Enhanced nattokinase production by optimized fermentation conditions of douchi-derived Bacillus subtilis natto NK11 and evaluation of its thrombolytic activity.

Nattokinase is considered effective in preventing and treating thromboembolic diseases due to its significant fibrinolytic activity, thus demonstrating great application potential in the fields of pharmaceuticals and functional foods. In this study, nattokinase-producing strains were isolated and identified from traditional fermented soybean products. The fermentation conditions were optimized via orthogonal experiments, and the crude enzyme was then characterized by SDS-PAGE, zymogram analysis, and in vitro blood clot thrombolytic assay. The results indicated that a strain of Bacillus subtilis NK11 from fermented douchi was isolated and identified. The optimal fermentation conditions were determined to be an initial pH of 6.0, an inoculant concentration of 3%, and fermentation at 36 ℃ for 72 h. These fermentation conditions provided the highest enzyme production and activity (1874.94 ± 77.34 IU/mL), which represented a 1.26 times increase compared to non-optimized. SDS-PAGE analysis revealed that the molecular weight of the crude enzyme was approximately 27 kDa, and the fermentation supernatant contained a limited variety of proteins, with nattokinase as the major component, thereby facilitating purification. Zymogram analysis and blood clot lysis tests demonstrated that the crude enzyme possessed proteolytic activity and exhibited significant thrombolytic activity in vitro. These results lay the foundation for nattokinase production using B. subtilis natto NK11 and its further application.