Enzymes in Brewing MORRIS A. POZEN The Brewing Research Laboratories, New York, N. Y.
This discussion is confined to the more important functions performed by enzymes in the various phases of the brewing process, rather than to a consideration of the mechanics of enzyme action. I t is made clear that the quality of the .finished beer depends not merely on the use of sound materials but more especially on. the careful regulation of the several series of enzyme actions involved in its production.
T
WO groups of enzymes, which are not active simultane-
ously, are chiefly responsible for the reactions essential to brewing. The first enzyme system is derived from the malt, is active during malting and mashing operations, and is completely inactivated when the wort is boiled with hops in the brew kettle. The second group of enzymes is introduced when the yeast is added to the cooled wort in the fermenters, and is active during fermentation, storage, and maturing of the beer. Successful brewing is largely dependent on regulated enzyme actions. Even newspaper and radio advertisements proclaim this to the public. Enzyme activity is responsible for the production of those substances which give to beers and ales the characteristic properties distinguishing them from all other beverages. Regulation of enzyme actions is necessary to produce a properly balanced wort, by which the type of beer is chiefly determined; to insure a nitrogen distribution delicately proportioned between yeast nutrients on the one hand and foam-producing and palate-active protein derivatives on the other; to obtain a suitable ratio of fermentable sugars to nonsugars in the wort; to assure effective filtration and clarification; to produce a clean fermentation; to assist the maturing process; and finally to stabilize the finished product against the adverse conditions it must encounter in commercial handling. In a word, the activities of enzymes are controlling factors a t practically every stage of the brewing process.
Courtesy, Brewery Age
CEREAL COOKERS (ABOVE)m MASHTuss (BELOW) IN A BREWHOUSE
grains, brewers’ malt is customarily produced from selected varieties of barley. The malting process is intended to simulate the changes occurring during normal germination; the process is controlled in such a way as to secure a maximum production of enzymes, a minimum loss of substance, and a suitable modification of the physical and chemical structure of the grain, so that extraction of soluble constituents during mashing operations will be facilitated. Malt is made by a regulated artificial germination of barley, followed by slow drying a t low temperatures, and terminated by kilning. The latter phase assists in the development of color, aroma, and flavor. During the germination there is a marked secretion of enzymes by the embryo in order to enable it to utilize the reserve materials by converting them into soluble substances. Some of the important enzymes involved are cytase, amylase, protease, peptidase, oxidase, and phytase. The oxidase functions chiefly as an activator in connection with the respiration of the growing plant. The cytase exercises an important action by attacking the walls of the starch-containing cells, thus making it possible for the amylase to permeate the walls and to have access to the starch grains. The amylase MALTING is then able to catalyze the conversion of some of the starch The role of enzymes in the production of a given brew to maltose and dextrins. The proteolytic enzymes hydrolyze often begins months before the actual brewing of the beer, a portion of the complex insoluble proteins into simpler soluand a t a place perhaps thousands of miles distant from the ble derivatives. Phytase liberates phosphorus in inorganic brewery in which the beer is produced. This follows be- form by hydrolysis of phytin. This is of value during fercause malt, the chief raw material in brewing, aside from mentation of the wort. water, is customarily aged for varying periods before use, and Pectase has also been found in malt and it may be of also because, with the exception of some of the largest brew- greater significance than is now appreciated because of the eries, most brewers do not produce their own malt but pur- possibility that one of the factors involved in the instability chase it from maltsters. of beers is the presence of small amounts of pectinous subWhile malt may be made from a number of different cereal stances. 1127
1128
I N I > U S T R I A L A N D E N G I N E E I< I N ti C 1%E M IS T 1%Y
It has been reported (2,15)tliat malt contains sisto-amy1:rac which iiiactivab amylase (the amylolytic fraction less than the dextrinizing and liquefying fractions) presumably by adsorbing the enzyme in insoluble form. This anti-enzyrrie is less potent in well-kilned malts than in malts cured a t low temperatures. The loss in amylolytic activity due to tlie cfrects of high kilning temperature is partially compensated by a similar reduction in tlie activity of sisto-amylase. Malt also c o n t a i n s peptones and other protein d e r i v a t i v e s which counteract the effects of sisto-ainylase both in the malting process and in tlie mash tub. AS t e r germination os t h e b a r l e y lias proceeded to the desired extent, the drying process is begun and is first conducted a t low temperatures u n t i l m o s t of t h e moisture has been removed. F i n a l l v . dependingon the color d e s i r e d in t h e dry malt, is resorted to; t h i s kilning termin a b the germination, reduces the moisture content still farther, rendering the malt more stable and resistant to the activities of microorganisms duringstorage, makes the physical condition of tlie malt more suitable for grinding, and develops color, aroma, and flavor. It is interesting to observe some of the effects of kilning temperatures on the malt enzymes and their action. Tliere is evidence (19) that some reversals of enzyme action, involving both carboliydretes and proteins, occur. Among the carbohydrates there is a part,ial reversion of invcrt sugar to sucrose and of fermentable sugars to dextrins. Some of the simpler protein derivatives revert to true coagulable proteins. There is likewise some destrnction of enzymes. The cytase is t.otally inactivated, the proteolytic enzymes are less seriously affected, while the amylase, especially the amylolytic fraction, appears tu withstand the kilning temperatures most successfully. As a result, a good brewers’ malt is capable of converting not only its own starch, but also several times its own weight of gelatinized starch derived from other sources, to sugars and dextrins under suitable conditions. This property is especially valuable in American brewing practice vhich makes fairly general use oi from 20 to 35 per cent of malt adjnnct8 such as raw or unmalted cereals, containing large proportions of starch. MAsarNc;
h malt is usually produced by comniercial mmltsters, the first step in the brewing process actually practiced in most breweries is mashing. This is essentially an aqueous digestion of the cruslied malt plus the precooked malt adjunct under controlled conditions of time, of temperature, and frequently of pH. The objects of the mashing operations Itre to continue the hydrolytic solubilizing enzyinc actions , to extrnot, the sduble b e y n during the malting pro products, and to secure a I,alaneed wort siiitalk Sor prodocing the type of beer desired. Tlie master brewer also aims to secure the maximum possible yield of extractives from Iiis
Vol. 26,No. 11
Tlie accomplishmcnt of these objectives is dependent on regulated enzyme actions. Tile mashing operation is conducted in the mash tub, a cylindrical vessel made of copper, of other metals, or occasionally of wood. The mash tub is provided with stirrers or rakes, liot and cold water arid stearn supplies, temperatureregulating devices and recording tliermometers. Most mash tobs iiirve a nietal slotted false bottom which acts as a sumort for the g r a i n s - a n d permits the wort to flow t h r o u g h from above ur water tu enter from below. T h e preference of tlie American yublic for l i g h t , very pale, b r i l l i a n t beers has led to the developmeiit of a peculiarly American t y p e of beer. This, in turn, has affected themashi n g operations. It was f o u n d that allmalt beors made from American malt are t o o satiating. This is due largely to the high protein content of American m a l t s which yield beers containing high proportions of soluble protein derivatives. This high content of nitrogen compounds causes the beer, in the language of the brewmaster, to “lie heavy on the stomach.” Another disadvantage is that a high-protein beer is more likely to contain undesirable proteins which can affect its stability. For these reasons most American beers are produced from mixtures of 65 to 80 per cent of malt with 20 to 35 per cent of malt adjuncts, which may consist of unmalted cereals such as corn grits, corn meal, refined grits, or rice; various partially precooked refined cereals, such as corn flakes; and finally, various sugars and sirups. The use of these materials riot only reduces the protein content of the wort but makes possible tlie production of paler beers and ales. By the judicious choice and use of malt adjuncts i t is also possible to develop distinctive types of brews. Assuming tliat the brewinaster lias decided on the type of beer he is to produce, the character of the malt, the nature and proportion of the malt adjunct, the alcoholic content, and the extract content of his finished beer, Consideration is then given to tlie conversion temperature. This is the temperature at which the mash will be lield to permit the amylase to prodnce fermentable sugars and dextrins. While other enzymic actions will proceed ab this temperature, the brewniastcr thinks only in ternis OS sugar and dextrin production in deciding on Iris conversion temperature. The malt amylase is most active in the temperature range 60” to 66” C., but, when unmalted cereals are used as malt adjuncts, the mashing operation is begun a t a much lower t.emperature. This is becaiise the raw eerezls must first be boiled with water in a cooker to gelatinize the starch before they are added to tho mash. Tlic temperature of the main mash is so regulated that, when the cooker mash is added, it will not raise the temperature of the niixture above the desired conversion temperature, n u t even when prepared cereals which can be added directly tu the mash tub are employed, most American brewmasters prefer to start the mashing a t lower temraw materials.
INDUSTRIAL AND ENGINEERING
November, 1934
peratures of from 45" to 50" C. This range of temperature is usually referred to as the "peptonization" stage, as most of the proteolysis is assumed to occur a t this point. The effect of temperature in actual brewery mashing operations on the nature of the amylolytic action is as follows (ir): CONVXIRBION
TEMP.
c.
RATIOOF SUQAR TO NONBUQAR
66 64
l : o 31 1:0.40
68
1:0.48 1:0.52
io 72
1:0.57
CHEMISTRY
1129
mashing time generally increased the production of colloidal nitrogen. When the mashing time was kept constant, the optimum production of the various nitrogen fractions occurred essentially as follows: colloidal nitrogen fraction at 60" to 65" C.; magnesium-sulfate-precipitable fraction a t 70°; tannin-precipitable fraction a t 60" to 70". There is evidence of the reversion of simpler protein derivatives a t 70" C. Most brewery control in connection with proteolysis, for obvious reasons, makes use of less detailed separation of the fractions. A fairly accurate picture may be had by consideration of the total nitrogen, tannin-precipitable, coagulable, and formaldehyde-titratable nitrogen. With the aid of such control methods the mashing procedure may be modified to give the nitrogen distribution found to be desirable in practice, I n addition to time and temperature the pH value of the mash is an important factor in regulating enzyme actions in the mash tub. It has been shown (8) that there are certain definite p H ranges which are optimal for various enzyme functions in the mash. Some of these are as follows:
It is thus apparent that by proper attention to his conversion temperature the brewmaster can control the diastatic action and can secure in the wort a predetermined ratio of fermentable sugars to dextrins. For low-alcohol beers high in residual extract, he employs a high conversion temperature; for high-alcohol beers relatively low in unfermentable extract, a low conversion temperature is used. The behavior of malt amylase in the mash tub indicates that the dextrinizing and liquefying fractions, which may be identical FUNCTION PH IN MABH (fl),are more resistant to high temperatures than the sacMax. yield of extract 5.1-5.2 charogenic fraction. The brewmaster follows the course of Max. sugar formation 5.0-5.5 Max. total nitrogen 5.1-5.2 the amylase activity with iodine test solution. hlax. amino nitrogen 4.5-5.0 5.4 The master brewer is also interested in the nitrogen disLiberation of phosphatee by phoaphatases tribution of his wort because of its important bearing on The p H of the mash is greatly influenced by the mineral certain essential properties of the beer. This depends largely on the activities of the protease and peptidase during mash- salts in the malt, chiefly acid phosphates; with a neutral ing. The aim is to produce and extract enough soluble pro- mash water these yield a slightly acid mash, the p H value tein derivatives to give body and character to the beer and to varying from 5.1 to 6, or slightly higher. The value is usuregulate the proteolytic action so that enough nitrogen deriva- ally nearer the upper limit. Other buffers in the malt are tives of low molecular weight are formed to serve as yeast significant factors. The composition of the brewing water nutrients during the fermentation stage. At the same time is also important. I n general, alkaline carbonate waters are there must be produced a suffiundesirable as they tend to prevent the mash from attaining a pH value cient proportion of protein cleavage within t h e r a n g e f o r o p t i m u m p r o d u c t s of h i g h e r m o l e c u l a r weight t o i n s u r e t h e d e s i r a b l e enzyme action. Worts made with palatefullness or Vollmundigkeit, such waters may show a reduced and to assist in foam production yield, unsatisfactory clarification, and foam stabilization. The brewpoor filtration, and instability in master who is oversolicitous about the finished beer. On the other the well-being of his yeast in perhand, waters containing calcium mitting excessive protein cleavage and magnesium sulfates and chloduring mashing may displease his rides usually exert a favorable effect customers who will find the beer on the pH by reacting with phosmade from such a wort lacking in phate buffers. Such w a t e r s are body and without that rich creamy prized for brewing purposes. Some head which they expect in a good of the most famous brews are made beer. The degree of dispersion of with waters naturally rich in the the colloidal protein particles is an permanent-hardness type of salts. important factor in foam producSuch waters have been simulated tion (6),a medium range appearby the addition of so-called BurCourtesy, Nathan Institute, Inc. i n g t o be o p t i m a l . P e r h a p s t o n i z i n g s a l t s . The beneficial Alexander's concept of a zone of effects of this treatment are due to PUREYEASTCULTUREPLANT maximum colloidality ( 1 ) applies Unite are &sa-lined and provided with jackets permitting the effect on the pH value rather heating or refrigeration. here. I n any event, excessive prothan to the inherent mineral charteolysis must be avoided. acter of the added materials It Kolbach and Buse (9) studied the effects of temperature has also been suggested (16) that the effects of using different and time on proteolysis during mashing. The analytical brewing waters may be due in part to variations in their procedure included determination of colloidal nitrogen (ad- concentration of heavy water. The first methods used to adjust the p H of the mash were sorbed with active carbon, about 50 to 60 per cent of the total nitrogen) ; formaldehyde-titratable nitrogen; and ni- of biological origin and consisted of inoculating the mash or trogen precipitated by magnesium sulfate (chiefly true pro- wort with a vigorous culture of lactic acid bacilli or in adding teins of high molecular weight), by tannin (about 25 per cent a biologically acidified wort. I n present day practice it is of the total permanently soluble nitrogen, including coagu- more common and more convenient to decarbonate the mash lable nitrogen, albumose nitrogen, and peptone nitrogen), water or to add lactic or sulfuric acid to the water. A special and by uranyl acetate (true proteins of medium and high malt has been developed which has been allowed to undergo molecular weight). At a given temperature increasing the a controlled lactic acid fermentation before drying and
1130
I
N DUSTK IAL
A N D E N G I N E E !I I N G C If E M I S T K Y
kilning. Tliedry malt contains about 2 per cent of lactic acid arid is used in the mash to the extent of 5 per cent of the total grains. Adjustoicnt of the pII has shown in actual brewery operations an increased yield of extract as high as 3 per cent in some instances, about two-thirds of the increase being siixars and dextrins, and one-third being protein derivatives.
Vol. 26, No. 11
a separate settling tank (lauter tub) is used, or a mash filter may be employed. Tlus procedure increases the capacity of the mash tub and the vield of extractives. The usual ery yield sometimes closely approaches the laboratory yield of 70 per cent or more. won^ BOILING
The boiling of the wort with hops is conducted in kettlev usually made of copper. The chief objectives of boiling the wort are to concentrate it, since it bas been diluted by the spargings; to inactivate all enzymes; to render the wort sterile; to extract soluble substances such as tannins, resins, and aromatic and hitter principles from the hops; to obtain a good separation of coagulable proteins; and to caramelize the sugar slightly (color development). The inactivation of the malt enzymes assures a wort of constant sugar-dextrin ratio for the fermentation, as yeast contains no amvlase. A sterile wort eliminates the Dossibility of undesir&e enzyme actions from foreign orga&m. The hop tannin facilitates more complete coagulation and reinoval of unstable proteins from the wort-an important consideration. Other hou extractives exert an antiseutic action against the activities of some organisms which may infect the beer at a later stage. The bitter and aromatic principles of hops contribute to beer character, while humulone also exerts a beneficial effect on head retention. The master brewer judges the effectiveness of his kettle operations lararely by the character of the "hot break"-that is, by the co&uiatibn of proteins in relatively large particles which settle readily and by the effective clarification of the wort which should be brilliant. After boiling, the wort is strained and the clear wort is passed through or over coolers. During thi., operation more unstable proteins are precipitated and the wort is Grated. In modem brewing practice, attempts are made to conduct the cooling operation in such a way as to protect the wort from infection and to bring it to the fermenters in sterile condition. I n fact, the modern trend is to maintain aseptic conditions in all stages subsequent to the kettle operations.
FSRMENTATION Clarilication and filtration were facilitated. These results are attributed to the beneficial effect of the pH adjustment on the activities of the malt enzymes. With the development of refinements in temperaturerecording instruments, temperature control, and plant methods for measnring pH values, the master brewer and the brewing technologist find it possible to secure closer regulation of enzyme actions during mashing operations. This has resulted in more accwate control over the composition of the wort. The last stage of the mashing process comprises a rest period during which the grains and the coagulated proteins settle out, while enzyme actions continue. The aqueous extract of the grains (wort) is then permitted to flow down through the layer of grains which acts as a filter bed and to run off through the slotted false bottom of the mmh tub. With a properly conducted mash, the wort filters rapidly and is perfectly clear. If enzyme actions during mashing have been improperly regulated, the wort may cause difficulties in filtration or may be turbid. The elear wort is pumped into the kettle, while the spent grains are extracted several times with hot rider (sparging) in order to recover additional extractives, the spargings being added to the kettle. Well-extracted grains usually contain less than 1 per cent of residual soluble extract. In some mashing methods the malt is ground more finely,
The cooled wort at a temperature of 6" to IO" C. is run into fermenters,which may be of the closed or open type, and are constructed of steel, lined with glass or other protective eoatinm. wood. of aluminum. or of other suit- , of Ditch-tined . able material. The fermentera are located in refrigerated. insulated rooms or cellam in which strict temperature control is maintained. In the Nathan brewing system the fermenter rnom is not cooled, hut each fermenter is individually refrigerated. To the cooled wort, yeast is added in the proportion of about 1 to 1.5 pounds of thick yeast per 31-gallon barrel. In some breweries the yeast is propagated in a pure culture apparatus, usually being derived from a single cell. In most breweries the yeast is of pure culture origin but has been propagated in actual brewery fermentations. In the ease of lager beers the yeast added is a bottomfermenting yeast, so called hecause it tends to settle to the bottom of the fermenter toward the end of fermentation. The fermentation temperatures range from 6" to 12" C., and the time of the principal fermentation is usually from 8 to 10 days. Ales and stouts are fermented with a topfermenting yeast which rises to the top of the fermenting liquid and is recovered by skimming. Top fermentations are conducted a t higher temperatures, 14" to 23' C., and the time of the principal fermentation is from 5 to 7 days. The characteristics which distinguish beers from ales are
November, 1934
INDUSTRIAL AND ENGINEERING CHEMISTRY
partly due to qualitative and quantitative differences in enzyme actions induced by the bottom- and top-fermenting yeasts, respectively. The introduction of the yeast brings into play the second group of enzymes which function during succeeding phases of the brewing process. Among the yeast enzymes are : zymase, sucrase, maltase, melibiase, carboxylase, catalase, oxidase, reductase, endotryptase, proteases, phosphatase, and others. The activity of zymase has been thoroughly investigated and will be discussed first, although from the standpoint of beer or ale character i t is questionable whether alcohol is the most important fermentation product. It may be pointed out that many of our citizens, conducting numerous “noble” experiments in recent years, found no difficulty in producing alcohol, but very few of the experimenters succeeded in obtaining beer as a n end product. The unfermentable extract, some of the by-products or secondary products of fermentation, and the changes resulting during maturing (largely the result of regulated enzyme actions) play important roles in contributing to beer character and quality. The alcohol is derived from the fermentable sugars present in the wort, which may include sucrose, maltose, dextrose, and levulose. It is assumed that the disaccharides are first hydrolyzed to hexoses by the enzymes sucrase and maltase, respectively. Recent work (18) indicates, however, that the fermentation of maltose can proceed without preliminary hydrolysis to hexose. I n any event, if hydrolysis of maltose is a prerequisite to fermentation, yeast provides the enzyme maltase to catalyze this reaction. The hexoses, including dextrose and levulose, are fermentable by zymase, levulose being fermented most rapidly. It is also believed (7) that levulose is the only sugar which is directly fermentable and that other hexoses, by the action of some unknown enzyme, are first converted to levulose before they are fermented, through a chain of reactions, to alcohol and carbon dioxide. There is evidence that zymase is itself a mixture of several enzymes, including a carboxylase and an enzyme responsible for oxidation-reduction reactions which are part of the fermentation process. Glutathione, which has been found in yeast, is believed to perform the latter function. The mechanics of alcoholic fermentation is a fertile field which has attracted many prominent investigators. The most recent theory on carbohydrate decomposition is due chiefly to Embden (3)and Meyerhof (IS, 14). Harden (6) has summarized this work and its significance. The following paragraphs are based largely on his r6sum6. I n the latest previous theory i t was assumed that the stages of fermentation proceeded in the sequence: Hexose + organic phosphate + hexose monophosphate +hexose diphosphate + methylglyoxal + pyruvic acid +aldehyde + carbon dioxide Methyl glyoxal + aldehyde +pyruvic acid + ethanol
1131
The following phase is the same as in the previous theory, carboxylase inducing the decomposition of the pyruvic acid into acetaldehyde and carbon dioxide: CHaC0,COOH
+ CHs.COH
+ COz
The means by which the acetaldehyde is assumed to be reduced to ethanol is also new. The concept of a n oxidationreduction reaction is again utilized but, in place of methylglyoxal, a triose phosphoric aldehyde, probably phosphoglyceraldehyde, is the compound oxidized :
CHZ(PO4Hz).CHOH.COH-o x i d a t i o n . + ~ ~ 2 ( ~ ~ , ~ z ) . ~ ~ CHsCOH reduction w CHsCHzOH The phosphoglyceric acid, which yields pyruvic acid, results from this reaction. As long as phosphoglyceraldehyde is furnished, the fermentation continues. This supply is derived from an enzymic reaction of dextrose and phosphate: 1 mole dextrose 2 moles phosphate +2 moles phosphoglyceraldehyde
+
The chain of reactions, once started, proceeds as long as sugar and phosphates are present, in the sequence:
-
Acetaldehyde reduction+ ethanol oxidation Phosphoglyceraldehyde phosphoglyceric acid + carbon dioxide + ethanol pyruvic acid + aldehyde ~
+
A trace of hexose diphosphate must be present as catalyst; otherwise the reaction ceases. Meyerhof refers to this phase as the stationary condition. Prior to this, an initial stage is assumed in which slightly different reactions produce the required concentrations of the reactants. I n this phase hexose diphosphate must be present and is actually consumed in the reaction: 1 hexose diphosphate 1 dextrose 2 phosphoric acid + 4 phosphoglyceraldehyde
+
+
Since no acetaldehyde has yet been produced in the first phase, i t cannot aid in the oxidation-reduction of the phosphoglyceraldehyde. Its place is taken by a second molecule of phosphoglyceraldehyde, one molecule being oxidized to phosphoglyceric acid and the other reduced to glycerophosphoric acid: 2CHz(PO,HZ).CHOH.COH +Hzo+ CH~(P04Hz)CHOH~COOH (phosphoglyceric acid) + CHa(PO4Hz).CHOHCH?OH (glycerophosphoric acid) When enough acetaldehyde has been formed, the reduction of the phosphoglyceraldehyde practically ceases. The slight amount of glycerophosphoric acid produced may be responsible for the small proportions of glycerol formed in alcoholic fermentations. Meyerhof summarizes the new fermentation theory as follows:
It was believed that methylglyoxal (CH,COCOH), one of the first decomposition products of sugars, passed by oxidation-reduction to pyruvic acid, which under the influence INITIAL PHASE of carboxylase was converted into carbon dioxide and acet1 dextrose 1 hexose diphosphoric acid 2 phosphoric acid aldehyde; the latter was reduced to ethanol in a reaction during which methylglyoxal was simultaneously oxidized +4 triose phosphoric acid -+ 2 glycerophosphoric acid 2 phosphoglyceric acid (1) t o pyruvic acid. 2 phosphoglyceric acid +2 pyruvic acid 2 hosphoric acid In the latest, theory the methylglyoxal phase is eliminated, 2 carbon dioxide 2 pEosphoric acid and it is assumed that p p ~ v i cacid, definitely established as +2 acetaldehyde (2) present at one stage of the chain of reactions, results from an enzymatic hydrolysis of phosphoglyceric acid (CH2(P04H2).STATIONARY CONDITION CHOHCOOH) . This compound was recently discovered 1 dextrose 2 acetaldehyde 2 phosphoric acid +2 triose by Embden ( 3 ) among the products of the action of yeast phosphoric acid 2 acetaldehyde +2 ethanol 2 phosphoand of muscle on carbohydrates. This reaction is repre- glyceric acid (3) sented by the equation: Reactions 1 and 2 represent the earlier stages of the ferCHz(PO4”2).CHOH.COOH+CHsCOCOOH H,PO, mentation. Reactions 2 and 3 represent the steady continua-
+
+
++
+
+
+
+
+
+
+
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INDUSTRIAL AND ENGINEERING CHEMISTRY
Vol. 26, No. 11
Esters: ethyl acetate, amyl acetate, amyl caproate. tion of the process. Reaction 3 requires hexose diphosphate Sulfur compounds: hydrogen sulfide, ethyl mercaptan (1). as catalyst. Nitrogen compounds: ammonia, trimethylamine. It is evident. whichever theorv we acceDt. that the alcoholic fermentation involves a chain of enzyme-catalyzed reMany of these compounds are volatilized and carried out actions. The fermentation is not part of the normal nutri- of the beer by the escaping carbon dioxide during fermentation of the yeast; but since yeasts are more resistant to alco- tion or are altered during the maturing process. That the aging process is a t least partially dependent on hol than many other organisms, i t is possible that the production of alcohol is the operation of a defense mechanism enzyme action is indicated bv the Dractical exDerience of against competing organisms. some brewers who find that maturing is accelerated by the addition I n addition to the fermentation phenomena, the nutrition and reof commercial enzyme preparations p r o d u c t i o n of the yeast involve to the young beer. The inventor enzyme actions. Protein derivaof a process which adds cell-free t i v e s , chiefly a m i n o a c i d s a n d yeast juice (rich in enzymes) to beer peptones, are assimilated by the claims t h a t t h e beer is ripened yeast from the wort, while nitrogerapidly ( I d ) . The Kathan system nous yeast derivatives are diffused of brewing ages beer in days ininto the fermenting wort. Mineral stead of weeks by k e e p i n g t h e young beer in constant motion for constituents are also taken up from s e v e r a l d a y s b y m e a n s of a solution. Most of the reactions insteady stream of purified carbon volved are enzymatic. It will be noted that in the ferdioxide. After the proper aging period, the mentation phase the master brewer beer may be r e m o v e d to special again makes use of the factors of finishing c e l l a r s , a l t h o u g h this time and temperature to regulate operation is more often conducted enzyme actions. Equally important, perhaps, is the use of a pure in the same cellar, where i t is permitted to stand under carbon dioxculture yeast which is more likely to show uniform qualitative and ide pressure, the gas being frequently recovered and purified ferquantitative enzyme a c t i vi t y a t Courtesy, H o f m a n Beverage Company m e n t a t i o n g a s , until the beer the same temperature and in the and Nathan Institute. Inc. “fixes” some of the gas and diss a m e medium. The precautions A MODERN AMERICAN BREWERY solves some of the balance. This taken to prevent i n f e c t i o n with is essential to good foam Droducforeign organisms also minimizes the possibility of unregulated and undesirable enzyme ac- tion and head retention. The beer is ;hen filterei under tions. I n some instances adjustment, of the pH is also prac- pressure and filled into trade packages (barrels), or goes ticed, although culture yeasts usually soon bring the wort to to the bottling department. The bottled beer is usually pasteurized a t temperatures of 60” to 65” C. for periods of 20 a favorable p H 4 . 2 to 4.5 (4). At the completion of the principal fermentation, the beer is to 30 minutes. pumped from the yeast sediment into storage tanks. The prompt separation of the young beer from the yeast after CHILLPROOFING fermentation also regulates enzyme action. Excessive conThe preference of the American public for ice-cold bottled tact of the beer with yeast results in undesirable changes, beer which must remain brilliant has led to considerable diflargely enzymic. ficulties. While pasteurization of bottled beer eliminates STORAGE AND MATURING most forms of microbiological spoilage, pasteurized beer is The beer is stored for periods varying from about 4 weeks more susceptible to cold, becoming hazy or turbid. The to several months. The temperature of the storage cellars same general instability was observed in bottled beers stored is maintained as close to 0 ” C. as practicable in order to fa- for some time a t ordinary temperatures or shipped over concilitate further precipitation of unstable proteins, resins, siderable distances. The cause of this behavior was generally and yeast, and also to inhibit the growth of any foreign or- believed to be the instability of certain of the proteins presganisms which may be present. I n addition to clarification, ent in the beer. In order to produce chill proof stable beers, brewers atslow reactions occur a t the prevailing low temperatures, resulting in the gradual disappearance of the relatively harsh tempted to remove undesirable proteins by prolonged storage character of young beer and the development of mellowness a t freezing temperatures, by the use of adsorbents and clarior maturity. There is some further alcoholic fermentation fying agents, by sharp filtration, by addition of chemical (secondary fermentation). The removal of the beer from protein precipitants, or by combinations of these methods. the main deposit of yeast and the maintenance of near-freez- Unfortunately none of these attempts was successful, as the ing temperatures during storage tend to regulate enzyme ac- stability was only slightly improved; in most instances, tions, which are a t least partly responsible for the maturing substances essential to beer quality were removed. The phenomena, and to prevent overripening of the beer. Among problem was finally solved by Wallerstein (20) who found the secondary or by-products of fermentation, derived from that a very small proportion of a proteolytic enzyme active sugars, proteins, and the yeast, some of which contribute to in slightly acid medium, added to the beer a t any stage of the brewing process after the boiling of the wort, rendered beer character, are (IO): the beer stable and chill-proof. As little as 1 gram of enzyme Alcohols: propyl, isopropyl, butyl, amyl, isoamyl, higher to 1 barrel of beer (about 1 part in 117,000) is sufficient for -~ alcohols, glycerol; this purpose. The enzyme appears to survive the pasteurizAldehydes: acetaldehyde, furfural. Acids: acetic. formic,. propionic. butyric, caproic, succinic, ing process, and its presence has been detected in pasteurized - bottled beers months later. The proteolytic enzyme process lmtic, sulfurous. ‘
I
November, 1934
I IC’ D U S T R I A L A N D E N G I N E E R 1 N G C H E M I S T R Y
for chill-proofing beer, which has been used successfully for many years in this country and in many others, is probably the outstanding American contribution to brewing technology.
“VITAMINIZED” BEER Some of the raw materials of brewing contain vitamins, and brewer’s yeast is an excellent source of vitamins B and G, but the finished filtered beer contains no nutritionally significant proportion of any vitamin. With the present fad for reenforcing various foods by the addition of vitamins or by irradiation, it is not surprising that processes have been proposed for “vitaminizing” beer. One of these processes (12) utilizes the cell sap separated from yeast by supercentrifugalization or by other methods. The inventor of this method says that the cell-free liquid contains 25 to 30 per cent of soluble yeast proteins, the vitamins and the enzymes. He proposes the addition of definite proportions of this cell sap to the beer after the main fermentation. The inventor asserts that the vitamins and enzymes mix with the beer and pass through the filter. It is quite likely that this treatment will add vitamins to the beer, but the matter of controlling the vitamin content in practice must be considered. Of still greater significance is the simultaneous addition of yeast proteins which may include unstable compounds, and of a high concentration of yeast enzymes. How the action of this battery of enzymes is to be regulated is not revealed. The importance of regulating their action in order to prevent undesirable changes in the beer constitumts is self-evident.
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ACKNOWLEDGMENT The author gratefully acknowledges his indebtedness to John Koenig for suggestions, criticism, and advice. LITERATURE CITED (1) Alexander, Jerome, J . Am. Chem. Soc., 43, 434 (1920); KoZZoidZ.. 36 334 11928). (2) Chrsaszcz, T., and Janicki, J., Biochem. Z., 263, 250 (1933); 264, 192 (1933). (3) Embden, Secaho, Deuticke, and Kraft, Klin. Wochschr., 12, 213 (1933). (4) Emslander, F., 2. ges. Brauw., 42, 127 (1919). (5) Geys, Wochschr. Brau., 44, 145 (1927). (6) Harden, d.,J. Inst. Brewing,39, 644-6 (1933). (7) Henrici, A. T., “Yeasts, Molds and Actinomycetes,” p. 216, John Wiley & Sons., New York, 1930. (8) Hopldns, R. L., J.Inst. Brewing, 31, 399 (1925). (9) Kolbach, P., and Buse, R., Wochschr. Brau., 50, 265, 273, 281 (1933). (10) Luers. H.. and Leiss. F.. Ibid.. 50. 373. 381 (1933). (11) Luers; H.; and Rummier, W., I & , 50, 297-‘301 (1933). (12) LUX,Fritz, Brewers’ Tech. Rev.,9, 15 (1934). (13) Meyerhof. O., and Kiessling, W., Biochem. Z., 267, 313 (1933). (14) Meverhof. 0.. and McEachern. Ibid.. 266, 417 (1933). (15) Petit, P., Brasserie et Malterie, 16, 49, 65, 81 (1920). (16) Raux, M. J., Ibid., 24, 65-8 (1934). (17) SchlichtinE, E., and Winther, H., “Practical Points for Brewers,” p. 40, sational Brewers Academy, New York, 1933. (18) Sobotka, H., and Holzman, M., Biochern. J . , 28, 734-9 (1934). (19) Wallerstein, Leo, J . Franklin Inst., 183, 531-56, 715-34 (1917). (20) Wallerstein, Leo, U. S.Patents 995,820 and 995,824 (1911). ,
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RECEIVED September 15, 1934. Presented as part of the Joint Symposium on the Chemistry of the Enzymes before the Divisions of Agricultural and Food Chemistry and of Biological Chemistry a t the 88th Meeting of the American Chemical Society, Cleveland, Ohio, September 10 to 14, 1934.
Water Purification in the Modern Brewery and Distillery F. J. LAXIHERS, International Filter Co., Chicago, Ill.
W
ATER forms the greatest portion of all the raw ma- beverages of the highest possible quality under efficient conterials used by either breweries or distilleries. In ditions solely through the utilization of modern water-purifiaddition, it influences product quality a t practically cation plants. every stage of manufacture because the great majority of THE BREWERY process reactions in brewing and distilling take place in aqueOnly occasionally is the water supply available to a brewous solution and are therefore directly subjected to the effects of chemical characteristics of the water. Finally, ery suitable for all plant purposes without any treatment water plays another important but entirely independent role whatsoever. The chemical characteristics of a good brewing in plant operation as utilized for cooling, for washing, or for water are quite different from those of a water satisfactory boiler feed purposes; here water quality has a basic and di- for boiler feed and cooling purposes; and the water for general washing or rinsing may constitute another individual requirerect relation to operating and maintenance costs. One of the outstanding achievements offered to progressive ment. breweries and distilleries is the modern water-purification BREWINGWATER plant, developed and technically perfected during the time of The water going directly into the brewing process-forming prohibition. At the inception of prohibition, water purification was just entering its most progressive development stage. 85 to 90 per cent of the finished beer-is used for the mashing In the following decade and a half both chemical and engi- and sparging operations that extract the desired organic conneering phases have undergone remarkable advancement ; stituents from the grain and hops. Since the physical charpractical and theoretical research, together with organized acteristics of the water, such as suspended matter, taste, or scientific development work, has revolutionized former prac- odor, might easily be carried directly into the finished prodtices and treatment methods. As a result, the economic uct, it is obvious that the water must be satisfactory in its and production aspects of plant operation can be benefited physical and esthetic characteristics; furthermore, its chemiin large measure through correct application of modern cal constituents-both organic and inorganic-must be conwater-purification methods; this is especially true in brew- sidered since these may affect the finished beer biologically ing and distilling fields a t the present time. The importance or chemically. CLARITY.Although filtration may form a part of the of these advantages is in some degree indicated by the fact that many plants, which otherwise would have been inoper- beer-production process a t one or more subsequent stages, it able because of adverse water conditions, are now producing is important that the brewing water itself be clear and devoid
