Optimization of the Medium Components by Statistical Experimental Methods to Enhance Nattokinase Activity

Natto, a traditional Japanese soy food, contains nattokinase, a potent fibrinolytic enzyme with fibrinolytic properties. Statistical experimental methods were used to find the optimal conditions of nattokinase production by Bacillus natto. Effects of various nitrogen, carbon, and inorganic salt sources were investigated to select the optimal medium components. In the variable screening, a 2^5–1 fractional factorial design was used to examine the experimental variables. Screening experiments showed that glucose, KH₂PO₄, and MgSO₄ significantly affected the enzyme activity of nattokinase. Based on these results, the Box-Behnken design and response surface methodology were applied to find the optimal conditions. A regression model was built by fitting the experimental results with a second-order polynomial. The regression model was statistically significant since the coefficient of determination (R²) was 0.9352. By analyzing the response surface plots and their corresponding contour plots with the regression model, the optimal variable conditions were obtained as follows: glucose, 0.065%; KH₂PO₄, 0.0016%; and MgSO₄, 0.0016%. The corresponding maximal activity of nattokinase was 12.34 FU/mL. Confirmation experiments were performed and the results showed that the difference between the predicted and experimental values of nattokinase activity were within 15%. A theoretical approach calculated from numerical calculation is in agreement with the experimental data.