Enhancing natto quality by co-fermentation with Pediococcus pentosaceus PP04 and Bacillus velezensis BV702: Nattokinase activity, nutritional profile, and flavor characteristics

<i>Bacillus</i> spp. are the main nattokinase (NK) producers, but monoculture fermentation often causes an unpleasant ammoniacal odor. This study first selected <i>B. velezensis</i> BV702 as a starter strain based on NK activity and sensory evaluation, and then introduced probiotic <i>Pediococcus pentosaceus</i> PP04 for co-fermentation to improve natto quality. The results demonstrated that under optimal conditions (strain ratio 1:2 [PP04:BV702], inoculation amount 5 %, fermentation temperature 37 °C, time 24 h), NK activity (3050 U/g) and sensory scores of natto were significantly enhanced. Mixed fermentation increased the essential amino acid content (582.79 mg/100 g) while reducing bitter amino acids. Volatilomics analysis revealed elevated levels of esters and characteristic flavor compounds (e.g., acetylpyrazine and 2-ethyl-3,5-dimethylpyrazine) in co-fermented natto. Notably, fruity, sweet, and nutty flavors intensified, whereas fatty and waxy odors diminished. This study provides insights into probiotic applications in natto production.