Use of Bacillus subtilis to enrich isoflavone aglycones in fermented natto

BACKGROUND: Natto is a food made by fermenting cooked soybeans with Bacillus subtilis. Soybean isoflavones are reported to provide many health benefits, including oestrogenic effects. However, isoflavone aglycones may be absorbed faster and in higher amounts in the human intestine than their glucosides. This study aimed to investigate the content of isoflavone components in commercial natto products as well as the use of B. subtilis strains to ferment cooked soybeans to produce a high level of isoflavone aglycones in natto. RESULTS: The content and composition of isoflavones in commercial natto products were predominantly (>76%) isoflavone glucosides. Fermentation of cooked soybeans with B. subtilis BCRC 14718 at 37 °C for 48 h was more effective in converting glucosides to aglycones than with other strains of B. subtilis, increasing the proportion of isoflavone aglycones from 12 to 68% of the total isoflavones in the fermented natto. The proportions of the isoflavone aglycones daidzein and genistein in cooked soybeans fermented with B. subtilis BCRC 14718 for 48 h increased from 6 to 54% and from 5 to 13% respectively. CONCLUSION: Bacillus subtilis BCRC 14718 incubated with cooked soybeans produces higher levels of isoflavone aglycones, which may enhance health benefits over traditional fermented natto. Copyright © 2008 Society of Chemical Industry     

纳豆食品发酵条件的优化

纳豆是日本的传统发酵食品,由大豆经纳豆菌纯种发酵后制成。因其不但营养价值丰富,而且含有多种生物活性物质,使其成为保健食品开发的热点之一。该文以成品纳豆在我国发展的限制条件为依据,使纳豆口味能够为我国居民更好地接受,对其发酵条件的影响因素,即浸泡时间、蒸煮时间、接种量、发酵时间和温度进行了优化,得出纳豆的最佳发酵条件是:浸泡时间为18h,121℃蒸煮40min,接入10%培养12h的种子液,35℃发酵18h,于4℃冰箱后熟24h。     

Volatile compounds in Bacillus‐fermented soybeans

Thua nao, a traditional, proteolytic, fermented soybean condiment of northern Thailand, was prepared from cooked whole soybeans by natural flora fermentation. The microbial flora during the fermentation was dominated by Bacillus species. The formation of volatile compounds during the fermentation was studied. In addition, the volatile compounds of two samples of commercial dried thua nao and two samples of commercial Japanese natto were analysed. Fermentation led to a large increase in the concentration of total volatile compounds, from 35 µg kg^?1 wet weight in cooked soybeans to 3500 µg kg^?1 wet weight in 72 h fermented material. The major volatile compounds in fermented beans were 3‐hydroxybutanone (acetoin), 2‐methlybutanoic acid, pyrazines, dimethyl disulphide and 2‐pentylfuran. Sun drying of 72 h fermented material resulted in the loss of 65% of total volatiles, including important aroma compounds. The commercial dried thua nao samples had low concentrations of total volatile compounds (380 µg kg^?1 wet weight). It is suggested that improved drying/preservation methods are needed to retain aroma compounds in the traditional products. The natto samples were devoid of aldehydes, aliphatic acids and esters, and sulphur compounds, whereas the thua nao samples contained a diversity of these compounds. Previous investigators have reported these compounds in natto and it is not possible to suggest the existence of systematic differences between the volatile compounds in traditional thua nao prepared with an undefined, mixed microbial flora and those in natto fermented with Bacillus subtilis. © 2001 Society of Chemical Industry     

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.     

苦荞对发酵豆乳纳豆激酶活力、风味及抗氧化活性影响

相较于纳豆,以纳豆芽孢杆菌发酵获得的发酵豆乳食用更方便,但同样存在纳豆激酶活性低、风味不良等缺点。作者首先研究了苦荞、糙米、薏米、藜麦、燕麦粉对纳豆芽孢杆菌产纳豆激酶能力的影响。结果表明,添加苦荞的效果最好,当添加质量分数为3%时,纳豆芽孢杆菌液体发酵的酶活力由原来的5 803.12 U/mL提高到23 326.23 U/mL;然后研究了苦荞粉对纳豆芽孢杆菌发酵豆乳的纳豆激酶活力、风味及抗氧化活性的影响。结果显示,当添加的苦荞质量为大豆质量的50%时,发酵豆乳的纳豆激酶活力、挥发性盐基氮质量浓度和总抗氧化能力分别为 4 460.28 U/mL、3.45 mg/dL和16.34 mmol/L,分别为纯大豆发酵所得豆乳的161.42%、79.84%和196.39%。添加苦荞能够明显提高发酵豆乳中纳豆激酶活力,并且改善风味和抗氧化活性。该研究可为开发富含纳豆激酶、食用方便的纳豆食品提供借鉴。     

黑豆纳豆发酵工艺的研究

为给进一步开发利用黑豆纳豆提供一定的理论和依据,以黑豆为原料、以纳豆芽孢杆菌为菌种,对影响黑豆纳豆的发酵工艺如浸泡时间、蒸煮时间、接种量、发酵时间和纳豆产品制作最佳工艺条件等因素进行研究。通过单因素实验、正交试验和产品感官评定,得到的黑豆纳豆发酵的最佳条件为：黑豆浸泡时间为20 h,添加NaC l 0.5%,葡萄糖2%,蒸煮时间为35 m in（121℃）,接种量为2.5%,发酵温度为37℃,最佳发酵时间为24 h,在4℃下后熟24 h。     

纳豆芽孢杆菌固态发酵小米糠产抗氧化肽工艺优化

为提高小米糠蛋白资源的利用率,为小米糠的深加工提供参考,采用纳豆芽孢杆菌对小米糠进行固态发酵以获得小米糠抗氧化肽。在单因素试验的基础上以小米糠发酵后水提液的总抗氧化能力(total antioxidant capacity,T-AOC)为指标,使用Plackett-Burman试验对发酵条件进行筛选,然后使用响应面法对发酵条件进行优化,并测定优化后小米糠水提液的多肽含量和1,1-二苯基-2-三硝基苯肼(1,1-diphenyl-2-picrylhydrazyl,DPPH)自由基清除能力。结果显示最适发酵条件为:小米糠平均粒径0.22 mm(60~80目)、菌液接种量0.4 mL(约108 CFU/mL)、初始pH 6.7、发酵时间26 h、发酵温度35℃。此条件下小米糠固态发酵后提取液的T-AOC实际值为(344.51±8.02)U/g小米糠,多肽提取量为(68.37±0.92)mg/g小米糠,清除DPPH自由基IC50为0.12 mg/mL。该研究表明,小米糠固态发酵条件经过优化后能够获得具有较高抗氧化能力的生物活性肽。     

苦荞纳豆酱的抗氧化活性

为研究苦荞纳豆酱总黄酮和总酚的体外抗氧化活性,测定DPPH自由基清除率、ABTS+·清除率、·OH清除率、O2－·清除率和Fe3+还原能力,并且与黄豆酱、甜面酱进行抗氧化活性比较研究。结果表明：苦荞纳豆酱的DPPH自由基清除率、ABTS＋·清除率、·OH清除率、O2 － ·清除率均高于黄豆酱和甜面酱;Fe3+还原能力略低于甜面酱。被测样品的抗氧化活性与总黄酮、总酚的含量有显著相关性（P＜0.01）。     

中式纳豆制备技术的研究

为探索出符合中国人口味且纳豆激酶酶活力高的中式纳豆加工技术,该研究从市售的4种纳豆中分离出4株纳豆芽孢杆菌（Bacillus natto）,并筛选出一株产纳豆激酶能力最强的菌株NK3。以牛奶培养基替代传统的种子液培养基培养纳豆菌种,通过单因素试验考察发酵温度、时间、接种量、加水量对纳豆激酶酶活力的影响,采用正交试验优化纳豆发酵条件。结果表明,以牛奶为培养基培养的纳豆菌生长良好,在发酵温度27 ℃、发酵时间1.5 d、接种量5%、加水量6%条件下,菌株NK3发酵制备出的纳豆无氨臭味,有淡淡的奶香味,且纳豆激酶酶活力高达668.5 U/g。     

纳豆菌生产微量生理活性物质的研究

高桥和宫城纳豆菌接种于大豆豆渣或大豆煮汁粉培地都能增殖。固体培养时,只有宫城菌在20%豆渣培地能生产出2,6-吡啶二羧酸。液体培养时,0.5%大豆煮汁粉培地比2%大豆煮汁粉培地生产了多量的2,6-吡啶二羧酸;同样的液体培养条件,高桥菌比宫城菌能生产出更多的维生素K2(MK-7)。     

米糠饮料发酵条件的研究

采用纳豆芽孢杆菌对米糠进行发酵,通过交互试验比较接种量、发酵温度和发酵时间对纳豆激酶活性和饮料感官评分的影响,确定最佳发酵条件为:接种量4%、发酵温度35℃和发酵时间24h。在此条件下可以获得酶活性达到621.5U/mL、口感较好、兼具米糠营养成分和纳豆营养产物的新型发酵饮料。     

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.     

纳豆加工工艺研究

为了开发纳豆这种保健食品,对纳豆蒸煮及发酵工艺进行了研究。以大豆感官品质为指标,确定大豆蒸煮条件为121℃高压蒸煮40 min;研究了蔗糖加量、纳豆芽孢杆菌接种量、发酵温度和发酵时间对溶栓酶活性的影响。结果表明,这些因素对溶栓酶活性影响显著,最佳的发酵条件为蔗糖添加量2.0%、接种量1.5%、发酵温度36℃、发酵时间32 h,在此条件下溶栓酶活力为1 892 U/g。     

Role of Carbohydrate-Cleaving Enzymes in Phenolic Antioxidant Mobilization from Whole Soybean Fermented with Rhizopus oligosporus

Previous research has suggested a relationship between free phenolic content and β-glucosidase activity in solid-state fermented food substrates and to amylase activity in germinating soybeans. This study was undertaken to examine the role of a number of carbohydrate-cleaving enzymes in phenolic antioxidant mobilization from whole soybean during solid-state fermentation. In addition to total soluble phenolic content, α- and β-glucosidase, α-amylase, and β-glucuronidase activities were measured in extracts of soybean fermented with a food-grade fungus, Rhizopus oligosporus. Antioxidant activity of the extracts was determined as 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging ability. Our results demonstrate that while total soluble phenolic content increased 120–135% in the extracts, increased antioxidant activity (+61%) was limited to the early fermentation period, with activity decreasing with increased culture time. Higher antioxidant activity was linked to increased glucosidase and glucuronidase activities, while high total phenolic content partly linked to increased amylase activity. The overall results (enzymatic activities and phenolic antioxidant contents) suggest the possible involvement of lignin remobilization and/or degradation activities, as well as phenolic detoxification activities, by Rhizopus oligosporus in phenolic antioxidant mobilization from fermented whole soybean.     

纳豆固态发酵工艺优化

为了得到高品质的纳豆,以优质黄豆为原料,采用纳豆激酶含量和黏液产率为评价指标,研究初始含水量、接种量、发酵温度、发酵时间对纳豆激酶含量和黏液产率的影响。根据单因素试验结果,对纳豆固体发酵工艺进行正交旋转组合设计的优化,得到纳豆固体发酵工艺的最佳参数为初始含水量为51%,接种量为0.15%,发酵温度为43 ℃,发酵时间为24 h,该条件下纳豆激酶含量为0.076 mg/mL,黏液产率为17.60%。     

鹰嘴豆纳豆发酵过程中挥发性成分分析

采用顶空固相微萃取-气质联用技术（HS-SPME-GC-MS）对鹰嘴豆纳豆挥发性成分进行了测定与分析。结果表明,发酵24 h、36 h、48 h和60 h的鹰嘴豆纳豆分别鉴定出54、50、39和37种挥发性成分,包括醇类、醛类、酮类、酸类、酯类、吡嗪类、烃类、氨气和其他类物质,其中吡嗪类、酸类、酯类是鹰嘴豆纳豆的主要挥发性成分。吡嗪类物质在发酵36 h时相对含量大幅提高,其中2,3,5,6-四甲基吡嗪在48 h时含量最高,为17.95%;酸类物质相对含量随着发酵时间延长先减少后增加,其中反式阿魏酸含量在48 h时最高,为15.16%;酯类物质随发酵时间的延长逐渐降低,从发酵24 h的17.54%减少到60 h的2.89%。     

利用纳豆菌种发酵奶液的研究

纳豆激酶具有溶解血栓的作用,纳豆激酶是由纳豆菌发酵大豆产生,纳豆有比较特殊的味道,利用纳豆芽孢杆菌进行奶液发酵,能够获得较高的纳豆激酶活力,而且没有特殊的气味,本文针对不同发酵时间对利用纳豆菌发酵奶液在色泽、口感、pH、芽孢杆菌数、纳豆激酶酶活力进行测定,结果表明利用纳豆菌对奶液进行发酵,成熟只需16h,就能得到较高的纳豆激酶酶活力,并能在4℃冰箱中保存5天,酶活的减少率低于10%。     

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.     

纳豆芽孢杆菌发酵菜用大豆中总黄酮的提取及其降血脂效果评价

本研究以菜用大豆等外品为原料,通过纳豆枯草芽孢杆菌发酵来提高其总黄酮含量,为提高菜用大豆产品的附加值提供理论支持。实验利用纳豆芽孢杆菌对菜用大豆等外品进行发酵,以发酵产品中总黄酮含量为评价指标确定纳豆发酵的最优条件,通过D101型大孔树脂对提取的总黄酮进行纯化,采用芦丁比色法测定总黄酮含量。体内实验分为肥胖预防实验和缓解实验:以C57BJ/6L小鼠为模型,预防实验在饲喂高脂饲料的同时,灌胃总黄酮提取物溶液（50 mg/kg·d）;缓解实验是利用高脂饲料建立起肥胖小鼠模型后,灌胃低、中、高剂量的总黄酮提取物溶液（分别为20、50和100 mg/kg·d）。通过小鼠体重、血脂以及促炎因子等指标评价总黄酮溶液的降脂效果。结果表明,菜用大豆发酵生产黄酮的最佳条件为:枯草芽孢杆菌接菌量为1.15 × 1011 CFU/100 g菜用大豆,发酵温度37 ℃,发酵时间24 h,后熟时间12 h,此时纳豆总黄酮得率为4.30 mg/g菜用大豆;测得该粗提物中黄酮含量为22.1%;预防实验组给予黄酮处理后,小鼠体重和Lee's指数与正常对照组相比均无显著差异(P >0.05)。缓解实验中高、中、低剂量黄酮提取物处理组小鼠血清中甘油三酯（TG）含量分别为313.10 ± 10.12、310.39 ± 31.76和310.1 ± 10.20 nmol/L,与正常对照组（330.80 ± 34.36 nmol/L）无显著差异（P>0.05）;黄酮各组灌胃干预后,小鼠血清总胆固醇（TC）、白介素-6（IL-6）和肿瘤坏死因子-α（TNF-α）含量均较高脂模型组显著下降（P<0.05）。结论:利用纳豆芽孢杆菌发酵菜用大豆提高了其总黄酮含量,该黄酮提取物具有预防和缓解高脂饮食小鼠血脂升高的效果。     

响应面法优化以豆渣为基质发酵纳豆菌

为提高豆渣的附加值,以豆渣为基质液体发酵纳豆菌;首先用Plackett-Burman方法对影响纳豆菌发酵因素的效应进行评价并筛选出了具有显著效应的三个因素:初始pH值、发酵温度和麸皮含量,其他因素对纳豆菌发酵无显著影响;然后采用最陡爬坡实验逼近最大响应区域,最后由响应面实验确定了主要影响因素的最佳条件:初始pH6．02、发酵温度36．4℃、麸皮0．31%;在优化培养条件下,纳豆菌发酵液活菌数达到4．35×10~9CFU/ml。