Content of Total Phenolics and Anthocyanins and the
Antimutagenicity of Aspergillus awamori-Fermented
Black Soybean after Heat Treatment
YEN-JU WANG AND CHENG-CHUN CHOU*
Graduate Institute of Food Science and Technology, National Taiwan University, Taipei, Taiwan (R.O.C.)
(Received: January 21, 2008; Accepted: April 22, 2008)
ABSTRACT
The Aspergillus awamoir-fermented black soybean, possessing enhanced functional properties, was previously suggested to be a potentially functional ingredient in the formation of healthy foods. In this study, the effect of heat treatments (40-100°C for 30 min) on the changes of total phenolics and anthocyanin contents, mutagenicity and the antimutagenicity of fermented black soybeans against 4-nitroquinoline-N-oxide, a direct mutagen, and benzo[a]pyrene, an indirect mutagen, on Salmonella Typhimurium TA100 and TA98 were examined.
Results revealed that the heated-fermented black soybean showed no mutagenicity. Heating the fermented black soybean at 80 or 100°C for 30 min resulted in a reduced antimutagenicity with the methanol extract. Reduction in antimutagenicity varied with the test strains of S. Typhimurium and the type of mutagens examined. However, the fermented black soybean still possessed anti-mutagenicity after exposure to 100°C for 30 min. Contents of total phenolics and anthocyanins reduced significantly (p < 0.05) as the heating temperature was raised to 40 and 80°C, respectively. Reduction in antimutagenicity of the fermented black soybeans after heating might be due to the lower anthocyanin content.
Key words: Fermented black soybean, anthocyanin, total phenolics, antimutagenicity, heat treatment
INTRODUCTION
Similar to normal soybeans, black soybeans, Glycine max (L.) Merr, is considered a nutritionally rich food-stuff. Compounds possessing biological activity such as anthocyanin, saponin, vitamin E and isoflavone can be found in black soybeans(1-4). The functional prop-erties of black soybeans have been examined by vari-ous investigators. Riberio and Saloadori(5) reported
that black soybeans effectively reduce the incidence of DNA damage by cyclophosphamide. Takahashi et al.(6)
observed that black soybeans inhibited low density lipo-protein oxidation. Moreover, Rodriguez-Bűrger et al.(7)
suggested that combining rice with the Rhizopus azygos-porus-fermented black soybeans is a viable method to develop a nutritious weaning food. In our laboratory, we have previously noted that content of aglycone, the bioactive isoflavone, was increased through fermen-tation with fungi(8). Furthermore, fermentation was
found to enhance certain functional properties such as the antioxidative and antimutagenic activities of black soybeans(9,10). For these reasons, it is clear that
ferment-ed black soybeans are potentially useful as a dietary adjunct or ingredient in the formation of healthy food.
In the food industry, heating is commonly used to enhance the preservation of food products. Alternation in the functional properties, e.g. antimutagenicity, of food materials as a result of heating has been observed by various investigators(11-14). Furthermore, the heating of food has also been reported to result in the induction or enhancement of mutagenic activity(15,16). To assess the
functional properties that contribute to health in ferment-ed black soybeans, this study examinferment-ed the antimuta-genicity of fermented black soybeans across various heat treatments. Moreover, the effect of heat treatment on the total phenolics and anthocyanin content of fermented black soybeans was also examined.
MATERIALS AND METHODS
I. Materials
Black soybeans were purchased from the local market. The test strains of S. Typhimurium including TA98 and TA100 were obtained from the Bioresourc-* Author for correspondence. TEL: +886-2-3366-4111;
es Collection and Research Center (BCRC), Hsinchu, Taiwan. Professor Yu, Graduate Institute of Food Science and Technology, National Taiwan University provided A. awamori used to prepare fermented black soybeans.
4-Nitroquinoline-N-oxide (4-NQO) and benzo[a]pyrene (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 concentra-tions of 0.5 and 20 µg/mL for 4-NQO and B[a]P, respec-tively. 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 to the method of Maron and Ames(17) and used for metabolic activation of B[a]P.
II. Preparation of Heat Treated-fermented Black Soybean and Methanol Extract
The fermented black soybeans were first prepared according to the procedures described by Lee and Chou(8). Briefly, a solid fermentation of the steamed
black soybeans with A. awamori was performed at 30°C and 95% RH for 3 days. The fermented black soybeans were then lyophilized in a freeze-drier (Free Dry System/Freezone® 4.5; Labconco Co., Kansas, Missouri, USA) and were ground to 30-mesh powder screen using a grinder (Model HF-365, Shivn Feng Enterprise Co. Ltd., Taipei, Taiwan). The powder was first heated in an electric oven at 40, 60, 80 or 100°C for 30 min, and then extracted with methanol (1:10, w/v) by refluxing at 25°C for 24 h with gentle shaking. The extracts, after filter-ing through Whatman No.1 filter paper, were vacuum concentrated and freeze dried.
III. Mutagenicity and Antimutagenicity Assay
Detailed procedures for the assay of mutagenic-ity and antimutagenicmutagenic-ity were described in our previous paper(9). Essentially, mutagenicity was assayed using S. Typhimurium TA98 or TA100 in the absence or pres-ence of S9 mix. The antimutagenicity against 4-NQO and B[a]P was assayed by incubating with 0.625-0.5 mg extract of the treated- or untreated sample per plate, using S. Typhimurium TA98 or TA100. S9 mixture was also added when B[a]P was tested as the mutagen. After incubating at 37°C for 48 h, the His+ revertant colonies
were counted. In the preliminary study, the doses of the tested samples of fermented black soybean extract were found to show no toxicity against S. Typhimurium.
Each assay was performed in triplicate, and antimu-tagenic activity was expressed as a percentage of muta-genic inhibition using the formula:
Inhibition (%) = 1-[(A-E)/(B-D)]× 100;
where A and B are the numbers of mutagen-induced
revertants in the presence and absence of sample, respec-tively. E and D are the number of spontaneous revertants observed with sample and control, respectively.
IV. Measurements of Total Phenolics and Anthocyanins The content of total phenolics, expressed in mg of gallic acid/g dried fermented black soybeans, was deter-mined according to that described in the paper of Lee and Chou(8). The method described by Abdel-Aal and
Hucl(18) was followed to determine the content of total anthocyanin, which was expressed as cyaniding 3-gluco-side equivalents (mg/g dried fermented black soybeans). V. 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 Insti-tute, Gary, NC, USA).
RESULTS AND DISCUSSION
I. The Effect of Heat Treatment on the Mutagenicity of Fermented Black Soybean Extract
Heating food may introduce mutagenic and carci-nogenic compounds such as heterocyclic aromatic amines(15). Surono and Hosono(16) also reported that
heating increased the mutagenicity of Terasi, a traditional fermented product of Indonesia. Therefore, mutagenic-ity of the fermented black soybean subjected to various heat treatments was evaluated. As shown in Table 1, the revertants in presence of the heated fermented black soybean extract (0.625-5.0 mg/plate) for S. Typhimuri-um TA98 with or without the S9 mixture were close to those for the negative control (spontaneous revertants in absence of heated fermented black soybean extract) and were less than twice that of spontaneous revertants. This indicated that no mutagenic factor formed in the ferment-ed black soybeans after the various heat treatments. Similar phenomenon was also noted with S. Typhimuri-um TA100 (data not shown). Therefore, extracts of the heated fermented black soybean showed no mutagenic effect on both strains of S. Typhimurium.
II. The Effect of Heat Treatment on the Antimutagenicity of Fermented Black Soybean Extract
Variation in the effect of heat treatment on the antimutagenic activity of antimutagens has been stud-ied. Vis et al.(14) reported that heat-denaturated oval-bumin showed a strong increase in antimutagenicity against MNNG compared to undenaturated ovalbumin. An enhanced antimutagenicity against MNNG was also
noted with the aqueous and methanol extracts of soybean after heating at 225°C for 12 min by Oshite et al.(13) On
the other hand, Hosono et al.(11) reported that heating the
cultured milk fermented by Lactobacillus bulgaricus or Streptococcus thermophilus at 55°C for 10 min resulted in a decreased antimutagenicity against 4-NQO and 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide. A similar phenom-enon of reduced antimutagenicity was also observed by Hsieh et al.(19) on lactic fermented soymilk after
expo-sure to 55°C for 10 min.
Table 2 shows the antimutagenicity of the unheat-ed- and heatunheat-ed-fermented black soybean extracts at a dose of 0.625-5.0 mg/plate against 4-NQO and B[a]P in S. Typhimurium TA98. Regardless of heat treatment, the antimutagenic effect of the fermented black soybeans extract increased as the dosage increased. At the same dosage level, the antimutagenicity exerted by the extract of the fermented black soybean heated at 40 or 60°C for 30 min showed a profound reduction compared with the extract of unheated fermented black soybean. For ple, at the highest dosage of 5.0 mg extract/plate exam-ined, the extract of unheated fermented black soybean exhibited an antimutagenicity of 82.46 and 85.25% against 4-NQO and B[a]P respectively in S. Typhimurium TA98. In comparison, significantly lower (P < 0.05) anti-mutagenicity of only 50.87 and 59.01% was noted with the extract of the 80°C heated-fermented black soybean.
Using S. Typhimurium TA100 as the test strain, the effect of heat treatment on the antimutagenicity of fermented black soybean against 4-NQO and B[a]P (Table
3) was found similar to that noted with S. Typhimurium TA98 (Table 2). Compared with the unheated fermented black soybean extract, a substantial reduction in the anti-mutagenicity was observed with the extracts of ferment-ed black soybeans heatferment-ed at 80°C or higher. The param-eter of IC50, the efficient concentration of test samples
that inhibited 50%, was obtained by polynomial adjust-ment of a second grade equation analysis of data shown in Tables 2 and 3 to further elucidate the effect of heat treatment on the antimutagenicity of the fermented black soybean extract. As shown in Table 4, the IC50
for the antimutagenicity of the fermented black soybean extract against 4-NQO and B[a]P in both test strains of S. Typhimurium was relatively stable at a temperature up to 60°C. However, significantly (P < 0.05) increased IC50 or
reduced antimutagenicity was noted with the extract of fermented black soybean heated at 80℃ or higher for 30 min when compared with that of the control (unheated-fermented black soybean). For example, the extract of the unheated-fermented black soybean showed an IC50 of
1.17 and 1.68 mg/plate against the mutagenesis induced by 4-NQO and B[a]P, respectively in S. Typhimurium TA98. On the other hand, the extract of the 80°C heated-fermented black soybean showed significantly larger IC50
of 4.93 and 3.69 mg/plate, for 4NQO and B[a]P, respec-tively. These results demonstrated that the antimuta-genic factors present in the fermented black soybean were thermolabile at 100°C. Nevertheless, extracts of the fermented black soybean still possess considerable anti-mutagenicity after heating at 100°C for 30 min.
Table 1. Mutagenicity of the extracts of fermented black soybean after heating at different temperatures for 30 min in S. Typhimurium TA98
Extracts (mg/plate)
Unheated Heated (°C)
40 60 80 100
Revertants
(CFU/plate) (CFU/plate)Revertants (CFU/plate)Revertants (CFU/plate)Revertants (CFU/plate)Revertants ………–S9……… Control 25 ± 5a 25 ± 5 25 ± 5 25 ± 5 25 ± 5 5 25 ± 3 23 ± 1 25 ± 3 28 ± 7 31 ± 2 2.5 30 ± 3 29 ± 5 30 ± 3 29 ± 3 29 ± 6 1.25 28 ± 7 28 ± 5 28 ± 7 32 ± 7 29 ± 8 0.625 24 ± 3 28 ± 3 24 ± 3 28 ± 5 32 ± 4 ………+S9……… Control 34 ± 2 34 ± 2 34 ± 2 34 ± 2 34 ± 2 5 55 ± 8 56 ± 5 54 ± 6 54 ± 6 54 ± 6 2.5 50 ± 9 52 ± 3 53 ± 4 53 ± 4 48 ± 6 1.25 52 ± 6 56 ± 4 48 ± 3 48 ± 3 44 ± 8 0.625 53 ± 3 46 ± 4 47 ± 3 47 ± 1 45 ± 1
III. The Effect of Heating on the Content of Total Phenolic Compounds in Fermented Black Soybean
Phenolic compounds are commonly found in plants. Dry beans contain many polyphenolic phytochemicals that are known to possess antioxidants, as well as antimu-tagenic and anticarcinogenic properties(1). Different from soybeans, black soybeans contain anthocyanin which is a kind of flavonoid. Anthocyanins are found to significant-ly suppress the growth of cultured tumor cells and have been shown to have greater inhibitory effect than other flavonoids(20). Previously, we noted that fermentation of
black soybeans with fungi resulted in higher content of total extractable phenolics and anthocyanins(10).
The effect of heat treatment on the total phenolics in fermented black soybeans is shown in Figure 1. Heat treatment, regardless of heating temperature, resulted in significantly decreased (P < 0.05) total phenolics content in the fermented black soybean, while the total phenolics content in the various heated fermented black soybean were similar.
Despite the association of phenolic compounds with antimutagenic activity, the extent of reduced mutagenic-ity of the fermented black soybean (Tables 2-4) did not
correspond precisely with the degree of reduced total phenolics (Figure 1) caused by the heat treatments exam-ined. For example, the total phenolics content of the vari-Table 2. Antimutagenicity effect exerted by extracts of unheated- and heated-fermented black soybeans against 4NQO or B[a]P in S.
Typhimurium TA98 Extracts (mg/plate) Unheated Heated (°C) 40 60 80 100 Revertants (CFU/plate) Inhibition a
(%) (CFU/plate)Revertants Inhibition (%) (CFU/plate)Revertants Inhibition (%) (CFU/plate)Revertants Inhibition (%) (CFU/plate)Revertants Inhibition (%) ………4NQO………
Control 57 ± 6b 57 ± 6 57 ± 6 57 ± 6 57 ± 6
5 D10 ± 1bc A82.46a D15 ± 1b A73.69b D12 ± 3b A78.95b C28 ± 1a A50.87c C26 ± 4a A54.39c 2.5 C19 ± 1b B66.67a C22 ± 3b B61.41b C22 ± 3b B61.41b B41 ± 5a B28.07c B41 ± 1a B28.07c 1.25 B26 ± 2b C54.39a B30 ± 1b C47.37b B28 ± 3b C50.88ab B42 ± 4a C26.31c A45 ± 3a C21.05d 0.625 A35 ± 2b D38.60a A34 ± 1b D40.35a A 38 ± 1b D33.33b A50 ± 5a D12.28d A46 ± 6a D19.29c ………B[a]P………
Control 61 ± 5 61 ± 5 61 ± 5 61 ± 5 61 ± 5
5 C9 ± 2a A85.25ab D10 ± 1a A83.60b B8 ± 1a A86.88a B25 ± 6a A59.01c B28 ± 1a A54.09d
2.5 B24 ± 4b B60.65b C22 ± 1ab B63.93a B24 ± 1ab B60.65b AB37 ± 3a B39.34c AB37 ± 1a B39.34c 1.25 A35 ± 1a C42.62b BC27 ± 2a C55.73a A37 ± 3a C39.34b A47 ± 1a C22.95d A44 ± 6a C27.86c 0.625 A38 ± 3a D37.70a AB44 ± 3a D27.86b A42 ± 5a D31.15b A50 ± 1a D18.03c A53 ± 1a D13.11d a Result are presented as means ± SD from three separate experiments.
b Number of revertants reported is total His+ revertants minus spontaneous His+ revertants per plate. Spontaneous revertants were those observed with sample or control. Statistical differences were calculated by Duncan’s multiple range test. Value in the same row (a, b, c, d) and column (A, B, C, D) with same letters are not significantly different (p > 0.05).
c Inhibition (%) = [1–number of induced revertants in the presence of sample / number of induced revertants in the absence of extract of sample]×100. Unheated Temperature (°C) 40°C 60°C 80°C 100°C 0 10 20 30 40 b b b a b
Total phenolics (mg gallic acid/g koji)
Figure 1. Total phenolic content of fermented black soybeans after
heating at different temperatures for 30 min. Means (bar values) with different letters are significantly different by Duncan’s multiple range test (p < 0.05).
ous heated fermented black soybeans showed no signifi-cant difference regardless of heating temperature (P > 0.05) (Figure 2), while the antimutagenic effect exerted
by the 80 or 100°C-heated fermented black soybeans was significantly less than other heated- and unheated fermented black soybeans. This suggested that antimu-tagenic factors other than phenolic compounds present in the fermented black bean were inactivated during heating at 80 and 100°C and thus lead to the significantly lower level of antimutagenicity observed. On the other hand, various types of phenolic compounds present in the test sample might also affect the observed antimutagenicity. These findings merit further investigation.
IV. The Effect of Heating on the Anthocyanin Content in Fermented Black Soybean
Figure 1 shows the content of anthocyanins in the fermented black soybean after heating at various temper-atures for 30 min. The total level of anthocyanin in the 40 or 60°C heated-fermented black beans was similar to that in the unheated fermented black soybean while a significantly lower (P < 0.05) anthocyanin content was noted in the fermented black soybean after heat-ing at 80°C or higher. In addition, it was noted that as the heating temperature increased from 80 to 100°C, the anthocyanin content further decreased. These results Table 3. Antimutagenicity effect exerted by extracts of unheated- and heated-fermented black soybeans against 4NQO or B[a]P in S.
Typhimurium TA100 Extracts (mg/plate) Unheated Heated (°C) 40 60 80 100 Revertants (CFU/plate) Inhibition a
(%) (CFU/plate)Revertants Inhibition (%) (CFU/plate)Revertants Inhibition (%) (CFU/plate)Revertants Inhibition (%) (CFU/plate)Revertants Inhibition (%) ………4NQO………
Control 311 ± 11b 311 ± 11 311 ± 11 311 ± 11
5 C70 ± 1bc A77.49a D83 ± 7b A73.31b A70.73b D173 ± 10a A44.37c D183 ± 1a A41.15c
2.5 B117 ± 11b B62.37a C131 ± 10b B57.87b B53.05b C276 ± 9a B11.25d C231 ± 1a B25.72c
1.25 B166 ± 8b C46.62a B177 ± 7b C43.09ab C39.54b B261 ± 1a C16.07c B265 ± 2a C14.79c
0.625 A182 ± 6b C41.47a A205 ± 5b D34.08b D29.58c A280 ± 2a D9.96d A285 ± 1a D8.36d
………B[a]P………
Control 177 ± 2 177 ± 2 177 ± 2 177 ± 2 177 ± 2
5 D46 ± 2a A74.01a C45 ± 3b A74.57a D44 ± 1b A75.14a C99 ± 6a A44.06c D84 ± 2a A52.54b
2.5 C69 ± 3c B61.01a B69 ± 2bc B61.01a C73 ± 10ab B58.75b C115 ± 3ab B35.02d C109 ± 1a B38.41c 1.25 B115 ± 4a C35.03ab A109 ± 2a C38.41a A122 ± 8a C31.07b B160 ± 2a C 9.60d B144 ± 3a C18.64c 0.625 A131 ± 1a D25.98a A128 ± 1a D27.68a A145 ± 9a D18.07b A169 ± 2a C4.51c A174 ± 2a D 1.69d a Result are presented as means ± SD from three separate experiments.
b Number of revertants reported is total His+ revertants minus spontaneous His+ revertants per plate. Spontaneous revertants were those observed with sample or control. Statistical differences were calculated by Duncan’s multiple range test. Value in the same row (a, b, c, d) and column (A, B, C, D) with same letters are not significantly different (p > 0.05).
c Inhibition (%) = [1–number of induced revertants in the presence of sample / number of induced revertants in the absence of extract of sample]×100.
Figure 2. Anthocyanin content of black soybean koji after heating at
different temperatures for 30 min. Means (bar values) with different letters are significantly different by Duncan’s multiple range test (p < 0.05). Unheated Temperature (°C) 40°C 60°C 80°C 100°C 0.0 0.2 0.4 0.6 0.8 1.0 a a a b c
indicated that anthocyanin in fermented black soybean is thermolabile at 80°C or higher. Reduction in the anthocyanin content as observed in the present study is overall comparable with reports of Aparicio-Fernández
et al.(1) and Abdel-Aal and Hucl(18). The former
report-ed a significant rreport-eduction in the content of total antho-cyanin in the common bean after typical home cooking (100°C for 2.5 h). The latter observed that the degrada-tion of anthocyanin in blue wheat slurries is greater at 95°C followed by 80 and 65°C. Delgado-Vargas et al.(21)
suggested that heating opened the structure of anthocy-anin to form chalcones, which were degraded further to form brown products. This process possibly led to a similar reduction in the total anthocyanin content in the heated fermented black soybean that was observed in the present study. Finally, it was noted that the trend in the reduction of anthocyanin content caused by heat treat-ment is, in general, similar to that observed on antimuta-genicity (Table 4)
CONCLUSIONS
In conclusion, this study demonstrated potential reduction in both antimutagenicity and content of total phenolics and anthocyanins of fermented black soybeans following heat treatment. The reduction of antimuta-genicity is generally related to the reduced anthocyanin content in the heated fermented black soybean. Note that components of the fermented black soybean showed no mutagenicity, but still possessed antimutagenicity after exposure to 100°C for 30 min. These findings are valu-able when fermented black soybean is further processed and utilized as an ingredient for the formulation of healthy foods.
ACKNOWLEDGEMENTS
This research was financially supported by The National Science Council, ROC (Taiwan). (NSC 95-2313-B-002-017). The starter organism provided by Prof. Yu, R. C. is deeply appreciated.
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![Table 2 shows the antimutagenicity of the unheat- unheat-ed- and heatunheat-ed-fermented black soybean extracts at a dose of 0.625-5.0 mg/plate against 4-NQO and B[a]P in S](https://thumb-ap.123doks.com/thumbv2/9libinfo/8862159.245326/3.892.90.818.156.554/table-antimutagenicity-unheat-unheat-heatunheat-fermented-soybean-extracts.webp)
