Food Microbiology,

1984, 1,339-347

Selection of micro-organisms
of soy sauce*
Shin-ichi
Kikkoman
USA

for use in the fermentation

Sugiyama
Foods, Inc., P.O. Box 69, Walworth,

Received 14 August

Wisconsin 53184,

1984

Micro-organisms
related to soy sauce production are mainly koji mold (Aspergillus
oryzae or Aspergillus sojae), lactic acid bacteria (Pediococcus halophilus) and
osmophilic yeasts (Saccharomyces rouxii, Candida (Torulopsis) versatilis, Candida

(Torulopsis) etchellsii).
The selection parameters
are as follows.

(I 1 Aspergillus

of these micro-organisms

for use in soy sauce production

oryzae or Aspergillus

sojae. (a) Do not produce mycotoxins; (b)

amylolytic, and macerating enzyme levels.
(2) Pediococcus halophilus. (a) Produce lactic acid and other useful organic acids in
a high concentration
of salt brine; (b) do not possess tyrosine decarboxylase and
histidine decarboxylase.
(3) Saccharomyces rouxii. Produce alcohol and excellent flavor substances in a high
salt concentration brine.
(4) Candida (Torulopsis) versatilis and C. etchellsii. Produce excellent flavor
substances in a high salt concentration brine.

possess high proteolytic,

lntroductidn
Fermented soy sauce is a traditional
oriental food. Most people in Japan,
for example, consume substantial quantities of soy sauce every day. In recent
years, soy sauce has become popular
among many people in the world for its
excellent flavor and taste.
There are many varieties whose characteristics depend on the types and
ratios of raw materials used, the microorganisms employed, and the conditions
of preparation. Most varieties are made
from mixtures of soybean and wheat.
The Japanese government recognizes

* Paper presented at the symposium Selection Parameters of Micro-organisms

for Use
in the Fermentation ofPlant Foods and Beverages.
0740%0020/841040339

+ 09 $02.OOiO

five kinds of soy sauce: koikuchi, usukuchi, tamari, shiro, and saishikomi. Over
85% of the soy sauce consumed in Japan
and the USA is the koikuchi type, which
means it is dark in color.
Fermented soy sauce is prepared by
enzymatic hydrolysis of plant proteins
and carbohydrates using the koji mold
enzymes followed by months of slow
fermentation by salt-tolerant lactic acid
bacteria and yeasts.
This paper is concerned about properties and selection parameters of the
micro-organisms related to fermented
soy sauce production.

The Manufacturing Process of

Fermented Soy Sauce
The steps in making fermented soy sauce
are shown in Fig. 1. The first step is koji
making. Defatted soybean grits are
0 1984 Academic

Press Inc. (London) Limited

340

S. Sugiyama

(-iGi--)
Brine fermentation

Condido versafihs
Condfdo eichellsi
I

Pressing
t

Rawsov sauce

Fig. 1. The manufacturing

processof fermented soy sauce.

moistened and cooked under steam pressure. The other main ingredient, wheat,
is roasted at 170-180C for a few
min, and then broken into four to five
pieces. The cooked soybean grits and
roasted wheat are mixed in approximately equal amounts and then inoculated with a pure culture of Aspergillus
oryzae or Aspergillus sojae. This mixture
is aerated in special bins for 2-3 days at
approximately 30C. This stage is called
koji. This allows the mold to grow
throughout the mass and provide amylase and proteinase needed to hydrolyze
the starch and protein in the raw
materials.
The second step is brine fermentation.
After the mold has developed sufficiently
in the koji, this material is suspended in
a strong salt solution which then
becomes moromi, and is then transferred to large fermentation tanks. The
moromi mash is held for 6-8 months at
25C with occasional brief aeration to

mix the contents and to stimulate microbial growth. During the fermentation
period, enzymes from the koji mold
hydrolyze the protein into amino acids
and low molecular weight peptides.
Starch is converted into sugars which
are fermented into lactic acid, alcohol,
and flavor substances. The pH drops
from an initial value of 6.5-7.0 to 4-74.8. The high salt concentration of the
moromi effectively limits the growth of
undesirable bacteria.
At the first stage of moromi, a pure
culture of Pediococcus halophilus
is
added. This osmophilic lactic acid bacterium grows very quickly and produces
lactic acid, which causes a drop of the pH.
When this pH level reaches approximately 5.0, a pure culture of Saccharomyces rouxii is added and results in an
alcoholic fermentation.
At the middle and last stage of brine
fermentation, a group of salt-resistant
yeasts, normally known as Torulopsis

Soy sauce fermentation

species (but now classified as species of
Candida (Yarrow and Meyer 19781, often
grow; these strains produce phenolic
compounds and add some aroma to soy
sauce. These species are not active in the
moromi at the same time as S. ro&i.
After 6-8 months of fermentation,
matured moromi is filtered under high
pressure through a cloth and moves on to
the next step of refining.
In refining, the salt content is adjusted
to 1617% (w/v), sodium benzoate is
added as a preservative, and the soy
sauce is then heated to 70-80C for an
hour or so. This heat treatment is
necessary to develop the desired
characteristics in the product. After
heating, the soy sauce is clarified by
sedimentation, and the clear supernatant can be packed immediately.

341

carboxy or amino terminal of peptides.

So far, four kinds of carboxy peptidases
and seven kinds of aminopeptidase have
been isolated from koji (Nakadai et al.
1972a,b,c,d,e,
Nakadai
et
al.
1973a,b,c,d,e). The pH optima of the
carboxy peptidases are in the acidic
region and, therefore, they should be
called acid carboxy peptidases. All of the
amino peptidases had high specificity to
the amino-terminal leucine and, therefore, they can be called leucine amino
peptidase.
The role of each enzyme in koji during
protein hydrolysis is shown in Fig. 2
(Fukushima
1982). In addition to
proteinase and peptidase, glutaminase
is an important enzyme. Generally,
vegetable proteins such as soybean and
wheat contain a large amount of glutamine as well as glutamic acid. Glutamine
Koji Mold
is liberated by the peptidase and is
partially
converted to glutamic acid by
Koji mold is comprised of A. oryzae or A.
the
action
of the glutaminase in koji. The
sojae. The digestion of protein and starch
balance
of
glutamine is readily converin the raw materials is the main function
ted
into
pyroglutamic
acid, which is not a
of the koji mold; strains of A. oryzae or A.
flavor
component.
Therefore,
glutamisojae which have high activities of
nase
is
necessary
in
the
enzymatic
digesproteinase, peptidase, and amylase are
tion
of
cereal
proteins
to
improve
the
required. Particularly, proteolytic activmost
important
flavor
components
of
the
ity is the most important character for
hydrolyzates
(Mizunuma
1983,
Nasuno
koji mold. Many detailed studies about
Aspergillus
proteinase and peptidase and Nakadai 1977, Yamamoto and
have been reported (Fukushima 1982, Hirooka 1974a,b).
The macerating enzymes such as pectiHayashi et al., 1970, Nakadai 1977a,b,
Nasuno and Nakadai 1977, Nakadai et nase, cellulase, and hemicellulase are
al. 1972a,b,c,d,e,
Nakadai
et al. also important for soy sauce production.
1973a,b,c,d,e,fg,h,
Sekine 1976, Sekine If macerating enzymes are not active in
et al. 1970). The proteolytic enzymes of the fermentation liquid, the solid-liquid
is
almost
impossible.
koji mold are summarized in Table 1. It separation
has been found that koji is composed of Macerating enzymes also bring proteolyseven kinds of proteinases with four tic enzymes and the proteins into contact
different pH optima. As shown in Table through the disruption of the cell wall
1, the major protinases for soy sauce koji structure of both soybean and wheat,
leading to the increase in the degree of
are alkaline proteinase and neutral
proteinase 1 (Mizunuma 1981, Sekine et protein hydrolysis (Mizunuma 1983,
Nakadai 1977b).
al. 1970). Koji also contains various
kinds of exopeptidases, which succesThe main characteristic for selection
sively liberate the amino acids from the of A. oryzae or A. sojae is the high

342 S. Sugiyama
Table

1. Main

proteolytic

enzymes

in koji (Aspergillus

MWIlO

Enzyme type
1. Proteinase
Alkaline
Semi-alkaline
Neutral I
Neutral II
Acid I
Acid II
Acid III
2. Acid carboxy
I
II
III

Optimum
PH

sojae).
Activity

Enzyme
concentrationtl

23
32
41
19
39
I100
31

10.5
8.3
7.0
6.0
3.2
3.0
3.0

929
55
80
9
44
10
5

418

120
105
61

3-4
3-4
3

10
19
62

43

3-4

0.15c
0.18d
O.Olc
0.05d
0.02r
O.lld

27
61
55
130
100
39
170

8.5
5-8
8.0
7.0
-

131
152
617

peptidase

IV
3. Leucine amino peptidase
I
II
III
IV
V
VI
VII

0.108
0.25
0.15
0.15
0.11
0.01
0.03

a Unit, (against casein) gr koji

b Koji, pg g--l.
c Substrates: Cbz-Ala-Glu.
d Substrates: Cbz-Glu-Tyr.
e Substrates: Leu-Gly-Gly.
Proteins
+ Mocerotmg enzymes
+
+
+
f

Alkolme protemase
Semi-alkolme proteinose
Neutml proteinose I and I I
Acid proteinase I -II I

.
PeptIdes
+ ACIU carboxypephdose I-IV
+- Leucme ammapeptldase I-VII
l

Ammo acids
I
Glutamine

Glutomic
acid
v---+
Glutommase

I
Pyroglutamic
acid

Fig. 2. The role of each enzyme in koji during

protein hydrolysis. (From Fukushima (1982j.j

activities of proteolytic enzymes as described above; however, the molds must

not produce toxic substances. It is obviously necessary that the koji mold not
produce mycotoxins such as aflatoxin,
ochratoxin, and sterigmatocystin under
any conditions. The industrial koji cultures of aspergilli have been tested thoroughly for mycotoxin productivity and
have been found negative (Aibara and
Miyaki 1966, Hesseltine et al. 1966,
Kikkoman
Foods 1977, Yokotsuka
1967).

Brine Fermentation
Lactic acid bacteria
In the second stage of manufacturing,
the moromi is transferred to deep fer-

Soy sauce fermentation

mentation tanks and P. halophilus
is
added. The main purpose of adding the
lactic acid bacteria is to reduce the pH to
its final value of 4.7-48 from its initial
value of 6.5-7.0. This prepares for
growth of the yeast such as S. rouxii.
Many different types of P. halophilus
strains have been reported Wchida
1982a,b), especially as they relate to
the pattern of acid formation from carbohydrates. According to Buchanan and
Gibbons (19741, acid can be produced
Table

2. Fermentation

pattern

of Pediococcus

Substrate
Group

Arabinose

from arabinose, galactose, maltose,

mannitol,
and
cr-methylglucoside,
dextrin; however, many strains which
can not form acid from arabinose or
mannitol are known (Uchida, 1982a).
The multiplicity
of the fermentation
patterns
for
various
strains
of
Pediococcus isolated from soy sauce is
shown in Table 2 (Uchida 1982a).
According to Uchida (1982a), 28 groups
of strains were found in natural moromi,
defined on the basis of the pattern of acid
strains

isolated

+
+
+
+
+
+
+
+

Melibiose

+
+
+
+
+
+
+
-

+
+
+
+
+
+
+
+

(1982a1.

from moromi.

and acid formation

Sorbitol

Mannitol

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
Total
a From U&da

Lactose

343

+
+
+
+
+
+
+
A-

+
-

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

+
+
+
+
+
+
+
+
+
+
+

No. of
strains
221
167
12
96
169
223
16
17
21
132
6
3
26
67
60
113
0
6
74
31
42
16
1
0
2
39
0
1
5
15
0
3
1586

c/r
13.9
10.5
0.8
6.1
10.7
14.1
1.0
1.1
1.3
a.3
0.4
0.2
1.6
4.2
3.8
7.1
0
0.4
4.7
2.0
2.6
1.0
0.1
0
0.1
2.5
0
0.1
0.3
0.9
0
0.2
100

344 S. Sugiyama
formation
from several carbohydrates.
In natural moromi, a pure culture of P.
halophilus
is not added; the naturally
occurring
Pediococcus strains in soy
sauce production shown in Table 2 are
believed to be P. halophilus
Wchida
1982 (a). Strains that produce acid only
from mannitol are the most numerous.
This indicates the complexity and diversity of the soy sauce fermentation.
The growing conditions
of P. halophilus are shown in Table 3. They are
able to grow in 24% salt solution and in a
medium with an a, of 0,808. Certain
vitamins and amino acids are required
for growth. As shown in Table 3, folinic
acid is required in the medium with salt,
and uracil is required by some strains.
There are many kinds of Pediococcus
species capable of growth in the moromi,
raising the question as to the traits
desired in strains to be used for pure
culture addition to moromi. Obviously,
the selection of the proper strain is
critical for manufacturing
of soy sauce,
and strain selection is based on the
totality
of desired characteristics.
Of
primary importance is the ability of the
strain to form lactic acid in a high salt
concentration
present in moromi. In
addition, a strain which does not possess
amino acid decarboxylase is necessary. If
Table 3. Growing

conditions

a From Mizunuma (1981).

b Promoting effect.
c Required
d Required

in the medium with salt.

by some strains.

Yeasts
The predominant
yeast of soy sauce
fermentation
is S. rouxii. Saccharomyces
rouxii begins to grow when the pH value
has decreased to around 5.0. It produces
2-3% of ethyl alcohol and around 1%
glycerol in addition to many of the flavor
components
of moromi
(Mizunuma
1983). The growth conditions of S. rozbi
are shown in Table 4 [Mizunuma
1981).
This yeast can be grown in a 25% salt
solution and in 80% glucose. As shown in
Table 4, pH, temperature,
substrates of
fermentation,
and nutritional
requirement vary with the existence of salt. The
important points for selection of S. rouxii
strains are the production of alcohol and
excellent flavor substances in a high salt
concentration
brine.
Candida (Torulopsis) species begin to
grow after alcohol fermentation
and produce specific flavor substances in soy
sauce. Over 270 flavor substances have
been isolated by Yokotsuka
and other
researchers (Yokotsuka et al. 1980). Normally, two species exist as natural flora

of Pediococcus halophilus.a

Maximum concentration of NaCl

Minimum water activity
Temperature: maximum
optimum
pH range
Nutritional requirements
Vitamins
Amino acids
Base

the strain has decarboxylases of tyrosine

or histidine, undesirable amines such as
tyramine and histamine are produced in
the moromi (Rice and Koemler 1976;
Uchida 19826).

24% (w/v)

0.808
42C
2530C
55-9.0

Biotin, vitamin Be, nicotinic acid,

pantothenic acid, riboflavin,b
folinic acidc
Glu, Arg, His, Ile, Leu, Phe, Val, Try
Uracild

Soy sauce fermentation

Table 4. Growing

conditions

of Saccharomyces

Maximum concentration of NaCl

Maximum concentration of glucose
Minimum water activity

345

rouxii.~
24-26% (w/v)
80% (w/v)
0.787-0.8

With NaCl(l8% w/v) Without NaCl

pH range
Temperature; max.
Can be fermented

4-5
40C
Glucose

Nutritional

Biotin
Thiamine
Pantothenic acid
Inositol

requirement

3-7
35C
Glucose
Maltose
Biotin
Thiamine
Pantothenic acid

a From Mizunuma (1981).

Table 5. Growing

condition

of Candida versatilka

Maximum concentration of NaCl

Minimum water activity
Optimum water activity

26% (w/v)
0.787
0.975-0.84
With NaCl(18% w/v) Without NaCl

pH range
Temperature: max.
Nutritional requirement

4-5
35C
Biotin
Thiamine
Inositol

3-7
30C
Biotin
Thiamine

a From Mizunuma (19811.

in moromi (Mizunuma 1981) and their

industrial characteristics are almost the
same. The growth condition of Candidu
(Torulopsis) versatilis is shown in Table
5 (Mizunuma 1981). These Candida
species are more aerobic than S. rouxii.
They can grow in a 26% salt solution as
can S. rowcii. The pH range, temperature
range, and growth factors vary with the
existence of salt. Inositol is an essential
factor for the growth in the medium in
the presence of salt. Usually, natural
flora of Candida species are used for
fermentation, and the most important
property of these yeasts is to produce a
flavor
large amount of excellent
substances in a high salt concentration
brine.

The prospects of improvement of

micro-organisms
by new techniques

In recent years, many studies regarding

improvement
in industrial
microorganisms by use of the protoplast fusion
technique have been reported (Croft and
Dales 1983: Morgan 1983; Ushijima and
Nakadai 1983,1984). As described previously, an Aspergillus
strain which possesses high activities of both proteinase
and glutaminase is an outstanding koji
mold for soy sauce production. The possibility of introducing such strains by the
technique of protoplast fusion has been
studied Wshijima and Nakadai 1983,
1984). To date, a commercially useful
strain has not been produced by
protoplast fusion because of instability of

346

S. Sugiyama

the constructed hybrids. Therefore, the

prospect for improvement
in microorganisms used for soy sauce production
by this new technique is dependent on
the stabilization
of these hybrids.
Integration
of the protoplast
fusion

technique
with the more traditional
methods of sexual hybridization
and
mutagenesis
is necessary. With this
marriage, the future of improvement of
micro-organisms
appears promising.

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