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Absence of diacetyl in fermenting wort
COMMUNICATIONS
454
able2-a fact which should not be minimizedthese results suggest the possiblity that the Sprotein may have suffered moderate changes in its secondary structure relat,ive to the solution structure of RNase. After this note was submitted for publication a detailed study and analysis of the circular dichroism spectra of RNase, RNase-S, and S-protein has been published by Pflumm and Beychok (23). These authors have found t.hat the RNase spectrum in the ultraviolet, peptide-absorbing region could be satisfactorily fitted by assuming reasonable estimates of a-helical, p-structured, and random coil conformations somewhat different from ours, and by the inclusion of a small positive dichroic band centering at 226 mp. REFERENCES 1. MEN~NDEZ; C. J., AND HERSKOVITS, T. T., Abst. Biol. 136, 156th Meeting Am. Chem. in press. SOL, Sept., 1968; Biochemistry, 2. RICHARDS, F. M., AND VITHAYATHIL, P. J., J. Biol. Chem. 234, 1459 (1959). 3. SCHELLMAN, J. A., AND LOWE, M. J., J. Am. Chem. Sot. 90, 1070 (1968). A. M., SCATTURIN, A., AND 4. TAMBURRO, MORODER, L., Biochem. Biophys. Acta 164, 583, (1968). 5. URNES, P. J., AND DOTY, P, Advan. Protein Chem. 16, 401 (1961). 6. CATHOU, R. E., HAMMF,S, C. G., AND SHIMMEL, P. R., Biochemistry 4, 2687 (1965). 7. SIMPSON, R. T., AND VALEF,, B. L., Biochemistry 6.2531 (1966). 8. JIRGENSONS, B., J. Am. Chem. Sot. 89, 5979 (1967). 9. SIMMONS, E. R., AND BLOUT, E. R., J. Biol. Chem. 243, 218 (1968). 10. TOMIMATSU, Y., VITELLO, L., AND GAFPI~LD, W., Biopolymers 4, 653 (1966). 11. GREENFIELD, N., DAVIDSON, B., AND FASMAN, G. D., Biochemistry 6, 1630 (1967). 12. KARTHA, G., BELLO, J., AND HARKER, D., Nature 213, 862 (1967). 13. WYCKOFF, H. W., HARDMAN,K. D., ALLEWELL, N. M., INAGAMI, T., JOHNSON, L. N., AND RICHARDS, F. M., J. Biol. Chem. 242, 3984 (1967). 14. WOODY, R. W., PH. D. THESIS, University of California, 1962. 15. TINOCO, I. JR., WOODY, R. W., AND BRADLEY, D. F., J. Chem. Phys. 38, 1317 (1963). 16. BEYCHOK, S., In “Poly-a-Amino Acids,” (G. D. Fasman, ed.), Marcel Dekker, New York.
17. IIZUICA, E., AND YANG, J. T., Biochemistry 3, 1519 (1964). 18. ROSENBERG, A., J. Biol. Chem. 241, 5119 (1966). 19. SARKAR, P. K., AND DOTY, P., Proc. Nail. Acad. Sci., U.S. 66, 981 (1966). 20. IIZUKA, E., AND YANG, J. T., Biochemistry 7, 2218 (1968). 21. HOLZWARTH, G., AND DOTY, P., J. Am. Chem. Sot. 87, 218 (1965). 22. TOWNEND, R.,KUMOSINSKI,T. F., TIMASHEFF, S. N., FASYAN, G. D., AND DAVIDSON, B., Biochem. Biophys. Res. Commun. 23, 163 (1966). 23. PFLUMM, M. N., AND BEYCHOK, S., J. Biol. Chem. 244, 3973 (1969). THEODORE T. HERSKOVITS CELIA J. MEN~?NDEZ& Department of Chemistry Fordham University, New York iO458 Received June 12, 1969, accepted September lY, 1969 4 Present address: Department of Chemistry and Biological Sciences, Columbia University, New York.
Absence
of Diacetyl
in Fermenting
Wart
The presence of diacetyl in fermenting wort seems unquestioned, though the amount found has differed markedly depending on the analytical method used for its determination (14). The reason for the different values was attributed to a transitory metabolic intermediate present in fermenting wort, and converted into diacetyl during distillation (1, 2, 5, 6). We and our eoworkers recently identified this material as a-acetolactic acid, which is formed by yeast cel1.s as an intermediate in valine biosynthesis and excreted into the medium (7, 9, 13). We also showed that this acid was convert.ed into diacetyl by oxidative decarboxylation during distillation (7-9). 2,3-Pentanedione (1, 5) and a-aceto+ hydroxybutyric acid (14) were also reported to be present in fermenting wort together with diacetyl and a-acetolactic acid. a-Acetolactic acid and a-aceto-cY-hydroxybutyric acid are easily converted into the corresponding vicinal diketones, diacetyl, and 2,3pentanedione by oxidative decarboxylation (7, g-11), so it is very difficult to measure the exact amount of the vicinal diketones present with the a-aceto-ol-hydroxy acids. Various methods (1,
COMMUNICATIONS TABLE
455
I
cr-aceto-or-hydroxy acids are stable. As equimolar diacetyl and 2,3-pentanedione give the same value in this determination, concentrations of these four CONTENT IN FERMENTING WORT compounds concerned were expressed as ppm of diacetyl. Step 1. The yeast was removed from fermenting The vicinal diketone content of pasteurized wort by centrifugation at 3,000~ for 10 min at beer with increasing amount of added a-aceto-ol0”. The resulting clear supernatant was then hydroxy acid was measured by this method, as immediately used in the following determinashown in Table II. This method is accurate since tions. added a-aceto-or-hydroxy acid was quantitatively Step 2. To 100 ml of the supernatant was added recovered after evaporation of the vicinal dikeenough 1 N sodium hydroxide to neutralize tones (column 3), and the vicinal diketone content the same volume of degassed supernatant to did not vary on addition of cu-aceto-a-hydroxy pH 7.0. acid (column 4). Step 3. The solution is evaporated to about half Changes in the vicinal diketone and the OLvolume under 20-30 mm Hg (for 45 min) at a aceto-or-hydroxy acid content during primary bath-temperature below 50”. fermentation of bottom fermenting brewers’ yeast Step 4. After evaporation, the residue is adjusted were measured by this method (Fig. 1). The to pH 4.2 with 10 N sulfuric acid and made up to vicinal diketones were only present at the beginthe original volume. The solution is shaken in ning of fermentation. This result is in contrast to an Erlenmeyer flask to expose it to air and kept at 60” for 30 min in a loosely plugged flask. OL- our previous results (7, 8, 13) and those of other investigators (l-5), which indicated the presence Acetolactic acid and ol-aceto-cu-hydroxybutyric of the vicinal diketones during active fermentation acid are quantitatively converted into the of brewers’ yeast. Table II shows that this discorresponding vicinal diketones by this treatcrepancy is due to the analytical methods used. ment. Step 5. The vicinal diketone content in 20 ml of When the vicinal diketones are collected by this solution is measured as ppm of diacetyl by TABLE II the modification (8) of the “micro method” of VICINAL DIKETONE CONTENT OF BEER Owades et al. (3). CONTAINING a-ACETO-G-HYDROXY Step 6. Another 100 ml portion of the supernatant ACID MEASURED BY VARIOUS is treated as indicated in Steps 4 and 5, byMETHODS passing Steps 2 and 3. Step 7. The vicinal diketone content in the fera-*ceto- ~~~ceto- Vicinal diketone determined menting wort is calculated from the difference m-AC&oa;%ds: a;$dgy between the oc-aceto-a-hydroxy acid plus vicinal rr-hydroxy vicinal By the d&et,,ne termind By the By “acidacid diketone content (determined at Step 6) and added’ determined @ Step ’ me*od of?l!%!I at Step 6 m Table of T;ble but Step ’ ;:::; the a-aceto-a-hydroxy acid content (determined in Tab e I 2was 9 at Step 5). omitted
METHOD FOR ESTIMATING THE VICINAL DIKETONE
2,5), including our own (7,8), have been proposed for measuring the amount of the vicinal diketones in the presence of the or-aceto-a-hydroxy acids, but they have all failed to prevent oxidative decarboxylation of the rr-aceto-a-hydroxy acids. We have studied the influences of the redox potential (7,13), temperature (7,8), time (7, S), presence of metal ions (6, ll), and pH value (10) on oxidative decarboxylation of the a-aceto-a-hydroxy acids in fermenting wort and developed a method for measuring the vicinal diketones without any oxidative decarboxylation of coexisting cu-acetoa-hydroxy acids. Experimental details are given in Table I. In this method, the vicinal diketones are evaporated off from the centrifuged supernatant of fermenting wort at neutrality and at low temperature, under which conditions the
Ob 0.14 0.34 0.68 0 0.14 0.34 0.68
0.03 0.19 0.36 0.71
a-Acetolactic acid 0 0.03 0.16 0.03 0.33 0.03 0.68 0.03
a-Aceto-cu-hydroxybutyric 0.03 0 0.03 0.19 0.16 0.03 0.37 0.34 0.03 0.71 0.68 0.03
0.03 0.05 0.07 0.12
0.03 0.12 0.22 0.40
acid 0.03 0.05 0.07 0.09
0.03 0.10 0.18 0.24
a cu-Acetoa-hydroxy acids, which were prepared by the method of Krampitz (12), were buffered with ~/30 potassium-phosphate buffer, pH 7.0, and contaminating vicinal diketones were removed by vacuum distillation. b All figures are expressed as ppm of diacetyl.
456
COMMUNICATIONS
0
I
2
3
FERMENTATION
4
5
6
IN DAYS
FIG. 1. Changes in the vicinal diketone and the a-aceto-a-hydroxy acid content during primary fermentation of beer. (0) : Vicinal diketones; (0) : ol-aceto-cx-hydroxy acids; (A) : yeast in suspension; (A) : reducing sugars. distillation or sweeping at 65”, almost all of the or-aceto-or-hydroxy acids present will be oxidatively decarboxylated, because on incubation at 60” for 30 min, added ol-aceto-ol-hydroxy acid was quantitatively converted into the corresponding vicinal diketone (column 2). Even on distillation under reduced pressure (at low temperature), unless the sample solution was neutralized (column 5), the vicinal diketone content increased on increasing the amount of ol-aceto-a-hydroxy acid added, indicating that the acid was oxidatively decarboxylated during determinations. The head space techniques (1, 14) and the vacuum distillation methods (4, 5) reported so far were carried out at low temperature, but not at neutrality. Column 6 shows that the ‘Lacid-treatment” we proposed (7, 8) also failed to prevent oxidative decarboxylation of the cY-aceto-or-hydroxy acids. We consider that the absence of the vicinal diketones during active fermentation will hold true with other fermentation systems than a continental lager fermentation system studied here. The vicinal diketones in finished beer are formed after primary fermentation is complete (10) by oxidative decarboxylation of the a-acetool-hydroxy acids, which were formed by yeast cells and passed into the fermenting medium (7, 13). The question recently raised by Lewis (15) about t,he mechanism we proposed for diacetyl formation (7, 13) is thus answered by the present findings. ACKNOWLEDGMENT We thank Dr. Y. Okuda for helpful suggestions, and also Mr. A. Takahashi and Mr. T. Suzuki of Kirin Brewery Co., Ltd. for permission to publish
this paper. We are also grateful to Dr. Y. Kuroiwa for his guidance and encouragement, and Miss K. Yamada for her technical assistance. REFERENCES 1. HARRISON, G. A. F., BYRNE, W. J., AND COLLINS, E., J. Inst. Brewing 71,336 (1965). 2. MAULE, D. R., PINNEGAR, M. A., PORTNO, A. D., AND WHITEAR, A. L., J. Inst. Brewing 72, 488 (1966). 3. OWADES, J. L., AND JAKOVAC, J. A., Am. Sot. Brewing Chemists, Proc. 22 (1963). 4. LATIMER, R. A., GLENISTER, P. R., KOEPPLE, K. G., AND DALLOS, F. C., Tech. Quart. Master Brewers Ass. Am. 6, 24 (1969). 5. SHIGEMATSU, N., KITAZAWA, Y., AND YABUUCHI, S., Bulletin Brew. Sci. 10, 45 (1964). 6. SHIGEMATSU, N., AND YABUUCHI, S., Bulletin Brew. Sci. 12, 53 (1966). 7. INOUE, T., MASUYAMA, K., YAMAMOTO, Y., OKADA, K., AND KUROIWA, Y., Am. Sot. Brewing Chemists, Proc. 158 (1968). 8. INOUE, T., MASUYAMA, K., YAMAMOTO, Y., AND OKADA, K., Rept. Res. Lab. Kirin Brewery Co., Ltd. No. 11, 1, (1968). 9. MASUYAMA, K., INOUE, T., YAMAMOTO, Y., OKADA, K., AND KUROI~A, Y., Rept. Res. Lab. Kirin Brewery Co., Ltd. No. 11, 9 (1969). 10. INOUE, T., AND YAMAMOTO, Y., Unpublished data. 11. DE MAN, J. C., Res. Trav. Chim. 78, 480 (1959). 12. KRAMPITZ, L. O., Arch. Biochem. 17.81 (1948).
COMMUNICATIONS 13. INOUE, T., MASUYAMA, K., YAMAMOTO, Y,. AND OKADA, K., Rept. Res. Lab. Kirin Brewery Co., Ltd. No. 11, 17 (1968). 14. SUOMALAINEN, H., AND RONKAINEN, P., Nature 220, 792 (1968). 15. LEWIS, M. J., The Brewers Digest, Sept. 74 (1968).
The Research Ltd.
457
Laboratories
TAKASHI INOUE YASUSHI YAMAMOTO of Kirin Brewery Co.,
Takasaki, Gunma Pref., Japan Received July 22, 1969, accepted September 1969
13,
454
able2-a fact which should not be minimizedthese results suggest the possiblity that the Sprotein may have suffered moderate changes in its secondary structure relat,ive to the solution structure of RNase. After this note was submitted for publication a detailed study and analysis of the circular dichroism spectra of RNase, RNase-S, and S-protein has been published by Pflumm and Beychok (23). These authors have found t.hat the RNase spectrum in the ultraviolet, peptide-absorbing region could be satisfactorily fitted by assuming reasonable estimates of a-helical, p-structured, and random coil conformations somewhat different from ours, and by the inclusion of a small positive dichroic band centering at 226 mp. REFERENCES 1. MEN~NDEZ; C. J., AND HERSKOVITS, T. T., Abst. Biol. 136, 156th Meeting Am. Chem. in press. SOL, Sept., 1968; Biochemistry, 2. RICHARDS, F. M., AND VITHAYATHIL, P. J., J. Biol. Chem. 234, 1459 (1959). 3. SCHELLMAN, J. A., AND LOWE, M. J., J. Am. Chem. Sot. 90, 1070 (1968). A. M., SCATTURIN, A., AND 4. TAMBURRO, MORODER, L., Biochem. Biophys. Acta 164, 583, (1968). 5. URNES, P. J., AND DOTY, P, Advan. Protein Chem. 16, 401 (1961). 6. CATHOU, R. E., HAMMF,S, C. G., AND SHIMMEL, P. R., Biochemistry 4, 2687 (1965). 7. SIMPSON, R. T., AND VALEF,, B. L., Biochemistry 6.2531 (1966). 8. JIRGENSONS, B., J. Am. Chem. Sot. 89, 5979 (1967). 9. SIMMONS, E. R., AND BLOUT, E. R., J. Biol. Chem. 243, 218 (1968). 10. TOMIMATSU, Y., VITELLO, L., AND GAFPI~LD, W., Biopolymers 4, 653 (1966). 11. GREENFIELD, N., DAVIDSON, B., AND FASMAN, G. D., Biochemistry 6, 1630 (1967). 12. KARTHA, G., BELLO, J., AND HARKER, D., Nature 213, 862 (1967). 13. WYCKOFF, H. W., HARDMAN,K. D., ALLEWELL, N. M., INAGAMI, T., JOHNSON, L. N., AND RICHARDS, F. M., J. Biol. Chem. 242, 3984 (1967). 14. WOODY, R. W., PH. D. THESIS, University of California, 1962. 15. TINOCO, I. JR., WOODY, R. W., AND BRADLEY, D. F., J. Chem. Phys. 38, 1317 (1963). 16. BEYCHOK, S., In “Poly-a-Amino Acids,” (G. D. Fasman, ed.), Marcel Dekker, New York.
17. IIZUICA, E., AND YANG, J. T., Biochemistry 3, 1519 (1964). 18. ROSENBERG, A., J. Biol. Chem. 241, 5119 (1966). 19. SARKAR, P. K., AND DOTY, P., Proc. Nail. Acad. Sci., U.S. 66, 981 (1966). 20. IIZUKA, E., AND YANG, J. T., Biochemistry 7, 2218 (1968). 21. HOLZWARTH, G., AND DOTY, P., J. Am. Chem. Sot. 87, 218 (1965). 22. TOWNEND, R.,KUMOSINSKI,T. F., TIMASHEFF, S. N., FASYAN, G. D., AND DAVIDSON, B., Biochem. Biophys. Res. Commun. 23, 163 (1966). 23. PFLUMM, M. N., AND BEYCHOK, S., J. Biol. Chem. 244, 3973 (1969). THEODORE T. HERSKOVITS CELIA J. MEN~?NDEZ& Department of Chemistry Fordham University, New York iO458 Received June 12, 1969, accepted September lY, 1969 4 Present address: Department of Chemistry and Biological Sciences, Columbia University, New York.
Absence
of Diacetyl
in Fermenting
Wart
The presence of diacetyl in fermenting wort seems unquestioned, though the amount found has differed markedly depending on the analytical method used for its determination (14). The reason for the different values was attributed to a transitory metabolic intermediate present in fermenting wort, and converted into diacetyl during distillation (1, 2, 5, 6). We and our eoworkers recently identified this material as a-acetolactic acid, which is formed by yeast cel1.s as an intermediate in valine biosynthesis and excreted into the medium (7, 9, 13). We also showed that this acid was convert.ed into diacetyl by oxidative decarboxylation during distillation (7-9). 2,3-Pentanedione (1, 5) and a-aceto+ hydroxybutyric acid (14) were also reported to be present in fermenting wort together with diacetyl and a-acetolactic acid. a-Acetolactic acid and a-aceto-cY-hydroxybutyric acid are easily converted into the corresponding vicinal diketones, diacetyl, and 2,3pentanedione by oxidative decarboxylation (7, g-11), so it is very difficult to measure the exact amount of the vicinal diketones present with the a-aceto-ol-hydroxy acids. Various methods (1,
COMMUNICATIONS TABLE
455
I
cr-aceto-or-hydroxy acids are stable. As equimolar diacetyl and 2,3-pentanedione give the same value in this determination, concentrations of these four CONTENT IN FERMENTING WORT compounds concerned were expressed as ppm of diacetyl. Step 1. The yeast was removed from fermenting The vicinal diketone content of pasteurized wort by centrifugation at 3,000~ for 10 min at beer with increasing amount of added a-aceto-ol0”. The resulting clear supernatant was then hydroxy acid was measured by this method, as immediately used in the following determinashown in Table II. This method is accurate since tions. added a-aceto-or-hydroxy acid was quantitatively Step 2. To 100 ml of the supernatant was added recovered after evaporation of the vicinal dikeenough 1 N sodium hydroxide to neutralize tones (column 3), and the vicinal diketone content the same volume of degassed supernatant to did not vary on addition of cu-aceto-a-hydroxy pH 7.0. acid (column 4). Step 3. The solution is evaporated to about half Changes in the vicinal diketone and the OLvolume under 20-30 mm Hg (for 45 min) at a aceto-or-hydroxy acid content during primary bath-temperature below 50”. fermentation of bottom fermenting brewers’ yeast Step 4. After evaporation, the residue is adjusted were measured by this method (Fig. 1). The to pH 4.2 with 10 N sulfuric acid and made up to vicinal diketones were only present at the beginthe original volume. The solution is shaken in ning of fermentation. This result is in contrast to an Erlenmeyer flask to expose it to air and kept at 60” for 30 min in a loosely plugged flask. OL- our previous results (7, 8, 13) and those of other investigators (l-5), which indicated the presence Acetolactic acid and ol-aceto-cu-hydroxybutyric of the vicinal diketones during active fermentation acid are quantitatively converted into the of brewers’ yeast. Table II shows that this discorresponding vicinal diketones by this treatcrepancy is due to the analytical methods used. ment. Step 5. The vicinal diketone content in 20 ml of When the vicinal diketones are collected by this solution is measured as ppm of diacetyl by TABLE II the modification (8) of the “micro method” of VICINAL DIKETONE CONTENT OF BEER Owades et al. (3). CONTAINING a-ACETO-G-HYDROXY Step 6. Another 100 ml portion of the supernatant ACID MEASURED BY VARIOUS is treated as indicated in Steps 4 and 5, byMETHODS passing Steps 2 and 3. Step 7. The vicinal diketone content in the fera-*ceto- ~~~ceto- Vicinal diketone determined menting wort is calculated from the difference m-AC&oa;%ds: a;$dgy between the oc-aceto-a-hydroxy acid plus vicinal rr-hydroxy vicinal By the d&et,,ne termind By the By “acidacid diketone content (determined at Step 6) and added’ determined @ Step ’ me*od of?l!%!I at Step 6 m Table of T;ble but Step ’ ;:::; the a-aceto-a-hydroxy acid content (determined in Tab e I 2was 9 at Step 5). omitted
METHOD FOR ESTIMATING THE VICINAL DIKETONE
2,5), including our own (7,8), have been proposed for measuring the amount of the vicinal diketones in the presence of the or-aceto-a-hydroxy acids, but they have all failed to prevent oxidative decarboxylation of the rr-aceto-a-hydroxy acids. We have studied the influences of the redox potential (7,13), temperature (7,8), time (7, S), presence of metal ions (6, ll), and pH value (10) on oxidative decarboxylation of the a-aceto-a-hydroxy acids in fermenting wort and developed a method for measuring the vicinal diketones without any oxidative decarboxylation of coexisting cu-acetoa-hydroxy acids. Experimental details are given in Table I. In this method, the vicinal diketones are evaporated off from the centrifuged supernatant of fermenting wort at neutrality and at low temperature, under which conditions the
Ob 0.14 0.34 0.68 0 0.14 0.34 0.68
0.03 0.19 0.36 0.71
a-Acetolactic acid 0 0.03 0.16 0.03 0.33 0.03 0.68 0.03
a-Aceto-cu-hydroxybutyric 0.03 0 0.03 0.19 0.16 0.03 0.37 0.34 0.03 0.71 0.68 0.03
0.03 0.05 0.07 0.12
0.03 0.12 0.22 0.40
acid 0.03 0.05 0.07 0.09
0.03 0.10 0.18 0.24
a cu-Acetoa-hydroxy acids, which were prepared by the method of Krampitz (12), were buffered with ~/30 potassium-phosphate buffer, pH 7.0, and contaminating vicinal diketones were removed by vacuum distillation. b All figures are expressed as ppm of diacetyl.
456
COMMUNICATIONS
0
I
2
3
FERMENTATION
4
5
6
IN DAYS
FIG. 1. Changes in the vicinal diketone and the a-aceto-a-hydroxy acid content during primary fermentation of beer. (0) : Vicinal diketones; (0) : ol-aceto-cx-hydroxy acids; (A) : yeast in suspension; (A) : reducing sugars. distillation or sweeping at 65”, almost all of the or-aceto-or-hydroxy acids present will be oxidatively decarboxylated, because on incubation at 60” for 30 min, added ol-aceto-ol-hydroxy acid was quantitatively converted into the corresponding vicinal diketone (column 2). Even on distillation under reduced pressure (at low temperature), unless the sample solution was neutralized (column 5), the vicinal diketone content increased on increasing the amount of ol-aceto-a-hydroxy acid added, indicating that the acid was oxidatively decarboxylated during determinations. The head space techniques (1, 14) and the vacuum distillation methods (4, 5) reported so far were carried out at low temperature, but not at neutrality. Column 6 shows that the ‘Lacid-treatment” we proposed (7, 8) also failed to prevent oxidative decarboxylation of the cY-aceto-or-hydroxy acids. We consider that the absence of the vicinal diketones during active fermentation will hold true with other fermentation systems than a continental lager fermentation system studied here. The vicinal diketones in finished beer are formed after primary fermentation is complete (10) by oxidative decarboxylation of the a-acetool-hydroxy acids, which were formed by yeast cells and passed into the fermenting medium (7, 13). The question recently raised by Lewis (15) about t,he mechanism we proposed for diacetyl formation (7, 13) is thus answered by the present findings. ACKNOWLEDGMENT We thank Dr. Y. Okuda for helpful suggestions, and also Mr. A. Takahashi and Mr. T. Suzuki of Kirin Brewery Co., Ltd. for permission to publish
this paper. We are also grateful to Dr. Y. Kuroiwa for his guidance and encouragement, and Miss K. Yamada for her technical assistance. REFERENCES 1. HARRISON, G. A. F., BYRNE, W. J., AND COLLINS, E., J. Inst. Brewing 71,336 (1965). 2. MAULE, D. R., PINNEGAR, M. A., PORTNO, A. D., AND WHITEAR, A. L., J. Inst. Brewing 72, 488 (1966). 3. OWADES, J. L., AND JAKOVAC, J. A., Am. Sot. Brewing Chemists, Proc. 22 (1963). 4. LATIMER, R. A., GLENISTER, P. R., KOEPPLE, K. G., AND DALLOS, F. C., Tech. Quart. Master Brewers Ass. Am. 6, 24 (1969). 5. SHIGEMATSU, N., KITAZAWA, Y., AND YABUUCHI, S., Bulletin Brew. Sci. 10, 45 (1964). 6. SHIGEMATSU, N., AND YABUUCHI, S., Bulletin Brew. Sci. 12, 53 (1966). 7. INOUE, T., MASUYAMA, K., YAMAMOTO, Y., OKADA, K., AND KUROIWA, Y., Am. Sot. Brewing Chemists, Proc. 158 (1968). 8. INOUE, T., MASUYAMA, K., YAMAMOTO, Y., AND OKADA, K., Rept. Res. Lab. Kirin Brewery Co., Ltd. No. 11, 1, (1968). 9. MASUYAMA, K., INOUE, T., YAMAMOTO, Y., OKADA, K., AND KUROI~A, Y., Rept. Res. Lab. Kirin Brewery Co., Ltd. No. 11, 9 (1969). 10. INOUE, T., AND YAMAMOTO, Y., Unpublished data. 11. DE MAN, J. C., Res. Trav. Chim. 78, 480 (1959). 12. KRAMPITZ, L. O., Arch. Biochem. 17.81 (1948).
COMMUNICATIONS 13. INOUE, T., MASUYAMA, K., YAMAMOTO, Y,. AND OKADA, K., Rept. Res. Lab. Kirin Brewery Co., Ltd. No. 11, 17 (1968). 14. SUOMALAINEN, H., AND RONKAINEN, P., Nature 220, 792 (1968). 15. LEWIS, M. J., The Brewers Digest, Sept. 74 (1968).
The Research Ltd.
457
Laboratories
TAKASHI INOUE YASUSHI YAMAMOTO of Kirin Brewery Co.,
Takasaki, Gunma Pref., Japan Received July 22, 1969, accepted September 1969
13,