[advances in applied microbiology] advances in applied microbiology volume 17 volume 17 ||...

Microbiology and Biochemistry of Soy Sauce Fermentation

I . I1 .

I11 . IV . V .

VI . VII .

VIII . IX .

X . XI .

XI1 . XI11 . XIV . xv .

F . M . YONG’ AND B . J . B . WOOD Department of Applied Microbiology.

Uniuersity of Strathclyde. Glasgow. Scotland

Introduction ..................................... 157 Fermented Soy Products .......................... 159 History of Soy Sauce Production .................. 161 Chemical Composition of Soy Sauce ................ 163 Raw Materials .................................. 165 A . Soybeans .................................... 165 B . Wheat ...................................... 167 C . Ratio of Soybeans to Wheat .................. 167 D . Salt ......................................... 168 E . Substitute Raw Materials ...................... 168 Treatment of Raw Materials ....................... 169 Koji ........................................... 171 Culturing the Koji ............................... 173 Mash (Moromi) ................................. 175 A . Preparation (Mashing) ........................ 175 B . Control of the Mash .......................... 176 C . Aging ...................................... 177 D . Microbiology of Mash ........................ 177 Pressing ........................................ 182 Pasteurization ................................... 182 “Chemical” Soy Sauce ............................ 183 Semichemical Soy Sauce. or Shinshiki S h o p ......... 184 Future Development in the Soy Sauce Industry ...... 185 Conclusions ..................................... 188 References ...................................... 188

I . Introduction

Soy sauce has occupied a place of honor as a condiment in orientaI cuisine since time immemorial and is now finding its way into the occidental kitchen . In the course of our studies on the biochemistry and microbiology of this fascinating fermentation. we became aware that there is no complete modern review of the subject readily available to the Western worker . The sequel is an attempt to correct this lack .

True soy sauce is the product of a complex fermentation in which a mixture of soybeans and wheat flour are inoculated with a mold; the mixture is incubated for about 3 days. and. when a good growth of mold mycelium has taken place. the resulting mass is placed in a brine . An anaerobic fermentation. in which yeasts and bacteria partici- pate. then occurs . The liquid obtained from this stage is the soy sauce of commerce .

Present address: Singapore Institute of Standards and Industrial Research. Republic of Singapore .

158 F. M. YONG AND B. J. B . WOOD

Inferior products which lack the rich, satisfying flavor of true soy sauce are prepared by acid hydrolysis of bean and wheat mixtures; unfortunately a substantial proportion of the “soy sauce” sold in the United Kingdom is of this variety.

The fermentation may perhaps owe its origin to the need, in time of plenty, for preserving excess foodstuffs against periods of scarcity. Drying and smoking of meat were man’s first methods of conserving his food supplies, discovered purely by chance. Later, it is likely he discovered the use of salt (sodium chloride) in combination with the drying and smoking methods. Not knowing what actually did take place one may speculate that it is also by accident that mankind discovered that meat and plant materials could be preserved for long periods by the addition of salt. Thus, probably the first fermentations were discovered accidentally when salt was incorporated with the food material and the salt selected certain harmless microorganisms which fermented the raw material to give a nutritious and acceptable food. Speculating along these lines, we might expect that the first fermented product in the Orient may have been fish sauce.

With the advent of religions in which meat was excluded from the diet, the use of salt and fermentation techniques was adapted to plant materials. The discovery of soy sauce fermentation may have been due to the introduction of Buddhism to China, Korea, and Japan.

Soy sauce is the term applied to a salty condiment made by a two-stage fermentation, in which the first stage is aerobic and the second stage is anaerobic. In the first stage, a mixture of soybean and wheat is inoculated with an Aspergillus culture. The mold grows and releases enzymes that break down the proteins and oligosaccharides present in the substrate, The second stage is begun when the mold growth has reached a desired level. The material is then added to a salt solution and an anaerobic fermentation is initiated in which lactobacilli and yeasts predominate. At the end of this stage, the liquid which is pressed off and clarified is soy sauce.

Soy sauce fermentation was at one time a household art and secret. The people at that time had no idea what happened except that soybeans could be fermented into a product with a completely different odor, appearance, and taste, but the product remained wholesome.

Although soy sauce fermentation is still to some extent carried out as small operations in the traditional way in the home, the art being passed on from father to son, it is one of the most modern and sophisti- cated fermentation industries in the Orient, especially in Japan. Most of the research on soy sauce has been and is being conducted in Japan. As early as the thirteenth century, especially during the Muromachi period ( 1328-1573), many investigations were carried out on the kinds

SOY SAUCE FERMENTATION 159

and relative quantities of raw materials and on the fermentative process, with the result that the product was improved considerably (Yokotsuka, 1960). There is currently an extensive amount of published work on different aspects of soy sauce fermentation.

Judging from recent reviews by Umeda (1957)) Mogi and Iguchi (1958, 1965, 1966), Mogi (1959), Yokotsuka (1960), Akira (1967), Onishi ( 1967, 1968), and personal communications with colleagues from Korea, Japan, and Hong Kong, the modern methods now in use there are only ad hoc modifications of the traditional process. The present method is still time-consuming, low in efficiency, and without adequate microbiological control.

The authors are of the opinion that the next few years will see rapid progress in the development of improved technology for the production of soy sauce, leading to a process which is much more economical of time, space, and labor, but without any sacrifice of the flavor and quality of the product.

Soy sauce fermentation is at present a two-stage batch process involving the biochemical activities of three types of microorganisms : mold, bacteria, and yeast. The first stage is done by growing Aspergillus oryzae or Aspergillus soyae on a mixture of soybeans and wheat. The extracellular amylases and more importantly the proteases hydrolyze the carbohydrates and proteins in the raw materials, respectively. When mold growth has reached the desired level, the mixture of soybean and wheat covered by mold mycelium is placed into 18% w/v sodium chloride solution. The mixture undergoes lactic acid and yeast fermentations for at least one year at ambient temperatures to give a product of good quality.

ti. Fermented Soy Products

The use of various bacteria, yeasts, and fungi to make excellent fermented foods from soybeans or a mixture of soybeans and cereals, such as rice or wheat, has been known in the Orient for centuries. Hesseltine ( 1965, 1966), Hesseltine and Wang ( 1967), and Gray ( 1970) have given comprehensive accounts of the different kinds of fermented soybean, soybean-and-wheat, and soybean-and-rice products; the types of micrcq- organisms; and the processes of making these foods. Table I, adapted from Hesseltine (1985)) lists the name( s ) of the fermentation, the chief organism( s ) used, the fermentation substrate, the nature of the product, and the country or countries where the fermentation is now carried on. From Table I, two very evident conclusions can be drawn: ( a ) fungi are the organisms most frequently used to process soybeans; and ( b ) the areas where the articles mentioned are of commercial interest lie in the Orient.

160 F. M. YONG AND B. J. B. WOOD

TABLE I FOOD FERMENTATIONS"

Nature of Area of com- Name of food Organism(s) used Substrate product mercial use

Of fungal origin

Tempeh

Sufu (Soy- bean cheese)

Miso (break- fast food and soup base)

Shoyu (Soy sauce)

Rhizqpus

Principally ( R . oligosporus)

Actinomucor ele- gans, Mucor species

Aspergillus oryzae, Saccharom yces rouzii

Aspergillus oryzae, Lactobacillus, Hansenula, and Saccharom yces

Of bacterial origin

(condiment)

Natto Bacillus subtilis

Soybeans

Soybeans

Rice and other cereals plus soybeans

Soybeans, wheat

Soybeans

Solid Indonesia and

Solid China, Formoea vicinity

Paste Japan, China, and some other parts of the Orient

Liquid China, Japan, Philippines, and some other parts of the Orient

Solid Japan

a Adapted from Hesseltine (1965).

The Orient is distinguished from the Occident not only on geographical and ethnological grounds, but also on the basis of the types of food that are processed by fungi. In the Occident, fungi are used in the processing of milk protein (i.e., certain cheeses) whereas in the Orient fungi are used mainly to process soybeans, although a variety of other materials, including rice, wheat, peanuts, copra, and fish, are also so processed. Moreover, the major past and present contributions of fungi to man's food supply must be sought in the Orient (Gray, 1970).

Of the many different fermented foods from soybeans, the soy sauce fermentation is both very typical and probably the most widespread. It is an article of immense commercial importance in the Orient and is gradually but certainly being accepted by the peoples of the Occident. The total annual production of soy sauce in Japan was estimated by Yokotsuka (1964) to be about one million kiloliters. This would mean that the yearly consumption per capita is about 10 liters in Japan. Unfor- tunately no details are available with regard to the total annual production in the People's Republic of China, Taiwan, and the nations of South-


east Asia. It has, however, been reported that soy sauce represents one of China’s largest uses of soybeans (Smith, 1949). Soy sauce is used daily in the kitchen in the preparation of food and also as a table condiment. At most meals a dish of soy sauce is placed on the table, and certain foods are dipped in the soy sauce for seasoning. The sauce contains about 18% w/v of salt, hence serving as a salting agent as well as being a flavor accentuator.

Although soy sauce is the product of the Oriental practice of modifying soybeans with fungi, lactic acid bacteria, and yeasts, which is best known in America and Europe, very few scientific reports on soy-sauce fermentation are available in Western journals.

What actually is soy sauce? Hesseltine (1965), Hesseltine and Wang (1967), and Gray ( 1970) have described soy sauce under traditional fermented foods. However, in the strict sense of the word, soy sauce is not a food. It is not a solid nourishment for human consumption. It is a liquid food condiment which is used to add flavor and color to the bland, basic Oriental diet including rice, raw fish, beancwd, fermented beans, and boiled vegetables. To be more specific, it is a combination of hydrolytic products of soybeans alone or soybeans and wheat. Among the Chinese soy sauce is also known as Ch‘au yau “drawing oil,” or Pak yau “white oil” (Groff, 1919). The term s h o p is preferred in Japan and it appears for the first time in Japan in 1596 and in China in 1618 (Yokosuka, 1964). In the territories formerly known as the Dutch East Indies and Malaysia, soy sauce is known as ketjap (Stahel, 1946).

111. History of Soy Sauce Production

The written records of the Chinese show that they have been using soy sauce for over three thousand years. In the book of Chau Lai (one of the Thirteen Classics of Confucius) written before 10o0 BC, we read that the king’s cook used twenty jars of soy sauce for the ceremonial rites of the Chau Dynasty (Groff, 1919). Hesseltine (1965) noted that, according to information supplied by a major shoyu manufacturer, shoyu has been produced in Japan for over lo00 years. Soy sauce fermentation probably started in Japan as a result of the introduction of Buddhism from China and the consequent change to a vegetable diet in 552 AD (Hesseltine, 1985). Buddhism was already well established in China and Korea by the fourth century before it was introduced into Japan between 500 and 600 AD (Bush, 1959).

Although the making of soy sauce originated in China, Japan has probably the largest soy sauce plant in the world, the Noda Mati plant of the Kikkoman Shoyu Company Limited which was founded in 1764.


It has been estimated that the annual production of shoyu at Noda in 1948 was as much as 23 million gallons, consuming 30,000 metric tons of soybeans, 27,000 metric tons of wheat, and 29,000 metric tons of salt (Smith, 1949). He also notes that it had been claimed that the general occurrence, throughout the small city of Noda, of the mold used in fermenting shoyu suppresses other microbial life and maintains a high health standard for the city.

Stahel (1946) made reference to the soy sauce made in the former Dutch East Indies but gave no indication as to when the fermentation of soy sauce was first started there.

Descriptions of the process of producing soy sauce by the traditional, or orthodox, fermentative procedure are to be found in books written in Chinese more than 1500 years ago (Lockwood and Smith, 1950-1951). However, Hoffmann (1874) was the first to make available to the Occi- dental nations, directions, though very brief, for the manufacture of soy sauce by the fermentation process. Thereafter short essays appeared in Western journals on soy sauce and its manufacture (Kellner, 1888; Kita, 1913; Prinsen-Geerlings, 1917a,b; Waksman and Davidson, 1926; AIlen, 1926; Morikawa, 1926; Dyson, 1928; Ramsbottom, 1936; Minor, 1945; Stahel, 1946; Blaisten, 1947; Lockwood, 1947; Lockwood and Smith, 1950-1951; Hoogerheide, 1954; Hesseltine, 1965; Sakurai, 1965; Hesseltine and Wang, 1967,1968; Gray, 1970).

In 1919 Groff gave a very comprehensive description of the then current procedures of soy sauce manufacture in Kwangtung, China. Thirty years later Smith (1949) published a report on Oriental methods of using soybeans as food. In his report he gave figures which illustrated the importance of soy sauce in the diet of people in China, Japan, and Korea, and also compared the state of technology in soy sauce making in these countries. Church (1923) published perhaps the most detailed piece of work on soy sauce found in Western literature. Yokot- suka (1960) has given the best review article on soy sauce to date in English. In this review he covered not only the various ingredients in soy sauce that are responsible for giving the sauce its particular meaty flavor and aroma, but also the technology and microbiology of soy sauce production in Japan.

Formerly, the technology of soy sauce fermentation was a closely guarded family art passed on from father to son. Even now some manufacturers point with pride to the fact that their factories have been operated as family enterprises for 5 centuries. The technology was developed before the biochemical processes involved in the degradation of the raw materials by microorganisms to give soy sauce were known. Even now there is still much to be learned about the microbiology of soy sauce fermentation. The major steps involved in the manufacture


Whole wheat grains Soybeans (50 parts) Mold (Aspergillus oryzae) grown on 1 polished rice; 1-2 parts of approx. S d a y (50 parts)

Soaked in wat.er for culture used 1 Roasted

1 1 12 hours 1

Boiled until soft -------

Mixed 1

Spread on trays to a depth of 1-2 inches .1

Incubated for approximately 72 hours in 30" room

1 Transferred to 50-gallon capacity

earthen vessel 1

Approximately 20 % salt brine added (200 parts)

1 Lactic acid and yeast fermentation

lasting for 1-3 years 1

Filtered -+ Residue -+ Used in animal feed 1

Filtrate .1

Pasteurized 2

Soy sauce

FIG. 1. Flow sheet for the fermentation of soy sauce by the conventional method.

of soy sauce are no longer a secret, but the finer and important points are.

Soy sauce is manufactured by two basic processes. The traditional method involves a fermentation technique; the quality of the product will in this case depend greatly on the personal experience of the human involved. The other, chemical method, came into existence at a much later date.

Prior to 1912 soy sauce took one to three years to mature. It is therefore a time-consuming and expensive process (Yokotsuka, 1960). One can understand why it took such a long time by looking at Fig. 1, which gives all the essentials of the fermentation process.

IV. Chemical Composition of Soy Sauce

There are two major kinds of shoyu in the Orient, the Chinese type and the Japanese type. In China most kinds of shoyu are made from

164 F. M. YONC AND B. J. B. WOOD

soybeans alone or from a mixture of soybeans and wheat with a higher percentage of the soybeans; Japanese shoyu is made from equal amounts of soybeans and wheat. Table I1 (from Smith, 1949) shows the chemical composition of Chinese and Japanese soy sauce. Japanese law has regu- lated the quality of the shoyu exported from Japan since 1950 (Yokot- suka, 1964). The inspection of quality consists of both organoleptic and analytical tests involving the determination of specific gravity (degrees Baumk ), sodium chloride, extract, and total nitrogen. Good quality Japa- nese shoyu is said to contain about 1.5% w/v total nitrogen; of this about 45% is lower peptides, 45% is amino acids (including every essential one), and the remaining 10% is ammonia nitrogen. Yokotsuka (1964) is of the opinion that although shoyu is so rich in amino acids, its absorp- tion by the gastrointestinal tract is limited by its high salt content, but this opinion has never been corroborated by experiments, and it would be interesting to know more of the extent of its nutritional contribution when it is a regular component of the diet. Good quality Chinese-type soy sauce is of high specific gravity and viscosity and high nitrogen con-

TABLE I1 CHEMICAL COMPOSITION OF CHINESE AND JAPANESE SOY SAUCE"

Item

Chinese Japanese soy sauce soy sauce

from from Nanking Noda

(gm/100 mi) (gm/100 ml)

Total solids 32 38 Mineral matter NDc 20 Sodium chloride 16 18 Phosphoric acids (as PIOa) ND 0.50 Total nitrogen 1 1 .5 Protein nitrogen ND 0.1 Nonprotein nitrogen ND 1.4 Amino nitrogen ND 0.7 Volatile acids (m acetic) 0.5 0 .1 Nonvolatile acids (as lactic) ND 0.6 Total acidity 0.7 ND Sugar (as glucose) 2 6 Dextrins ND 1 Vkcosityb ND 5 Hydrogen ion concentration (pH) ND 4.5 Specific gravity (at 15°C) 1 1

a Modified, after Smith (1949). b By Ostwald's U-tube viscometer at 25°C.

Not determined.

SOY SAUCE FERMENTATION 165 tent; it is dark in color and is sometimes sweetened with cane sugar. This is different from Japanese-type shoyu, which is lower in viscosity and in total nitrogen content and is a lighter (but beautiful) red. Yokot- suka (1964) claims that in spite of the lower nitrogen content of Japanese shoyu, amino acids (especially glutamic acid) content is higher than in the Chinese type. Most characteristic of Japanese shoyu is its aroma and flavor. The Japanese have attributed this to the use of much wheat as raw material and to the strong yeast fermentation.

V. Raw Materials

The raw materials used-soybeans, wheat, and salt-are relatively cheap and easily available.

A. SOYBEANS

The soybean known botanically as Glycine m x ( L ) Merr. is also called soya bean, soja bean, Chinese pea, and Manchurian bean. It is native to Eastern Asia. The name soya came from the Chinese through the Japanese. It is taken from Chinese “Chiang-yiu,” which means soy sauce and is pronounced “show-yu” in Japanese. The Japanese contracted it into “so-ya” but with the fundamental characters for “chiang-yiu.” “So-ya” was further corrupted into “soy-a” or “soya” and in English into soybean ( Markley, 1950).

There are slight differences in the chemical composition of the beans from different countries, as shown in Table III (adapted from Yokotsuka,

Either whole beans or defatted soybean meal can be used in sauce 1980).

TABLE I11 COMPOSITION OF SOYBEANS FROM VARIOUS SOURCES“

Water Total Protein Crude Invert content nitrogen nitrogen6 fat sugars

Country (%) (%) (%) (%) ( %)

Japan 13 6 38 16 1 5 China 11 6 38 19 19 U.S.A. 10 6 38 20 17

(I Adapted from Yokotsuka (1960). The carbohydrate content waa not determined by Yokotsuka (1960) but Kawamura (1967) has found it to be about 34% based on dry basis.

Obtained by multiplying total nitrogen with the factor 6.25 (Lillevik, 1970).


manufacture. When whole beans are used an oily fraction can be separated from the fermented soy mash. This fraction is known as “soy-sauce oil,” which is used for making lower grade soap and as a source of linoleic acid. It was found by Kubo (1947) that soy sauce oil contains esters, chiefly ethyl esters of higher fatty acids produced during fermentation by the exchange of glycerol with ethyl alcohol. Soy sauce oil also contains 3040% free fatty acids (Yokotsuka, 1964). These esters and free fatty acids are inhibitory to yeasts. The soy sauce oil is separated from soy sauce by decantation before pasteurization. Little, if any, work seems to have been done on the changes in soybean oil occurring during the fermentation. For exampIe, it would be interesting to know if any change in degree of unsaturation of the fatty acids occurs.

For the past 60 years defatted soybeans have been used to replace whole beans in the production of soy sauce. At first, pressed soybean meal was used to replace whole beans; now, however, solvent-extracted soybean meal is widely used. In recent years defatted soybean meal has replaced whole beans in about 75% of the shoyu production in Japan (Hesseltine and Wang, 1968). The main reason for using defatted soybean is that the cost is very much lower. Furthermore the utilization of nitrogen in defatted soybeans is higher. It has been found by practical experience that the fermentation of defatted soybean meal requires a shorter period; it takes about 15 months at ambient temperatures for whole beans, but only about 10 months for defatted soybean meal (Yokot- suka, 1960). The difference in this rate of fermentation has never been explained; it has only been observed. It may be due to the surface layer of oil affecting the rate of transfer of oxygen through the fermented soy mash, thus slowing down yeast growth, or to the yeast-static compounds that result when whole soybeans are used (Fukai, 1928), or to whole beans being less rapidly digested than the more finely divided meal.

It is important to note that soy sauce made from whole beans is said to be more stable than that produced from defatted soybean meal. Yokotsuka (1960) did not explain exactly what he meant by stability. Presumably stability here would mean resistance to microbiological spoil- age rather than chemical deterioration of the soy sauce on storage. Good-quality fermented soy sauce does not have a white (yeast) film on the surface when exposed to the atmosphere because it contains “yeast-static” or “yeast-cidal” compounds. Yokotsuka et al. ( 1958) recognized that soy sauce produced from whole beans has a greater resistance to yeast invasion than that produced from defatted bean meal. They found that raw soy sauce made from whole bean (meaning soy sauce that had not undergone any treatment either by heating or addition of preservatives) has the same storage stability as a pasteurized product.


Fukai (1928) and Fukai and Komatsu (1934a,b) studied the ethyl esters of fatty acids and free fatty acids, such as caproic acid, in soy sauce, and the toxicity of these compounds toward undesirable yeasts which may infect soy sauce.

B. WHEAT

Either whole wheat or wheat flour may be used. Usually only soft wheat flour, i.e., wheat flour with about 8-9% w/w protein, is used. When wheat bran is used as a substitute for wheat flour or whole wheat, the resulting soy sauce is not satisfactory (C. T. Yeow, personal communication). Smith (1949) has reported that when whole wheat or wheat flour was unobtainable or too expensive in China, the Chinese substituted wheat bran, oats, kaoliang ( a root vegetable), and rye. Barley was not favored. Rice as a substitute was tried by Yenko and Baens-Arcega (1940) in the Philippines. The different starting materials modify to some extent the flavor of the resulting soy sauce. It is preferable to have the grain rich in starch and low in fiber.

Yokotsuka (1964) listed the roles of wheat in soy sauce manufacture as follows: (1) To make the moisture content of the material to be cultured with mold just adequate for mold growth; it must be about 45% in order to minimize the damage due to the growth of undesirable bacteria. Cooked soybeans have about 60% moisture, so roasted and crushed wheat serves to decrease the moisture of the material. (2 ) To assist in obtaining the highest proteolytic activity from the koji; the activity is highest when the starting material is an equal mixture of soybeans and wheat, along with greater growth of the mold. (3 ) TO serve as the major source of carbohydrates as the precursor of sugars, alcohol, and organic acids. ( 4 ) To serve as the source of lignin and glycosides, the precursors of vanillic flavor of shoyu. (5) To serve as a rich source of glutamic acid.

C. RATIO OF SOYBEANS TO WHEAT

The use of wheat decreases the total nitrogen content of soy sauce but it contributes aroma, flavor, and glutamic acid. The best soy sauce is generally believed to be made from a soybean-to-wheat ratio of 50:50 by weight based on the weight of the raw materials as received. The present method of adjusting the ratio of soybean-to-wheat has certain disadvantages. These arise because the moisture content and, more importantly, nitrogen and carbohydrate contents of each consignment may differ; perhaps it would be better to have a ratio based on protein-to- carbohydrate.


Considerable work has been done to find out the working ratio of soybeans-to-wheat that would provide conditions favorable for maximum mold growth but unfavorable for bacterial growth, so giving a good quality soy sauce ( Church, 1923; Ohara and Moriguchi, 1955a,b).

D. SALT

Sodium chloride acts as a preservative and exerts a selective action on the microorganisms which grow in the fermentation. If salt were not present, a dangerous anaerobic bacterial fermentation would occur in the traditional process, and one might expect such organisms as Botuli- nus to grow on such neutral or slightly alkaline mixtures, just as they can on imperfectly processed cans of green beans, for example. But the role of salt is not confined to the purely negative one of preventing the growth of dangerous organisms. It is clear from many studies, including our own (Yong, 1971), that the salt is necessary to permit the exclusive development of the flavor and aroma-forming yeasts and lactic acid bacteria.

Usually commercial, not chemically pure, sodium chloride is used for making soy sauce. Church (1923) noted that according to Japanese authority, experimental work had been successful with purified sodium chloride only occasionally, and commercial practice, never. It may well be that raw salt will carry a larger inoculum of halophilic and halotoler- ant bacteria than pure salt. In commercial salt a larger quantity of essential “impurities” necessary for yeast and bacterial growth may be present. Foreign substances other than basic salts of calcium and mag- nesium, often found in even a fair grade of bulk commercial salt, do not seem to interfere with shoyu making. Sea salt is used as a rule in Japan. In our own studies (Yong, 1971)) we employed British Drug Houses Limited’s “Laboratory Reagent Grade” sodium chloride, and found it to be entirely successful on a laboratory scale. The fact that we employed fermentations with added pure cultures of yeasts and Lactobacilli may, however, have influenced our results.

E. SUBSTITUTE RAW MATERIALS There have been several attempts to make “soy sauce” using raw

materials other than soybeans and wheat, but they were unsuccessful commercially. Church (1923) experimented with peanut press cake instead of the soybean and wheat mixture. The sauce obtained was called peanut sauce, not soy sauce, since “the taste of peanut was retained to such an extent that those accustomed to judging peanut products by tasting were not deceived, even when uninformed as to the ingredients


of the sauce.” The fermentation of peanut press cake on an industrial scale would not be easy. The control of bacterial growth and activity of bacteria harmful to the process is more difficult in a koji of peanut press cake than in a soybeans and wheat mixture.

Soybeans are produced in the Philippines only in limited quantities; to meet the requirements of the manufacturers of soy sauce, soybean milk, and soybean oil, considerable quantities of beans are annually imported ( Baens-Arcega, 1969). As a cheap source of proteinaceous material Baens-Arcega (1966) claimed successful use of a 50-50 mixture of copra meal and soybeans in the mold process of manufacturing soy sauce.

Kato and Matsumoto (1964) used soybean hull to make soy sauce. The Japanese were so desperate for raw materials during the Second World War that they even tried to produce soy sauce from garbage (Tsukahara, 1948). Oda et al. (1949) prepared a “substitute soy sauce” using acorns and wheat with an Aspergillus culture which produces tannase.

VI. Treatment of Raw Materials

Whole soybeans or defatted soybean meal can be used. Soybean flour is seldom used and would involve the preparation from the flour of tiny cubes before it could be used for fermentation (Yamaguchi, 1958). The whole beans or defatted soybean meal are best prepared by soaking in water for 10-12 hours at 29°C. The soaking can be done either by using running water or with still water which is changed every 2 or 3 hours. Unless the water is changed during soaking a rapid, undesirable fermentation, due to spore-forming rods (Church, 1923), occurs. These bacilli as spores are found on the beans as they come from the field. Beans soaked in unchanged water become warm, even hot, and sour at the bottom of a mass 5-6 inches deep in 2 or 3 hours at an initial temperature of 22°C. Such beans, even after autoclaving, are sour to the taste. It is the customary factory practice in Japan to soak the beans with changes of water at intervals of several hours. Nakaya (1934) found that beans should preferably be soaked until there is a 2.10 to 2.15 times increase in weight.

Before the introduction of the autoclave the soaked beans were cooked in a large open iron pan until they were soft enough to be easily pressed flat between the thumb and finger. This desired softness can be obtained by autoclaving at 10 psi for 1 hour as against the much longer cooking in an open pan. Autoclaving under pressure has the additional advantage of sterilizing the material. A slight excess of water, just more than enough to cover them, is added to the beans before autoclaving. The beans


are drained as soon as the autoclave pressure runs down. The pressure and time used for autoclaving the beans are very important. Kawano (1938) found that the digestibility of soybeans decreased as the pressure in the autoclave was increased. Evans et al. (1961) have reported that autoclaving soybean protein formed lysine-aspartic acid and lysine-glutamic acid linkages that are resistant to mild hydrolysis. Yokotsuka (1957) studied the influence of cooking soybeans on the quality of soy sauce and found that too long a cooking time causes a decrease in total, amino, ammonia, and tannin-precipitable nitrogen, acidity, volatile acids, and glycerol. Recently Tateno and Umeda (1955) and the Noda Soy Sauce Co., Ltd. (1955), claimed that total nitrogen in the soybean is best utilized when the beans are soaked in water for 10-12 hours at room temperature and then autoclaved at 10-13 psi for about 1 hour. Immedi- ately after cooking the material was cooled to less than 4OOC.

Rapid cooling of the beans on an industrial scale is done by spreading out the cooked beans to cool in about a 1-foot layer on a large traylike platform, the beans being turned over from time to time to hasten the cooling. Or they may be spread in wire trays and cooled with the draft of air from an electric fan. The rapid cooling of the hot sterile beans prevents the growth of microorganisms collected from utensils and han- dling after the beans are taken out of the autoclave. Details of the autoclaving process differ slightly depending on whether roasted cracked wheat or steamed wheat flour are to be employed. In the former case, the soaked beans are autoclaved immersed in water; in the latter case the soaked beans are drained before autoclaving, so giving a dryer product.

Either whole wheat, wheat flour, or wheat bran, may be used; the choice will depend on their availability and price. When whole wheat is used it is first roasted and then coarsely crushed. The roasting should be continued until the wheat is crisp but not tough, and is browned to give a slight charred flavor. It is said that some manufacturers of soy sauce roast the wheat only slightly whereas others char the cereal. The browned wheat is believed by the Japanese to add flavor to the finished product through the formation of maltol due to the activity of the yeasts during the moromi stage of the fermentation (Church, 1923). It also adds a desired brown color. In the roasting of the wheat, practically all microorganisms present on it are killed. After roasting, the wheat is crushed, the crushing being carried to the extent of breaking the grains into large pieces. Furthermore, the crushing should be of such a character as to reduce some portions of the kernel to a fine powder or wheat dust. A supply of roasted wheat may be kept on hand and crushed as needed. Wheat bran and wheat flour on the other hand are generally steamed instead of being roasted. The properties of the


finished sauce will of course differ, depending on whether roasted grain or steamed flour has been employed, the latter giving a paler sauce of milder flavor.

Asao and Yokotsuka (1957) reported on the changes produced in phenolic compounds in 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. These phenolic compounds 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 psi for 10, 20, 30, and 60 minutes, 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 minutes. Paper chromatography of the ether extract of cooked wheat showed the presence of vanillin, ferulic acid, vanillic acid, and 4-ethylguaiacol, Among these, vanillin was the most abundant. These workers concluded that the phenolic compounds came from the degradation of lignin and glycosides by heating.

The sterile softened beans and the crushed wheat are mixed in large trays or on mixing tables under nonsterile conditions. The beans are cooled below 28°C before being mixed with the wheat. The beans and the wheat need to be thoroughly mixed in such a way that the beans are held apart. The angular pieces of wheat when evenly and thoroughly distributed among the beans serve as a mechanical means of separating the wet smooth beans which would naturally pack much closer. The interstices are filled with the finer wheat particles to a certain extent, but not enough to check aeration. It is well to have the wheat, rather than the beans, on the surface. Furthermore, these two ingredients need to be thoroughly mixed so that the wheat dust may form a coat over each bean. The surfaces of the beans treated in this manner have a lower water content than when the precaution of thorough mixing is not taken. The dry wheat dust takes up the moisture readily. The lower water content thus induced on the exterior of the beans is better adapted to mold growth than to bacterial growth.

VII. Koji

Etymologically the word “koji” is an abbreviation of “kabi-tachi,” meaning something like “bloom of mold (Tamiya, 1958). Koji is just an enzyme preparation produced by growing a mold, such as AspergiZlus orynae, on steamed rice or other cereals or sometimes on steamed pulses. Historical documents show that Japan learned the use of koji from China more than 1700 years ago (Ono, 1941). The use of koji is analogous

172 F. M. YONG AND B. J . B. WOOD

to the use of malt in the Western nations. Its use as a starter is not exclusive to the shoyu industry, it is used in the brewing of the Japanese rice-wine (sake) and fermentation of “miso” (a sort of porridge or mush made by mixing mashed steamed soybeans, salt, and koji, and by allow- ing the mixture to ferment).

Seed koji is produced by culturing A. soyae or A. oryzae on either steamed, polished rice (usual practice in Japan) or a mixture of wheat bran and soybean flour (China). It is usually incubated for 72 hours in small boxes or trays in a warm room. To avoid confusion the authors would like to use the term seed koji for the starter used to inoculate the very much larger quantity of soybean-wheat mixture to be fermented and koji for the large quantities of inoculated soybean-wheat mixture. The term koji is currently used to designate the starter as well as the whole mixture of soybean-wheat after being inoculated without any dis- tinction and will cause confusion to someone learning about soy sauce fermentation for the first time. The seed koji after being incubated for about 72 hours is added to the soybean-wheat already prepared and thoroughly mixed. Usually 1-2% w/ w of the seed koji is used as the starter.

Some morphological comparison of the Aspergillus strains used by the soy sauce industry has been reported by Kibi (1926), Sakaguchi and Yamada (1945a,b), and others. Besides A. oryzae and A. soyae, A. ochraceus, A. mellius, and A. niger have been tested in laboratories but have shown no practical value (Yokotsuka, 1960). A good culture mold must have high proteolytic activity and be easy to culture. Yokot- suka (1960) has also stated that a good culture mold must give the characteristic aroma and flavor to the soy sauce. This requirement would therefore imply that the mold is responsible for imparting the flavor and aroma to soy sauce. The authors have not been able to find any conclusive experiments to support this contention. There can, however, be no doubt as to the practical importance of the extracellular hydrolases produced by the mold.

The mold Aspergillus oryzae was first isolated from koji in 1878 by a teacher of natural history from Germany, Dr. Ahlburg (Ahlburg and Matsubara, 1878). A strong diastatic activity was first demonstrated in 1881 by a teacher of applied chemistry from England, Dr. Atkinson (Atkinson, 1881). Takamine succeeded in improving the diastatic activity of the mold and prepared from it a drug having a strong digestive activity-Takadiastase ( Takamine, 1894).

One of the major components of koji mold is a starch-liquefying amylase, Taka-amylase A. It was crystallized by Akabori (Akabori et al., 1951, 1953a,b, 1954) and was also purified by Sawasaki ( 19eOb). Okazaki ( 1954, 1955) and Sawasaki (1960a) claimed to have purified a saccharo- genic amylase, Taka-amylase B, although its crystallization was not achieved.

SOY SAUCE FERMENTATION 173 Many investigators ( Kibi, 1951; Matsushima, 1954; Kageyama and

Sugita, 1955; Sakamoto and Shuzui, 1957; Miura, 1958; Matsushima, 1959; Suzuki et al., 1959; Morimoto and Terui, 1960; Matsushima and Shimada, 1962; Yasui, 1964) have studied the proteases produced by Aspergillus oryzae and tried to identify the components of the mixture. Nowadays, it seems to be clear that three kinds of proteases-acid, neutral, and alkaline-exist in crude enzyme preparations of A. oryzae. Some workers (Morimoto and Terui, 1960; Matsushima and Shimada, 1962; Nunokawa, 1963; Morihara et al., 1!368) have reported on the fact that there are two kinds each of acid and neutral proteases. Matsu- shima (1959) indicated the presence of a “semi-acid” protease, but it has not been isolated in any form as yet, so its existence appears to be still in doubt. Recently, Misaki et at. (1970) reported the isolation of a “semi-alkaline” protease. Thus the constitution of A. oryxae proteases is so complicated and diverse that the separation and purification of the proteases have not been sufficiently performed except in the case of alkaline protease (Crewther and Lennox, 1950; Akabori et al., 1953a,b; Miura, 1955; Hayashi et al., 1967b). Tsujita (1967) has succeeded in crystallizing acid protease, but in the case of neutral protease there is‘little information in respect to its purification.

The mixture of proteases from Aspergillus sojae have also been extensively studied (Sakaguchi and Yamada, 1944; Yamamoto, 1957b,c; Haya- shi et al., 1967a,c; Sekine et al., 1970). The constitution of A. sojae proteases is very similar to that of A. oryxae.

Iguchi (1949, 1950a,b, 1951, 1952a,b,c, 1953, 1955a,b, 1956) and Iguchi and Yamamoto (1955a,b) obtained by X-ray irradiation a mutant strain of A. sojae K.S. which had two or three times the proteolytic activity of the other strains tested. More recent workers had tried to increase even further the protease productivity of this mutant (Sekine et al., 1969; Nasuno et al., 1971).

Although a lot of work had been done on the isolation and purification of the proteases and amylases of A. oryzae and A. sojae, and the mutation of parent strains to produce higher levels of proteases, there is very little work on the levels of proteases and amylases in the koji and SOY

mash throughout its whole period of fermentation ( Sakaguchi, 1958a).

VIII. Culturing the Koji

The effects of physical environmental factors such as temperature and moisture on the culturing of mold has been investigated by Kinoshita and Matsumoto (1935), Murakami (1951a,b), Harada (1951), and Yamamoto ( 1957a,b,c).

In industrial practice the whole mass of koji is distributed in small flat trays, made from bamboo strips, stacked above each other but sep-


arated by a gap of about 4 inches. Each tray is filled up to a depth of about 2-3 inches. The trays are placed in tiers along the walls of the room. In the incubation of koji, Church (1923) has stressed that “favorable aeration . . . is extremely important in shoyu-koji fermentation because moisture and the lack of oxygen induce the development of Mucors and bacteria, and are said to cause the diastatic enzyme to develop at the expense of the proteolytic enzyme.” The control of temperature and moisture is equally important ( Yokotsuka, 1960; Kibi, 1938). The temperature of the culturing room is usually kept at 2535°C. When the mold grows, the temperature of the material rises. When the temperature becomes too high, that is, above 40”C, the culture must be cooled. Cooling is usually done twice, at about 20 and 40 hours after inoculation. A rather high moisture content is required at the beginning, when the mold is growing rapidly; lower moisture content is desirable at the later stages, when spores are being formed. Kinoshita and Matsumoto (1935) reported that the cooling must be done before the temperature rises above 40°C since mold growth is inhibited above this temperature. Harada (1951) studied koji cultured under various temperature conditions and found that the highest proteolytic activity was obtained when the culture was cooled rapidly during the second cooling operation.

Cooling is done by stirring the koji, the bottom being brought to the top and vice versa. Thorough stirring is also necessary, especially on the first day of culture when the beans are bound together into a mass by the mycelia or white threads of the shoyu mold.

Usually 3 days of mold growth will be sufficient; with a shorter incubation period the enzymes produced would be inadequate. On the other hand, if the growth period is extended, excessive sporulation occurs and undesirable flavor may be imparted to the sauce (Lockwood and Smith, 1950-1951; Baens-Arcega, 1970). Mature koji has a clear yellow to yellowish-green color on the surface and throughout the whole mass.

The koji may at times become infected with Rhizopus nigricans if the atmosphere of the koji chamber is moist to the point of condensation as drops. A little Mucor or Rhizopus is disregarded in the material, unless a bad flavor or odor is also present. It is poor practice, however, to allow the Rhizopus to enter. If allowed to gain a foothold, its fruiting to any extent may be prevented by breaking up the koji into chunks and turning these chunks bottomside up. Instead of exposing a large surface area as in the case of bacterial infection, care should be taken to have only surfaces where Rhizopus has secured no firm footing exposed to the air. “Trays of koji infected with Rhizopus should be stacked in a cool, dry place until the material is mature or needed for the shoyu- moromi” ( Church, 1923).


The koji stage of mold fermentation brings about the enzymatic breakdown of proteins to peptides and amino acids by proteolytic enzymes, especially by neutral and alkaline proteases. Starches are hydrolyzed to dissaccharides and monosaccharides by a-amylase secreted by the mold. There have been no reports on the breakdown of soybean oil at this stage of fermentation. Maximal proteolytic activity and hence maximum degradation of insoluble protein into soluble protein, polypep- tides, peptides, and amino acids, is obtained from a koji of equal parts of wheat and soybeans (Yamamoto, 1957b). We have found that there is a rapid increase in reducing sugar during the very early stages of mold growth. This is due to the activity of extracellular invertase hydrolyzing the considerable quantities of sucrose present in the beans (Yong, 1971). There is evidence for the production of @-amylase and cellulase by the mold (S. K. Goel and B. J. B. Wood, unpublished data). Our studies have shown that Aspergillus isolated from soy koji also produces variable but appreciable amounts of lipase ( Yong, 1971).

Apparently it is not yet known whether the deamination of amino acids to yield the free ammonia, which is always present in small amounts in mature koji and may attain considerable proportions in overripe koji, is the result of intra- or extracellular enzyme activity, and this is among our current subjects of study.

Continuous or mechanically controlled mold culturing is greatly desired but as yet has not been successful (Yokotsuka, 19f30).

IX. Mash (Moromi)

The mature shoyu-koji is mixed with about an equal amount of brine to form the mash, or “moromi.” The mash is kept in a large container, which can be either a wooden tub or a concrete tank such as is used in modern Japanese factories (Sakurai, 1965), for about one year at ambient temperatures, or for 3-4 months if warmed. “Ordinarily the mash is stirred by compressed air” (Yokotsuka, 1960). Major chemical changes in this process are degradations of protein and carbohydrate caused by enzymes derived from koji. According to Yokotsuka, a lactic acid 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.

A. PREPARATION (MASHING) Formerly, equal volumes of salt water (17-19% w/v of sodium chloride)

and koji were used, but recently the volume of salt water has been increased to 110-120% of the raw material. Mixing koji with more water


causes a better utilization of the total nitrogen of the raw material; however, this may have some undesirable effect on the composition of the soy sauce. It is regarded as dangerous to use a solution of less than 16% because of the danger of putrefaction. Usually the solution of salt added to the koji is controlled by the specific gravity of the saline solution, the range being between 20° and 22OBaumB (Be). Ohara and Moriguchi (1955a,b) mixed salt solutions of 19O, 20°, and 23OBe with koji, and found that the lower the specific gravity the better the utilization of total nitrogen and amino nitrogen, also the better the fermentation; in addition, less residual sugar and higher acidity were obtained in the soy sauce.

B. CONTROL OF THE 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; if warmed to about 3540°C it will take only 3-4 months. Recently, S. I. Tan (personal communication, 1970) has successfully made soy sauce of good quality at the Singapore Institute of Standards and Industrial Research by incubating the mash at 4OoC for 1 month. Some lactobacilli will grow at temperatures up to 47°C in more norma1 media, but it seems surprising that they will tolerate the combination of high temperature and high salinity. It is even more remarkable that yeasts can survive such conditions, especially when one considers 'the low pH (4.5-5) which they require in order to grow at all in these saline conditions. In our own studies, we routinely employed a temperature of 4OoC, and once the mash pH had dropped from its initial level of 6.5 to around 4.8 the yeast grew rapidly, increasing in number of viable cells 30-60 times in the space of 3-4 days. It would be most interesting to examine the effects of yet higher temperatures.

In the traditional method or in some cottage industries, the mash is stirred by wooden paddles once each day in the initial stage of the fermentation, then once a week, and finally less frequently toward the end of the fermentation. In modern factories in Japan, the mash is aer- ated and agitated by compressed air at 6-10 psi for 5-10 minutes. Yokot- suka (1960) did not specify the frequency of the stirring and aeration by compressed air nor any other related data. Yamada and Furusaka (1954) have reported that too much stirring, especially in summer, hin- ders the fermentation of mash. Stirring the mash is very important and a difficult procedure, and no final conclusion has been reached regarding the frequency of stirring.


C. ACING

The initial pH of the new mash is usually 6.0-7.0. It decreases rapidly, especially in summer, to about 4.5, the point at which alcoholic fermentation begins if the temperature is suitable. Usually, in 3 or 4 months at room temperatures, the total nitrogen dissolved in the mash increases to 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 at ambient temperature. He found under the conditions used by him that glutamic acid was at a maximum after 15 months, and then it started to decrease. Udo concluded that the optimum aging period could be determined by analyzing for free glutamic acid content. He was using whole beans and, more recently, Umeda et aE. (1953), working with defatted soybeans, reported the same finding. The 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 for only 1 month. Yokotsuka (1960) has noted that higher temperatures cause a rapid decrease of mash acidity and rapid inactivation of enzymes.

D. MICROBIOLOGY OF MASH

Yokotsuka (1960) stated that, besides Aspergillus oryzae or A. soyae, Monilia, Penicillium, and Rhizopus are sometimes found in the mash, but that these molds are believed to have no relation to proper aging. It is unlikely that these molds would be growing in such a high salt concentration, and it has been found that sodium chloride inhibits the growth of Aspergillus oryzae (Ichikawa, 1954a). It is likely that the molds exist as spores in the mash, not in the vegetative form, which would most likely have autolyzed in 18% wlv salt solution. There are two conflicting reports on the effect of koji on the flavor of soy sauce. Sugita ( 1956) studied the relationship between the organoleptic evalua- tion of cultured koji and the quality of soy sauce made from it, and recognized a fairly good relation between them. However, Sakaguchi (1959a) fermented soy sauce in a microbiologically closed system in which koji mold (that is, either A. oryzae or A. sojae) was the only microorganism participating and obtained a product which has no characteristic flavor and taste. It might be possible that the cultured koji obtained by Sugita was contaminated with "flavor-producing" microorganisms. The amino acids, especially glutamic produced by the mold fermentation will, however, contribute to the taste, and hence quality, of


the product obtained. Our own studies support the views advanced by Sakaguchi. Soy sauce made with mold alone, or mold plus Lactobacil- lus, lacked the full, rich aroma of a good sauce. The participation of the yeast was essential, and the best flavor was invariably obtained from fermentations where the moromi had been either soured with lactic acid, then fermented with yeast, or fermented with a mixture of yeast and lactobacilli. A mold-koji was, of course, employed in all cases.

It was realized as early as 1915 that “complicated chemical changes are going on during the ripening process of shoyu-moromi” and that “. . . these chemical changes are entirely due not only to the various enzymic actions of Aspergillus oryzae in koji, but also to the vegetative and autolytic actions of many kinds and numbers of microbes in moromi” (Yukawa, 1915). The “many kinds and numbers of microbes” refer to the bacteria and yeasts present in the soy mash. Much of the published work on the microbiology of soy mash has been concerned with the isolation and identification of bacteria and yeasts from soy mashes obtained from the factories ( Mogi, 1949).

1. Yeasts

The first report on soy yeasts appeared in 1906, when Saito isolated five strains of salt-tolerant yeasts from soy mashes in the Choshi district in Japan and classified them as Saccharomyces soja, Zygosaccharomyces japonicus, Pichia farinosa, Mycoderma sp. and Torula sp. Subsequently, many taxonomic studies were conducted on soy yeasts (Mitsuda, 1910; Nishimura, 1910a-g, 1911, 1912a,b; Kita, 1911; Ishimaru, 1935; Sakasai and Yoshida, 1966; Onishi and Suzuki, 1970). These workers also isolated Z. major, 2. sulsus, Torulopsis, and Monilia species from soy mash. However, none of these observations explained which yeast( s ) played an important role in the ripening of soy mashes and how the yeast( s ) improves the quality of the product. Takahashi and Yukawa (1911-1915) showed that 2. maior and Z. soyae were useful in ripening soy mashes, giving the characteristic taste and flavor through their fermentation. Z. soyae and Z. major are believed to be the most indispensable yeasts in normal fermentation (Asao et al., 1969).

Takahashi and Yukawa (1911, 1914), Ishimaru (1935), and Mogi et al. (1951, 1952) showed that three kinds of yeasts are harmful to soy sauce on keeping: film-forming yeasts, such as Zygosaccharomyces s u h s , 2. japonicus, and Pichia; ring-forming Torulopsis which grows on the surface of the soy sauce around the edge of the container; and bottom yeasts belonging to Zygosaccharomyces.

2. mafor, 2. sofa, 2. s u h s , and 2. japonicus were included in one species, Saccharomyces rouxii, by the Lodder and Kreger-van Rij’s system (1952). These yeasts are also known as soy yeasts. They are found


in soy mashes because of their tolerance to high concentrations of sodium chloride and osmotic pressure.

Reliable information on the biochemical activity of the yeasts in the soy mash has not yet been obtained. In recent years, extensive studies has been carried out by Onishi (1954a,b, 1957a,b,c, 1958a,b,c, 1960a-d, 196l), Onishi, et al. ( 1961), and Onishi and Saito ( 1961, 1962). The growth of soy yeasts was found to involve a process of physiological adaptation. The nutritional requirements for nitrogen compounds and vitamins in two contrasting media (sodium chloride-free and a high concentration of sodium chloride) were also studied. Probably the most striking finding on the applied aspects of soy yeasts was that the pH range for the growth of soy yeasts in a sodium chloride-free medium is very wide (pH 3.0-7.0), while that in an 18% w/v sodium chloride medium is very narrow (pH 4.05.0). Saccharomyces rouxii was found to ferment glucose and maltose but not galactose, saccharose, and lactose. In a high sodium chloride medium (under aerobic conditions) S . rmxii produced high yields of glycerol whereas it produced only small amounts of glycerol in an ordinary medium. As much as 4030% of the glucose fermented was converted into glycerol in the saline medium under aerobic conditions.

Yamada and Furusaka (1954) claimed that more than 10% w/v of sodium chloride is necessary for the growth of 2. major in soy mash. Obata (1955), however, found that soy sauce aroma is produced when the sodium chloride content of the medium is higher than 5% w/v.

Takeda (1954) has reported that the addition to soy mash of a yeast inoculum grown up in shake-flask culture, would produce a relatively good quality soy sauce. However, Yauchi et al. (1955) observed that excessive fermentation induced by cultured yeasts could result in inferior soy sauce.

Recently, Yokotsuka et al. (1967a) isolated 300 strains of yeasts from several kinds of shoyu mash which were considered to have good flavor. Thirteen strains of non-film-forming yeasts were divided into three groups according to the speed of alcohol fermentation in soy-koji extract supplemented with 5% wlv glucose and 18% wlv sodium chloride. The three groups are: ( I ) good and rapid fermentative; (11) bad fermentative; and (111) slow but good fermentative. The strains of groups I and I11 were evaluated as superior strains with respect to good flavor production in actual fermentation of soy mash. The strains of group I11 were observed to produce a characteristic flavor like that of aged soy mash. The production of this characteristic flavor was showed by Yokotsuka et al. (1967a) to be due to the formation of 4-ethylguaiacol, 4-ethylphenol, and 2-phenylethanol. Torulopsis species were the only type of yeast that produced these alkyl phenols during the fermentation


of soy sauce (Asao et al., 1967; Yokotsuka et al., 1967b). The alkyl phenols are not produced in soy mash fermented by S. rouxii alone ( Asao et al., 1969).

Furfuryl alcohol, one of the components producing flavor in soy sauce, was found to be produced by yeasts, for example, S. rouxii and Pichia furinosa, by reduction of furfural present in koji extract (Morimoto and Matutani, 1969).

Mori and Onishi ( 1967) have reported successful diploid breeding of salt-tolerant S. rouxii, a heterothallic haploid, for soy sauce and miso fermentation. The factors affecting the stability of the diploids obtained were also investigated ( Mori and Onishi, 1969).

It is clear that, as with so many other aspects of the product of soy sauce, the reports in the literature on the functions of yeasts are confusing and sometimes completely contradictory. This may reflect the influence of local or regional differences in the practice of soy sauce manufacture, but there clearly exists a great need for detailed studies to resolve these problems.

There are also conflicting reports on the ecology of mold, yeasts, and bacteria in the fermentation of soy sauce. Lockwood and Smith (1950-1951) are of the opinion that a good yeast growth in the koji before the mold growth becomes apparent will result in a product of superior quality. In this case, yeast would be added to the steamed soybeans about a day before mixing them with parched wheat, and the yeast would start to grow before the mold gets under way. It has been the normal accepted procedure to let the mold grow and degrade the raw materials before mixing the koji with brine and inoculating with yeasts. Since the mold fermentation is not under aseptic conditions in practice, one would expect yeasts and bacteria to be present in the koji. The majority of workers consider that these microorganisms have not been shown to be of importance in the making of a good quality koji, thus disagreeing with the contentions of Lockwood and Smith.

Yokotsuka (1960) and Onishi (1963) among many other Japanese workers are of the opinion that a lactic acid fermentation is followed by a yeast fermentation. This hypothesis is supported by the fact that after lactic acid bacteria have propagated in the young moromi of pH 6.0 to 7.0, lowering the pH to below 5.0, the yeasts will grow vigorously. I t has been found by Onishi (1957b, 195810) that the yeast isolated from soy sauce mash would not grow at a pH above 4.5 in artificial media containing a high concentration of salt. It is possible that the growth of yeast in soy mash which has an approximate salt concentration of 18% is controlled by the pH value of the mash. Hesseltine and Wang (1968) noted that it was not known for sure where the lactic acid bacteria and the yeast enter the fermentation.


On balance, therefore, the most likely sequence of dominant microorganisms would seem to be fungus, then lactic acid bacteria, and finally yeasts. Further work is needed ( a ) to establish this sequence, ( b ) to investigate whether it is better to have mixtures rather than using pure strains of each of the major classes of microorganisms, and ( c ) to deter- mine whether the numerically less important organisms have a part to play, or are merely contaminants exerting no significant effect on the fermentation. In our own work, convenience constrained us to use pure cultures of a few selected species. Even so, employment of A. oryzae under aseptic conditions for the koji, and of one strain each of S . rouxii and of Lactobacillus delbrueckii under aseptic conditions for the moromi stage, gave a product judged entirely acceptable by ourselves and by colleagues from Hong Kong, Malaysia, Korea, and Thailand.

2. Bacteria

Saito (1907) first isolated homofermentative (that is, giving lactic acid as sole product of the fermentation of glucose), salt-tolerant, tetrad- forming cocci from soy mash. He named the organism Sarcinu hamu- guchiae. A similar coccus has also been isolated by Sugimori and Joze (1970) and they called it Tetracoccus soyae. Ishimaru (1930) reported the presence of Pediococcus acidihctici var. soya in soy mash and described its effect on soy-sauce brewing. Sakaguchi (1954, 1958b) studied the same kind of bacterium isolated from soy mash and proposed the name P . soyae for it. Independently, and at almost the same time, Iizuka and Yamazato (1959a,b) proposed the same species name for an identical organism which they isolated from a so)- mash.

The growth factor requirements of P . soyae have been extensively examined by Sakaguchi. He has isolated from soy-koji extract, a peptide which increased the organism’s rate of cell division and the cell yield (Sakaguchi, 1959c,d). Addition of glycylbetaine or of carnitine to defined media also increased the growth rate and cell yield (Sakaguchi, 1960a). He has also examined the requirements for amino acids and vitamins exhibited by the organism ( Sakaguchi, 1960b); the effect on its growth of highly concentrated solution of inorganic salts; and the organism’s role in soy sauce fermentation (Sakaguchi, 1959a,b), which he found to be entirely concerned with the production of acid.

Sakaguchi and Mori (1969) have suggested that P. soyae, P. halophilus [which was isolated from salted anchovy pickles by Mees (1934)], and P. homari [which was isolated from meat curing brines by Deibel and Niven ( 1960)] are sufficiently similar in their morphological, physiological, and nutritional characteristics to place them into a single species.

Matsumoto (1925) isolated various Bacillus strains from soy mash,


and Ishimaru (1933a,b) also reported on many species of salt-tolerant bacilli and a few strains of Micrococcus in soy mash. Sakaguchi (1958a) found very few vegetative cells of Bacillus in soy mash and he therefore assumed that bacilli mainly grow in the koji and survive in the soy mash as spores.

Lockwood (1947) and Lockwood and Smith (1950-1951) reported on the use in soy sauce fermentation of Lactobacillus delbrueckii which was isolated from soy mash. The Japanese workers report on the presence of Lactobacillus in the moromi, but do not seem to attach much signifi- cance to it, whereas Lockwood (1947) claims to have produced good soy sauce using L. delbrueckii as the only bacterium in the fermentation.

The growth of lactic acid bacteria in soy mash would result in the formation of organic acids and make the mash acidic, a condition which is necessary for “sound fermentation, to remove undesirable flavors, and add indispensible good flavors to the soy-sauce” ( Sakaguchi, 195913). However, the lowering of the pH will most probably also result in the depression of neutral and alkaline proteinase activities, followed by their inactivation; thus the percentage of protein solubilization would decrease.

X. Pressing

In ancient China and in more primitive factories, the liquid is removed by “drawing” (Groff, 1919) or siphoning off the liquid on top. If the method of drawing is used, fresh salt solution is added to the “teng shi” or the beans remaining in the jar from the first drawing, and lactic acid and yeast fermentations are allowed to occur for another 1-2 months before the second drawing. This method of drawing may be done 4 times, and each “drawing” represents a different grade; the first drawing being the best and the last drawing the cheapest and poorest grade.

In modern factories the liquid part of the mash is separated from the residue with a hydraulic press. The residue, which has a moisture content of about 40%, is sometimes used as animal feed. The oily layer of the filterate from soy mash is separated from the

aqueous layer by decantation.

XI. Pasteurization

Raw soy sauce is pasteurized, and this process not only kills the vegetative form of microorganisms but also denatures enzymes and brings about the sedimentation of incompletely degraded protein compounds by coagulation. Matsumoto (1923)) Matsumoto and Murakami (1941),


Ohara and Orishi ( 1951), Ueno and Omori ( 1951), and Kosaka (1956) have reported on the compounds coagulated by pasteurization.

Usually the raw soy sauce is pasteurized at 65OC. Alum (1 ounce to every 80 gallons) or kaolin (1 ounce to 1 gallon) is then added for clarification. The precipitate is allowed to settle overnight, and the sauce is finally filtered ( Baens-Arcega, 1970). It is not clear what steps, if any, are taken to prevent the kaolin from introducing an inoculum of undesirable microorganisms.

Chemical preservatives are very often added to pasteurized soy sauce to prevent growth of microorganisms, especially film-forming yeasts, such as Zygosaccharornyces sulsus, 2. japonicus, and Pichia; ring-forming Torulopsis which grows on the surface of soy sauce around the edge of the container; and bottom yeasts belonging to the Zygosaccharornyces. The latter two species may be present in dilute soy sauces. Reports on these organisms had been made by Takahashi and Yukawa (1911, 1914), Ishimaru (1935), and Mogi et al. (1951, 1952). The chemical preservative most widely used in Japan is butyl-p-hydroxybenzoate at a concentration of 0.005% An alternative choice is usually sodium benzo- ate which is used in the region of 0.02% concentration.

XII. “Chemical” Soy Sauce

In order to lower production costs, several attempts were made to shorten the long fermentation period required to produce a relatively good soy sauce and at the same time to obtain a product of more constant quality. At first various lots of sauces which had been fermented for different periods of time were blended and sold. It needs no one to point out that this was not an improvement in soy sauce technology. The Japanese, unlike their Chinese rivals in the same industry, did not cling tenaciously to their traditional technology, which was established when the role of the beneficial “contaminants” of mold, lactic acid bacteria, and yeast was still yet unknown to them, but were more courageous and aggressive in trying to modify the fermentation process then in existence. Whereas the Chinese process was carried out in 50-gallon earthen jars out in the open, the Japanese as early as 1948 were construct- ing special cement tanks housed in large buildings, so providing cover and a degree of control over the environmental conditions (Smith, 1949).

Early in this century experiments began to be made on acid rather than enzymatic hydrolysis of the mixture of soybeans and wheat. Chow (1935) has given a review of the chemistry and manufacture of chemical soy sauce in mainland China.

Then from 1950 onward (Yokotsuka, 1960), there was a growing ten- dency to make soy sauce by acid hydrolysis in order to increase the


Soybeans Wheat

Hydrolyzed with 20% hydrochloric acid

1 Filtered

1 Fil t,rate

1 Neutralized wit,h sodium hydroxide

1 Pasteurized

1 Soy sauce

FIG. 2. Flow sheet for production of chemical soy sauce.

yield and reduce production costs. This chemical method is illustrated in Fig. 2.

According to Minor (1945), a mixture of soybean (meal) and wheat is hydrolyzed by refluxing with constant boiling hydrochloric acid (20% solution) until a maximum concentration of amino acid nitrogen has been obtained. Sufficient hydrolysis would have taken place after 12-16 hours. After hydrolysis, the preparation is neutralized gradually with a 50% sodium hydroxide to pH 4-5. The sauce is then ready to be placed in hardwood storage tanks for aging prior to bottling. When properly manufactured, the sauces will have a final salt concentration of 18% w/v sodium chloride.

The greatest problem here is that the carbohydrate in the raw materials is not oiily hydrolyzed faster than the protein, but also a small, but important, percentage of it is converted into undesirable compounds such as “dark color of humus,” levulinic acid, and formic acid. Moreover, the complete decomposition of tryptophan, the formation of an excess of lower boiling point sulfur compounds, giving bad odors, and the lack of fermented flavor, are further major disadvantages of this method. There is no market for a purely acid-hydrolyzed product in Asia, although it finds a market in Europe.

XIII. Semichemical Soy Sauce, or Shinshiki Shoyu

The next move made by the Japanese was to take the advantage

The idea of hydrolyzing the raw materials first and then subjecting of both methods-fermentative and chemical.

SOY SAUCE FERMENTATION 185 the whole to fermentation was known in the 1930’s; however, at that time the microorganism added to ferment the hydrolyzed substrate was a mold of the Aspergillus flauus-oryxae group (Kodama and Kimura, 1931; Snelling, 1937; Itikawa and Huzi, 1939). This method was not very successful in producing a good quality soy sauce, and therefore was not seriously taken up.

Noda Shoyu Company Limited managed to develop a process involving chemical hydrolysis followed by fermentation with lactic acid bacteria and yeast just after World War I1 (Yokotsuka, 1964). In this method, defatted soybeans are first partially degraded by using dilute hydrochloric acid, (about 7-8$), then neutralized with sodium hydroxide. Next rather large amounts of wheat bran or copra-meal koji are added to the partially hydrolyzed defatted soybeans. Only yeast is used in the fermentation. Using this “intermediate” method the fermentation period was shortened to 2 or 3 months, but a small amount of the characteristic furfurol and sulfide-like odors were unavoidable in the final products. This method is widely applied in Japan, and the amount of shoyu made by this method is estimated to be 20% of the total production ( Yokotsuka, 1964).

The soy sauce made by first hydrolyzing the raw materials and then fermenting them with bacteria and yeasts is called “shinshiki” or “semichemical” soy sauce, Investigations have been conducted into the best conditions for hydrolyzing the raw materials, either together or sepa- rately, and the components present in this type of soy sauce (Ueno and Kuramochi, 1960, 1961; Ueno and Nobuhara, 1960a-d; Ishigaki and Nagase, 1964a,b ) .

XIV. Future Developments in the Soy S a u c e Industry

When it was realized that the fermentation process was definitely essential in producing a superior product with superior quality, efforts were made to shorten the traditional or conventional fermentation process. Soybeans which were once boiled in an open pan are now autoclaved, and a lot of work went into getting the best conditions for autoclaving to get the maximum digestibility of soybeans (Kawano, 1938; Tateno and Umeda, 1955; Noda Soy Sauce Co. Ltd., 1955).

In recent years defatted soybeans have replaced whole soybeans in about 75% of the shoyu produced in Japan. This is because defatted soybeans are much cheaper, and, more importantly, the fermentation period is much shorter. The use of defatted soybeans reduced the fermentation period from about 15 months to approximately 10 months.

A lot of work has been done by Japanese workers, such as Kinoshita and Matsumoto ( 1935), Harada ( 1951), and Murakami (1951a,b), on


elevated and controlled temperature conditions of incubation to reduce the fermentation period. In the early days of soy sauce fermentation, the soy mash was left out in the sun during the summer to mature and there was no device to maintain it around 37°C for fermentation to take place faster and uninterrupted by variations of temperature resulting from seasonal variations of climate. Work on the effect of the moisture content of koji throughout the 72 hours of incubation had been conducted by Yamamoto (1957b), and this factor has been found to be of some importance in producing the correct soy sauce flavor and of great importance in preventing excessive bacterial contamination. He found that a higher moisture content (50-75%) favored mycelial growth, and a lower moisture favored spore formation.

At the very beginning of soy sauce fermentation, chance inoculation from the surroundings was relied upon to provide the necessary useful microflora. This would in a way account for the 3-year fermentation period required in the early days of fermentation. When it was realized and proved that soy sauce making is essentially a microbial process, attempts, which were successful, were made to isolate, identify and to use appropriate organisms as pure inocula (Lockwood, 1947; Lock- wood and Smith, 1950-1951). The use of pure culture inocula was a really significant step in understanding the process and has the following two main advantages: ( a ) Contaminating microorganisms are not carried over from one fermentation to another. ( b ) Since most types of soy sauce require at least 6 months to mature, it is apparent that an old moromi would be deficient in the appropriate microorganisms, giving rise to problems if it were used to inoculate a new moromi. With pure cultures, young, active cells in the correct proportions can be used to inoculate each new batch of moromi.

It had been found that sodium chloride at such high concentrations as 18% wlv, will not only decrease the rate of growth of lactic acid bacteria and yeasts, but also their physiological activities. The fermentation of soy sauce may actually have come into existence as a result of efforts to preserve cooked soybeans with a strong solution of sodium chloride. It has been found by Ichikawa (1954a,b) that fermentation of the koji in a medium without sodium chloride would result in a soy sauce with excessive amounts of ammonia due to uncontrolled growth of mold in the soy mash. If a process could be developed whereby one need not use Aspergillus orzyzae or A. soyae to break down the proteins and oligosaccharides in the raw materials, and also whereby the use of sodium chloride could be reduced or eliminated during moromi fermentation, the fermentation period might be drastically reduced. The use of acids to hydrolyze the raw materials is too drastic and results in the production of undesirable compounds such as levulinic


and formic acids which would give an inferior quality product (Yokot- suka, 1964). The use of commercially available enzymes to cause effec- tive, selective, and rapid hydrolysis of the raw materials is certainly a very interesting method for replacing the use of molds in the koji stage.

If a liquid multistage continuous soy sauce fermentation process can be developed, the production cost will be reduced by more than one-half because the mold fermentation stage takes up about 40% of the production cost and another 40% goes into the filtration and processing of the mash. The high cost of filtration results from the fact that it is necessary to press-filter the soy mash at very high pressure. Using a lower pressure means a longer filtration time for expression of the raw soy sauce and subjects the raw soy sauce to greater chances of oxidation of the oxidiz- able ingredients of the shoyu, which are the flavor-producing agents in shoyu.

To develop a multistage continuous soy sauce fermentation process, one needs to have some basic and important data on soy sauce fermentation. There have been a lot of investigations into soy sauce fermentation, mainly by the Japanese, and therefore there is a voluminous amount of literature on soy sauce in Japanese journals. However, in spite of and because of this superabundance of literature, one gets confused when one first begins to read about soy sauce fermentation. The investigations were mainly carried out on koji or soy mashes obtained from the factories rather than those fermented under controlled conditions in the laboratories.

Published research on soy sauce fermentations can be divided into approximately eight areas :

1. The isolation and identification of the beneficial microorganisms from koji and soy mash, and maintaining pure cultures of the isolates.

2. The mutation of molds to give highly proteolytic strains to degrade the proteins in the soybeans.

3. The use of raw materials other than wheat and soybeans. 4. Preservation. 5. The development of technology to improve yield and quality; the

modifications have so far only resulted in alterations in the equipment already present in the factory, without any daring and far-sighted attempt to develop an entirely new process which will require radically new equipment and processes.

6. Possible production of aflatoxin by molds used in soy sauce manufacture. Hesseltine et al. (1966) and Hesseltine (1966) reported on tests carried out on extracts of soy sauce from Japan and Taiwan for aflatoxin at the levels of 3-5 parts per thousand million and have found it to be negative. They also found that none of 53 strains of A. oryzae


used in food fermentations, such as miso and soy sauce, produced detect- able quantities of aflatoxin.

7. The flavor of soy sauce. Work in this particular aspect of soy sauce fermentation has been carried out since 1887 by Tawara (1887), Yukawa ( 1916), Ikeda and Kawaguchi ( 1922), Ishida ( 1925a,b), Taira ( 1925, 1926), Shoji (1927), Udo (1931a,b), Akabori and Kaneko (1936), Kihara (1940), Nakajima et aZ. (1949), Yokotsuka (1949, 1951a-d, 1952a,b,c, 1953a-d, 1954, 1957, 1960), Obata and Yamanishi ( 1951a,b,c, 1953), Asao and Yokotsuka ( 1958, 1961a,b, 1963), Yokotsuka ( 1960), and others.

8. Color of soy sauce. The color of soy sauce is very important since it is said to be associated with flavor. It has been studied by Kurono and Katsume (1927) and Kurono et aZ. (1927). They found that the color of soy sauce is caused by the so-called “nonenzymatic browning action.” Very recently, Okuhara et aZ. (1969a,b, 1970), Okuhara and Saito ( 1970a,b), and others have carried out extensive investigations on the color of soy sauce.

XV. Conclusions

It is clear that soy sauce both in its production and its chemistry is a most complex material, offering much to fascinate the biologist, microbiologist, chemist, biochemist, and chemical engineer. Our own studies (Yong, 1971; Yong and Wood, 1973) are an attempt at a syste- matic attack on some of these problems. Yet soy sauce is only one of a range of similar products, mostly unfamiliar to the Occidental palate, but affording the investigator challenges and problems very similar to those described for soy sauce (Hesseltine, 1965; Wood and Yong, 1973).

We are convinced that, so far as soy sauce is concerned, the application of modern technology will yield a process for the production of soy sauce by continuous fermentation techniques, employing enzyme extracts to replace the present koji stage, and continuous yeast fermentation under aseptic conditions, to give a low-cost product of very high quality indeed.

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