Bacillus velezensis M4 from Northeast Chinese Soybean Paste Combines Nattokinase and Antibacterial Activities

A bacterial strain M4 exhibiting high nattokinase (NK) activity and favorable antibacterial properties was isolated from fermented soybean paste in Northeast China. Based on morphological observation, physiological and biochemical characterization, 16S rDNA sequence analysis, and whole-genome sequencing, the strain was identified as Bacillus velezensis. Its probiotic potential and safety were systematically evaluated using a combination of in vitro assays and genome mining. Genomic analysis revealed that M4 possessed a complete genome consisting of a single circular chromosome of 4,473,838 bp with a GC content of 46.94%, encoding 4516 predicted proteins. Functional domain annotation identified four proteins (XLQ58132.1, XLQ58158.1, XLQ59409.1, and XLQ59873.1) containing both the Peptidase inhibitor I9 and Peptidase S8 domains, confirming the presence of the typical molecular signature of NK. Furthermore, the genome harbored 132 genes encoding carbohydrate-active enzymes, 37 biosynthetic gene clusters, and 142 genes encoding proteolytic enzymes. Comparative genomic analysis revealed a close phylogenetic relationship with other B. velezensis strains and identified 98 strain-specific genes. Safety assessment demonstrated that M4 exhibited no hemolytic activity, was susceptible to eight commonly tested antibiotics, and lacked genes encoding high-risk virulence factors. Probiotic characterization indicated that M4 exhibited certain levels of gastrointestinal tolerance, acid resistance, bile salt resistance, antioxidant activity, and antibacterial properties. In conclusion, B. velezensis M4 shows potential for development as a functional strain.