| Summary: | 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 <i>Bacillus subtilis</i> GUTU09 and <i>Bifidobacterium animalis subsp. lactis</i> 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 <i>Bacillus subtilis</i> GUTU09 and <i>Bifidobacterium</i> 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, <i>Bacillus subtilis</i> GUTU09 to <i>Bifidobacterium</i> 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.
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