Effect of Fermentation Parameters on Natto and Its Thrombolytic Property

Abstract

Natto is a popular food because it contains a variety of active compounds, including nattokinase. Previously, we discovered that fermenting natto with the combination of Bacillus subtilis GUTU09 and Bifidobacterium animalis subsp. lactis BZ25 resulted in a dramatically better sensory and functional quality of natto. The current study further explored the effects of different fermentation parameters on the quality of natto fermented with Bacillus subtilis GUTU09 and Bifidobacterium BZ25, using Plackett-Burman design and response surface methodology. Fermentation temperature, time, and inoculation amount significantly affected the sensory and functional qualities of natto fermented with mixed bacteria. The optimal conditions were obtained as follows: soybean 50 g/250 mL, triangle container, 1% sucrose, Bacillus subtilis GUTU09 to Bifidobacterium BZ25 ratio of 1:1, inoculation 7%, fermentation temperature 35.5 °C, and fermentation time 24 h. Under these conditions, nattokinase activity, free amino nitrogen content, and sensory score were increased compared to those before optimization. They were 144.83 ± 2.66 FU/g, 7.02 ± 0.69 mg/Kg and 82.43 ± 5.40, respectively. The plate thrombolytic area and nattokinase activity both increased significantly as fermentation time was increased, indicating that the natto exhibited strong thrombolytic action. Hence, mixed-bacteria fermentation improves the taste, flavor, nattokinase activity, and thrombolysis of natto. This research set the groundwork for the ultimate manufacturing of natto with high nattokinase activity and free amino nitrogen content, as well as good sensory and thrombolytic properties.

Keywords: combination fermentation; natto; nattokinase; thrombolytic property.

Figure 1

Effects of NaCl addition (a₁,b₁,c₁) and sucrose addition (a₂,b₂,c₂) on natto fermentation. Different letters above each bar means significant differences.

Figure 2

Effects of strain proportion (a₁,b₁,c₁) and inoculation volume (a₂,b₂,c₂) on natto fermentation. Different letters above each bar means significant differences.

Figure 3

Effects of fermentation temperature (a₁–c₁) and fermentation time (a₂–c₂) on natto fermentation. Different letters above each bar means significant differences.

Figure 4

Effects of after-ripening time on natto (a–c). Different letters above each bar means significant differences.

Figure 5

Response surface diagram of NK activity and FAN content as a response to the interaction of: (A₁,A₂) fermentation temperature and fermentation time (inoculation amount = 6%), (B₁,B₂) inoculation amount and fermentation time (temperature = 34 °C), (C₁,C₂) inoculation amount and fermentation temperature (time = 30 h).

Figure 6

Response surface diagram of sensory score as a response to the interaction of: (A) fermentation temperature and fermentation time (inoculation amount = 6%), (B) inoculation amount and fermentation time (temperature = 34 °C), (C) inoculation amount and fermentation temperature (time = 30 h).

Figure 7

(A₁,A₂₎ Comparison of the effect of single-bacteria and mixed-bacteria fermented natto on fibrin plate dissolution circle; (B) changes in nattokinase activity during fermentation; (C) prothrombin condensation inhibition ability.