Dual-purpose Bacillus subtilis fermentation: enhanced nattokinase production via oxygen-enriched fed-batch cultivation and natto starter preparation from harvested biomass.

Nattokinase (NK) is one of the most important functional components in natto, but its content is low. In this study, the fermentation conditions using Bacillus subtilis JZ08-02 for high-yield NK production were investigated, and the residual bacterial pellets were used to prepare a natto starter. Batch fermentation of NK was conducted using a 5 L fermenter, and soybean milk and glucose were used as the substrates. When the stirring speed was increased from 450 to 650 rpm with air supply at 1.0 vvm, NK was increased from 4859 ± 142 to 12,294 ± 226 IU/mL. When pure oxygen was supplied, 15,013 ± 550 IU/mL of NK was obtained. When fed-batch fermentation was conducted, the titer was further elevated to 18,014 ± 112 IU/mL, which was increased by about 76% compared with the previous result. The experimental findings revealed that aeration control and nutrient feeding regimens exerted pronounced effects on NK productivity during submerged fermentation. The crude enzyme supernatant was obtained by centrifugation and the precipitate was collected. With optimized protectant, the bacterial pellets were freeze-dried with 90.1% cell survival rate. Using economical and edible feedstocks, this study achieved a significant enhancement in NK fermentation yield via oxygen-enriched fed-batch cultivation. At the same time, a natto starter was prepared as a by-product using the residual cell waste.