Abstract: The submerged fermentation conditions of buckwheat for nattokinase production in shake flasks and a 2.5 L fermentor using Bacillus subtilis natto were optimized. Changes in bacterial growth and the metabolism of nutrients (nattokinase, soluble protein, reducing sugar, phenolics and antioxidants) during fermentation using soybean protein isolate hydrolysate and commercial soybean peptone as a supplementary nitrogen source were comparatively studied. The optimized fermentation conditions in a 2.5 L fermentor were obtained as follows: 6 h of buckwheat soaking, pulping at a solid-to-water ratio of 1:10 (g/mL), addition of 0.4% α-amylase, heating for 40 min at 90 ℃, supplementation of 12 h hydrolyzed soybean protein isolate (SPI) with a commercial protease preparation (NS37071), pH adjustment to 7.0, inoculum size 3%, ventilation rate 3.5 L/min, stirring speed 300 r/min, medium volume 1.2 L, and 36 h fermentation. Supplementation of SPI hydrolysate prolonged the logarithmic growth phase and led to increased consumption of reducing sugar and soluble protein as compared with soybean peptone. Residual reducing sugar and soluble protein tended to level off at 36 h of fermentation with supplementation of SPI hydrolysate, and simultaneously the maximum nattokinase activity was obtained. In addition, the specific dissolution rate of phenolics and the specific formation rate of antioxidants reached a maximum at 12 and 6 h, respectively. The nattokinase activity of the fermentation products obtained under the optimized conditions was 152.5 FU/mL, which were rich in phenolics (0.109 mg/mL) and antioxidants (27.43 μmol/mL).

Key words: nattokinase, submerged fermentation, buckwheat, soy protein isolate hydrolysates, antioxidant