市售川产水豆豉分离株中高产蛋白酶与β-葡萄糖苷酶菌株的菌属鉴定与产酶能力评价

Identification of the Strain Which Highly Produces Protease and β-D-glucosidase Isolated from Shuidouchi Produced in Sichuan and Evaluating Its Ability of Producing Protease

目的筛选并鉴定72株川产水豆豉分离株中高产蛋白酶及β-葡萄糖苷酶的菌株,为深入研究水豆豉的营养价值及开发其发酵菌株提供实验依据。方法采用酪蛋白法和对硝基苯-β-D半乳糖吡喃糖苷(pNPG)法分别测定菌株产蛋白酶和β-葡萄糖苷酶的能力,选出优势菌株,通过形态学、生化特征、16S rRNA和基质辅助激光解析-电离飞行时间质谱(MALDI-TOF-MS)对其进行鉴定。绘制菌株生长曲线和产酶曲线并测得其遗传稳定性。结果选择产蛋白酶能力前10名菌株中β-葡萄糖苷酶活力最高者(0.084 U/L)作为优势菌株,表型及分子生物学法鉴定其为枯草芽孢杆菌(Bacillus subtilis)。菌株生长曲线、产酶曲线符合枯草芽孢杆菌特性,遗传稳定性结果显示其产酶能力能稳定遗传至第9代并于第10代开始下降。结论从72株川产水豆豉分离株中筛选出的高产蛋白酶和β-葡萄糖苷酶菌株经鉴定为枯草芽孢杆菌。

Objective To select and identify the bacterium which highly produces protease andβ-D-glucosidase from 72 strains of Shuidouchi from Sichuan,and to provide evidence for further research on its nutritional value and fermentation strain exploiting.Methods Casein degradation test and pNPG chemical test were applied respectively to detect the capacity to produce protease andβ-D-glucosidase of each strain.Characteristics of morphology,biochemistry,16 S rRNA and MALDI-TOF-MS were used to identify the fermentation strain,which genetic stability,curves of growth and enzyme producing were also obtained.Results The strain with the highest enzyme activity ofβ-D-glucosidase(0.084 U/L)among the top 10 strains for producing protease was selected as the fermentation strain and was identified as Bacillus subtilis,which curves of growth and enzyme producing conformed as well.The result of genetic stability showed that capacity of enzyme producing was stable until the 10 th generation.Conclusion The fermentation strain which highly produced protease andβ-D-glucosidase was selected from 72 strains of shuidouchi from Sichuan and was identified as Bacillus subtilis.