On Various Factors Affecting Formation of Isobutanol and Isoamyl Alcohol During Alcoholic Fermentation

[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. of excess of these amino acids, but insufficiency To eliminate any effect of deficiency of vita of these amino acids for yeast growth may mins and minerals which may be caused from stimulate synthesis of these amino acids,, dilution, sufficient amounts of these materials through which higher alcohols are formed as were added in every medium. As shown in the branched reaction products of these Table VII, the fermented media contained syntheses. much amino acids contained much higher These findings may lead to the following alcohols and their A/B ratio were lower than consideration that, since the formation of those of the fermented media contained less higher alcohols through different pathway is. amino acids. From these results it may be controlled by contents of these amino acids thought that difference of amino acids content contained in the fermenting medium, dif between grain wort and fruit juice is one of ference of amino acids content between wort the most important factors affecting the com- and fruit, as shown in Table VI, may cause position of higher alcohols of the alcoholic the different composition of higher alcoholss beverages produced. between a grain-made-alcoholic beverage such as beer and whisky and a fruit-made-alcoholic DISCUSSION beverage such as wine and brandy. The Recently many works have been done to results of analysis of higher alcohols in variouss know the mechanism of higher alcohols for alcoholic beverages, as shown in Tables V mation and it has been elucidated that higher and VII, that yeast inoculated in the amino alcohols are formed mainly through two path- acid-rich medium produced larger amounts. ways, one of which is well-known Ehrlich's of higher alcohols and lower A/B ratio com- scheme, i.e., an amino acid undergoes deamina pared with that in the amino acid-poor medi tion followed by decarboxylation to an alde um ascertain the reality of the consideration. hyde, which is reduced to an alcohol having As shown in Fig. 4, since the amounts of one carbon atom less than that of the parent the higher alcohols formed per the amounts amino acid. Another pathway includes forma of amino acids reduced in the amino acid-rich tion of an ƒ¿-keto acid from glucose or some 6) K. Yoshizawa, This Journal, 26, 494 (1962); 27, 162 amino acids through the synthetic pathway (1963); 28, 801 (1964); 28, 279 (1964); 29, 671 (1965). 7) W.C. Lawrence, Wallerstein Communications, 27, 124 of amino acids. The ƒ¿-keto acid formed is (1964). 640 Kiyoshi YOSHIZAWA

decarboxylation to form aldehydes, from which higher alcohols are formed. The composition of higher alcohols may depend on the ability of amino acids synthesis of yeast strain. For instance, A/B ratios of the fer mented amino acid-poor media by various yeast strains were 4.0-5.5; existence of suf ficient amounts of amino residue and am monium in the medium increases formation of amino acids and decreases higher alcohols formation. As shown in Table VIII, in the fermented medium contained 5 g/l of casamino acid, isobutanol/valine (%) was almost equal to isoamyl alcohol/leucine (%), and A/B ratio/ leucine/valine in the medium was about 1. FIG. 4. Isoamyl Alcohol and Isobutanol Formed per According to decreasing casamino acids con- Casamino Acids-N Reduced in the Fermented tentt added in the medium, isobutanol/valine Casamino Acid-Added Media. (%) and especially isoamyl alcohol/leucine (%) increased, and A/B ratio rose gradually. As a result, A/B ratio became higher than leucine/valine added in the medium according to the decreasing amounts of casamino acid R The medium contains 5g/1 of casamino acid P The medium contains 0.5g/1 of casamino acid added in the medium. The results that N The medium contains 0.5g/1 of casamino acid , in and 2.4 g/1 of NH.C1 the medium contained 2 g/l of casamino acid, the yield of isoamyl alcohol formed against leucine added in the medium was over 100%, medium were smaller than those in the amino contrary to the yield of isobutanol against acid-poor medium, it is considered that, in valine (80%), may show that isoamyl alcohol the amino acid-rich medium, higher alcohols is formed more easily than isobutanol from are formed mainly through Ehrlich's scheme from amino acids intactly incorporated into glucose through the synthetic pathway. yeast cells. The amount of each higher TABLE VIII. CHANGES OF A/B RATIO IN THE alcohol formed is regulated by the amount of FERMENTED MEDIA WITH VARIOUS the corresponding precursor amino acid in AMOUNTS OF CASAMINO ACID corporated into yeast cells or contained in the medium. For instance, A/B ratios of the fermented amino acid-rich media are 2.3-3.6, and leucine (with isoleucine)/valine added in these media is 2. In the amino acid-poor medium yeast may synthesize amino acids required for its growth from glucose through * The amount added in the medium . the pathways of amino acids synthesis . If there are insufficient amounts of amino residue SUMMARY and ammonium required for amination of the 1. Effects of amino acids content , pH, f accumulated keto acids, the keto acids undergo ermentation temperature, aeration of the On Various Factors Affecting Formation of Isobutanol and Isoamyl Alcohol 641 fermenting medium and yeast strain on the alcoholic beverage and a fruit-made-alcoholic formation of higher alcohols were studied. beverage. 2. The contents of amino acids and am 3. Fermentation temperature, aeration of monium affected largely the composition of the fermenting medium and yeast strain used higher alcohols formed, and are considered affect the composition of higher alcohols one of the most important factors making the formed. different A/B ratio between a grain-made