obiology 118 (2007) 62–68www.elsevier.com/locate/ijfoodmicro

International Journal of Food Micr

Mutagenic and antimutagenic effects of methanol extracts of unfermentedand 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, Taiwanb 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 includingAspergillus awamori, Aspergillus oryzae BCRC 30222, Aspergillus sojae BCRC 30103, Rhizopus azygosporus BCRC 31158 and Rhizopussp. No. 2 was performed. Mutagenicity and antimutagenicity of the methanol extracts of unfermented and fermented steamed black soybeansagainst 4-nitroquinoline-N-oxide (4-NQO), a direct mutagen and Benzo[a]pyrene (B[a]P), an indirect mutagen, on Salmonella TyphimuriumTA100 and TA 98, were examined. The methanol extracts of unfermented and fermented steamed black soybeans show no mutagenic activity foreither 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 soybeansextract varied with the starter organism, mutagen, and test strain of S. Typhimurium examined. Generally, the extracts of A. awamori-fermentedblack soybean exhibited the highest antimutagenic effect. With strain TA100, the inhibitory effects of 5.0 mg of A. awamori-fermented blacksoybean extract per plate on the mutagenic effects of 4-NQO and B[a]P were 92% and 89%, respectively, while the corresponding rates for extractof unfermented were 41% and 63%, respectively. With strain 98, the inhibition rates were 94 and 81% for the fermented bean extract and 58% and44% for the unfermented bean extracts. Testing of extracts prepared from black soybean by A. awamori at temperatures 25, 30 and 35 °C and fortimes of 1–5 days revealed that, generally, the extract prepared from beans fermented at 30 °C for 3 days exhibited the greatest inhibition againstthe 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 andcarcinogenesis (Maron and Ames, 1983; Ferguson et al.,2004). Various investigators have attributed the relatively lowrates of breast, prostate and colon cancers in Asian countriesto the consumption of soy, which contains antimutagenicfactors (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

et al., 2003; Trock et al., 2006). Avoiding exposure tomutagens and eating an adequate supply of nutritious foodscontaining antimutagens may then reduce the rate of mutationand incidence of cancer in humans (Ribeiro and Saloadori,2003; Ferguson et al., 2004).

Black soybeans (Glycine max (L.) Merr.) are a nutritionallyrich foodstuff. The seed coats of black soybeans containanthocyanin and so they are darker than the seed coats of otherstrains of other soybean (Choung et al., 2001). They alsocontain isoflavone, vitamin E, soaponin and anthocyaninwhich 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 soybeansfermented by filamentous fungi are further processed to make

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traditional fermented condiments such as In-yu black sauceand In-si or Ttou-si, the dried by-product of black soybeansauce (Su, 1980). The beneficial effects of the black soybeanwas described in Ben-Tsao Gong Mu, an ancient ChineseBotanical Encyclopedia, dating back to the early 16th century(Li, 1990).

Recently, black soybeans have been reported to inhibit lowdensity lipoprotein (LDL) oxidation (Takahashi et al., 2005) andto effectively reduce the incidence of DNA damage by cyclo-phosphamide (Ribeiro and Saloadori, 2003). In addition,combining the Rhizopus azygosporus-fermented black soybeanwith rice has also been suggested as a way to develop anutritious weaning food. (Rodriguez-Bűrger et al., 1998).

In an attempt to develop healthy food possessing functionalproperties, a series of studies on black soybean has been con-ducted in our laboratory. We noted that black soybean possessedantioxidative activity, including α-α-diphenyl-2-picyl-hydoxylradical-scavenging effect, Fe2+-chelating ability, and reducingactivity, which was enhanced by fermentation with fungi (Leeet al., 2007). Additionally, fermentationwas also noted to increasethe content of aglycone, the bioactive isoflavone (Lee and Chou,2006).

To further evaluate those qualities, the mutagenic andantimutagenic effects of the methanol extracts of black soybeanand 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 arecommonly used as starter organisms for the preparation oftraditional, oriental fermented food products, were used toferment cooked black soybeans. All the test organisms wereobtained from the Bioresources Collection and ResearchCenter (BCRC), Hsinchu, Taiwan except A. awamori andRhizopus sp. No. 2, which were provided by Professor Yu,Graduate Institute of Food Science and Technology, NationalTaiwan University. Strain markers and bacterial survival wereroutinely 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 indimethylsulfoxide (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 homogenatetreated with Aroclor 1254 was purchased from MP Bio-medicals, Inc. (Solon, Ohio, USA). S9 mix (S9 fraction ofliver homogenate with cofactors) was prepared according

to the method of Maron and Ames (1983) and used formetabolic activation of B[a]P.

2.3. Solid fermentation of black soybean

Black soybeans obtained from a local market, were firststeam-cooked at 121 °C, for 15 min in an autoclave. Solid statefermentations of the steamed black soybeans was thenperformed at 30 °C and 95% RH for a period of 3 days.When effects of fermentation temperature and time wereexamined, the steamed black soybeans were fermented by A.awamori at 25, 30, and 35 °C for a period of 0–5 days. Thefermentation procedures have been described (Lee and Chou,2006).

2.4. Preparation of methanol extracts

The unfermented and fermented steamed black soybeans, weredried at 60 °C for 24 h, then they were ground to 30-meshpowders screen using a grinder (Model HF-365; Shivn FengEnterprise Co., Ltd., Taipei, Taiwan). The powders were extractedwith methanol (1:10, w/v) by refluxing at about 25 °C for 24 hwith gentle shaking. After filtering through Whatman No. 1 filterpaper, the extracts were vacuum concentrated and dried using afreeze-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 mutageniceffect of the extracts. Briefly, 0.1 ml of a 10 to 11 h culture ofS. Typhimurium TA 100 or TA98 was added to 0.1 ml DMSOcontaining 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 for20 min. After incubation, 2.0 ml top agar was added to andvirtexed with the tube contents. The mixture was poured onto aplate of minimal glucose agar and colonies were counted afterincubation for 48 h at 37 °C. It was found that revertantappeared in plates containing extract came close to that found inplate without extract in the preliminary study. Therefore, thedoses of extracts tested were not toxic to S. Typhimurium(Waleh et al., 1982).

The antimutagenic effects of the extracts were examinedunder the same conditions as were used for mutagenicity testingexcept with the addition of 0.1 ml mutagen to each test systembefore incubation at 37 °C.

Each assay was performed in triplicate, and antimutagenicactivity was expressed as a percentage of mutagenic inhibitionusing the formula:

Inhibitionð%Þ ¼ 1� ½ðA� CÞ=ðB� CÞ� � 100;

where A and B are the numbers of mutagen-induced revertantsin the presence and absence, respectively, of an extract, and C isthe number of spontaneous revertants.

Table 1Effects 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 ofextracts(mg/plate)

Unfermented black soybeanextracts

Extracts of black soybean fermented with

A. awamori A. sojae A .oryzae R. azygospous Rhizopus sp. No.2.

Revertants(CFU/plate) a

Inhibitionrate(%) b

Revertants(CFU/plate)

Inhibitionrate(%)

Revertants(CFU/plate)

Inhibitionrate(%)

Revertants(CFU/plate)

Inhibitionrate(%)

Revertants(CFU/plate)

Inhibitionrate(%)

Revertants(CFU/plate)

Inhibitionrate(%)

4-NQONone 549±96 549±96 549±96 549±96 549±96 549±965 D388±15 41d D189±4 92a D229±9 82b C247±6 77b C333±10 55c D230±10 82b2.5 C461±5 23d C242±6 79a C261±8 74a B338±20 54b B358±10 49c C313±8 61b1.25 B506±13 11e B462±20 22d B281±8 69a A381±14 43b A425±3 32c B363±8 48b0.625 A541±10 2d A587±7 17c A309±6 62a A405±4 37b A413±4 35b A424±8 32b

B[a]PNone 271±12 271±12 271±12 271±12 271±12 271±125 C180±13 63b C143±6 89a C146±10 87a C155±6 81a C146±9 87a D149±9 85a2.5 C191±14 55d B154±4 81a C152±9 82a C163±9 75b C152±9 83a C178±11 65c1.25 B229±23 29e A165±5 74a B174±20 67b B178±12 65b B198±12 51c B214±11 40d0.625 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 (pb0.05).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 2Effects of the unfermented and fermented black bean extracts against the mutagenic effects of 4-NQO and B[a]P on S. Typhimurium TA98

Mutagen Amountsof extracts(mg/plate)

Unfermented black soybeanextracts

Extracts of black soybean prepared with

A. awamori A. sojae A .oryzae R. azygospous Rhizopus sp. No.2.

Revertants(CFU/plate) a

Inhibitionrate(%) b

Revertants(CFU/plate)

Inhibitionrate(%)

Revertants(CFU/plate)

Inhibitionrate(%)

Revertants(CFU/plate)

Inhibitionrate(%)

Revertants(CFU/plate)

Inhibitionrate(%)

Revertants(CFU/plate)

Inhibitionrate(%)

4-NQONone 98±8 98±8 98±8 98±8 98±8 98±85 C53±6 58d D26±2 94a C27±3 92a D51±3 61c D55±3 56d C41±4 74b2.5 BC57±3 54b C58±3 52b B33±2 85a C61±2 48c C66±3 42c B55±4 56b1.25 B63±3 45b B74±6 32c B36±2 81a B72±2 34c B74±3 31c A71±3 35c0.625 A76±5 28b A91±4 10d A45±3 69a A84±2 18c A83±4 19c A76±4 28b

B[a]PNone 80±7 80±7 80±7 80±7 80±7 80±75 C55±6 44c C34±6 81a C35±6 80a C32±6 84a C33±4 82a C44±6 64b2.5 B63±4 30d B42±3 67a B44±5 64a B42±3 67a B49±5 54b B56±6 42c1.25 B67±4 23c A46±3 59a A56±9 43b B49±3 54a B58±7 39b A66±7 25c0.625 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 (pb0.05).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 3Inhibition of the mutagenic effects of 4-NQO and B[a]P on S. Typhimurium bythe methanol extracts of fermented black soybean (5 mg/plate) fermented by A.awamori at different temperatures for 3 days

Fermentationtemperature(°C)

Inhibition rate(%) a

S. Typhimurium TA98 S. Typhimurium TA100

4-NQO B[a]P 4-NQO B[a]P

Not fermented 58Cb 44D 41C 63C25 °C 92A 62B 85B 87A30 °C 94A 81A 92A 89A35 °C 72B 51C 80B 78B

a Inhibition rate (%)=[1−number of induced revertants in the presence ofextract of black soybean or fermented black soybean /number of inducedrevertants in the absence of extract of black soybean or fermented blacksoybean]×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 differentupper case letters A, B, C, D are significantly different (pb0.05).

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2.6. Statistical analysis

The mean values and the standard deviation were calculatedfrom the data obtained from three separate experiments. Meanswere compared using Duncan's multiple range test method inSAS, 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/perplate of the extracts) were not mutagenic for strains TA100 andTA98, since the number of revertants colonies obtained with orwithout 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 redbean fermented with A. oryzae or methanol extracts of soybeanfermented 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 againstthe mutagenic effects of 4-NQO and B[a]P on S. TyphimuriumTA100 and TA 98. 4-NQO exerts potent intracellular oxidativestress, and its metabolic product binds to DNA predominantly atguanine residues (Kanojia and Vaidya, 2006). It shows a highpotential ability to reduce oxidative stress, which contributes totumor promotion (Nunoshiba and Demple, 1993). Previously, avariety 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 thatleads to its activation as a reactive benzo[a]pyrene dihydridialexposide, which is an electrophilic species capable of binding toDNA, RNA and some other macromolecules (Josephy et al.,1992).

Extracts of unfermented and fermented steamed blacksoybeans inhibited the mutagenic effects of 4-NQO and B[a]Pon S. Typhimurium TA100 (Table 1). Compounds such asvitamin E, saponin, phytic acid, linoleic acid, genistein anddaizein, commonly found in bean and bean products, have beenreported 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 antimutageniceffect (Cardador-Martinez et al., 2002; Aparicio-Fernándezet al., 2005). Therefore, Such materials may all contribute to theobserved antimutagenic effect of the soybean extracts. Theantimutagenic effect of the extracts increased as dosageincreased. At all the doses tested, fermented black soybeanextracts showed higher antimutagenic effects than those extractsof 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 antimutagenicactivity against the mutagens tested on S. Typhimurium TA98(Table 2). However, the inhibition exerted by the extract withsame dose level for S. Typhimurium TA98 (Table 2) was notexactly the same as that for TA100 (Table 1). These results showthat the antimutagenic effect exerted by the extract can varywith strain of S. Typhimurium used for testing.

The greater antimutagenic effects obtained by fermentationof beans, as found in the present study, is in accordance withobservations of other investigators. Among those, Park et al.(2003) found that doenjan, the Korean fermented soy paste,exerted higher antimutagenic activity than raw soybean; Hsiehand Chou (2006) reported that fermentation with bifidobacteriaand lactic acid bacteria, alone or together, significantlyenhanced the antimutagenic activity of soy milk; and Lin andChou (2006) reported findings for fermented soybeans similarto the findings of this study.

The flavonid glycosides are less antimutagenic than thecorresponding aglycones such as daidzein, glycitein andgenistein (Edenharder and Tang, 1997). The fungi used as thestarter organisms in the present study were capable of producingβ-glucosidase, which promotes cleavage of the β-glycosyl bondin the black soybean glucoside isoflavones to form aglycones(Lee and Chou, 2006; McCue and Shetty, 2003). It was foundthat the fermentation of black soybean with the starter organismsexamined caused a marked increase in the content of aglyconeand total anthocyanin (Hung, 2006; Lee and Chou, 2006).Therefore, these changes may all lead to the enhancedantimutagenicity activity observed with the extracts of fermentedblack soybean. However, formation of other bioactive com-pounds and increased linoleic acid contents in bean substratesfollowing fermentation, as reported by Park et al. (2003), mayalso contribute to the enhanced antimutagenicity.

Table 4Inhibition of the mutagenic effects of 4-NQO and B[a]P on S. Typhimurium bythe methanol extract of fermented black soybean (5 mg/plate) fermented by A.awamori during fermentation at 30 °C

Fermentationtime (days)

Inhibition rate(%) a

S. Typhimurium TA98 S. Typhimurium TA100

4-NQO B[a]P 4-NQO B[a]P

0 58Db 44C 41C 63C1 63C 38C 74B 69B2 67C 60B 92A 77B3 94A 81A 92A 89A4 68C 70A 93A 83A5 74B 65B 95A 85A

a Inhibition rate (%)=[1−number of induced revertants in the presence ofextract of black soybean or fermented black soybean /number of inducedrevertants in the absence of extract of black soybean or fermented blacksoybean]×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 differentupper case letters A, B, C, D are significantly different (pb0.05).

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3.4. Fermentation temperature and time affect the antimutageni-city of fermented black soybean extract

Previously, fermentation temperature was reported to affect theantioxidative activity of the methanol extract of the fermentedblack soybean (Lee et al., 2007). As shown in Table 3, theA. awamori-fermented black soybean extract exhibited variousdegrees of inhibition of the mutagenic effects of 4-NQO or B[a]Pon the both strains of S Typhimurium depending on fermentationtemperatures. Generally, the methanol extract of fermented blacksoybean fermented at 30 °C showed the greatest inhibition ofmutation by of 4-NQO and B[a]P, regardless of the test organism.

Table 4 shows the effect of fermentation time on theantimutagenic activity of the A. awamori-fermented blacksoybean extracts, In general, the antimutagenic activity of thefermented black soybean extract increased as the fermentationtime was extended from 1 to 3 days at 30 °C. Extending thefermentation time to 5 days did not significantly (pN0.05)increase the antimutagenic effects of the black soybean extract forS. Typhimurium TA 100, while these extracts had a decreasedantimutagenic effects for S. Typhimurium TA 98.

The relatively high antimutagenicity activities noted in thefermented black soybean extracts is generally associated with thehigher contents of total phenolics, anthocyanin and aglycones infermented black soybean (Lee and Chou, 2006; Lee et al., 2007).However, it is also possible that formation of other antimutagenicmetabolite (Park et al., 2003) as influenced by fermentationcondition occurs during the fermentation process. They may allcontribute to observed antimutagenicity of the fermented blacksoybean extract and merit further investigation.

The results of the present study show that fermentation with anappropriate starter organism and under appropriate cultivationconditions may relatively enhance the antimutagenic activity ofblack soybeans. Information provided in this study, along withknowledge of the enhanced content of aglycone, the bioactiveisoflavone (Lee and Chou, 2006) not only support the suggestionof using, further provide a better cultivation condition to prepare

the fermented black soybean as an ingredient for the formulationof healthy food.

Acknowledgement

This research was financially supported by The NationalScience Council, ROC (Taiwan). (NSC 95-2313-B-002-017).

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