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.     

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.     

Enhanced Antioxidative Activity of Chinese Traditionally Fermented Okara (Meitauza) Prepared with Various Microorganism

In order to investigate the antioxidative activity of Meitauza koji (Meitauza, a Chinese traditionally fermented okara), the okara was fermented with Aspergillus oryzae 3.951, Rhizopus oligosporus 3.912, Actinomucor elegans 3.118 and Bacillus subtilis B2 as kojis. All the methanol extract yields and peptide content of the kojis were higher than that of non-fermented okara. Moreover, the Meitauza kojis displayed enhanced antioxidative activities in comparison with the non-fermented okara. Among the four kinds of kojis tested, those fermented with B. subtilis B2 exhibited the highest levels of 2,2′-azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals scavenging activities and reducing power. The ABTS and DPPH radicals scavenging activities of Meitauza koji fermented with B. subtilis B2 were ca. 12.53 and 7.92 folds that of the non-fermented okara. 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 koji extract. In addition, the peptide content of the Meitauza koji prepared with B. subtilis B2 increased from 8.6 mg to 34 mg per 100 mg extract. These results suggest the potential for developing traditional functional food with okara fermented by B. Subtilis B2.     

Fermentation temperature affects the antioxidant activity of the enzyme-ripened sufu, an oriental traditional fermented product of soybean

In this study, sufu, a Chinese traditional fermented product of soybean, was prepared by ripening salted tofu cubes in the mash of Aspergillus oryzae-fermented rice-soybean koji possessing various hydrolytic enzymes at 25°C, 37°C and 45°C. Antioxidant activity including 2,2-diphenyl-2-picylhydoxyl (DPPH) radical-scavenging activity, Fe(2+)-chelating ability and reducing power exerted by the methanol extract of sufu was determined and compared with that of the non-fermented tofu extract. It was found that antioxidant activity of the sufu extracts was, generally, higher than the non-fermented tofu extract. Ripening temperature and the duration of ripening period affected the antioxidant activity of the sufu extracts. Taking into account of extraction yields, the sufu product ripened at 45°C for 16 days showed the most profound enhancement in the DPPH radical-scavenging effect and Fe(2+)-iron-chelating ability, which is 3.4 and 11.5 folds, respectively, that noted with the non-fermented tofu.     

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.     

Mutagenic and antimutagenic effects of methanol extracts of unfermented and fermented black soybeans

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 degrees C and for times of 1-5 days revealed that, generally, the extract prepared from beans fermented at 30 degrees C for 3 days exhibited the greatest inhibition against the mutagenic effects of 4-NQO and B[a]P.     

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.     

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.     

Antioxidant activity of lactic-fermented Chinese cabbage

Lactic-fermented cabbage, similar to Kimchi in Korea, is a very popular fermented vegetable product in Taiwan and China. In this study, fermented cabbage prepared by a dry-salt method was first extracted with water and methanol. Antioxidant activity such as DPPH radical scavenging effects, reducing power and Fe²⁺-chelating ability of the solvent extracts of fermented cabbage was determined and the effect of fermentation on the change of antioxidant activity, total phenolic and total flavonoid content was also investigated. Results revealed that antioxidant activity observed on the Chinese cabbage mixture may vary with extraction solvents and fermentation. Generally, the methanol extract of the cabbage mixture showed a higher DPPH radical scavenging activity and reducing activity than the water extract. Although, fermentation did not alter the Fe²⁺-chelating ability and reducing activity of the methanol extract of the cabbage mixture, it reduced these same antioxidant activities in the water extract. Amongst the various extracts examined, the methanol extract of fermented cabbage showed the highest DPPH radical scavenging effect. On the other hand, the highest Fe²⁺-ion chelating and reducing activities were exerted by the methanol extracts of both the cabbage mixture and the fermented cabbage, which showed no significant difference (p < 0.05). Additionally, the type of solvent and fermentation were also found to affect the total phenolic and flavonoid content of the extracts. Fermentation increased the total phenolic content of the methanol extract, whilst reducing the total flavonoid content of the water extract. Furthermore, changes in the antioxidant activity observed on the extracts of the cabbage mixture and fermented cabbage did not coincide exactly with the total phenolic and total flavonoid content.     

Microbial Changes in Fermented Peanut and Soybean Pastes Containing Kojis Prepared using Aspergillus oryzae and Rhizopus oligosporus

Microbiological profiles of peanut and soybean misos containing 6 and 12% NaCl and kojis prepared with Aspergillus oryzae and Rhizopus oligosporus were monitored over a 90-day period of fermentation. Although total plate counts in misos containing R. oligosporus koji fluctuated while counts in misos containing A. oryzae koji remained constant during the fermentation period, the final microbial populations in sixteen test formulae were similar to the initial counts. Mold populations in mists containing two types of kojis declined similarly during fermentation. There was little difference in populations of Saccharomyces rouxii in misos containing the two types of mold kojis during the first month of fermentation; however, higher numbers of S. rouxii were noted in mitis containing R. oligosporus koji compared to those containing A. oryzae koji during later stages of fermentation. The final pH values of misos were similar, even though misos containing A. oryzae koji had a larger magnitude of drop in pH than did misos containing R. oligosporus koji over the 90-day test period.     

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.     

Effect of mixed kojis on physiochemical and sensory properties of rapid‐fermented fish sauce made with freshwater fish by‐products

The objective of this study was to investigate the effect of mixed kojis on essential indices of fish sauce. Fish sauce inoculated with mixed kojis (FSB, Aspergillus oryzae koji:Aspergillus niger koji = 3:1) and A. oryzae koji (FSA, control) was prepared. At the end of fermentation, the content of amino nitrogen, total soluble nitrogen, Free amino acid (FAA) and glutamic acid in FSB increased by 7.50%, 7.84%, 9.80% and 28.43% compared to FSA, due to higher acid protease activity in mixed kojis. Moreover, flavour groups including phenols, ketones and nitrogen‐containing compounds in FSB were also improved. According to the results of sensory evaluation, FSB showed higher intensity of umami and caramel attributes, lower intensity of ammonia and sour attributes. All the results suggested that the usage of mixed kojis to produce fish sauce was an effective method that can accelerate the fermentation process and improve the flavour of fish sauce made with freshwater fish by‐products.     

菌种接种方式对黄豆酱品质的影响

以沪酿3.042米曲霉、AS 3.35黑曲霉、AS 3.324甘薯曲霉、沪酿3.130毛霉、AS 3.972红曲霉、米根霉和枯草芽孢杆菌为菌种进行制曲、发酵,通过测定发酵7 d后蛋白酶活力和氨基酸态氮含量,研究单菌种制曲发酵、曲料混合发酵和多菌种混合制曲发酵方式对黄豆酱品质的影响。结果表明:曲料混合发酵产蛋白酶活力和氨基酸态氮含量效果最好,其次为多菌种混合制曲发酵和单菌种制曲发酵。曲料混合发酵和多菌种混合制曲发酵中,2菌种和3菌种组合产蛋白酶活力和氨基酸态氮含量整体较高,其中曲料混合发酵5号(沪酿3.042米曲霉、AS 3.35黑曲霉和枯草芽孢杆菌)产蛋白酶活力和氨基酸态氮含量最佳,分别达到713 U/mL和0.89 g/100 g。研究发现,沪酿3.042米曲霉成曲、AS 3.35黑曲霉成曲和枯草芽孢杆菌成曲按照曲料混合发酵方式接种更有利于得到品质较优黄豆酱。     

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.     

Effect of fermentation on the antioxidant activity of red beans (Phaseolus radiatus L. var. Aurea) ethanolic extract

The antioxidant activities of 50% ethanol extracts from red bean non-fermented and fermented by Bacillus subtilis or Aspergillus oryzae were determined using Sprague–Dawley rats as a testing model. Oral administration of all the extracts decreased the malondialdehyde (MDA) level in the liver but not in the brain tissue. Bacillus subtilis fermented extract increased the levels of ascorbic acid, α-tocopherol and glutathione (GSH) and the superoxide dismutase (SOD) activity in the liver tissue, while it increased only the ascorbic acid level in the brain tissue. Aspergillus oryzae fermented extract increased the levels of GSH and the SOD activity in the liver tissue, and it also increased GSH and ascorbic acid in the brain tissue. In general, the extracts from fermented red bean were more effective than the non-fermented extract in raising the antioxidant levels in the liver tissue.     

提高郫县豆瓣甜瓣子品质的工艺条件优化

以郫县豆瓣生产过程中分离到的米曲霉（Aspergillus oryzae）、黑曲霉（Aspergillus niger）、根霉（Rhizopus）为出发菌种,以感官评价和氨基酸态氮及综合酶活力为评价指标,筛选出功能米曲霉（Aspergillus oryzae）SICC3.1083和SICC3.930进行提高甜瓣子品质研究。通过单因素试验,筛选出混合种曲比例、发酵温度、盐分3个显著影响因素,采用响应面法优化混合种曲发酵工艺条件。结果表明,最佳发酵工艺为米曲霉SICC3.1083和SICC3.930质量比1.0∶1.0、发酵温度40 ℃、发酵时间25 d、盐分12%。在此优化工艺条件下,甜瓣子感官评分为88分,氨基酸态氮含量为0.78 g/100 g,比优化前分别提高了17%和20%,感官评分和理化指标均优于传统发酵工艺。     

Physical and Chemical Changes in Fermented Peanut and Soybean Pastes Containing Kojis Prepared Using Aspergillus oryzae and Rhizopus oligosporus

Studies were made to determine and compare physical and chemical changes occurring during 90 days of fermentation of miso-like products containing peanuts and soybeans as well as a combination of these oilseeds. Two koji molds (Aspergillus oryzae and Rhizopus oligosporus) and two levels of NaCl (6 and 12%) were evaluated. The L color values decreased more rapidly in misos containing A. oryzae koji compared to misos containing R. oligosporus koji and changes occurred earlier in low-salt formulae than in high-salt formulae. The type of mold koji had no apparent effect on changes in viscosity. The free fatty acid content of misos increased dramatically during the first 4 days of fermentation. Misos containing A. oryzae koji had higher soluble nitrogen and free amino acid content in the final product than did those containing R. oligosporus kojis. Peanut miso products had higher soluble nitrogen contents than did respective soybean products; however, the type of oilseed had little effect on accumulation of free amino acids.     

菌种耦合发酵米渣生酱油的品质和抗氧化活性

以高盐稀态法酿造米渣生酱油,探究菌种耦合对其品质和抗氧化活性的影响。4组不同菌种耦合发酵的米渣生酱油品质测定结果表明其各项质量指标均达到GB 18186—2000《酿造酱油》中一级酱油的标准,其中S4生酱油(米曲霉耦合黑曲霉和鲁氏酵母发酵)游离氨基酸总量达到41.81 g/L,4种米渣生酱油的总氨基酸、必需氨基酸、呈味氨基酸和抗氧化氨基酸的质量浓度均高于同时发酵的大豆生酱油S5生酱油(米曲霉发酵)。S3生酱油(米曲霉耦合鲁氏酵母发酵)和S4生酱油的抗氧化活性普遍高于S1生酱油(米曲霉发酵)和S2生酱油(米曲霉耦合黑曲霉发酵),各结果均表明S3的抗氧化活性最强,清除羟自由基的能力达到等质量浓度VC的最高175.42倍,对1,1-二苯基-2-三硝基苯肼自由基清除能力达到VC的2.16倍。抗氧化活性受发酵温度影响明显,分别在35℃和50℃发酵时出现峰值。多菌种耦合发酵对米渣生酱油品质和抗氧化活性均有明显改善,其中鲁氏酵母发挥了明显作用。     

7株米曲霉与沪酿3.042发酵制备郫县豆瓣用甜瓣子的对比研究

为了筛选高效的具有潜在应用价值的郫县豆瓣生产专用米曲霉菌株,研究了7株高产蛋白酶、α-淀粉酶的米曲霉试验菌株(24M-1、21M-2、25M-1、19M-2、BM-2、18M-1、DM1)与沪酿3.042米曲霉在制备甜瓣子中的发酵性能差异,考察了不同菌株发酵的种曲中中性蛋白酶、酸性蛋白酶、α-淀粉酶的酶活力差别;进一步评价了由试验菌株制作的种曲发酵的甜瓣子的氨基酸态氮、总酸、挥发性风味物质、感官评价等发酵特性指标。结果表明,菌株24M-1、DM1发酵甜瓣子的各项发酵特性指标表现突出,甜瓣子的综合品质较佳,明显优于沪酿3.042米曲霉发酵的甜瓣子,可进一步优化菌株培养条件及发酵工艺参数,为其在郫县豆瓣加工中的应用奠定了基础。