Caballero B. (ed.) Encyclopaedia of Food Science, Food Technology and Nutrition. Ten-Volume Set

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carbonated soft drinks. The traditional beverages

suffer from a number of technical defects, especially

variable quality, short shelf-life and inappropriate

packaging. These defects are not insurmountable.

For example, bottled, pasteurized sorghum beer

has been developed. More importantly, however,

traditional beverages have a poor image, especially

among affluent younger people. The sorghum beer

industry in southern Africa is addressing both these

problems by attempting to raise the technical stand-

ard and status of its personnel to that in the lager beer

industry. The CSIR and the University of Pretoria in

South Africa run training courses in sorghum malting

and sorghum beer brewing technologies. Several hun-

dred people from all over southern and eastern Africa

have attended these course and the sorghum beer

technology course is becoming a requirement for

brewers in the industry.

0030 Because sorghum and millets are uniquely suited to

cultivation in the semiarid tropics, there is consider-

able interest in Africa and India in using them as a

replacement for imported barley in the production of

lagers, stouts, and distilled beverages. In 1988 the

government of Nigeria banned the importation of

barley malt. Today in Nigeria, lager beers are pro-

duced using unmalted sorghum grain as the sole

cereal source. Commercial amylases and proteases

are used to produce sugars and free amino nitrogen

from the sorghum grain starch and protein. In add-

ition, research and development is being undertaken

worldwide to produce clear lager-type beers using

malted sorghum, which would itself provide the

necessary hydrolytic enzymes.

See also: Alcohol: Metabolism, Beneficial Effects, and

Toxicology; Alcohol Consumption; Beers: History and

Types; Biochemistry of Fermentation; Lactic Acid

Bacteria; Maize; Malt: Malt Types and Products;

Chemistry of Malting; Millets; Sorghum; Starch:

Structure, Properties, and Determination; Sources and

Processing; Functional Properties; Yeasts

Further Reading

Daiber KH and Taylor JRN (1995) Opaque beers. In:

Dendy DAV (ed.) Sorghum and Millets: Chemistry and

Technology, p. 299. St Paul: American Association of

Cereal Chemists.

Haggblade S and Holzapfel WH (1989) Industrialization of

Africa’s indigenous beer brewing. In: Steinkraus KH

(ed.) Industrialization of Indigenous Fermented Foods,

p. 191. New York: Marcel Dekker.

Hallgren L (1995) Lager beers from sorghum. In: Dendy

DAV (ed.) Sorghum and Millets: Chemistry and Tech-

nology, p. 283. St Paul: American Association of Cereal

Chemists.

Holzapfel WH (1989) Industrialization of mageu fermenta-

tion in South Africa. In: Steinkraus KH (ed.) Industrial-

ization of Indigenous Fermented Foods, p. 285. New

York: Marcel Dekker.

Inyang CU and Dabot YA (1997) Storability and potability

of pasteurized and sterilized ‘‘kunun-zaki’’: a fermented

sorghum beverage. Journal of Food Preservation and

Processing 21: 1–7.

Murty OS and Kumar KA (1995) Traditional uses of sor-

ghum and millets. In: Dendy DAV (ed.) Sorghum and

Millets: Chemistry and Technology, p. 185. St Paul:

American Association of Cereal Chemists.

Novellie L (1986) Sorghum beer and related fermentations

of southern Africa. In: Hesseltine CW and Wang HL

Table 1 Nutrient composition and contribution to recommended dietary allowance (RDA)

of commercial sorghum beer and lager

beer

Nutrient (mg l

1

unless otherwise stated) Sorghum beer Lager beer Percentagecontribution to RDA l

1

Sorghumbeer Lagerbeer

Alcohol (% w/w) 2.50 3.59

Energy (kJ l

1

) 1651 1605 15 14

Protein (g l

1

) 5.40 2.80 10 5

Thiamin 0.24 0.03 17 2

Riboflavin 0.39 0.19 24 12

Nicotinic acid 2.93 4.72 16 26

Cu 0.21 0.09 7 3

Fe 1.90 0.07 19 0.7

Zn 1.40 0.10 9 0.7

Mn 1.60 0.16 40 4

Ca 48 44 6 6

Mg 137 83 39 24

K 312 357 8 9

Na 20 69 1 3

P 245 246 31 31

No RDA.

Reprinted from van Heerden IV. In defence of sorghum beer. SA Food Review supplement, Feb/Mar 1988, pp. 83–84 with permission.

2358 FERMENTED FOODS/Beverages from Sorghum and Millet

(eds) Indigenous Fermented Food of Non-western

Origin, p. 219. Berlin: Cramer.

Novellie L and De Schaepdrijver P (1986) Modern develop-

ments in traditional African beers. In: Adams MR (ed.)

Progress in Industrial Microbiology, vol. 23, p. 73.

Amsterdam: Elsevier Science.

Palmer GH, Etokakpan OV and Igyor MA (1989) Review:

sorghum as brewing material. MIRCEN Journal 5:

265–275.

Rooney LW, Kirleis AW and Murty DC (1986) Traditional

foods from sorghum: their production, evaluation, and

nutritional value. In: Pomeranz Y (ed.) Advances in

Cereal Science and Technology, vol. 8, p. 317. St Paul:

American Association of Cereal Chemists.

South African Bureau of Standards (1970) Standard Test

Method for the Determination of the Diastatic Power

of Malts Prepared from Kaffircorn (Sorghum) Including

Bird-proof Varieties, and from Millet. SABS Method

235. Pretoria: South African Bureau of Standards.

South African Bureau of Standards (1979) Standard Speci-

fication for the Production of Mageu. SABS 1199–1979.

Pretoria: South African Bureau of Standards.

Tomkins A, Alnwick D and Haggerty P (1988) Fermented

foods for improving child feeding in eastern and south-

ern Africa: a review. In: Alnwick D, Moses S and

Schmidt OG (eds) Improving Young Child Feeding in

Eastern and Southern Africa: Household-level Food

Technology, p. 136. Ottawa: International Research

Development Centre.

Van Heerden IV (1989) Sorghum beer – a decade of nutri-

tional research. Proceeding of the Second Scientific and

Technical Convention, p. 293. Johannesburg: The Insti-

tute of Brewing Central and Southern African Section.

World Health Organization (1996) Fermentation: Assess-

ment and Research. Report of a joint FAO/WHO work-

shop on fermentation as a household technology to

improve food safety. Food Safety Unit, Division of

Food and Nutrition. Geneva: WHO.

Soy (Soya) Sauce

M Sasaki and N Nunomura, Kikkoman Corporation,

Noda City, Japan

Background

0001 ‘Shoyu’ is the Japanese name for soy sauce. It is made

mostly by fermentative methods and is used mainly as

an all-purpose seasoning in Japanese cuisine.

0002 In Japan the total production is about 1.04 million

kiloliters (kl), against a total population of 125

million. The annual consumption of shoyu per capita

is about 8.3 l, of which 3.5 l are consumed in the

home and the remaining 4.8 l institutionally and in-

dustrially (1999). The consumption volume in Japan

is decreasing slightly, but that in American and Euro-

pean area is increasing.

0003There were 5363 manufacturers in 1957, but by

1999, the number had decreased to 1611. Five large

companies together produce about one-half of the

total shoyu. Five different types of shoyu are available

in Japan: koikuchi, usukuchi, tamari, shiro, and

saishikomi.

0005Koikuchi is made from a mixture of soya beans and

wheat kernels in almost equal amounts, and is char-

acterized by a deep reddish brown color and a strong,

pleasant aroma. Low-salt shoyu of koikuchi is also

enjoyed, and its consumption is growing. It contains

half the salt of regular koikuchi shoyu, and it is con-

sidered to be healthier.

0006Usukuchi is characterized by a lighter brown color

and milder flavor than koikuchi. It is used mainly for

cooking, especially when the original color and flavor

of the foodstuff are to be maintained.

0007Tamari is characterized by a high content of amino

acids, and has a dark brown color and distinctive

flavor. It is made from soya beans with only a small

amount of wheat in the ratio 10:1–2. It is only con-

sumed around the Nagoya region of Japan as a

cooking and dipping sauce for several typical Japan-

ese dishes, such as raw fish (‘sasimi’ in Japanese) and

broiled eel (‘kabayaki’ in Japanese). One of the char-

acteristics of Japanese-type shoyu is the use of almost

equal amounts of soya beans and wheat as raw

materials. Tamari represents the Chinese type and

originates from China. In contrast, shiro is made

from wheat with only a small amount of soya beans

in the ratio 10:1–2. It is characterized by a very light

yellow color, and it has a very low amino acid content

and very high sugar content. It is used mainly for

cooking and is only consumed around the Nagoya

region. Saishikomi is produced from equal amounts

of wheat and soya beans in the koji, and raw shoyu is

used instead of brine. It is used mainly as a dipping

sauce.

tbl0001Table 1 Production of six kinds of shoyu (Japan Agricultural

Standard (1999))

Type Amount (kl) Market share (%)

Koikuchi 686 171 82.9

Usukuchi 115 848 14.0

Tamari 15 233 1.8

Shiro 5 949 0.7

Saishikomi 4 610 0.6

Total production 827 811 100.0

FERMENTED FOODS/Soy (Soya) Sauce 2359

0008 The annual production of the five kinds of shoyu,

recognized as being of a quality equal to the Japan

Agricultural Standard by the Japan Soy Sauce Inspec-

tion Institute in 1999, is shown in Table 1.

0009 As shown in Table 1, about 83% of all shoyu is of

the koikuchi type, and thus this type of soy sauce is

regarded as the typical Japanese shoyu. The prepar-

ation of koikuchi is described below.

History

0010 The origin of shoyu is considered to be from a

Chinese food, called ‘sho’ in Japanese. The first de-

scription of sho was found in the book ‘Shurai’ (in

Japanese), which was written in China 3000 years

ago. According to this book, sho was made by aging

a mixture of mold culture, foxtail millet, dried

meat, and a liquor in a bottle for 100 days. The

final product was in the form of a mash or ‘miso,’

which is now one of a number of typical fermented

foods in Japan (a semisolid salty food made from soya

beans, rice or barley, and salt by fermentation). Soya

beans are not described in the book as one of the raw

materials of sho, but it is quite possible that soya

beans were used, because it is known that soya beans

were widely cultured in China 4000 years ago. The

first documentation for the clear liquid part of sho is

in the Chinese book ‘Chin-Min-Yao-Shu’ (Saimin-

Yojutsu in Japanese) written in the sixth century ad.

Sho or hishio was also made from fish and salt at the

beginning of Japanese history. Sho made from soya

beans is presumed to have been introduced from

China into Japan along with other foods by the Chi-

nese Buddhist priest Ganjin (ad 1254). The first

record of the name ‘shoyu’ appeared in ‘Ekirinbon-

suyoshu’ (ad 1595), and it is likely that the basic

manufacturing process of today’s Japanese-type

shoyu was derived by the early seventeenth century.

In the Edo era (1603–1867) of Japan, the technology

for preparing shoyu developed dramatically and the

scale of production increased. However, remarkable

improvements in the processing of shoyu have taken

place since 1950, with dramatic advances in both

biochemistry and technology.

0011 Shoyu was exported to The Netherlands from the

port of Nagasaki for the first time in 1668, and, at

present, it is exported to 94 countries as a seasoning

of world-wide appeal.

Outline of the Manufacturing Process of

Koikuchi Shoyu

0012 The process for production of koikuchi consists of

five major steps: treatment of raw material, koji

making, mash fermentation and aging, pressing and

refining, and pasteurization. Among the steps, koji

making, mash fermentation, and pasteurization are

the most important and difficult processes in shoyu

production.

0013The manufacturing process is shown in Figure 1.

Raw Materials

0014The amounts of raw materials used for shoyu produc-

tion in Japan in 1997 are listed in Table 2.

0015Soya beans Soya beans originated in the East, and

the cultivated soya beans, e.g., Glycine max, have

been derived from Glycine ussuriensis, which is a

wild species growing in eastern Asia. (See Soy (Soya)

Beans: The Crop; Processing for the Food Industry.)

0016Whole and defatted soya beans contain approxi-

mately 33 and 45% (w/w) protein, respectively. The

key component for shoyu production is protein, and

about three-quarters of the total nitrogen in shoyu

originates from soya beans.

0017At least 60–70% of the total protein in soya beans

is accumulated in particles called ‘protein bodies’ or

aleurone grains in the developing cotyledons. About

90% of the seed protein (about 36% of seed dry

weight) is composed of globulins. The globulins are

a complex mixture of compounds, and the major

difference between the proteins is the molecular size,

as separated by ultracentrifugation. The 7S and 11S

fractions make up about 70% of the total globulins;

the minor constituents are 2S (15%) and 15S (9.1%).

The 7S fraction consists of b-andg-conglycinin

(35%), as classified by serological characteristics,

whilst the 11S fraction consists of glycinin (41.8%

of the total globulins). (See Protein: Chemistry.) The

major components of the soluble carbohydrate

fraction in the mature soya bean seed are: sucrose

(41.3–67.5%), stachyose (12.1–35.2%) and raffinose

(5.2–15.8%). Soya bean oil consists of 94–97% fatty

acid glycerides, together with 1.6–2.3% phospho-

lipids. (See Carbohydrates: Classification and Proper-

ties; Fatty Acids: Properties; Phospholipids:

Properties and Occurrence.)

0018Wheat Wheat belongs to the genus Triticum of

the family Gramineae (the grasses). The origin of the

wheat is not entirely certain, but cultured species

Table 2 Raw materials used for shoyu production in Japan

(1999)

Raw material Amount (tonnes)

Soya beans, whole 29 587

Soya beans, defatted 154 872

Wheat 154 727

Salt 190 466

2360 FERMENTED FOODS/Soy (Soya) Sauce

originated in prehistory. Cultivation of wheat is be-

lieved to have originated in the mountainous regions

of Syria and the area formerly part of Palestine of the

Middle East. (See Wheat: The Crop; Grain Structure

of Wheat and Wheat-based Products.)

0019 Wheat is commercially divided into seven classes

on the basis of botanical species, habit of growth and

color: hard red winter wheat, hard red spring wheat,

soft red winter wheat, soft red spring wheat, hard

white winter wheat, hard white spring wheat, soft

white winter wheat, and soft white spring wheat.

0020 Hard wheat is more suitable for making shoyu,

because the protein content is higher than in soft

wheat. The carbohydrates in wheat include starch,

cellulose, and sugar. Starch, which is by far the most

abundant constituent of the carbohydrate fraction, is

important chiefly as a source of energy for the koji

mold in the koji-making process. The proteins of the

starchy endosperm are largely the gluten-forming

proteins gliadin and glutenin, both of which are insol-

uble in water and in aqueous salt solutions. About a

quarter of the total nitrogen in shoyu originates from

the proteins in the wheat. (See Starch: Structure,

Properties, and Determination.)

0021Salt The salt should contain as little iron and man-

ganese as possible, because oxidative browning of

shoyu is synergistically accelerated by the presence

of these elements.

0022Water Water for shoyu should be clean bacterio-

logically and chemically. For the same reason as

with salt, low iron and manganese contents are

preferable.

Raw material...

Treatment of raw material

(1) Soaking in water

(2) Autoclaving

Mixture

(Moisture 40−50%)

Seed culture of A. oryzae or A. sojae

(0.1−0. 2% weight of raw materials)

Koji (800 kg, moisture 25−30%, pH 6.5−7.0)

Brine (NaCI content 25%, 1200 litres)

(New moromi mash)

(1) Enzyme degradation

(2) Lactic acid fermentation

(3) Yeast fermentation

Shoyu oil (2−3 litres)Cake (150 kg)

SedimentRefined shoyu

Products (pH 4.7−4.9)

or Soya beans (400 kg) or Soya beans (50~60 litres)

Bottling

Pasteurization

Pressing

Brine fermentation

(6−8 months)

Koji making

(48−72 h)

Matured mash

Raw shoyu (1650 litres, total nitrogen 1.6−1.8%)

(2) Crushing

(1) Roasting

Defatted soya beans (330 kg) Wheat (355 kg) Salt (295 kg) Water (1094 litres)

fig0001 Figure 1 Manufacturing process of Koikuchi shoyu.

FERMENTED FOODS/Soy (Soya) Sauce 2361

Treatment of Raw Materials

Soya Beans

0023 The protein in raw soya beans is native and cannot be

hydrolyzed by the enzymes of the koji mold, and it is

necessary to denature the protein to render it suscep-

tible to enzyme action. Conditions for denaturing the

soya beans affect digestibility, i.e., the ratio of the

total nitrogen of a shoyu to that of the raw materials,

and hence denaturing methods have been vigorously

investigated, including the development of machines.

As a result, protein digestibility has increased from

60 to 90%.

0024 There were many problems to be solved in improv-

ing the digestibility. For example, if soya bean

proteins remain partly undenatured, shoyu becomes

cloudy when it is diluted with water or heated for

cooking. This cloudiness diminishes its commercial

value. However, over-denatured soya protein loses

accessibility for enzyme reactions; as a result, digest-

ibility is low. The methods of cooking can be classi-

fied into five groups, according to the stages of

historical development from the first generation to

the fourth generation, as described below. (See Pro-

tein: Digestion and Absorption of Protein and Nitro-

gen Balance.)

0025 First generation (traditional method) Before the

nineteenth century, soya beans were soaked and

then boiled or steamed at atmospheric pressure. At

that time, the digestibility was about 60% for whole

soya beans.

0026 Second generation Around 1938, the method of

cooking was changed to autoclaving under pressure.

Soya beans or defatted soya bean grits were sprinkled

with hot water and then cooked at a gauge pressure of

0.8 kg cm

2

for about 1 h. After stopping the steam,

the cooked soya beans were kept in the cooker over-

night with the valve unopened.

0027 The digestibility was, at best, about 66% for defat-

ted soya beans, because of overdenaturation of the

protein.

0028 Third generation The method that characterizes the

third generation was invented by Tateno and co-

workers (1955) and is called the ‘NK method.’

0029 Thoroughly moistened soya beans or defatted soya

bean grits were cooked in a rotary cooker under a

pressure of 0.8 kg cm

2

for 45 min, and then immedi-

ately cooled to below 40



C by reducing the inside

pressure with a jet condenser. This technique consid-

erably increased the protein digestibility from 66 to

87% for defatted soya beans.

0030This method contributed greatly to the develop-

ment of techniques for treating the raw material.

0031Fourth generation (high-temperature/Short-time

(HTST) method) (1970) For convenience, this tech-

nique is divided into the following two methods.

0032Method 1 Thoroughly moistened soya beans are

cooked under a higher pressure and a shorter time

than with the NK method, using saturated steam, i.e.,

2.0–7.0 kg cm

2

for 15 s to 5 min.

0033The protein digestibility using this method is 84.24

(2 kg cm

2

, 5 min) to 89.70% (6 kg cm

2

, 0.5 min)

for defatted soya beans.

0034Method 2 Soya beans are cooked without water

using superheated steam at a gauge pressure of 4–

8kgcm

2

at 220–287



C for atleast 15 s.

0035The protein digestibility is almost the same as that

recorded for method 1 using saturated steam, but this

method has the advantage of making it possible to

store the heat-treated raw materials.

0036Others The treatment of soya beans with water

containing ethanol was investigated, but this method

was not used because of a large number of problems.

0037The extruder method has been used and has been

found to be suitable for small quantities of material.

Wheat

0038Wheat kernels are roasted without adding water. In

the past, roasting was carried out in an iron pan, but

this method was replaced with a continuous roaster at

the beginning of the 1900s. Nowadays, the HTST

method, described above, is employed. Control of

the process is important. If the wheat is insufficiently

roasted, the raw starch cannot be digested by the

amylase from the koji mold. However, if the wheat

is over-roasted, the protein digestibility decreases.

0039The roasted wheat kernel is coarsely crushed into

flour or fine pieces.

Koji Manufacture

0040The main purpose of this process is to produce the

enzymes needed for hydrolysis of the raw materials.

Many nutrients for the yeasts and lactobacilli that

multiply in the next stage, and some flavor compon-

ents that influence the quality of shoyu are produced

in this process.

Procedure

0041Nearly equal amounts of cooked soya beans and

roasted, crushed wheat (6:4–4:6) are mixed and

inoculated with a pure starter culture of Aspergillus

2362 FERMENTED FOODS/Soy (Soya) Sauce

oryzae or A. sojae – the so-called koji starter or seed

mold. The inoculated mixture is then transferred to

the equipment for koji cultivation, and spread on to

large perflorated stainless steel trays (3 (wide) 12

(long) 0.6 (high) m) to a depth of 30–40 cm, and

then incubated in a room at 25–30



C for 2–3 days.

During this period, the temperature, moisture, and

aeration are controlled to allow the seed mold to

grow on the mixture, and to promote the production

of enzymes. A temperature above 35



C leads to a

decrease in enzyme production and, sometimes, to

death of the koji mold. The temperature of the koji

is controlled by stirring. The first stirring is performed

at about 20 h, and the second at about 25 h, after

inoculation. The resulting end product (clear yellow

to yellowish green in color) is ‘koji.’

0042 The method described above is a batch system. In

recent years, an advanced system for continuous

koji cultivation has been developed and employed

industrially.

Koji Molds

0043 The koji molds used for shoyu are A. oryzae and

A. sojae. These species differ not only in their conidial

morphology, but also in several physiological charac-

teristics of shoyu-manufacturing importance. Gener-

ally, A. oryzae is characterized by a high productivity

of a-amylase, and A. sojae is characterized by a high

productivity of protease. A. oryzae is used not only

for shoyu production, but also for the other Japanese

fermented foods, such as ‘miso’ and ‘sake.’ However,

the use of A. sojae is limited to shoyu manufacture.

However, about four times as many manufacturers

use A. oryzae than A. sojae.

0044 The breeding of koji molds has been investigated,

using mutation, crossing, and protoplast fusion, in an

attempt to increase their enzyme productivities. At the

present time, several koji molds with a high enzyme

productivity of protease and amylase are used to

ensure a high productivity of the whole process.

0045 Since the discovery of aflatoxin, the question has

arisen as to whether or not koji molds produce this

carcinogenic mycotoxin. The results of a large

number of investigations have proved that no Japan-

ese industrial strains of koji mold produce this toxin.

(See Mycotoxins: Occurrence and Determination.)

Mash Manufacture and Aging

Procedure

0046 The mixing of koji and brine is called ‘shikomi’ in

Japanese, and the mixture is called the ‘moromi’

mash. The salt content in the brine is 23–25%, and

the ratio of brine to koji is usually 1:1.2–1:1.3 (v/v).

0047The koji and brine are measured simultaneously,

and the mixture is pumped into deep fermentation

vessels. Wooden casks of 5–10 kl, or 10–20 kl con-

crete tanks, were once used as fermentation vessels,

but recently, they have been replaced with resin-

coated, iron tanks of 50–300 kl or fiber-glass tanks

of 30–100 kl. This moromi mash is stirred by air

pressure to prevent salt-tolerant lactobacilli and

wild yeasts from growing.

0048At one time, shoyu was manufactured over the

four seasons. Koji and the mash were prepared in

February or March at a room temperature of

5–15



C, and the mash was fermented and aged

from spring to autumn. The shoyu that was brewed

under these conditions contained larger amounts of

total nitrogen and amino acids, but only a moderate

amount of lactic acid. On organoleptic evaluation,

it was considered better than shoyu brewed in the

other seasons. Nowadays, in modern shoyu factories,

in order to produce an excellent-quality shoyu and

reduce the fermentation period, the temperature of

the mash is controlled; the control simulates the

temperature changes of a mash brewed from spring

to autumn. The koji is first mixed with brine at

about 0



C to keep the temperature of the mash

below 15



C for several days. Selected salt-tolerant

lactic acid bacteria (Tetragenococcus halophilus)

are added as a starter, and then the temperature is

gradually raised to 28–30



C after 20–30 days.

During this period, a salt-tolerant yeast, such as a

pure culture of Zygosaccharomyces rouxii, is added.

After the vigorous alcoholic fermentation has fin-

ished, the temperature is dropped again and kept at

around 25



C over the final 2 months. The addition

of Candida (Torulopsis) yeasts is recommended to

obtain a good ‘volatile’ flavor in the final product.

A period of 6–8 months is necessary to complete

fermentation and aging. In the past, fermentation

was carried out without the addition of lactic acid

bacteria or yeasts. Natural conditions controlled the

fermentation by adventitious bacteria and wild

yeasts. (See Lactic Acid Bacteria.)

Chemical Changes in Mash Fermentation

0049In the early stages of mash fermentation, proteins

from the raw materials are hydrolyzed to lower-

molecular-weight peptides and free amino acids.

0050Approximately 20% of the starch from wheat is

consumed by the molds during koji fermentation, and

the rest is retained for the moromi mash. Most of this

remaining starch and the other carbohydrates from

wheat are converted to hexoses and pentoses in

the moromi mash. A proportion of these sugars is

subsequently metabolized into lactic acid, acetic

FERMENTED FOODS/Soy (Soya) Sauce 2363

acid, and other organic acids by lactobacilli. As a

result, the pH drops from an initial value of 6.7–7.0

to 4.7–4.8.

0051 In the next stage, vigorous alcoholic fermentation

(from the action of the yeast Z. rouxii) occurs, and the

remaining sugars are metabolized into ethanol and a

number of minor flavor compounds. For example,

Candida versatilis or C. echellsii converts ferulic

acid and 4-hydroxycinnamic acid from wheat to 4-

ethylguaiacol and 4-ethylphenol, which enhance the

quality of shoyu.

0052 As described above, the dominant species of yeasts

found in the mash are Tetragenococcus halophilus,

Z. rouxii, C. versatilis, and C. echellsii. The major

properties of these microbes are listed in Tables 3

and 4.

Pressing

005 3 The fermented mash is put into a cloth, and the liquid

part is squeezed out under hydraulic pressure, which

sometimes reaches 100 kg cm

2

, for 1–3 days. The

difficulty of pressing shoyu mash is due to the viscos-

ity of more than 300 cps.

0054 The liquid part of the mash is called raw shoyu, or

‘nama-shoyu’ in Japanese. The residue from the press-

ing is called shoyu cake, or ‘shoyu-kasu’ in Japanese.

This can be used as an additive in animal feed. The

final moisture content of shoyu-kasu is less than

25%. At one time, pressing required large amounts

of labor compared with the other processes, but

today, very efficient and automatic machines are

available.

Refining

0055The raw shoyu is stored in a tank and separated into

three layers: (1) sediment at the bottom, (2) clear

shoyu in the middle, and (3) an oily layer at the top.

The oil layer is called shoyu oil, or ‘shoyu-abura’ in

Japanese, and is removed by decanting. The middle

layer, clear shoyu, is heated at 115–120



C for a few

seconds in a heat exchanger in order to kill any vege-

tative microbial cells, denature enzymes, coagulate

proteins, develop the agreeable reddish brown color,

and generate aroma.

0056The clear shoyu is then filtered, bottled, and

marketed.

tbl0003 Table 3 PropertiesofZygosaccharomycesrouxii,Candidaversatilus,andCandidaechellsii

Z.rouxiiC.versatilisC.echellsii

GrowthOptimumGrowthOptimumGrowthOptimum

Temperature(



NaCl0%<3525–30<3525<3025

NaCl18%<4225<32–3525–30<33–3525

NaCl0%3.0–7.04.5–6.03.0–7.04.0–5.53.0–7.05.0

NaCl18%3.5–5.54.0–5.03.0–7.04.0–5.03.0–7.04.0–5.0

NaCl(%,w/v)<24–26<26<26

Wateractivitv(a

) 0.787–0.81 <0.787 0.787

NaCl (%) NaCl (%) NaCl (%)

Vitamin requirement 0 18 0 18 0 18

Thiamine N N, A E E A E

Calcium pantothenate E E N N, A N A, E

Biotin E E E E E E

Inositol A E N, A A N N, A

E, essential; A, accelerative; N, nonessential.

From Hisaoy (1977) Microbial Ecology, No. 4, p. 34. Tokyo: Japan Scientific Societies Press.

tbl0004Table 4 Properties of Tetragenococcus halophilus

1. Gram-positive Micrococcus, diameter 0.6–0.9 mm

2. Halophilic

3. Facultative anaerobe

4. Catalase-negative

5. Indole is not formed

6. Nitrates are not reduced

7. Nonmotile

8. Spores are not formed

9. Growth Growth Optimum

Temperature (



C) 20–42 25–30

pH 5.5–9.0 7.0

Water activitv 0.94–0.808 0.99–0.94

NaCl (% w/v) 5–24 5–10

Vitamin requirement: Biotin, vitamin B

, nicotinic acid,

pantothenic acid

Amino acid requirement: Leucine, isoleucine, valine, glutamic

acid, arginine, histidine, tryptophan, phenylalanine

2364 FERMENTED FOODS/Soy (Soya) Sauce

Retail Product

Chemical Composition

0057 The chemical compositions of various types of shoyu

are presented in Table 5.

Aroma and Flavor Compounds

0058 All the production processes are related to the forma-

tion of flavor compounds, i.e., the heat treatment of

raw material, koji culturing by molds, lactic acid

fermentation, yeast fermentation, aging of the

moromi mash, and pasteurization. Among these pro-

cesses, yeast fermentation in the moromi mash and

pasteurization contribute most to aroma and flavors.

(See Sensory Evaluation: Aroma; Taste.)

0059 The first attempt to identify the flavor compounds

of shoyu was made by Tahara in 1887. Since then,

many studies on shoyu flavor have been reported,

and, to date, over 300 volatiles have been detected.

Among them, caramel-like aroma compounds, such

as furanones, and phenolic compounds contribute

most to the flavor of Japanese shoyu. (See Phenolic

Compounds.)

0060 4-Hydroxy-2(or 5)-ethyl-5(or 2)-methyl-3(2H)-

furanone (HEMF) was isolated for the first time

from the natural product (1976). It has an intense,

sweet aroma and a shoyu-like flavor. As it is present

at high levels (100 p.p.m.) in shoyu and has the lowest

threshold of detection in water (less than 4 10

5

p.p.m.) among the compounds found in shoyu, the

aroma value (the concentration of component/the

threshold concentration of component in water) is

calculated to be > 5 10

. Accordingly, it is a signifi-

cant contributor to shoyu flavor and is believed to be

a ‘character impact compound’ of shoyu.

0061 In the 1990s, HEMF has been found in various

foods such as miso (1991), Emmental cheese (1994),

lager beer (1996) and coffee (1993), but the levels

in these foods are much lower than that in shoyu.

The homologs of HEMF, 4-hydroxy-2,5-dimethyl-

3(2H)-furanone (HDMF), and 4-hydroxy-5-methyl-

3(2H)-furanone (HMMF) have also been isolated

from shoyu. The amount of HDMF in shoyu is very

low (about 10 p.p.m.), but HMMF is present at high

levels (100 p.p.m.). HDMF is found in many food-

stuffs, such as pineapple, strawberries, roasted

almonds, and beef broth, and in various model

systems, such as the degradation of fructose, the pyr-

olysis of d-glucose, and roasting of alanine and

rhamnose. Similarly, HMMF is found in many food-

stuffs, such as beef broth, wild raspberries, and

guava fruit, and in various model systems, such as

the degradation of l-dehydroascorbic acid, and

roasting of glycine and xylose. The formation of

these compounds by a nonenzymatic reaction has

been proved.

0062HDMF and HMMF in shoyu are probably formed

from sugars and amino acids during heating pro-

cesses, such as the heat treatment of the raw materials

and the pasteurization of the liquid part of the mash.

However, HEMF is presumed to be biosynthesized

through the pentose phosphate cycle by shoyu yeasts.

Not only shoyu yeasts but also the other yeasts

employed for alcoholic beverages and single-cell pro-

tein can change intermediates, such as d-ribulose 5-

phosphate, to HEMF. Moreover, it has been proved

that d-xylulose 5-phosphate, one of the intermediates

in the pentose phosphate cycle, is present in both the

enzymatic hydrolysates of isolated soya bean protein

and in shoyu moromi mash before the growth of

yeasts in shoyu fermentation, and is the precursor

for HEMF.

0063The amount of HEMF produced depends greatly

on the concentration of sodium chloride in the

shoyu. The highest productions occurred at a sodium

chloride concentration of 17–18%.

0064HEMF has also been reported to show antitumor

activity.

0065Phenolic compounds, such as 4-ethylguaiacol and

4-ethylphenol, have an important relationship with

the aroma of shoyu. These compounds are derived

from ferulic acid and 4-hydroxycinnamic acid in koji,

respectively, by the action of Candida (Torulopsis)

yeasts. Based on the results of sensory evaluations,

the optimum concentration of 4-ethylguaiacol has

been found to be 0.8 p.p.m. in shoyu.

0066Many aldehydes, such as isobutyraldehyde, isova-

leraldehyde, and so on, increase during pasteurization,

as a result of the aminocarbonyl reaction and Strecker

degradation. The aroma derived from pasteurization is

called ‘fire aroma,’ or ‘Higa’ in Japanese.

0067The typical quantitative analysis of koikuchi shoyu

is shown in Table 6.

0068Shoyu flavor consists of HEMF and other minor

components, and, whilst HEMF gives the fundamen-

tal flavor, many minor components such as 4-ethyl-

guaiacol give the product character.

Color

0069The color of fresh koikuchi-type shoyu is deep red-

dish brown, and it is very sensitive to oxidation. The

color of shoyu packed in bottles or cans is usually

stable but darkens rather quickly after opening the

seals. During the brewing process, the development of

shoyu color derives mainly from nonoxidative

browning reactions, but after opening the seals, it is

mainly due to oxidative and nonenzymatic browning

reactions between amino acids and sugars, which are

FERMENTED FOODS/Soy (Soya) Sauce 2365

tbl0005 Table 5 Typical principal compositions of different kinds of shoyu

Koikuchi Usukuchi Tamari Shiro Saishikomi

Be Baume degree 21.55 22.24 25.14 20.54 25.08

NaCl (%, w/v) 16.25 18.60 17.50 17.45 14.30

Total nitrogen (%, w/v) 1.60 1.19 2.58 0.53 2.17

Ethanol (%, v/v) 2.60 2.96 3.32 4.19 3.21

Color (Y value) 1.32 22.14 0.08 75.93 1.25

pH 4.80 4.83 4.93 4.62 5.00

Ammonia (mg ml

1

) 1.29 1.06 2.17 0.59 1.65

Organic acids (mg ml

1

)

Pyroglutamic acid 2.62 1.34 5.55 0.79 6.61

Lactic acid 6.76 5.26 11.10 nd 3.45

Acetic acid 1.09 0.94 2.37 0.73 0.93

Formic acid 0.05 nd 0.14 nd 0.04

Citric acid 1.00 0.15 1.99 0.30 3.18

Succinic acid 0.33 0.32 0.41 0.06 0.63

Amino acids (mg ml

1

)

o-Phosphoserine 0.72 0.54 1.25 0.18 1.44

Taurine nd nd 0.58 nd nd

L-Aspartic acid 1.85 4.39 7.12 0.53 5.61

L-Threonine 2.81 2.07 4.02 0.51 3.33

L-Serine 4.00 2.95 5.48 0.49 4.88

L-Glutamic acid 12.67 10.51 13.80 6.06 11.99

L-a-Aminoadipic acid 0.41 0.45 0.45 0.10 0.32

Glycine 2.11 1.60 2.84 0.68 2.58

L-Alanine 6.88 2.48 5.80 1.07 6.01

L-Citruline 0.17 0.21 0.66 nd nd

L-Valine 4.09 2.95 5.73 1.21 5.15

L-Methionine 0.95 0.73 0.85 0.32 0.94

L-Cystathione 0.10 0.10 0.21 0.03 0.20

L-Isoleucine 3.64 2.72 3.64 0.88 3.77

L-Leucine 5.57 4.29 4.40 1.56 5.07

L-Tyrosine 0.48 0.27 0.62 0.37 0.49

L-Phenylalanine 3.40 2.69 3.88 0.68 4.16

b-Alanine 0.53 0.28 0.60 0.14 0.85

L-b-Aminoisobutyric acid nd nd nd nd nd

g-Aminobutyric acid 0.33 0.15 0.52 0.21 0.73

L-plus allo-d-hydroxylysine 0.38 0.40 0.91 0.08 1.65

L-Ornithine 0.33 0.40 0.94 0.03 0.00

L-Lysine 3.84 3.01 5.77 0.84 4.88

L-Histidine 1.01 0.90 1.00 0.42 1.15

L-Carnosine 0.67 0.54 1.34 0.12 1.34

L-Arginine 3.91 3.18 4.19 1.26 5.71

L-Proline 3.33 1.78 3.88 1.28 3.71

Sugars (mg ml

1

)

Sucrose nd nd nd nd nd

Ribose nd nd nd nd nd

Mannose 0.47 nd 0.66 nd 0.43

Fructose nd 13.11 0.93 0.74 nd

Arabinose 2.78 6.57 1.31 4.65 5.49

Galactose 3.32 3.15 3.15 1.12 5.55

Xylose 0.46 1.31 0.74 5.37 0.74

Glucose 11.91 13.67 8.16 58.66 7.75

lons (p.p.m. w/v)

K 6140 5210 9980 1450 6700

Mg 655 510 1000 154 934

Ca 170 111 163 59 224

Fe 9.2 6.4 10.3 2.3 10.6

4590 3750 7110 2040 5970

94 90 217 72 103

nd nd nd nd nd

Diluted with water to four times.

nd, not detected.

From Sasaki (unpublished).

2366 FERMENTED FOODS/Soy (Soya) Sauce

themselves related to flavor in shoyu. In particular,

these oxidative reactions give rise to an inferior or-

ganoleptic quality of shoyu, in that the existence of

oxygen leads to a decrease in the number of ‘good’

flavor compounds, such as HEMF and HMMF, and

an increase in the number of off-flavors resulting

from isobutyric acid, isovaleric acid, and many pyr-

azine compounds. To preserve flavor and color, it is

important to store shoyu at a cool temperature. (See

Browning: Nonenzymatic.)

Safety

0070 The main concern with the safety of Japanese shoyu is

the long-term effects of consumption, and whether or

not shoyu contains mycotoxins and mutagens.

0071 Mycotoxins As strains of A. oryzae or A. sojae are

taxonomically related to aflatoxin-producing molds,

the question has arisen as to whether or not the molds

produce aflatoxins. Fortunately, chemical, enzymatic,

and molecular biological research has shown that

none of the koji molds produce aflatoxins. Moreover,

the possible presence of other mycotoxins, e.g.,

ochratoxins, sterigmatocystin, patulin, cyclopiazonic

acid, and penicillic acid, in shoyu has been checked,

and results have indicated that, with the exception of

very few strains that produce cyclopiazonic acid, koji

molds do not produce such toxins.

0072Long-term effects of shoyu consumption The long-

term effects of feeding rats shoyu and its effects on the

gastric mucosa have been evaluated. Animals fed

shoyu were smaller than the controls, but were

healthier, more active, and lived longer. Breast tumors

developed in 10 control rats, but none developed in

those fed shoyu, and it was concluded that shoyu did

not appear to be a carcinogen in rats. Moreover, the

acute toxicity of shoyu was explained as being due to

its sodium chloride content.

0073Mutagens After an investigation of mutagens in

shoyu using the Ames test, it has been found that

shoyu treated with nitrite at a concentration level

detected in the human mouth, i.e., less than

50 p.p.m., did not induce any mutagenicity, though

mutagenic material was formed in shoyu when

2000 p.p.m. of nitrite was present at pH 3.0. It was

concluded that shoyu cannot be an inducer of muta-

genicity at the level of human consumption.

South-east Asian Countries

0074China The annual production of soy sauce in China

was estimated to be 500 000 kl in 1993.

0075The manufacture of soy sauce in Peking and Shang-

hai nowadays is different to the traditional method

and is as follows. The koji is prepared by a usual

method on a large scale, culturing Aspergillus oryzae

with a mixture of steamed soybeans and wheat or

wheat bran (6–8:4–2), and then mixed with salt

water to make a hard mash, with a moisture content

of about 80% and a salt concentration of about 6–

7%. This hard and low-salt mash is kept at 45–50



for 20–30 days for enzymatic digestion. The digested

mash is extracted with hot salt water (90



C, Be 17–

19) and then with hot water. The salt-free residue is

suitable for animal and bird feed. There is no alco-

holic fermentation of mash or pressing of the mash, as

there is in Japanese shoyu manufacture. The yield of

soy sauce on a nitrogen basis is 70–80%. About 80%

of Chinese soy sauce is made by this process. There

are four governmental standard grades of soy sauce in

China, and the standard of the highest grade is as

tbl0006 Table 6 Typical analysis of flavor constituents in Japanese

shoyu

Flavorcomponent Concentration

(p.p.m.)

Ethanol 31 501.10

Lactic acid 14 346.57

Glycerol 10 208.95

Acetic acid 2 107.74

4-Hydroxy-5-methyl-3 (2H)-furanone (HMMF) 256.36

2,3-Butanediol 238.59

Isovaleraldehyde 233.10

4-Hydroxy-2 (or 5)-ethyl-5-(or 2)-methyl-3

(2H)-furanone (HEMP)

232.04

Methanol 62.37

Acetol 24.60

Ethyl lactate 24.29

2,6-Dimethoxyphenol 16.21

Ethyl acetate 15.13

Isobutyraldehyde 14.64

Methyl acetate 13.84

Isobutyl alcohol 11.95

Furfuryl alcohol 11.93

Isoamyl alcohol 10.01

Acetoin 9.78

n-Butyl alcohol 8.69

4-Hydroxy-2,5-dimethyl-3 (2H)-furanone (HDMF) 4.83

Acetaldehyde 4.63

2-Phenylethanol 4.28

n-Propyl alcohol 3.96

Acetone 3.88

Methionol 3.65

2-Acetylpyrrole 2.86

4-Ethylguaiacol 2.77

Ethyl formate 2.63

4-Butanolide 2.02

Methional 1.42

4-Ethylphenol 0.34

Dimethyl sulfide 0.04

From Yokotsuka T, Sasaki M, Nunomura N and Asao Y (1980) Journal of

the Brewing Society of Japan 75: 717.

FERMENTED FOODS/Soy (Soya) Sauce 2367