LWT - Food Science and Technology 42 (2009) 56–62

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LWT - Food Science and Technology

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Antimutagenic activity of Aspergillus awamori-fermented black

soybean response to simulated digestive juice treatments
and its antimutagenic mechanisms
Yu-Hsiang Hung, Yen-Ju Wang, Cheng-Chun Chou*
Graduate Institute of Food Science & Technology, National Taiwan University, 59, Lane 144, Keelung Road, Sec. 4, Taipei, Taiwan

a r t i c l e i n f o a b s t r a c t

Article history: Aspergillus awamori-fermented black soybeans, a potential healthy food ingredient, possess anti-
Received 28 January 2008 mutagenicity against 4-nitroquinoline-N-oxide (4-NQO); a direct mutagen and benzo[a]pyrene (B[a]P;
Received in revised form 2 June 2008 an indirect mutagen). Results of this study revealed that antimutagenic, desmutagenic, and blocking
Accepted 2 June 2008
effects all contributed to the antimutagenicity of the methanol of fermented black soybean. However, the
desmutagenic effect of the fermented black soybean extract on B[a]P is not caused by the interaction of
Keywords:
intact B[a]P or S9 mixture with the extract. Instead, it is due to the interaction of B[a]P metabolites with
Antimutagenicity
the antimutagenic factors in the extract. Exposure to simulated gastric juice (pH 1.2) for 30 min or in-
Fermented black soybean
Mechanism
testinal juice for 2 h resulted in a reduced antimutagenicity of fermented black soybean extract. Nev-
Simulated digested treatment ertheless, the simulated gastric juice treated-fermented black soybean extracts and the simulated
intestinal juice treated-fermented black soybean extracts still possessed a considerable antimutagenic
effect against 4-NQO and B[a]P.
Ó 2008 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved.

1. Introduction
The black soybean (Glycine max (L.) Merr.) is a nutritionally rich
food. Certain components of black soybeans such as isoflavone,
sopponin, anthocyanin, and vitamin E have been shown to exert
biological activity (Aparicio-Fernández, Manzo-Bonilla, & Loarca-
Pina, 2005; Cardador-Martinez, Castano-Tostado, & Loarca-Pina,
2002; Miyazawa, Sakano, Nakamura, & Kosaka, 1999; Rao & Sung,
1995). In China, black soybeans were used to prepare traditional
fermented products such as in-yu black soybeans, and in-si or tou
si, the dried by-product of the mash black soybean sauce. The
beneficial effects of black soybean were described in Ben-Tsao Gong
Mu, an ancient Chinese Botanical Encyclopedia as early as the 16th
century (Li, 1990).
Recently, researchers have also demonstrated that black
soybeans inhibit low density lipoprotein oxidation (Takahashi et al.,
2005) and reduce the incidence of DNA damage by cyclophospha-
mide (Ribeiro & Salvadori, 2003). Moreover, using Rhizopus
azygosporus-fermented black soybean with rice has also been
suggested as a way to develop a nutritious weaning food (Rodri-
guez-Bu} rger, Mason, & Nielsen, 1998).
* Corresponding author. Tel.: þ886 2 3366 4111; fax: þ886 2 2362 0849.
E-mail address: fstcchou@ntu.edu.tw (C.-C. Chou).
In the past few years, a series of studies of black soybean have
been conducted in our laboratory in an attempt to develop healthy
food that possesses beneficial properties. It was found that
fermentation by filamentous fungi enhanced the antioxidative
activity (Lee, Hung, & Chou, 2007) and the content of the bioactive
isoflavone, aglycone, which has been linked to health benefits and
well being (Lee & Chou, 2006). Additionally, fermentation was also
found to enhance the antimutagenic effect of black soybeans
against 4-NQO and B[a]P (Hung, Huang, & Chou, 2007). Further, it
was also noted that the antimutagenicity of the fermented black
soybean varied with the starter organism, mutagen, and the test
strain of Salmonella typhimurium. These observations further
support the suggestion of using fermented black soybean as an
ingredient for the formulation of healthy food.
Dietary components, after ingestion, are exposure to gastric and
intestinal juices. The chemical structure and properties of the
dietary components may change in the presence of gastric juice,
with its low pH, bile acid, and digestive enzymes, thus altering their
functional properties. For example, it has been observed that the
antimutagenicity of onion changes after exposure to stimulated
gastric juice (pH 1.2) or intestinal juice, with increases or decreases
in antimutagenicity depending on the kinds of onion, mutagen and
the test strain of S. typhimurium. Lo, Yu, Chou, and Tsai (2002) also
reported that the antimutagenic activity of probiotics changed after
bile and acid treatments. Moreover, the exposure of lactic
fermented soymilk to acid (pH 2.0) or bile salt has been reported to

0023-6438/$34.00 Ó 2008 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.lwt.2008.06.001
 Y.-H. Hung et al. / LWT - Food Science and Technology 42 (2009) 56–62
reduce antimutagenicity (Hsieh & Chou, 2006). Additionally, Vis,
Plinck, Alink, and van Boekel (1998) observed that bile acid was
a strong comutagenic to N-methyl-N-nitro-N-nitrosoguanidine
(MNNG). In the present study, the possible mechanism of the
antimutagenic effect of methanol extract of the Aspergillus
awamori-fermented black soybean, which exhibited the most
significant enhancement in antimutagenicity compared to the
unfermented black soybean (Hung et al., 2007), was further
explored. Finally, the antimutagenicity of the fermented black
soybean extract in the presence of simulated gastric and intestinal
juices was also examined.
2. Materials and methods
2.1. Microorganisms and chemicals
In the present study, S. typhimurium TA98 and TA100, obtained
from the Bioresources Collection and Research Center (BCRC),
Hsinchu, Taiwan, were used as the test organisms. S. typhimurium
TA100 results from the substitution of a leucine (GAG/CTC) by
a proline (GGG/CCC). Mutagens that cause base-pair substitution
mutations primarily at one of the GC pairs will revert this mutant to
the wild-type state. In contrast to S. typhimurium TA100, S. typhi-
murium TA98 is a 1 frameshift mutation which affects the reading
frame of a nearby repetitive –C–G–C–G–C–G–C–G– sequence
(Mortelmans & Zeiger, 2000). A. awamori was provided by Professor
Roch-Chui Yu, Graduate Institute of Food Science and Technology,
National Taiwan University. S. typhimurium strain markers and
bacterial survival were routinely monitored for each experiment
(Maron & Ames, 1983).
4-NQO and B[a]P were obtained from Sigma-Aldrich Co. (St.
Louis, MI, USA). Both mutagens were dissolved in dimethylsulf-
oxide (DMSO, Wako Pure Chemical Industries, Ltd., Osaka, Japan) at
concentrations of 0.5 and 20 mg/ml, for 4-NQO and B[a]P,
respectively. Rat liver-S9 homogenate treated with Aroclor 1254
was purchased from MP Biomedicals, Inc. (Solon, OH, USA). S9 mix
(S9 fraction of liver homogenate with cofactors) was prepared
according to the method of Maron and Ames (1983) and used for
metabolic activation of B[a]P.
2.2. Preparation of fermented black soybean and methanol extract
Procedures described by Lee and Chou (2006) were followed to
prepare the fermented black soybean and its methanol extract.
Briefly, a solid state fermentation of the steamed black soybeans
with A. awamori at 30  C and 95% RH for a period of 3 days was
performed to obtain the fermented black soybeans.
To prepare the methanol extract, the fermented steamed black
soybeans, after drying at 60  C for 24 h, were ground to 30-mesh
powders through a screen using a grinder (Model HF-365; Shivn
Feng Enterprise Co., Ltd., Taipei, Taiwan). The powders were then
extracted with methanol (1:10, w/v) by refluxing at ca 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, MO, USA).
2.3. Treatments of fermented black soybean extract
with simulated gastric and intestinal juices
Preparations of simulated gastric and intestinal juice (USP) and
the digested fermented black soybean extracts were performed
according to those described by Shon, Choi, Kahng, Nam, and Sung
(2004). The simulated gastric fluid consisted of 3.2 g of pepsin
(Sigma-Aldrich Co., St. Louis, MI, USA) and 2.0 g of NaCl, dissolved in
7.0 ml HCl (1 mol equiv/l). The solution was combined with distilled
water to make 1.0 l and the pH adjusted to 1.2. The simulated
57
intestinal fluid consisted of 10 g of pancreatin (Sigma-Aldrich Co.,
St. Louis, MI, USA) dissolved in 400 ml of distilled water and 190 ml
of 0.2 mol equiv/l NaOH. The pH of the intestinal fluid was 7.5.
To prepare the digested fermented black soybean extracts,
a 0.1 ml aliquot of DMSO solution containing various amounts of
fermented black soybean extract was mixed with 0.1 ml of mutagen
and 0.2 ml of simulated gastric juice for 30 min or 0.2 ml intestinal
fluid for 2 h. The digested fermented black soybean extracts were
then analyzed for mutagenic activity.
2.4. Assay for mutagenic and antimutagenic effect
Using S. typhimurium TA98 and TA100 as test strains and with 4-
NQO and B[a]P as mutagens, mutagenic and antimutagenic effects
of fermented black soybean extract dissolved in DMSO were eval-
uated according to the Ames test of Maron and Ames (1983) with
minor modification. Detailed procedures were described in our
previous paper (Hung et al., 2007). This assay system, using Sal-
monella strains with preexisting mutation as test organism, is
employed world-wide as an initial screen to determine the
mutagenic potential of new chemical and drug because there is
a high predictive value for rodent carcinogenicity when a muta-
genic response is obtained (Mortelmans & Zeiger, 2000).
In the preliminary study, the doses of extracts tested (0–5.0 mg
treated- or untreated-fermented black soybean extract/plate) were
found to be non-toxic to S. typhimurium. The antimutagenic effect
of the extracts was expressed as a percentage of mutagenic
inhibition using the formula:
Inhibition ð%Þ ¼ 1  ½ðA  CÞ=ðB  DÞ  100;
where A and B are the number of mutagen-induced revertants
observed in the presence and absence, respectively, of sample, and
C and D are the number of spontaneous revertants observed in the
sample and control, respectively. Furthermore, an extract concen-
tration providing 50% inhibition (IC50) was calculated from the
regression equation of inhibition percentage against extract
concentration.
2.5. Analysis of bio-antimutagenic effect
Methods described by Chen and Yen (1997) and Sato, Ose,
Nagase, and Hayase (1987) were followed to examine the bio-
antimutagenicity of fermented black soybean extract. The over-
night-cultured S. typhimurium TA98 or TA100 (5.0 ml) was washed
twice by means of centrifugation with a cold 1/15 M phosphate
buffer (PB, pH 7.0) at 4  C and re-suspended in cold PB (4.0 ml). This
cell suspension (3.0 ml) was added with 0.5 ml of 4-NQO or 0.5 ml
of B[a]P and incubated at 37  C for 1 h with gentle shaking. In the
case of B[a]P, S9 mix (3.0 ml) was also added. The treated bacteria
were washed twice with centrifugation and re-suspended in cold
PB. The treated S. typhimurium TA100 (0.1 ml) was mixed in a tube
with fermented black soybean extract (0.1 ml, 5.0 mg) or DMSO
(0.1 ml). After 20 min of incubation at 37  C in a rotary shaker, the
molten top agar (2.0 ml) was added. The mixture was poured onto
minimal glucose agar plate. The colonies were counted after 48 h of
incubation at 37  C.
2.6. Analysis of desmutagenic effect
The desmutagenicity of fermented black soybean extract was
investigated by examining the effect of fermented black soybean
extract on intact mutagen, S9 mix and B[a]P metabolites according
to the procedures described by Yen and Hsieh (1994). In these
analyses, DMSO (0.1 ml) containing no fermented black soybean
extract served as control.
58 Y.-H. Hung et al. / LWT - Food Science and Technology 42 (2009) 56–62
To examine the effect of fermented black soybean extract on
intact mutagen, fermented black soybean extract (0.1 ml, 5 mg/
plate) and mutagen (0.1 ml, 0.05 mg/plate for 4-NQO; 2.0 mg/plate
for B[a]P) were mixed in a tube and incubated at 37  C for 0, 20 or
40 min. They were then added to the overnight culture of
S. typhimurium TA98 or TA100 (0.1 ml) and PB (0.5 ml). In the case
of B[a]P, S. typhimurium (0.1 ml) and S9 mix (0.5 ml) were added.
They were then incubated at 37  C with gentle shaking for 20 min.
After adding top agar (2.0 ml) and vortexing the tube, they were
pour plated onto minimal glucose agar plate. After 48 h of in-
cubation at 37  C, revertant colonies were counted.
When the effect on B[a]P metabolites was investigated, the
metabolites were first prepared by preincubation of B[a]P (0.1 ml)
and S9 mix (0.5 ml) together at 37  C for 20 min with gentle
shaking. Fermented black soybean extract (0.1 ml) was then added
to the reaction mixture (B[a]P metabolites) and incubated for 0, 20
or 40 min at 37  C. Finally, they were combined with either
S. typhimurium TA98 or TA100 (0.1 ml) and incubated at 37  C for
another 20 min with gentle shaking. Further cultivation and colony
counting were performed as mentioned above.
To examine the effect on S9 mix, fermented black soybean ex-
tract (0.1 ml) and S9 mix (0.5 ml) were preincubated together at
37  C for 0, 20 or 40 min. S. typhimurium TA98 or TA100 and B[a]P
were added. They were incubated at 37  C for another 20 min and
were assayed for antimutagenicity as mentioned above.
2.7. Analysis of the blocking effect
To evaluate the blocking effect of fermented black soybean
extract, on equal volume of, S. typhimurium TA98 or TA100 and
DMSO with or without fermented black soybean extract were first
incubated together at 37  C for 1 h. The bacteria were then washed
three times with nutrient broth No. 2 with centrifugation. Then,
mutagenicity of 4-NQO and B[a]P was assayed with the fermented
black soybean extract-treated or untreated (control) S. typhimurium
TA98 or TA100.
2.8. Statistical analysis
The mean values and the standard deviation were calculated
from the data obtained from three separate experiments. These
data were then compared by the Duncan’s multiple range method
(SAS, 2001).
3. Results and discussion
3.1. Bio-antimutagenic effect
It was reported that antimutagens may exert a bio-
antimutagenic effect by modulating cellular mutagenic processes,
i.e., by acting on DNA replication and repair processes after DNA is
Table 1
Bio-antimutagenic effect of fermented black soybean extract on 4-NQO and B[a]P assayed in Salmonella typhimurium TA98 and TA100a
Sample Hisþ revertants/plate
4-NQO
TA98
b
Revertant/plate % Of control
Controlc 83  6 Ad 100.0
Fermented black soybean extract 57  8 B 68.7
a
4-NQO or B[a]P plus S9 mix was preincubated with bacterial suspension at 37  C for 1 h before performing the antimutagenicity assay with fermented black soybean
extract.
b
Revertants were the number of total Hisþ revertants minus spontaneous Hisþ revertants.
c
The control was with mutagen but without fermented black soybean extract.
d
Results are presented as means  SD for three experiments, and data in the same column with same letter are not significantly different (p > 0.05) according to Duncan’s
multiple range test.
damaged by the mutagen (Chen & Yen, 1997; Kada, Kaneko, Mat-
suzaki, & Matsuzaki, 1985). Previously, Kada et al. (1985) reported
that epigallo–catechin–gallate, isolated from Japanese green tea
(leaves of Camellia sinensis) exerted bio-antimutagenic effect by
altering DNA-polymerase III in a mutator strain of Bacillus subtilis.
Using UV-induced mutation of Escherichia coli B/r WP2 as the test
organism, Nakamura, Suganuma, Kuyama, Sato, and Ohtsuki (1998)
demonstrated that extracts of various common vegetables such as
Kamo eggplant (Solanum melongena), Fushimi sweet pepper (Cap-
sicum annum var. grossum), Kitsura oriental pickling melon (Cucu-
mis melon var. conomon var. conomon) and shishga tani pumpkin
(Cucurbita moschata) showed bio-antimutagenic effect.
In the present study, the method described by Chen and Yen
(1997) was followed to evaluate the bio-antimutagenic activity of
the methanol extract of fermented black soybean against 4-NQO
and B[a]P in test strains of S. typhimurium TA98 and TA100. As
shown in Table 1, the fermented black soybean extract significantly
(p < 0.05) reduced the number of revertants of S. typhimurium TA98
and TA100 when 4-NQO was tested as the mutagen. The number of
revertants appearing in the plate containing fermented black soy-
bean extract was significantly (p < 0.05) less than that observed in
the plate containing no fermented black soybean extract. With
B[a]P as the mutagen, a similar reduction in the number of re-
vertant was also noted in the plate containing the fermented black
soybean extract. They all demonstrated that the mutagenicity of
both mutagens examined was diminished by the fermented black
soybean extract in the bio-antimutagenic test, which implied that
DNA damaged by 4-NQO or B[a]P was repaired by treatment with
fermented black soybean extract. Furthermore, it was noted that
the extent of reducing revertants or the reduced mutagenicity of
a mutagen was, in general, greater with the test strain of TA100
than TA98. This indicated that the bio-antimutagenic effect of fer-
mented black soybean extract may vary with test strains of S.
typhimurium. A possible cause of this effect is that black soybeans
contain polyphenol compounds (Hung et al., 2007) which may
contribute to the antimutagenic effect of the fermented black
soybean extract observed.
3.2. Desmutagenic effects
Antimutagens may exert a desmutagenic effect on mutagens by
either directly inactivating mutagens, or affecting their precursors by
suppressing the activity of metabolic enzymes (Chen & Yen, 1997;
Kada et al., 1985). The antimutagenicity of various substances such as
lignin on benzo[a]pyrene (Sato et al.,1987), amino acid–sugar Maillard
reaction products on 2-amino-3-methylimidazo[4,5-f]quinoline (Yen
& Hsieh,1994; Yen & Tsai,1993) and Taiwanese oolong tea on activated
Trp-p-2 (Kojima, Miwa, Mori, Osaki, & Konishi, 1989) has been
suggested to be due to a desmutagenic effect.
Being a direct-acting mutagen, 4-NQO requires no hepatic micro-
somal enzyme activation to induce mutation. The desmutagenicity of
B[a]P
TA100 TA98 TA100
Revertant/plate % Of control Revertant/plate % Of control Revertant/plate % Of control
87  19 A 100.0 59  5 A 100.0 141  15 A 100.0
11  11 B 12.6 40  11 B 67.8 44  13 B 31.2
 Y.-H. Hung et al. / LWT - Food Science and Technology 42 (2009) 56–62 59

Table 2
Effects of preincubation of fermented black soybean extract with B[a]P, S9 mix, B[a]P metabolites for various periods on the mutagenicity of B[a]P and 4-NQO assayed in
Salmonella typhimurium TA98 and TA100

Incubation time (min)a TA98 TA100

Revertant/plateb % Of inhibitionc Revertant/plate % Of inhibition

Control Fermented black soybean extract Control Fermented black soybean extract
4-NQO
0 123  22 72  15 41.4 A 264  58 227  16 14.0 A
20 165  9 61  12 63.0 B 221  47 119  15 46.2 B
40 139  5 51  19 63.3 B 287  31 86  28 70.0 C

B[a]P
0 182  12 155  30 14.8 A 80  13 57  11 28.8 A
20 186  11 153  13 17.7 A 91  7 63  20 30.8 A
40 193  11 157  24 18.7 A 101  8 72  19 28.7 A

S9 mix
0 167  8 145  24 13.2 A 78  13 58  11 25.6 A
20 169  17 146  16 13.6 A 96  11 71  18 26.0 A
40 192  18 161  22 16.1 A 103  13 74  20 28.2 A

B[a]P metabolites
0 156  17 136  24 12.8 A 75  4 58  7 22.7 A
20 159  9 87  6 45.3 B 94  7 65  7 30.9 B
40 190  7 92  13 51.6 B 110  12 69  9 37.3 B
a
Fermented black bean extract was preincubated with B[a]P, S9 mix, B[a]P metabolite or 4-NQO for 0–40 min, before the mutagenesis assay was performed.
b
Results were presented as mean  SD for three experiments. Number of revertants reported is the total Hisþ revertants minus spontaneous Hisþ revertants per plate.
c
Inhibition (%) ¼ [1  number of induced revertants in the presence of fermented black soybean extract/number of induced revertants in the absence of fermented black
soybean extract]  100. Within the column, means with different letters are significantly different (p < 0.05) according to Duncan’s multiple range test.

fermented black soybean extract against 4-NQO was examined by
preincubation of a test sample with mutagen at 37  C for 0–40 min.
Next, the antimutagenicity of the fermented black soybean extract was
determined. As shown in Table 2, it was noted that the
antimutagenicity against 4-NQO in S. typhimurium TA98 significantly
increased (p < 0.05) from 41.4 to 63.0% as the preincubation time was
increased from 0 to 20 min although no significant increase in the
antimutagenicity was noted with the further extension of
preincubation to 40 min. In S. typhimurium TA100, extending the
preincubation time of the test sample with 4-NQO was also found to
result in a significant increase (p < 0.05) in the antimutagenicity of the
fermented black soybean extract. These observations clearly demon-
strated that a desmutagenic effect, i.e., a direct interaction of
fermented black soybean extract with the mutagen may contribute to
the antimutagenicity of the fermented black soybean extract against
4-NQO.
To elucidate the actual desmutagenic effect of fermented black
soybean extract against the indirect-acting mutagen, a B[a]P, test
sample was preincubated with S9 mix, intact B[a]P or B[a]P me-
tabolite for a period of 0–40 min before an antimutagenicity assay
was performed. As shown in Table 2, increasing the preincubation
time of fermented black soybean extract with B[a]P or S9 mix did
not result in a significant change (p > 0.05) in antimutagenic effect
in S. typhimurium TA98 or TA100. It is therefore suggested that
direct interaction between fermented black soybean extract with
B[a]P or the inactivation of the activity of the hepatic microsome is
not likely to be the main cause of the antimutagenic effect of the
fermented black soybean extract observed. Nevertheless, the anti-
mutagenicity of the fermented black soybean extract increased as
the preincubation period of the sample with B[a]P metabolite was
increased. As shown in Table 2, an increased inhibition of 45.3 and
51.6%, respectively, in TA98 was noted when fermented black soy-
bean extract was preincubated with B[a]P metabolite for 20 and
40 min compared to an inhibition of 12.8% observed without pre-
incubation. A similar trend in increasing inhibition with extending
preincubation time was also noted when S. typhimurium TA100 was
tested. These results showed that the interaction of B[a]P metab-
olite with the antimutagenic factors in the fermented black soy-
bean extract may have contributed to the observed desmutagenic
activity of the fermented black soybean extract against B[a]P in
both S. typhimurium TA98 and TA100.
3.3. Blocking effect
Antimutagens may exert a blocking effect, adjusting the func-
tion of bacterial cells to reduce the DNA mutagen induced by the

Table 3
Mutagenicity of 4-NQO and B[a]P to Salmonella typhimurium TA98 and TA100 treated with fermented black soybean extract

Treatmenta Hisþ revertants/plate

4-NQO B[a]P

TA98 TA100 TA98 TA100

b
Revertant/plate % Of control Revertant/plate % Of control Revertant/plate % Of control Revertant/plate % Of control
Control 493  26 Ac 100.0 1305  25 A 100.0 83  14 A 100.0 113  14 A 100.0
Fermented black soybean extract 473  20 A 95.9 1028  22 B 78.8 47  6 B 56.6 86  13 B 76.1
a
S. typhimurium TA98 and TA100 and DMSO containing fermented black soybean extract were preincubated at 37  C for 1 h. The bacteria were washed three times with
nutrient broth and were then mixed with 4-NQO or B[a]P and S9 mix at 37  C for 20 min before mutagenesis assay was performed. The bacteria preincubated with DMSO
without fermented black soybean extract served as control.
b
Number of revertants reported is the total Hisþ revertants minus spontaneous Hisþ revertants per plate.
c
Results are presented as means  SD for three experiments. Within the column, means with different letters are significantly different (p < 0.05) according to Duncan’s
multiple range test.
60 Y.-H. Hung et al. / LWT - Food Science and Technology 42 (2009) 56–62

Table 4 significantly reduced by treatment with fermented black soybean

Mutagenicity of the digested fermented black soybean extracts in Salmonella extract. Therefore, fermented black soybean extract might exert
typhimurium TA98 and TA100
a blocking effect on the mutagenicity of 4-NQO in TA100. On the
Extracts Treated with gastric juice Treated with intestinal juice other hand, the blocking effect might also have led to the reduced
(mg/plate) mutagenicity of B[a]P in both S. typhimurium TA98 and TA100.
TA98 TA100 TA98 TA100
a
Revertants Revertants Revertants Revertants
(CFU/plate) (CFU/plate) (CFU/plate) (CFU/plate) 3.4. Mutagenicity of the digested fermented black soybean extract
S9
Control 35  5 101  6 45  4 101  6 Table 4 shows the results of the mutagenicity test of the digested
5 60  5 107  6 57  6 78  10
fermented black soybean extract (0.625–5.0 mg/plate) on S. typhi-
2.5 57  4 105  6 60  5 78  2
1.25 52  8 99  2 64  6 82  6 murium TA98 and TA100. The number of revertants in the presence
0.625 55  3 109  11 57  7 83  9 of the digested fermented black soybean extract for both test
þS9
strains of S. typhimurium, with and without the S9 mixture, was
Control 46  3 92  6 46  6 96  3 found to be similar to that for the negative control (spontaneous
5 66  6 96  9 65  3 100  13 revertant in absence of digested fermented black soybean extract)
2.5 64  5 98  4 64  5 96  18 and was less than twice that of spontaneous revertants. This result,
1.25 62  3 90  10 63  4 106  9
similar to that observed on lactic fermented soymilk (Hsieh et al.,
0.625 58  10 91  12 64  11 105  5
2007), demonstrated that both gastric juice treated- and intestinal
a
Results are presented as means  SD from three separate experiments. juice treated-fermented black soybean extracts, at the dosage levels
tested, had no mutagenic effect on S. typhimurium.

mutagens instead of bio-antimutagenesis and desmutagenesis
(Chen & Yen, 1997). Hsieh, Fang, Yu, and Chou (2007) noted that
a blocking effect was one of the mechanisms of the antimutagenic
effect exerted by fermented soymilk against 4-NQO and B[a]P. DNA
aducts with chlorophyll and chlorophyllin were also found to
exhibit antimutagenicity by reducing the DNA damage induced by
mutagens (Tajmir-Riahi, Neault, & Diamantoglou, 2004). Also,
ellagic acid was reported to exert a blocking effect on the muta-
genicity of 2-amino-3-methylimidazo[4,5-f]quinoline by modifying
the binding site of mutagens on DNA to avoid the DNA damage
(Ayrton, Lewis, & Walker,1992).
To examine if the fermented black soybean extract exerted
a blocking effect on 4-NQO and B[a]P, test organisms of S. typhi-
murium TA98 or TA100 were preincubated with fermented black
soybean extract at 37  C for 1 h before mutation was induced by the
test mutagens. As shown in Table 3, revertants of the fermented
black soybean extract-treated and non-treated (control) S. typhi-
murium TA98, respectively, were 473 and 493 revertants/plate,
showing no significant difference (p > 0.05) when test with 4-NQO.
While a significantly reduced level of revertants (p < 0.05) was
found with the treated-TA100 compared to the control. Further-
more, the revertants induced by B[a]P in both TA98 and TA100 were
3.5. Antimutagenicity of the digested fermented black soybean
extract
The inhibitory effect of the gastric juice treated- and intestinal
juice treated-fermented black soybean extract at a dosage of
0–5 mg/plate against the mutagenicity of 4-NQO and B[a]P in
S. typhimurium TA98 and TA100 is shown in Table 5. At all the dosage
levels examined, the fermented black soybean extract, regardless of
digestion treatment, showed antimutagenic effect against the mu-
tagens tested in both test strains of S. typhimurium. Oshite, Oda, and
Nguyen (1996) reported that the antimutagenicity of the heated
aqueous extract of soybean remained unchanged after exposure to
0.1 N HCl for 1 h. Krul et al. (2001) observed that the antimutagenic
activity of green tea and black tea extract against 2-amino-3,
4-dimethylimidazo[4,5-f]quinoline (MeIQx) was reduced in an in
vitro gastrointestinal model. We observed that the digested fer-
mented black soybean extract at all the dosage levels examined
showed less antimutagenicity than did the non-treated fermented
black soybean extract. For example, the non-treated fermented
black soybean extract at a dosage of 5 mg/plat exhibited an
antimutagenicity of 82.5% against 4-NQO in S. typhimurium TA98.
While a lower level of antimutagenicity of 62.8 and 60.3% was noted

Table 5
Antimutagenicity of digested fermented black soybean extracts against 4-NQO or B[a]P in Salmonella typhimurium TA98 and TA100

Extracts (mg/plate) Non-treatment Treated with gastric juice Treated with intestinal juice

Revertants (CFU/plate)a Inhibition (%)b Revertants (CFU/plate) Inhibition (%) Revertants (CFU/plate) Inhibition (%)

TA98 TA100 TA98 TA100 TA98 TA100 TA98 TA100 TA98 TA100 TA98 TA100
4-NQO
None 57  6 311  11 78  1 286  2 73  5 398  15
5 10  1 D 70  1 C 82.5 77.5 29  2 C 203  5 D 62.8 58.1 29  2 C 199  12 D 60.3 58.1
2.5 19  1 C 117  11 B 66.7 62.4 39  1 BC 235  2 C 50.0 43.4 34  1 BC 230  7 C 53.4 47.2
1.25 26  2 B 174  8 B 54.4 46.6 49  5 AB 260  5 B 37.2 32.2 34  3 AB 288  11 B 41.2 28.5
0.625 35  2 A 182  6 A 38.6 41.5 54  5 A 269  8 A 30.8 22.6 48  2 A 295  13 A 34.3 26.4

B[a]P
None 61  5 177  2 65  1 310  3 73  3 314  14
5 92 C 46  2 D 85.3 74.0 32  3 C 213  2 D 50.8 31.3 24  3 B 139  1 D 67.1 54.3
2.5 24  4 B 69  3 C 60.7 61.0 37  1 BC 250  1 C 43.1 19.4 35  2 AB 203  2 C 52.1 35.4
1.25 35  1 A 115  4 B 42.6 39.5 44  1 AB 275  4 B 32.3 11.3 44  4 A 230  3 B 33.7 26.8
0.625 38  3 A 131  1 A 37.7 26.0 51  6 A 289  4 A 21.5 6.8 48  1 A 272  2 A 29.3 13.4
a
Result are presented as means  SD from three separate experiments. Number of revertants reported is the total Hisþ revertants minus spontaneous Hisþ revertants per
plate. Within the column, means with different letters are significantly different (p < 0.05) according to Duncan’s multiple range test.
b
Inhibition (%) ¼ [1  number of induced revertants in the presence of fermented black soybean extract/number of induced revertants in the absence of fermented black
soybean extract]  100.
 Y.-H. Hung et al. / LWT - Food Science and Technology 42 (2009) 56–62
Table 6
Half-inhibition (IC50) and reduction of the antimutagenicity of the digested fermented black soybean extracts against 4-NQO or B[a]P in Salmonella typhimurium TA98 and
TA100
Treatment TA98
4-NQO B[a]P
a c
IC50 (mg/plate) Reduction (%) IC50 (mg/plate)
Control 1.11  0.47 Cb 0.0 1.78  0.47 C
Gastric juice 2.90  0.50 A 61.7 4.33  0.50 A
Intestinal juice 2.34  0.67 B 52.6 2.91  0.67 B
a
IC50 is the efficient concentration of the test samples that inhibit 50% mutagenic activity. IC50 was obtained by interpolation from linear regression analysis.
b
Result are presented as means  SD from three separate experiments. Means with different letters in the same column are significantly different (p < 0.05) according to
Duncan’s multiple range test.
c
Reduction (%)¼[1  IC50 of control/IC50 of treated sample]  100. Reduction of antimutagenicity of control corresponds to 0.0%.
for the gastric juice treated and the intestinal juice treated-fer-
mented black soybean extract, respectively. Additionally, the
treated- and non-treated fermented black soybean extract showed
a dose-dependent antimutagenic activity against the mutagens
tested in S. typhimurium TA98 and TA100 (Table 5). To further ex-
plore the effect of digestion on the antimutagenicity of the fer-
mented black soybean extract, the efficient concentration of the test
samples that inhibit 50% mutagenic activity (IC50) was obtained by
interpolation from regression analysis. Table 6 showed the IC50 of
the digested and non-treated fermented black soybean extract
against 4-NQO and B[a]P. The IC50 of the digested fermented black
soybean extract was all significantly less (p < 0.05) than that of the
non-digested extract. For example, the intestinal juice treated ex-
tract and the non-digested extract were 2.34 and 1.11 mg/plate, re-
spectively. This shows that the antimutagenicity of the former
against 4-NQO in S. typhimurium TA98 is only about 0.38 fold that of
the latter. Thus it implies digestion reduces the antimutagenicity of
the fermented black soybean extract. Furthermore, it was also noted
that the degree of reducing antimutagenicity of the fermented black
soybean extracts after simulated digestive juice treatments varied
with mutagens and strains of S. typhimurium examined (Table 6).
Nevertheless, the extract of fermented black soybean extract still
possesses its antimutagenic potential in an in vitro simulated di-
gestion model. The antimutagenic compounds from the fermented
black soybean extract were not completely inactivated by gastric
acid and intestinal juice, and were readily available for absorption.
4. Conclusion
Based on the data obtained from the present study, it is
concluded that bio-antimutagenic, desmutagenic, and blocking
effects all contributed to the antimutagenicity of the methanol of
fermented black soybean against 4-NQO and B[a]P in both
S. typhimurium TA98 and TA100. On the other hand, exposure to
simulated gastric juice (pH 1.2) for 30 min or intestinal juice for 2 h
resulted in a reduced antimutagenicity of fermented black soybean
extract. Nevertheless, these treated-fermented black soybean
extracts still possessed a considerable antimutagenic effect against
4-NQO and B[a]P.
Acknowledgement
This research was financially supported by The National Science
Council, ROC (Taiwan) (NSC 95-2313-B-002-017).
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