With the dramatic increase in mortality of cardiovascular diseases (CVDs) caused by thrombus, this has sparked an interest in seeking more effective thrombolytic drugs or dietary nutriments. The dietary consumption of natto, a traditional Bacillus-fermented food (BFF), can reduce the risk of CVDs. Nattokinase (NK), a natural, safe, efficient and cost-effective thrombolytic enzyme, is the most bioactive ingredient in natto. NK has progressively been considered to have potentially beneficial cardiovascular effects. Microbial synthesis is a cost-effective method of producing NK. Bacillus spp. are the main production strains. While microbial synthesis of NK has been thoroughly explored, NK yield, activity and stability are the critical restrictions. Multiple optimization strategies are an attempt to tackle the current problems to meet commercial demands. We focus on the recent advances in NK, including fermented soybean foods, production strains, optimization strategies, extraction and purification, activity maintenance, biological functions, and safety assessment of NK. In addition, this review systematically discussed the challenges and prospects of NK in actual application. Due to the continuous exploration and rapid progress of NK, NK is expected to be a natural future alternative to CVDs.

The gut microbiota of soymilk intervention is beneficial to maintaining human health. Bacillus subtilis fermented soymilk has brought much interest, due to its richness of thrombolytic nattokinase and the strain of potential probiotic properties. In this study, soymilk was fermented by B. subtilis BSNK-5, and the BSNK-5-fermented soymilk (SMF) on the production of short chain fatty acids (SCFAs) and the regulation of fecal microbiota was initially evaluated by in vitro fecal fermentation. SMF supplementation obviously increased the levels of SCFAs from 32.23 mM to 49.10 mM, especially acetic acid, propionic acid, and isobutyric acid. Additionally, SMF changed the composition and microbial diversity of gut microbiota. After 24 h of anaerobic incubation in vitro, SMF decreased the Firmicutes/Bacteroidota ratio favoring weight loss, increased Lachnospiraceae_UCG-004 and the other beneficial bacteria producing SCFAs, as well as suppressing pathogenic Streptococcus genus. These results revealed the potential use of BSNK-5-fermented soymilk as a potential candidate to promote gut health.

Bacillus subtilis spores have important biological applications; however, high spore-cell densities and sporulation efficiencies in fermentation is poorly reported. This study systematically analyzed the spore densities and formation efficiency of B. subtilis BSNK-5 in different culture substrates. A response surface regression equation was established based on the results of single factor and Box–Behnken experimental designs. The optimal medium formulation, as predicted from the equation, consisted of soluble starch at 3 g·L⁻¹, soybean flour at 12 g·L⁻¹, and MgSO₄ at 5 g·L⁻¹. The spore yield reached 2.43 × 10⁹ CFU·mL⁻¹, and the sporulation rate was 83.3%, which was nearly three times higher than before optimization using an optimized medium at 36 °C and 200 rpm for 60 h.