International Journal of Food Microbiology 118 (2007) 62 – 68

www.elsevier.com/locate/ijfoodmicro

Mutagenic and antimutagenic effects of methanol extracts of unfermented

and fermented black soybeans
Yu-Hsiang Hung a , Hui-Yu Huang b , Cheng-Chun Chou a,⁎
a
Graduate Institute of Food Science and Technology, National Taiwan University, 59, lane 144, Keelung Rd., Sec. 4, Taipei, Taiwan
b
Department of Food Science, Nutrition & Nutraceutical Biotechnology, Shih Chien University, Campus No.70 Ta-Chih Street, Chung-Shan District, Taipei, Taiwan
Received 8 December 2006; received in revised form 1 June 2007; accepted 1 June 2007

Abstract

In this study, solid fermentation of steamed black soybean with various GRAS (Generally recognized as safe) filamentious-fungi including
Aspergillus awamori, Aspergillus oryzae BCRC 30222, Aspergillus sojae BCRC 30103, Rhizopus azygosporus BCRC 31158 and Rhizopus
sp. No. 2 was performed. Mutagenicity and antimutagenicity of the methanol extracts of unfermented and fermented steamed black soybeans
against 4-nitroquinoline-N-oxide (4-NQO), a direct mutagen and Benzo[a]pyrene (B[a]P), an indirect mutagen, on Salmonella Typhimurium
TA100 and TA 98, were examined. The methanol extracts of unfermented and fermented steamed black soybeans show no mutagenic activity for
either test strains at the doses tested. The extracts inhibited mutagenesis by either 4-NQO or B[a]P in S. Typhimurium TA100 and TA98.
Fermentation with fungi also enhanced the antimutagenic effect of black soybean while the antimutagenic effect of the fermented black soybeans
extract varied with the starter organism, mutagen, and test strain of S. Typhimurium examined. Generally, the extracts of A. awamori-fermented
black soybean exhibited the highest antimutagenic effect. With strain TA100, the inhibitory effects of 5.0 mg of A. awamori-fermented black
soybean extract per plate on the mutagenic effects of 4-NQO and B[a]P were 92% and 89%, respectively, while the corresponding rates for extract
of unfermented were 41% and 63%, respectively. With strain 98, the inhibition rates were 94 and 81% for the fermented bean extract and 58% and
44% for the unfermented bean extracts. Testing of extracts prepared from black soybean by A. awamori at temperatures 25, 30 and 35 °C and for
times of 1–5 days revealed that, generally, the extract prepared from beans fermented at 30 °C for 3 days exhibited the greatest inhibition against
the mutagenic effects of 4-NQO and B[a]P.
© 2007 Elsevier B.V. All rights reserved.

Keywords: Mutagenicity; Antimutagenicity; Black soybean; Solid fermentation

1. Introduction et al., 2003; Trock et al., 2006). Avoiding exposure to

There is a close correlation between mutagenesis and
carcinogenesis (Maron and Ames, 1983; Ferguson et al.,
2004). Various investigators have attributed the relatively low
rates of breast, prostate and colon cancers in Asian countries
to the consumption of soy, which contains antimutagenic
factors (Adlercreutz, 1990; Barnes et al., 1995; Yamamoto
⁎ Corresponding author. Tel.: +886 2 3366 4111; fax: +886 2 2362 0849.
E-mail address: fstcchou@ntu.edu.tw (C.-C. Chou).
0168-1605/$ - see front matter © 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.ijfoodmicro.2007.06.005
mutagens and eating an adequate supply of nutritious foods
containing antimutagens may then reduce the rate of mutation
and incidence of cancer in humans (Ribeiro and Saloadori,
2003; Ferguson et al., 2004).
Black soybeans (Glycine max (L.) Merr.) are a nutritionally
rich foodstuff. The seed coats of black soybeans contain
anthocyanin and so they are darker than the seed coats of other
strains of other soybean (Choung et al., 2001). They also
contain isoflavone, vitamin E, soaponin and anthocyanin
which have shown to exert biological activity (Rao and Sung,
1995; Miyazawa et al., 1999; Cardador-Martinez et al., 2002;
Aparicio-Fernández et al., 2005). In China, black soybeans
fermented by filamentous fungi are further processed to make
 Y.-H. Hung et al. / International Journal of Food Microbiology 118 (2007) 62–68
traditional fermented condiments such as In-yu black sauce
and In-si or Ttou-si, the dried by-product of black soybean
sauce (Su, 1980). The beneficial effects of the black soybean
was described in Ben-Tsao Gong Mu, an ancient Chinese
Botanical Encyclopedia, dating back to the early 16th century
(Li, 1990).
Recently, black soybeans have been reported to inhibit low
density lipoprotein (LDL) oxidation (Takahashi et al., 2005) and
to effectively reduce the incidence of DNA damage by cyclo-
phosphamide (Ribeiro and Saloadori, 2003). In addition,
combining the Rhizopus azygosporus-fermented black soybean
with rice has also been suggested as a way to develop a
nutritious weaning food. (Rodriguez-Bűrger et al., 1998).
In an attempt to develop healthy food possessing functional
properties, a series of studies on black soybean has been con-
ducted in our laboratory. We noted that black soybean possessed
antioxidative activity, including α-α-diphenyl-2-picyl-hydoxyl
radical-scavenging effect, Fe2+-chelating ability, and reducing
activity, which was enhanced by fermentation with fungi (Lee
et al., 2007). Additionally, fermentation was also noted to increase
the content of aglycone, the bioactive isoflavone (Lee and Chou,
2006).
To further evaluate those qualities, the mutagenic and
antimutagenic effects of the methanol extracts of black soybean
and fermented black soybean on the mutagenicity of 4-
nitroquinoline-N-oxide (4-NQO) and benzo[a]pyrene (B[a]P)
in S. Typhimurium were studied.
2. Materials and methods
2.1. Bacterial strains and chemicals
The test strains of S. Typhimurium were TA98 and TA100.
The filamentous fungi Aspergillus oryzae BCRC 30222, As-
pergillus sojae BCRC 30103, Aspergillus awamori, R.
azygospous 31158 and Rhizopus sp. No. 2, which are
commonly used as starter organisms for the preparation of
traditional, oriental fermented food products, were used to
ferment cooked black soybeans. All the test organisms were
obtained from the Bioresources Collection and Research
Center (BCRC), Hsinchu, Taiwan except A. awamori and
Rhizopus sp. No. 2, which were provided by Professor Yu,
Graduate Institute of Food Science and Technology, National
Taiwan University. Strain markers and bacterial survival were
routinely monitored for each experiment (Maron and Ames,
1983).
2.2. Mutagen and S9 mix preparations
4-NQO and B[a]P were obtained from Sigma-Aldrich Co.
(St. Louis, MI, USA). Both mutagens were dissolved in
dimethylsulfoxide (DMSO, Wako Pure Chemical Industries,
Ltd., Osaka, Japan) at concentrations of 0.5 and 20 μg/ml,
for 4-NQO and B[a]P, respectively. Rat liver-S9 homogenate
treated with Aroclor 1254 was purchased from MP Bio-
medicals, Inc. (Solon, Ohio, USA). S9 mix (S9 fraction of
liver homogenate with cofactors) was prepared according
63
to the method of Maron and Ames (1983) and used for
metabolic activation of B[a]P.
2.3. Solid fermentation of black soybean
Black soybeans obtained from a local market, were first
steam-cooked at 121 °C, for 15 min in an autoclave. Solid state
fermentations of the steamed black soybeans was then
performed at 30 °C and 95% RH for a period of 3 days.
When effects of fermentation temperature and time were
examined, the steamed black soybeans were fermented by A.
awamori at 25, 30, and 35 °C for a period of 0–5 days. The
fermentation procedures have been described (Lee and Chou,
2006).
2.4. Preparation of methanol extracts
The unfermented and fermented steamed black soybeans, were
dried at 60 °C for 24 h, then they were ground to 30-mesh
powders screen using a grinder (Model HF-365; Shivn Feng
Enterprise Co., Ltd., Taipei, Taiwan). The powders were extracted
with methanol (1:10, w/v) by refluxing at about 25 °C for 24 h
with gentle shaking. After filtering through Whatman No. 1 filter
paper, the extracts were vacuum concentrated and dried using a
freeze-dryer (Mode 77500-00 M; Labconco Co., Kansas,
Missouri, USA).
2.5. Assay for mutagenic and antimutagenic effect
The pre-incubation method of Maron and Ames (1983)
with minor modification was employed to study the mutagenic
effect of the extracts. Briefly, 0.1 ml of a 10 to 11 h culture of
S. Typhimurium TA 100 or TA98 was added to 0.1 ml DMSO
containing 0 to 5.0 mg of an extract and 0.5 ml PBS or S9 mix.
The entire mixture was incubated at 37 °C in a rotary shaker for
20 min. After incubation, 2.0 ml top agar was added to and
virtexed with the tube contents. The mixture was poured onto a
plate of minimal glucose agar and colonies were counted after
incubation for 48 h at 37 °C. It was found that revertant
appeared in plates containing extract came close to that found in
plate without extract in the preliminary study. Therefore, the
doses of extracts tested were not toxic to S. Typhimurium
(Waleh et al., 1982).
The antimutagenic effects of the extracts were examined
under the same conditions as were used for mutagenicity testing
except with the addition of 0.1 ml mutagen to each test system
before incubation at 37 °C.
Each assay was performed in triplicate, and antimutagenic
activity was expressed as a percentage of mutagenic inhibition
using the formula:
Inhibitionð%Þ ¼ 1  ½ðA  CÞ=ðB  CÞ  100;
where A and B are the numbers of mutagen-induced revertants
in the presence and absence, respectively, of an extract, and C is
the number of spontaneous revertants.
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Y.-H. Hung et al. / International Journal of Food Microbiology 118 (2007) 62–68
Table 1
Effects of the unfermented and fermented black bean extracts against the mutagenic effects of 4-NQO and B[a]P on S. Typhimurium TA100
Mutagen Amounts of Unfermented black soybean Extracts of black soybean fermented with
extracts extracts
A. awamori A. sojae A .oryzae R. azygospous Rhizopus sp. No.2.
(mg/plate)
Revertants Inhibition Revertants Inhibition Revertants Inhibition Revertants Inhibition Revertants Inhibition Revertants Inhibition
(CFU/plate) a rate(%) b (CFU/plate) rate(%) (CFU/plate) rate(%) (CFU/plate) rate(%) (CFU/plate) rate(%) (CFU/plate) rate(%)
4-NQO
None 549 ± 96 549 ± 96 549 ± 96 549 ± 96 549 ± 96 549 ± 96
5 D388 ± 15 41d D189 ± 4 92a D229 ± 9 82b C247 ± 6 77b C333 ± 10 55c D230 ± 10 82b
2.5 C461 ± 5 23d C242 ± 6 79a C261 ± 8 74a B338 ± 20 54b B358 ± 10 49c C313 ± 8 61b
1.25 B506 ± 13 11e B462 ± 20 22d B281 ± 8 69a A381 ± 14 43b A425 ± 3 32c B363 ± 8 48b
0.625 A541 ± 10 2d A587 ± 7 17c A309 ± 6 62a A405 ± 4 37b A413 ± 4 35b A424 ± 8 32b

B[a]P
None 271 ± 12 271 ± 12 271 ± 12 271 ± 12 271 ± 12 271 ± 12
5 C180 ± 13 63b C143 ± 6 89a C146 ± 10 87a C155 ± 6 81a C146 ± 9 87a D149 ± 9 85a
2.5 C191 ± 14 55d B154 ± 4 81a C152 ± 9 82a C163 ± 9 75b C152 ± 9 83a C178 ± 11 65c
1.25 B229 ± 23 29e A165 ± 5 74a B174 ± 20 67b B178 ± 12 65b B198 ± 12 51c B214 ± 11 40d
0.625 A264 ± 12 5e A179 ± 9 64a A249 ± 7 16d A214 ± 12 39b A256 ± 8 10d A241 ± 13 21c
a
Data were expressed as means ± SD (CFU/plate) of three separate experiments. Statistical differences were calculated by Duncan's multiple range test. Value in the same row with different lower case letters (a, b, c, d)
and column with upper case letters (A, B, C, D) are significantly different (p b 0.05).
b
Inhibition rate (%) = [1 − number of induced revertants in the presence of extract of unfermented black soybean or fermented black soybean / number of induced revertants in the absence of extract of unfermented
black soybean or fermented black soybean] × 100. Numbers of spontaneous revertants were 159 ± 9 and 127 ± 2, respectively, when studies on 4-NQO and B[a]P were performed.
 Y.-H. Hung et al. / International Journal of Food Microbiology 118 (2007) 62–68
Table 2
Effects of the unfermented and fermented black bean extracts against the mutagenic effects of 4-NQO and B[a]P on S. Typhimurium TA98
Mutagen Amounts Unfermented black soybean Extracts of black soybean prepared with
of extracts extracts
A. awamori A. sojae A .oryzae R. azygospous Rhizopus sp. No.2.
(mg/plate)
Revertants Inhibition Revertants Inhibition Revertants Inhibition Revertants Inhibition Revertants Inhibition Revertants Inhibition
(CFU/plate) a rate(%) b (CFU/plate) rate(%) (CFU/plate) rate(%) (CFU/plate) rate(%) (CFU/plate) rate(%) (CFU/plate) rate(%)
4-NQO
None 98 ± 8 98 ± 8 98 ± 8 98 ± 8 98 ± 8 98 ± 8
5 C53 ± 6 58d D26 ± 2 94a C27 ± 3 92a D51 ± 3 61c D55 ± 3 56d C41 ± 4 74b
2.5 BC57 ± 3 54b C58 ± 3 52b B33 ± 2 85a C61 ± 2 48c C66 ± 3 42c B55 ± 4 56b
1.25 B63 ± 3 45b B74 ± 6 32c B36 ± 2 81a B72 ± 2 34c B74 ± 3 31c A71 ± 3 35c
0.625 A76 ± 5 28b A91 ± 4 10d A45 ± 3 69a A84 ± 2 18c A83 ± 4 19c A76 ± 4 28b

B[a]P
None 80 ± 7 80 ± 7 80 ± 7 80 ± 7 80 ± 7 80 ± 7
5 C55 ± 6 44c C34 ± 6 81a C35 ± 6 80a C32 ± 6 84a C33 ± 4 82a C44 ± 6 64b
2.5 B63 ± 4 30d B42 ± 3 67a B44 ± 5 64a B42 ± 3 67a B49 ± 5 54b B56 ± 6 42c
1.25 B67 ± 4 23c A46 ± 3 59a A56 ± 9 43b B49 ± 3 54a B58 ± 7 39b A66 ± 7 25c
0.625 A75 ± 6 9e A53 ± 5 47a A69 ± 3 19d A61 ± 8 34b A68 ± 3 22c A77 ± 8 6e
a
Data were expressed as means ± SD (CFU/plate) of three separate experiments. Statistical differences were calculated by Duncan's multiple range test. Value in the same row with different lower case letters (a, b, c, d)
and column with upper case letters (A, B, C, D) are significantly different (p b 0.05).
b
Inhibition rate (%) = [1 − number of induced revertants in the presence of extract of unfermented black soybean or fermented black soybean / number of induced revertants in the absence of extract of unfermented
black soybean or fermented black soybean] × 100. Numbers of spontaneous revertants were 21 ± 1 and 23 ± 2, respectively, when studies on 4-NQO and B[a]P were performed.

65
66 Y.-H. Hung et al. / International Journal of Food Microbiology 118 (2007) 62–68
2.6. Statistical analysis
The mean values and the standard deviation were calculated
from the data obtained from three separate experiments. Means
were compared using Duncan's multiple range test method in
SAS, version 8 (SAS Institute, Gary, NC, USA).
3. Results and discussion
3.1. Mutagenic activities of black soybean extracts
It was found that the test concentrations (0.625–5.0 mg/per
plate of the extracts) were not mutagenic for strains TA100 and
TA98, since the number of revertants colonies obtained with or
without extracts in the system were similar (data not shown).
These results are in agreement with reports of Chou et al. (2002)
and Lin and Chou (2006) who found that ethanol extract of red
bean fermented with A. oryzae or methanol extracts of soybean
fermented with various fungi did not show mutagenic activity.
3.2. Antimutagenicity against 4-NQO and B[a]P in S. Typhimur-
ium TA 100
Table 1 shows the inhibitory effect of various extracts against
the mutagenic effects of 4-NQO and B[a]P on S. Typhimurium
TA100 and TA 98. 4-NQO exerts potent intracellular oxidative
stress, and its metabolic product binds to DNA predominantly at
guanine residues (Kanojia and Vaidya, 2006). It shows a high
potential ability to reduce oxidative stress, which contributes to
tumor promotion (Nunoshiba and Demple, 1993). Previously, a
variety of dietary substances such as soybean (Lin and Chou,
2006), lactic acid bacteria and bifidobacteria-fermented milk
(Hsieh and Chou, 2006), yoghurt (Nadathur et al., 1995),
doenjang, a traditional Korean fermented soypaste (Park et al.,
2003) were found to suppress on the mutagenesis induced by 4-
NQO. On the other hand, B[a]P is a polycyclic hydrocarbon.
Being an indirect mutagen, it undergoes metabolic change that
leads to its activation as a reactive benzo[a]pyrene dihydridial
exposide, which is an electrophilic species capable of binding to
DNA, RNA and some other macromolecules (Josephy et al.,
1992).
Extracts of unfermented and fermented steamed black
soybeans inhibited the mutagenic effects of 4-NQO and B[a]P
on S. Typhimurium TA100 (Table 1). Compounds such as
vitamin E, saponin, phytic acid, linoleic acid, genistein and
daizein, commonly found in bean and bean products, have been
reported to exhibit antimutagenic activity (Tavan et al., 1997;
Miyazawa et al., 1999; Park et al., 2003). Additionally, antho-
cyanin and phenolic compounds of common black bean
(Phaseolus vulgaris) were also found to exert antimutagenic
effect (Cardador-Martinez et al., 2002; Aparicio-Fernández
et al., 2005). Therefore, Such materials may all contribute to the
observed antimutagenic effect of the soybean extracts. The
antimutagenic effect of the extracts increased as dosage
increased. At all the doses tested, fermented black soybean
extracts showed higher antimutagenic effects than those extracts
of unfermented soybeans.
3.3. Antimutagenicity against 4-NQO and B[a]P in S. Thphimur-
ium TA 98
The extracts also exhibited a dose-dependent antimutagenic
activity against the mutagens tested on S. Typhimurium TA98
(Table 2). However, the inhibition exerted by the extract with
same dose level for S. Typhimurium TA98 (Table 2) was not
exactly the same as that for TA100 (Table 1). These results show
that the antimutagenic effect exerted by the extract can vary
with strain of S. Typhimurium used for testing.
The greater antimutagenic effects obtained by fermentation
of beans, as found in the present study, is in accordance with
observations of other investigators. Among those, Park et al.
(2003) found that doenjan, the Korean fermented soy paste,
exerted higher antimutagenic activity than raw soybean; Hsieh
and Chou (2006) reported that fermentation with bifidobacteria
and lactic acid bacteria, alone or together, significantly
enhanced the antimutagenic activity of soy milk; and Lin and
Chou (2006) reported findings for fermented soybeans similar
to the findings of this study.
The flavonid glycosides are less antimutagenic than the
corresponding aglycones such as daidzein, glycitein and
genistein (Edenharder and Tang, 1997). The fungi used as the
starter organisms in the present study were capable of producing
β-glucosidase, which promotes cleavage of the β-glycosyl bond
in the black soybean glucoside isoflavones to form aglycones
(Lee and Chou, 2006; McCue and Shetty, 2003). It was found
that the fermentation of black soybean with the starter organisms
examined caused a marked increase in the content of aglycone
and total anthocyanin (Hung, 2006; Lee and Chou, 2006).
Therefore, these changes may all lead to the enhanced
antimutagenicity activity observed with the extracts of fermented
black soybean. However, formation of other bioactive com-
pounds and increased linoleic acid contents in bean substrates
following fermentation, as reported by Park et al. (2003), may
also contribute to the enhanced antimutagenicity.
Table 3
Inhibition of the mutagenic effects of 4-NQO and B[a]P on S. Typhimurium by
the methanol extracts of fermented black soybean (5 mg/plate) fermented by A.
awamori at different temperatures for 3 days
Fermentation Inhibition rate(%) a
temperature
S. Typhimurium TA98 S. Typhimurium TA100
(°C)
4-NQO B[a]P 4-NQO B[a]P
Not fermented 58C b 44D 41C 63C
25 °C 92A 62B 85B 87A
30 °C 94A 81A 92A 89A
35 °C 72B 51C 80B 78B
a
Inhibition rate (%) = [1 − number of induced revertants in the presence of
extract of black soybean or fermented black soybean / number of induced
revertants in the absence of extract of black soybean or fermented black
soybean] × 100. Data are expressed as means of three separate experiments.
b
Statistical differences were calculated by Duncan's multiple range test.
Value in the same row for the same test strain of S. Typhimurium with different
upper case letters A, B, C, D are significantly different (p b 0.05).
 Y.-H. Hung et al. / International Journal of Food Microbiology 118 (2007) 62–68
Table 4
Inhibition of the mutagenic effects of 4-NQO and B[a]P on S. Typhimurium by
the methanol extract of fermented black soybean (5 mg/plate) fermented by A.
awamori during fermentation at 30 °C
Fermentation Inhibition rate(%) a
time (days)
S. Typhimurium TA98 S. Typhimurium TA100
4-NQO B[a]P 4-NQO B[a]P
0 58D b 44C 41C 63C
1 63C 38C 74B 69B
2 67C 60B 92A 77B
3 94A 81A 92A 89A
4 68C 70A 93A 83A
5 74B 65B 95A 85A
a
Inhibition rate (%) = [1 − number of induced revertants in the presence of
extract of black soybean or fermented black soybean / number of induced
revertants in the absence of extract of black soybean or fermented black
soybean] × 100. Data are expressed as means of three separate experiments.
b
Statistical differences were calculated by Duncan's multiple range test.
Value in the same row for the same test strain of S. Typhimurium with different
upper case letters A, B, C, D are significantly different (p b 0.05).
3.4. Fermentation temperature and time affect the antimutageni-
city of fermented black soybean extract
Previously, fermentation temperature was reported to affect the
antioxidative activity of the methanol extract of the fermented
black soybean (Lee et al., 2007). As shown in Table 3, the
A. awamori-fermented black soybean extract exhibited various
degrees of inhibition of the mutagenic effects of 4-NQO or B[a]P
on the both strains of S Typhimurium depending on fermentation
temperatures. Generally, the methanol extract of fermented black
soybean fermented at 30 °C showed the greatest inhibition of
mutation by of 4-NQO and B[a]P, regardless of the test organism.
Table 4 shows the effect of fermentation time on the
antimutagenic activity of the A. awamori-fermented black
soybean extracts, In general, the antimutagenic activity of the
fermented black soybean extract increased as the fermentation
time was extended from 1 to 3 days at 30 °C. Extending the
fermentation time to 5 days did not significantly (p N 0.05)
increase the antimutagenic effects of the black soybean extract for
S. Typhimurium TA 100, while these extracts had a decreased
antimutagenic effects for S. Typhimurium TA 98.
The relatively high antimutagenicity activities noted in the
fermented black soybean extracts is generally associated with the
higher contents of total phenolics, anthocyanin and aglycones in
fermented black soybean (Lee and Chou, 2006; Lee et al., 2007).
However, it is also possible that formation of other antimutagenic
metabolite (Park et al., 2003) as influenced by fermentation
condition occurs during the fermentation process. They may all
contribute to observed antimutagenicity of the fermented black
soybean extract and merit further investigation.
The results of the present study show that fermentation with an
appropriate starter organism and under appropriate cultivation
conditions may relatively enhance the antimutagenic activity of
black soybeans. Information provided in this study, along with
knowledge of the enhanced content of aglycone, the bioactive
isoflavone (Lee and Chou, 2006) not only support the suggestion
of using, further provide a better cultivation condition to prepare
67
the fermented black soybean as an ingredient for the formulation
of healthy food.
Acknowledgement
This research was financially supported by The National
Science Council, ROC (Taiwan). (NSC 95-2313-B-002-017).
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