纳豆激酶高产菌株的选育及固态发酵技术

Strain Improvement of Bacillus subtilis for Enhanced Production of Nattokinase and Optimization of Solid-State Fermentation Conditions

以枯草芽孢杆菌为出发菌株,选用超声波和紫外线进行诱变处理,根据致死率、突变率与诱变剂量的关系选择合适的诱变剂量,以筛选高产纳豆激酶的优势菌株。实验表明：最佳诱变条件为：超声波45 k Hz、2 80 W,时间60 min,紫外线照射距离30 cm,时间120 s。经多次初筛、复筛,选育出一株命名为BSCZ-4的优势菌株,其纳豆激酶酶活力为原始菌株的1.96倍。对该菌种进行连续20代培养,测得其溶解圈直径稳定在18~19 mm之间,表明该突变株遗传特性稳定。并采用Box-Behnken响应曲面法优化其固态发酵工艺条件,结果为：魔芋精粉添加量5%、接种量8%,34℃发酵24 h,纳豆激酶活力可达（4 087.83±93.75）U/g。

In this study, the nattokinase-producing capacity of Bacillus subtilis subsp. subtilis BS21076 was improved by sequential mutagenization with ultrasonic followed by UV light at the appropriate doses determined according to death and mutation rates. The optimal mutation conditions of strain BS21076 were established as 60 min ultrasonic treatment at 45 k Hz and 280 W followed by 120 s UV irradiation at a distance of 30 cm. A high nattokinase-producing mutant strain named BSCZ-4, producing 1.96 times higher nattokinase than BS21076, was obtained after several rounds of primary and secondary screening. After 20 consecutive passages, the diameter of the zone of dissolution produced by BSCZ-4 remained stable in the range of 18–19 mm, suggesting good genetic stability. Using Box-Behnken response surface methodology, the optimal culture conditions for the enhanced production of nattokinase by BSCZ-4 in solid-state fermentation were determined as addition of 5% konjac power to the culture medium, an inoculum size of 8%, and 24 h culture at 34 ℃, resulting in a nattokinase activity of（4 087.83 ± 93.75） U/g.