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, KH 2 PO 4 , and MgSO 4 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 ( R 2 ) 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%; KH 2 PO 4 , 0.0016%; and MgSO 4 , 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.