Journal of industrial Microbiology, (1995) 14, 467-471

9 1995 Societyfor IndustrialMicrobiology0169-4146/95/$12.00

Industrial production of soy sauce

B.S. Luh
Dept of Food Science & Technology, University of California, Davis, CA 95616, USA
(Received 27 June 1994; accepted 22 September 1994)

Key words: Soy sauce; Fermentation; Aspergillus oryzae; Aspergillus sojae; Pediococcus halophilus; Zygosaccharomyces rouxii;
Candida species

SUMMARY

Soy sauce is a seasoningagent with a salty taste and a distinctaroma suggestiveof meat extracts. The sauce is made by fermentationof a combinationof
soy beans and wheat in water and salt. This paper covers the method for productionof fermented soy sauce, and that for acid-hydrolysisof defatted soy bean
proteins. The microorganismsinvolvedin soy sauce production,and biochemicaland chemical changes in soy bean and wheat during fermentationinfluence
greatly the sensoryattributes and qualityof soy sauce. Recentprogress in industrializationof soy sauce manufactureis discussed.

INTRODUCTION ties of soy sauce, and recent progress in technology concerning

soy sauce production are also presented.
Of the many Oriental fermented products, soy sauce is the
one most widely consumed in China, Japan, Korea and other PRODUCTION OF FERMENTED SOY SAUCE
Asiatic countries as a condiment and coloring agent in prep-
aration of foods and for table use. It is a dark brown liquid, Soy sauce is made by fermentation of a combination of soy
stable at ambient temperature, which does not require refriger- beans, wheat grain, water and salt [8,17]. The processes for
ation during storage due to its low water activity and high salt production of fermented soy sauce consist of three major steps,
content. Soy sauce and miso are flavoring agents having simi- namely, Koji production, brine fermentation, and refining. A
lar aroma and flavor. Soy sauce is a liquid whereas miso is a flow sheet for manufacture is shown in Fig. 1.
paste. Each is made by a two-step fermentation process from
wheat flour and soy beans with a mixture of molds, yeasts Koji production
and bacteria. The first step involves fermentation with mold Koji is a source of proteolytic enzymes for converting soy
to produce proteolytic and amylolytic enzymes in the Koji bean proteins into peptides and amino acids, and amylase for
which is a culture starter. A good culture starter must give hydrolyzing gelatinized starch into simple sugars. The sub-
characteristic aroma and flavor to the soy sauce, have high stances converted by the enzymes in Koji become the nutrients
proteolytic and amylolytic activities, and must be easy to cul- for yeasts and lactic bacteria in the subsequent brine fermen-
ture. This is followed by a second fermentation with yeast and tation. In Koji production, defatted soybean flakes or soy beans
bacteria in the presence of 18-20% salt. The microorganisms are soaked in water to increase the moisture content and then
used in these fermentation steps are not inoculated at the same cooked under pressure in a retort. Formerly, the soaked soy
time, but are applied sequentially. beans containing 60% moisture were cooked with saturated
Literature on soy sauce manufacture is largely in Chinese, steam at 0.8-1.0 kg cm -2 gauge pressure for 40-45 rain in a
Japanese, and other Oriental languages. Several references batch type pressure cooker. Currently, the soaked beans con-
have been published by Fukushima [4,5,6], Noda [10], Nuno- taining 30-45% moisture are cooked at 6-7 kg cm-2 gauge
mura and Sasaki [11], Wang and Hesseltine [20], Onaga et al. pressure (about 170 ~ for 20-30 s in a continuous cooker
[13], Reed [15], Skinner et al. [16], Sugimori [18] and Yokot- which allows high pressure and short time cooking. The wheat
suka [19]. 'This paper covers the methods for production of contains 8% moisture and is heated in a continuous roaster
soy sauce made by fermentation of wheat and soy beans, and with hot air at 150 ~ for 30-45 s at atmospheric pressure. It
by acid hydrolysis of defatted soy beans. The chemical proper- is then cracked in a machine into 4 or 5 pieces per kernel
accompanied by smaller particles of wheat flour. In making
regular soy sauce, the cooked soy beans (or defatted soy
flakes) are mixed with an equal amount of roasted wheat and
This paper is dedicated to Professor Herman Jan Phaff in honor of then inoculated with 0.1-0.2% of starter mold (Aspergillus
his 50 years of active research which still continues. oryzae or Aspergillus sojae) in wooden trays, each loaded with
Correspondence to: B.S. Luh, Dept of Food Science & Technology, a 3-5 cm thickness of the fermenting Koji. The ratio of wheat
University of California, Davis, CA 95616, USA. to cooked soybean may vary, depending on the type of soy
 Industrialproductionof soysauce
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468
Wheat Soybeans Brine fermentation
(50 parts) (50 parts) The second step in making fermented soy sauce is brine
fermentation. It utilizes the lactic bacterium, Pediococcus hal-
Starter Soaked ophiIus and the yeasts Zygosaccharomyces rouxii and Candida
Roasted
Aspergill~s o0'~ A so]oo 1 species both of which tolerate a salt concentration of 20 g per
(Mixed strains) Steamed 100 ml. The brine effectively prevents growth of undesirable
1 microorganisms. The harvested Koji is mixed with 20% salt
Crush lightly I
Mixed Cooled
brine, and transferred by means of a spiral pump into deep
Incubationroom fermentation steel tanks coated with epoxy resins on the
(25.35~ 72 hr.) interior. The resultant mixture is called moromi mash. It is
important to control the microorganisms in the brine fermen-
Mold mixture tation. The specially selected P. halophilus is cultured and
(Koji) added to the mash. To control its growth rate it is necessary
Salt solution ,, ,l to keep the fermenting mixture at 15 ~ for the first month,
(20%, 200 parts) Mash (17-19% salt) allowing the pH of the mash to decrease slowly from 6.5 to
(Moromi) 5.0. Then cultures of Z rouxii and Candida species are added
as a starter. The temperature of the moromi is allowed to rise
Lactic acid fermentation slowly to nearly 28 ~ until vigorous alcoholic fermentation
' (Pediococcus halophilus) starts. The temperature in the tank can be controlled by coil-
Yeast fermentation type heat exchangers with mixing devices, thermocouples, and
(Zygosaccharomyces rouxi~ CaMida sp.} control systems.
(12 mo. at ambient temperaturesor 15~ for 1 too., After the alcohol fermentation is finished, the temperature
28~ for 4 mo. and 15~ for 1 mo.) is kept at 25 ~ Aeration stimulates microbial growth and
1 mixes the contents. During the fermentation period, proteolytic
Pressed enzymes from Koji hydrolyze the proteins in soy bean and
Cake ~ Filtered ~ Liquid wheat to form amino acids and low molecular weight peptides.
Starch is converted to simple sugars which are fermented
Animal feed Pasteurized primarily to lactic acid, ethanol and carbon dioxide. During
the brine fermentation, the pH of the mixture drops from 6.5
Soy sauce to 5.0 in the first month at 15 ~ This is followed by fermen-
Fig. l. Flowsheet for manufacture of soy sauce. tation at 28 ~ for four months. Sometimes it is necessary to
add more pure cultures of P. halophilus and Z. rouxii and
Candida species to the moromi mash during the fermentation.

sauce m be prepared. After incubation at 25 ~ for 72 h, the Refining

The final process in soy sauce fermentation is refining
Koji becomes a greenish yellow mass as a result of mold
growth and sporulation. It is necessary to stir the contents, to which includes pressing, filtration, pasteurization and pack-
remove the metabolic heat formed, and to stir further to control aging.
the temperature at 25-28 ~ The important factors are selec- The aged moromi is pressed in a vertical automatic press
tion of the best strains of A. oryzae and A. sojae and con- to separate the soy sauce from the residue. After pressing, the
trolling the product temperature in that range during the Koji filtered raw soy sauce is pasteurized in a heat-exchanger at
fermentation process. More recently, the Koji manufacturing 70-80 ~ for a few minutes to ensure clarity, to inactivate
process has been changed from a manual process to an auto- residual enzymes, and to inactivate any undesirable microor-
matic equipment process. This includes a continuous cooker ganisms. It may be necessary to clarify the soy sauce addition-
for the soy beans, a continuous wheat roaster, mixer, cooler, ally by centrifugation or sedimentation. The sauce is treated
automatic inoculator, mechanical mixer, temperature control- with caramel as a coloring agent, and then packaged either in
lers, conveyors, and mechanical devices for turning the sub- clean glass bottles, enameled gallon cans or in plastic con-
strates during incubation. The inoculated mixture is put into tainers.
large shallow perforated vats in closed chambers and forced The residue from the press can be extracted with more 20%
air is circulated through the mass. After 3 days, A. oryzae or salt brine to increase the yield. Much expertise is needed to
A. sojae grows and the culture mixture becomes green-yellow produce a soy sauce that is attractive in flavor and taste, stable
in color as a result of mold growth and sporulation. The auto- on storage at room temperature, and acceptable to the con-
matic Koji-making system increases the protease activity of sumer. The quality assurance group must check the pH, acid-
Koji and protects it from infection by undesirable microorgan- ity, amino nitrogen, salt content, color, microbial contami-
isms. This new device reduces the labor cost by 85-90% com- nation, and sensory attributes: color, aroma and flavor of the
pared with the conventional manual method. product.
 Industrial production of soy sauce
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469
SOY SAUCE PRODUCTION BY THE ACID- reddish brown color. More than 56% of Japanese soy sauces
HYDROLYSIS PROCESS are of this type.
The 'Upper grade' or 'Usukuchi shuyu' is used mainly in
In addition to the fermentation method, soy sauce is also the western part of Japan. The 'Standard grade' or 'Tamari
made by acid hydrolysis of defatted soya beans with food shuyu' sauce is characterized by a higher total nitrogen and
grade hydrochloric acid as a catalyst in a pressure cooker. The formol nitrogen compared with the other varieties. It is con-
hydrolysate is neutralized with sodium hydroxide or sodium sumed largely in the Nagoya region of Japan. The Saishikomi
carbonate under careful supervision so that excessive alkali and Shiro soy sauces are produced and consumed only in iso-
does not come into contact with the neutralized product. The lated localities or for special industrial uses.
acid-hydrolyzed soy sauce is refined by sedimentation, treat- The amino acid composition of the soy bean, wheat and
ment wtih active carbon, filtration through a filter press to a representative Koikuchi soy sauce is presented in Table 2.
remove undesirable substances, and high vacuum to remove Glutamic and aspartic acids are the major amino acids present
undesirable volatile compounds. However, the acid- in the fermented soy sauce. A total of 18 amino acids are
hydrolyzed soy sauce is less attractive in aroma and flavor present in the sample. Arginine, lysine, phenylalanine, serine,
because of lack of aromatic substances such as esters, alcohols threonine, leucine, isoleucine, valine, alanine and proline are
and carbonyl compounds which are derived from the fermen- present in significant quantities in the sauce. A comparison of
tation process. In some countries, a combination of fermen- the amino acids present in the fermented soy sauce with those
tation and acid-hydrolysis procedures are used for making less- in the soy bean and wheat indicates the following changes: a)
expensive soy sauce. High quality soya sauce is made exclus- arginine is converted into ornithine in the fermentation pro-
ively by the fermentation process. cess; b) tryptophan and cystine may have been used during
the fermentation as nutrients; and c) tyrosine decreases in the
Grades of soy sauce moromi mash.
In Japan, 'Special grade' soy sauce or 'Koikuchi' is made Sugars in fermented soy sauce have been reported by Yok-
exclusively by the fermentation process, and is marketed under otsuka [19]. A representative Japanese fermented soy sauce
the Japanese Agricultural Standard (JAS) for this grade. It is contains the following sugars % (w/w): arabinose, 0.08%; glu-
an all-purpose seasoning characterized by a pleasant aroma, cose, 2.05%; mannose 0.06%; galactose, 0.17%; xylose,
complex flavor, and a deep reddish brown color. It comprises 0.06%; disaccharide, 0.65%; polysaccharide, 1.15%; unidenti-
more than 56% of the annual soy sauce production in Japan. fied sugar 0.23%; total sugar, 4.45% (as glucose). Organic
A second type is 'Upper grade' or 'Usukuchi shoyu' which acids: acetic acid, 0.16%; citric acid, 0.04%; formic acid
is characterized by a lighter reddish brown color and milder 0.02%; lactic acid, 0.68%; succinic acid, 0.05%; total 0.95%.
aroma and flavor, and comprises about 24% of the total pro-
duction. The product is used mainly for cooking when one Volatile flavor compounds
wishes to preserve the original flavor and color of the food- Forss and Sugisawa [3] reported 150 volatile compounds
stuff itself. in soy sauce. Aishima [1] presented gas chromatographic pro-
A third type is 'Standard grade' or 'Tamari shoyu' which files of soy sauce aroma. He observed more than 200 peaks
is characterized by a slightly higher amino acid content, but in the chromatograms by use of distillation-extraction tech-
lacks in aroma. The distinguishing feature of 'Standard grade' niques and using various types of glass and fused silica capil-
is that soy bean is the main raw material and wheat is not lary columns. These volatile compounds included: 1) hydro-
used or is used only in small amounts. This grade comprises carbons, 2) alcohols, 3) esters, 4) aldehydes, 5) acetals, 6)
8% of the total production. The remainder belongs to the ketones, 7) furans, 8) lactones, 9) furanones, 10) pyrones, and
'Non-JAS' grade. 11) pyrazines. Of these aromatic compounds, HEMF (4-hy-
The 'Special' and 'Upper' grades are derived mainly from droxy-2 (or 5)-ethyl-5 (or 2)-methyl-3-(2H)-furanone) appears
the use of equal amounts of wheat and soy beans as the raw to be a very important flavor component of fermented soy
materials of Koji. The Japanese Soy Sauce Association indi- sauce. HEMF possesses a strong flavor and resembles the
cates an annual production of 1.2 million tons of soy sauce aroma of the moromi mash of Koikuchi soy sauce. Nunomura
in Japan. and Sasaki [11] reported that HEMF is the most abundant
aroma component, comprising 200-300 p.p.m., in Koikuchi
CHEMICAL COMPOSITION OF SOY SAUCE soy sauce.
The volatiles are formed by microorganisms during the
In general, good quality soy sauce contains 1.0-1.65% total Koji and brine fermentation as well as the heating and pasteur-
nitrogen (w/v), 2-5% reducing sugars, 1-2% organic acids, ization process. The same authors reported on the quantitative
2.0-2.5% ethanol and 17-19% sodium chloride (w/v). About aspects of 12 major flavor components in Japanese soy sauces
45% of the total nitrogen is found in simple peptides, and 45% as follows: isobutyl alcohol, 3.07-18.35 p.p.m.; n-butyl alco-
in amino acids. hol, 1.41-11.48p.p.m.; isoamyl alcohol, 4.47-22.0p.p.m.;
The properties of some Japanese soy sauces are shown in acetoin, 5.08-8.44p.p.m.; ethyl lactate, 7.35-27.12p.p.m.;
Table 1. Koikuchi is a representative of the fermented soy furfuryl alcohol, 4.35-10.07 p.p.m.; methionol, 2.60-
sauce popular in Japan [4]. It is an all-purpose seasoning 4.47 p.p.m.; 2-phenylethanol, 3.71-10.25 p.p.m.; HDMF (4-
characterized by a strong aroma, attractive flavor, and a deep hydroxy-2,5-dimethyl-3(2H) furanone), 1.83-5.39p.p.m.; 4-
 Industrial production of soy sauce
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470
TABLE 1
Typical compositions of five varieties of soy sauce

Be NaC1 Total Formol Reducing Alcohol pH

(g per nitrogen nitrogen sugar (vol per
100 ml) (g per (g per (g per 100 ml)
100 ml) 100 ml) 100 ml)

Koikuchi 22.0 16.9 1.57 0.94 3.0 2.3 4.7

Usukuchi 22.2 18.9 1.19 0.80 4.2 2.1 4.8
Tamari 29.9 19.0 2.55 1.05 5.3 0.1 4.8
Saishikomi 26.9 18.6 2.39 1.11 7.5 pace 4.8
Shiro 26.9 19.0 0.50 0.24 20.2 trace 4.6

Source: [4].

TABLE 2 soy sauce volatiles as related to organoleptic characteristics by

Distribution of amino acids in soy bean, wheat and Koikuchi soy multivariate analysis.
sauce
BIOTECHNOLOGY IN SOY SAUCE RESEARCH
Amino acid Soybean Wheat Raw Koikuchi-
(%) (%) materials shoyu
of (%) Biotechnology and genetic engineering have been widely
koikuchi- used by food researchers [9], microbiologists [12], and geneti-
shoyu cists [2]. Osaki et al. [14] investigated a shortening of the brine
(%) fermentation period through fermentation in a bio-reactor on
a pilot plant scale. The first step in this process is digestion
Arginine 8.42 4.71 7.58 2.6 of the defatted soy beans by proteolytic and amylase enzymes
Histidine 2.55 2.12 2.45 2.5 in Koji at 55 ~ in a salt brine 10% (w/v). The resultant
Lysine 6.86 2.67 5.90 6.5 hydrolysate is fermented in bio-reactors with cells of P. halo-
Tyrosine 3.90 3.19 3.74 1.0 philus entrapped in calcium alginate beads [14] or in a mixture
Tryptophan 1.28 1.13 1.25 - of gel beads of alginate and silica [7]. In the reactor with
Phenylalanine 5.01 4.43 4.88 4.2
immobilized P. halophilus, the viable cells in gels at steady
Cystine 1.58 1.80 1.63 0.9
state showed a decimal increase of 2.5. The number of viable
Methionine 1.56 1.74 1.60 1.4
Serine 5.57 5.22 5.49 5.3 cells in gels increased 1000-10 000 times over numbers in the
Threonine 4.31 2.76 3.96 4.2 effluent with a holding time of 40 h. The production of lactic
Leucine 7.72 6.52 7.45 7.3 acid reached a satisfactory level of 10 g L -1 after 28 days.
Isoleucine 5.10 3.78 4.80 4.8 In the subsequent alcoholic fermentation by Z. rouxii or
Valine 5.38 4.69 5.22 5.5 Candida versatilis, using a bioreactor, the difference in the
Glutamic acid 21.00 29.30 22.89 22.5 number of viable cells between gels and effluents decreased
Aspartic acid 12.01 4.85 10.38 10.5 gradually with time. About 2% ethanol was produced continu-
Glycine 4.52 3.94 4.39 3.9 ously in 40-80 h while the pH level reached 4.8-5.2. These
Alanine 4.51 3.37 4.25 4.4 results indicate that both lactic acid and alcoholic fermentation
Proline 6.28 9.94 7.11 6.5
can be achieved in a very short time with a satisfactory level
Ornithine - - - 5.7
of production. However the aroma pattern of the fermented
samples differed slightly from that of commercial soy sauce.
The bioreactor samples were higher in isobutyl alcohol,
EG (4-ethyl-2-methoxyphenol(4-ethylguaiacol)), 1.12-3.67 isoarnyl alcohol and 2-phenylethanol, but lower in HEMF
p.p.m.; HEMF (4-hydroxy-2 (or 5)-ethyl-5 (or 2)-methyl- which is an important aroma component of Koikuchi soya
3(2H)-furanone), 177.8-418.7 p.p.m.; HMMF (4-hydroxy-5- sauce.
methyl-3(2H)-furanone), 84.54-153.6 p.p.m. Despite the reduction in time of brine fermentation in the
Color, flavor and aroma are three important criteria for eva- bioreactor, the Japanese Government has ruled that soy sauce
luating the sensory quality of soy sauce [13]. These authors produced without the aging process is not a fermented product,
applied chemical and physical tests to analyze the salt content, because the product does not meet the criteria for 'Special'
amino acids, sugars, total nitrogen, and % amino nitrogen in grade soy sauce. It is hoped that soy sauce researchers will
samples collected in California. Similar approaches were used continue to work on the subject to improve the protease and
by Forss and Sugisawa [3], Fukushima [4], and Yokotsuka amylase activity of Koji through protoplast fusion, mutation,
[19]. Aishima [1] studied the gas chromatographic profiles of and genetic engineering studies of the genes involved in soy
 Industrial production of soy sauce
B8 Luh
471
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Proc. First International Conf. on Food Science and Technology,
Wuxi, Jiangsu Province, People's Rep. of China, pp. 1-13.
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