 5 to 7 mg, tannins 200 to 300 mg, mash 0.15 to 0.2 g.
By-products of alcoholic fermentation
Higher Alcohols (Fusel Oils)
The formation of higher alcohols either by the Ehrlich mechanism or by a pathway common to the synthesis of either amino acids or higher alcohols has already been described. For beer fermentations it has also been shown that the Ehrlich mechanism alone cannot account for all the amounts of higher alcohols formed. Maule found no clear relationship between the nitrogen content or the amino acid content of the wort and the formation of higher alcohols. If 33 % sucrose was included in the mash bill more isobutanol and more isoamyl alcohol were formed, and less n-propanol. The resulting beer had a more "alcoholic" flavor.
However, the presence of individual amino acids affects formation of the corresponding alcohols via the Ehrlich mechanism. Addition of leucine (in glucose solution) to brewers' wort increased the concentration of amyl alcohols in the beer, 3-methyl butanol being the dominant isomer. Addition of isoleucine also increased the amyl alcohol fraction significantly, and 2-methyl butanol was the dominant isomer. Addition of valine had no effect on the formation of amyl alcohols but increased the concentration of isobutanol.
For some yeast strains there is good correlation between the formation of higher alcohols and the original gravity of the wort. Formation of higher alcohols occurred during the active primary fermentation and followed the disappearance of sugars closely, as shown in Fig. 15. There was no increase in the level of isoamyl or d-amyl alcohol during the lagering period. But increases of the total higher alcohol fraction from 5 to 20% during this period have been reported by Wellhoener.
Table. 7 shows the levels of higher alcohols formed in top- and bottom-fermented beer. About 50 to 66% of the higher alcohols consist of isoamyl alcohol, followed in descending order by active amyl alcohol, isobutyl alcohol, and n-propanol. The concentrations of n-butanol and n-amyl alcohol are negligible. None of the alcohols are normally present in quantities higher than those shown in the columns headed. Threshold concentrations." But they may approach these concentrations, and it is more than likely that the total higher alcohols contribute to beer flavor. It has been noted that threshold values give at best an approximation of the concentrations which can be detected in beer or in de-gassed beer. Values given by Harrison represent concentrations of these alcohols which can be perceived in beer if they are added to the concentrations already present. Engan measured the effect of combinations of flavor compounds on threshold values and found a definite additive effect. The results of his threshold determinations in beer are in general agreement with those shown in Table 7. Phenylethyl alcohol, which has a pronounced rose-like odor, significantly affects the flavor of beer. Its addition to stabilize beer flavor has been suggested.
Temperature of fermentation has a pronounced effect on the formation of higher alcohols, the concentration increasing with a rise in temperature. However, there are differences in the effect on individual alcohols; n-propanol concentration increases sharply from 6° to 12° or 15°C, though for higher temperatures the increase was slight or insignificant. But isobutyl and amyl alcohol concentrations increased significantly with temperatures above 15°C up to 27°C. Rate of formation of higher alcohols is at 28°C, but maximum yield is obtained at 20°C. Fig. 16 shows the dependence of fusel oil formation on temperature and its relation to the formation of biomass. The production of 2-phenylethanol also increases significantly with rising temperature.
Aeration also increases the concentration of higher alcohols, probably because of its stimulating effect on yeast growth. In some of the experiments conducted, the effect of stirring cannot be readily separated from the effect of aeration. Maule showed that stirring of a batch fermentation increased the levels of all the aliphatic alcohols; stirring plus aeration increased the concentration of n-propanol but not that of isobutanol or of the amyl alcohols. Makinen and Enari concluded that increased production of fusel oils in continuous fermentations must be due to aeration since unaerated continuous fermentations gave concentrations equal to those obtained in batch fermentations.
As one would expect, the strain of yeast exerts the major effect on the formation of higher alcohols. Some strains produce as little as 40 ppm higher alcohols while others under the same conditions produce as much as 200 ppm. But these are extremes; for yeasts used in actual practice the variations are less. Wellhoener found a range from 49 to 122 ppm for flocculent, bottom-fermenting yeasts, and from 62 to 94 ppm for nonflocculent yeasts. Narrower ranges were reported by Drews et al. and Kamiyami and Nakagawa. Occasionally, strains have been found which produce high proportions of a particular alcohol. For instance, Kamiyami and Nakagawa worked with a strain which produced 33% of the higher aliphatic alcohols as n-propanol. There appears to be no correlation between flocculence of a yeast and formation of higher alcohols. Continued use of a yeast for re-pitching of successive batches does not affect production of higher alcohols. The effect of pitching rate is not very pronounced. Starting pH of the wort had little effect on the formation of higher alcohols and pasteurization of the beer had no effect.
Ester formation takes place during active growth of yeast, and little ester formation occurs during the lagering period. This makes it unlikely that esters are formed by chemical reaction between the acids and alcohols in beer. The reaction probably takes place within the yeast cell between the activated acid and an alcohol:
CH3-CO CoA + CH3CH2OH ® CH3 - CO-O-CH2-CH3
acetyl coenzyme A ethanol ethyl acetate
Similar reactions take place in the formation of other esters, and both acids and alcohols compete in these enzyme-catalyzed reactions within the cell. Knowledge of the mechanism of ester formation is largely based on the work of Nordstrom, which has been reviewed by this author and by Rainbow.
The total amount of esters is generally expressed as ethyl acetate. In American lager beer it is between 25 and 50 ppm. However, ethyl acetate, though the predominant ester, accounts for less than half the total. A large number of esters has been identified but quantitative determination of individual esters is quite difficult. Table. 7 shows the concentrations of some of the esters as well as flavor threshold values. It is thought that ethyl acetate, isoamyl acetate, and phenylethyl acetate contribute an estery or fruity flavor to the beer, but the contribution of other esters cannot be excluded. Such estery flavors are somewhat desirable in top-fermented beer and in dark beer, but are definitely undesirable in light lager beer. Esters containing acids with higher numbers of carbon atoms are increasingly adsorbed on the surface of the yeast cells and, therefore, it has been concluded that esters higher than ethyl caproate do not occur in beer in significant amounts.
The extent of ester formation depends very much on the yeast strain. It is usually associated with the concentration of higher alcohols. Beer high in concentrations of isoamyl alcohols is also likely to show higher concentrations of isoamyl acetate. Conversely, the occurrence of individual fatty acids influences the formation of their esters. For instance, addition of butyric acid to experimental fermentations resulted in the formation of high levels of butyrate esters.
It has been mentioned that ester concentration does not increase much during the lagering period. Hashimoto and Kuroiwa and Kepner et al. report increases of about 10 to 20% in the level of ethyl acetate and isoamyl acetate during the lagering period. The concentration of phenylethyl acetate did not increase at all.
Ester formation, like fusel oil formation, is highly dependent on temperature. Bavisotto et al. found a 60% increase in the concentration of ethyl acetate when the temperature of the fermentation was raised from 12.5 to 25°C. Isoamyl acetate concentration increased by about 30%. Generally a temperature optimum of 20 to 25°C has been reported. Hence, top-fermented beers contain much higher proportions of esters than bottom-fermented beers. Maximum ester formation in synthetic media occurred at a pH of 4.5 but the starting pH had little effect. Pasteurization does not affect ester concentration significantly.
Information on the effect of aeration on ester formation is somewhat contradictory. Nordstrom reported an increase in total ester concentration as the result of aeration, but a decrease in isoamyl acetate and ethyl caprylate. He thought that some losses occurred as a result of volatilization. Maule observed a drastic decrease in the concentration of ethyl acetate and isoamyl acetate in stirred, aerated fermentations (vs. stirred, unaerated fermentations).
Diacetyl, Acetoin, 2, 3-Butanediol, and 2, 3-Pentanedione
These related compounds are of considerable interest in the investigation of beer flavor. Particularly, diacetyl affects beer flavor at very low concentrations. It gives a cheesy or buttery off-flavor if it is present in concentrations exceeding 0.45 ppm. In 9.4% (w/w) ethanol solutions Salo found an odor threshold of 0.0025 ppm. In beer, flavor thresholds of 0.2 ppm, 0.1 ppm and 0.162 ppm have been reported by Drews et al, Harrison and Sega et al, respectively.
Diacetyl, acetoin and the diketones are formed during yeast growth. The mechanism of diacetyl formation is still subject to controversy. It had generally been accepted in the brewing industry that diacetyl is formed by decarboxylation of a-acetolactate to acetoin and oxidation of acetoin to diacetyl. However, the reduction of diacetyl bydiacelylh reductase is irreversible and diacetyl is not formed from acetoin. Currently two theories are under consideration: one involves spontaneous decomposition of a-acetolactate to diacetyl by oxidative decarboxylation, the reaction taking place at relatively high pH values and being strongly temperature-dependent; the other theory suggests formation of diacetyl via pyruvic acid and the acetaldehyde thiamine pyrophosphate complex (active acetaldehyde). The active acetaldehyde reacts with acetyl coenzyme A to yield diacetyl. It is well known that the addition of valine to worts suppresses diacetyl formation. This effect may be explained by either of these theories on the basis of feedback inhibition.
Studies of diacetyl formation in beer have been greatly complicated by analytical difficulties. Interference of related compounds in coloimetric methods and the formation of diacetyl from a-acetolactate during distillation are serious sources of error. Hence, some of the earlier work on diacetyl is difficult to interpret. It seems that the very high peak of diacetyl during the early phases of fermentation is an artifact. Analysis of diacetyl in the final beer does not appear to be subject to the same doubts. Brenner and Kamimura suggested that diacetyl is bound to yeast alcohol dehydrogenase during fermentation, and that diacetyl is slowly reduced by this enzyme rather than by diacetyl reductase.
The effect of valine in suppressing diacetyl production was first demonstrated by Owades et al, and elaborated by Portno. Worts with a low ratio of amino acids to fermentable carbohydrates yield more diacetyl, and diacetyl production starts when valine is exhausted from the wort. pH affects diacetyl production. In a stirred batch fermentation diacetyl became detectable only when the pH dropped below 4.6. There is considerable variation in the production of diacetyl by different yeast strains. It has been reported that under identical conditions strains of S. carlsbergensis yield more diacetyl than strains of S. cerevisiae.
The effect of temperature on diacetyl formation is great: at 21°C diacetyl production twice that at 13°C, but at temperatures of 8°C it is negligible. Aeration affects diacetyl production positively; but the effect may well be secondary since aeration also affects yeast growth.
It is well known that yeast in suspension reduces diacetyl levels if it is kept in contact with the wort and if access of air is prevented. This is correct, even though much of this work is based on fermentations in which the reliability of the analytical methods is open to doubt. Yeast cells in suspension reduce diacetyl to acetoin and 2,3-butanedoil. When diacetyl at a concentration of 35 ppm was added to a yeast slurry and kept at 24°C for 24 hr and then at 0°C for 48 hr the level of diacetyl was reduced to 0.15 ppm, while 1.35 ppm of acetoin and 35.6 ppm of 2,3-butanediol were recovered. In a similar experiment addition of acetoin led to a gradual reduction of the compound and quantitative recovery of butanediol. Diacetyl may be removed from beer by filtering it through a diatomaceous earth filter impregnated with yeast cells or by the use of enzyme extracts from yeast cells. Bakers' yeast also appears to be suitable for the removal of diacetyl.
Practical suggestions for the production of beer with low diacetyl levels have been made by Lewis, Latimer et al., Brenner and others. Basically these are as follows: Conduct the fermentation at low temperatures; use a yeast strain which produces little diacetyl, and prevent contamination with wild yeasts or with bacteria of the genera Pediococcus andLactobacillus; provide adequate nutrition with wort nitrogen (particularly valine); avoid stimulation of yeast growth by aeration, and avoid access of air during the latter stages of fermentation and during the transfer of fermented wort to the lagering cellars. In recent years the problems relating to the mechanism of diacetyl formation have been reviewed extensively.
Actual diacetyl concentrations in the final beer vary greatly in some instances it is impossible to find any diacetyl with available analytical techniques. Thorne reported the presence of traces up to 2 ppm in lager beer; while Drews et al. found from 0.15 to 0.25 ppm. Harrison found about 0.05 ppm in top-fermented beer.
The formation of acetoin and 2,3-butanediol will not be discussed. Actual concentrations in beer vary widely but are generally well below flavor threshold concentrations. 2,3-Pentanedione is often determined together with diacetyl as "vicinal diketones." It may be formed according to a scheme similar to the formation of diacetyl, that is, by oxidative decarboxylation of a-acetohydroxybutyric acid.
Concentrations of acetaldehyde rarely exceed flavor threshold values, which are given as 25 ppm by Drews et al. and as 50 ppm by Harrison. Mandl found an average of 11.3 ppm (range 7.4 to 18.8) in light German lager beer, and 10.0 ppm (range 3.5 to 14.2) in light export beer. Fermentation temperature has some effect on acetaldehyde formation, and the effect of O2 tension is great. This has been well investigated with continuous fermentations. Cowland and Maule found 0.5 ppm acetaldehyde at an O2 tension of 0.05 mm Hg, and 11 ppm at 74 mm Hg. Similar observations have been made in batch fermentations. The production of acetaldehyde has been made in batch fermentations. The production of acetaldehyde has been studied more extensively in the wine industry where the customary addition of SO2 to musts stimulates acetaldehyde formation.
Ronkainen et al. as well as Harrison noted the presence of higher aldehydes, such as the C3, C4 and C5 aldehydes as well as furfuraldehyde. Palamand and Hardwick found that the addition of 12.5 ppm acetaldehyde to beer depressed the aroma and produced a sweet estery note.
Glycerol is produced as a regular by-product of alcoholic fermentation in amounts of about 3% based on the weight of the sugar fermented. On this basis one would expect to find about 0.2% glycerol in beer. Parker and Richardson found that most Canadian beers contain between 0.15 and 0.2% glycerol with a total range of from 0.11 to 0.25%. The threshold value for the detection of glycerol in beer was about 1%. Mandl found glycerol values in German beer in good agreement with those reported above. Nordstrom has discussed the mechanism of glycerol formation during yeast growth.
The pH of fermenting wort is lowered by the formation of CO2 and organic acids. Of the latter lactic, acetic, pyruvic, citric and malic acids predominate. The fatty acids are present in lower concentrations, but their flavor contribution may be considerable. Table 8 shows values reported for the acids of the tricarboxylic acid cycle and some fatty acids.
Palamand and Hardwick find that most of these acids at concentrations as low as 5 ppm affect beer flavor. All the fatty acids from C1 to C10 as well as some higher fatty acids have been identified in beer. The formation of some of these acids is related to amino acid uptake and amino acid synthesis, similar to that of the formation of the corresponding alcohols. Addition of L-valine to a synthetic medium increased the formation of isobutyric acid severalfold, and addition of L-leucine similarly increased the formation of isovaleric acid.
Unsaturated fatty acids present in wort have an O2-sparing action and affect yeast viability. Apparently, these acids are required by yeast and they are not synthesized under anaerobic conditions. This could account for the O2 -sparing effect. Addition of lipids containing unsaturated fatty acids also increase yeast viability.
About 46 ppm sulfur was present in Japanese lager beer. Of this amount 94% was in the form of nonvolatile compounds, and sulfite sulfur accounted for most of the remaining 6%. Very low concentrations of H2S and of thiocarbonyl compounds (thioformaldehyde, thioacetone, and dithioformaldehyde) were detected. The non-volatile fraction consisted largely of sulfate sulfur, inactive S-S-compounds, reducible -S-S-compounds and very low concentrations of -SH compounds. These compounds are derived from the wort where sulfates and sulfur-bearing amino acids constitute the bulk of the sulfur compounds. Hydrogen sulfide is present in wort but it is readily driven off during the kettle boil. A great part of this sulfide is probably formed by decomposition of sulfur-containing amino acids.
Some sulfur-containing volatile compounds are formed during fermentation (H2S, SO2 and thiol compounds). They are of great interest because of their potential effect on beer flavor. The taste threshold values of some of these compounds are extremely low. Harrison and Drews et al. reported the following values: H2S, 5 and 10 ppb; ethane thiol, 10 and 5 ppb; dimethyl sulfide, 60 and 35 ppb; and diethyl sulfide, 3 and 30 ppb, respectively. The concentrations of these compounds in beer are usually below the threshold values. Analytical detection of the compounds is difficult and distillation techniques may result in the artificial formation of sulfur compounds during the preparation of samples for analysis. In a recent review of sulfur compounds in beer, Lewis and Wildenradt noted that "confusion dominates the field," and ascribe this confusion to the minute concentrations of these sulfur compounds and to their instability. Ryan described the formation of mercaptans during fermentation but Hashimoto et al. were unable to find any mercaptans in Japanese beer. Lawrence reported the presence of tertiary butyl mercaptan (probably pre-existing in wort) but found no other thiols or thiocarbonyl compounds.
It is not surprising, therefore, that the role of sulfur compounds in beer flavor has not been determined with any precision. It is true that high concentrations of H2S or of the thiol compounds produce undesirable odors. However, it has not been definitely established whether or not minute concentrations of these compounds contribute a desirable flavor note; for instance, Thorne observed that H2S at concentrations of 5 ppb had a desirable effect on beer flavor, but at 50 ppb he obtained a definite "rotten egg" odor.
Lewis and Wildenradt have considered the pathways by which H2S can be formed. The sulfate of the wort is not a likely source of H2S if methionine or pantothenate is present. Cysteine, which is present in the wort or which is synthesized during fermentation, is more likely to be decomposed to H2S. Jansen observed an increase in H2S concentration during pasteurization of beer.
A careful analysis of volatile sulfur compounds in pale ale by Sinclair et al. showed the presence of 0.8 to 39.2 ppb H2S, of 0 to 10 ppb dimethylsulfide, and of 1 to 6 ppb volatile thiols. The total volatile sulfur was equal to the sum of dimethylsulfide and thiol sulfur, indicating good quantitative recovery during the analysis. The more volatile sulfur compounds are also swept out of beer by escaping CO2, and the level of H2S drops during the lagering period.
The compound is also formed in beer fermentations; in contrast to H2S its concentration is not lowered during the lagering period. Most of the SO2 is in bound form. The compound has received less attention in brewing than in wine making, because it it's not as widely used as an additive by breweries. Its concentration in beer is generally well below levels of 50 to 100 ppm, at which it can be readily recognized by taste. Values of 5 to 50 ppm have been reported by Thorne. Hashimoto et al., who studied its development during fermentation, report a final SO2 value of about 6 ppm in beer.
The so-called sunstruck flavor, a well-known beer defect, is due to the formation of 3-methyl-butene-1-thiol by reaction of a side chain of isohumolone with H2S or an -SH compound. The role of sulfur compounds in beer flavor and the formation of various sulfur compounds have been reviewed by several authors. Some of the findings reported have to be qualified by the analytical difficulties mentioned above.
The general scheme of the brewing process has been shown in Fig. 1. There is no need to dwell on the details of the process or to stress its engineering aspects. However, some of the aspects closely related to yeast performance will be considered.
Generation of Heat
It is not possible to make an accurate calculation of the amount of heat energy liberated during anaerobic fermentation. But an estimate may be made as follows: In the anaerobic fermentation of glucose to ethanol and CO2 the loss of free energy is 50 to 52 kcal per mole glucose. Since 2 moles of energy-rich phosphate bonds in the form of ATP, are formed per mole glucose fermented, an estimated 28 kcal will be conserved inside the yeast cell (assuming the DF of hydrolysis of the terminal phosphate group of ATP to be -14,000 cal per mole). Thus the difference (50 kcal minus 28 kcal = 22 kcal) represents the heat energy liberated per mole glucose fermented under anaerobic conditions.
Recently, the actual production of heat during the fermentation of brewers' wort was determined by Lejsek in a calorimeter. He found 135 kcal per kg of extract, which corresponds to 24.3 kcal per mole glucose if all the fermentable sugar is assumed to be in the form of glucose. For practical reasons one may assume a maximal fermentation of 1% of the extract per day for lager beer fermentations, and in that case 24.3 kcal/hl/day or 28.5 kcal/bbl/day would appear as heat of fermentation. In ale fermentations and especially in continuous fermentations the generation of heat per day would be greater depending on the faster rate of fermentation.
Production of Bacterial Extracellular Enzymes by Solid State Fermentation
The solid state fermentation (SSF) process is a technique in which moist, water-insoluble solid substrate is fermented by microorganisms in the absence of any free water. Although this technique has been in practice since ancient times for some fermented foods, cheese making and in composting, little progress has been made in this field as compared to submerged fermentation (SmF), technique. The SSF process has numerous advantages like superior productivity, simple technique, low capital investment, low energy requirement and less waste output, better product recovery, lack of foam build-up etc. over SmF. However, this process suffers from certain problems such as difficulties in scale up, metabolic heat generation and heat transfer limitations.
Bacterial strains belonging to the genus Bacillus are known to produce extracellular enzymes such as a-amylase, alkaline protease, xylanase and others in submerged fermentation. Some species of Bacillus are also reported to produce extracellular enzymes in SSF; this has been attributed to their ability to adhere to the substrate particles, to produce filamentous cells for penetration, and water activity requirements. In this investigation, an attempt has been made to find out the amenability of some Bacillus spp. to produce extracellular enzymes and to optimize culture parameters in SSF.
Materials and methods
Bacillus coagulans B49, B. licheniformis S 40, B. licheniformis A 99, B. circulans and Exiguobacterium aurantiacum were isolated from soil/compost samples, identified and maintained as described earlier.
Enzyme Production in SSF
Erlenmeyer flasks (250 mL) containing 10 grams of wheat bran (WB) and 25 mL salt solution (g/L, K2HPO4, 11.0; NaH2PO4. 6.1; KCL 3.0; MgSO4; 7H2O, 0.2; pH, 7.0) were autoclaved at 15 lb psi for 45 min, cooled and inoculated with 10% (v/w) 48 h-old inoculum and incubated at 50°C (37°C for B. circulans and E. aurantiacum) in an incubator. The incubator was humidified by means of sterile water. The flasks were periodically removed and the contents were mixed by gentle tapping. At the desired intervals, the flasks were taken out and the contents were extracted with buffer.
Bacillus coagulans was also grown in an double jacketed glass reactor (24 cm length, 3 cm inner diameter) filled with 30 g of moist wheat bran. Air was passed through a filter and fed into the column through a perforated aluminium cylinder fitted inside the column. Moisture level was maintained by injecting sterile water into the air inlet. Temperature (50°C) was maintained by circulating hot water using a thermo bath (TB-85, Shimadzu Corp. Japan).
Amylase Production vs. Incubation Period
Ten grams of WB was moistened with 25 mL of salt solution (g/L, (NH4) 2HPO4, 1.0; MgSO4, 7H2O, 0.5; K2HPO4 0.1; CaCl2, 0.1; FeSO4, 0.1; MnCl2, 0.1; pH 7.2) in 250 mL flasks, autoclaved, inoculated and incubated as described above. The contents of the flasks were harvested at 24 h intervals and assayed.
Effect of Moisture Level
The influence of moisture level on the enzyme titre was evaluated by varying the ratio of wheat bran to the selected salt solution (w/v; 1: 1.5, 1:2.0, 1: 2.5).
Effect of Various Additives
The effect of glucose, maltose, starch and mustard oil cake (1 and 2%) on enzyme titre was studies by supplementing wheat bran with these substrates. In another experiment, mustard oil cake, maize bran with these substrate. In another experiment, mustard oil cake, maize bran, gram bran and cassava (tapioca) starch were used instead of whet bran.
Solid State Cultivation in Trays
Enamel coated metallic trays (23 × 18 × 4 cm) with 40 and 80 grams of wheat bran, and trays (30 × 26 × 5 cm) with or without aluminium mesh bottom containing 300 grams wheat bran and salt solution were covered with aluminium foil, autoclaved, cooled, inoculated and incubated.
The dextrinizing activity of a-amylase was assayed by the starch iodine method at 70°C. Alkaline protease was assayed by caseinolysis (9) at 60°C and pH 10, and xylanolysis at 60°C (B. licheniformis A 99)/40°C (B. circulans) and pH 9.0.
One dextrinizing amylase unit is defined as the amount that leads to 10% reduction in the intensity of the blue colour of starch iodine complex in 10 min. One unit of alkaline protease and xylanase are defined as the amount required for liberating one micromole of tyrosine/zylose per ml under the assay conditions.
All experiments were conducted in triplicate and the average of the three are represented as number of units of enzyme produced per gram of dry bacterial bran.
Table 1. Production of extracellular enzymes by solid state fermentation wheat bran
Organism Enzyme Units g-1 dry Improvement
bacterial bran over submerged
B. licheniformis Alkaline protease 3.5 17
B. licheniformis Cellulase free 2050 250
A 99 xylanase
B. Circulans Alkaline xylanase 97 50
Exiguobacterium Alkaline a-amylase 598 -
Bacillus coagulans a-amylase 27000 38
Results and discussion
In contrast with the general belief that the SSF technique is not suitable for bacterial cultivation because of the their requirement for higher water activity. Bacillus coagulans B49, B. licheniformis S 40, B. licheniformis A 99, B. circulans and E. aurantiacum could be cultivated in SSF where they excreted extracellular enzymes. The enzyme titres produced in SSF were higher than those in SmF. Similar observations were made by other workers.
The production of amylase was high in wheat bran and maize bran as compared to that in mustard oil cake, tapioca and gram bran. Wheat bran was found to be the best substrate and suitable for necessary manipulation.
All the strains produced high titres of enzymes in 72 h, as reported by others, high enzyme titres were attained when the initial moisture level was 1: 2.5 (w/v) in comparison with that at low levels. The critical importance of moisture level in SSF media and its influence on the biosynthesis and secretion of enzymes can be attributed to the interference of moisture in the physical properties of the solid particles. The higher moisture level decreases porosity, change in wheat bran particle structure, development of stickiness, reduction in gas volume and decreased diffusion that results in lowered oxygen transfer. While lower moisture content causes reduction in the solubility of nutrients of the solid substrate, lower degree of swelling and higher water retention.
Table 2. Solid state fermentation in trays
Amount of wheat bran (g)
Enzyme (organism) 10** 50 100 200 300
Enzyme production (U/g DBB)
a-amylase 24208 22100 ND ND 16007
Alkaline protease 3.0 2.2 3.1 ND ND
Xylanase 1950 1618 1635 1546 1500
** In flask
ND Not detrmined.
Alkaline protease production by B. licheniformis S 40 was greater at 10% inoculum level, while the titre of cellulase-free xylanase of B. licheniformis A 99 was relatively high at 15% inoculum level, as compared to that at low or high inoculum levels. When the bacterial strains were cultivated in trays with more amount of substrate than that in flasks, the titre remained unaffected or slightly declines. With improved aeration, it could be possible to maintain the same enzyme titres in flasks as well as in trays. In the glass reactor, a-amylase production was slightly higher than that in flasks.
This investigation clearly indicated that SSF is suitable not only for fungal cultivation but also for the cultivation of some bacterial strains.