JOURNAL OF FERMENTATION AND BIOENGINEERING

Vol. 71, No. 3, 163-167. 1991

Esterase Activity in Soy Sauce M o r o m i as a Factor

Hydrolyzing Flavor Esters
TAKESHI HIGUCHI,* TERUMICHI AOKI, AND KINJI U C H I D A
Research and Development Division, Kikkoman Corporation, Noda-shi, Chiba 278, Japan
Received 5 April 1990/Accepted4 November 1990

In order to elucidate the reason for the meager occurrence of volatile esters in soy sauce, the ester-decompos-
ing activities of microorganisms concerned in soy sauce fermentation were examined. Soy yeasts showed at
least 10 times higher esterase activity than the other yeasts used for fermented beverages. The yeast esterase was
not greatly affected by the pH or NaCI concentration. Soy koji cultured with Aspergillus sojae or A. oryzae
showed very high ester-splitting activity. By gel-filtration of koji esterase, the i-amylacetate (i-AmAc) decom-
posing fraction was obtained. This fraction showed a decrease of activity at lower pH or higher NaCI concen-
tration. Koji esterase decreased its activity in moromi but remained over the entire moromi period. Koji
esterase exhibited a higher activity than yeast esterase in fermenting moromi. These strong esterase activities
are thought to be one of the causes of the low concentration of ester flavor in soy sauce.

Volatile esters like i-amylacetate are distributed in
various fermented beverages such as beer, sake or wine,
and are known to contribute greatly to the production of
their aromas (1). In the brewing of beer (2) or sake
(Ishikawa et al.: Abstr. Annu. Meet. Agric. Chem. Soc.,
Japan, p. 408, 1979, Ishikawa et al. Abstr. Annu. Meet.
Soc. Ferment. Technol., Japan, p. 147, 1980) i-amyl-
acetate is mainly synthesized from acetyl-CoA and i-
amylalcohol by alcohol-acetyltransferase (AATFase) in
yeasts. Soy sauce is also a complexly-fermented product,
in which, nearly 300 kinds of flavor components have so
far been detected (3). In soy sauce, however, i-amylacetate
is usually not detected, or it is contained only at a very low
level, even though the precursor, i-amylalcohol, is present
in a certain amount (4). The reason for the absence or the
very low concentration of i-amylacetate in soy sauce has not
been explained clearly. In sake or beer fermentation, the
esterase of the yeast is thought to be a factor in decompos-
ing esters. Yanagiuchi et al. (5) measured the esterase activ-
ities of the yeasts and koji used for sake production, and
showed that the yeast esterase could decompose flavor
esters while koji esterase was inactive in the condition of
sake fermentation.
In this paper, investigations into the factors concerned
in ester-formation and decomposition in soy sauce fermen-
tation are described. The soy yeasts and koji culture were
found to have strong esterase activities, and this feature
could be responsible for the decomposition of the flavor-
esters in the process of soy sauce brewing.
MATERIALS AND METHODS
Organisms and culture conditions Aspergillus sojae
2048 (6) and Aspergillus oryzae 1299 (koji mold), Pediococ-
cus halophilus 7117 (7) (lactic acid bacteria for soy sauce),
Zygosaccharomyces rouxii NISL 3355 (8) (a salt-tolerant,
soy yeast), Candida versatilis NISL 3722 (8) (a salt-
tolerant, 4-ethyl guaiacol producing yeast), Pichia
farinosa AHU 4134 (8) (a salt-tolerant, pellicle-producing
yeast), and wine yeasts (WE 432, ATCC 4098, IAM 4274
* Corresponding author.
163
(9)) were stock cultures in this laboratory. Brewer's yeast
(IFO 1167), distillery yeast (IFO 0216), and baker's yeast
(IFO 0555) were obtained from the Institute for Fermenta-
tion, Osaka (IFO). YM medium [0.5% Polypepton, 0.3%
yeast extract, 0.3% malt extract and 1.0% glucose
(pH 5.2)], YM medium supplemented with 10% NaC1, and
YPG medium (7) were used for salt-intolerant yeasts, salt-
tolerant yeasts, and lactic acid bacteria, respectively. They
were cultured statically at 30°C in test tubes containing
5 ml of medium or flasks containing 100 ml medium.
Koji and moromi (soy sauce mash) were prepared ac-
cording to the method described by Sekine and Sigeta (10).
E n z y m e assays Alcohol-acetyltransferase (AAT-
Fase) was assayed with permeabilized cells of the yeast
(11). The reaction mixture contained 1 ml of 2.4mM
acetyl-CoA, 1 ml of 0.1% i-amylalcohoi and 1 ml of
permeabilized cell suspension. All were dissolved or
suspended in 0.1 M phosphate buffer (pH 7.0). In a 5 ml
test tube with a screw cap, the reaction mixture was incu-
bated with gentle shaking for 60 min at 25°C. Then, synthe-
sized i-amylacetate (i-AmAc) was extracted and measured
gas-chromatographically as described below. Esterase ac-
tivity was assayed by a modified method of Toshimitsu et
al. (12) with p-nitrophenylacetate (p-NPA activity) or a-
naphthylacetate (a-NA activity) as the substrates. Reac-
tion mixture containing 0.5 ml of sample solution or
suspension, 3.5 ml of 0.1 M phosphate buffer (pH 7.0) and
0.5 ml of 1 mM substrate dissolved in 1/°o' methanol were
incubated for 30 min at 30°C, then if needed, cells were
eliminated by centrifugation at 2,000 g for 5 min. In the
case of a-NA substrate, reaction mixtures were mixed with
0.5 ml of Fast Blue B reagent which consisted of 5 parts of
1°/oo Fast Blue B salt in 100mM phosphate buffer (pH 7.0)
and 2 parts of 5°//00sodium dodecylsulfate, and centrifuged
at 2,000 g for 5 min if necessary. Liberated p-nitrophenol
and a-naphtol were measured with the absorption at
400 nm and 600 nm, respectively. Esterase activity toward
i-AmAc (i-AmAc activity) was assayed as follows. The re-
action mixture containing 1 ml of 0.1% i-AmAc, i ml of
0.1 M phosphate buffer (pH 7.0), and 1 ml of sample solu-
tion or suspension was incubated for 60min at 30°C,
then liberated i-amylalcohol was analyzed gas-chromato-
164 HIGUCHI ET AL.
graphically. Esterase activities toward higher-alcohol
esters other than i - A m A c were assayed in the same way.
The reverse reaction o f esterase was assayed in 100 ml o f
m o r o m i juice (MJ (13)) or p h o s p h a t e buffer ( 1 0 0 m M ,
p H 5 . 0 ) containing 0.1% i-amylalcohol, 0.5°/00 acetate,
15%o NaC1 and yeast suspension (final concentration 107
cells/ml). The reaction mixture was incubated for 24 h at
30°C, centrifuged at 1,500g for 10min to eliminate cells
and then the i - A m A c synthesized was measured.
The esterase activity in m o r o m i was assayed with i-
A m A c as the substrate. The reaction mixture contained
20 g of m o r o m i and 50 pl o f i - A m A c . F o r an estimation o f
exclusive yeast esterase of k o j i esterase, l07 cells/ml (final
concentration) o f the yeast was added to a moromi-sample
after heat-treatment at 100°C for 60 min. The mixture was
incubated for 24 h at 30°C, mixed thoroughly with the
internal standard, and liberated i-amylalcohol was meas-
ured.
Protease was assayed by the azocasein method (14).
Time course of esterase activity in m o r o m i Moromi
was sampled each month, filtered through filter paper,
then 0.1 ml o f i - A m A c was added to 3 ml o f the filtrate.
Liberated i-amylalcohol was measured gas-chromato-
graphically.
Extraction, precipitation and gel filtration of esterases
from k o j i One kg o f k o j i cultured with A . sojae 2048
was mixed with 1.5 / o f distilled water and held at r o o m
temperature for 6 0 m i n . The mixture was filtered first
through cloth, then through a S t a n d a r d Super Cell bed,
and Millipore filter (pore size 0 . 2 2 p m ) . To the filtrate,
8 0 ~ saturated (NH4)2SO 4 was added. The precipitate was
collected by centrifugation at 12,000g for 15 min, then
solved in 50 ml of the buffer (pH 7.0, 50 m M sodium phos-
phate). Gel filtration was p e r f o r m e d by H P L C equipped
with a TSKgel 3000SW column. The applied sample
volume was 13.5 ml. The flow rate was 9 m l / m i n . The elu-
tion buffer contained 50 mM sodium p h o s p h a t e (pH 7.0)
and 0.3 M NaCl. A f t e r 38.5 min from the start o f the elu-
tion, 27 fractions were collected every 6 min.
Extraction of volatile materials Higher alcohols and
their ethyl esters were analyzed by a modified method o f
Y o k o t s u k a el al. (15). To 3 ml of sample, 1 g o f NaC1 and
100pl o f the internal standard containing 1 m M o-xylenol
and 1 m M d i m e t h y l m a l o n a t e in 50%o ethanol, were added.
It was extracted with 1 ml o f methylacetate by liquid-liquid
o o.,os
Soy Yeast NISL 3355
(Zggosaccharomgces rouxii)
Brewer's Yeast IF0 1167
Distillery Yeast IFO 0216
Sake Yeast Kyokai no. 7
Baker's Yeast IFO 0555
Wine Yeast IAM 4274
Wine Yeast ATCC 4098
Wine Yeast WE 432
FIG. 1. Esterase activities of several fermentation yeasts. All yeasts were incubated in 5 ml of YM medium (without NaCI) for 2 d. Yeasts
were collected and assayed as described in the text.
J. FERMENT.B1OENG.,
extraction in a test tube with a screw cap for 20 min at
30°C by the use of a continuous shaker (Thermomixer
TM 121). The methylacetate layer separated above the aque-
ous solution by centrifugation at 1,500g for 10 min was
collected. If necessary, the extracted solution was concen-
trated into about 30 pl by the blowing o f N 2 before analy-
sis by g a s - c h r o m a t o g r a p h y . Volatiles formed in the reverse
reaction o f esterase were extracted with fleon-I 1 for 20 h
and then concentrated to 3 0 p l at 45°C according to the
method o f Rapp (16). The concentrated sample was ana-
lyzed by gas-chromatography. Volatiles in m o r o m i were
directly extracted with methylacetate. Twenty g of a
moromi-sample was mixed thoroughly with 10 ml of dis-
tilled water, 0.5 ml o f the internal standard and 5 ml o f
methylacetate. The extraction mixture was stirred by a
voltex mixer at full speed for 30 s and let stand statically
for 30s. This operation was repeated three times. The
methylacetate layer was then separated by centrifugation
at 3,000g for 10min.
Analysis by gas-chromatography Gas-chromatogra-
phy o f methylacetate-extracts was performed with a Shi-
madzu GC-14A g a s - c h r o m a t o g r a p h equipped with an F I D
and a silica capillary column (0.25 m m i.d. × 15 m length)
coated with PEG20M; the oven temperature was pro-
g r a m m e d as follows: 50°C for 10rain, 50-220°C at
4 ° C / r a i n and then 220°C for 7.5 min; inlet temperature,
240°C; carrier gas, N2 (1 m l / m i n ) , split rate, I : 30. Gas-
c h r o m a t o g r a p h y o f the extracts was performed according
to the method o f A o k i et al. (8).
Other methods Cell numbers o f yeasts and bacteria
were determined by a haematometer.
Protein concentration was determined by the method o f
Lowry (17) with bovine serum albumin as a standard.
RESULTS
Esterase activities of organisms occurring in m o r o m i
First, A A T F a s e activities o f a soy yeast, Z. rouxii
NISL3355, and o f a sake yeast, S. cerevisiae Kyokai-no. 7
were measured to estimate the ester synthesizing activity.
Appreciable A A T F a s e activity was detected in S. cere-
visiae Kyokai-no. 7 as reported, but not in Z. rouxii
NISL3355.
To examine the possibility that esters formed by A A T -
Fase are decomposed by any esterases co-existing with
Activity (nmol/min/107cells)
o.lo o.ls 0.,85 0.90
VOL. 71, 1991
A A T F a s e , i - A m A c splitting activities of various yeasts
were measured. The activity in Z. rouxii N I S L 3355 was
more than 10 times that f o r m e d in S. cerevisiae Kyokai-no.
7 or in other yeasts for fermented beverages (Fig. 1). The
esterase activities o f several yeasts concerned in moromi
(soy sauce mash) fermentation were also measured (Table
1). C. versatilis N I S L 3722 and P. farinosa A H U 4134 had
esterase activities as high as Z. rouxii N I S L 3355. The re-
sult showed that these yeasts might decompose volatile
esters in soy sauce fermentation.
Koji molds (A. sojae and A. oryzae) and lactic acid bac-
terium (P. halophilus) are also concerned in soy sauce
fermentation in a d d i t i o n to the yeasts. The esterase activ-
ities o f koji, l m o n t h aged moromi (just before the addi-
tion o f yeast) and lactic acid bacteria (P. halophilus 7117)
are shown in Table 1. High esterase activities were found
in both the koji and moromi, but not in cells of P.
halophilus. To m a k e moromi, koji is mixed with nearly
twice its weight o f saline. Usually, about 1 month after the
mixing, yeasts are added. At the m a x i m u m growth, the
moromi contains 107 cells/ml o f yeasts and 2 × 108 cells/ml
o f lactic acid bacteria. The activity in 1/3 g koji was higher
than that o f 107 yeast cells, and even at the stage just be-
fore the a d d i t i o n o f yeast the esterase activity in 1 g o f
moromi was higher than that o f 107 yeast cells. The activity
o f 2 × 108 cells o f P . halophilus was less than 1% of the ac-
tivity o f 107 cells o f the yeast. These results indicated that
both the yeasts and koji molds had high esterase activities,
and these enzymes might decompose esters under the condi-
tions o f moromi fermentation.
The ester-synthesizing activities o f these esterases of the
yeast and in moromijuice were also determined in the pres-
ence o f excessive concentrations o f the substrates (0.5%0
acetate and 0 . 1 % i-amylalcohol) c o m p a r e d with those in
soy sauce. Esterase in the moromi juice is thought to be
derived from koji, since the juice was prepared from
young-stage moromi before the addition o f yeast. Only a
negligible a m o u n t o f i - A m A c , 1.8 and 1.1 p p m for the
yeast suspension and the moromi juice respectively, was
synthesized t h r o u g h the reverse reaction o f esterases. Thus
the equilibrium is thought to decline towards hydrolysis o f
i - A m A c in the moromi conditions.
Properties of yeast esterase Some properties o f the
yeast esterase were examined with intact yeast cells. As the
yeast esterase was not secreted into the m e d i u m (data not
shown), it is thought to act intracellularly in moromi.
Activities to decompose several volatile esters are shown
TABLE 1. Esterase activities of microorganisms concerned in
soy sauce fermentation
Organisms Activity
(nmol/min/107 cells)
Z. rouxii NISL 3355 2.3
C. versatilis IFO 10038 2.0
P. farinosa AHU 4134 3.6
Pedio. halophilus 7117 ~0.01
Activity (nmol/min/g kojl)
A. sojae 2048 (koji) 262.4
Activity (nmol/min/ml moromi juice)
A. sojae 2048 (moroml) 6.1
A. oryzae 1299 (moromO 10.4
Koji was extracted by 10 times the amounts of water for 1 h.
Moromi was extracted just before the addition of yeast (aged for
1 month). Yeasts and bacteria were incubated in 100 ml of medium
for 3d.
ESTERASES IN SOY SAUCE MOROMI 165
TABLE 2. Decomposing activities of soy yeast and koji esterases
towards volatile esters
Relative activities (~)a
Substrates
Yeast Koji
i-Amylacetate 100 100
i-Butylacetate 88 89
fl-Phenethylacetate 74 72
Ethylcaproate ll7 8
a The activity for i-amylacetate is shown as 1009/00.
in Table 2. The yeast esterase split i-butylacetate, ]L
phenethylacetate and ethylcaproate as well as i-AmAc. Soy
sauce moromi is fermented at a relatively low p H (about
p H 5) and a high NaC1 concentration (about 17%), so the
effects o f these factors on the esterase were examined. The
yeast esterase activity was not so seriously affected by pH
or NaC1 concentration (data not shown).
Gel filtration of kofi esterases It is generally known
that esterase activity is composed o f several enzymes. The
extract o f koji was c h r o m a t o g r a p h i c a l l y fractionated to
separate the esterases involved and to determine the en-
zyme responsible for the decomposition o f volatile esters.
Distilled water extract o f koji was precipitated by 80%
saturated (NH4)2SO4, followed by fractionation by gel
filtration with TSKgel G3000SW. I - A m A c - , p - N P A - , tr-
N A - activities and protease activity were measured on each
fraction. The results are shown in Fig. 2-a, b. One i - A m A c
activity peak, one a - N A activity peak, two p - N P A peaks
and two protease peaks are apparent. The two p - N P A
peaks a p p e a r e d in the same fractions as the i - A m A c peak
and a - N A peak. Both the protease peaks were in fractions
different from those o f the esterase described above. It is
assumed that the esterase activity in koji was based on at
least two enzymes which had different substrate-speci-
ficities.
We obtained a fraction that could decompose i-AmAc,
and examined the properties of the koji esterase with this
fraction.
Properties of koji esterase The activities o f the i-
A m A c - d e c o m p o s i n g fraction on several volatile esters are
shown in Table 2. It split i-butylacetate and fl-phen-
ethylacetate as well as i-AmAc. The koji esterase also
split ethylcaproate but its activity was only 6°//oo o f the i-
A m A c decomposition. The activities o f the a - N A - d e c o m -
posing fraction in the koji extract on these esters were less
than 5% o f each o f the i - A m A c - d e c o m p o s i n g fractions.
Hence, most o f the volatile esters were thought to be
decomposed by the i - A m A c - d e c o m p o s i n g activity.
The effects o f p H and NaC1 concentration on the
esterase were examined. Esterase activity was found to de-
crease with a decrease o f p H or increase of NaC1 concentra-
tion. The koji esterase showed the m a x i m u m activity at
p H 7-9 and the activity at p H 5 was about 109/o o f the max-
imum. The activity at 17°//00 NaCl was about 1/3 of that
TABLE 3. Comparison of koji and soy yeast esterase under
moromi conditions (pH 5, NaCI 17%)
Activity
Origin (nmol/h/ml)
Koji (Moromi just before yeast addition) 20.5
Yeast (Heated rnoromi+ 107 yeast cells) 9.6
Control (Heated moromi only) 0.0
166 H1GUCHI ET AL. J. FERMENT.BIOENG.,

r-4
E
r~ (a) m I00~
i00

20 e ~ 801~
r~ ~ 6o
o >,

>~ J
>
.r4
>' 40 \
>
50
> i0 ~ 20 "
.,-4 <
4J Z <
i

1 2 3 4 5 6
Time (month)
Z
I
-,-t
I
FIG. 3. Time course of the esterase activity in moromi through
aging.
(b)
0.4 300~e
Ob the highest at the start of the moromi period, after which it
O decreased gradually. However, even after 5 months of ag-
ing, the moromi was able to decompose 7 ppm of i-AmAc
O
200'~ within 24 h. This suggests that if some volatile esters are
lq
produced at any stage of soy sauce fermentation, such
esters will finally be decomposed during the long aging
4
0.2 4J

period with the remaining esterase activity.

: ' I00
c~ o c
©©- .~
© content of volatile esters in soy sauce, LAmAc-synthe-
5 i0 15 20 25
Fraction number
FIG. 2. Gel filtration of the koji esterase on a TSKgel 3000SW
column by HPLC. Symbols: (a) e , i-AmAc activity; • ,p-NPA ac-
tivity; A, a-NA activity. (b) [], protease activity; O, protein concen-
tration. Koji cultured with A. sojae 2048.
without NaC1 (data not shown).
C o m p a r i s o n of kofi a n d y e a s t esterases in moromi
simulated conditions Fermenting moromi contains
both esterases originated from koji mold and yeast. To esti-
mate the actual contributions of each esterase to the decom-
position of volatile esters in rnoromi, their i-AmAc activ-
ities were measured separately in conditions simulated to
those of fermenting mororni. Mororni just before the addi-
tion of yeast was used as a source of the koji esterase
devoid of yeast esterase. The moromi was heated at 100°C
for 1 h to inactivate the esterases originating from the koji
mold, and then 107 cells/ml of yeast cells cultured sepa-
rately were added. This was used as the sample for the
yeast esterase. The heat-treated moromi without the yeast
addition was used as the control. The pH of all these sam-
ples was adjusted to 5.0.
The results are shown in Table 3. Both the yeast and the
koji-mold esterases decomposed i-AmAc, whereas the con-
trol did not. The koji-mold esterase was found to be more
active than the yeast one. Thus the esterase originating
from the koji-mold contributed much more than the yeast
esterase to the decomposition of volatile esters.
T i m e c o u r s e o f esterase activity in m o r o m i a g i n g
Usually, soy sauce moromi is held for more than half a
year for aging. The time course of esterase activity in
moromi during aging is shown in Fig. 3. The activity was
DISCUSSION
in order to clarify the reason for the unexpectedly small
sizing and decomposing activities in soy sauce yeast, Z.
rouxii, were measured. I-AmAc-synthesizing activity in Z.
rouxii could not be detected, contrary to the result in S.
cerevisiae. I-AmAc-decomposing activity in Z. rouxii was
more than 10times that in S. cerevisiae. Therefore, i-
A m A c might be decomposed immediately, even if it is syn-
thesized. Whether i-amylacetate-synthesizing activity was
too weak to be detected, or the ester was once synthesized
but decomposed immediately, is u n k n o w n , and it would
be difficult to determine.
The esterase activity originating from koji-mold was
higher than that due to Z. rouxii under the same condi-
tions as moromi, while the activity of the lactic acid bacte-
rium was negligible.
In the case of sake manufacturing, it was reported (5)
that the effect of the koji esterase was very small and the
main i-AmAc-decomposing factor was the yeast esterase,
although its activity was fairly weak compared with the
esterase in soy yeasts. The pH of sake moromi is around 4,
while that of soy sauce moromi is about 5. According to
the pH dependence curve of the soy sauce koji esterase, the
activity partially remained at pH 5 but not at pH 4. Thus
the difference in moromi pH between sake and soy sauce is
thought to be one of the factors that affects the contribu-
tion of koji esterases during their moromi fermentation.
Soy sauce koji is well k n o w n for its strong proteases
(18). The possibility that these proteases decompose the
esters could be feasible. Gel filtration of the koji extract
showed an i-AmAc decomposing peak, an a - N A decom-
posing peak and two protease peaks. The esterases and pro-
teases were eluted separately from each other. Thus, these
two proteases were thought not to be concerned in the
esterase activity. However, as it is k n o w n that at least
seven different proteases are present in soy koji (18), it can
VOL. 71, 1991
n o t be c o m p l e t e l y e l i m i n a t e d that the esters were d e c o m -
p o s e d by a p r o t e a s e u n d e t e c t a b l e in o u r e x p e r i m e n t .
T h e results o f gel filtration also i n d i c a t e d t h a t there were
t w o esterases s h o w i n g different substrate-specificities. It
has b e e n r e p o r t e d with f i l a m e n t o u s f u n g i (19), yeast (20)
a n d h i g h e r a n i m a l s (21, 22) that one o r g a n i s m h a d m o r e
t h a n t w o esterases. In the case o f sake koji, K u r i y a m a et
al. (19) f r a c t i o n a t e d k o j i esterases i n t o ones t h a t c o u l d
d e c o m p o s e e t h y l c a p r o a t e or p - N P A .
R e c e n t l y , several studies h a v e been u n d e r t a k e n with the
aim o f f o r t i f y i n g A A T F a s e or d i m i n i s h i n g the esterase o f
yeast f o r m a n u f a c t u r i n g ester-enriched sake (23, 24). In
the case o f soy sauce, h o w e v e r , it is t h o u g h t difficult to
m o d i f y the v o l a t i l e flavor by the s a m e a p p r o a c h since
s t r o n g esterase activity exists in the m o r o m i t h r o u g h o u t
the f e r m e n t a t i o n process. T h u s , in o r d e r to enrich flavor-
esters in soy sauce, it m i g h t be necessary to d i m i n i s h b o t h
the yeast a n d k o j i esterases s i m u l t a n e o u s l y .
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