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AROMA AND FLAVOR OF JAPANESE SOY SAUCE BY TAMOTGU YOKOTSUKA No& Institute for Scientific Re.rcnrch, atld Research ltlstitirtc of Noda Soy Sauce Company. Lfd., Nocla.shf. Chfba.ken. Japan Page I . Introduction . . . . . . . . . . . . . 75 I1 . Composition . . . . . . . . . . . . . 77 A . Analysis . . . . . . . . . . . . . 77 B . Nitrogen-containing Compounds . . . . . . . . 78 C . Carbohydrates. Sugar. Alcohols. and Extractive . . . . . 82 D . Acids. Salts. and Related Compounds . . . . . . . &3 E . Color . . . . . . . . . . . . . . 87 I11 . Chemical Components of Flavor . . . . . . . . . 87 A . Fraction I . . . . . . . . . . . . . 96 B . Fraction I1 (b.p. < 78°C.) . . . . . . . . . 97 C . Fraction I11 (b.p. >78”C.) . . . . . . . . . 99 D . Fraction IV (The Aretal Fraction) . . . . . . . g9 E . Fraction VI . . . . . . . . . . . . 10 IV . Sources of Aroma and Flavor Development . . . . . . 104 A . Raw Materials . . . . . . . . . . . . 104 B.Processing . . . . . . . . . . . . . loFl C . Koji . . . . . . . . . . . . . . 108 D . Mash (Moromi) . . . . . . . . . . . 111 E . Pasteurization . . . . . . . . . . . . 115 V . Flavor Ingredients as Natural Preservatives . . . . . . 119 A . Natural Yeast-static and Bactericidal Compounds . . . . . 119 B . Artificial Preservatives for Soy Sauce . . . . . . . 121 VI . Summary . . . . . . . . . . . . . 121 Acknowledgments . . . . . . . . . . . . 123 References . . . . . . . . . . . . . 123 I . INTRODUCTION The four major characteristics of Japanese soy sauce. which differs from other types produced in the Orient. are aroma. flavor. color. and stability . About 60 years ago. certain chemical procedures were intro- duced into the Japanese soy sauce industry. and since then the fermen- tation period has been shorter . Although these chemical procedures have come into general use. the basic method of manufacture is almost unchanged . 75

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Page 1: [Advances in Food Research] Advances in Food Research Volume 10 Volume 10 || Aroma and Flavor of Japanese Soy Sauce

AROMA AND FLAVOR OF JAPANESE SOY SAUCE

BY TAMOTGU YOKOTSUKA

No& Institute for Scientific Re.rcnrch, atld Research ltlstitirtc of Noda Soy Sauce Company. Lfd., Nocla.shf. Chfba.ken. Japan

Page I . Introduction . . . . . . . . . . . . . 75 I1 . Composition . . . . . . . . . . . . . 77

A . Analysis . . . . . . . . . . . . . 77 B . Nitrogen-containing Compounds . . . . . . . . 78 C . Carbohydrates. Sugar. Alcohols. and Extractive . . . . . 82 D . Acids. Salts. and Related Compounds . . . . . . . &3 E . Color . . . . . . . . . . . . . . 87

I11 . Chemical Components of Flavor . . . . . . . . . 87 A . Fraction I . . . . . . . . . . . . . 96 B . Fraction I1 (b.p. < 78°C.) . . . . . . . . . 97 C . Fraction I11 (b.p. >78”C.) . . . . . . . . . 99 D . Fraction IV (The Aretal Fraction) . . . . . . . g9 E . Fraction VI . . . . . . . . . . . . 1 0

IV . Sources of Aroma and Flavor Development . . . . . . 104 A . Raw Materials . . . . . . . . . . . . 104 B.Processing . . . . . . . . . . . . . loFl C . Koji . . . . . . . . . . . . . . 108 D . Mash (Moromi) . . . . . . . . . . . 111 E . Pasteurization . . . . . . . . . . . . 115

V . Flavor Ingredients as Natural Preservatives . . . . . . 119 A . Natural Yeast-static and Bactericidal Compounds . . . . . 119 B . Artificial Preservatives for Soy Sauce . . . . . . . 121

VI . Summary . . . . . . . . . . . . . 121 Acknowledgments . . . . . . . . . . . . 123 References . . . . . . . . . . . . . 123

I . INTRODUCTION

The four major characteristics of Japanese soy sauce. which differs from other types produced in the Orient. are aroma. flavor. color. and stability . About 60 years ago. certain chemical procedures were intro- duced into the Japanese soy sauce industry. and since then the fermen- tation period has been shorter . Although these chemical procedures have come into general use. the basic method of manufacture is almost unchanged .

75

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76 T A M OTSU Y OKOTSU K A

Oiir ancestors knew tlici tcwhnicliies of enzymatically hydrolyzing cer- tain protein foods into amino acids to make them more appealing. For example, since ancient times the people in the Orient have enriched the flavor of fish and meiit by fermenting it in the presence of high salt concentrations. These foods, formerly cidled fish soy or merely SOY, can still be seen today in “nuoc-mam” in Java, or “shottsuru” in Japan, and itre believed to be antecedents of the soy sniice now in use. Years ago several kinds of pulses and corn were used as raw materials; now, however, only soybeans and wheat are used.

The mold Aspergillrts soyne is the source of the proteolytic enzymes. The first soy sauce produced was cnlled “soy,” ”sho,” or “mislio,” and was very different from soy sauce produced today. It resembled the present day “miso” ( Ilitrtl mitsh ) or “moromi” (miish ) and W B S consumed without further treatment. During the Muromachi period ( 1~28-1573), much research was done on the kinds and relative quantities of raw materials and on the fermentative process, with the result that the product was improved considerably.

Today three types of soy sauce are available in Japan: “koikuchi,” “usukuchi,” and “tamari.” About 90% of the Japanese soy sauce produc- tion is of the koikiichi type. In China, most of the soy sauces belong to the tamari type; that is, thcy are made of soybeans or with a greater amount of soybeans than wheat. High-quality Japanese fermented soy sauce is made from equitl amounts of soybeans and wheat. The product consists not only of amino acids, but also of many flavor inpedients derived from the alcoholic fermentation which is part of the manufac- turing process. Wheat bran also plays an importiint role in the develop- ment of flavor and aroma.

Chemical studies begitn to supplement sensory methods during the period from 1867 to 1912. In addition, autoclaves and hydraulic presses came into iise in its manufacture. At that time, the soy mash was usually fermented at ambient temperatures for 1 to 3 years. Next various lots of sauce (which had been fermented for different periods) were blended and sold. Several attempts were made to shorten the long fermentation period and hence lower the cost of production. Thus, dur- ing the last decade there hits been a growing tendency to make soy siiiice by acid hydrolysis, in order to increase the yield and reduce pro- duction time. A chemical method, announced by the Noda Soy Sauce Co., Ltd. (1955), is now being commonly used by soy sauce manu- factiircrs. Even where the fermentation method is still used, soybean Iiydrolyzates are added to the fermented soy sauce as a flavor booster.

The flavor siibstanccs of true Jiipancse soy saiice produced by the traditional fermentation method will be discussed in this review,

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AROMA AND FLAVOR OF JAPANESE SOY SAUCE 77

II. COMPOSITION

A. ANALYSIS Most of the Japanese soy sauces are the koikuchi type, containing

5@% or more of i\ chemical hydrolyzatc of defatted soybeans. Only a few of the large manufacturers produce the bestquality product, which requires aging for more than a year. Good-quality fermented soy sauce contains 1.5 g. of total nitrogen, and 18 g. of sodium chloride per 100 ml., and appropriate proper amounts of amino acids, sugar, alcohol, glycerin, and organic acids. It should have a high buffer capacity, stability, and good aroma, flavor, and color. Typical composition of koikuchi soy sauce samples is shown in Table I and of a high-quality

TABLE I COMPOSITION OF KOIKUCHI SOY SAIJCI?'

Specific Year and gravity Sodium Total Amino Total Alcohol

grade BC Extract" chlorideb nitrogenh nitrogcnb Sugarb acidsb (% vol. )

1937 23.84 20.50 18.46 1.52 0.65 5.19 0.63 1.63 1949 19.10 7.16 17.70 0.73 0.39 0.79 0.30 0.41 1957

Grade A 22.75 18.15 18.50 1.49 0.79 4A5 0.90 2.00 1957

Grade B 22.38 16.19 18.25 1.28 0.68 3.02 0.86 0.88

From Nuda Soy Sauce Company, 1,iniitcd ( 1958). Data cxprcsscd a'i g./lM nil.

fermented soy SilUCe in Table 11. The composition of tamari and usukuclii soy swces is shown in Tables I11 and IV.

Sodium chloride in soy sauce is usunlly determined by titration with silver nitrate, using potassium chromate as nn indicator. Accurate re-

TABLE 11 COMPOSITION OF A TYPICAL HIGH-QLMLITY FERMENTED SOY SAUCE"

Specific gravity, B6 24.00 Extract, g./100 ml. Total nitrogen, g./lOO ml. 1.51 Protein nitrogen. g./100 ml. Amino nitrogen, g./lOO ml. 0.70 Reducing sugar, g.1100 ml. Dextrin, g./lOO ml. 1.06 Viscosity Total acid as lactic, g./lOO ml. Alcohol, % 2.00 Glycerin, g./lOO ml. Inorganic substances, % 19.70 Sodium chloride, g./lOo ml. PH 4.02

0.48 Volatile arid, g./lOO ml.

From No& Soy Snwe Company, Limitrd (1957).

38.13 0.09 5.99 3.50 0.17 1.00

18.02

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78 TAMOTSU YOKOTSUKA

specific gravity Sodium Total ( n o chlorideb nitrogen* Extracth

Sample 1 25.90 16.43 2.86 31.78 Sample 2 23.45 19.29 1.80 19.42

From Noda Soy Sauce Company, Limited ( 1957). b Data expressed as g./lOO ml.

sults are obtainable only when the sample is first ashed; otherwise, cer- tain corrections are necessary, depending on the nature of the sample. Sato (1955) proposed the use of bromophenol blue as an indioator for

TABLE IV cOhfPOSI1 ION OF LrSUKUCIXI SOY SAW<:@

specific gravity Sodium Total Amino Total (Be) chloride* nitrogenb nitrogenb Sugarb Alcoholb acid9 pH

~~

22.65 20.13 1.05 0.53 2.94 0.34 0.76 4.71

‘1 From Noda Soy Sauce Coinpuny, Limited ( 1957). *Data expressed as g./lOO ml.

this titration without ashing the siimple. Snkasai and Yokotsuka ( 1956) tried to measure sodium chloride by electrometric titration to avoid the obstacle imposed by the deep color of soy sauce.

l3. NITROCEN-CONTAINING COMPOUNDS The Kjeldahl method is widely used for the determination of total

nitrogen in soy sauce. The official method long used in Japan has been to digest 5 ml. of soy sauce with 20 ml. of sulfuric acid and 3 g. of cata- lyst, consisting of 9 parts potassium sulfate and 1 part cuprous sulfate. However, a semimicro method involving digestion of 1 ml. of a sample with 5 ml. of sulfuric acid and 1 g. of catalyst has also been commonly used.

Yokotsuka et al. (1955) made a thorough study of the kind and amount of catalyst, and concluded that mercurous compounds are the best. Reproducible results were also obtained by using 0.2 to 0.3 g. of a mixture of cuprous sulfate and dipotassium phosphate and 5 ml. of sulfuric acid per milliliter of soy sauce. The results obtained by this method were 2% higher than those obtained by the official method,

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AROMA AND FLAVOR OF JAPANESE SOY SAUCE 79

These investigators reported also that almost 9& of the total nitrogen was easily converted by the ordinary Kjeldahl method, in which only cuproiis srilfate was used as the catalyst; however, the remaining 2% was very resistant to digestion. They attributed this resistance to the nitrogen-containing phenolic fraction, which increases when soy saiice is heated. Since the quality of a soy sauce may be relnted to its total nitro- gen content, this observation is important. Accordingly, faster and more accurate methods of analysis are needed.

The nitrogen compounds consist of about 40 to 50% amino acids, 10 to 15% ammonia, 40 to !50% peptides and peptones, and less than 1% proteins. Takada (1934) stated that 45% subpeptone and peptone, but no protein or metaprotein are present. Tsunoda and Ishizuka (1952) also claimed the absence of protein on the basis that no precipitate was obtained with sodium tungstate or trichloroacetic acid. These workers are believed to have studied commercially pasteurized soy sauce in which the proteins would have been precipitated by heat.

Sakasai and Yokotsuka (1957) divided the total nitrogen of unheated soy sauce into: ( 1) tannin-coagulatable, ( 2) phosphomolybdate-pre- cipitable, and (3) residual nitrogen. The quantity of each fraction was 0.2 to 1.5%, 45.9 to 46.88, and 52.6 to 53.3%, respectively. The ammonia content was 8.8 to 11.2%, the difference between formol nitrogen and ammonia nitrogen was 34.1 to 43.11, and the free glutamic nitrogen was 3.8 to 4.9% of the total nitrogen. These ratios varied depending on the extent to which the raw materials had been cooked.

The ratio of amino nitrogen to total nitrogen has been considered as a criterion for judging quality, with a high ratio indicating high quality. Usiially the amino nitrogen content has been reported to be 50 to 6oR, of the total; however, the imperfect formol titration method that has been used has caused erroneous results because of the ammonia present. Moreover, because of the intense color of soy sauce, it is difficult to obtain a sharp end point by formol titration. Ohara and Moriguchi (1954) tried to determine formol nitrogen by electrometric titration at pH 8.0 without adjusting the pH of the sample. Sakasai and Yokotsuka (1957) also reported a method of determining the formol nitrogen in soy sauce by electrometric titration; the ammonia nitrogen con- tent was determined by the Conway diffusion method. The difference between the formol and ammonia nitrogen was regarded as amino nitro- gen. By using this method, the amino nitrogen content of ordinary soy sauces was found to be about 40 to 45% and ammonia nitrogen about 10 to 15% of the total nitrogen. Sakasai & Yokotsuka (1958) recovered almost loolxI of the amino acids from a synthetic mixhire (similar to natural soy sauce) by formol titration at pH 8.5.

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80 TAMOTSIJ YOKOTSUKA

The pH value seems to be important with the Conway method of determining ammonia. These same authors rcprted that lower alkalinity failed to recover all of the ammonia conjugated to the weak acids present in soy sauce, such as benzoic or isovaleric. On the other hand, with higher alkalinity (pH values above 12), glutamine decomposes into ammonia. The Van Slyke gasometric method for amino acid deter- mination is used with good results; however, it recovers only 25% of the ammonia nitrogen present and does not measure proline or oxyproline nitrogen. Form01 titration, on the other hand, measures all of the am- monia nitrogen and 25% of the nitrogen present in proline and oxypro- line. Yoshino and Takano (1956) proposed a colorimetric method for measuring amino acids using ninhydrin, and obtained results similar to those using the Van Slyke method. However, since the color developed with ninhydrin varies with the kind of amino acid, these results are subject to question.

1. Amino A& The individual amino acids present in soy sauce have been identified

and are the subject of many reports. Alanine, proline, leucine, glutamic acid, oxyglutamic acid, aspartic acid, lysine, arginine, cystine, phenyl- alanine, and methionine were detected by Omura ( 1931), Udo (1931a), and Kaneko (1939). Glutamic acid was identified for the first time by Udo (1931b), who also showed its importance as a flavoring agent in soy sauce. He isolated crystalline dutamic acid and aspartic acid, which were mostly in the conjugated form, and concluded that the chief in- gredient responsible for the delicious taste of soy sauce is glutamic acid and its salt. Tomiyasu (1939) claimed the existence of P-hydroxy- glutamic acid in unpasteurized soy sauce. Yoshino ( 1951a,c), using paper chromatography, found threonine, valine, glycine, and histidine by precipitating them as carbamino acid mercurous acetates.

Tsunoda and Ishizuka (1952) determined the amino acids present in soy sauce by bioassay (see Table V). According to their results, 50% or more of the total nitrogen present is in the form of free amino acids. They also studied the peptides of glutamic acid, aspartic acid, and glycine. Fifty per cent of the glutamic and 40% of the aspartic acid were ehimated to be in the conjugated form. Yoshino (1953) identified 14 amino acids, excepting serine and tryptophan, from 16 amino acids which had been identified by Tsunoda and Ishizuka. Hori et al. (1956) reported that the peptide form of glutamic acid present is less than 10%, and that 50% of the gliitamic acid in the hydrolyzate is pyroglutamic acid. They found almost equal amounts of glutamine and free glutamic acid in the mash at the beginning of the fermentation but hardly any

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AROMA AND FLAVOR OF JAPANESE SOY SAUCE a1

glutamine in commercial soy sauce. They concluded that glutamic acid is changed into pyroglutamic acid in the earlier stage of fermentation. The asparagine content of commercial soy sauces was about 0.1%. Fuji- wnra et ul. (1958) reported on the amino acids content of many kinds of soy sauce as measured by a microbioassay method.

TABLE V ALflNO ACID CONTENT OF SOY SAUCE"

Filtrate after sodium tungstate

Amino acid Before hydrolysisb After hydrolysisb precipitntionb

Arginine 6.60 6.39 6.43 Aspartic 4.73 7.60 6.96 Cystine 0.26 0.93 0.59 Glutamic 12.08 21.10 20.00

Histidine 1.42 1.62 1.52 Isoleucine 3.88 3.34 3.78 Leucine 8.78 7.04 7.04 Lysine 3.68 5.67 5.76 Methionine 1.99 1.38 1.36 Phenylalanine 3.70 3.4s 3.45 Proline 6.97 8.87 5.89 Serine 9.40 4.76 4.85 Threonine 3.08 3.01 2.99 Tryptophan 0.61 0.15 0.08 Tyrosine 0.72 0.96 0.79 Valine 4.93 5.21 5.35

GI ycine 2.90 4.15 3.44

From Tsunoda and Ishizuka ( 1952). b Total nitrogen of the sample 1.62 g./lOO ml.; all data based on bioassay, and

Ion exchangers, chromatography, bioassay, and enzymes have been widely used for determining glutamic acid. Among these, the enzymatic method, using microbial cells, seems to be most convenient and reliable (Seidman and Blish, 1957).

expressed as mg./ml.

2. Orgunic Bases Yamada (1926) obtained 0.16 g. of putrescine and 0.4 g. of cadaver-

ine from 2 liters of soy sauce (as picrates). Udo (1931b) identified lysine, putrescine, cadaverine, a base with the empirical formula C,H,N,, adenine, choline, and betaine, as picrate or platinum salts in the fraction containing glutamic acid. Yukawn ( 191%) identified tyra- mine ( p-hydroxyphenylethylamine ) , and Kuninaka ( 195458) detected adenine, hypoxanthine, xanthine, guanine, cytosine, and uracil as the

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82 TAMOTSIJ YOKOTSUKA

constituents of soy sauce by using chemical methods, electrophoresis, paper chromatography, and ultraviolet spectroscopy. These compounds were considered to be derived from nucleic acid, produced by the en- zymes present in the mold.

C. CARBOHYDRATES, SUGAR, ALCOIIOLS, AND Exrrtrcrm

Yoshino (1951a,b) studied sugars in soy sauce by paper chromato- graphic methods. He found almost no difference in the sugar content between two mashes that were 1 and 12 months old. Hamada et al. (1958) identified the following compounds in soy sauce by paper chromatography, paper electrophoresis, and carbon column chromatog- raphy: four sugars (arabinose, xylose, glucose, and galactose), two sugar alcohols derived from glycerol and mannitol, and one nonre- ducing oligosaccharide. They determined the composition of the oligo- saccharide by the Anthrone method and obtained the following results: xylose, 0.0824341%; arabinose, O.lf3&0.387%; glucose, 1.ooeO.7W; and galactose, 0.4824302%

Usually the sugar content of soy saucc is expressed as glucose equiv- alent as determined by Bertrand’s method. Interfering substances in soy sauce, such as proteins, amino acids, and other reducing compounds, may cause errors in the analysis of sugars, especially when changes in sugar content during fermentation are studied. This problem has been discussed by Yamada and Ishimaru (1930). Kandachi and Nishi (1949) tried to obtain accurate results by first removing protein from the sample. It is evident that this procedure alone is not sufficient to over- come the difficulty since it is caused by the presence of many sub- stances. Yoshino and Takano ( 1954) determined the reducing sugar content of soy sauce by colorimetric method, using 3,5-dinitrosalicylic acid for samples diluted 50 to 100 times. Kobayashi and Tabuchi (1954) studied a modification of the method of Somogii and recognized that results were effected by the sugar/amino acid ratio but not by dilution of the sample. Cystine, tryptophan, tyrosine, serine, glycine, lysine, methionine, arginine, and threonine increased the value, and generally higher results were obtained by Somogii’s method than by Bertrand’s. Yoshino and Miyauchi (1956) also performed the analysis colori- metrically by chromotropic acid in the error range +3.5%. Total hexose was determined for the sample which was pretreated with Amberite IR 120, and the remainder of it from total carbohydrate was considered to be total pentose. Almost any decrease of total pentose was seen in mash life, though much of that was seen for total hexose.

Glycerin is one of the key compounds differentiating a soy sauce prepared with whole soybeans from that made with defatted soybeans.

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AROMA AND FLAVOR OF JAPANESE SOY SAUCE 83

Okuhara and Yokotsuka (1956) ;ittempted to isolate glycerin from soy sauce by precipitating the proteins, and cxtracting with methanol, acetone, or ethyl acetate, or by distilling undcr vacuum. They practiced successive vacuum distillation, adding water before each distillation. In this case no 2,3-butylene glycol was detected in the distillate by paper chromatography although this compound was later identified in soy sauce by one of the same authors. The amount of glycerin in the dis- tillate was determined by the chromotropic acid method. They found 0.4 to 0.5% glycerin in a soy sauce sample prepared from defatted soy- beans, and 1.0 to 1.2% in one prepared from whole beans, and con- 6rmed the theory that the presence of 0.5% or more of glycerin by weight in a soy sauce is organoleptically detectable because of its sweet taste.

D. ACIDS, SALTS, AND RELATED COMPOUNDS The acidic substances in soy sauce are important to the aroma,

flavor, color, and storage quality of the product. Acetic, lactic, succinic, and phosphoric acid are the major acids, and they are discussed here mainly as they affect taste, Udo (1932) found 0.1% succinic acid in soy sauce, 84% in conjugated form. He listed histidine, arginine, lysine, pu- trescine, cadaverine, ammonia, aspartic acid, glutamic acid, lactic acid, succinic acid, and formic acid as the compounds which were largely present in a conjugated form at pH 4 to 5. Under the same conditions, leucine, alanine, phenylalanine, tyrosine, glucose, and galactose were present in the free form.

This worker then synthesized many kinds of salts and classified them according to their tastes (delicious, bitter, and tasteless). All of the following compounds, arginine, histidine, lysine, putrescine, cadaverine, and ammonia, when conjugated with glutamic acid were classed as delicious. Arginine, histidine, lysine, putrescine, cadaverine, and choline, when conjugated with succinic acid had a good taste, as did some of the salts of acetic, lactic, and phosphoric acid. All of the salts of tyramine and choline were bitter. In general, the salts of lactic, formic, phos- phoric, and acetic tasted bitter, but were sweeter than magnesium chloride or other inorganic salts.

The barium salt of the nonvolatile part of the ether extract, in- soluble in 80% alcohol, had been regarded as the succinate, and the remainder as lactate. Formic acid was determined from the steam distillate of the ether extract by the mercurous chloride method; the remaining substance was regarded as acetic acid. Using this procedure,

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84 TAMOTSIJ YOKOTSUKA

Kihara (1938) determined the organic acid content of soy sauces to be as tabulated:

Organic Arid Content ( i f Soy Sauce

Lactic Succ i n k Arctic. Formic Acid (9) ( X I ( % I ( 9 )

mill Free Conjugated

1.24 0.11 0.10 0.003 0.47 0.02 0.06 - 0.77 0.09 0.04 -

Udo (1932) obtained almost the same results. Hori et al. (1957) separated the acidic acids from a soy sauce

sample on a silica gel column and obtained the results shown in the tabulation:

Separation of Acid$ from Soy Sauce on Silica Gel Column

Hydrochloric acid hydrolyzate of

Fermented soy sauce soybean meal Acid (g./g. total N ) ( g . / g . total N )

Levulinic Tram Citric Small amount hlalic Small amount Pyroglu tamic 0.3SB Lactic 0.859 succinic n.028 Oxalic 0.004

n.61~1 0.380 0.012 0.045 0.015

Small amount 0.016

Titratable acidity has been measured by titrating to pH 7.0, using an indicator. Because of the intense color of soy sauce, however, it is hard to get a sharp end point. Sakasai and Yokotsuka (1956) measured acidity by potentiometric titration to pH 8.2. At this pH, 100% of mono- carboxylic acids and two-thirds of the phosphoric acid were titrated. Typical titration curves of a soy sauce are given in Fig. 1, showing the great buffer capacity. This is effective in ameliorating the salty and acidic tastes and in promoting the stability of soy sauce.

Ueda et at. (1958) analyzed the organic acid content of many kinds of soy sauce, using silica gel partition chromatography. Three samples of their data are shown in Table Va.

Tanaka and Ueda (1958) reported the content of inorganic ingre- dients such as total ash, sodium chloride, calcium, magnesium, iron, phosphorus, and potassium in several kinds of soy sauce.

Ueda et d. (1958) studied the changes in organic acids in the

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AROMA AND FLAVOR OF JAPANESE SOY SAUCE 85

course of mash aging ( propionic, levulinic, acetic, pyruvic, fumaric, for- mic, a-ketoglutaric, succinic, lactic, malonic, pyroglutamic, glycolic, malic, and citric acid), using the silica gel partition chromatography. A new mash was made in April of 1957 and analyses were performed every month till May of 1W. Lactic and acetic acid are the major organic acids in raw soy sauce, accounting for 85% and 8%, respectively.

2 . 0 ~ I .o 0 2 4 6 8 10 12 14 16 18 20 22 24 26

N / 2 0 NOOH (rnl.1- FIG. 1. Titration curves of soy sauce and extracts thereof. A: Soy sauce pro-

duced from whole soybeans and wheat. B: Soy sauce produced from defatted soy- beans and wheat. C: Soy sauce produced by the scmi-chemical method. a,b,c: Ether extract of A,B, and C. al,b*,cl: Residue of extraction of A,B, and C.

Both of them increased rapidly in the early stages of fermentation and reached the maximum value in 3 to 4 months and in 7 to 8 months, respectively. They divided the fermentation into the following four stages:

(1) Beginning stage (0 to 1 month). In this period remarkable in- creases in organic acid were not observed, but citric and malic acids derived from raw material were apt to decrease.

(2) Fermenting stage (1 to 4 months). Almost every organic acid increases during this period.

(3) Middle fermenting stage ( 4 to 9 months). Propionic and formic acid increase and reach maximum values by the eighth month.

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TABLE Va ORGANIC ACID cOhmN?o OF SOY SAUCEb

Total ko- pro- a-Keto- Suc- -0- Sample nitro- Butyric butyric Un- pionic Levulinic Acetic Pyruvic Formic glutaric cinic Lactic glutaniic Glycolic Malic Citric

No.e gen acid acid known acid acid acid acid acid acid acid acid acid acid acid acid

1 1.5:3S 1.03 0.66 - 11.89 172.28 157.92 8.81 26.13 1.68 77.1.5 1175.25 8.81 18.32 4.21 7.49 2 1.245 2.39 4.56 0.80 18.60 153.93 114.41 4.20 18.14 4.26 53.32 1221.73 4.20 8.10 3.05 13.04 3 2.220 4.30 1.20 - 2.64 1244.51 122.54 - 287.80 4.67 - 29.53 - 4.16 3.28 25.27

“Data expressed as mg./100 ml. b Ueda et ul. ( 1958).

-

No. 1 and No. 2 are fermented soy sauces, and No. 3 is hydrochloric acid hydrolysate of soybean meal.

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AROMA AND FLAVOR OF JAPANESE SOY SAUCE 87

( 4 ) L’itst fcrmanting stag(: (!I to 18 rnontlis). Voli1tik ;wit1 content decreases; a small increasc is noted for ketonic acid.

Onago et d. (1957) compared seven soy sauce samples obtained from markets in California for flavor and chemical composition. The optimum concentration of salt seemed to lie between 18.7 and m.4 g./lOO ml. Total acidity also appeared to be important, and samples ranking higher in flavor were higher in total acidity. Nitrogen content was an important factor influencing flavor acceptance. Amino acids in soy sauce, as identified by two-dimensional paper chromatography, were found to be alanine, arginine, aspartic acid, glutamic acid, glycins, leu- cine, lysine, methionine, phenylalanine, proline, serine, threonine, and valine. A rapid ion exchange method to remove salt from soy sauce for paper chromatographic study is described.

E. COLOR

The color of soy sauce is very important since it is associated with flavor, It has been studied by Kurono and Katsume ( 1927), Kurono et al. (1927), Omata and Ueno (1953-55), and Nakano (1953); they found that the color development of soy sauce is chiefly caused by the so-called browning reaction. This browning reaction has two meanings for the flavor of soy sauce; many types of flavoring are made by this reaction, and on the other hand this reaction takes place among many kinds of important flavoring ingredients, such as amino acids and sugars.

Formerly the color of soy sauce was measured in a Lovibond tintom- eter. Many reports have been made on spectrophotometric measure- ments: Yamazaki ( 1951), Irie and Yamazaki ( 1951), Omata and Ueno (195355), Onuki and Chiba (1953), Furuta and Ohara (1954), and Ohara et al. (1953). According to Omata and Ueno (1953), the color of soy sauce represented by the C.I.E. system was a dominant wavelength 590 to 6u) mp, an excitation purity 86 to 8=, and a luminous transmit- tance of 0.14 to 0.17. Recently the color of soy sauce has been generally measured by comparing it with a standard color issued by the Soy Sauce Society of Japan. This standard is made of chemical pigments so as to match with the C.I.E. value of soy sauce. Umeda and Saito (1956) carried out numerous experiments in preparing this standard color.

111. CHEMICAL COMPONENTS OF FLAVOR

Research on the chemistry of the odor and flavor constituents of Japanese soy sauce has been conducted in Japan since 1887. Among the Japanese scholars who have studied this subject are: Tawara (1887), Ishida (1929), Yukawa (1916), Ikeda and Kawaguchi (1922), Taira

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88 TAMOTSIJ YOKOTSUKA

(1925), Slioji (1927), Akabori iintl Kancko (1936), Kiliara (1940), Nakajirria ct ul. ( 1949), Asao and Yokotsuka ( 1957a,b), Yokotsuka (194957), Obata and Yamilnishi ( 1951-52), and others. The following methods have been used: drying, steam distillation, vacuum distilla- tion, and solvent extraction of soy sauce or soy cake. The synthesis of flavor substances hiis been investigated as well as their isolation from natural products. The compounds identified or considered to be present in soy sauce are listed in Table VI.

The fust study of the flavor of soy sauce was made by Tawara (1887), who isolated from the ethanol-soluble part of dry matter of the neutralized soy sauce a crystalline compound with typical flavor. Later, Ishida (1925) claimed that this was lcucine.

Yukawa ( 1916) demonstrated the presence of tyrosol (p-hydroxy- phenylethylalcohol ) and tyramine ( p-hydroxyphenylethylamine ) in commercial soy sauce by first adding ethanol to the concentrate of soy sauce. After the sodium chloride was filtered off, the filtrate was made alkaline and extracted with ether. The ether was removed, and the di- benzoate of tyrosol, melting at 111"C., was then prepared. Tyramine was isolated from the filtrate of tyrosol and identified as the platinum salt. Yukawa (1917a,b) claimed that tyrosol is associated with the bit- terness of soy sauce.

Ikeda and Kawaguchi (1922) steam-distilled soy sauce and found that the major part of its flavor existed in the higher-boiling-point (b.p.) fractions. They obtained a fraction boiling at 50" to 55°C. (1 mm. Hg) and determined the empirical formula to be C,H,,O,. It was a pale- yellow oil; its flavor was easily changed by alkali. It gave a silver mirror test and also yielded a crystalline compound with bromine. From these reactions, it was considered to be an unsaturated ketone or aldehyde. Besides this compound, they established the existence of butylaldehyde and valeraldehyde. Kodama (1922a) steam-distilled 400 liters of soy sauce, extracted the distillate with ether, and found 278 g. of water- containing oil which had a strong aroma and flavor resembling soy sauce. By fractional distillation, followed by washing with sodium bisul- fite and barium bicarbonate, 13.57 g. of oil with a b.p. of 3 5 O to 81OC. (15 mm. Hg) were obtained. Since the oil was not pure, fractional dis- tillation was repeated. A third fraction, boiling at 60" to 62°C. (15 mm. Hg) was a pale-yellow oil with a very strong odor, perceptible at 1 X g. in 1 ml. of air. It was neutral to litmus, soluble in water and ordinary organic solvents. It was given the empirical formula C,H,,O,. The molecular refraction agreed more with compounds having conju- gated double ketone groups than with compounds containing two ketone groups. The empirical formula CH,CO CH : CH CHO was established.

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AROMA AND FLAVOR OF JAPANESE SOY SAUCE 89

The fifth fraction was a yellow-green oil with a stronger aroma and flavor than the third fraction, and had the formula CH,CH,CO*CH : CH CHO.

Taira (1925) extracted 54 liters of heated soy sauce with ether and obtained 98 g. of oil. Ethyl acetate, amyl acetate, valeraldehyde, and butylaldehyde were detected. Later he isolated normal-2,3-butylene glycol from a fraction with a b.p. of 90" to 91°C. (15 mm. Hg). He con- sidered this to be one of the major ingredients of soy sauce aroma and flavor.

Yamada (1928-29) pointed out that the aldehyde derived from the soy sauce fermentation is acetaldehyde. Butylaldehyde and valeralde- hyde are derived from the heating of the soy sauce. Soy sauce was steam-distilled, extracted with ether, and distilled into 16 fractions by that worker. Yamada also noticed that the furfural-like compounds in- creased with heating.

Shoji (1927-36) and Shoji and Onuki (1932) distilled an ether extract of soy sauce into three fractions, with b.p. of 150" to 160°C. (18 mm. Hg), 120" to 160°C. (9 mm. Hg), and a residue. For the first time, phenolic compounds with ( presumably) two aromatic hydroxyl groups were indicated as major ingredients of the aroma and flavor of soy sauce. The basis for this hypothesis was as follows: After it had been washed with sodium bicarbonate, the ether extract had a flavor resembling that of guaiacol or vanillin; the flavor disappeared after treatment with sodium hydroxide and reappeared with acidification. Other qualitative tests for phenolic compounds were also performed. About 15 g. of essential oil from the chloroform extract of 64 liters of soy sauce was then divided into three fractions: a small amount of acidic, 10 g. of phenolic, and 4 g. of neutral fractions were obtained by ordinary procedures. Caproic acid, methyl alcohol, ethyl or propyl alcohol, isobutyl alcohol, isoamyl alcohol, hexyl alcohol, methyl-n- nonyl carbinol, and methyl-n-undecyl carhinol were considered to be present. Later, the unheated sample was used to replace the hented soy sauce, and phenylacetic acid and guaiacol were detected. The differ- ence in flavor between the heated and unheated soy sauce was quite noticeable.

Kurono and Fukai (1928) isolated 9.4 g. of aldomedon (m.p. 94" to 95OC.) treating 25 liters of steam distillate obtained from 36 liters of commercial soy sauce with dimedon. A warm alkaline solution of this culture had the flavor of soy sauce. Ry elementary analysis, an empirical formula of C,;H,,,O, was assigned, and the compound named "soyanal." However, it gave naphthylmethan, a remarkable iodoform reaction product, succinic acid by permanganate oxidation; and absorbed one-

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TABLE VI VOLATILE COMPOUND6 ISOLATED FROM SOY SAUCE AND SOY CAKE

Origin and author

Name Formula Raw soy HeSted

mice soy sauce Soy cake

Ethyl alcohol Amy1 alcohol Hexyl alcohol Meth ylnon ylcarbinol 2,3-Butyleneglycol

Aliphatic cnmpounda CHsCHtOH C J L C H d H Cjf;,CH&H CHiCHOHCeHii CHaCHOHCHOHCHi

Acetaldehyde Propiooaldeh yde .V-Butyaldchyde Isobu tj-rsldehyde

CHaCHO C,H&HO CHi(CH*)tCHO (CHa)zCHCHzCHO

A--Valeraldehyde CHs(CH2)aCHO

Isovakraldehyde (CHa)sCHCHtCHO

Furfural

5-Hydroxymethylfurfurrrl HOCHJ JCHO

n Acetoin Maltol

CH~CHOHCOCH, 0 II

Yamada (1926) - - -

- - Shoji (1927) - Shoji (1927) Taira (1926) Ahbor i (l!436) Yokotauka (1957) Akabori (1036) Tokotsuka (1949)

- Tokoteuka (1949) Kodama (1922) Tokotsuka (19-19) Nakajimrr and 'I-okotpuka (1953)

Takei (19.19a.b) Akabori (1936) Ikeda and

Kawagrichi fl022)

Aksbori (1936) Yokoteuka (19.19) Nakajima and

Takei (1949a,b) Akabori (1936) - Yamada (1927)

- -

-

- Yokobuka (19.19)

Akahri (1936) - Kihara (1940) - Nakajima and

Takei (1949a.b)

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Shoyualdeh yde

Isobutj-rglyoxal(?)

Imvderaldehydedi ylacetal a-Hydroxyisocaproaldehyde diethylacetal Formic acid Acetic acid

Propionic acid Butyric acid

Isovderic acid

Capmic acid

oxslic acid

Ethyl acetate

Amy1 acetate

Ethyl lacetate Ethyl palmitate

Ethyl b i a t e

Ethyl oleate

Iaovderate

Caproate

CHaCOCH=CHCHO CHsCHrCOCH=CHOH

(~HICOCHZCHICH~CHO) c m ( C H a C v H C H O )

CbHI&

CHs

- -

Yokohka (1953) Yokotsuka (1953)

Yokoteuka (1953) Yokotsuka (1953)

Yokotaukn (1953)

Yokotsuka (1953)

Asso and Yokot- euka (1957)

Taira (1926) Akabon ( 1936)

;Utabori (193fi)

-

-

YokotPuka (1953)

Yokotauka (1953)

- Yokotsuka (1949)

- Yokotauka (1949) - Yokotsuka (1951) - Yokotauka (1953)

Nakajima and - - Yokotwka (1953)

Nakajirna and Yokotsuka (1953)

- Yokotauka

- Fokotauka

Takei (1919a,b)

Takei (1949a,h)

( 1952aJb,c)

(1952a,h,c) - -

- Yokotsuka

- Yokotsuka

- Yokotsuka

- Yokotauka

- Yokotsuka

- Yokotsuka

(19%hc) -

( 195Za,b,c)

( 1952aJb,c)

( 1952a,b,c)

(1952a,bJc)

(1952a,b,c)

8

s

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92 T

AM

OT

SIJ

YO

KO

TSU

KA

-r, I

Gx

/ \ 0

-03-

0

w

G OZ

-u3-

h

v

- P. ." H B U

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AR

OM

A AND F

LAVOR OF

JAP

AN

ES

E

SOY

S

AU

CE

I I I

43

f!

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TABLE VI-(Continued)

Origin and author

Raw soy Heated Name Formula sauce soy sauce Soy cake

Vanillate OOCR Tokotfiuka (1953) ,

Tyrosol ester COOR

Yokotsuka I t C O C b - ~ --CH,CH.OOCR (1953)

\-i

Benzoate

Trimethylgallat e I

I COOK

Yokotsiika (1953)

Asao and Yokot8uks (1957)

C ~ H S O ~ (m.p. 126"-8"C.) C H 6 0 ~ (m.p. 350"-260"C.~ CWH~OO (m.p. 330°C.) C!SH~~.I~O (m.p. 95'4°C.)

Unknown roompounds - - Yokotauka (1957) -

- Yokotsuka (1957) - A.sao and Yokot -

- - I'ohUtSLdid (19u) - - -

- suka (1957)

Aeao nnd Yokot- suka f 1957)

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AROMA AND FLAVOR OF JAPANESE SOY SAUCE 95

half mole of bromine for each mole of compound, From these results, the following constitutional formula was assigned:

The amount of soyanal present in ordinary soy sauce (as determined by the iodoform reaction of the chloroform extract) was reported to be 0.04% Shoji (1927; 1932) denied the existence of soyanal in soy sauce from the results of polarographic studies.

Akabori (1936) extracted 90 liters of heated soy sauce concentrate with ether and obtained 240 g. of an oil; 0.2 g. of succinic acid crystal- lized from this oil. The filtrate from a barium carbonate treatment was extracted with ether, and unknown crystals with an m.p. of 310" to 311OC. were separated. After the crystals were removed, the filtrate was fractionally distilled. A fraction with a b.p. of 81" to 82°C. (10-11 mm. Hg) in an empirical formula C,HI,Oz was established. The phenyl- urethane and p-nitrobenzylidencyclo-acetal derivatives of P-7-butylene glycol were prepared from the fraction.

Akabori and Kaneko (1936) distilled !?32 liters of soy sauce <6O"C. at 20 to 30 mm. Hg. This distillate was extracted with ether after sodium chloride had been added, then most of the ethyl alcohol was distilled off. The residue was washed with potassium carbonate and distilled into 7 fractions: Fractions IV to VII (boiling at 60" to 99"C./15 mm. Hg) contained sulfur, and Fractions I11 and IV (boiling at 54" to 60", and No to 63OC./15 mm. EIg) were levorotory. From Fraction 111, acetoin was identified as the nickel glyoximine, and optically active lactic acid as the zinc salt. From the ether solution of Fraction V (boiling at 63' to 87"C./15 mm. Hg), methionol, y- ( methylmercapto) -propylalcohol, was obtained. Later, Akabori and Kaneko synthesized /3-methylmercapto- propionaldehyde, and found that its flavor was similar to that of heated soy sauce, but stronger than that of methionol. They believe that soy sauce flavor contains these two compounds.

Kihara (1940) extracted 100 liters of raw soy sauce with ether and obtained 400 ml. of oil, 285 ml. of which were ethanol and ethyl ace- tate. The alcohol content of the soy sauce was thus 0.7%. The extract was then fractionally distilled, yielding 70 ml. of a higher-boiling por- tion and 30 ml. of the highest-boiling portion, It was then separated by chemical methods into acid, neutral, and phenolic fractions. Maltol (C,H,,O,) was identified from a higher-boiling fraction as the ferrous chloride double salt (m.p. 159OC.). This compound was claimed to have the aroma of soy sauce. The highest boiling fraction showed phe- nolic properties.

Yokotsuka (194953) studied the flavor substances of soy sauces. One ton of freshly pressed and cnished soy cake was steam-distilled,

CH$( OH) = CH CH&HzCHO CHaCO CH,CH:CHzCHO.

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96 TAMOTSU YOKOTSUKA

l-stcam-distilled

again-

The distillate was cooled with water, and the uncondensed gases werc absorbed in sodium bisulfite or 2,4-dinitrophenylhydrazine solution. They were then passed through a saturated solution of sodium bicarbonate. The yield data are shown in Table VII.

TABLE VII YIELD AND COhWOSITlON OF VARIOUS FHACTIONS OBTAINED BY

STEAM DISTILLATION OF SOY CAKE"

( water-insoluble layer sep- arated from the distillate)

(large amount of yellow aqueous solution which came over after Fraction IV).

-Fraction V

-Fraction VI

Soy cak- 1.OOO kg. steam distillation

Fraction I (10 g.)- Carbonyl compounds 1- (run through the cooler IISulfur-containing compounds and caught in several traps 1

-Fraction I1 (13 1.)- (first distillate was clear; major constituent was ethanol)

-Fraction I11 (230 1.). (clouded distillate, coming over after

(1.2 g.)

-Separated by functional

-Fractional distillation: groups

lower-boiling part (0.6%) ; ethanol (89%); water ( 6%) ; and higher-boiling fraction (2%)

-Upper layer from salting-out process (140 8.)

A. FRACTION I

This consisted of the lower-boiling fraction (b.p. < 70°C.). The un- condensed gases contained carbonyl and sulfur-bearing compounds. When 2,4-dinitrophenylhydrazine solution was used as a trap, 30 g. of yellow precipitate was obtained; acetaldehyde, propionic aldehyde, and isobutylaldehyde were identified from the precipitate. The compounds not absorbed in the trap were absorbed in solutions of sodium bicarbon- ate, mercuric chloride, gold chloride (AuCI,), and silver nitrate, and barium hydroxide. They were practically all trapped in a saturated sodium bicarbonate solution, which was then treated with sulfuric acid

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AROMA AND FLAVOR OF JAPANESE SOY SAUCE 97

to give 1.2 g. of oily substance. This was washed with diluted methanol, then distilled under vacuum. The compound in these two subfractions was thought to be isobutylaldehyde-dimethylmercaptal or acetaldehyde- diethylmercaptal. Since this substance came out at almost the same rate during distillation, it was considered to have been formed during the steam-distillation process.

B, FRACTION I1 (b.p. < 78°C.)

One liter of Fraction I1 was separated into three parts: (1) 86 ml. boiling at 20" to 77°C.; ( 2 ) 750 ml. boiling at 78" to 80°C.; and (3) 150 ml. of residue. Part (1) was separated again several times, and the principal constituents of each fraction were identified by their deriva- tives. Results are shown in Table VIII. From Fractions G and H, the

TABLE VIII FLAVOR COMWNENTS OF SOY SAUCE:

CHARACTERISTICS OF FRACTION I1 (b.p. < 78"C.)a

Boiling Frac- point Yieldh Chemical tion "C.) ( g . ) Color Flavor component PH

21-22 0.4 40-47 0.3

50-72 0.3

72-72.5 3.0

73-74.5 3.0

7&77 3.0

77-78 30.0

Residue 7.0

Colorless Colorless

Colorless

Yellow

Paler

Colorless

Colorless

Yellow

than C

Stimulative Very

stimulative Slightly

stimulntive Like soy

sauce Like soy

sauce Alcohol-

like Almhol-

like Like soy

sauce

Acetaldehyde Propionaldehyde

Butyraldehyde

C,Hl,O,

Mixture

Isovaleraldeh yde

Ethanol, etc.

Mixture

2.2 2.4

3.2

3.4

,3.4

4.6

4.6

<1.2

0 From Yokotsuka ( 194W3) . * Low yields attributed to repeated distillation. Aldehydes identified as 2.4-

dinitrophenylhydrazone. Sample D easily soluhle in water; sample E formed emul- sion easily; remainder slightly soluble.

2,4-dinitrophenylhydrazone of isovaleraldehyde was obtained. To catch the lowest-boiling, sulfur-containing compounds, 6 liters of Fraction I1 was distilled into four fractions, with b.p. < 78°C. From these fractions, a compound thought to be ethylmercaptan was isolated. The residue (Table VIII, sample K) contained a small amount of esters, which

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TABLE IX FLAVOR COMPONEXTS OF Sou SAUCE:^

REDISTILLMTON OF FRA-ON 111 (b.p. > 78"C.)b Boiling Pres-

Frac- point sure Yield Appear- Sulfur tion ("C.) (mm.) (8 . ) Flavor ance present Chemical components

L 2- 68 7.0 Resembles Liquid - Mainly ethanol, isovaleraldehyde ( la%), and low-

M 48-50 22 9.5 Sweet,flavor Liquid + Amy1 alcohol, amyl acetate, isobutyl alcohol, and 4

N 67-71 2216 9.8 Resembles Liquid + Benzaldehyde, benzoates isovalerianate and n- 0 d

0 73 4 3.3 Terpene- Liquid + + y-Methylmercaptopropylalchohol, an unknown * sulfur-containing compound, isovaleric acid, n-

P 9.3-103 4 5.2 Soycake- Liquid ++ caproic acid, vanillic acid, palmitic acid, and 2 v)

l-hydroxy-2-methoxy-4-ethylbenzene. ( These c Q 10.3-165 4 2.0 Soycake- Partly + compounds were isolated from the mixture of >

R 163 4 16.7 Soy cake- Completely + Ethyl palmitate like crystallized

S 183 4 19.5 Soycake- Partly - Ethyl linolate and ethyl oleate like crystallized

T Residue 7.0 Soy cake- Viscous - Resembles Fraction S like liquid

I and J boiling esters (30%).

like sauce hydroxymethyl furfural. r C

M capronate.

like

like

like crystallized Fractions 0. P, and Q.)

x

' I From Yokotsuka ( 194L53). a Fractions 0, P, and Q were mixed together, and fractionated again by distillation.

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AROMA AND FLAVOR OF JAPANFSE SOY SAUCE 99

when hydrolyzed gave acetic, butyric, isovaleric, and vanillic acids, as identified by paper chromatography.

C. FRACTION 111 (h.p > W C . )

Fraction I11 was fractionated by Staudiiigcr's inctlitxl iiito ilcid, alcohol, ester, and carbonyl fractions. Several phenols, isovaleric wid, and n-caproic acid were detected from the hydrolyzate by paper chro- matography. Ninety grams of Fraction I11 were redistilled under vacuum. The results are shown in Table IX. From Fractions 0, P, and Q, 4- ethylguaiacol was also separated as a phenylurethane (m.p. 104°C.) and identified by a mixed m.p. determination. It was a major com- ponent of these fractions and had a very strong flavor, resembling soy sauce. The four compounds in Scheme I were also believed to be present:

OCOCHI OCOCeH, OCOCFII OCOCaHr QOCHs Q:Ha QOCIh

Sffh O O c S l ~ ~ OOCsH, SCHEME I

Then the benzoate, isovalerianate, and n-caproate esters of acetovanil- Ion (3-methoxy-4-hydroxyacetophenone ) or isoacetovanillon were identi- fied. Among these compounds, the phenol esters were unstable and appeared to decompose during heating to give a pasteurized flavor to the soy sauce. There were some very difficultly saponifiable compounds present in these fractions, presumably aromatic ethers, although the de- tails were not reported.

D. FRACXION IV (THE ACETAL FFUCXION) Fraction IV, a yellow, oily layer separated from the steam distillate

of Fraction 111, seemed to be important because of its high yield and strong odor. An elementary analysis showed the major constituents of this fraction to have the empirical formulas C,H,,O, (b.p. 33"C./lU) mm. Hg) and C,,H,,O, (b.p. 3 3 O to &3.SoC./105 mm. Hg). The former compound was obtained when the distillation was carried out without special care, while the latter was obtained only when sufficient considera- tion was given for light and heat. From elementary analysis, freezing- point depression, and degradation by heat, hydrochloric acid, potassium permanganate, or silver oxide and light, the compounds were thought to be: isovaleraldehydediethylacetal ( CH., ) ,CIICH,CH (OC,H, ) and a-hydroxyisocaproaldehydediethylacetal ( conjugated to two molecules of water), (CH,),CHCH,CHOHCH( OC,H,), + 2H,O. The latter com-

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100 TAMOTSIJ YOKOTSUKA

pound was very unstable under sunlight and easily decomposed into two fractions, water-soluble and insoluble. At the same time, the yellow color faded; from the decomposed compounds, isovaleraldehyde, ethanol, and water were identified. The unstable compound reacted with alkali at room tcmperature, to form C,H ,,,CO and C,H,,COOH. These were identificd i i s ii 2.4-dinitroplic~nylI~ytlriizonc~ with an m.p. of llO"C., a~ ld an anilidc with an m.p. of 149°C. 'IIiv compounds were iissilmcd to hi the components of heated soy sauce derived from leucine and its precursor Fraction V was not investigated.

E. FRACTION VI

The lower layer which had been separated from the acetill fraction, was treated with activated carbon; the latter was then eluted with ether. The extract was fractionated by distillation under reduced pressure, and the 5 fractions (U-X) were obtained. Although the aromas of W and X were very good, they contained no sulfur and were a mixture of free acids and phenols, plus small amounts of furfurals and aldehydes. This indicates that, contrary to the opinion of some, good soy sauce aroma contains no sulfur, and it is associated with phenolic compounds.

Nakajima et al. (1949) extracted 330 liters of soy sauce with ether; the extract was distilled and divided chemically into various fractions. From the lower-boiling fraction, isobutylaldehyde and isovaleraldehyde were identified as the 2,4-dinitrophenylhydrazones. The lower-boiling portion was distilled into eight fractions. The neutral compounds were repeatedly fractionally distilled to give a yellow oil with a b.p. of 71" to 1OOOC. (18 mm. Hg). It was claimed to be the major flavor ingredient of soy sauce. Methionol was identified and removed from the fraction by the procedure of Akabori et al. (1938). Elementary analysis gave the empirical formula of COHO02 for a compound originally isolated by Kodama (1922b). By chemical methods, the structural formula CH,-CO-CCH,=CH-CHO was assumed for shoyualdehyde, al- though identification by organic synthesis was not reported.

Akabori ( 1931a,b) studied the reaction compounds between sugars and amino acids, and between amino acids and furfural derivatives and suggested a relationship to soy sauce flavor. He identified dimethyl- barbituric acid and furfural-dimethylbarbituric acid from L-glutamic acid and glucose; isovaleraldehyde from L-leucine and glucose or fruc- tose, or from leucine and arabinose; acrtaldehyde from DL-alanine and glucose; phenylacetaldehyde from phenylalanine and glucose; and iso- butyraldehyde from DL-valine and glucosti. In the same way he ob- tained isovaleraldehyde from leucine and furfural or oxymethylfurfural; phenylacetaldehyde from phenylalanine and oxymethylfurfural; acetal-

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AROMA AND FLAVOR OF JAPANESE SOY SAUCE 101

tldiydc from iilunine and furfiirid; ibo~)iityr;iICICJIy~I(~ from isovidinc ; I I M ~

galactose; n-caproaldehyde from n-;uninoinc~thylhc'ptylic acid iuitl glu- cose; and acetone from a-aminoisobutyric acid and furfural.

Akabori and Kaneko ( 1937) identified p-methylmercaptopropionalde- hyde in the heated material of nwthioninc* and glucose and claimcd that this compound is associiited witti the strong flavor which develops when soy sauce is boiled.

Obata et al. (195158) gave a good deal of attention to sulfur- containing compounds. They investiguted the flilvor of soy siiiice chiefly by synthetic procedures. The odor that develops in the fermentation of yeast juice mixed with various kinds of amino acids was noted. A soy sauce-like flavor, attributable to 7-methylmercaptopropylalcohol, could be detected when L-methionone was added. The same odor was ob- tained from cystine and from L-methionine. The odor of half mercaptals and mercaptals was also noted. Those with a flavor most like that of soy sauce were: acetaldehyde-half-mercaptal, isovaleraldehyde-half- mercaptal, furfural-half-methylmercaptal, acetaldehyde-methylmercap- tal, isovaler-aldehyde-methylmercaptal, furfural-mercaptal, acetalde- hyde-ethylmercaptal, and isobutyraldehyde-ethylmercaptal, and mercap- tals of isovaleraldehyde. The condensation product from crotonaldehyde and methylmercaptan had an aroma similar to that of soy sauce. Several sulfur-containing ketones and methyltliioethers were synthesized. y- Methylmercaptopropylpyruvate had an aroma closely resembling that of soy sauce. Later, 16 mercaptals and mercaptols were noted. An aroma somewhat similar to that of soy sauce was recognized in 1,l-di-( methyl- mercapto) -butanol-( 3) and 1,l-di-( methylmercapto) -butene-( 2).

In order to study the flavor compounds resulting from fermentation, Yokotsuka (1953a,b,c,d) extracted the best-quality raw soy sauce with ether or chloroform, and found that the majority of the aromatic com- pounds of soy sauce can be extracted by these solvents. The ether ex- tract was washed with saturated barium hydroxide, and sufficient petroleum ether was then added to bring about precipitation. Needle- like crystals with an m.p. (decomposing) of about 320O"C. were obtained from an ethanol solution of the compound. The filtrate had a strong aroma and flavor. When it was passed through an aluminum oxide col- umn, most of the aromatic compounds were absorbed. They were eluted with ether, ether-alcohol, 1% sulfuric acid, and 1% sodium hy- droxide, successively. The results are shown in Table X. The free acidic components isolated in soy sauces by paper chromatography are given in Table XI.

Asao and Yokotsuka (1957a,b) reported on the acid components of raw soy sauce. They separated these compounds countercurrently with

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102 TAMOTSIJ YOKOTSUKA

TAI~LE X ~ ~ l I l ~ O h l A l C M . ~ A I ' l i l ~ S E I B A I M I I O N OI" N I . U ' I l l A l , b ' l lA<XiON OF k;I I I b I I 1';kIItAc I

OF h W SOY SAU('E ON AI.UhfINA COI.UMN"

Acidic components in hy- Flavor drolyzate identified in

Frac- Solvent Color of inten- Fluo- Yield pure crystalline form, tion solution sity rescence ( g.) except liquid substance

C,,H,,O (map. 320°C.),

- 4-Ethyl-guaiaco1, palmit- ic acid

El Ether Pale ++ E2 Yellow ++ E3 Yellow + ++ - 0.09 4-Ethyl-guaiacol, tyrosol,

acetic acid, palmitic acid, and (benzoic acid? )

E4 Pale + ++ - 0.01 l-Benzoyloxy-2-methoxy-

(benzoic acid, 4-ethyl- guaiacol? )

-

0.12 Acetic acid -1 yellow

yellow 4-ethyl-benzene and

EA1 Ether and Pale + ++ - 0.01 4-Ethylguniacol and

EA2 Yellow + + EA3 Orange ++ - EA4 Pale ++ - Trace -

ethanol ( 1 : 1 ) yellow vanillic acid Vanillic acid -1 0.04

yellow

S1 Eluted with 1% Pale + - Trace Vanillic acid

S2 extracted with Red 4- + + + 0.03 \'anillic arid S3 ether Yellow +++ ++ 0.03 C,H,;O, (n1.p. 250"-

s4 Pnle + - Trnce -

H,SO,, then yellow

2rnO"C.)

yellow

N 1 Eluted with 1% Pale + + Tracts

N2 fied and then Yellow NaOH, acidi- yellow

extracted orange 0.07 Vnnillic acid + ++I + N3 with ether Yellow + a From Yokotsuka ( 1953).

a mixture of ether and chloroform (SO:%) and 0.2 N Sorenson's phos- phate buffer (pH 6.0). The acids were further purified on an aluminum oxide column. The results are shown in Table XII. Oxalic, benzoic, vanillic, and syringic acids were separated in pure crystalline form. They were identified by m.p. determinations, paper chromatography,

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AROMA AND FLAVOR OF JAPANESE SOY SAUCE 103

Nainc

Flavor Color Effect of inten- inten- heating sity sity arid Hcagcnt R I

Acids Oxalic acid Succinic acid Lactic acid Acetic acid Propionic acid Isobutyric acidh Benzoic acidb Isovaleric acidb n-Caproic acidb

0 O.O.u).04

0.11 0.20 0.33 0.39 0.42 0.58 0.87

- + + + + +

Phenols Guaiacol 0.96-0.98 + 4-Ethylguaiaa)l 0.920.94 +++ Tyrosol 0.86-0.88 -

Vanillates Vanillin( ?) 0.47-0.49 ++ + Unknown compoiind 0.05 ++

Vanillic acid 0 . 0 ~ . 0 9 - Unknown compound 0.2-34.25 ?

Tyrosol esters 0.786.78 -

Phenolic acids

+ ++ ++ +++ + + + +

-c

+ + ++ + +++ + + + -t +

- ( A ) : 0.3% Bromo- - phenol blue and - 0.2% citric acid in + H,O - - & - -

(B) : 10% Fe,(SO,), + in H,O and 0.5% + K,Fe(CN),, in * H,O t

+ - + Both ( A ) and (B) r?r

a From Yokotsuka ( 1953). The separation of the four acids is difacult by this method, but their existence

Temperature: 20°C. after 6 hr.; solvent: butanol, ethanol, water, 3!5% ammonia is evident as shown in the author's previous reports.

in water ( 10: 1 :2:0.5).

and infrared spectroscopy. Syringic acid and ferulic acid and trimethyl- gallic acid ( 3,4,5-trimethoxybenzoic acid) were isolated and identified. Asao and Yokotsuka pointed out that a new series of aromatic com- pounds and several syringyl compounds exist in soy sauce in addition to the several guaiacyl and phenylene compounds which they had pre- viously isolated. They attributed the origin of these compounds to lignin substances in the raw materials.

Yokotsuka (1951a,b,c,d) and others have examined the flavor com- ponents of soy sauce, especially their chnngc- tlriring hcating. In addi- tion to the increase in acidic compoimds, the increase in ether-soluble sulfur compounds was considered one of the most important character- istics of heated soy sauce. Yokotsuka also rocokmized that the constit-

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104 TAMOTSIJ YOKOTSUKA

TABLE XI1 FLavori COMPONENTS I N s o u SAW< E: Ac Inlr: C0MPl:UNI s’’

Solvent layer Biiffer layer Components con- Srpa- Coinponcwts isolated hidered to be pres- rating ( Frac- (Frar- in crystalline form ent by paper funnel t ion) (g.)

1 B1

2 B2

3 B3

4 R4

5 B5

6 B6

7 B7

8 B8

9 BY 10 B10

-

-

-

0.18

0.21

0.15

0.14

- Trace

tion )

c1

c 2

c3

c 4

c 5

C6

c 7

C8

c9 c10

(g. ) a i d identified chromatography ~

Oxiilic acid an- hytlride ( B l )

-

0.07 Vanillic acid (C4,

0.07 BS )

0.09

0.14 Syringic acid

0.19 Bcnzoic acid (C7, B7)

(C8)

0.34 Unknown crystals C,H , 0 -1?03

- _-

ni.p. 95-96°C.

Acetic, lactic,

or succinic ( B l ) acetic, caproic (C2)

Acetic, propionic, caproic, valeric (€33)

Acetic, propionic, bntyric (C4)

Ferulic, oantUtc, syringic (B5)

Ferulic, vanillic, syringic (C6)

Syringic ( B 7 )

Benzoic (B8, C8)

( I From Asao iind Yokotsuka ( 1957).

uents of heated soy sauce were more complex than those of the raw. This has also been observed by Shoji (1936a,b) and others.

IV. SOURCES OF AROMA AND FLAVOR DEVELOPMENT

A. RAW MATERIALS

1. Soybean

The typical differences in chemical composition of the beans from different countries are shown in Table XIII. Soybean oil is changed into soy sauce oil or the ethyl esters of the fatty acids during fermentation. For the past 50 years, defatted soyhei1ns have been used to replace whole beans in the production of soy siiiice. At first, pressed soybean oilmeal was used; now, however, solvent-extracted oilmeal is widely used. The reason for using defatted instead of whole beans is that cost

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AROMA AND FLAVOR OF JAPANESE SOY SAUCE 105

of the dcfatted prodiict is lower. Furthermorc, tlw utilization of nitrogen is sometimes higher in dcfatted soybeans. There is a great composition difference between soy sauces produced from defatted and whole beans.

A comparison of soy sauces from defatted and whole beans should be based on fermentation period, cost, quality, and stability. The nitro- gen content of whole soybeans is Iower than that of defatted beans;

TABLE XI11 COMPOSmON OF SOY BEANS F R O ~ I \'AIUOUS SOUnCES"

H20 Total Crude Invert No. of content nitrogen fat sugars

Country samples ( % a ) (9) ( a ) (9)

Japan 12 12.97 5.901 15.59 14.54 China 7 10.70 5.928 18.58 18.68 U S A . 21 9.54 8.229 19.70 16.91

a From Noda Soy Sauce Company, Limited ( 1957).

whole beans have a slower rate of fermentation (it takes about 15 months at ordinary temperature for whole beans, but only about 10 months for defatted beans); soy sauce made from whole beans is more stable than that produced from defatted beans.

2. Wheat and W?ieat Bran

Wheat from Canada or Australia is widely used for soy sauce pro- duction in Japan. It is the carbohydrate source for culturing the mold and in the fermentation of the mash. Wheat bran is indispensable in producing the specific flavor of Japanese soy sitlice, but too much of it causes both inferior flavor and excessive color.

3. Ratw of Soybeans to Wheat

The use of wheat decreases the nitrogen content of soy sauce, but it contributes aroma and flavor. The best soy sauce is generally believed to be made from a soybean-to-wheat ratio of 50:50 by weight, or 52:48 by volume. Ohara and Moriguchi (1955a,b) reported that, in order to obtain a product of higher total nitrogen, amino acid, and total acidity content, the ratio of defatted soybean to wheat should be increased. These same authors studied the ratio of wheat and bran and found that with a higher proportion of wheat, aroma and flavor were better, acidity was higher, but color was less intense. There was no difference in total nitrogen, amino nitrogen, and residual sugar.

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106 TAMOTSIJ YOKOTSUKA

1. Wholc Soybcm

In the manufacture of sauce, the soybeans are soaked in water for about 15 hr. at room temperature and then autoclaved. According to Nakaya et al. (1934), good results are obtained when the beans in- crease 2.10 to 2.15 times in weight, and 2.20 to 2.25 times in volume during this period. Kawano (193s) found that the digestibility of auto- claved soybeans decreased as the pressure in the autoclave increases (see Table XIV). This may be caused by the browning reaction. Re-

TABLE XIV DICESTII~ILI I'Y OF COOKED SOYDEANS"

Nitrogen content of the raw material Digestible nitrogen Cooking __

( ks. 1 (16) pressure Total Amino Total Amino

0.0 3.310 1 .of30 48.44 15.52 0.5 3.812 1.145 58.23 18.03 1 .o 3.409 1.045 53.27 16.19 1.5 3.322 0.990 50.34 15.34 2.0 2.937 0.881) 40.58 13.95

0 From Kawano ( 1938).

cently Tateno and Umeda (1955) and the Noda Soy Sauce Co., Ltd. (1955), claimed that total nitrogen in the soybeans is best utilized when the beans are soaked in water for 10 to 12 hr. at room temperature and then autoclaved at 10 to 13 lb./sq. in. for about 1 hr. Immediately after cooking, the material was cooled to <40°C. This method marks a change in the soy sauce industry of Japan in that manufncturers are now taking the beans out of the autoclave immediately instead of allow- ing them to remain for an additional 12 hr.

Yokotsuka (1957) studied the influence of cooking soybeans on the quality of soy sauce. Too long a cooking time causes a decrease in total, amino, ammonia, and tannin-precipitable nitrogen, acidity, vola- tile acids, glycerin, and the ratio of various nitrogen constituents, Some chemical changes in the soybean caused by cooking under different pressures have been reported by Kawano (1938).

2. Defatted Soybeans Tateno et al. (1950) reported that the utilization of total and amino

nitrogen increases with increases in moisture content of the defatted

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AROMA AND FLAVOR OF JAPANESE SOY SAUCE 107

bean. For carbohydrate utilization, the reverse was true. Yokotsuka and Tekimoto (1958) studied the chemical changes the defatted soy- beans undergo when cooked at various pressures, times, and moisture content of raw material. They also studied the digestibility of the p r d - uct by in oitro enzymatic action and also the effect on yield and chemical composition of the soy sauce. In the high moisture range, the moisture content of defatted soybeans had no influence on the digesti- bility of the cooked product by mold enzymes. In the low moisture range, a decrease in digestibility was noted. A higher moisture content in the soaked bean seemed to be beneficial for the mold culture. The effect of cooking time and steam pressure on the yield and chemical composition of soy sauce was approximately the same for both defatted and whole beans.

3. Wheat and Wheat Bran

In soy sauce manufacture the wheat is usually first roasted, then crushed. It is generally believed that roasted wheat contributes aroma and flavor to soy sauce. On the other hand, wheat bran is generally steamed, instead of being roasted. Asao and Yokotsuka (195%) re- ported on the changes in phenolic compounds in the wheat and wheat bran by steaming. The characteristic products produced by cooking wheat were the guaiacyl series compounds, such as vanillin, vanillic acid, and ferulic acid (Scheme 11). These were claimed to be associated with the aroma and flavor of soy sauce. Wheat bran (which had been subjected to ether extraction) was cooked under 13 Ib. pressure for 10, 20, 30, and 60 min., and the free and conjugated phenolic compounds in the cooked bran were determined colorimetrically. The free phenols increased with heating, but the conjugated phenols were at a minimum when cooked for 20 min. Paper chromatography of the ether extract of cooked wheat showed the presence of vanillin, ferulic acid, vanillic acid, and 4ethylguaiacol. Among these, vanillin was the most abundant. These workers concluded that the phenolic compounds came from the degradation of lignin and glycosides by heating.

OH OH OH OH

0 4 C H . I ~$-OCHS b - O C H . w8 1

G H h ‘coo11 I

CII=CHCOOH Vanillin Ferulic acid Virnillic wid 4-Ethyl&aitwol

SCHEME I1

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108 TAMOTSU YOKOTSUKA

C. KOJI

Koji is a mixture of wheat bran and soybean flour cultured with the mold Aspergillus oryzae or A. soyae. It is usually cultured for 72 hr. in small boxes or trays in it warm room. During this period, the material is cooled twice by hand mixing. Continiious or mechanically controlled mold culturing is greatly desired but as yet has not been successful.

1. Seed Mold First, 0.1 to 0.2% of the sccd mold is mixed with the material. Some

morphological comparison of the strains used by the soy sauce industry has been reported by Kibi ( 1926), Sakaguchi and Yamada (1945a,b), and others. Resides Aspergillus or!izae and A. soyae, A. ochraceus, A. mllius, and A. niger have been tested in laboratories but have shown no practical value. A good culture mold must give the characteristic aroma and flavor to the soy sauce, have high proteolytic activity, and must he easy to culture.

Iguchi (194956) and Iguchi and Yamamoto (1955) obtained a mutant by X-ray irradiation wliich hid two or three times the proteolyt- ic activity of other strains tested. Sakaguchi and Ishitani (195254) reported on a natural mutation of A. oryzne.

2. Ctcltrrring Koji

It is most important in cultiiring koji to control temperature and moisture. The temperature of the cultnring room is usually kept at 25" to 35OC. U'hen the mold grows, the temperature of the material rises. When the temperature becomes too high, the culture must be cooled. Cooling is usually done twice, about 20 and 28 hr. after inoculation. A rather high moisture content is required at the beginning, when the mold is growing rapidly; lower moisture is desirable at the later stages, when spores are being formed. Kinoshita et al. (1935) reported that the cooling must be done before the temperature rises >40"C. Harada ( 1951 ) studied koji cultured under various temperature conditions and found that the highest proteolytic activity was obtained when the cul- ture was cooled rapidly during the second cooling operation. He kept the two lots of koji at the samc- tczmperatiire prior to the second cooling period. One was cooled rapidly <!35"C., while the other one was not. The proteolytic activities of the two lots of koji were almost identical in this case; howtwer, thc moisture contcnt of the former was 5% higher, and the utilization of total nitrogen was 7% greater (in the soy sauce). He concluded that it was necessary to kcep the water content of koji at 27 to 37% by adequate cooling during cidhiring. Murakami (1951a,b)

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AROMA AND FLAVOR OF JAPANESE SOY SAUCE 109

also observed that lower tcmperatiirc-s :ire prcfcrable for the mold cul- ture of miso; however, it was necessary to maintain the higher tempera- ture, i.e., about 42"C., for less than 5 hr. after the second cooling period.

Yamamoto (1957a,b,c) used a new method for separating the me- dium from the mold mycelia by a thin net of nylon. The optimum temperature for both mycelium weight and spore production was 35°C. with A. soyue (KS) on wheat bran. A higher moisture range of 50 to 75% favored mycelial growth. For spore production, a lower moisture content was favored. When wheat bran and defatted soybeans were used, a higher C/N (carbon-to-nitrogen) ratio caused more mycelial growth and an increased greenish spore. The maximum proteolytic ac- tivity was obtained when wheat bran and soybean flour were used in equal amounts. Yamamoto also identified a neutral protease, an acid protease (optimum pH 3 to 4) , and an alkaline protease (optimum pH 7 to 10) in the cultured koji. He recognized that the lower the cultural temperature the higher the protease activity will be so long as the mold is growing. Eventually, protease activity reaches a maximum. It seems to be the current trend to obtain high proteolytic activity by increasing the moisture content of koji. The results are shown in Figs. 2 and 3.

pH 7.0 10

1.2

10

0.8

0.6

0 4

0.2

I 2 3 4 5 6

Tim. in daya

FIG. 2. Effeeas of temperature and pl l on the production of alkaline protease during the wheat bran koji culturing.

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110 TAMOTSU YOKOTSUKA

- r, c

oa 0 0

0 V

- .- ; 0.6 0 - 2. c

’$ 0 4 e 0 0

0

0 :: 0.2 c

. e a

00 0 5 10 15 20

Time in days

FIG. 3. Effect of cultural temperature on the growth and production of mold protease activity, - - - - - - - dried protease in the paste culture;

mycelial weight.

- P

0 I 2 3 4

Time in d a y s

FIG. 4. Effect of cultural temperature on the prodrwtion of mold amylase in the paste culture.

The reverse was true for the production of mold amylase (Fig. 4) . Chemical changes during koji culturing were studied by Kawamori

(193843), who observed a loss of carbohydrate and an apparent in- crease in nitrogen content. A higher moisture content caused more rapid growth of the mold and greater loss of carbohydrates.

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AROMA AND FLAVOR OF JAPANESE SOY SAUCE 111

It is very difficult to estimate the effect of koji on the flavor of the soy sauce. Commonly, the volume of spores, tightness, color, flavor, etc., are observed organoleptically; the moisture content, acidity, the potency of protease and amylase, etc., are determined chemically. Sugita (1956) studied the relationship between the organoleptic evaluation of cultured koji and the quality of soy sauce made of it, and recognized a fairly good correlation between them. However, the potency of protease had very little meaning, and the analysis for digestion was preferable for anticipating the quality of soy sauce. Asao and Yokotsuka (195%) studied the chemical changes of the phenolic compounds, especially the guaiacyl series, during mold culturing. They used A. oyzue (KS) on ether-extracted, steamed wheat bran for 24, 48, 72, and 96 hr., re- spectively. Both free and conjugated phenolic compounds in the ether extract were determined colorimetrically. The amount of the free phenol- ic compounds reached a maximum before spore formation, while that of the conjugated phenolic compounds decreased gradually. By paper, chromatography, a rapid disappearance of vanillin, a gradual decrease of ferulic acid, and an apparent increase of vanillic acid were observed. The spot corresponding to 4-ethylguaiacol increased gradually. From these results, it was concluded that these important aromatic ingredi- ents were already present at the mold culturing stage. The importance of wheat bran to soy sauce aroma and flavor was claimed. In this ex- periment, ferulic acid was identified for the first time in the material resembling soy sauce. It was isolated as needle crystals, melting at 167" to 168°C.

Soy sauce production by submerged mold culture has been recently studied by Shiyota and Sakaguchi (1949), Ichikawa (1954), and Mogi (1958a,b,c,d). Soy sauce which is almost similar to that ordinarily fer- mented in flavor and chemical composition has come to be produced by this method. However, many problems remain unsolved.

D. MASH (MOROMI)

The koji is mixed with about an equal amount of saline water to form the mash, or moromi. The mash is kept in a large container for about 1 year at ordinary temperatures, or for 3 to 4 months if warmed. Ordinarily the mash is stirred by compressed air. Major chemical changes in this process arc? degradations of protein and carbohydrate caused by the enzymes derived from koji. A lactic fermentation occurs in the first stage, an alcoholic fermentation by yeasts in the second stage, and an aging or completion of fermentation in the last stage, The color of the mash gradually becomes more intense.

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112 TAMOTSIJ YOKOTSUKA

1. Preparation ( Mashing)

Formerly, equal volumes of salt water and koji were used, but re- cently the volume of salt water hits bcen increitsed to 1101 to 12M of the raw material. Mixing koji with more water causes a better utiliza- tion of the total nitrogen of the raw material; however, this may have some undesirable effect on the composition of the soy sauce. Sodium chloride in the mash is usually 17 to 19%. It is regarded as dangerous to use a solution <16% because of the danger of putrefaction. Ohara and Moriguchi (19%) mixed salt solutions of 19", 2l0, and 23" Baumk (Be) with koji, and found that the lower the Bk the better the utiliza- tion of total nitrogen and amino nitrogen, also the better the fermenta- tion; in addition, less residrinl sugar i1Ild higher acidity were obtained in the soy sauce. I

2. Control of Mash

In natural fermentation, where the temperature of the mash is not controlled, a fermentation period of over 12 months is necessary, since the mash must be fermented by yeasts for one summer to give good aroma and flavor. To shorten the fermentation period, the temperature must be controlled artificially.

Formerly, the mash was stirred occasionally with a rod. Nowadays it is stirred with compressed air at 6 to 10 lb. pressure for 5 to 10 min. Too much stirring, especially in suinmer, hinders the fermentation of mash, according to Yamada and Ftlrtisilka (1954). Stirring the mash is very important and a difficult procidure, and no c.onclusion has been reached regarding the frequency of stirring.

3. Aging

The initial pH of the new mash is usually 6.0 to 7.0. It decreases rapidly, especially in summer, to about 4.5, the point at which alcohol fermentation begins if the temperature is proper. Usually in 3 or 4 months ilt normal temperatures, the total nitrogen dissolved in the mash attains a constant value. Amino acid content increases at almost the same rate as the total nitrogen.

Udo (1931b) analyzed the chemical composition of mashes aged for varying periods (see Table XV). These are typical figures for the de- velopment of mash from whole beans at ambient temperature. Glutamic acid was at a maximum after 15 months and then started to decrease. Udo concluded that the optimum aging period could be determined by analyzing for free glutiunic acid content. Recently, Umeda et al. ( 1953), working with defatted soybeans, reported the same finding.

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AROMA AND FLAVOR OF JAPANESE SOY SAUCE 113

Tho maximum glutamic nitrogen content was obtained in 10 or 11 months. In another experiment, they found that the glutamic nitrogen content of a mash aged 58 months was equal to that of a mash aged

Tmm XV

WHOLE BEANS AND WHEAV CHEMICAL CHANCES OCCURIUNC IN SOY SAUCE MASH PREPARED FROM

Date of Total prepara- Aging nitro-

tion (months) genb

Aug. 1 1.274 J ~ Y 2 1.485 June 3 1.463 May 4 1.469 April 5 1.571 March 6 1.469 Feb. 7 1.501 Jan. 8 1.406 DW. 9 1.336 Nov. 10 1.5QQ OCt. 11 1.488 June 15 1.476 April 17 1.493 June 27 1.644

-~ ~

Protein nitro- gene

~~ ~

Amino Am- nitro-

moniab genb

Nitrogen organic Reducing Clutamic basesb sugarse acidbsc

0.043 0.043 0.043 0.043 0.088 0.045 0.045 0.039 0.041 0.039 0.041 0.035 0.039 0.036

~~

0.070 0.758 0.113 0.848 0.114 0.865 0.145 0.945 0.132 0.872 0.155 0.964 0.156 0.958 0.157 0.956 0.150 0.888 0.178 0.917 0.171 1.025 0.156 0.731 0.169 0.919 0.153 0.882

~~~ ~~

0.311 15.45 0.027 0.358 5.36 '0.290 5.18 0.033 0.254 5.22 0.367 5.01 0.120 0.327 4.91 0.343 5.10 0.119 0.235 4.72 0.344 5.39 0.242 0.352 5.20 0.367 5.31 0.289 0.276 5.68 0.379 0.289 4.94 0.156 0.307 4.82 0.220

a From Udo ( 1931). b Data expressed as g./lOO ml. CValues rather high, presumably because of lack of specificity in analytical

method, especially due to aspartic acid.

only 1 month. The nitrogen utilization is always 5 to 10% lower in mashes that are

begun in the summer, according to Noda Soy Sauce Co., Ltd. (1955). This is believed to explain the poorer quality of koji produced in the summer. Higher temperatures cause a rapid decrease of mash acidity and rapid inactivation of enzymes.

Kubo (1947) found that soy sauce oil, consisting chiefly of ethyl esters of higher fatty acids, was produced during fermentation by the exchange of glycerin with ethyl alcohol.

4. Microbiology Besides Aspergillus otyzae or A. soyae, Monflia, Penicillium, and

Rhixopus are sometimes found in mash; however, these molds are be- lieved to have no relation to proper aging.

There are many reports concerning the yeast found in mash, Many

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114 TAhfOTSLJ YOKOTSUKA

Zygosaccharomyces are present because of their resistance to higher osmotic pressure. Zygoswcharninyccs s o p , Z. japonicus, Z . maior. Z . s u h , Pichia farinosa, Torulu, Mycodermu and Monilinia have been identified by Mitsuda (1910), Nishimura ( 1910-12), Kita ( 1911), Taka- hashi and Yukawa (1911-14), and Ishimaru (1935). Among these, Z. s u b s , 2. japnlcus, Pichia, and Torula are considered to be harmful in soy sauce fermentation. Zygosaccharomyces major and 2. soyae are be- lieved to take part in the normal fermentation.

Most of the earlier studies on soy yeasts have been chiefly concerned with taxonomic problems. Reliable information on the biochemical ac- tivity of the yeasts in the mash has not yet been obtained. Recently, extensive studies have been conducted by Onishi (195458). From the ecological point of view, he studied the methods for determining viable yeast count in the presence of molds, and methods for determin- ing viable yeast count in soy mashes (which differ significantly from other mashes in their high content of sodium chloride and nitrogen com- pounds). During the course of these experiments, it became apparent that the growth of soy yeasts involves a process of physiological adapta- tion. Salt-tolerant yeasts were isolated from soy mashes, and the taxonomic studies carried out. Changes in the microflora during soy brewing were observed. The nutritional requirements for nitrogen com- pounds and vitamins in two contrasting environments (sodium chloride- free and a high concentration of sodium chloride) were studied. It was found that the pH range for the growth of soy yeasts in a sodium chloride-free medium is very wide (pH 3.0 to 7.0), while that in an 18% sodium chloride medium is limited (pH 4.0 to 5.0).

A striking increase in permeability of the cell membrane of Sac- chromyces rouxii, a typical salt-tolerant yeast, occurred when culti- vated in saline. This change, however, did not occur with a concen- trated sugar solution. There it appears that this increase in permeability should be attributed to the specific effect of sodium chloride rather than to an osmotic effect. This increase in permeability must be one of the most important factors to explain the salt-tolerant property of cer- tain yeasts. The influence of temperature on the growth and death of osmophilic yeasts in environments of both ordinary and high concen- trations of sodium chloride have been investigated; it was found that many of these yeasts are able to grow in the saline medium at tempera- tures as high as 40°C., whereas in a sodium chloride-free medium no growth occurs at this temperature. Saccharomyces rouxii was found to ferment glucose, giving good yields of glycerol in a high-saline medium (under aerobic conditions ) , even though it produced only small amounts of glycerol in an ordinary medium. As much as 40 to 50% of

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AROMA AND FLAVOR OF JAPANESE SOY SAUCE 115

thc glucosc! fermcmted Wits convertcd to glyccrol unclcr aerobic condi- tions.

Takeda (1954) reported that good results were obtained by adding to the mash, yeasts which had been cultured under aerobic conditions. This practice is used in some plants; however, Yauchi et 01. (1955) recognized that excessive fermentation induced by cultured yeasts could result in inferior soy sauce.

Yamada and Furusaka (1954) claimed that more than 1W of sodium chloride is necessary for the growth of 2. major in soy sauce mash. Obata (1955) found that soy sauce aroma is produced only when the sodium chloride content of the medium is higher than 5%.

A great number of bacteria belonging to the Subtilis or Micrococ- cus groups have been isolilted from soy sauce mash by Matsumoto (1925) and Ishimaru (1933a,b). These bacteria have been found to be active in the soy-koji making stage; they survive in the soy mash as resting spores. Kenji Sakaguchi (195p59) has reported on the activity of bacteria in soy sauce fermentation. A new species of salt- tolerant lactic acid bacteria, Padiococcus soyue nov. sp., was found to be the sole bacterium vigorously growing in soy mash. Its proteases, transaminases, and the roles played in the brewing were studied. The organism could grow in solutions of high osmotic pressure (e.g., in solutions of many inorganic salts and sugars, reaching to 130 atm.). It required betaine and inorganic salts specifically, as well as known vita- mins, bases, and amino acids. In the soy mash, salt-sensitive lactic acid bacteria decrease rapidly within a period of 2 months. On the other hand, Pea. soyae grows well in ah 18% salt mash (starting at 10Z-lOq/ml. and reaching to 10"-lOO/ml. at the fourth month of fermentation). The pH value of soy mash was lowered when Ped. soyue reached lO"/ml.

5. Pressing The liquid part of the mash is removed with a hydraulic press. The

residue (soy cake) has a 25 to 3W moisture content. The use of filtering equipment, such as the filter press or the Oliver filter, has not succeeded in decreasing the moisture content of the residue below about 40%. The oily layer of the press fluid is separated by decantation. The yield of soy sauce oil from 1 kl. of whole bean and wheat mash is about 60 liters (see Table XVI).

E. PASTEURIZATION Raw soy sauce is heated to destroy microorganisms and enzymes

and to bring about the sedimentation of protein compounds by coagu- lation. Concerning the compounds coagulated by pasteurization, there

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116 TAMOTSU YOKOTSUKA

arc rcports of hlutsiimoto ( 1923), hlatsiiinoto and hliirakami (1941), Ohara and Onislii ( 1951 ), Ucw) iiiI(1 Omori ( 1951 ), iind Kosaka ( 19%). The temperature of heating now i n iisc i s ilhoiit 80^(:.. althoiigh 70°C. was formerly used. The highcr tcnipcrature is generally believed to in- crease the solubility and dispersion of biityl-),-hydros)..benzoate, widely used as a preservative in Japan. Umeda et al. (1951-52), Sugita and Ohara (1957-58), Ohara (1958), and Yokotsuka et al. (1958) have re- ported on dissolving and dispersing but) I-p-hydroxybenzoate. Soy sauce pasteurization has actually been studied very little. Akabori and Kaneko (1936) found an increase in aldehydes during the heating of soy sauce (Fig. 5 ) . No increase in isovaleraldehydc was observed when the sauce was heated <60°C. (20-30 mm. Hg).

TABLE XVI CO~IPOSITION ov SOY CAKE^

Moisture content Sodium chloride Total nitrogen Invert sugars Crude fat

Soy cnke from whole hems and wheat

($1

25.20 5.24 3.862

12.00 10.41

Soy cake from defatted beans and wheat

( 9 )

25.80 5.00 3.853

13.78 5.77

a From Noda Soy Sauce Cornpiny, Limited ( 1958).

Yokotsuka and Takimoto (1956) studied the subject by refluxing raw soy sauce in a flask at 80°C. in a water bath. At 5hr . intervals, 350-ml. sampjes were taken out for 35 hr. The chemical changes in these samples are shown in Table XVII. Total titratable acidity, ether- soluble nitrogen-containing acids, ether-soluble organic acids, phenolic substances in the conjugated form, and the extinction value at 470, 530, and 610 mp increased with hating. Free phenolic substances reach a maximum during heating. The ether-soluble acids in conjugated form and total nitrogen did not change, but amino nitrogen and reducing sugars decreased. Iodine absorption, potassium permanganate con- sumption by the ether extract, and sulfur content of the ether extract increased on heating. Furfural mnteiit of the ethcr extract increased remarkably immediately after heating but decreased rapidly thereafter. Free glutamic acid decreased by about 5% after heating for 5 ht. Phos- phoric acid decreased with heating.

The change in the ultraviolet absorption spectra of the weakly acidic (phenolic), acidic, and neutral fractions of the ether extract of

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AROMA AND FLAVOR OF JAPANESE SOY SAUCE 117

soy sauce was investigated, The maximum absorption of the weak acidic fraction shifted from 285 mp to about 280 mp on heating, and its con- tent exceeded the maximum value in the course of heating. This phe- nomenon was believed to reflect a change in 4-ethylguaiacol content, which was identified by the authors as i1n important flavor ingredient of soy sauce. All of the acid fractions had the same maximum absorption

50 ““t

20 60 100 140 180 220 260

Volume of distillate (ml.) -c

FIG. 5. Increase in aldehyde during the heating of soy sauce; -0--0- vacuum distillate of soy sauce. -x--x- repeated distillation of the above distillate.

wavelength at about 265 mp, but they differed from the phenolic frac- tions. The ultraviolet absorption of the neutral fraction increased gradually with heating, always showing the maximum value at about 290 mp, which seemed to indicate the change in furan components.

Yokotsuka and Takimoto (1956) evaluated the effect of heating on color and acidity in terms of the stabilization of the soy sauce. They listed the causes of the increase in acidity of heated soy sauce as: oxidation of unsaturated oil or aldehyde compounds, hydrolysis of esters or phenolesters, decomposition of glycosides, browning reaction, pyrolysis of sugars, and formittion of pyroglutamic acid from glutamic acid. These acidic compounds, including organic acids and phenols, constitute an important part of the so-called “heated” flavor. The other

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c c. m

TABLE XVII E F F ~ OF HEATING ON CHEMICAL COMPOS~ION OF SOY SAW@

Ether extractives

Length Free form Conjugated forma heating Total

80°C. TotalN AminoN sugar sub- Organic substancec TotalN Volatile Organic Phenolse Total N period at Reducing acidic Phenolic Li

E (b.1 (I) ($1 (%) stancesb acidsb ( 9 ) (%) acidsb acidsb (%) ( 9 ) 3

0 5

10 15 20 25 30 35

1 .so 1.51 1.50 1.52 1.51 1.52 1.52 1.52

0.97 2.73 25.0 11.7 0.017 0.006 0.56 25.7 0.007 0.006 3

5 0.96 2.47 28.0 12.8 0.048 0.007 0.60 25.1 0.007 0.006 g 0.94 2.10 33.0 1.3.8 0.060 0.009 8.35 0.012 0.007 0.91 1.90 35.0 14.4 0.057 0.009 9.10 0.012 0.007 0.89 1.84 0.89 1.59 36.0 17.1 0.055 0.012 9.65 25.4 0.015 0.010 0.85 1.44 0.83 1.34 37.0 17.9 0.051 0.01s 10.80 25.8 0.015 0.012

‘l From Yokotsuka and Takimoto ( 1956). b Milliliters of 0.05 N sodium hydroxide per 10 ml. soy sauce. C Measured by Felin-cioCalteu method. d Extracted at pH 3.0 for 60 hr. after removing free forms at pH 4.5 (natural) by ether extraction for 60 hr.

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AROMA AND FLAVOR OF JAPANESE SOY SAUCE 119

'heated" flavor components of soy sauce art* aldehydes, mercaptans, and their interreaction compounds.

The evaporation of the lower-boiling compounds, which occurs when soy sauce is heated, is another important factor cwising flavor changes. The vapor that rises from the surfilce of heated soy sauce contains mercaptans, mercaptals, lower-boiling aldehydes, ethanol, ethyl acetate, isovaleraldehyde, and traces of higher-boiling compounds. It is clear that while adequate heat is necessary for flavor development, excessive heat is harmful.

V. FLAVOR INGREDIENTS AS NATURAL PRESERVATIVES

A. NATURAL YEAST-STATIC AND R ACTEFUCIDAL COMPOUNDS

A good-quality fermented soy sauce does not have a white (yeast) film on the surface when open to the air because it contains yeast-static compounds. Three kinds of yeasts are widely known to be harmful to finished soy sauce. Film-forming yeasts, such as Zygosucchuromyces suhts, Z. juponicus, and Piclziu; ring-forming To~lops is which grows on the surface of soy sauce aroiind the edge of the container; and bot- tom yeast belonging to Zygosaccharomyces. The latter two may be present in dilute soy sauces. Reports on these organisms have been made by Takahashi and Yukawa (1911), and Ishimaru (19,3535) and Mogi et al. (195152).

Yokotsuka et al. (1958) recognized that soy sauce produced from whole beans has a greater resistance to yeast invasion than that pro- duced from defatted beans. Raw soy sauce has the same tendency as a pasteurized product. The yeast-static power of an ether extract of soy sauce produced from whole beans and defatted beans against Z. sulsus in synthetic medium was compared. Potency was expressed in terms of concentration of butyl-p-hydroxybenzoate which was used as the con- trol. Whole hems had a potency corresponding to 0.033% of butyl-p- hydroxybenzoate, and that of the defatted beans was 0.025%. After heat- ing for 5 hr. at 8O"C., the former rose to 0.037% and the latter to 0.050RI. This shows that raw soy sauce produced from whole beans is more resistant to yeast invasion than that from defatted beans. This was re- versed for heated soy sauce. Residue from the ether extract of the two heated soy sauces was reheated and extracted with ether. The potency of this extract was determined similarly; the values obtained were 0.008% for the soy sauce produced from whole beans and 0.013% for that from defatted beans. The results show that the yeast-static com- pound was derived from the degradation of the ether-insoluble portion of soy sauce. On the other hand, yeast-static compounds increased

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120 TAMOTSIJ YOKOTSUKA

through ht-at degr;rdirtion of tlw ctlrc.r-soluble portion. wliieh differed with the dcbgrec- of hciIti11g. Aftcr licating at 70" and 90°C. for 5 hr., the potencics were 0.03% and 0.0472, rcspectively, for whole soybean soy sauce, and 0.047% and 0.059% for soy sauce made from defatted

TABLE XVIII

FILM-FORMING YEAST$ ON DILUTED SOY SAUCER CONCENTRATION OF COMPOUNDS NECE~SARY TO PREVENT GIlOWTH OF

Minimum effective concentration Compounds probably present

Prcservative equivalrnt in soy sauce _______ _ _ _

($1

0.01 4-Etliyl-giiniac.ol Isobutyl benzoate 0.02 n-Caproic acid Isobutyl vanillate 0.04 Ethyl vanillate Propylbenzoate

Ethyl benzoate Isoamyl acetate

Benzaldehydc Ethylisovaleriana te

0.06 Benzoic acid Benzaldehyde diethylacetal

0.08 Vanillin 0.1 Isovaleraldeh yde

Isovaleracetal Vanillic acid

0.1 1-Benzoyloxy-2-methoxy- Isobutylacetal 4ethylbenzcne

Tyrosol Acetic acid Propionic acid Isobutyric acid Isovaleric acid Ethyl acetate Ethyl lactate Ethyl pnlmitate Ethyl oleate Ethyl linolate Ethyl mercaptan Isoamyl alcohol Butanol Ethanol (1%)

=From Yokotsnka ( 1954).

beans. These silme authors determined the minimum concentration that inhibits film-forming yeasts in diluted soy sauce. The results are shown in Table XVIII. As may be seen, it was found that 4-ethylguaiacol and n-caproic acid have the highest potency, bcing effective at 0.02%. After comparing the potencies of the neutral and acidic fractions of the ether extract of soy sauce by bioassay, they found that the major yeast-static

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AROMA AND FLAVOR OF JAPANESE SOY SAUCE 121

powcr of heatcd and iinhcatcd soy s;iiicc exists in tlic acidic fraction. When the iicidic part was dividcd into sodium bicarhonatc-soluble and -insoluble parts, the grcater potency wiis found in the former. On the basis of these experiments, it is concluded that the major sources of yeast-static power in soy sauce' are organic acids, such as n-caproic acid. Although the unit potencies of n-caproic acid and 4-ethylguaiacol are equal, there is much more of the former present.

Ujiie and Yokoyama (1956) studied the bactericidal nature of soy sauces, using several kinds of pathogenic bacteria. They named the following as the reason for bactericidal power of soy sauce as being: (1) acidity, (2) high osmotic pressure, and (3) chemical preservatives present such as butyl-p-hydroxybenzoate.

B. ARTIFICIAL PRESERVATIVES FOR SOY SAUCE Butyl-p-hydroxybenzoiite is the chemical prcservative for soy sauce

now most widely used in Japan. However, sodium benzoate is used for soy sauce to be exported since the use of biityl-p-hydro~yhenzoate is not officially permitted in some countries. Usually 0.005'x of the former and 0.02% of the latter is used for soy sauce. Such artificial preservatives as the fatty acids (caproic, capric, and lauric acid), furanacrylic acid, benzoic acid, benzoate, methyl, ethyl, iind isobutyl-p-hydroxybenzoate, mustard oil, rhodan esters, vitamin K.3, p-aminoazobenzol, dehydro- butylacetic acid, several vanillates, i d sorbic acid have been studied (Fukai, 1928; Fukai and Komatsu, 19+34a,b; Frikai et al., 1951; Yabuta and Kobe, 1928; Kurono, 1934; Tomiyasu and Toyomizu, 1952; Tera- mot0 and Hashida, 1954; Tsukamoto et al., 1M8; Umeda and Ikura, 1951; and Umeda and Ito, 1952). Fujikawa et al. (1934-44), Ota (1934), Umeda and Hatano (1948), Akatsu (1950-54), and Yoshida et al. (1952) have reported that many kinds of natural pigments, lichen constituents, a substance produced by certain Torulopsis, and japonic acid (an antibiotic produced by Aspergillus juponicus) are natural pre- servatives for soy sauce.

VI. SUMMARY

The chemistry and composition of Japanese soy sauce has been re- viewed, with emphasis on the flavor and aroma constituents. Over 90% of Japanese soy sauce is the koikuchi type, containing about equal amounts of fermcnted saiicc and a chemical hydrolyzate, obtained from defatted soybeans.

The nitrogen compounds in soy siiiice consist of about 40 to 502 amino acids, 10 to 15% ammonia, 40 to 50% pcptides and peptones, and less than 1% proteins. The ratio of amino to total nitrogen is as a cri-

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122 TAMOTSU YOKOTSUKA

terion of quality, with a high ratio indicating higher quality. Seventeen amino acids present in soy sauce are: arginine, aspartic acid, cystine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. Quantitative i d y s e s for free iltld conjugatrd amino acids have been presented. Glutamic acid is the major amino acid. The organic bases present are adenine, hypoxanthine, xanthine, guanine, cytosine, and uracil. They are considered to be decomposition products of nucleic acid, produced by enzymes present in the mold culture.

Glucose, galactose, maltose, arabinose, and xylose are the sugars present in soy sauce. Glycerin is one of the key compounds that differ- entiate a soy sauce prepared with whole soybeans from that made with defatted beans. The organic acids of soy sauce are lactic, pyro- glutamic, succinic, and acetic. Lactic acid is the main organic acid present. Vanillic, syringic, ferulic, and trimethyl gallic acids are present in soy sauce as indicated by chromatography of the ether-chloroform extract on an aluminum oxide column or on paper.

The volatile compounds in raw soy sauce and in soy cake were classified as: aliphatic carbonyl series; aliphatic esters, alcohols, and acids; sulfur-containing compounds; phenols; and other aromatic com- pounds. Some workers maintain that the specific aroma of soy sauce is due to sulfur-containing compounds. However, most of the aroma and flavor of sov sauce exists in the acid fraction. The guaiacyl and syringyl series of phenolic compounds, 4-ethylguaiacol, has a strong flavor that resembles soy sauce. Tyrosol or p-hydroxyphenylethyl alcohol is found in commercial soy sauce and is associated with the bitterness of soy sauce.

The mercaptals having a flavor resembling soy sauce are: acetalde- hyde- and isovaleraldehyde-half-mercaptal; furfural-, acetaldehyde-, and isovaleraldehyde-methylmercaptals; acetaldehyde-, isobutylaldehyde- mercaptals. Methylmcrcaptopropyl pyruvate, and the condensation product from crotonaldehyde and methylmercaptan have an aroma similar to that of soy sauce.

The characteristic flavor ingredients produced by cooking soy sauce are the guaiacyl compounds; namely vanillin, vanillic acid, ferulic acid and 4-ethylguaiacol. Among these, vanillin was the most aromatic. The increase in these phenolic compounds is noted when heating the wheat previously extracted with boiling ethanol, and when boiling an ethanol extract of wheat. The phenolic compounds come from the degradation of lignin and glycosides by heating.

The following factors related to the aroma and flavor of soy sauce have been discussed: raw materials, i.e., soybeans, defatted beans,

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AROMA AND FLAVOR OF JAPANESE SOY SAUCE 123

wheat, wheat bran and sult; koji, which is a mixture of wheat bran and soybean flour cultured with Aspergillus oryzae or A. soyae; condition of fermentation, especially the volume of salt water relative to koji, salt concentration and aging period; and microorganisms involved in diges- tion and fermentation.

ACKNOWLEDGMENTS The author wishes to express his h e w thanks to Professor Dr. Y. Sumiki of

Tokyo University, for kind guidance in this work, and also to Professor Dr. K. Sakaguchi for his assistance. I am also greatly indebted to the members of our laboratory: Director Dr. M. Mogi, colleagues K. Takinioto, Y. Asao, T. Sakasai, and A. Okuhara. I am also grateful to the authorities of Noda Soy Sauce Carnpany Limited, especially to K. Mogi and I. Nakaya. for their permission to publish this review, Finally, I wish to express my bincere thanks to Dr. B. S. Luh, Professor Dr. E. M. Mrak, Professor Dr. G. F. Stewart, and Mr. M. Yaguchi, of the Uni- versity of California at Davis, and to Professor Dr. T. Ohara of the Tokyo University of Education for their kindness in assisting thc publication of this review.

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Akabori, S. 1938. Studies on the flavoroub ingredients of soy sauce. I. J. Chem. SOC. Japan, Pure Chem. Sect. 51. 828431.

Akabori, S. 1938. Studies on the flavorous ingredients of soy sauce. IV. On the 8-y-butylen-glycol and its pinacolin-rearrangement. J. Chem. SOC. Japan, Pure Chem. Sect. 59, 11321154.

Akabori, S., and Kaneko, T. 1938. Studies on the flavorous ingredients of soy sauce. 11. Isolation and synthesis of a snlphur-containing compound from soy sauce. 1. Agr. Chem. SOC. lapan. 57, 832-838.

Akabori, S., and Kaneko, T. 1937. Studies on the flavornus ingredients of soy sauce. 111. On the P-methylmercapto-propionaldehyde. 1. C h . Soc. Japan, Pure Chem. Sect. 58. 238, 237.

Akabori, S., Kaneko, T., and Mochizuki, S. 1938. Studies on the flavorous ingredients of soy sauce. V. On the synthesis of methionol and its relating compounds. J . Chem. SOC. Japan, Pure Chem. Sect. 59, 1135-1138.

Akatsu, K. 1950. Studies on the antibiotics substance produced by Asperglllua faponkw. I. 1. Agr. Chem. soc. lapan. 25, 243-348.

Akatsu, K. 1952. Studies on the antibiotics substance prodriced by AsperglUua japonfcus. 11. Molecular formula, toxicity, and some practical tests. J . Agr. Chem. SOC. Japan. 25, 517419.

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XVI. Flavoroils iiigr~dit.iits i i i raw soy s;iiicv. ( 5 ) . Acidic colnporinds. R d . Agr. Chein. Soc. Japcin 32. (j17-622.

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Fujikawa, F. 1939. Uber die antiseptishe Wirkung der Verbindungen der Olivetol- reihe auf Shoyu (soy sauce). VII. Uber die antiseptische Wirkung der Phenole, Phenolcarbonsiiuren, ails denrn die Flechtmstaffe zusammengesetzt sind, sowie deren Ester. I. Phum. SOC. Japan 59, 240-242.

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Fukai, T. 1928. The relation between soy sauce fermentation and fats and oils. I. On the ethylesters of fatty acids and frcc fatty acids in soy sauce oil. J . A g t . Chem. SOC. Japan 5. 458-470.

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Fukai, T. and Komatsu, S. 1934b. The relation between soy sauce fermentation and fats and oils. VI. The toxic action of capric acid on the propagation of several fermentation microorganisms. Rept. Goof. l a . Brewing Japan 119, 219-229.

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AROMA AND FLAVOR OF JAPANESE SOY SAUCE 125

Iguchi, N. 1949. Studies on Aspergilli. I. Mutation of Asp. sojae Sakaguchi et Yamada by irradiation of ultraviolet ray. J. Agr. C h . Soc. Japan 23, 1U8.

Iychi , N. 1950a. Studies on Aspergilli, 11. Mutation of ASP. sdue Sakasuchi d Yamada by irradiation of ultraviolet ray (cont.). J . Agr. Chem. SOC. lopun 23, 16-18.

Iguchi, N. 1950b. Studies on Aspergilli. 111. The effect of ultraviolet radiation on power of enzyme activities and production of kojic acid in ASP. SOW. J . Agr. Chem. SOC. Japan 24. 283-286.

Iguchi, N. 1951a. Studies on Aspergilli. IV. The production of ultraviolet induced mutations in Arp. soiue (Supplement to part 2). J. Agr. Chem. SOC. Japan 25. 81-84.

Iguchi, N. 1951b. Studies on Aspergilh. V. The production of X-ray induced muta- tions in Asp. soiue. J. Agr. Chem. SOC. Japan 25, 454-465.

Iguchi, N. 195%. Studies on Aspergilli. VI. Nutritional requirements of Asp. sobe mutants for vitamin and amino acids. J. Agr. Chrrn. SOC. Japan 26, 146.

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134 TAMOTSU YDKOTSUKA

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