On Various Factors Affecting Formation of Isobutanol and Isoamyl Alcohol During Alcoholic Fermentation
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[Agr. Biol. Chem., Vol. 30, No. 7, p. 634641, 1966] On Various Factors Affecting Formation of Isobutanol and Isoamyl Alcohol during Alcoholic Fermentation By Kiyoshi YOSHIZAwA Research Institute of Brewing Received January 14, 1966 In the previous reports, we remarked that the ratio of isoamyl alcohol content (includes active amyl alcohol) to that of isobutanol (A/B ratio) in the alcoholic beverage made from grain is lower than that from fruit juice. In this rseport, the reason why these differences occur was examined. It was certain that the differences of amino acids content and yeast strain used for fermentation are the most effective factors affecting the difference of A/B ratio among alcoholic beverages. INTRODUCTION ally contains less higher alcohols and its A/B In the previous reports,1•`3) we noted that ratio is lower comparing with the latter. there exists a remarkable difference in the The results are summarized in Table I. For composition of higher alcohols between an instance, except American whisky A/B ratio alcoholic beverage made from grain such as of brandy is higher than that of whisky. beer and whisky and that made from fruit Furthermore, there exists a remarkable dif such as wine and brandy. The former gener ference in A/B ratio among whisky types, i.e., TABLE I. HIGHER ALCOHOLS CONTENT IN VARIOUS ALCOHOLIC BEVERAGES * It includes the amount of active amyl alcohol . ** Average value . *** Standard deviation . 1) K. Yoshizawa, "Gas Chromatography", Kagaku no Scotch 1.2, American 5.4, Canadian 3.7, Irish Ryoiki No. 46, Vol. 2, p. 121 (1961). 2) -K. Yoshizawa and M. Yamada, J. Soc. Brewing, Japan, 1.6 and Japanese special class 2.5. A/B ratio 59, 629 (1964). seems to be one of the most useful signs 3) K. Yoshizawa, K. Otsuka and S. Imai, I. Soc. Brewing, Japan, 60, 263 (1965). distinguishing the type of these alcoholic On Various Factors Affecting Formation of Isobutanol and Isoamyl Alcohol 635 beverages each other. TABLE III. THE COMPOSITION OF THE CASAMINO The characteristic difference of this ratio ACID-ADDED MEDIUM may be come from the difference of raw materials and procedures among these alcoholic beverages. Table II summarizes main dif ferences between production of grain-made- alcoholic beverages such as beer and whisky and that of fruit-made-alcoholic beverages such as wine and brandy. Besides, Crowell and Guymon4) reported that, in wine making, an aerated condition increased formation of isobutanol and diacetyl. We noted also the same phenomenon in whisky makings' TABLE II. MAIN DIFFERENCES IN PROCEDURE BETWEEN ALCOHOLIC BEVERAGES MADE FROM GRAIN AND THOSE FROM FRUIT This report deals with investigations on the effects of amino acids content and pH in the tained 5 g/l of casamino acid as a sole nitrogen source fermenting medium, aeration, fermentation (it is called the amino acid-rich medium), another temperature and yeast strain used for fermen 0.5 g/l of casamino acid (amino acid-poor medium). tation upon the amounts of higher alcohols Besides, the medium containing 2.4g of ammonium formed and A/B ratio. chloride and 0.5 g of casamino acid in 1 1 (ammonium- added medium) was used to examine the effect of EXPERIMENTAL ammonium ion on formation of higher alcohols. Yeast. To examine the effect of two types of The fermented medium was separated from yeast by centrifugation and analyzed. yeast on formation of higher alcohols, two strains of brewery yeast, Saccharomyces cereaisiae (Bass beer yeast Analysis. The higher alcohols formed were IAM 4839) and Saccharomycescarlsbergensis (IAM 4729), identified and determined their amounts by gas and two strains of wine yeast, OC No. 2 and Johan chromatography described in the previous report.2) nesberger II, were used. Amino acids were identified and determined their amounts by Kjeldahl method and column chromato Medium and Fermentation. One platinum ear of yeast cells was added in a 500 ml Erlenmeyer graphy. flask containing 150 ml of medium, and incubated at RESULTS 15°C and 30°C, respectively. As shown in Table III, I. Effects of casamino acid content, yeast two types of medium were used, one of which con- strain and fermentation temperature on the 4) E. A. Crowell and J. F. Guymon, Am. f. Enol. Viticulture, formation of higher alcohols 14,214 (1963). 5) K. Yoshizawa, K. Otsuka, S. Uehara, S. Shibata, T. To examine the effects of amino acids con- Tezuka, S. Imai and H. Okubo, J. Soc. Brewing, Japan, 58, tent in the medium, which is considered one 1205 (1963). 636 Kiyoshi YOSHIZAWA of the most dominant differences between the making process of alcoholic beverages made from grain and those made from fruit, yeast cells were inoculated in the media and fer mented at 15°C and 30°C, respectively. In the amino acid-rich medium every yeast grew vigorously, but in the amino acid-poor medium every yeast, especially Saccharomyces carlsbergensis at 30°C, showed decrease in growth and fermentation velocity. Generally speaking, wine yeasts grew and fermented more vigorously than brewery yeasts. As FIG. 1. Ethanol Formed in the Fermented Casamino shown in Tables IV and V, the amount of Acid-Added Media. casamino acid reduced and higher alcohols formed differed largely among the fermented media. Every yeast strain incubated in the amino acid-poor medium formed a smaller R The medium contains 5g/1 of casamino acid amount of higher alcohols than in the amino p The medium contains 0.5g/1 of casamino acid N The medium contains 0.5 g/1 of casamino acid acid-rich medium. A/B ratio in the fermented and 2.4 g/1 of NH4C1 TABLE IV. FERMENTATION OF GLUCOSE WITH CASAMINO ACID DIFFERED IN AMOUNTS * Expressed as g/100 ml . R The medium contains 5g/1 of casamino acid. P The medium contains 0.5 g/1 of casamino acid. N The medium contains 0.5 g of casamino acid and 2.4 g of NH4C1 in 11. TABLE V. ISOBUTANOL AND ISOAMYL ALCOHOL FORMED FROM FERMENTATION OF GLUCOSE WITH CASAMINO ACID DIFFERED IN AMOUNTS * Expressed as mg/100 ml . R The medium contains 5g/1 of casamino acid. P The medium contains 0.5g/1 of casamino acid. N The medium contains 0.5 g of casamino acid and 2.4 g of NH4C1 in 11. On Various Factors Affecting Formation of Isobutanol and I soamyl Alcohol 637 FIG. 2. Isoamyl Alcohol and Isobutanol, and A/B Ratio in the Fermented Casamino Acid-Added Media. R The medium contains 5g/1 of casamino acid P The medium contains 0.5g/1 of casamino acid N The medium contains 0.5 g/1 of casamino acid and 2.4 g of NH4CI amino acid-poor medium was larger than beverage, which contains far less assimilable that in the amino acid-rich medium. In the amino acids in its raw material than the ammonium-added medium the formation of former. So the content of amino acids and higher alcohols decreased largely. ammonium in the fermenting wort and must These results coincide well with the findings is thought to be one of the most important that A/B ratio of a grain-made-alcoholic bever factors which affect the composition of higher age is lower than that of a fruit-made-alcoholic alcohols, such as A/B ratio, in the alcoholic 638 Kiyoshi YOSHIZAWA beverage produced. Yeast strain and fermen of the medium inoculated with wine yeast tation temperature are also important factors OC No. 2. Higher alcohols formation, es affecting the composition of higher alcohols pecially isobutanol formation, increased and formed. A/B ratio decreased. II. Effects of pH and aeration on the formation III. Effects of amino acids content in wort and of higher alcohols grape must on the formation of higher Bass beer yeast and wine yeast OC No. 2 alcohols were inoculated in the media, which had As shown in Table VI, the composition of various pH values, and fermented at 30°C. amino acids in the casamino acid used differs As shown in Fig. 3, some definite difference from that of wort and grape must, from could be observed among the composition of which beer, whisky, wine and brandy are higher alcohols formed in the fermented made. To ascertain if the composition of media. amino acids in the fermenting medium such An aeration condition was made by shaking as wort and grape must affects the formation a 500 ml Erlenmeyer flask containing 100 ml of higher alcohols, the wort diluted with 4 vol. of water and supplemented with a TABLE VI. THE COMPOSITION OF AMINO ACID IN THE CASAMINO ACID-ADDED MEDIUM COMPARED WITH THAT IN WORT AND GRAPE JUICE FIG. 3. Isoamyl Alcohol, Isobutanol and A/B Ratio in the Fermented Media with Various pH Values and Aeration Conditions. * The medium contains 5g of casamino acids in 11 . On Various Factors Affecting Formation of Isobutanol and Isoamyl Alcohol 639 TABLE VII. HIGHER ALCOHOLSFORMED IN THE FERMENTED WORT AND GRAPE JUICE WITH DIFFERENT AMINO ACID CONTENTS * Sugar content , 10 g/100 ml as glucose. ** Sugar content , 16 g/100 ml as glucose. deficient amount of amino acids was fermented decarboxylated to an aldehyde, which un and the content of higher alcohols formed dergoes reduction to an alcohol."" Thus was compared with that using the original yeast can produce isobutanol from valine,. wort. Besides, the grape must fortified with isoamyl alcohol from leucine, n-propanol from 2 g/1 of casamino acid was fermented and the threonine and a-amino-n-butyric acid and content of higher alcohols formed was com- active amyl alcohol from isoleucine in existence pared with that using the unfortified one.