Products for Haze-Forming Proteins of Beer

Agric. Biol. Chem., 48 (5), 1139~1146, 1984 1139

Affinity of Proanthocyanidins and Their Oxidation Products for Haze-forming Proteins of Beer and the Formation of Chill Haze1 Katsuhiko Asano, Keiji Ohtsu, Kyoko Shinagawa and Naoki Hashimoto Brewing Science Laboratory, Kirin Brewery Co., Ltd., Takasaki, Gumma370-12, Japan Received August 8, 1983

Amongthe polyphenols found in beer, proanthocyanidins were found to have a specific affinity for the haze-forming proteins in beer and retained their capacity to form a chill haze. Proanthocyanidins pentamer and tetramer had the highest affinities for the haze-forming proteins, followed by the trimer, dimer and catechins. During the brewing process, proanthocyanidin trimer and polymeric proanthocyanidins easily formed insoluble complexes with proteins in the wort as a result of their high affinity for proteins; consequently, these were not found in finished beer, whereas such proanthocyanidin dimers as procyanidin B3, and catechin survived in finished beer. Procyanidin B3 and catechin, when stored in beer or a buffer solution, seemed to undergo oxidative polymerization and increased their affinity for the haze-forming proteins to form a extensive chill haze.

It is generally recognized that proteins and polyphenols in beer. Proanthocyanidin is a polyphenols are mainly responsible for the typical oligomeric or polymeric polyphenol. non-biological haze of beer. In a previous The term 'proanthocyanidin' refers to the paper,1} we characterized those haze-forming polymer consisting of linked flavan 3-ol units proteins whose high affinity for polyphenols and a particular polymer in this group is brought about the formation of chill haze in commonly referred to as procyanidin or beer and showeda possible mechanismfor the prodelphinidin. In beer, however, very few combination of these haze-forming proteins proanthocyanidins, except for the proan- with polyphenols. However, the polyphenols thocyanidin dimers, have been characterized which tend to combine with haze-forming until now. The nature of and mechanismby proteins still remain uncharacterized. Beer which polyphenols participate in the for- contains a complex mixture of polyphenols mation of beer haze have been intensively and they are thought to be involved in the investigated by British workers.2~10) Since formation of beer haze. These polyphenols can Gramshaw2~4)suggested the important roles be classified as (1) monomeric polyphenols and of catechin and proanthocyanidin dimer in the (2) oligomeric and polymeric polyphenols. formation of beer haze, much attention has One of the dominant and well-characterized been directed to proanthocyanidins and they monomeric polyphenols in beer is catechin. are thought to be the main precursors of beer Structurally, this is a flavan 3-ol. Phenolic haze. In particular, such a proanthocyanidin alcohols such as resorcinol, pyrocatechol and dimer as procyanidin B3 seems to be readily phloroglucinol, and phenolic acids such as oxidized and polymerized to increase their protocatechuic acid, gentisic acid, caffeic acid affinity for proteins. The mechanism of oxi- and gallic acid are also classified as monomeric dative polymerization and the nature of the Turbidity Formed in Beer at LowTemperatures. 1140 K. Asano et al. oxidation products of the proanthocyanidins, Protein preparations. The haze-forming proteins were however, still remain uncertain. prepared from beer as described previously1* and the haze- The present paper describes the specific af- forming proteins fraction II was used throughout this finity of proanthocyanidins for the haze- study. Poly-L-proline, poly-L-lysine and papain were pur- chased from Sigma Chemical Go. and gliadin from Tokyo forming proteins and shows that oxidative Kasei Kogyo Co., Ltd. polymerization increases the affinity of proanthocyanidins for these proteins and induces Measurement of haze-forming capacity. The polyphenol sample dissolved in 2.0ml of 0.02m sodium phosphate the formation of chill haze. buffer, pH 4.2, containing 3.6% ethanol (100 or 200mg/liter) was mixed with an equal volume of protein MATERIALS AND METHODS solution (1,000mg/liter in the phosphate buffer) in a test tube (1.5 x 15.6 cm). Twoblanks were run simultaneously. Polyphenol preparations. Procyanidin B3 and prodel- One contained 2.0 ml of sodium phosphate buffer and an phinidin B3 were extracted from 504g of barley husk, and equal volumeof protein solution, and the other contained procyanidin C2 likewise from 400 g of hops with 3,000ml 2.0ml of the sample solution and an equal volumeof of 75%acetone. The extraction was repeated twice and the sodium phosphate buffer. The mixture was chilled at 0°C extracts werecombined. After removingthe acetone under for 40 min and the chill haze was measurednephelometri- reduced pressure, each extract was defatted with 1,400ml cally with a Zeiss-Pulfrich photometer. Readings in ab- of petroleum benzin (bp 40~60°C) three times. solute units were corrected by subtracting the blank values Proanthocyanidins in the aqueous phase were adsorbed on and converted to EBCformazin units.12) 1 50 g ofPolyamide Woelm®(for chromatography, Woelm Pharma GmbHand Co.) and, after washing the polyamide Measurement of polyphenols combined with proteins. with deionized water to remove unadsorbed materials, Following the haze-forming capacity measurement de- were eluted with 2,500ml of 75% acetone. The eluate was scribed above, the chill haze was removed by centri- evaporated to dryness under reduced pressure, dissolved in fugation at 18,000rpm at 0°C for 20min. The polyphenol 5ml of 75%methanol and then applied to a Toyopearl content in the supernatant was measured according to HW-40F column (2.2x100cm, Toyo Soda Man- Analytica EBC.13) The amount of polyphenols combined ufacturing Co., Ltd.) which had been preequilibrated with the proteins was calculated from the decrease in the with 75% methanol. The column was eluted with 75% polyphenol level in the supernatant. methanol at a flow rate of 0.9ml/min and the eluates was collected in 20ml fractions. Fractions of the eluates con- High performance liquid chromatography (HPLC). taining procyanidin B3 and prodelphinidin B3, and pro- Proanthocyanidins in the wort, fermenting wort and beer cyanidin C2 were combined and dried under reduced were analyzed by high performance liquid chromatog- pressure. Each fraction, after having been dissolved in 1 ml raphy. The 2.0 ml sample was evaporated to dryness under of 70% ethanol, was fractionated and purified on a reduced pressure and then dissolved in 0.4ml of 1/15m Sephadex LH-20 column (1 x 100cm) with 70% ethanol as sodium phosphate buffer, pH 2.5, containing 5% acetoni- the eluent at a flow rate of 16 ml/hr. The eluates containing tril. After removing the sediments by filtration, a 100/il each proanthocyanidin were evaporated to dryness under aliquot was pumped on to an octadecylsilica column reduced pressure to yield 150, 130 and 25mg of pro- (0.3 x 15cm, Toyo Soda LS-410 SIL, 5ftm). The column cyanidin B3, prodelphinidin B3 and procyanidin C2, re- was eluted at a flow rate of 0.9ml/min with 1/15m spectively. Proanthocyanidin tetramer was extracted from phosphate buffer, pH 2.5, containing 5% acetonitril for 200 g of hops, which had been pretreated with petroleum 5 min, and then with a stepwise gradient concentration of benzin (bp 40~60°C) to remove resinous materials, with acetonitril in the phosphate buffer as follows: 7.5% for 1,200 ml of 80% ethanol. Proanthocyanidin pentamer was 15min, 20% for 10min, 30% for 5min and 50% for 5min. extracted from 200g of hops by boiling with 6,000ml of The eluate was mixed with a stream of 0.1% 4-dimethyl- deionized water for 90min. The extract was then con- aminocinnamaldehyde (DAC) dissolved in methanol con- centrated under reduced pressure and ethanol was added taining 25%sulfuric acid and monitored colorimetrically to a final concentration of 80% by volume. Each extract at 638nm. was purified according to the procedure of Matsuo and Itoo11} to yield 683 and 70mg of proanthocyanidins Thin layer chromatography. Proanthocyanidins stored at tetramer and pentamer, respectively. 50°C in 0.02 m sodium phosphate buffer, pH 4.2, contain- ( + )-Catechin, ( -)-epicatechin and caffeic acid were pur- ing 3.6% ethanol (100 mg/liter) were analyzed by thin layer chased from Sigma Chemical Co., resorcinol from Kishida chromatography. One ml of the sample solution was Chemical Co., Ltd. and other mono- and polyphenols evaporated to dryness under reduced pressure at 35°C and were from Tokyo Kasei Kogyo Co., Ltd. then dissolved in 0.1 ml of ethanol. Ten /il aliquots were spotted on a pre-coated silica gel 60 plate 0.2mm in Affinity of Proanthocyanidins for Haze-forming Proteins of Beer 1141

thickness (10x 10cm, E. Merck) and developed with toluene-acetone-formic acid (4: 6 : 1) over a distance of monophenols had none at all. 7.5cm. The plate was then sprayed with vanilin-HCl reagent (10% vanilin in ethanol and cone. HC1were mixed Affinity ofproanthocyanidins for proteins in the ratio 5: 3 before use). Because the haze-forming capacities of proanthocyanidins and catechins varied widely, RESULTS the differecne in their affinity for proteins was examinedin a system of proanthocyanidins or Haze-forming capacities ofpolyphenols catechins (lOO mg/liter) and proteins of various Various mono- and polyphenols were found proline content (500 mg/liter) in the phosphate in beer and chill haze.5~7'9'10'14~19) The haze- buffer. As wehave shownpreviously,1* the forming capacities of these mono- and poly- proline-rich proteins had a higher affinity for phenols were measured in a system consisting polyphenols than proteins with little or no of mono- or polyphenol (lOOmg/liter) and proline. Table II shows that proantho- haze-forming proteins II (500mg/liter) in cyanidins tetramer and pentamer combined 0.02m sodium phosphate buffer, pH 4.2, con- with such proline-rich proteins as poly-L- taining 3.6% ethanol. Table I shows that the proline, haze-forming proteins II and gliadin, proanthocyanidins, such as procyanidin B3, in the largest amounts, followed by.proan- prodelphinidin B3, procyanidin C2, proanthocyanidin trimer (procyanidin C2), pro- thocyanidins tetramer and pentamer, and to anthocyanidin dimers (prodelphinidin B3 a muchlesser extent catechins (catechin and and procyanidin B3) and catechins. epicatechin) had haze-forming capacities, but Proanthocyanidin pentamer also combined the other monomeric polyphenols including with the proteins containing little or no proline

Table I. Haze-forming Capacities of Polyphenols H a z e-fo rm in g P o ly p h en o ls c a p a c ity (EBC f.u.fl)

M o n o p h en o l z?-H y d r o x y b e n z o ic ac id 0 V a n illic a cid 0 F er u lic ac id 0 S y rin g ic a cid 0 P o ly p h e n o l M o n o m e rs R e so rcin o l 0 P y ro c a te ch o l 0 P ro to c a te ch u ic ac id 0 G e n tisic ac id 0 C af e ic a c id 0 C h lo ro g en ic a c id 0 P h lo ro g lu cin o l 0 G a llic a c id 0 R u tin 0 ( + )-C atec hin 0 .02 ( - VE pi cat ec hi n 0.4 2 D im e rs Pr o cy a ni d i n B 3 5.5 Pr od e lp hi n id in B3 1 8.2 T rim e r Pr o c ya n id i n C 2 7 9 .3 T e tr am e r P ro a n th o c y a n id in tetra m e r 2 6.5

P en ta m er P ro a n t h o cy a n i di n p e nt a m e r 3 8.2

EBCformazin units. Table II. Combination of Proanthocyanidins with Proteins and the Formation of Chill Haze Combined proanthocyanidins (mg/g protein) Haze-forming capacity (EBC formazin units) P roanth ocyan idin s P oly -L-P ro H aze-form 'ng G liad in P apFa in Po lyJ -L-L yJsproteinsIIP o ly-L-P ro . TITI- G liadin P apain P oly-L-L ysproteins * > C atechin 12 .2 1 8 .8 32 . 2 6. 2 0 .5 0. 0 2 4 .8 in E p icatechin 25 . 6 1 9. 4 2 6 . 4 3. 4 0 . 0 3 0 . 4 4 . 9 z> o Procyanidin B3 1 1 7 . 6 4 4 . 8 1 6 . 4 4 . 6 5 2 . 5 5 . 5 1 9 . 6 0 . 0 5 <^ Prodelphinidin B3 1 3 5 . 6 2 2 . 6 2 6 . 0 3 . 4 5 4 . 7 1 8 . 2 3 7 . 2 0 . 1 & Procyanidin C2 1 8 3. 4 83 . 4 4 7. 6 1 6 .6 4 0. 8 7 9. 3 40 . 3 0 .3 P ro anthocyanid in 19 0. 4 1 8 4. 6 12 1. 4 5 6 .0 1 6. 2 2 6. 5 4 0 .5 4 . 3 tetram er P ro anthocyanid in 1 7 8 .8 1 7 3 .2 15 0 .8 5 7 . 8 3. 4 2 0 . 0 3 8 .2 5 9. 4 8 . 2 0 . 0 7 p en tam er Affinity of Proanthocyanidins for Haze-forming Proteins of Beer 1143 such as papain and poly-L-lysine, and formed with a high affinity for proteins such as pro- chill haze. Proanthocyanidin tetramer com- anthocyanidins tetramer and pentamer im- bined with papain but not with poly-L-lysine. mediately formed insoluble complexes with the On the other hand, such proanthocyanidin proteins in the worts and were not found in the dimers as procyanidin B3 and prodelphinidin clarified worts. Proanthocyanidin trimer and B3, and catechins did not combine appreciably dimers, and to a lesser extent catechin, also with these proteins. formed insoluble complexes in the worts but could still be found in appreciable amounts in The fate ofproanthocyanidins in the brewing soluble form. After the worts had been boiled, process however, the levies of proanthocyanidin trimer To examine the effect of the large difference and dimers decreased significantly, mostly to in affinity of the proanthocyanidins for pro- zero, and only procyanidin B3 and catechin teins on the fate of proanthocyanidins in the survived in the finished beer. During the wort brewing process, laboratory scale brewing was boiling, small amounts of uncharacterized pro- conducted in the following manner. To por- ducts from the proanthocyanidins and cat- tions of 30ml of sweet wort, which had been echins were formed and were found in the pre-treated with 150 mg of polyvinylpolypyr- finished beer. rolidone (PVPP) to remove polyphenols in the wort, proanthocyanidins and catechins were Changes in the haze-forming capacities of pro- added at a concentration of lOOmg/liter. The anthocyanidins by oxidation worts were boiled at 102°C for 90min and then The formation of chill haze is accelerated on cooled to 8°C. After removing the precipitates, storing the beer at higher temperatures, pro- 10ml of the boiled wort was fermented with bably bacause of the oxidation of proan- bottom fermenting yeast at 8°C for 9 days, thocyanidins in the beer. To confirm this, we then cooled to 0°C for 10 days. The beer was examined the changes in the haze-forming filtered through a membrane filter (0.45 /mi). capacities of proanthocyanidins and catechins The changes in the composition and content of present in both the beer and phosphate buffer. proanthocyanidins and catechins in the wort The proanthocyanidins and catechins were and beer were analyzed by HPLC. dissolved in beers pretreated with 500ppmof Table III shows that proanthocyanidins PVPP at a concentration of 50mg/liter.

Table III. Fate of Proanthocyanidins during the Brewing Process Content of proanthocyanidin (mg/liter)a P ro anthocyanidin s A fter ad dition After wort boiling After fermentation A fter storage to the wort (C ool ed wor t) ( You ng bee r) (B eer)

C atechin 83 54 (28) 59 (2 3) 46 (26) E picatechin 100 0 (79) 0 (70) 0 (70) Procyanidin B3 59 ll (20) 8 (17) 10 (14) Prodelphinidin B3 3 5 trace (6) 0 (4) 0 (3) Procyanidin C2 44 0 (14) 0 (14) 0 (12) Proantho cyan idin 0 0 0 0 tetram er P roanth ocyan idin 0 0 0 0 pen tam er

Proanthocyanidins were added to the wort at levels of lOOmg/liter. Figures in parentheses represent the content of uncharacterized products of proanthocyanidins during wort boiling. 1144 K. Asano et al

i « r. i i ià" .. « r^ -i- ^ Proanthocyanidin Proanthocyanidin . Catechin Epicatechin Procyanidin B3 Prodelphimdin B3 Procyamdin C2 tetramer pentamer

CDO<^> CDC^CD o 0 0 o o o o 0 0 9 2 SQ So OQO ° o 00 ao 00- OO . .»O gg à"à":> © f? OP H - OQ Q O O D O . ::v? I! trH . PQ n f?f? nf???- a_b o_y y_y _b_s- «-a o-a-o-u_6_^_i 0 4 7 0 4 7 0 4 7 0 4 7 0 4 7 0 4 7 0 4 7 Storage Period at 50°C ( day) Fig. 1. Changes in the Composition of Proanthocyanidins Stored in Phosphate Buffer.

Table IV. Changes in Haze-forming Capacities Proanthocyanidin dimers and catechins were of Proanthocyanidins in Beer during Storage partially transformed to higher molecular Haze-forming capacity weight materials of lower Rf values and the (EBC formazin units) amountof these higher molecular weight ma- P roanth ocyan idin s Storage period at 50-C (day) terials increased with increases in the storage period. Proanthocyanidin trimers weretrans- 0 4 7 formed to both higher and lower molecular

C atech in 0. 1 4 .4 1 2. 1 weight materials, whereas proanthocyanidins E p icatechin 22.3 tetramer and pentamer were degraded to lower molecular weight materials during storage. Procyanidin B3 2. 5 1 7. 8 23 .9 Table V shows the change in the affinity of Prodelphinidin B3 5.1 ll.1 14.9 proanthocyanidins and catechins for haze- Procyanidin C2 1 5.0 25.6 26.4 forming proteins II during storage. The P roan th ocyanid in 59. 0 42 .9 36 .0 amounts of proanthocyanidin dimers and tetram er P roan th ocyanid in catechins which combined with haze-forming 33. 8 25.5 24.5 pentam er proteins II were little or none initially, but increased with increases in the storage period. In contrast, large amounts of proanthocyanidins trimer, tetramer and pentamer initially Portions of 10ml of the mixtures were put combined with haze-forming proteins II, but in glass tubes (1.8 x 7cm) closed with a septum then lost this affinity gradually during stor- cap, stored at 50°C for 4 and 7 days and then age. Figure 2 shows that the polymerization chilled at 0°C for 40min. The chill haze was of procyanidin B3 was induced by oxygen and measured nephelometrically with a Zeiss- wassupressed by an anti-oxidant such as sulfite. PulfrichTable IVphotometer.shows that proanthocyanidin dimers and catechins greatly increased their DISCUSSION haze-forming capacities with increases in the storage period. In contrast, proanthocyanidins In this work, the dominant roles of pro- tetramer and pentamer lost their haze-forming anthocyanidins in the formation of chill haze capacities gradually with increases in the stor- in beer were confirmed. The affinity of pro- age period. anthocyanidins for the haze-forming pro- Figure 1 shows the changes in the com- teins in beer were found to be related to the position of proanthocyanidins and catechins molecular size of the proanthocyanidins. stored in the phosphate buffer (lOOmg/liter). Proanthocyanidins of higher molecular weight Affinity of Proanthocyanidins for Haze-forming Proteins of Beer 1145

Table V. Changes in Affinity of Proanthocyanidins for the Haze-forming Proteins and the Formation of Chill Haze

Combined proanthocyanidins Haze-forming capacity (mg/g protein) (EBC formazin units) P roanth ocyanid ins Storage period at 50-C (day) Storage period at 50-C (day)

0 4 7 0 4 7

C atech in 5.9 0.4 E p icatech in 2.6 0.6 Procyanidin B3 1 .3 5. 0 1 4. 1 0. 3 3 . 1 7 .9 Prodelphinidin B3 15 .2 41 .1 46 . 8 15 . 6 4 3 .9 3 9 . 7 Procyanidin C2 33 . 3 2 8 .0 33 . 3 76 .3 5 6. 9 46 .7 P roan th ocyanid in tetram er 98 .8 8 2 .6 7 7. 2 2 0 . 5 1 7 . 0 1 5 . 4 Proanthocyanidin pentamer 90 .2 8 1 .6 8 0. 4 28.1 19.3 16.8

that the o-dihydroxyl groups of proanthocyanidins were the sites for combination with proteins and showed that proan- © © thocyanidin trimer, having three sites for such a combination, had a greater ability to precipitate proteins than proanthocyanidin dimer 6 q> o - with only two sites for combination. In the brewing process, however, proanthocyanidin trimers and polymeric proan- 1 2 3 thocyanidins easily formed insoluble com- Fig. 2. Effects of Oxygen and Sulfite on the plexes with proteins in the wort and con- Composition of Procyanidin B3 Stored in Phosphate sequently could not be detected in the finished Buffer. beer. Therefore, it seems probable that pro- 1, stored with lOmg/liger of sulfite ; 2, stored without any anthocyanidins surviving in beer are those treatment; 3, stored after bubbling oxygen gas at a flow having a low affinity for proteins such as rate of 50ml/min for 5min. procyanidin B3 and catechin. Although procyanidin B3 and catechin did not appreciably had a higher affinity for the haze-forming form chill haze due to their low affinity for the proteins and readily formed chill haze. The haze-forming proteins in beer, they were trans- high abilities of proanthocyanidin oligomers formed to higher molecular weight materials to precipitate proteins along with their strong when stored in beer or phosphate buffer, and astringency were observed by Bate-Smith.20) formed considerable amounts of chill haze. Assuming that proanthocyanidins combine Because the formation of higher molecular with proteins through multipoint hydrogen weight products of procyanidin B3 was in- bonds between the hydroxyl groups of pro- duced in the presence of oxygen, oxidative anthocyanidins and carbonyl oxygen of the polymerization seemed to occur during stor- peptide bond,21 ~23) higher molecular weight age. Bate-Smith25) and Gramshaw4) have pro- proanthocyanidins having a large number of posed a mechanismfor the oxidative polymeri- hydroxyl groups maybe able to combine more zation of proanthocyanidins via quinoid easily and strongly with proteins than those of intermediates, but this has not been demon- low molecular weight. Haslam24) has suggested strated until now. The polymerized products 1146 K. Asano et al

of procyanidin B3, although still remaining 7) J. D. McGuinness, D. R. J. Laws, R. Eastmond and R. J. Gardner, /. Inst. Brew., 81, 237 (1975). uncharacterized, must have a large number of 8) J. D. McGuinness, R. Eastmond, D. R. J. Laws and hydroxyl groups and therefore a high affinity R. J. Gardner, /. Inst. Brew., 81, 287 (1971). for the haze-forming proteins in beer. 9) I. McMurrough, Eur. Brew. Conv. Proc. Congr., Based on these observations, we concluded 17th, Berlin, 1979, p. 309. that procyanidin B3, and to a lesser extent 10) I. McMurrough, G. P. Hennigan and M. J. Loughrey, J. Inst. Brew., 89, 15 (1983). catechin, were important precursors of chill ll) T. Matsuo and S. Itoo, Agric. Biol. Chem., 45, 1885 haze in beer. Both procyanidin B3 and cat- (1981). echin, because of their low affinity for proteins, 12) L. R. Bishop, /. Inst. Brew., 66, 388 (1960). survived in the finished beer. They, and partic- 13) European Brewery Convention, Analytica EBC, 3rd ularly procyanidin B3, were readily oxidized ed. Schweizer Brauerei-Rundschau, Zurich, 1975, p. and polymerized during storage of the beer to E64. 14) D. E. F. Gracy and R. L. Barker, J. Inst. Brew., 82, increase their affinity for the haze-forming 72 (1977). proteins in beer and formed a large amountof chill haze. 15) R. Vancraenenbroeck, H. Gorissen and R. Lontie, 16th Amsterdam, Eur. Brew. Conv., Proc. Congr., 1977, p. 429. Acknowledgments. We wish to thank the manage- 16) R. Vancraenenbroeck, M. Kara-Zaitri and A. ment of Kirin Brewery Co., Ltd. for permission to publish Devreux, Eur. Brew. Conv., Proc. Congr., 17th, this work. Weare grateful for the continuous encourage- Berlin, 1979, p. 293. ment of Dr. S. Kubo, Senior Managing Director, Dr. Y. 17) J. Jerumains, Eur. Brew. Conv., Proc. Congr., Kuroiwa, the former director of the Research 17th, Berlin, 1979, p. 309. Laboratories and Dr. E. Kokubo, Director of the Brewing 18) M. Dadic and G. Belleau, /. Am. Soc. Brew. Chem., Science Laboratory. We thank Dr. B. L. Moller, Carlsberg 38, 154 (1980). Laboratory (Denmark), for kindly providing authentic 19) G. Leupold and F. Drawert, Brauwissenschaft, 34, samples of proanthocyanidins. Weare most appreciative 205 (1981). of the excellent technical assistance of Mr. H. Murayama 20) E. C. Bate-Smith, Phytochemistry, 12, 907 (1973). and Ms. J. Tsunoda. 21) S. G. Shuttleworth, A. E. Russell and D. A. Williams-Wynn, J. Soc. Leather Trades''Chem., 52, REFERENCES 486 (1968). 22) C. F. Van Sumere, J. Albrecht, A. Dedonder, H. De 1) K. Asano, K. Shinagawa and N. Hashimoto, J. Am. Pooter and I. Pe, "The Chemistry and Biochemistry Soc. Brew. Chem., 40, 147 (1982). of Plant Proteins," ed. by J. B. Harborne and C. F. 2) J. W. Gramshaw, /. Inst. Brew., 73, 455 (1967). Van Sumere, Academic Press Inc., London, 1975, p. 3) J. W. Gramshaw, /. Inst. Brew., 75, 61 (1969). 211. 4) J. W. Gramshaw, Tech. Quart. Master Brew. Ass. 23) A. E. Hagerman and L. G. Butler, /. Biol. Chem., Amer., 1, 167 (1970). 256, 4494 (1981). 5) R. Eastmond, /. Inst. Brew., 80, 188 (1974). 24) E. Haslam, Biochem. J., 139, 285 (1974). 6) R. Eastmond and R. J. Gardner, /. Inst. Brew., 80, 192 (1974). 25) E. C. Bate-Smith, Nature, 179, 1283 (1957).