Food Sci. Technol. Res.

, 9 (3), 237241, 2003

Preparation of Low Salt Miso-Like Fermented Seasonings Using Soy-Oncom and

Okara-Oncom (Fermented Soybeans and Okara with Neurospora intermedia) and Their
Antioxidant Activity and Antimutagenicity

Masako MATSUO and Tokuo TAKEUCHI

Faculty of Home Science, Gifu Womens University, 80 Taromaru, Gifu 501-2592, Japan

Received July 29, 2002; Accepted April 9, 2003

To develop a new type of Miso, low salt miso-like fermented seasonings were prepared with soybean Oncom(S-
Oncom) and Okara Oncom (O-Oncom): those were fermented soybeans and fermented Okara with Neurospora inter-
media, respectively. Their antioxidant activity and antimutagenicity were analyzed and both Oncom had higher
-glu-
cosidase activity than Koji (cooked rice molded with Aspergillus oryzae) with or without the presence of 4% salt and
2% ethanol. The strongest 1,1-diphenyl-2-picryl-hydrazyl radical scavenging, superoxide anion scavenging and anti-
mutagenic activities were detected in the extracts prepared from the seasonings with S-Oncom, O-Oncom and Koji
with both 70% ethanol and with water. These activities of O-seasoning are attributable to the isoflavone aglycones
present and to water soluble substances. Based on these results, O-seasoning is deemed to be healthier than other low
salt miso-like fermented seasoning prepared with steamed soybeans and Koji. Furthermore, the dishes prepared using
O-seasoning were evaluated as acceptable.

Keywords: low salt miso-like seasoning, antimutagenicity, superoxide scavenging, Oncom, Neurospora intermedia

Miso has been found to be useful in suppressing hypertension
(Kanda et al., 1999), colonic aberrant crypt foci (Ohara et al.,
2002), cerebrovascular diseases (Kanazawa et al., 1995) and ele-
vation of plasma cholesterol (Horii et al., 1990), and has been
used for several centuries in Japan, where it is known to contrib-
ute to good health. However, the consumption of Miso in Japan
is currently decreasing due to the popularization of western food
and also because of its high salt content. In Indonesia, many
kinds of Oncom, a traditional unsalted food fermented from
pressed peanut cake or Okara (the solid waste of soybean milk
production) by Neurospora intermedia, have been developed for
consumption (Sastraatmadja et al., 2002). Neurospora belongs to
a different subdivision (mycotina) than Aspergillus, the fungus
used in fermentation of Miso; specifically, Neurospora belongs
to the subdivision Ascomycotina while Aspergillus belongs to
Deuteromycotina. If these fungi were used together in ferment-
ing Miso, its physiological functions might be improved via a
collaborative effect of these fungi. The purpose of this study was
to develop new directions for Miso by fermenting three kinds of
low salt miso-like seasoning using soybeans, soybean Oncom (S-
Oncom) and Okara Oncom (O-Oncom). These three seasonings
were then judged for their sensory effects as seasoning, and their
functions as antioxidants and antimutagens were examined.
Materials and Methods
Preparation of O-Oncom Okara powder (30 mesh) was
provided by Misuzu Co. (Nagano, Japan). O-Oncom was pre-
E-mail: matsuo@gijodai.ac.jp
Abbreviations: S-Oncom, soybean-Oncom; O-Oncom, Okara-Oncom; C-sea-
soning, Control seasoning; S-seasoning, seasoning used S-Oncom; O-sea-
soning, seasoning used O-Oncom; O2
, superoxide anion; DPPH, 1,1-di-
phenyl-2-picryl-hydrazyl; IC50, fifty percent inhibition.
pared by the following method described by Matsuo (1997a):
Okara containing 60% moisture was autoclaved for 20 min under
steam pressure at 0.18 Mpa, and pressed at 1.1 g/cm2 to a thick-
ness of 2.5 cm. Oncom starter containing spores of Neurospora
intermedia FGSC 2559 (Fungal Genetics Stock Center, Kansas
City, KS) was then inoculated to the Okara surface to give a final
concentration of 0.3%. The inoculated Okara was incubated at
30C for 18 h tightly covered with plastic sheeting. After the
spores had germinated, the Okara was fermented uncovered at
27C for another 19 h under 65% relative humidity.
Preparation of S-Oncom S-Oncom was prepared by the
method described above with the exception that dehulled four-
cut soybeans (Fukuyutaka, Gifu, 2000) were used instead of
Okara.
Preparation of miso-like seasonings Koji (cooked rice
molded with Aspergillus oryzae) was provided by Marusanai
Co., Ltd. (Aichi, Japan). Control seasoning (C-seasoning) con-
sisting of 1 kg steamed soybeans and 0.5 kg Koji were mixed
well, and salt and ethanol were added to the mixture at the final
concentration of 4% and 2%, respectively. Then the mixture was
fermented at 30C. S-Oncom seasoning (S-seasoning) and O-
Oncom seasoning (O-seasoning) were prepared following essen-
tially the same method except that the steamed soybeans were
replaced with S-Oncom or the mixture of 90% S-Oncom and
10% O-Oncom, respectively. During fermenting, pH, titratable
acidity, reducing sugars and formol nitrogen of each seasoning
were periodically monitored. The fermenting period until these
values reached a constant level was 6 weeks for C-seasoning and
5 weeks for S-seasoning and O-seasoning.
Component analysis of the seasonings Titratable acidity,
formol nitrogen, soluble nitrogen and reducing sugars were de-
termined by the standard analytical methods for Miso as de-
238
scribed by the Ministry of Agriculture and Forestry (1977). Pro-
tein solubility and protein hydrolysis were calculated individual-
ly from the percentage of soluble nitrogen and formol nitrogen to
the total nitrogen, respectively. Organic acids were analyzed
using a Finepak SIL-5 column (4.5250 nm, Jasco Co., Tokyo)
and 0.5% phosphoric acid as a mobile phase with monitoring by
absorbance at 210 nm (Tachi et al., 1993). Ethanol, acetaldehyde
and ethyl acetate were analyzed by a PEG 6000 glass column
(25% 545U, 6080 mesh, 3 mm2 m, GLC Science, Tokyo) at
80C and detected by FID (GC-15A PFsc, Shimadzu, Tokyo)
(Honma et al., 1973). Color was defined according to the Hunter
color scale using a color difference meter (ND-1000 DP, Nippon
Densyoku, Tokyo), and hardness was measured by a creep meter
(RE-3305, Yamaden Co., Ltd., Tokyo) at compressibility 30%
and speed 5 mm/s.
Enzyme activities The supernatants of 20% homogenate
of Koji, S-Oncom and O-Oncom were dialyzed against water for
use as the respective enzyme solution. The activities of protease
(E.C. 3.4.21.14), leucine aminopeptidase (E.C. 3.4. 11.1) and
glutaminase (E.C. 3.5.1.35) were assayed by the methods for soy
source (Nakadai, 1985), and lipase (E.C. 3.1.1.3) activity was
assayed by following the procedure described by the Worthing-
ton enzyme manual (Lilian, 1977). For -glucosidase (EC
3.2.1.21), we followed the method described by Iwashita et al.
(1998). Specifically, the mixture of 0.1 M acetate buffer (pH 4.5)
0.25 ml, 8 mM p-nitrophenyl -glucoside 0.25 ml and enzyme
solution 0.25 ml was incubated at 40C for 30 min, and then the
reaction was stopped by addition of 0.1 M Na2CO3 2.5 ml.
Enzyme activity was calculated from the increase of absorption
which developed by p-nitrophenol at 400 nm. One unit (U) of -
glucosidase activity was defined as the amount of -glucosidase
which produced 1 mol of p-nitrophenol per min in the reaction
mixture.
Preparation of extract of a seasoning For unheated
extract, the lyophilized seasoning was shaken with 5 volumes of
n-hexane for one night at room temperature. The residue was
shaken with 5 volumes of 70% ethanol, and the residue of 70%
ethanol extract was subsequently shaken with 5 volumes of water
at room temperature. The seventy percent ethanol extract and the
water extract were individually concentrated. For heated extract,
the lyophilized seasoning was shaken with the same kinds of sol-
vents as the unheated extract for 12 h at 60C, and each extract
was individually concentrated.
Pretreatment of miso-like seasoning before cooking The
seasonings were heated for 10 min at 80C to inactivate their
enzymes produced by A. oryzae and N. intermedia, followed by
homogenization by a grinder mill (Millser 300DG, Iwatani Inter-
national Corp., Osaka).
Recipe for custard cream cooked with miso-like season-
ing Twenty grams egg yolk and 10 g miso-like seasoning were
blended together, and 50 g sugar was added. The mixture was
beaten until smooth, and 13 g sifted soft flour was added, 180 ml
milk was added gradually, and the mixture was boiled gently for
1 min.
Recipe for cupcakes cooked with miso-like seasoning One
gram of baking powder was added to 50 g sifted soft flour. Fifty
grams unsalted butter and 20 g miso-like seasoning were
creamed until soft. The butter mixture was mixed with 40 g sugar
until it was light and fluffy, and then blended with 50 g whole
M. MATSUO & T. TAKEUCHI
egg and beaten well. The butter and flour mixtures were beaten
together until smooth, poured into fluted aluminum baking cups,
and baked in a moderate oven at 135C for 33 min.
Recipe for sesame miso sauce cooked with miso-like sea-
soning Sixty grams miso-like seasoning, 25 g ground sesame
seeds and 30 g sugar were beaten together until smooth, and then
mixed well with 22 ml water. The mixture was brought to a boil
to melt the sugar.
Recipe for oden miso sauce cooked with miso-like season-
ing One hundred grams miso-like seasoning, 30 ml sake, 20 g
honey, 20 g sugar and 5 ml water were beaten together until
smooth, and the mixture was boiled for 1 min.
Sensory evaluation In the sensory analysis of the un-
heated miso-like seasoning, 24 female students (2122 years
old) were asked to judge specified items. For the test of the
dishes cooked with miso-like seasoning, 28 female students (21
22 years old) were asked to evaluate organoleptically using a 5-
point rating scale: good (2), somewhat tasty (1), neither good
nor poor (0), rather poor (
1), poor (
2).
The data from the sensory test on the unheated miso-like sea-
soning were analyzed by the paired preference test (Roessler et
al., 1978) and the data from the taste tests of the dishes were ana-
lyzed by the Tukeys multiple range test using the statistical anal-
ysis system SPSS. Other data were expressed as the means
standard error. The differences between the values were analyzed
by Tukeys multiple range test
Antioxidant activity and 1,1-diphenyl-2-picryl-hydrazyl
(DPPH) radical scavenging activity The seasoning extract (0.3
ml) was added to a mixture of 0.1 M acetate buffer (pH 5.5, 0.3
ml) and 0.2 mM DPPH in ethanol, and the decrease in absor-
bance at 517 nm for 1 min was measured at room temperature.
Fifty percent inhibitory concentration (IC50) was assayed as the
weight of the seasoning to scavenge 50% DPPH radical.
Superoxide anion (O2
) scavenging activity Following the
method described by Oyanagi (1984), a mixture of 65 mM phos-
phate-borate buffer (pH 8.2, 0.1 ml), 0.5 mM xanthine (0.1 ml),
10 mM hydroxylamine (0.05 ml), the seasoning extract, and xan-
thine oxidase (EC 1.2.3.2, Wako Pure Chemical Industries, Ltd.,
Osaka) diluted 100-fold with 50 l water was incubated for 30
min at 37C. Naphthylethylenediamine reagent (1 ml) was added
to the mixture, and the absorbance at 550 nm was measured.
Antimutagenicity Antimutagenicity against N-nitrosodim-
ethylamine (NDMA) was assayed by a modified Ames test using
Salmonella typhimurium TA 100 in the presence of S9 pretreated
with acetone and fasting (Ebata & Furukawa, 1997). The His
revertant colonies were counted after incubation for 48 h at 37C,
and the suppression ratio was calculated as follows: 1
(rever-
tants/test plate
spontaneous mutants/test plate)/(revertants/con-
trol plate
spontaneous mutants/control plate)100. The data
presented in Table 7 show the meansstandard error of three
measurements.
Isoflavone analysis The extract from the seasoning with
70% methanol was analyzed by HPLC with Crestpack C18S (Jas-
co Co. Tokyo) using a linear gradient from 10% to 60% metha-
nol containing 4% acetate as a mobile phase and detected at 262
nm (Matsuo, 1997b).
Results and Discussion
Chemical components of steamed soybeans, S-Oncom and
Preparation of Low Salt Miso-Like Fermented Seasonings 239

O-Oncom The chemical components of steamed soybeans, S- soft, and the high activities of xylanase and cellulase of N. inter-
Oncom and O-Oncom were compared and the results of this media (Matsuo, 1997a) are assumed to have contributed to the
comparison are shown in Table 1. The protein content of S- hydrolysis of the soy cell wall. The chemical components of S-
Oncom was higher than that of the steamed soybeans. The pro- seasoning were intermediate between O-seasoning and those of
tein content of O-seasoning, in which 10% O-Oncom was substi- C-seasoning.
tuted for S-Oncom was higher than that of C-seasoning.
Enzyme activities of Koji, S-Oncom and O-Oncom Four
percent salt and 2% ethanol were the minimum amounts essen-
tial for fermentation of Miso (Okada & Takeuchi, 1977). Prior to Table 3. Physical and chemical characteristics of the miso-like seasonings.
the preparation of the miso-like seasoning, the enzyme activities Miso-like seasoning
Item
of Koji, S-Oncom, O-Oncom, as well as their tolerance to 4% C-Seasoning S-Seasoning O-Seasoning
salt and 2% ethanol were compared (Table 2). The lipase and - Water (%) 52.9 52.0 51.8
glucosidase activities of O-Oncom and S-Oncom were higher Salt (%) 3.8 3.7 3.8
pH 5.2 5.8 5.7
than those of Koji, and this ranking did not change under the Titratable acidity I (ml) 11.3 9.1 8.9
presence of 4% salt and 2% ethanol. Soy isoflavone glycosides Titratable acidity II (ml) 11.0 13.2 12.8
are hydrolyzed to aglycones by -glucosidase, and the aglycones Total sugar (%) 16.9 17.5 18.0
Reducing sugar (%) 12.1 13.4 14.5
show higher antioxidant activity than that of glycosides (Hsieh & Sugar hydrolysis (%) 71.9 76.3 81.0
Graham., 2001). If S-seasoning is fermented with the substitution Total nitrogen (%) 1.9 2.1 2.0
of S-Oncom for soybeans, the antioxidant activity of the season- Soluble nitrogen (%) 1.1 1.1 1.2
Formol nitrogen (%) 0.5 0.6 0.7
ing is higher than that of C-seasoning. The -glucosidase activity Protein solubility (%) 56.7 53.0 56.4
of O-Oncom was the greatest among the three kinds of fer- Protein hydrolysis (%) 22.6 27.5 32.5
mented products. The antioxidant activity of O-seasoning, which Ethanol (%) 2.38 1.93 1.57
Acetaldehyde (mg%) 10 19 20
is fermented with a partial substitution of O-Oncom for S- Ethyl acetate (mg%) 7 9 9
Oncom, is stronger than that of S-seasoning . Citric acid (mg%) 107 142 135
Lactic acid (mg%) 112 93 91
Components of seasoning Three kinds of low salt miso- Acetic acid (mg%) 223 230 219
like seasoning were prepared, and their components were com- Pyroglutamic acid (mg%) 29 36 34
pared (Table 3). The titratable acidity of O-seasoning was low Color L 35.2 24.3 26.3
a 9.5 10.9 10.8
and the amount of lactate production was little. The hydrolysis b 16.5 12.6 14.0
ratio of both protein and saccharide was high, and the presence Hardness (103 N/m2) 12.2 4.3 4.0
of enzymes of N. intermedia can be attributed to this hydrolysis
during both the preparation of O-Oncom and the fermentation of
O-seasoning. During the fermentation, the ethanol level de-
creased, but the levels of acetaldehyde and ethylacetate in-
creased. The color was slightly reddish and dark, indicating that a Table 4. Sensory analysis of the unheated miso-like seasonings.
Maillard reaction with reducing sugars and formol nitrogen Responder number
Question Items
might occur during the fermentation. Hardness was extremely S-Seasoning O-Seasoning
Texture 5 19*
Color 16 8
Which do you like? Gloss 5 19*
Table 1. Chemical components of steamed soybean, S-Oncom and O- Flavor 16 8
Oncom. Taste 19* 5
Total evaluation 18* 6
Components Steamed soybean S-Oncom O-Oncom
Sweet 15 9
Moisture 63.5 59.8 50.9 Salty 10 14
Crude protein 13.8 17.4 13.1 Which do you feel strongly? Sour 8 16
Crude fat 7.0 9.0 4.5 Bitter 7 17*
Carbohydrate 13.9 11.8 29.7 Umami 19* 5
Ash 1.8 2.0 1.8 Twenty-four female students (2122 years old) were asked to judge speci-
Total 100.0 100.0 100.0 fied items. *p0.05.

Table 2. Effect of 4% salt and 2% ethanol on the enzyme activities of Koji, S-Oncom and O-Oncom.
Salt Ethanol Enzyme activity (U/g) Enzyme activity (%)
Enzymes
(%) (%) Koji S-Oncom O-Oncom Koji S-Oncom O-Oncom
Protease 0 0 65.2 19.7 35.7 100 30 55
4 2 45.4 15.5 9.5 70 24 15
Leucine aminopeptidase 0 0 4.2 2.1 3.0 100 50 71
4 2 2.3 1.4 0.0 54 33 0
Glutaminase 0 0 0.15 0.15 0.30 100 100 200
2 2 0.05 0.17 0.23 30 110 150
Lipase 0 0 0.087 0.131 0.357 100 151 410
4 2 0.011 0.025 0.032 12 29 37
-Glucosidase 0 0 113 3000 7750 100 266 688
4 2 87 2650 7420 77 235 658
240 M. MATSUO & T. TAKEUCHI

Table 5. Sensory evaluation scores of the dishes prepared with the miso-like seasonings.
Western style dishes Japanese style dishes
Evaluation scores Custard cream Cupcake Sesame-Miso sauce Oden-Miso sauce
C-Seasoning O-Seasoning C-Seasoning O-Seasoning C-Seasoning O-Seasoning C-Seasoning O-Seasoning
Total 33 26 43 30 50 45 50 48
Mean 1.2 0.9 1.5 1.1 1.8 1.6 1.8 1.7
Twenty-eight female students were asked to evaluate organoleptically using a 5-point rating scale: good(2); somewhat tasty(1), neither good nor poor(0);
rather poor(
1), poor(
2).

Table 6. DPPH-IC50 and O2
-IC50 of the miso-like seasonings extract with 70% ethanol and water.
DPPH-IC50a) O2
-IC50b)
Extracts (mg seasoning/ml) Extracts (mg seasoning/ml)
Miso-like seasoning
70 % Ethanol Water 70% Ethanol Water
Unheated Heatedc) Unheated Heatedc) Unheated Heatedc) Unheated Heatedc)
C-Seasoning 16.2 5.9 263 140 31.1 45.8 70.0 31.1
S-Seasoning 11.6 5.5 91 61 29.7 35.7 33.1 14.4
O-Seasoning 7.9 5.0 81 55 17.1 28.5 25.3 11.3
a)
The fifty percent inhibitory concentration of 1,1-diphenyl-2-picryl-hydrazyl radical and superoxide anion.
b)
The fifty percent inhibitory concentration of superoxide anion.
c)
The defatted lyophilized seasoning was shaken with 5 volumes of 70% ethanol and water for 12 h at 60C.

Table 7. Antimutagenicity of the miso-like seasonings extract with 70% ethanol and water.
70% Ethanol extract Water extract
Miso-like seasoning Unheated Heated Unheated Heated
(2 mg seasoning/plate) (2 mg seasoning/plate) (4 mg seasoning/plate) (4mg seasoning/plate)
C-Seasoning 22.92.0b 25.62.4b 38.30.2a 23.35.0b
S-Seasoning 35.62.0a 29.92.0b 36.31.2ab 26.37.0ab
O-Seasoning 40.02.0a 40.72.0a 48.62.0a 30.12.6a
Data were expressed as meansSE (n3). Means having the same superscripts in each row do not differ significantly (p0.05).

Sensory character of unheated seasoning The character Table 8. Isoflavone content of the miso-like seasoning.
of unheated S-seasoning and O-seasoning was compared by a Isoflavones Miso-like seasoning
sensory test (Table 4). O-seasoning was preferable to S-season- (mg%) C-Seasoning S-Seasoning O-Seasoning
ing in texture and gloss, and was judged to be slightly more bitter Daidzin 4.30 1.7 N.D.
and to have less flavor than S-seasoning. Genistin 2.03 0.5 N.D.
Daidzein 3.93 6.3 8.7
Sensory evaluation of dishes cooked with miso-like Genisrein 4.93 8.6 15.0
seasoning A sensory evaluation test of the dishes cooked with The extract from the seasoning with 70% ethanol was analyzed by HPLC
the miso-like seasoning was performed and the results are shown with Crestpak C18S using a linear gradient from 10% to 60% methanol con-
taining 4% acetate and detected at 262 nm. N.D., not detected.
in Table 5. All evaluation scores on both western and Japanese
dishes were positive, namely all dishes cooked with O-seasoning
were evaluated as acceptable. No significant difference was de-
tected between the scores of O-seasoning and C-seasoning. It is
therefore expected that O-seasoning would be popular as a useful enging activities of both DPPH radical and O2
increased nota-
seasoning for both western and Japanese dishes. bly. These antioxidant activities of O-seasoning were highest in
Antioxidant activity Since miso generally shows high the 3 kinds of seasonings. The browning substances produced by
antioxidant activity, the antioxidant activities of the miso-like Maillard reaction, melanoidines, showed strong antioxidant
seasonings were compared according to their DPPH-IC50 and activity (Yamaguchi & Fujimaki, 1973). The increase of these
O2
-IC50 (Table 6). In both extracts with 70% ethanol and water, scavenging activities in water extract from the seasonings is
the scavenging activities of DPPH radical and O2
were highest caused by melanoidines produced during heating.
for O-seasoning, followed by S-seasoning, and finally C-season- Antimutagenicity The antimutagenicity of Miso has been
ing. O-Oncom used as a submaterial of the seasoning remarkably previously reported (Kiyosawa et al., 1995), and the antimutage-
improved the antioxidant activities of S-seasoning. The antioxi- nicity of these seasonings was measured for comparison (Table
dant activities of extracts from these seasonings with 70% etha- 7). All extracts of these seasonings showed high antimutagenici-
nol were stronger than those with water. Heating the water ty, and the activity of the 70% ethanol extracts in particular was
extracts from these seasonings caused them to darken, and scav- higher than that of water extracts. All antimutagenicity measure-
Preparation of Low Salt Miso-Like Fermented Seasonings
ments of O-seasoning were stronger than those from other sea-
sonings. During heating, the antimutagenicity of the 70% ethanol
extracts did not change, but that of the water extracts decreased.
Some melanoidines have been reported to be antimutagens
(Powrie et al., 1986), however, we do not consider the water sol-
uble antimutagens of the miso-like seasoning to be melanoidines.
Isoflavone component Soy isoflavones are soluble in 70%
ethanol, and the antioxidant activity of their aglycones is stronger
than that of glycosides (Pratt & Birac, 1979). Daizein and
genistein are potent antimutagens of Miso (Kiyosawa et al.,
1995). Isoflavones in the miso-like seasonings were analyzed and
the results are shown in Table 8. 6-O-malonyl--glucoside, the
main isoflavone of soybeans (Toda et al., 2000), was not detected
in these seasonings at all. O-seasoning was apparently rich in
isoflavone aglycones due to the high activity of -glucosidase of
O-Oncom (Table 2). The O2
scavenging activity and anti-
mutagenicity of O-seasoning (Tables 6 and 7) may be contribut-
ed collaboratively by the isoflavone aglycones and some water
soluble substances of O-Oncom. Based on these results, we con-
sider O-seasoning to be more beneficial for health than C-sea-
soning.
Acknowledgments This work was supported in part by a grant from the
Salt Science Research Foundation, Japan. We thank Ms. Hisayo Murase,
Ms. Shiori Yamauchi, Ms. Mayuko Hiraide, Ms. Yuki Nakahashi, Ms. Aya
Onda and Ms. Naho Yokochi for their valuable technical assistance.
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