Enginnyring
Progress in
Process
development
LEO WALLERSTEIN, LEONARD T. SALETAN,
AND
PHILIP P. GRAY
Wallerstein laborafaries, 7 80 Madison Ave., New York, N. Y.
I
N 1935 the American brewing industry was just entering the first stages of recovery following a long period of involuntary
inanition, during which technological and scientific advances could hardly have been expected. Sufficient progress, however, had already been made in the United States in the first two decades of the century to establish a firm base for the production and the distribution of stable packaged beer as i t is known today. Three papers published i n 1935 concerning brewing ( I % , 159, 164) dealt respectively with the biochemistry of alcoholic fermentation, a description of the brewing process, and the application of oxidation-reduction potential to brewing control, all of course, in terms and with the emphasis consistent with the state of the brewing a r t a t t h a t time. Any list of uniquely American developments which have made possible the marked superiority of our packaged beers must include factors such as use of malts from American G-rowed barleys, use of adjuncts, adoption of filter aid filtration, advances in mechanical refrigeration, and enzymatic chillproofing of beer, Fundamental also for the enormous growth which has taken place in the bottling of beer was the introduction of crowns and the early emphasis on mechanization in the bottleshop, namely, automatic soaking, filling, pasteurizing, and labeling of the packaged product. All of these were fairly well advanced by 1935, the beginning of the period under review. The brewing industry in today’s complex technological environment covers a n extremely broad field. The authors have chosen t o cover what they consider the more important advances and trends in the industry b y treating the subject under several general topic headings such as the biochemistry of brewing, the product itself, and trends in processing and equipment. Finally, characteristic of brewing science and technology in this country, as of other fields of food technology, has been the attention paid t o the development of techniques for large scale production of a uniform, attractive, stable packaged product. I n the case of brewing, the research activities of the authors’ laboratories, dating back almost exactly 50 years, have played a significant part in developments making such a product possible. Therefore special consideration will be given t o aspects of progress affecting the stability of the finished product, especially as increasing shelf life has continued to be a major goal of the industry.
Advances in Brewing Science Since one is compelled to be highly selective in a review of this kind, many contributions will perforce have to be neglected. Some omissions in particular fields will be deliberate, namely, hops, utilization of by-products, and malting, because other papers cover these fields (49,117, la0). Selected as warranting special attention under the heading of scientific advances are starch chemistry and the malt amylases; recent investigations on the @-globulin of barley and its possible
role in the colloidal stability of beer; alcoholic fermentation; and finally nitrogen assimilation by yeast and yeast genetics. STARCH AND AMYLASE ACTION. Early brewing chemists laid much of the groundwork for the present knowledge in the field of carbohydrate chemistry, notably, Cornelius and James O’Sullivan (reviewed in 144) on maltose and invert sugar, Brown on starch (JO-Sd),and Lintner on amylase (113). Their investigations served as a foundation for more recent strides which have been made in elucidating understanding both of starch chemistry and the nature and the action on starch of the malt amylases. The action of the amylases is so intimately connected with the structure of starch, especially from the point of view of the transformations occurring during brewing t h a t it would be difficult t o discuss advances in the knowledge of starch without immediately being concerned with the malt amylases. It is now- generally agreed t h a t starch-and this applies to the starch introduced from the malt endosperm as well as through cereal adjuncts such as rice and corn commonly employed in American brewing practice-consists of two components, the linear polymer, amylose, and the branched polymer, amylopectin. Both fractions are polymers of glucose. The amylose fraction consists of long unbranched chains of glucose units connected together by cy-l,-l-glucosidic linkages. The length of the glucose chains may vary, depending on the source of the amylose, with a chain length of 200 to 300 glucose units being typical. The amylopectin fraction is also made up of glucose units, most of which are joined by ~~-1,4-glucosidic linkages. but with branches formed by cy-1,G linkages. There is some disagreement as to the degree and the nature of branching in the amylopectin molecule. The linear form, amylose, and two conceptions of the branched form, amylopectin, are shown in Figure 1. Like starch, malt amylase is now known to be of dual nature, owing its activity essentially to two components. The concept t h a t starch hydrolysis by malt diastase is the result chiefly of the combined action of two enzymes has as its origin observations made over GO years ago. I n 1879 Marker (115) confirmed O’Sullivan’s observation (140) t h a t dextrin predominated in the hydrolytic products from the action of mildly heated malt extract on starch, whereas maltose was the main product of the action of unheated extract. Additional evidence was contributed by Brown and Heron (SO) and Bourquelot (27). I n 1890 Wijsman (WOR), by differential diffusion in a starch-gelatin medium-a technique reminiscent of modern chromatographic procedures-was able to demonstrate the actual physical separation of two amylolytic components in malt, differing in their mode of action and extent of attack on starch. Later workers, notably Ohlsson (136, 137), confirmed the existence of the two amylases by selectively inactivating the alpha component a t acid p H values (about 3.3) and the beta component a t a higher temperature (about 70” C.). The terminology “cy- and P-amylase” is due to Kuhn (100, 101). Further studies during the period under review culminated not only in the separation but the actual iso-
2768
November 1952
INDUSTRIAL AND ENGINEERING CHEMISTRY
lation and crystallization of both the a-amylase (160)and the Pamylase (121) of malt. The action of p-amylase on starch is characterized by the rapid formation of maltose as the sole low molecular weight product, and there is no rapid change in the viscosity of the starch paste as occurs following the action of a-amylase. Hydrolysis of the starch molecules by p-amylase alone ceases when about 60% of the theoretical yield of maltose has been formed, a t a point where a blue-violet color with iodine is still obtained. It is generally considered that p-amylase attacks the starch molecule by splitting off 1 maltose unit a t a time, beginning with the nonreducing end of the chain and continuing until stopped by a nearby 1,6-glucosidic branching point-see, e.g., (40, 99). This concept of the mode of attack by p-amylase also serves to explain the formation of the residual dextrin termed a-amylodextrin, The action of malt a-amylase on starch is more complex. The a-amylase rapidly liquefies the starch, disintegrates it into dextrins of low molecular weight which eventually cannot produce a coloration with iodine, and renders i t susceptible to the further action of p-amylase. More slowly and overlapping the initial reaction, maltose, glucose, and maltotriose are formed, until about 80% of the starch is in the form of these sugars (mostly maltose), and the remainder is limit dextrin. Detailed studies of the limit dextrins produced from starch by the amylases have been made by Myrblck and coworkers (1%). It is now considered that dextrinization and liquefaction of starch are both accomplished by a-amylase, although formerly it had been thought that a separate enzyme, amylophosphatase, was responsible for liquefaction (116, 191). Contrary to the mode of attack of the p-amylase, the marked effect on viscosity of the a-amylase is explained on the basis of random attack by the enzyme on ~-1,4-glucosidiclinkages, including the more centrally located ones. While of much greater interest in connection with the use of distillers’ malt for grain alcohol production than for brewing, report of the presence of small proportions of limit dextrinases in malt as related to increased alcohol yields in fermentation should also be cited (95). As indicated, the three chief manifestations of amylase activity are liquefaction, dextrinization, and saccharification; and methods for determination of aflylase activity based on each have been proposed. Dextrinizing methods are largely an outgrowth of the original Wohlgemuth procedure (909). One of these, developed by Sandstedt, Kneen, and Blish (166),with later modifications-e.g., (18, 94, 138, 143)-involves the measurement of a-amylase activity in the presence of an excess of p-amylase, the degree of activity being estimated on the basis of the length of time necessary to reach a reddish-brown end point equivalent to the color of the reaction product of iodine with a specially prepared reagent dextrin. The most generally used saccharifying method still employed for measurement of combined amylase activity (chiefly beta) is based on the original Lintner procedure; it involves measurement by reduction of either cupric or ferricyanide ions of the reducing substances obtained from the hydrolysis of starch (5, 93). Determinations of both a- and p-amylase in malt may be carried out by estimating both saccharifying and dextrinizing activity (93) and correcting for the saccharifying activity of the alpha component. Graesser and Dax (63) described a method for a-and p-amylase in malt based on determination of saccharifying activity before and after differential inactivation of the pamylase by heating the extract a t 70” C. in the presence of calcium ions. FRACTIONATION OF BARLEYGLOBULINS AND SEPARATION OF “CHILLHAZEMATERIAL.” Notable contributions to the knowledge of the complex proteins in brewing barleys during the period under review have been made by a group of Scandinavian investigators, particularly Sandegren (161-164, Quensel (I@), and Sliverborn, Danielsson, and Svedberg (166),who studied the
2769
individual barley globulin fractions, their work throwing light on the possible effect of these fractions on the colloidal stability of beer. They established that the globulin in barley consists of four fractions. One of these, with a molecular weight of about 100,000, designated as p-globulin, was found to be characteristic of barley and was not found, except perhaps in trace amounts, in other cereal grains (44, 166). The p-globulin remains unaffected during malting and is relatively resistant to proteolysis during mashing, undergoing changes only during kettle boiling. pGlobulin is of interest in relation to conclusions of some of these
Figure 1.
Structure of Amylose and Amylopectin
Upper /eft: linear form amylose Right: amylopectin formula with multiple branching (Meyer) Lower: amylopectin laminated formula (Hawarth)
investigators as to its role in development of chill haze in beer. I n this connection it should perhaps be emphasized that it appears that the work on the globulins has been carried out on Kenia and similar 2-rowed barleys in use in European brewing practice and apparently has not been extended to American 6rowed barleys. Also the beers under consideration in these studies were, of course, processed according to European methods, as contrasted with American practice. Present understanding of the composition of the chill haze substance is due to the researches of a number of investigators, prominent among them being Hartong (76, 78), Quensel (I&), Sandegren (161,152), and St. Johnston (169-171). It is now generally accepted that the chill haze substance is a proteintannin complex. The protein fraction seems to be composed of the cleavage products of p-globulin and has a molecular weight of about 30,000 The chill haze material contains in general about 60 to 65 % of protein and 35 to 40% of tannin, the proportions depending upon the nature of the wort as influenced by the type of malt used and the hopping rate. I n essence this material appears to correspond t o the chill-sensitive protein-tannin compound separated from wort by St. Johnston and designated as fraction T. I n studies by St. Johnston it was found that protein T was completely soluble above a critical temperature of about 60’ C., but that upon cooling i t separates as a fine haze. Hartong (76) considers the protein-tannin complex as a coacervate, accounting for its unusual solubility characteristics and to some degree also its variability in composition. There are some indications also from the work of Sandegren and St. Johnston as to the formation of the haze substance which may be of practical importance in the control of beer stability. Thus Sandegren suggests the possibility of oxidation of sulfhydryl groups with consequent formation of sulfur-sulfur linkages and of molecules of larger size and hence lower solubility. I n addition to such oxidation of the protein moiety, oxidation of the tannin portion of the molecule has also been postulated (118). In addition, St. Johnston introduces the concept of mutual floc-
INDUSTRIAL AND ENGINEERING CHEMISTRY
2170
culation between various protein fractions in wort and beer. He separated three protein frsctions from both unhopped and hopped wort by fractional precipitation with ammonium sulfate; protein T , the protein-tannin fraction involved in chill haze formation, already discussed; protein C, a coagulable protein found in hot trub; and protein 0, apparently a n oxidized form of protein C. Protein 0 may be related to a nucleoprotein which had been obtained from unhopped wort (87). Proteins T and C have isoelectric points a t p H 6.4 and 6.0, respectively, while protein 0 has an isoelectric point a t pH 3.9, so peprdes that a t the pH of wort (about 5.2), T and C a r e positively charged while 0 is negatively charged. Interactions in the wort stage among these oppositely Ehrlih b charged colloids, ded~~~ deamination ammonia pending upon kettle acids boiling and cooling conditions, surface exposure, oxidation, etc., are assumed to play a large part in determining , the subsequent stability of the beer. Xorueaq yeasr nucfeic mally traces of proproteins acids tein 0 remain in the Figure 2. Scheme Showing Nitrogen beer! and Oxidation Assimilation by Yeast (According to haze may appear as Thorne) a result of mutual flocculation between fractions T and 0 or simply by precipitation of fraction 0. These recent contributions t o the knowledge of the chill haze compound serve to explain the relationship of chill haze formation t o nitrogenous fractions recognized in a practical way as long ago as 1910 when the proteolytic enzyme treatment for chillproofing was introduced by the senior author (195). ALCOHOLICFERMEXTATION. The fundamental advances in the biochemistry of fermentation during the period under review are b y now familiar. The zymase system of yeast, once thought of as a single entity, is in reality a complex mixture of enzymes and coenzymes. The transformation of glucose to ethyl alcohol is now known to comprise a set of 14 definite reactions, catalyzed by a series of enzymes and coenzymes, as shown in Table 1. Some of the coenzymes are related to the B vitamins, such as Cozymase I (diphosphopyridinenucleotide,in which the pyridine
I
Table 1. Reaction
Summary of Reaction Scheme of Alcoholic Fermentation of Glucose"
Substrate
Product
Enzyme
Dextrose
Glucopyranose-6-phosphate
Hexokinase
Fructofuranose-6-phosphate Fructofuranose-1,6-diphosphate Phosphodihydroxyacetone a n d d-3-phosphoglyceraldehyde 3-Phosphoglyceraldehyde 1, 3-Diphosphoglyceraldehyde
Oxoisomerase (phosphohexoisomerase) Phosphohexokinase Zymohexaee (aldolase)
Triosephosphatedehydrogenase Phosphokinase (ATP-phosphoglyceric transphosphorylase) PhosDhoelvceromutase
2 3
C;:ucop).rariose-4-pl.opkllarC t'rucrof lira nose-d-pt.osphn t r
4
rrccroirirnnose-1 ,ti-digho-phnrr
5 6
Phosphodihydroxyacetone 3-Phospho~lyceraldehydea n d inorganic phosphate 1 3-Diphosphoglyceraldehyde l:3-Diphosphoglyceric acid
1, 3-Diphosphoglyceric acid 3-Phosphoglyceric acid
3-Phosphoglyceric acid 2-Phosphoglyceric acid 2-Phosphoenol pyruvic acid
2-Phosphoglyceric acid 2-Phosphoenol pyruvic acid a n d water Enol pyruvic acid
Enol pyruvic acid Pyruvic acid Acetaldehyde
Pyruvic acid Acetaldehyde a n d carbon dioxide Et,hyl alcohol
9
10 11 12 13 14 0
group is nicotinic acid amide) and cocarboxylase, which is the pyrophosphoric acid ester of thiamine (vitamin B,). NITROGEN AssIhfILATIos BY YEAST. The mechanism of nitrogen assimilation by yeast has been the subject of some study, much of it in recent years by Thornc (179-185). His investigations n-ere made primarily with top-fermentation yeast, but in the main his findings are in agreement with those of Nielsen (129) and Hartelius (74) who used bottom-fermentation yeast. The development of modern analytical techniques such as the quantitative microbiological assay of amino acids and the application of paper partition chromatography has been most helpful in recent investigations-e.g., (16)-and holds great promise for the future. For many years the Ehrlich hypothesis of deamination had been generally accepted, according t o which an amino acid such as leucine is transformed, in this case, into isoamyl alcohol, in the course of the assimilation of its nitrogen by yeast (46, 127, 128). I n a critical examination of this hypothesis, Thorne found that various binary mixtures of amino acids were almost always supcrior in nutrient effect t o the combined mean of the two components, some of the binary mixtures being much superior to others, I n the case of some pairs of amino acids this enhancement could be explained on the basis of the Stickland reaction (167, 168) between members of two classes of amino acids. one the hydrogen donator and the other the hydrogen acceptor, resulting in the oxidation of one amino acid by the other with liberation of ammonia. However, it was also found that there were significant growth increases in binary mixtures M here the Stickland mechanism cannot operate. Moreover, the nutrient value of malt wort was significantly superior to that of ammonium phosphate or most mixtures of amino acids. These and further considerations led t o the theory of direct assimilation of amino acids. Figure 2, taken from Thorne (184))may be regarded as a summary of the present knowledge of nitrogen assimilation from wort. The relative thicknesses of the arrows are intended to indicate the proportional amount of nitrogen assimilated by the various paths. Peptides also appear to play a role in both total growth of yeast and in the outcropping in ale fermentations, although the mechanism is not as yet fully understood (16, 43). YEASTGENETICS. One of the areas where much interesting research on yeast has been accomplished in the past 20 years is in genetics, largely as a result of the efforts of Winge in Denmark (203-5208) and the Lindegrens (106-111) in this country. During this time it has generally been accepted t h a t yeast is subject to genetic inheritance in accordance with Mendelian principles, in common R ith other plants and animals. Sexual differentiation, t h a t is, the existence of two different mating types, was established. It has been found (99, 203) that it is part of the normal life cycle of yeast to undergo fertilization and reduction division, resulting in an alternation of generations involving a haplophase
1
7 8
Vol. 44, No. 11
Phosphotrioseisomerase Spontaneous (1)
Coenzyme Adenosine triphosphate potassium ion
.... . .,
Adenosine triphosphate
....... ........ . . .. . .
Oxidized cozyrnase I Adenosine diphosphate
.......
Magnesium ions Adenosine diphosphate
. . . . .. .
Cocarboxylase Reduced cozymase I
Reproduced from Amerine, M. A., and Joslyn, M. A., "Table Wines." p. 298, Berkeley a n d Los Angeles, Calif., University of California Press, 1951.
November 1952
INDUSTRIAL AND ENGINEERING CHEMISTRY
and a diplophase. When yeast sporulates, a diploid cell by meiosis (reduction division) produces haploid spores. During this process there is a segregation of the chromosomes of which, according to Lindegren (106) there are 6, the diploid cell having contained a double set and the haploid cells possessing only a singleset. Conjugationor fusion of 2 haploid cells must then occur in order t o produce the diploid state, after which the yeast again grows in its vegetative phase and continues t o maintain the diploid number of chromosomes by mitosis until once more sporulation occurs and the process is repeated. T h e ascospores (haploid cells) differ morphologically from the diploid in that they are smaller and rounder. By development of a micromanipulator technique, Winge and Laustsen crossed spores of different yeast genera and demonstrated Mendelian segregation of certain characteristics (106). Of great practical importance is the possibility of deliberate hybridization of yeast for development of desired properties. Hybridization of new strains of hrewers’ yeast does not so far a p pear too promising, owing to the difficulty in inducing sporulation with these yeasts. However, some new hybrids have been produced in bakers’ yeast, with which most of the work has been done. The work on yeast genetics has also focused some attention on the whole question of culture purity and stability in practical fermentations. Since most cultivated brewery yeasts apparently are either haploids or “illegitimate” diploids (110)and sporulate with difficulty if a t all, the opportunity under practical conditione for chance sporulation with the production of aberrant strains appears remote. This still leaves the possibility for mutation under some conditions, B possibility, however, which has not received much serious attention as yet, except as t o flocculation, a s in Thorne’s work (178),discussed below. While lager yeasts are not useful for hybridization, Thorne (178) in his study of the inheritance of flocculation as a genetic property, working with ale yeast, was able t o produce hybrids A number of brewing factors have been considered t o affect this characteristic. Thorne, however, demonstrated that a t least for the type of flocculence he considered, this property is controlled by a system of three pairs of polymeric genes. Of special interest in connection with this discussion is the fact that Thorne found t h a t his yeast tended towards spontaneous mutation of the dominant flocculent to the recessive nonflocculent type. Reverse mutation also occurred, b u t was rare. I n the same connection but along much more empirical lines, a paper was published by the authors’ laboratories (57) referring to changes in the bios requirements for growth of a brewery yeast after it had been in large-scale plant use for some time, Experiments with pure isolates of the initial and final strains indicated the likelihood t h a t the latter, under the conditions existing in the particular brewery, gradually became dominant, apparently because of more rapid growth and greater flocculence. The gradual increase in apparent extract of the brews with successive fermentations can be accounted for in this way, the decrease in attenuation resulting from inability of the second strain t o ferment residual maltotriose in the wort. Whether or not the change represented a genetic or mutational transformation or merely the gradual selection and concentration of a strain initially present in minor proportions was not determined. However, the findings indicated t h a t the bios-typing technique (12, 13, 33, 158) appears to represent one suitable means for following such changes. MISCELLANEOUS. There have been published a number of papers on determination of color in beer and wort, more recently by spectrophotometric methods (7, 23, 36, 79, 132, 148, 174, 177, 186). Some work has also been done on measurement of turbidity (3, 9, 36, 131). Improved methods have been described for determination of various metals in beer, including iron (6,41,56,69,61,130);cop-
271 1
per (8, 41,59, 173); tin (4,172); aluminum (4, 186‘); zinc (10, 11); etc., see also ( 2 0 , 4 9 ) . Also, improved methods for the estimation of silica (175) and tannin (37, 98, 175) have recently been reported. Application of the flame photometer for the detection of calcium, potassium, and sodium in brewing materials is the subject of a recent paper (17‘6). Various procedures for the objective measurement of the foam quality of beer have been proposed (24, 28, d9, 58, 83, 149). Increasing recognition has also been given to microbiological control, and many papers on this subject are worthy of note. The series of articles by Shimwell (163) should be cited. Reference might also be made to a study of the possibility of airborne contamination in the hrewery (69) and to the development of a differential procedure for determining bacteria in the presence of large numbers of yeast cells (70, ‘71).
tool 1935
37
I^
,/.
90
39
41
43
45
41
4s
50
Y E A R
Figure 3.
Beer Sales, Packaged vs. Draft, 1935 to 1950
Some papers have appeared dealing with the evaluation of malt for brewing purposes (29, 48, 76, 96), and advances have been made in the knowledge of the structure of hop resins (34, 35, 145, 146, 189) and in their separation by chromatographic procedures (51, 66, i 4 7 ) . The isolation of a trisaccharide from malt wort by BIom and coworkers at the Tuborg Brewery (25, 26), later confirmed by Barton-Wright et al. (17, 73) should be mentioned. Of considerable importance in quality control in widely diversified fields has been the application of statistical principles to taste evaluation. With the needs of the brewing industry in this respect of primary consideration, a n important contribution to the entire field as well, was the development of the triangular technique for differential taste testing (21, 84). This procedure was applied t o the study of the effect of oxidation on the taste of beer (66).
Product Trends According t o Treasury Department figures in 1935, total beer withdrawals in the United States amounted t o 45,000,000 barrels (188). Sales in 1951 were 84,000,000 barrels (187), representing about a 90% increase. On a per capita basis the 1951 figure corresponds t o a consumption of about 17 gallons or’slightly more than 0.5 barrel. Despite the considerable increase, American per capita consumption stili lags far behind, for example, t h a t of Belgium, where about 46 gallons or 1.5 barrels are consumed annually per person (193). The steady growth in production in the United States during the period under review is shown in Figure 3. Concomitantly and more significantly with the increase in production were changes in the way in which beer was packaged. I n 1935, 29,5y0
INDUSTRIAL AND ENGINEERING CHEMISTRY
2172
of the total beer produced was distributed in bottles and 70.5% was draft. I n 1951 the position was reversed, 73.6% being bottled or canned and 26.4% draft. The increase in production of bottled and canned beer in proportion to draft is also shown in Figure 3. For special mention in this period, of course, was the development in the canning of beer. I n fact, the beginning of the period under review, the year 1935, marks the introduction of cans as containers for beer. I n 1950 canned beer accounted for about 16% of total beer production. Use of nonreturnable bottles was also introduced in this period. The extended market for bottled and canned beer and the increased consumption of beer in the home brought with i t changes in the direction of a distinctly milder, lighter bodied, and paler product. There has also been a decreasing proportion of ale in the total output.
Table
II.
Changes in Compositional Characteristics of Beer, 1936 to 1951
Color, O Lovibond series 52 Apparent extract, % Alcohol 7%by weight Real exbract % Protein (nit&o$en X 6.25), % Original extract, %
1936
1951
5.4
3.1 3.03 3.70 4.69 0.30 11.9
4.20 3.72 5.87 0.52 13.0
The average composition of beer based on the production of the 10 leading breweries in 1951 compared with the composition for 1936 (139) is shown in Table 11, as taken from the authors’ laboratory records. As can be seen, there has been a considerable reduction in the original extract, as well as in the real extract, and present-day beer also has a lower protein content, reflecting a decrease in the ratio of malt t o adjunct employed. Today’s beer is also lighter in color. I n the direction of mildness, there has been a reduction in hopping rate, with the present average now being 0.4 pound per barrel, as against much higher values (0.7 pound per barrel) in 1935 (188). I n line with the generally greater recognition of the importance of vitamins in nutrition, the vitamin content of beer has received considerable attention (14, 86, 88, 103, 104, 105, 114, 133, 134). Beer may be regarded as a good source of riboflavin and nicotinic acid and a moderately good source of biotin and pyridoxin. Trends in Processing While the fundamentals of the brewing process have not changed in any important respect during the period, there have been a number of developments worth noting. Technology has played a greater role, and quality control, instrumentation, automatic controls, mechanization of handling of materials, and analytical control of the materials and the finished product are now commonplace. The general principles of food engineering common t o all food plants are receiving increasing application in the processing of beer. The Master Brewers and other personnel involved in beer production are now more frequently found to have been trained as chemists, chemical engineers, and food technologistP. The brewing technologist is now an important part of the production and quality control team. Almost all breweries now have fully automatic conveyors, including screw and bucket conveyors and pneumatic equipment, for unloading of the malt and transferring it from point to point within the plant. The importance of control of infestation in the stored grains is fully recognized, and greater attention is being given t o proper construction of silos and storage bins to prevent entry of insects. The use of volatile fumigants, such as ethylene oxide, ethylene dichloride, or various mixtures of these with carbon tetrachloride or carbon dioxide on the stored grains is now common practice.
Vol. 44, No. 11
I n mashing, the proper mineral content of the water has long been known to be of primary importance, and is especially so today for the production of the mild, pale beers now popular. The presence of calcium sulfate is of benefit in increasing the acidity of the wort through conversion of secondary phosphates t o primary phosphates, thus affording a more favorable p H range for the activity of the malt enzymes. Treatment of breuing water by the addition of calcium sulfate was patented as long ago as 1908 (199). At t h a t time it was noted that use of calcium sulfate resulted in better gelatinization and liquefaction and afforded some enzyme proteotion at the high temperatures in the cooker. Only much more recently has i t been recognized that such effects are due t o the protective influence of calcium ions on the malt a-amylase a t high temperatures (86, Q4,126). Today calcium is accepted as a n important brewing water constituent not only for such reasons but also because it is desirable in the removal of much of the oxalate during fermentation and storage, thus preventing delayed deposition of calcium oxalate in the finished product. Where the ram water supply is unusually hard or where i t contains excessive quantities of specific mineral constituents such as sodium chloride, demineralization by ion exchange may be resorted t o (1,2,19, 72,97,113, 167,201). Following demineralization, salts necessary to give the water the desired characteristics for brewing purposes are added. Some changes which have gradually taken place in wort handling in this period are worthy of note I n line with the trend towards a paler, milder product, there has been a reduction in the time of boiling the x o r t in the kettle. The reduction in the proportion of hops used also helps in this respect. The older type of hop strainer is being eliminated and new equipment, which removes the hops as the kettle is emptied, has been introduced for more rapid separation of the hops from the wort. The latest types employ a screw device or screen and scraper for rapid removal of the hops, I n the cooling of the wort the open type of cooler formerly in widespread use is being replaced by the closed heat exchanger, whether it be of the double pipe, shell and tube, or plate type (see 190). An increasing number of plants now remove the cold trub (protein-tannin coagulum) which separates from the wort after the temperature during cooling has reached about 9’ C. by filtration with diatomaceous earth which is slurried in as the wort leaves the cooler. Benefits claimed include improvement of taste qualities and clarity of the finished beer, more vigorous fermentation, cleaner yeast, and savings in cost (60,89, 133). Some European breweries remove part of the hot trub (largely coagulated albuminous material) by centrifuging the hot wort (47, 89, 98, 16g). However, although there have been some such practice has not experiments a t one or two breweries (60), been adopted here, as brewers in this country do not generally share the belief of European brewers that separation of hot trub with retention of the cold trub is particularly beneficial. Other factors which have prevented adoption of wort centrifugation here are primarily the limited capacity of the centrifuges and labor costs. I n the fermenting of beer there is increasing usage of closed fermentors, and carbon dioxide of over 99.9% purity is collected for re-use for carbonation and for counterpressure. The carbon dioxide gas may be put through scrubbers and in many plants is liquefied and stored in the liquid state. The liquefaction of carbon dioxide involves essentially the collection of the gas from the storage tanks, after which it is precooled and dehumidified and is then liquefied under pressure of about 250 pounds (45). The gas is stored in liquid form until needed when some of the liquid is drawn into one or more evaporators from which i t is utilized. Advantages claimed for such a system are high purity and uniformity of the evaporated gas, limited storage space requirements, and ready availability of the gas.
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INDUSTRIAL AND ENGINEERING CHEMISTRY
I n line with increasing awareness of the detrimental effect of oxidation on the stability of the finished product, various measures have been considered for minimizing opportunities for oxidation in cellaring. Among these are: use of carbon dioxide as counterpressure; limitation of transfers from one tank t o another in so far as possible; carbonation in tanks by Lamsentype stones for purging of air, and similar carbonation at every transfer; attention to equipment such as pumps, hoses, etc., t o prevent entrainment of air; and the suggested use of a special baffle (166) a t the base of the storage tanks to reduce turbulent flow as beer enters the tanks. Modern storage tanks are fabricated of steel, of one-piece, seamless construction, and are usually glass-lined (vitreous enamel). Some are coated with synthetic resins of various types, With modern controls, refrigeration, and filtration methods, storage time of the beer in the cellars has gradually been reduced, and the average storage period is now about 4 t o 6 weeks in most plants. One of the factors which has made this reduced storage period possible is the practically universal adoption in this country of the enzymatic process in finishing beer. Kraeusening is far less common than in the past. When beer is transferred from the storage to the finishing tank, it is now almost universally prefiltered with diatomaceous earth. Final filtration for the degree of brilliance demanded is ordinarily accomplished by means of pulp filters in which compressed cakes of filtermass are used. More recently, some breweries have resorted to use of diatomaceous earth filters for final filtration, no filtermass cakes being used anywhere in the filtration process. The development in this direction seems recently t o have been furthered by the availability of a special type of filter claimed to facilitate this operation (166). High speed fillers are now available for both bottles and cans. Fillers for flat top cans must be specially constructed; either low or high pressure fillers can be used (114). Immediately following filling of the flat top cans, and prior to sealing, the beer is injected with a fine stream of carbon dioxide, just below or over the surface, in order to eliminate air. The filling of cone top cans is not very different from t h a t of bottles. There have been a number of other processing trends and innovations in processing equipment which are related in large part t o the general technological progress of American food industries. Among these may be mentioned: use of stainless steel for tanks and screens; wider use of automatic controls as in refrigeration, humidity control, weighing and handling, and various phases of brewhouse operation; air conditioning of cellars replacing the older type of refrigerating coils; and introduction of straight line pasteurizers.
Shelf life and Stability From the point of view of its physical stability, beer may be regarded as a complex colloidal solution containing in rather delicate equilibrium a series of protein cleavage products, largely in combination with tannin, as well as dextrins, pentosans, and hop resins. When beer is sold in bottles or cans, it is handled in essentially the same manner as any nonperishable food product and is subjected to a variety of conditions which unfavorably influence its physical and taste stability, such as agitation, long storage on the shelf, exposure t o sunlight in the case of bottles, and extremes in temperature. Moreover, with the greatly increased production of the national or shipping breweries, beers are transported considerable distances, and the over-all time from filling t o consumption may be anywhere from a few days to months or longer. At the same time, consumer standards have become more exacting, and clarity and freshness of taste are expected as a matter of course. Accordingly, preservation of flavor and improvement in stability during the period have become increasingly important. The unfavorable influence of oxidation on beer stability and
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hence the importance of limiting air content had to some extent been recognized prior t o 1935. This is evidenced by the appearance in 1935 of a paper on oxidation-reduction potential (164), typical of the attention given in the literature of t h a t period to attempts t o apply the rH concept to the control of oxidation in brewingpractice (58, 77, 101,119,141,161). Beginning with a paper in 1936 (64) on the simultaneous determination of carbon dioxide and air in beer, a series of papers has been published by the authors’ laboratories dealing with various aspects of measuring and controlling oxidation in beer (56, 63, 64, 66, 67, 160, 194, 200). References 65, 68, 69, 61,64, 66-68, 160, 171, 174, 199, 194, 100,together with other papers on the general subject of beer stability, have been collected in book form under the title “Bottle Beer Quality. A 10-Year Research Record a t Wallerstein Laboratories,” published in 1948.
Figure 4. Trend in Air Content-Semplingby Selected Breweries
Every modern bottling line is now equipped with air-elimination devices, designed to create a foam in the neck of the bottle and thereby to displace the air in the head space prior to capping. The steady lowering of air content in the standard 12-ounce beer package is shown in Figure 4 taken from a review of some of the work from the mthors’ laboratories in this field (64) as brought up t o date to include data for 1951. The values shown were obtained by random selection of samples analyzed in their laboratories. It can be seen t h a t the mean air content has progressively decreased from 7.9 cc. in 1935 t o 2.2 cc. in 1951. Especially worthy of note is the marked drop between 1946 and 1951. I n many cases breweries have been able t o maintain much lower values; in fact some uniformly achieve an air content below l cc. per 12-ounce bottle. The maintenance of low air content depends upon systematic and regular determination of air in bottles taken from the filling line. This has been made possible by the development of simplified and compact apparatus adapted for rapid routine determination of carbon dioxide and air content (66). Efforts to reduce opportunities for oxidation during cellaring must also be considered. One of the papers (150) in the series from the authors’ laboratories already referred t o deals with a method for measuring colorimetrically the amount of dissolved oxygen in beer sampled in the tank. Measurement of dissolved air in tank beer has also become a n essential control procedure. Data on such determinations are not readily available, but generally, where proper precautions to minimize pickup can be taken, air has been found t o run at an equivalent of less than 0.5 cc. per 12-ounce bottle. No doubt, however, considerable variations are to be encountered not only from plant to plant but from tank t o tank even in any one brewery. In papers from the authors’ laboratories (65,100) attention was called to the fact t h a t because of the dependence of r H measure-
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ments on the presence of a poised. reversible oxidation-reduction equilibrium nonexistent in beer, a correct rH value cannot readily be determined As a consequence, the indicator time test (ITT) was described, and early efforts to measure rH have now been abandoned in favor of the relatively simple, practical ITT procedure. The ITT method depends upon the measurement of the rate of decoloratlon of an oxidation-reductlon indicator, 2, 6-dichlorobenzenone Indophenol. Because of apparent confusion in some of the literature, it should be pointed out that Hartong independently and simultaneously had worked out a similar procedure using the same indicator. Hartong’s paper was published in the December 29th, 1934, issue of Wochenschrrff r.f Brazierei; hile the indicator tlme test li-as first described in the specifications of a patent application. filed January 5, 1935, u hich later matured into U.S. Patent S o . 2,159,985, issued ?\lay 30, 1939. The indicator time test procedure has proved of particular value as a measure of the relative degree of oxidation during the course of processing of a given brew and has found Ridespread use in the industry. Within the past few years the addition of new antioxidants to beer has been attempted on a trial basis, the materials receiving chief consideration being ascorbic acid and related compounds (65). However, treatment has been limited, in part owing to the relatively high cost and in part to the fact that need for such treatment has diminished as control of air content has improved. Directly related to factors considerably affecting colloidal stability in beer is the presence of trace metals, some of which act as oxidation catalysts. Most commonly these metals are copper and iron, whose presence in more than normal traces is ascribable to pickup from brewing equipment surfaces Simplified rapid methods for determination of traces of these metals, giving results reproducible in the order of less than 0.1 p,p.m., have replaced the more lengthy and involved procedures available in 1935 and have done much to make feasible the detection and the limitation of metal entry. This is especially true for the method involving colorimetric determination of iron directly in the beer without prior ashing, through use of a,a’-dipyridyl (61). Copper is determined colorimetrically after ashing by reaction v i t h sodium diethyldithiocarbamate and extraction of the colored complex with amyl acetate, after eliminating iron by complexing with a, a’-dipyridyl (17 3 ) . Two other aspects of beer stability which must be mentioned are the phenomenon of gushing or wildness in beer and the development of the so-called sunstruck flavor. Whether or not gushing is associated with the characteristics of the malt, it has been found that certain conditions have a n unfavorable influence where gushing tendencies may exist (60, 198). These conditions apparently are related to development of colloidal particles of subniicroscopic size which act as nuclei for the sudden release of gas (60, 8%’).Of special importance in predisposing beer to gushing is the presence of certain metals in more than normal traces, especially in conjunction with high air content. Tendency towards gushing is also favored by agitation or vibration of bottles as in shipment or handling in the trade and by storage of bottles at low temperatures, particularly on their sides where greater opportunity for oxidation is afforded. The development of sunstruck flavor has been found to be a consequence of a photochemical reaction (68, 90, 156, %’IO), the portion of the spectrum most responsible for the photochemical effects being in the blue, violet, and ultraviolet regions. The odorous compounds formed are a mixture of mercaptans and volatile sulfides derived from more complex, nonodorous sulfur compounds normally present in beer. As part of technical efforts to improve beer stability, a series of patents issued to the senior author disclosing further developments in the enzymatic chillproofing process concerned with enzymatic combinations giving enhanced effects may be cited (196, 197). ildditional methods for stabilizing beers involving
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adsorption of trou’llesome protein fractions have also been proposed (81, 91, 198). Much of the research described in this section has had the practical effect of increasing the shelf life of packaged beer in the period under review While a direct comparison between the beers of 20 years ago and today is patently impossible, as an approximation it is ventured t h a t today’s beer has a shelf life about six times that of the product marketed 20 years ago. Conclusion
The part played by the rapid development of a number of organizations dedicated to the technical betterment of bre??ing technology in the United States during this period must be emphasized. For example, this period has seen the organization of educational and research groups working in a number of specialized fields, as in the improvement of barley varieties (Midwest Barley Improvement Assoc.), and research on malt (Malt Research Institute, with its excellent cooperative arrangement with the Barley and Malt Laboratory of the U. S. Department of A4griculturea t Xadison, Wis.). The Brewers Yeast Council has been extremely active in developing a worthwhile program encouraging brewers to salvage their waste yeast for food and feed purposes and recently has undertaken a n expansion in the research activities it sponsors, to encourage work of a much more fundamental nature. The American Society of Brewing Chemists, an organization which began its activity on a very limited scale in 1934 as the Malt Analysis Standardization Committee, has, since its real start in 1936, become an outstanding association of brewing chemists and scientists, promoting by its meetings, committee activities, and publications much of value to the industry. More recently, the Master Breu ers Assoc. of America initiated the formation of the Brewing Industries Research Institute, in which it has been joined by technical organizations such as the above and by a number of trade groups, the objective being to sponsor and support investigations on industry-wide problems a t various research institutions. The projects under consideration are to be selected not only on the basis of direct value to the brewing industry, but with due regard as well t o eventual broader scientific implications. While this paper has dealt primarily u ith American brewing conditions, the groups referred to have also had before them the example of a number of European organizations with similar objectives but with longer experience. Notably, the Institute of Brewing in England has had a well-organized research program for many years a t the University of Birmingham, a t Rothamsted, and other institutions. Recently this expanded into the formation of the British Brewing Industry Research Foundation \\ ith elaborate facilities in large, well-appointed research laboratories at Nutfield, England, under leadership of a world-famous chemist, Sir Ian Heilbron. The attention given to various aspects of brewing research by all these groups augurs well for the future. With the emphasis in educational institutions being placed on fundamental training programs to include food technology and bio- or biochemical engineering and with increasing familiarity with new scientific tools like microbiological assays, radioactive isotopes, and chromatography, etc., much can be expected from future research activities in the brewing field. Literature Cited (1) Adams, B. A., and Holmes, E. L., Brit. Patents 450,308; 450, 309 (July 13, 1936). (2) Adams, B. A,, and Holmes, E. L., Trans. SOC.Chem. Ind., 54, IT-6T (1935). (3) Ahrens, A. A., Freschmann, J , Stansbrey, J. J., and Scallet, B. L., Am. SOC.Brewing Chemists, Proc., 60-8 (1949). (I Alexander, ) 0. R., and Biske, V. >I., Ibid., 69-75 (1948) (5) Am. Sac. Brewing Chemists, “Methods of Analysis," 4th ed , Section 6, Malt, Sawyer, W E , 1944.
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(6) Am, SOC.Brewing Chemists, Subcommittee on Iron, Am. SOC. Brewing Chemists, Proc., 166-9 (1950). (7) Ibid., Subcommittee on Color, pp. 193-9. (8) Ibid., Subcommittee on Copper, pp. 147-52 (1951). (9) Ibid., Subcommittee on Turbidity, pp. 165-7. (10) Andrews, J., and Lloyd, R. 0. V., J . Inst. Brewing, 57, 368-73 (1951). (11) Andrews, J., and Stringer, W. J., Ibid., pp. 363-8. (12) Atkin, Lawrence, Paper presented before the Division of Agricultural and Food Chemistry a t the 112th Meeting of the AMERICAN CHEMICAL SOCIETY, New York, N. Y. (13) Atkin, Lawrence, Gray, P. P., Moses, William, and Feinstein, Miriam, European Brewery Convention, Congress Lucerne 1949, Vol. I, 96-112; Wallerstein Labs. Communs., 12, 15370 (1949). (14) Barton-Wright, E. C., Biochem. J . , 38, 314-9 (1944). (15) Barton-Wright, E. C., European Brewery Convention, Congress Lucerne 1949, Vol. I, 19-31. (16) Barton-Wright, E. C., J . Znst. Brewing, 57, 415-26 (1951). (17) Barton-Wright, E. C., and Harris, G., Nature, 167, 560 (1951). (18) Bawden, R. F., and Artis, W. G., Am. SOC.Brewing Chemists, Proc., 41-4 (1951). (19) Becker, K., Tech. Proc. Master Brewers Assoc. Am., 125-39 (1950). (20) Bendix, G. H., J. Assoc. Ofic. Agr. Chemists, 32, 158-61 (1949). (21) Bengtsson, K., and Helm, E., Wallerstein Labs. Communs., 9, 171-80 (1946). (22) Bishop, L. R., J . Znst. Brewing, 37, 345-59 (1931). (23) Zbid., 56, 373-82 (1950). (24) Blom, J., Ibid., 43, 251-60 (1937). (25) Blom, J., and Schwara, B., Ibid., 53, 302-5 (1947). (26) Zbid., 55, 240-2 (1949). (27) Bourquelot, E., Compt. rend., 104, 576 (1887). (28) Brenner, M. W., McCully, R. E., and Laufer, S., Am. SOC. Brewing Chemists, Proc., 63-81 (1950). (29) Brenner, M. W., McCully, R. E., and Laufer, S., Tech. Proc. Master Brewers Assoc. Am., 1950, 25-43. (30) Brown, H. T., and Heron, J., Liebigs Ann. Chem., 199, 165 (1879). (31) Brown, H. T., and Heron, J., Proc. Roy. SOC.,30, 393 (1880). (32) Brown, H. T., and Morris, G. H., J . Chem. Soc., 57,458 (1890). (33) Burkholder, P. R., Am. J . Botany, 30, 206-11 (1943). (34) Campbell, T. W., and Coppinger, G. M., J . Am. Chem. Soc., 75, 1849-50 (1951). (35) Carson, J. F., J . Am. Chem. Soc., 73, 4652-4 (1951). (36) Clendinnen, F. W. J., Wallerstein Labs. Communs., 12, 229-43 (1949). (37) Clerck, J. de, Congress 1947 of the Continental Brewery Centre. Papers to be discussed, 78-82. (38) Clerck, J. de, J . Znst. Brewing, 40, 407-19 (1934). 139) Clerck, J. de, Descamps, A., and Vandermeersch, E., Bull. assoc. anciens etud. brass. univ. Louvain, 43, 68 (1947). (40) Cleveland, F. C., and Kerr, R. W., Cereal Chern., 25, 133-9 (1948). (41) Clifcorn, L. E., Am. SOC.Brewing Chemists, Proc., 76-82 (1941). (42) Connery, F. E., and Wright, R., Paper presented as part of the Symposium on Malting and Brewing Technology before the Division of Agricultural and Food Chemistry a t the 121st Meeting of the AMERICAN CHEMICAL SOCIETY,Milwaukee, Wis. (43) Daml6, W. R., and Thorne, R. S. W., J . Inst. Brewing, 55, 13-8 (1949). (44) Danielsson, C. E., Biochem. J . , 44, 387-400 (1949). (45) DeMarkus, L. Co., “The De Markus COS System,” 26 pp., Pittsburgh, Pa. (46) Ehrlich, F., Ber., 40, 1027-47 (1907). (47) Elmfeldt, B., Svensk Bryggeritidskrift, 60, 35 (1945). (48) Enders, C., Congr. intern. tech. chim. ind. agr., Compt. rend., 6, No. 1, 235-44 (1939). (49) Essery, R. E., J . Inst. Brewing, 51, 185-8 (1945). (50) Glaeske, H. M., Wallerstein Labs. Communs., 12, 245-53 (1949). (51) Govaert, F., and Veraele, M., Fermentatio, Nos. 1/3, 1-16 (1948). (52) Govaert, F., and Veraele, M., Intern. Congress for Fermentation Industries, Lectures and Communications, Ghent 1947, 279-96. (53) Graesser, F. R., and Dax, P. J., Wallerstein Labs. Communs., 9, 43-8 (1946). (54) Gray, P. P., Modern Brewery Age, 39, No. 2, 45-6, 113-7; NO. 3, 49, 50, 100-5 (1948). (55) Gray, P. P., Wallerstein Labs. Communs., 1, No. 2, 21-32 (1938). (56) Zbid., NO. 4, 15-20 (1938). (57) Ibid.. 14, 185-97 (1951).
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(58) Gray, P. P., and Stone, I., Am. SOC. Brewing Chemists, Proc., 11-23 (1940); Wallerstein Labs. Communs., 3, 159-71 (1940). (59) Ibid., 62-74 (1942); Wallerstem Labs. Communs., 5, 193-9 (1942). (60) Zbid., 127-38 (1949); Wallerstein Labs. Conzmuns., 12, 311-23 (1949). (61) Gray, P. P., and Stone, I. M., IND.ENG.CHEX,ANAL.ED., 10, 415-7 (1938). (62) Gray, P. P., and Stone, I. M., J . Assoc. Ofic. Agr. Chemists, 19, 162-72 (1936). (63) Gray, P. P., and Stone, I., J . Znst. Brewing, 45, 253-63 (1939); Wallerstein Labs. Communs., 2, No. 5, 5-16 (1939). (84) Ibid., 443-52 (1939); Wallerstein Labs. Communs., 2, No. 6, 24-34 (1939). (65) Gray, P., and Stone, I., U. S. Patent 2,159,985 (May 30, 1939). (66) Gray, P. P., Stone, I., and Atkin, L., A m . SOC.Brewing Chenzists, Proc., 101-11 (1947); Wallerstein Labs. Communs., 10, 183-94 (1947). (67) Gray, P. P., Stone, I., and Rothchild, H., Wallerstein Labs. Communs., 2, No. 7, 49-60 (1939). (68) Ibid., 4, 29-40 (1941). (69) Green, 5. R., and Gray, P. P., Am. Soc. Brewing Chemists, Proc., 81-90 (1949); Wallerstein Labs. Communs., 12, 325-33 (1949). (70) Zbid., 19-31 (1950); Wallerstein Labs. Communs., 13, 357-68 (1950). (71) Green, S. R., and Gray, P. P., Arch. Biochem. Biophys., 32, 59-69 (1951); Wallerstein Labs. Communs., 14,289-95 (1951). (72) Griessbach, R., Angew. Chem., 52, 215-9 (1939). (73) Harris, G., Barton-Wright, E. C., and Curtis, N. S., J. Inst. Brewing, 57, 264-80 (1951). (74) Hartelius, V., Biochem. 2 ,299, 317-33 (1938). (75) Hartong, B. D., European Brewery Convention, Congress Lucerne 1949, Vol. I. 56-61. (76) Hartong, B. D., Wallerstein Labs. Communs., 3, 107-12 (1940). (77) Hartong, B. D., Wochschr. Brau., 51, 409-11 (1934). (78) Ibid., 54, 33-6 (1937). (79) Hartong, B. D., and van den Hoek, A. P., J . Znst. Brewing, 5 5 , 156-64 (1949). (80) Hartong, B. D., Mastenbroek, G. G. A , and Mendlik, F., European Brewery Convention, Congress Lucerne 1949, VOI. 11,250-1. (81) Heimann, E., and Meyer, J. F., U. S.Patent 2,291,624 (Aug. 4, 1942). (82) Helm, E., Brygmesteren, 8, 302-6 (1951). (83) Helm, E., and Richardt, 0. C., J . Znst. Brewing, 42, 191-205 (1936). (84) Helm, E., and Trolle, B., Wallerstem Labs. Communs., 9, 18194 (1946). (85) Hollenbeck, C. M., and Blish, M. J., Cereal Chem., 18, 754-71 (1941). (86) Hopkins, R. H., Wallerstein Labs. Communs., 8, 110-7 (1945). (87) Hopkins, R. H., and Berridge, N. J., J . Inst. Brewing. 55, 30615 (1949). (88) Hopkins, R. H., Wiener, S., and Rainbow, C., Ibid., 54, 264-9 (1948). (89) Hurlimann, H., Zbid., 57, 21-7 (1951). (90) Jacobsson, B., and Hogberg, B., Wallerstein Labs. Communs., 10, 5-16 (1947). (91) Joachim, H., U. S. Patent 2,416,007 (Feb. 18, 1947). (92) Kerr, R. W., Nature, 164, 757-9 (1949). (93) Kneen, E., and Sandstedt, R. M., Cereal Chem., 18, 237-52 (1941). (94) Kneen, E., Sandstedt, R. M., and Hollenbeck, C. M., Ibid., 20, 399423 (1943). (95) Kneen, E., and Spoerl, J. M., Am. SOC. Brewing Chemists, Proc.. 20-6 (1948). (96) Kolbach, P., Congr.’intern. tech. chim. ind. agr., Compt. rend., 6, NO. 1, 270-9 (1939). (97) Kominek, E. G., Brewers Digest, 19. 104T-6T (1944). (98) Krana, F., Schwtiz. Brau. Rundschau, 60, 103-7 (1949). (99) Kruis, K., and Satava, J. O . , 0 v3;voji R kliEeni sp6r jakoZ sexualit5 kvasinek, Nakl. C., Akad. Praha, 67 pp., 1918. (100) Kuhn, R., Ann., 443, 1 (1925). (101) Kuhn, R., Ber., 57B, 1965-8 (1924). (102) Laer, M. van, Bull. assoc. anciens elhues inst. sup&. fermentations Gand, 36, 6 (1935). (103) Laufer, L., Brenner, M. W., and Laufer, S., Proc. Food Conf. Zmt. Food Technol., 1940, 77. (104) Laufer, S., Davis, C. F., and Saletan, L. T., Am. SOC.Brewing Chemists, PTOC., 36-42 (1942). (105) Levine, H., Zbid., 43-8 (1941). (106) Lindegren, C. C., Am. Soc. Brewing Chemists, Proc., 76-82 (1946). (107) Lindegren, C. C., Ann. Mo. Bot. Garden, 32, 107-23 (1945). (108) Lindegren, C. C., Intern. Congr. Genetics, Proc., 8,338-55 (1948).
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(109) Lindegren, C. C., “The Yeaet Cell, Its Genetics and Cytology,” St. Louis, Educational Publishers, 1949. (110) Lindegren, C. C., Wallerstein Labs. Communs., 7, 153-68 (1944). (111) Lindegren, C. C., and Lindegren, G., Ann. Mo. Bot. Garden, 31,203-16 (1944). (112) Lintner, C. J., J . prakt. Chem., 34, 378 (1886). (113) Luers, H., and Fries, G., Wochschr. Brau., 56, 121-4 (1939). (114) Lynes, K. J., and Norris, F. TI‘,, J . Inst. Brewing, 54, 150-7 (1948). (115) Marker, M., Landw. Vera.-&., 23, 69 (1879). (116) Mayer, K., 2. physiol. Chem., 262, 29-36 (1939). (117) McClary, J. E., Paper presented as part of the Symposium on Malting and Brewing Technology before the Division of Agricultural and Food Chemistry a t the 121st Meeting of the AMERICAN CHEMICAL SOCIETY, Milwaukee, Wis. (118) Mendlik, F., J . Inst. Breuing, 56, 134-40 (1950). (119) Mendlik, F., Wochschr. Brau., 52, 417-22 (1935). (120) Meredith, W. 0. S., and Anderson, J. A,, Paper presented as part of the Symposium on Malting and Brewing Technology before the Division of Agricultural and Food Chemistry a t CHEMICAL SOCIETY, the 121st Meeting of the AMERICAX Milwaukee, \Tis. (121) Meyer, K. H., Fischer, E. H., and Piguet, A., Helv. Chim. Acta, 34, 316-24 (1951). (122) Michaelis, L., IND. ESG. CHERT., 27, 1037-42 (1935). (123) Moll, A., Schweiz. Brau. Rundschau, 60, 143-5 (1949). (124) Mutschler, W. J., Wallerstein Labs. Communs., 13, 115-20 (1950). (125) Myrback, K., in Pigman, W.W., and Wolfrom, M. L., “Advances in Carbohydrate Chemistry,” Vol. 3, pp. 251-310, New York, Academic Press, 1948. (126) Nakamura, H., J . Soc. Chem. I n d . ( J a p a n ) , 34, Suppl. Bdg., 265-8 (1931). (127) Neubauer, O., and Fromherz, K., 2. physiol. Chem., 70,326-50 (1911). (128) hTeuberg, C., and Hildesheimer, A , , Biochem. Z., 31, 170-6 (1911). (129) Xielsen, S . , Ergeb. Biol., 19, 375 (1943). (130) Nissen, B. H., Am. Soc. Brewing Chemists, Proc., 32-6 (1946). (131) Nissen, B. H., and Petersen, R. B., Ibid., 77-90 (1942). (132) Nissen, B. H., Petersen, R. B., and Koch, 8. R., Ibid., 151-8 (1949). (133) Norris, F. ST., Congress 1947 of the Continental Brewery Centre. Papers to be discussed, 111, 23-7. (134) Norris, F. W., Wnllerstein Labs. Communs., 13,141-56 (1950). (135) Obata, Y., and Yamanishi, T., Bull. Chem. Soc. Japan, 22, NO. 6; 247-50 (1949); NO. 4 (1950). (136) Ohlsson, E., Compt. rend. trav. lab. Carlsberg, 16, No. 7, 1-68 (1926). (137) Ohlsson, E., 2. physiol. Chern., 189, 17-63 (1930). (138) Olson, W.J., Evans, R., and Dickson, A. D., Cereal Chem., 21, 533-9 (1944). (139) Oser, B. L., Wallerstein Labs. Comrnuns., 1, No. 2, 5-9 (1938). (140) O’SuIlivan, C., J . Chem. Soc., 30, 125 (1876). (141) Preece, I. A . , J . Inst. Brewing, 42, 27-34 (1936). (142) Quensel, O., Inaugural dissertation, Gppsala, 1942. (143) Redfern, S.,Cereal Chem., 24, 259-68 (1947). (1441 Reillv D.. Wallerstein Labs. Communs., 14, 101-10 (1951). Riedi,’W.,’ Brauwissenschajt, No. 4, 52-3; S o . 6, 81-9 (1951). Ibid., No. 9, 133 (1951). Riabv. F. L., and Bethune, J. L., Am. Sot. Brewing Chemists, Proc., 1-7 (1950). Roey, G., van, Bull. assoc. anciens e‘tud. &ole sup&. brass. univ. Louvain, 45, 16-22 (1949). Ross, S., and Clark, G. L., Wallerstein Labs. Communs., 2, No. 6, 46-54 (1939). Rothchild, H., and Stone, I. M., J. Inst. Brewing, 44, 425-31 (1938): Wallerstein Labs. Communs., 1, No. 4, 21-8 (1938). ’ Sandenr’en. E.. Congress 1947 of the Continental Brewery CenGe. ‘Papers t o b e discussed, Vol. 111, 28-36. (152) Sandegren, E., European Brewery Convention, Congress Lucerne 1949, Vol. I, 78-90. (153) Sandegren, E., Rev. int. brass. et malt., 128-9 (1949-50). (154) Sandegren, E., Suominen, H. S., and Ekstrom, D., Acta Chem. Scand., 3, 1027-34 (1949). (155) Sandstedt, R. M., Kneen, E., and Blish, M. J., Cereal Chem., 16, 712-23 (1939). (1.56) . , Saverborn. S..Danielsson. K. E., and Svedberg, T., Svensk Kem. Tid., 56, 75-85 (1944). (157) Schmal, A., Schweir. Brau. Rundschau, 54, 131-5 (1943). (158) Schultz, A. S.,and Atkin, L., Arch. Biochem., 14, 369-80 (1947). (159) Schwara, R., IND.ENG.CHEM.,27, 1031-7 (1935). (160) Schwimmer, S., and Balls, A. K., J. Biol. Chem., 176, 465-6 (1948).
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(161) Segard, J., Bull. assoc. anciens elhues inst. s u p t . fermentations Gand, 36, 243 (1936). (162) Seidel, K.. Brauwelt, 4, 53-6; 5 , 72-4 (1949). (163) Shimwell, J. L., Wallerstein Labs. Communv., 10, 29-38, 11933, 195-207 (1947); 11, 27-39, 135-45 (1948); 12, 71-88, 187-7-94, 267-75, 349-64 (1949). (164) Siebel, F. P.. Jr., and Singruen, E., IXD.ENG.CHEhx., 27, 1042-5 (1935). (165) Smith, A. C., Jr., Tech. Proc. Master Brewers Assoc. Am., 1950, 57-66. (166) Sparkler Mfg. Co., private communication, 1952. (167) Stickland, L. H., Biochem. J . , 28, 1746-59 (1934). (168) Ibid., 29, 288-90, 889-98 (1935). (169) St. Johnston, J. H., Ibid., 54, 305-20 (1948). (170) St. Johnston, J. H., Wullerslein Labs. Communs., 13, 241-52 (I ~9.50). ----,
(171) Zbid., pp. 341-56. (172) Stone, I., I N D . ENG.CHEM., AiiAL. ED., 13, 791-2 (1941). (173) Ibid.. 14, 479-81 (1942). (174) Stone, I., and Gray, P.P., Am. Sot. Brewing Chemists, Proc., 40-9 (1946), Wallerstein Labs. Communs., 9, 209-16 (1946). (175) Ibid.,76-94 (1948); Wallerstein Labs. Communs., 11, 301-18 (1948). (176) Stone, I., Gray, P. P., and Kenigsberg, M., Ibid., 8-20 (1951); m‘cllerstein Labs. Communs., 14, 297-309 (1951). (177) Stone, I., and Miller, M. C., Ibid., 140-50 (1949); WalZerstein Labs. Communs., 12, 335-47 (1949). (178) Thorne, R. S.W., Compt. rend. I m z . lab. Carlsberg, Se’r. physiol., 25, 101-40 (1951). (179) Thorne, R. S. W., J . Inst. Brewing, 43, 288-93 (1937). (180) Ibid., 45, 13-32 (1939). (181) Ibid., 47, 255-72 (1941). (182) Ibid., 50, 186-98 (1944). (183) Ibid., 52, 5-14; 15-6 (1946). (184) Thorne, R. S. W.,Wallerstein Labs. Communs., 13, 319-40 11950). (185) Trolle, B., Syborg, P., and Buchmann-Olsen, B., J . Inst. Brewing, 57, 347-63 (1951). (186) Tullo, J. IT., Stringer, IT. J., and Harrison, G. A. F., Analyst. 74, 296-9 (1949). (187) L-. S. Brewers Foundation, Statistical Memo No. 251, Feb. 5 , 1952. (188) U. S. Treasury Dept., B u r e a u of Internal Revenue, Alcohol Tax Unit. (189) Verzele, AI., and Govaert, F.,Bull. soc. chim. Belges, 58, 432 (1949). (190) Vogel, E. H., Jr., Schwaiger, F. H., Leonhardt, H. G., and hIerten, J. A., “Practical Brewer. A Manual for the Brewing Industry,” Master Brewers Assoc. of America, 1946. (191) Waldachmidt-Leitz, E., and Mayer, K., 2. physiol. Chem., 236, 168-80 (1935). (192) Wallerstein Labs. Communs., 4, 189-91 (1941). (193) Ibid., 13, 195 (1950). (194) Wallerstein, L., Am. Brewer, 76, No. 1, 46-8, 80 (1943); Wallerstein Labs. Communs., 6, 43-9 (1943). (195) Wallerstein, L., U. S.Patent 995,820 (June 20, 1911). (Application filed Apr. 11, 1910.) (196) Wallerstein, L., U S. Patents 2,011,095; 2,011,096 (Aug. 13, 1935). (197) Wallerstein, L., U. S.Patents 2,077,446; 2,077,447; 2,077,448; 2,077,449 (Apr. 20, 1937). (198) Wallerstein, L., U. S. Patent 2,433,411 (Doc. 30, 1937). (199) Wallerstein, M., U. S. Patent 905,029 (Nov. 24, 1908). (200) Wallerstein, &I., Wallerstein Labs. Communs., 1, No. 1, 5-11 (1937). (201) Waroway, R. R., Brewers Digest, 24, lllT-3T, 117T (1949). (202) Wijsman, H. P., Jr., Rec. trav. chim., 9, 1 (1890). (203) Winge, 0 , Compt. rend. trav. lab. Carlsberg, S&. physiol., 21, 77-111 (1935). (204) Ibid., 24, 79-96 (1944). (205) Winge, O., Wallerstein Labs. Communs., 15, 21-44 (1952). (206) Winge, O., and Laustsen, O., Compt. rend. trav. lab. Carlsberg, Se‘r. phusioZ., 22, 99-119 (1937). (207) Ibid., 22, 235-47 (1938). (208) Winge, O., and Roberts, C., Ibid., 24, 263-315 (1948). (209) Wohlgemuth, J., Biochem. Z.,9, 1-9 (1908). (210) Yamanishi, T., and Obata, Y., Bull. Chern. SOC.Japan, 23, 125-7 (1950). RECEIVED for review -4pril 18,1952. ACCEPTED August 13, 1952. Presented as part of the Symposium on Malting and Brewing Technology before the Division of Agricultural and Food Chemistry at the 121st Meeting of the AMERICAN CHEMICAL SOCIETY,Milwaukee, Wis.
