纳豆芽孢杆菌发酵菜用大豆中纳豆激酶的提取及其性质分析  

Extraction and properties of nattokinase from vegetable soybean fermented with Bacillus natto

作　　者：李秀凉 李倩文[1,3] 朱玉 韩晓云 孙庆申 宋永[1,2] LI Xiuliang;LI Qianwen;ZHU Yu;HAN Xiaoyun;SUN Qingshen;SONG Yong(Engineering Research Center of Agricultural Microbiology Technology,Ministry of Education,Heilongjiang University,Harbin 150500,China;Key Laboratory of Molecular Biology,College of Heilongjiang Province,School of Life Sciences,Heilongjiang University,Harbin 150080,China;Key Laboratory of Microbiology,College of Heilongjiang Province,School of Life Sciences,Heilongjiang University,Harbin 150080,China)

机构地区：[1]黑龙江大学农业微生物技术教育部工程研究中心,黑龙江哈尔滨150500 [2]黑龙江大学生命科学学院黑龙江省普通高等学校分子生物学重点实验室,黑龙江哈尔滨150080 [3]黑龙江大学生命科学学院黑龙江省普通高等学校微生物重点实验室,黑龙江哈尔滨150080

出　　处：《中国酿造》2020年第12期106-112,共7页China Brewing

摘　　要：以菜用大豆为原料,直投式纳豆芽孢杆菌作为发酵剂,纳豆激酶活力作为评价指标,通过单因素和正交试验优化纳豆激酶发酵工艺条件;通过盐溶、硫酸铵分段盐析、二乙氨乙基(DEAE)阴离子交换柱分离纯化纳豆激酶,并采用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)确定其分子质量,并进行酶学性质和体外溶栓效果分析。结果表明,菜用大豆发酵生产纳豆激酶的最佳条件为:每100 g蒸煮菜用大豆接入1.15×10^11 CFU/g直投发酵剂,发酵温度37℃、发酵时间36 h、后熟时间18 h时,此时纳豆激酶活力可达2326.60 IU/g。纳豆激酶的分子质量介于25~35 kDa之间,最适作用温度、pH值分别为37℃、8.0,当温度低于40℃、pH值在6.0~8.0时较稳定;体外溶栓实验表明纳豆激酶具有良好的体外溶栓效果。Using vegetable soybean as the substrate,Directed-Vat-Set Bacillus natto as the starter,and nattokinase activity as evaluation index,the fermentation conditions of nattokinase were optimized by single-factor experiments and orthogonal experiments.Then the extracted nattokinase was isolated and purified by salting-in,salting-out with gradient ammonium sulfate solution and dicthylaminoethyl(DEAE)anion exchange column,and the molecular mass was determined through sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE).Finally,the enzymatic property and in vitro thrombolytic properties were also analyzed.The results shown that the optimal conditions for nattokinase production through vegetable soybean fermentation were obtined as follows:On 100 g vegetable soybean base,1.15×10^11 CFU/g Bacillus natto was inoculated as starter with fermentation temperature 37℃,fermentation time 36 h and post-ripening time 18 h.The purified nattokinase activity was up to 2236.60 IU/g with molecular mass between 25-35 kDa.The optimal temperature and pH for nattokinase activity was 37℃and 8.0,respectively.It showed good stability under 40℃and pH 6.0-8.0.The results of in vitro thrombolysis showed that nattokinase had good thrombolysis effect.

关 键 词：纳豆激酶 发酵 分离纯化 稳定性 体外溶栓 

分 类 号：TS201.3[轻工技术与工程—食品科学]