利用奶粉豆粕培养基,采用稀释平板分离法,以H/C(透明圈直径/菌落直径)为指标,从日本传统食品纳豆中分离出9株蛋白酶产生菌。将这些初筛菌株进行发酵培养,采用Folin-酚法测定酵上清液的蛋白酶活性,选出蛋白酶活性最高的菌株H2,并依据形态特点和主要生理生化特征,鉴定为芽孢杆菌纳豆亚种(Bacillussubtilis sub.Natto)。最后以蛋白酶活性为指标,考察培养基初始pH值、温度、装液量、接种量和种龄等因素对H2菌株液态发酵的影响。结果表明,该菌株液态发酵产蛋白酶的适宜条件是:培养基初始pH值7、培养温度40℃、装液量40 mL/250 mL、接种量3%、种龄25 h。

H/C(transparent circle diameter/colony diameter) taken as indicator,nine protease-producing strains were isolated from the Japanese traditional natto food,which cultured in soybean milk powder mixed with milk powder.Folin-phenol method used for detecting the protease activity of the fermented supernatant,and the highest protease activity strain H2 was selected,which was identified as Bacillus subtilis sub.natto according to morphological and physiological characteristics.In order to investigate the effect of initial medium pH,temperature,broth content,inoculation amount and seed age on protease activity of H2 strain,single factor experiment has been done.The results showed that the best conditions were: initial medium pH7,incubation temperature 40 ℃,broth content 40 mL/250 mL,3% inoculation amount and seed age 25h.