This study investigates the feasibility of using black turtle beans (Phaseolus vulgaris) as an alternative substrate for natto fermentation to produce nattokinase, an enzyme with substantial fibrinolytic activity. Given the allergenic concerns associated with soybean-based natto, black turtle beans offer a potential hypoallergenic alternative. Four indigenous Bacillus sp. strains (CO18, MO39, TO46, and MS05) were evaluated for their fibrinolytic enzyme production. Results showed that Bacillus subtilis TO46 exhibited the highest enzyme activity, with optimized fermentation conditions being determined using Response Surface Methodology (RSM) and Central Composite Design (CCD). Key factors such as initial pH, peptone concentration, bacterial density, and fermentation time were optimized to enhance nattokinase production. Under optimal conditions, a remarkable nattokinase activity of 418.32 FU/mL was achieved, surpassing that of traditional soybean-based natto. The findings suggest that black turtle beans are a promising alternative substrate for natto fermentation, offering potential benefits for individuals with soybean allergies while maintaining high fibrinolytic activity.

Index Terms- Bacillus subtilis; black turtle bean natto; fermentation optimization; nattokinase activity; soy allergies