鹰嘴豆纳豆液态发酵高产蛋白酶的培养基及发酵条件优化

为了提高纳豆中的蛋白酶活力,以纳豆芽孢杆菌为菌种,对鹰嘴豆纳豆液态发酵进行优化。以蛋白酶活力为指标,采用Plackett-Bnrmao法筛选出三个对蛋白酶活力影响最大的因素:装液量、转速和鹰嘴豆粉添加量。通过响应面优化鹰嘴豆纳豆液态发酵的培养基和发酵条件,建立二次回归模型的拟合度良好,对提高蛋白酶活力影响显著(p<0.005),所得最佳培养基为鹰嘴豆粉添加量5.9%,豆粕粉1.0%,葡萄糖0.6%,氯化钠0.5%,最佳发酵条件为:转速250 r/min,装液量76 mL/500 mL,温度37℃,发酵时间48 h。该条件下发酵所得蛋白酶活力达(3558.0±1.5) U/mL,相对于对照培养基的(2491.4±2.8) U/mL提高了42.8%,且纤维蛋白平板法验证的纳豆激酶溶解圈面积提高了108.6%。结果表明,优化后的鹰嘴豆纳豆液态发酵能有效提高蛋白酶活力。     

纳豆激酶高产菌的快速筛选及其发酵纳豆特性

通过纤溶活性筛选法和纳豆激酶基因筛选法,快速、准确筛选到一株纳豆激酶高产菌株MJ-1,经16S rRNA基因序列分析,该菌株被鉴定为枯草芽孢杆菌。以黄豆为原料进行固体发酵制备纳豆食品,考察了纳豆食品的纳豆激酶活力、抗凝活性和抗氧化活性。在初始含水量60%、发酵温度37 ℃的条件下,纳豆激酶发酵活力在20 h达到最大值（90.18 FU/g,以干质量计）,达到了商业化纳豆的酶活力水平。抗凝活性分析显示该菌株可合成抗凝成分,赋予纳豆食品抗凝活性,在提取物质量浓度为3 mg/mL时,抗凝效率达91%。同时,与未发酵黄豆相比,发酵过程显著提高了其抗氧化活性。     

混菌发酵纳豆的工艺研究

为改善纳豆风味,该研究以市售大豆为原料,采用纳豆发酵粉和乳酸菌粉混合发酵,并以感官评分、挥发性盐基氮含量和纳豆激酶活力为考察指标,采用单因素试验和正交试验对纳豆发酵工艺进行优化。结果表明,混菌发酵纳豆最优发酵工艺为菌粉比例（乳酸菌∶纳豆芽孢杆菌）1.0∶1.5,发酵温度40 ℃,发酵时间27 h。在此最优发酵工艺下,纳豆感官评分为9.0分,挥发性盐基氮含量为11.02 mg/100 g,纳豆激酶活力为438.90 U/g,此时纳豆色泽鲜亮,氨臭味明显降低,拉丝状态好,纳豆品质明显优于市售纳豆。     

一株纳豆芽孢杆菌的产酶条件优化

从一株实验室保藏的产纳豆激酶细菌出发,先对其液体发酵产酶的培养基和培养条件进行优化,在此优化基础上,利用Plackett-Burman试验筛选出影响纳豆芽孢杆菌产酶的3个主要因素:胰蛋白胨添加量、MgSO4添加量、发酵温度,利用Box-Behnken进行响应面试验设计,优化得到最优发酵条件。发酵培养基条件(g/L):胰蛋白胨26.6、乳糖10.0、Na2HPO45.0、NaH2PO41.0、CaCl20.2、MgSO41.35;发酵条件:种龄12h、接种量2%、发酵温度33℃、发酵时间56h、装液量50mL/250mL。此发酵条件下发酵液的纳豆激酶酶活力为743.65U/mL,比优化前提高了232.33U/mL。     

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%。     

纳豆芽孢杆菌发酵豆渣上清液的抗氧化功能研究

目的:为深度开发豆渣副食资源,考察了纳豆芽孢杆菌发酵豆渣的条件。方法:采用单因素和正交实验,以羟自由基清除率为衡量指标,对纳豆芽孢杆菌发酵豆渣的条件进行了优化。结果:以5%的豆渣为碳源,初始pH7.0、接种量4%、装瓶量25mL/250mL、温度为32℃、200r/min、发酵24h,纳豆芽孢杆菌发酵豆渣的抗氧化活力最高。结论:实验表明纳豆芽孢杆菌发酵豆渣能产生较强的抗氧化活力,在保健食品和绿色饲料方面具有开发潜力。     

纳豆菌液态发酵荞麦产纳豆激酶及其代谢特性分析

对纳豆枯草芽孢杆菌（Bacillus subtilis natto）液态发酵荞麦产纳豆激酶揺瓶、2.5 L发酵罐条件进行优化。对比自制大豆分离蛋白酶解物与商业大豆蛋白胨作为补充氮源时菌体生长规律以及代谢物质（酶、可溶性蛋白、还原糖、多酚及抗氧化物质）变化规律。纳豆菌液态发酵荞麦产纳豆激酶2.5?L发酵罐最优条件为荞麦浸泡6?h后,按料液比1∶10（g/mL）加水打浆,加入0.4%?α-淀粉酶,90?℃加热40?min,补充NS37071酶解12?h时所得酶解物,调节发酵培养基pH?7.0,接种量3%,通气量3.5?L/min,转速300?r/min,装液量1.2?L,发酵36?h。与商业大豆蛋白胨相比,补充大豆分离蛋白酶解物时,纳豆菌生长对数期较长,可溶性蛋白与还原糖的消耗量较大,在36?h趋于平稳,纳豆激酶活力持续上升至36?h达到最大值,酚类物质比溶出速率在12?h达到最大值,抗氧化物质比生成速率在6?h达到最大值。以荞麦为原料,补充自制大豆分离蛋白酶解物,通过优化纳豆菌液态发酵条件,可制备具有高纳豆激酶活力（152.5?FU/mL）、富含谷物多酚（0.109?mg/mL）且具有强抗氧化活性（27.43?μmol/mL）的发酵产物。     

纳豆菌液态发酵谷物产纳豆激酶及发酵产物抗氧化活性研究

本文研究了纳豆菌液态发酵时间对纳豆菌生物量、蛋白酶酶活、纳豆激酶酶活的影响。在此基础上,研究添加四种谷物(糙薏仁、玉米、荞麦和糙米)对纳豆菌液态发酵产蛋白酶酶活、纳豆激酶酶活、谷物总酚迁移率、总酚残留率以及发酵产物抗氧化活性的影响。结果表明:添加糙米和荞麦可显著促进纳豆菌产蛋白酶(2444.19、1813.71 U/mL)、纳豆激酶(719.67、681.38 U/mL),且以荞麦为底物所产发酵产物的谷物总酚迁移率最高(0.64 mg/g干谷物)、抗氧化活性最强(30.37μmol trolox equiv/mL);采用浸泡蒸煮处理四种谷物、延长发酵时间可显著提高其发酵产物的蛋白酶酶活、纳豆激酶酶活、谷物总酚迁移率以及抗氧化活性。采用浸泡蒸煮处理荞麦,利用纳豆菌液态发酵其48 h,可制备富含纳豆激酶、谷物多酚、肽类物质且具有强溶栓、抗氧化活性的功能性食品。     

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。     

高产纳豆激酶的枯草芽孢杆菌优选及发酵工艺条件优化

以黄豆为主料,添加不同辅料,采用不同处理方式,以产酶活力为指标研究三株枯草芽孢杆菌发酵性能,筛选优势菌株进行固态发酵,确定最优工艺条件。结果表明:在三株受试菌种中BS21076发酵性能最优,活菌数及纳豆激酶活力均较高;最优固态发酵条件为:大豆90℃下烘炒5min,破碎度为四瓣,魔芋精粉添加量3%,接种量为8%,37℃下发酵24h;在此条件下,酶活力可达(3582.48±83.13)U/g,较传统发酵NK活力极显著(p0.01)提高。     

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。     

Screening of a Bacillus subtilis strain producing both nattokinase and milk-clotting enzyme and its application in fermented milk with thrombolytic activity

Bacillus subtilis is a generally recognized as safe probiotic, which is used as a starter for natto fermentation. Natto is a functional food with antithrombus function due to nattokinase. Compared with natto, fermented milk is a more popular fermented food, which is commonly fermented by Lactobacillus bulgaricus and Streptococcus. However, there is no report on B. subtilis–fermented milk. In this study, to produce a functional fermented milk with antithrombus function, a B. subtilis strain (B. subtilis JNFE0126) that produced both nattokinase and milk-clotting enzyme was isolated from traditionally fermented natto and used as the starter for the functional fermented milk. In liquid fermentation culture, the peak values of thrombolytic activity and milk-clotting activity were 3,511 U/mL at 96 h and 874.5 Soxhlet unit/mL at 60 h, respectively. The optimal pH and temperature were pH 7.0 at 40°C for nattokinase and pH 6.5 and 55°C for milk-clotting enzyme, respectively. The thrombolytic activity in the fermented milk reached 215.1 U/mL after 8 h of fermentation. Sensory evaluation showed that the acceptance of the milk fermented by B. subtilis JNFE0126 was similar to the traditional milk fermented by L. bulgaricus and S. thermophilus. More importantly, oral intake of the fermented milk by the thrombosis-model mice prevented the development of thrombosis. Our results suggest that B. subtilis JNFE0126–fermented milk has potential as a novel, functional food in the prevention of thrombosis-related cardiovascular diseases.     

Characterization of fermented black soybean natto inoculated with Bacillus natto during fermentation

BACKGROUND: To make nutrients more accessible and further increase biological activity, cooked black soybeans were inoculatedwith Bacillus natto and fermented at 37 °C for 48 h. The changes in physiochemical properties of fermented black soybean natto were investigated. RESULTS: The inoculation procedure significantly increased moisture, viscosity, color, polyphenol compounds and anthocyanin, and significantly decreased hardness after 48 h fermentation. Fibrinolytic and caseinolytic protease, β‐glucosidase activities, TCA‐soluble nitrogen, and ammonia nitrogen contents in the inoculated samples significantly increased as fermentation time increased. Genistin and daidzin concentrations gradually decreased with increased fermentation time. However, genistein and daidzein increased with fermentation time, which reached 316.8 and 305.2 µg g^?1 during 48 h fermentation, respectively. DPPH radical scavenging activities of the fermented black soybeans increased linearly with fermentation time and concentration. Compared with the soaked black soybeans and cooked black soybeans, the fermented black soybeans with B. natto resulted in higher scavenging activity towards DPPH radicals, which correlated well with the content of total phenols (r = 0.9254, P < 0.05) and aglycone isoflavone (r = 0.9861, P < 0.05). CONCLUSION: Black soybean natto fermented by B. natto has the potential to become a functional food because of its high antioxidant activity. Copyright © 2010 Society of Chemical Industry     

纳豆菌分离、鉴定及纳豆激酶高产菌株的正向选育

从日本纳豆食品中分离到1株枯草芽孢杆菌纳豆亚种(Bacillus subtilis natto)Bs01-1菌株,根据纳豆芽孢杆菌产纳豆激酶与蛋白酶活性的正相关关系,采用紫外线直接照射涂菌蛋白平板进行诱变育种的,成功地筛选到2株突变株MBS04-6和MBS04-9,其溶纤活性分别比出发菌株提高了87%和69%,达到4 179 IU/mL和3788 IU/mL。该法筛选菌株的正向突变率达到10%。     

纳豆激酶高活性菌株的筛选及其发酵条件的优化

为开发具有特定生理活性的新型纳豆保健品,从24种豆制品中分离和筛选出3株有明显纤溶活性的芽孢杆菌菌株,其中BN-6菌株活性最高,达到900U/ml。通过单因素试验和正交实验确定了液体发酵纳豆激酶最佳条件培养液中豆粕粉2%,玉米粉1%,初始pH=6.5,温度37℃。     

富含纳豆激酶抹茶纳豆酸奶的发酵工艺研究

该研究以富含纳豆激酶（NK）的纳豆豆浆和复原乳为主要原料,研制富含NK的抹茶纳豆酸奶。分别探究纳豆豆浆、白砂糖、抹茶粉添加量、乳酸菌接种量对富含NK抹茶纳豆酸奶感官评分的影响,采用正交试验优化富含NK抹茶纳豆酸奶发酵工艺,并对富含NK抹茶纳豆酸奶的品质进行评价。结果表明,富含NK抹茶纳豆酸奶最适发酵工艺条件为：纳豆豆浆添加量6%,白砂糖添加量8%,抹茶粉添加量0.7%,乳酸菌接种量0.3%。在此优化条件下,酸奶的感官评分为88.6分,脂肪、蛋白质、非脂乳固体含量、酸度、NK活性及乳酸菌数分别为3.15 g/100 g、3.08 g/100 g、8.25 g/100 g、82 °T、688.6 U/mL、5.8×108 CFU/mL。其各品质指标均符合国标GB 19302—2010《发酵乳》要求,且具有一定的保健功能。