Suppression on the mutagenicity of 4-nitroquinoline-N-oxide by the methanol extracts of soybean koji prepared with various filamentous fungi

In this study, solid fermentation of soybean with various GRAS filamentous fungi including Aspergillus sojae BCRC 30103, Aspergillus oryzae BCRC 30222, Aspergillus awamori, Actinomucor taiwanesis and Rhizopus sp. was performed to prepare various soybean kojis. Toxicity, mutagenicity and suppression on the mutagenesis induced by a direct mutagen, 4-nitroquinoline-N-oxide (4-NQO) on Salmonella typhimurium TA 100, by the various methanol extracts of the prepared soybean koji and unfermented soybean were determined and compared. Results revealed that methanol extracts of unfermented soybean and kojis show no toxicity and mutagenic activity within the dose levels examined on test organism. On the other hand, antimutagenic activity against 4-NQO was observed with the extract of unfermented soybean. Furthermore, fermentation, regardless of the starter organism employed, resulted in an enhanced antimutagenic effect on the mutagenesis of 4-NQO by the extracts of the soybean koji. Across the dose range (0.625-5.0 mg/plate) tested, a dose-dependent antimutagenic activity was observed. Antimutagenic activities of the koji extracts varied with starter organism, with A. awamori-prepared koji extract exhibiting the highest rate of suppression on the mutagenicity of 4-NQO. Further study with A. awamori also revealed that fermentation temperature affected the antimutagenic activity of the prepared koji extract. In general, the extract of the A. awamori-soybean koji prepared at 30 degrees C showed a higher antimutagenic activity than those prepared at 25 or 35 degrees C.     

Antimutagenic activity of Aspergillus awamori-fermented black soybean response to simulated digestive juice treatments and its antimutagenic mechanisms

Aspergillus awamori-fermented black soybeans, a potential healthy food ingredient, possess antimutagenicity against 4-nitroquinoline-N-oxide (4-NQO); a direct mutagen and benzo[a]pyrene (B[a]P; an indirect mutagen). Results of this study revealed that antimutagenic, desmutagenic, and blocking 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 intact B[a]P or S9 mixture with the extract. Instead, it is due to the interaction of B[a]P metabolites with the antimutagenic factors in the extract. 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, 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.     

Mutagenicity and antimutagenic effect of soymilk fermented with lactic acid bacteria and bifidobacteria

In this study, soymilk was first fermented with lactic acid bacteria (Streptococcus thermophilus, Lactobacillus acidophilus) and bifidobacteria (Bifidobacterium infantis, Bifidobacterium longum) both individually and simultaneously. Mutagenicity and the suppression of fermented soymilk against the mutagenesis induced by 4-nitroquinoline-N-oxide (4-NQO), a direct-acting mutagen, and 3,2'-dimethyl-4-amino-biphenyl (DMAB), an indirect-acting mutagen, on Salmonella typhimurium TA 100, was then investigated. It was found that the fermented soymilk shows no mutagenic activity on Sal. typhimurium TA 100. Fermentation, in general, significantly (p<0.05) enhanced the antimutagenicity of soymilk. The levels of increased antimutagenicity of fermented soymilk varied with the starter organism and the type of mutagen tested. Although unfermented soymilk exerted lower antimutagenic activity against DMAB than 4-NQO, the fermented soymilk, generally, showed a higher antimutagenic activity against DMAB than 4-NQO. Among the various fermented soymilk tested, soymilk fermented with both Str. themophilus and B. infantis simultaneously exhibited the highest antimutagenicity of 85.07% and 85.78%, respectively, against 4-NQO and DMAB Further investigation on this fermented soymilk revealed that both the antimutagenic factors formed during fermentation and the cells of the starter organisms contributed to the increased antimutagenic activity against DMAB, while the former led to the increased activity against 4-NQO.     

Enhancements of isoflavone aglycones, total phenolic content, and antioxidant activity of black soybean by solid-state fermentation with Rhizopus spp.

In the present study, a solid-state fermentation process of black soybean was developed. Three filamentous fungi including Rhizopus oligosporus (BCRC 31996), R. oligosporus (NTU-5), and Rhizopus oryzae (BCRC 30894) were cultivated with steam-cooked intact and dehulled black soybeans. It was found that fermentation enhanced the total phenolic and isoflavone aglycone content as well as antioxidant activity of the black soybean methanol [80 % (v/v)] extracts. Among the three candidates, R. oligosporus BCRC 31996 was chosen as the working strain, and dehulled Tainan NO.3 black soybeans were used as the substrate based on the relatively high total phenolic content, isoflavone aglycone accumulation, and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging effect of the fermented extracts. In a 6-day fermentation, extracts of the fermented dehulled black soybeans yielded higher total phenolic content (2,876.2 μg/g) which was 1.16 times of the result from the intact soybean extracts. The DPPH scavenging effect of the fermented dehulled black soybean extract reached its maximum on the third day. The activity of β-glucosidase of dehulled black soybean samples increased with time and resulted in accumulation of isoflavone aglycones. The product of solid-state black soybean fermentation serves as a functional ingredient in the products of nutritional supplements and health foods.     

Fermentation with Bacillus spp. as a bioprocess to enhance anthocyanin content,the angiotensin converting enzyme inhibitory effect,and the reducing activity of black soybeans

Food possessing anthocyanins, angiotensin converting enzyme (ACE) inhibitory activity or reducing activity show beneficial effect on human health. To develop healthy food, black soybeans were fermented with either Bacillus subtilis BCRC 14715 or Bacillus sp. CN11, or a mixture of both Bacillus spp. in the present study. The anthocyanin content, the ACE inhibitory activity and the reducing power of the fermented black soybean were then examined. It was found that the ACE inhibitory activity of the extracts of bean and viscous material from the fermented black soybeans varied with extraction solvents and starter organism, yet increased as the fermentation period was extended, regardless of starter organism. After 18 h of fermentation, the water extract of bean showed less ACE inhibitory activity than did the respective 80% ethanol extract. While the water extract of viscous material showed a higher ACE inhibitory activity than the respective ethanol extract. With respect to extraction yield, it was found that the ACE inhibitor in the fermented black soybean could be extracted more efficiently with water than 80% ethanol. Fermentation with B. subtilis BCRC 14715 was also found to increase the anthocyanin content of black soybean and the reducing activity of the extracts. Finally, the 80% ethanol extract showed a higher reducing activity than the water extract.     

Enhancement of antioxidant activity,total phenolic and flavonoid content of black soybeans by solid state fermentation with Bacillus subtilis BCRC 14715

In the present study, a solid state fermentation of black soybeans with Bacillus subtilis BCRC 14715 was performed. The effect of fermentation on the changes of total phenolic and flavonoid content and antioxidant activities including 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging effect, and Fe²⁺-chelating ability exerted by various solvent (water, 80% methanol, 80% ethanol, 80% acetone) extracts of black soybeans was examined. It was found that fermentation enhanced the total phenolic and flavonoid content as well as antioxidant activity of the black soybean extract. Among the various extracts examined, the acetone extract of fermented black soybeans showed the highest total phenolic and flavonoid content. The acetone extract and the methanol extract of fermented black soybeans showed the highest DPPH free radical-scavenging effect and Fe²⁺-chelating ability, respectively. Analysis of extraction yields showed that the active principle associated with the DPPH radical-scavenging effect was most efficiently extracted from black soybeans using water, regardless of fermentation. Water and methanol effectively extract the Fe²⁺-chelating principles from non-fermented and fermented black soybeans, respectively.     

Effects of Ripening Process on the Mutagenicity and Antimutagenicity of Sufu, a Chinese Traditional Fermented Product of Soybean

In the present study, sufu, the cheese-like fermented product of soybean, was prepared by ripening salted tofu cubes in Aspergillus oryzae-fermented rice?Csoybean koji mash at 35?°C for a period of 16?days. This mixture was held at room temperature (ca. 25?°C) for another 1?month. The mutagenicity and antimutagenicity exerted by the methanol extract of non-fermented tofu and sufu against 4-nitroquinoline N-oxide (4-NQO), a direct mutagen, and 3, 2-dimethyl-4-amino-biphenyl hydrochloride (DMAB), an indirect mutagen, on Salmonella typhimurium TA 100 were examined. Results revealed that the methanol extract of non-fermented tofu and sufu samples collected at various ripening intervals exhibited neither toxicity nor mutagenicity, yet did display antimutagenicity against mutagens across the dose levels examined. Generally, a dose-dependent antimutagenic effect was noted across the dose range examined. The antimutagenicity of the tofu extract toward 4-NQO and DMAB was enhanced significantly (P?<?0.05) through fermentation. Generally, the antimutagenicity of the sufu extract increased with the extension of ripening period. Furthermore, the extract of the non-fermented tofu and sufu, regardless of ripening period, showed a significantly higher (P?<?0.05) antimutagenicity against DMAB than 4-NQO.     

Enhanced antioxidative activity of soybean koji prepared with various filamentous fungi

In the present study, soybean koji fermented with various GRAS filamentous fungi, including Aspergillus sojae BCRC 30103, Aspergillus oryzae BCRC 30222, Aspergillus awamori, Actinomucor taiwanensis and Rhizopus sp. These organisms are commonly used as starters in the fermentation of many traditional, oriental food products. The growth of starter organisms, total phenolic content, and antioxidative activities of the methanol extract of these kojis are compared with specific reference to alpha-diphenyl-2-picryl-hydrozyl (DPPH) radicals scavenging effects, Fe2+-chelating ability, and reducing power. Depending on starter organism, various extents of mycelia propagation (35.23-86.29 mg/g koji) were noted after 3 days of fermentation. Total phenolic content increased in soybean after fermentation. Koji also displayed enhanced antioxidative activates in comparison with the non-fermented soybean. Among the five kinds of koji tested, those fermented with Asp. awamori exhibited the highest levels of DPPH-free radicals scavenging activity, Fe2+-chelating ability and reducing power. The DPPH-free radicals scavenging activity and Fe2+-chelating ability of this soybean koji was ca. 8.9 and 6.7 fold that of the control. Analysis of the dose-response effect also revealed that before reaching a threshold point, there is a linear relationship between increases in antioxidative activity and increases in the concentration of the koji extract. These results show the potential for developing a healthy food supplement with soybean fermented by the GRAS filamentous fungi.     

Storage effects on the content of anthocyanin, mutagenicity and antimutagenicity of black soybean koji

Black soybean koji, possessing antimutagenicity, has been suggested as a potential ingredient for the preparation of healthy food. The objective of this research was to determine the potential mutagenicity and the changes of antimutagenicity of the extract of Aspergillus awamori-fermented black soybeans (koji) held at 4 and 25 °C with or without deoxidant and desiccant for a period of 120 days. It was found that no mutagenic compound formed during the storage period. Depending upon storage temperature and packaging condition, the antimutagenicity of the extract of koji reduced and was associated with a reduction in anthocyanin content during the storage period. Generally, koji stored at 4 °C with both deoxidant and desiccant retained the highest antimutagenicity among the various storage conditions examined. At the end of storage, the extract of black soybean stored at 4 °C with both deoxidant and desiccant retained 79.74 and 87.80% of its original antimutagenicity against 4-nitroquinoline-N-oxide in Salmonella Typhimurium TA98 and TA100, respectively. An extract of black soybean koji stored under similar storage condition showed an antimugenicity residual of 88.23 and 79.34% against benzo[a]pyrene in S. Typhimurium TA98 and TA100, respectively.     

Storage temperature and packaging condition affect the total phenolic content and antioxidant activity of black soybeans and koji

In this study, powders of steamed black soybeans and the Aspergillus awamori‐fermented black soybeans (koji) were subjected to storage at 4 °C and 25 °C with or without deoxidant and desiccant for 120 days. It was found that total phenolic content and the antioxidant activity including the DPPH radicals scavenging effect, Fe²⁺‐chelating ability and reducing activity of the methanol extracts from black soybeans and koji decreased as the storage period was extended. Furthermore, storage temperature and packaging condition affected the antioxidant activity of the methanol extracts of black soybeans and koji. After 120‐day storage, extract from black soybeans holding at 4 °C with deoxidant and desiccant exhibited the highest residual of DPPH radicals scavenging effect, Fe²⁺‐chelating ability and reducing activity of 71.78%, 72.66% and 70.04%, respectively. Meanwhile, the highest residual of 77.78%, 81.71% and 85.05% respectively, was noted with extract from koji held at 25 °C with deoxidant and desiccant.     

Possible mechanisms of antimutagenicity in fermented soymilk prepared with a coculture of Streptococcus infantis and Bifidobacterium infantis

The possible mechanisms of antimutagenicity against 4-nitroquinoline-N-oxide (4-NQO; a direct mutagen) and 3,2'-dimethyl-4-amino-biphenyl (DMAB; an indirect mutagen) were examined in fermented soymilk prepared with a coculture of Streptococcus thermophilus and Bifidobacterium infantis. The antimutagenicity in the fermented soymilk was not due to the bioantimutagenic effect of modulation of DNA repair processes. The mutagenicity of DMAB decreased with increased preincubation of fermented soymilk and the DMAB metabolite but not with intact DMAB or an S9 mixture. Mutagenicity of 4-NQO was not affected by preincubation of fermented soymilk with this mutagen. Mutagenicity of both 4-NQO and DMAB was reduced when Salmonella Typhimurium TA 100 was pretreated with fermented soymilk, indicating that fermented soymilk affected the function of the bacterial cell, which might also lead to reduced mutagenicity of the tested mutagens. Desmutagenic and blocking effects were the main mechanisms of antimutagenicity in the fermented soymilk against DMBA. In contrast, the antimutagenic effect of the fermented soymilk on 4-NQO was primarily due to a blocking effect.     

ANTIMUTAGENIC ACTIVITIES OF PHENOLIC PLANT FLAVONOIDS

Total phenolic plant flavonoids obtained from Thuia accidentalis L. and Cupressus sempevirens L. showed antimutagenic effects on benzo(a) pyrene [B(a)P] in Salmonella typhimurium strains TA100 and TA98. The n-butanol extract was more effective then chloroform and petrol eum-ether extracts, respectively. Aqueous extracts exhibited no inhibition in both strains. Total flavonoids from C. sempevirens showed more antimutagenic activity than flavonoids from T. accidentalis. None of the extracts revealed any cytotoxic or mutagenic activity on the bacterial tester strains.     

Isoflavone Conversion of Black Soybean by Immobilized Rhizopus spp.

Isoflavones exhibit many bioactive benefits, such as antioxidation, antimutagenic effects, and reduction of symptoms of postmenopause conditions. Aglycone forms of isoflavone have shown more biological effects over their glycosylated ones. Most isoflavones in nature, however, exist as glycosylated forms and cannot easily be absorbed in the intestines. The purpose of this study was to develop a fermentation process of black soybean to produce functional food using a Luffa cylindrical fiber biofilm reactor. Three filamentous-fungi, including Rhizopus oligosporus (BCRC 31996), Rhizopus oligosporus (NTU-5), and Rhizopus oryzae (BCRC 30894), were cultivated in black soybean medium. The assay using 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging effect of fermented broth and isoflavone aglycosylation rate of genistin and daidzin were evaluated as indicators to determine the most suitable strain. After a 2-day cultivation in flasks, DPPH radical scavenging effect of R. oligosporus NTU-5 reached 83%, which is the maximum value among three strains. R. oligosporus NTU-5 was subsequently chosen as target strain and used for the scale-up of fungal-based aerobic growth. The optimal cultivation condition obtained in a 5-L bioreactor with Luffa cylindrical fiber was 30°C, 0.1 vvm aeration, 100 rpm agitation, 10% inoculum volume, and 6% (w/v) black soybean as medium. After a 6-day cultivation, R. oligosporus NTU-5 converted isoflavones to their aglycones, daidzein and genistein, and these were 59.7 and 23.6 μM, respectively. Isoflavone aglycosylation rate of daidzin and genistin were 77.6 and 31.8%, respectively. The DPPH scavenging effect was 62%.     

Evaluation of antioxidative and mutagenic properties of 50% ethanolic extract from red beans fermented by Aspergillus oryzae

Various bean products fermented by microorganisms are commonly consumed in Asian diets; however, the safety or functional properties of fermented beans can vary with different microbial species and with different processes being applied to different beans. The objectives of this study were to evaluate the antioxidative and mutagenic properties of 50% ethanolic extracts from red beans fermented by Aspergillus oryzae. The extracts' antioxidative activities, including alpha,alpha;-diphenyl-beta-picryl-hydrazyl (DPPH) radical-scavenging effects, Fe(2+)-chelating ability, and reducing power, were studied in vitro. The antioxidative effects provided by the extracts depended strongly on their concentrations. In general, antioxidative activity increased with extract concentration to a certain point and then leveled off as the concentration further increased. The fermented red bean extracts showed less of a scavenging effect on the DPPH radical and less reducing power than the commercial antioxidants alpha-tocopherol and butylated hydroxytoluene, but better Fe(2+)-chelating ability. No mutagenicity or toxicity effect on any of the tested strains (Salmonella Typhimurium TA97, TA98, TA100, TA102, and TA1535) was found for the 50% ethanolic extracts of fermented red beans with the Ames mutagenicity assay. These results suggest that the 50% ethanolic extracts were not mutagenic.     

Mutagenic Safety and Fatty Liver Improvement of Nanonized Black Soybeans in Senescence-Accelerated Prone-8 Mice

Abstract: Nanotechnology, as a new enabling technology, has the potential to revolutionize food systems. However, much attention has been focused on nanoparticle foods due to their potential physiological properties. This study was aimed to evaluate the mutagenic safety and fatty liver improvement of black soybean in senescence-accelerated mice (SAMP8). The mutagenic activity of black soybeans was investigated using the Ames test (Salmonella Typhimurium TA98, 100, 102, and 1535). Furthermore, senescence-accelerated prone-8 mice (SAMP8) have been reported to display spontaneous fatty liver. Male SAMP8 mice were divided into control and supplemented with 10% micronized or nanonized black soybeans diet and fed for 12 wk. The results revealed that the Ames test of micronized and nanonized black soybeans exhibited no mutagenicity. Administration of black soybeans to mice showed no effects on food intake and body and organ weights. The nanonized black soybean group had a lower degree of spontaneous fatty liver, alanine aminotransferase, and thiobarbituric acid-reactive substance concentrations, and had enhanced superoxide dismutase, catalase, and glutathione peroxidase activities of livers when compared with the SAMP8 control and micronized black soybean groups. The mice fed with black soybeans had significantly lower triglyceride concentrations than the SAMP8 control group. The results of this study suggest that nanonized black soybeans have no side effects and, moreover, may minimize liver lesions in SAMP8 mice.     

Antimutagenic and antimicrobial activities of pu-erh tea

The biological action of water extract of pu-erh tea (WEPT) was evaluated by Salmonella mutagenesis assay and bacteria test. Like green tea, oolong tea and black tea, WEPT showed neither cytotoxicity and nor mutagenicity toward Salmonella typhimurium TA98 and TA100 with or without S9 mix (an external metabolic activation system). WEPT at 0.5-5 mg/plate expressed a dose-dependent inhibitory effect against both the mutagenicity of aflatoxin B₁ (AFB₁), an indirect mutagen which requires metabolic activation, and 4-nitroquinoline-N-oxide (NQNO), a direct mutagen, in Salmonella typhimurium TA98 and TA100. In general, the antimutagenic activity of WEPT against AFB₁ and NQNO was weaker than other tea extract because of the least amount of total catechin in WEPT. For antimicrobial action, WEPT, green tea, oolong tea and black tea at 2.0 mg/ml showed inhibitory effect on growth of Staphylococcus aureus and Bacillus subtilis, but no effect on Escherchia coli. Obviously, WEPT has potential antimicrobial effect on gram-positive Staphylcoccus aureus and B. subtilis than that of gram-negative E. coli. In addition, caffeine and epicatechin (EC), main polyphenolic compounds in WEPT, showed both antimutagenic and antimicrobial effect against strains mentioned above, which may partially account for the antimutagenic and antimicrobial action of pu-erh tea.     

Antigenotoxic effects of water extract from Korean fermented soybean paste (doen-jang)

Aflatoxin B1 is a major metabolite of the toxigenic molds Aspergillus flavus and Aspergillus parasiticus. In this study, a bacterial reverse mutation assay with Salmonella Typhimurium strains TA1535, TA1537, TA98, TA100, and TA102 and an in vitro chromosome aberration test with Chinese hamster lung (CHL) cells were used to investigate the genotoxicity of water extract from Korean soybean paste (doen-jang [dwen-jahng]) and its antigenotoxic activity against aflatoxin B1. The water extract itself did not exhibit cytotoxicity or mutagenicity. The extract significantly reduced the numbers of revertants when it was added to the assay system with Salmonella Typhimurium TA100 (P < 0.05). The extract also exhibited significant inhibitory effects on chromosome aberration in CHL cells (P < 0.05). Dose-response relationships were observed between the concentration of the water extract and both its antimutagenic effect and its suppression of chromosome aberration. The results of this work indicate that water extract from Korean soybean paste could have potential as an antigenotoxic substance.     

Effects of three biological thiols on antimutagenic and antioxidant enzyme activities

The antimutagenic and antioxidant enzyme activities of captopril (CAP), cysteine (CYS), and glutathione (GSH) were evaluated for finding concentration-dependent inhibitory effects against: (1) the mutagenicity of 2-amino-3-methylimidazo [4,5-f] quinoline (IQ), an indirect mutagen; and (2) N-methyl-N′-nitrosoguanidine (MNNG), a direct mutagen toward Salmonellatyphimurium TA98 and TA100. Of the three thiols, CYS and GSH exhibited better inhibitory effects against IQ-induced mutation toward TA98 and TA100, respectively. GSH also showed a protective effect against MNNG-induced mutation toward TA98 and TA100, meanwhile, CAP showed the least inhibitory effect. CYS, GSH, and CAP also dose-dependently increased the activities of glutathione transferase, glutathione peroxidase, and glutathione reductase in hepatic BNL cells. CAP showed the superior inducing effects on glutathione transferase activity. These data suggested that the bioactive properties of biological thiols might contribute to their effects of antimutagenic activities as well as regulation on activities of antioxidant enzymes.     

Bioavailability of Zinc in Fermented Soybeans

The bioavailability of zinc in soybean meal fermented by lactic acid-producing bacteria (LAPB) and by Rhizopus oligosporus in tempeh fermentation was evaluated with the rat bioassay, using the slope ratio method and log total femur zinc or weight gain versus zinc intake as response parameters. Fermentation significantly increased (P < 0.05) the RBV of zinc from 73.0% in boiled non-fermented soybeans (pH 6.7) to 84.5% in boiled LAPB fermented soybeans. The highest RBV (95.3%) was found in the autoclaved and LAPB fermented product (pH 6.5). Rhizopus oligosporus fermentation resulted in a RBV of 89.0%. Thus, fermentation in the processing of soybeans will improve zinc bioavailability in this food.     

Functional Properties of Freeze-Dried Powders of Unfermented and Fermented Aqueous Extracts of Legume Seeds

Functional properties of freeze-dried powders made from aqueous extracts of cowpea, peanut and soybean seeds were compared with those of a commercial cultured buttermilk powder product. Powders of seed extracts fermented with Lacrobacillus bulgaricus and Streptococcus thermophilus were also analyzed. The color of legume powders was similar to that of the commercial buttermilk product, and fermentation had a beneficial effect. Emulsion capacities of powders prepared from extracts were superior to the commercial product, as were foam capacity and stability. The nitrogen solubility profiles of powders from unfermented extracts were similar to those of powdered buttermilk; however, solubility of nitrogen in fermented extracts was less than that of unfermented products. The viscosity of rehydrated fermented powders was less than that of the controls but greater than that of the commercial product. The water adsorption capacity of various seed extract powders was similar, regardless of fermentation treatment.