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Mutagenic and antimutagenic effects of methanol extracts of unfermented and fermented black soybeans
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
b
Graduate Institute of Food Science and Technology, National Taiwan University, 59, lane 144, Keelung Rd., Sec. 4, Taipei, Taiwan 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 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: [email protected] (C.-C. Chou). 0168-1605/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2007.06.005
et al., 2003; Trock et al., 2006). Avoiding exposure to 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 cyclophosphamide (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 conducted 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 4nitroquinoline-N-oxide (4-NQO) and benzo[a]pyrene (B[a]P) in S. Typhimurium were studied.
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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
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, Aspergillus 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 Biomedicals, Inc. (Solon, Ohio, USA). S9 mix (S9 fraction of liver homogenate with cofactors) was prepared according
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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Mutagen Amounts of Unfermented black soybean extracts extracts (mg/plate) Revertants Inhibition (CFU/plate) a rate(%) b
Extracts of black soybean fermented with A. awamori
A. sojae
A .oryzae
R. azygospous
Rhizopus sp. No.2.
Revertants (CFU/plate)
Inhibition rate(%)
Revertants (CFU/plate)
Inhibition rate(%)
Revertants (CFU/plate)
Inhibition rate(%)
Revertants (CFU/plate)
Inhibition rate(%)
Revertants (CFU/plate)
Inhibition rate(%)
4-NQO None 5 2.5 1.25 0.625
549 ± 96 D388 ± 15 C461 ± 5 B506 ± 13 A541 ± 10
41d 23d 11e 2d
549 ± 96 D189 ± 4 C242 ± 6 B462 ± 20 A587 ± 7
92a 79a 22d 17c
549 ± 96 D229 ± 9 C261 ± 8 B281 ± 8 A309 ± 6
82b 74a 69a 62a
549 ± 96 C247 ± 6 B338 ± 20 A381 ± 14 A405 ± 4
77b 54b 43b 37b
549 ± 96 C333 ± 10 B358 ± 10 A425 ± 3 A413 ± 4
55c 49c 32c 35b
549 ± 96 D230 ± 10 C313 ± 8 B363 ± 8 A424 ± 8
82b 61b 48b 32b
None 5 2.5 1.25 0.625
271 ± 12 C180 ± 13 C191 ± 14 B229 ± 23 A264 ± 12
63b 55d 29e 5e
271 ± 12 C143 ± 6 B154 ± 4 A165 ± 5 A179 ± 9
89a 81a 74a 64a
271 ± 12 C146 ± 10 C152 ± 9 B174 ± 20 A249 ± 7
87a 82a 67b 16d
271 ± 12 C155 ± 6 C163 ± 9 B178 ± 12 A214 ± 12
81a 75b 65b 39b
271 ± 12 C146 ± 9 C152 ± 9 B198 ± 12 A256 ± 8
87a 83a 51c 10d
271 ± 12 D149 ± 9 C178 ± 11 B214 ± 11 A241 ± 13
85a 65c 40d 21c
B[a]P
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 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 Unfermented black soybean of extracts extracts (mg/plate) Inhibition Revertants rate(%) b (CFU/plate) a
Extracts of black soybean prepared with A. awamori
A. sojae
A .oryzae
R. azygospous
Rhizopus sp. No.2.
Revertants (CFU/plate)
Inhibition rate(%)
Revertants (CFU/plate)
Inhibition rate(%)
Revertants (CFU/plate)
Inhibition rate(%)
Revertants (CFU/plate)
Inhibition rate(%)
Revertants (CFU/plate)
Inhibition rate(%)
4-NQO None 5 2.5 1.25 0.625
98 ± 8 C53 ± 6 BC57 ± 3 B63 ± 3 A76 ± 5
58d 54b 45b 28b
98 ± 8 D26 ± 2 C58 ± 3 B74 ± 6 A91 ± 4
94a 52b 32c 10d
98 ± 8 C27 ± 3 B33 ± 2 B36 ± 2 A45 ± 3
92a 85a 81a 69a
98 ± 8 D51 ± 3 C61 ± 2 B72 ± 2 A84 ± 2
61c 48c 34c 18c
98 ± 8 D55 ± 3 C66 ± 3 B74 ± 3 A83 ± 4
56d 42c 31c 19c
98 ± 8 C41 ± 4 B55 ± 4 A71 ± 3 A76 ± 4
74b 56b 35c 28b
None 5 2.5 1.25 0.625
80 ± 7 C55 ± 6 B63 ± 4 B67 ± 4 A75 ± 6
44c 30d 23c 9e
80 ± 7 C34 ± 6 B42 ± 3 A46 ± 3 A53 ± 5
81a 67a 59a 47a
80 ± 7 C35 ± 6 B44 ± 5 A56 ± 9 A69 ± 3
80a 64a 43b 19d
80 ± 7 C32 ± 6 B42 ± 3 B49 ± 3 A61 ± 8
84a 67a 54a 34b
80 ± 7 C33 ± 4 B49 ± 5 B58 ± 7 A68 ± 3
82a 54b 39b 22c
80 ± 7 C44 ± 6 B56 ± 6 A66 ± 7 A77 ± 8
64b 42c 25c 6e
B[a]P
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.
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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
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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. Typhimurium 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 4NQO. 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, anthocyanin 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. Thphimurium 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 compounds 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 temperature (°C)
Inhibition rate(%) a
4-NQO
B[a]P
4-NQO
B[a]P
Not fermented 25 °C 30 °C 35 °C
58C b 92A 94A 72B
44D 62B 81A 51C
41C 85B 92A 80B
63C 87A 89A 78B
S. Typhimurium TA98
S. Typhimurium TA100
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 time (days)
0 1 2 3 4 5
Inhibition rate(%) a S. Typhimurium TA98
S. Typhimurium TA100
4-NQO
B[a]P
4-NQO
B[a]P
58D b 63C 67C 94A 68C 74B
44C 38C 60B 81A 70A 65B
41C 74B 92A 92A 93A 95A
63C 69B 77B 89A 83A 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 antimutagenicity 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
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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
b
Graduate Institute of Food Science and Technology, National Taiwan University, 59, lane 144, Keelung Rd., Sec. 4, Taipei, Taiwan 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 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: [email protected] (C.-C. Chou). 0168-1605/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2007.06.005
et al., 2003; Trock et al., 2006). Avoiding exposure to 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 cyclophosphamide (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 conducted 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 4nitroquinoline-N-oxide (4-NQO) and benzo[a]pyrene (B[a]P) in S. Typhimurium were studied.
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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
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, Aspergillus 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 Biomedicals, Inc. (Solon, Ohio, USA). S9 mix (S9 fraction of liver homogenate with cofactors) was prepared according
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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Mutagen Amounts of Unfermented black soybean extracts extracts (mg/plate) Revertants Inhibition (CFU/plate) a rate(%) b
Extracts of black soybean fermented with A. awamori
A. sojae
A .oryzae
R. azygospous
Rhizopus sp. No.2.
Revertants (CFU/plate)
Inhibition rate(%)
Revertants (CFU/plate)
Inhibition rate(%)
Revertants (CFU/plate)
Inhibition rate(%)
Revertants (CFU/plate)
Inhibition rate(%)
Revertants (CFU/plate)
Inhibition rate(%)
4-NQO None 5 2.5 1.25 0.625
549 ± 96 D388 ± 15 C461 ± 5 B506 ± 13 A541 ± 10
41d 23d 11e 2d
549 ± 96 D189 ± 4 C242 ± 6 B462 ± 20 A587 ± 7
92a 79a 22d 17c
549 ± 96 D229 ± 9 C261 ± 8 B281 ± 8 A309 ± 6
82b 74a 69a 62a
549 ± 96 C247 ± 6 B338 ± 20 A381 ± 14 A405 ± 4
77b 54b 43b 37b
549 ± 96 C333 ± 10 B358 ± 10 A425 ± 3 A413 ± 4
55c 49c 32c 35b
549 ± 96 D230 ± 10 C313 ± 8 B363 ± 8 A424 ± 8
82b 61b 48b 32b
None 5 2.5 1.25 0.625
271 ± 12 C180 ± 13 C191 ± 14 B229 ± 23 A264 ± 12
63b 55d 29e 5e
271 ± 12 C143 ± 6 B154 ± 4 A165 ± 5 A179 ± 9
89a 81a 74a 64a
271 ± 12 C146 ± 10 C152 ± 9 B174 ± 20 A249 ± 7
87a 82a 67b 16d
271 ± 12 C155 ± 6 C163 ± 9 B178 ± 12 A214 ± 12
81a 75b 65b 39b
271 ± 12 C146 ± 9 C152 ± 9 B198 ± 12 A256 ± 8
87a 83a 51c 10d
271 ± 12 D149 ± 9 C178 ± 11 B214 ± 11 A241 ± 13
85a 65c 40d 21c
B[a]P
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.
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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 Unfermented black soybean of extracts extracts (mg/plate) Inhibition Revertants rate(%) b (CFU/plate) a
Extracts of black soybean prepared with A. awamori
A. sojae
A .oryzae
R. azygospous
Rhizopus sp. No.2.
Revertants (CFU/plate)
Inhibition rate(%)
Revertants (CFU/plate)
Inhibition rate(%)
Revertants (CFU/plate)
Inhibition rate(%)
Revertants (CFU/plate)
Inhibition rate(%)
Revertants (CFU/plate)
Inhibition rate(%)
4-NQO None 5 2.5 1.25 0.625
98 ± 8 C53 ± 6 BC57 ± 3 B63 ± 3 A76 ± 5
58d 54b 45b 28b
98 ± 8 D26 ± 2 C58 ± 3 B74 ± 6 A91 ± 4
94a 52b 32c 10d
98 ± 8 C27 ± 3 B33 ± 2 B36 ± 2 A45 ± 3
92a 85a 81a 69a
98 ± 8 D51 ± 3 C61 ± 2 B72 ± 2 A84 ± 2
61c 48c 34c 18c
98 ± 8 D55 ± 3 C66 ± 3 B74 ± 3 A83 ± 4
56d 42c 31c 19c
98 ± 8 C41 ± 4 B55 ± 4 A71 ± 3 A76 ± 4
74b 56b 35c 28b
None 5 2.5 1.25 0.625
80 ± 7 C55 ± 6 B63 ± 4 B67 ± 4 A75 ± 6
44c 30d 23c 9e
80 ± 7 C34 ± 6 B42 ± 3 A46 ± 3 A53 ± 5
81a 67a 59a 47a
80 ± 7 C35 ± 6 B44 ± 5 A56 ± 9 A69 ± 3
80a 64a 43b 19d
80 ± 7 C32 ± 6 B42 ± 3 B49 ± 3 A61 ± 8
84a 67a 54a 34b
80 ± 7 C33 ± 4 B49 ± 5 B58 ± 7 A68 ± 3
82a 54b 39b 22c
80 ± 7 C44 ± 6 B56 ± 6 A66 ± 7 A77 ± 8
64b 42c 25c 6e
B[a]P
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.
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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
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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. Typhimurium 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 4NQO. 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, anthocyanin 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. Thphimurium 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 compounds 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 temperature (°C)
Inhibition rate(%) a
4-NQO
B[a]P
4-NQO
B[a]P
Not fermented 25 °C 30 °C 35 °C
58C b 92A 94A 72B
44D 62B 81A 51C
41C 85B 92A 80B
63C 87A 89A 78B
S. Typhimurium TA98
S. Typhimurium TA100
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 time (days)
0 1 2 3 4 5
Inhibition rate(%) a S. Typhimurium TA98
S. Typhimurium TA100
4-NQO
B[a]P
4-NQO
B[a]P
58D b 63C 67C 94A 68C 74B
44C 38C 60B 81A 70A 65B
41C 74B 92A 92A 93A 95A
63C 69B 77B 89A 83A 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 antimutagenicity 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
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