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.
Niuganba (NGB) is naturally fermented beef meat. It originated in the southwest region of China, and over the time it has become a popular delicacy of China (Li et al., 2017;Yu et al., 2020). NGB is prepared using high-quality meat from limbs, abdomen, and other parts of the beef carcass. The process involves marinating, drying, and fermenting beef meat until it acquires a unique taste and aroma.NGB contains a myriad of essential minerals and amino acids. When stored for a prolonged period, the texture of this fermented meat turns dry, hard, and crispy due to water loss. Due to its texture, this meat was named "Niuganba" (Yan et al., 2011).
Fibrinolytic enzymes are effective and highly safe in treating cardiovascular and cerebrovascular diseases. Therefore, screening fibrinolytic enzyme-producing microbial strains with excellent fermentation performance is of great value to industrial applications. The fibrin plate method was used in screening strains with high yields of fibrinolytic enzymes from different fermented food products, and the screened strains were preliminarily identified using molecular biology. Then, the strains were used for the solid-state fermentation of soybeans. Moreover, the fermentation product douchi was subjected to fibrinolytic activity measurement, sensory evaluation, and biogenic amine content determination. The fermentation performance of each strain was comprehensively evaluated through principal component analysis. Finally, the target strain was identified based on strain morphology, physiological and biochemical characteristics, 16S rDNA sequence, and phylogenetic analysis results. A total of 15 Bacillus species with high fibrinolysin activities were selected. Their fibrinolytic enzyme-producing activities were higher than 5,500 IU/g. Through molecular biology analysis, we found four strains of Bacillus subtilis, 10 strains of Bacillus amyloliquefaciens, and one strain of Bacillus velezensis. The principal component analysis results showed that SN-14 had the best fermentation performance and reduced the accumulation of histamine and total amine, the fibrinolytic activity of fermented douchi reached 5,920.5 ± 107.7 IU/g, and the sensory score was 4.6 ± 0.3 (out of 5 points). Finally, the combined results of physiological and biochemical analyses showed SN-14 was Bacillus velezensis. The high-yield fibrinolytic and excellent fermentation performance strain Bacillus velezensis SN-14 has potential industrial application value.
Natto is a high-value fermented soybean produced by B. subtilis. However, B. subtilis produces a pungent amine odor. This study compared the volatile organic compounds (VOCs), free amino acids (FAAs) and biogenic amines (BAs), nattokinase (NK) of natto made by two-strain fermentation with Bifidobacterium animalis BZ25 and Bacillus subtilis GUTU09 (NMBB) and that of natto made by single-strain fermentation with Bacillus subtilis GUTU09 (NMB). Compared with NMB, volatile amine substances disappeared, ketones and aldehydes of NMBB were reduced, and alcohols increased. Besides that, the taste activity value of other bitter amino acids was lowered, and BA content was decreased from 255.88 mg/kg to 238.35 mg/kg but increased NK activity from 143.89 FU/g to 151.05 FU/g. Correlation analysis showed that the addition of BZ25 reduced the correlation between GUTU09 and BAs from 0.878 to 0.808, and pH was changed from a positive correlation to a negative one. All these results showed that the quality of natto was improved by two-strain co-fermentation, which laid a foundation for its potential industrial application.
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