Enhancing menaquinone-7 biosynthesis by optimized fermentation strategies and fed-batch cultivation of food-derived Bacillus subtilis

Background: Menaquinone-7 (MK-7), a type of vitamin K₂ (VK₂), is considered to be a health-promoting functional ingredient in the current recommended dietary supply. However, it is a challenge to isolate high-yield MK-7-producing strains with reasonable safety from natural food, which further limits their use as starter culture.

Results: In this study, an MK-7 production strain derived from commercial natto with a yield of 2.33 ± 0.06 mg L^-1, Bacillus subtilis 4-b4, was screened using a selective medium containing rotenone combined with a VK₂ enzyme-linked immunosorbent assay kit, and high-performance liquid chromatography. This strain exhibited excellent safety with no hemolysis, excellent antioxidant activity, and sensitivity to Antibiotics. Furthermore, its fermentation strategies (fermentation condition and the supply of key precursor substances) were optimized to improve MK-7 biosynthesis. As a result, the yield increased 3.31-fold, reaching 7.70 ± 0.24 mg L^-1 at shake flask fermentation level. After scaling up through fed-batch fermentation, the yield further increased to 23.43 ± 0.41 mg L^-1 in a 5 L bioreactor.

Conclusion: The current study developed a strain with good safety and Antibiotic sensitivity for producing MK-7, and improved its biosynthesis through optimized fermentation strategies and scaling up the process, laying the foundation for the availability of biologically active MK-7 fermented food in the food industry. © 2025 Society of Chemical Industry.

Bacillus; fed‐batch; functional ingredient; menaquinone‐7; optimization.