采用酪蛋白平板初筛-摇瓶复筛的方法,从自然发酵大酱中筛选获得1株高产纳豆激酶(nattokinase,NK)的菌株SH21,经16S rRNA基因序列鉴定其为枯草芽孢杆菌。以NK酶活力及嘌呤含量为指标,通过单因素试验和响应面法优化枯草芽孢杆菌SH21液体发酵条件。在单因素试验的基础上,得出碳源、氮源、pH、接种量、发酵温度、发酵时间对菌株发酵液中NK酶活力和嘌呤含量的影响。采用 Plackett-Burman 设计筛选得到pH、接种量和发酵温度3个显著因素,结合 Box-Behnken设计中心组合试验并进行响应面分析。优化后的发酵条件为pH 6.0,接种量3%,发酵温度38 ℃,在此条件下,NK酶活力由166.99 U/mL提升到204.52 U/mL,提高了1.22倍,嘌呤含量由75.4 mg/L降低到51.27 mg/L,降低了32%。该研究为进一步开发高NK酶活性、低嘌呤产量的枯草芽孢杆菌发酵产品奠定了研究基础。
A strain with high nattokinase activity was screened from naturally fermented pastes with casein plate selecting and shaking culturing, the strain SH21 was identified as Bacillus subtilis by 16S rRNA gene sequencing. Using nattokinase activity (NK) and purine as indicators, the liquid fermentation conditions of Bacillus subtilis SH21 were optimized by single factor experiments and response surface methodology. The single factor tests were designed to investigate the influence of carbon sources, nitrogen sources, pH, inoculation, fermentation temperature and fermentation time on nattokinase activity and purine content. Then the Plackett-Burman test was designed, and three significant factors, pH, inoculation and fermentation temperature were obtained. Finally, the Box-Behnken design was used and the response surface analysis was performed. The optimized fermentation condition was pH 6.0, with the inoculation amount 3%, and fermentation temperature 38℃. The enzyme activity increased from 166.99 U/mL to 204.52 U/mL, which was improved by 1.22 folds. While the purine content decreased from 75.4 mg/L to 51.27 mg/L, reduced by 32% under the optimized condition. This study lays the foundation for the further development of B. subtilis fermentation products with high nattokinase activity and low purine production.
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