一株虹鳟源枯草芽孢杆菌产胞外蛋白发酵条件优化

Fermentation Condition Optimization for Extracellular Protein of Bacillus subtilis from Rainbow Trout(Oncorhynchus mykiss)

【目的】为提高虹鳟源枯草芽孢杆菌益生菌候选株RT-BS07胞外蛋白分泌能力,对其发酵条件进行了优化,并对发酵产物抑菌特性进行比较分析。【方法】以不同培养条件下RT-BS07株产胞外蛋白浓度为指标,结合单因素试验和正交试验对其发酵条件进行优化,并测定不同发酵条件下胞外蛋白对嗜水气单胞菌等5种水产养殖过程中常见条件致病菌的抑菌活性。【结果】初始pH值为7.0,盐度3%的LB培养基,33℃下振荡培养36 h,空气浴摇床转速150 r/min为菌株最佳蛋白分泌的发酵条件,在此条件下菌株胞外蛋白浓度可达(22.91±0.91)mg/mL,显著高于常规培养下的(12.74±0.26)mg/mL(盐度1%、初始pH值7.0,摇床转速120 r/min、28℃下培养24 h)。该胞外蛋白对鲁氏耶尔森菌、维氏气单胞菌、嗜水气单胞菌等水产常见条件致病菌具有较好的抑菌效果,抑菌面积分别为(13.87±1.03)mm^(2)、(11.78±0.46)mm^(2)、(7.93±0.21)mm^(2)。【结论】枯草芽孢杆菌RT-BS07株可分泌具有较强抑菌性能的胞外蛋白,并可通过优化发酵条件显著增加蛋白产量。试验结果将为虹鳟养殖过程中常见条件致病菌的生物绿色防治及抗菌制剂的研发应用提供实践数据和理论参考。

[Objective]To improve the extracellular protein secretion capacity for the candidate probiotic strain of Bacillus subtilis RT-BS07 from rainbow trout,the fermentation conditions were optimized,and the antibacterial properties for fermentation products were compared and analyzed.[Method]Adopting the concentration of extracellular protein under different fermentation conditions as the index,the fermentation conditions were optimized by combining single factor tests and orthogonal tests,and the antibacterial activities of extracellular protein under different fermentation conditions against five kinds of common conditionally pathogenic bacteria in aquaculture,such as Aeromonas hydrophila,were determined.[Result]The results showed that the optimal fermentation conditions for protein secretion were LB medium with an initial pH value of 7.0,salinity of 3%,33℃ for 36 h,and air bath shaker speed of 150 r/min.Under these conditions,the concentration of strains could reach(22.91±0.91)mg/mL.It was significantly higher than(12.74±0.26)mg/mL under the conventional culture.The extracellular protein had significant antibacterial effects on Yersinia ruckeri,A. veronil,A.hydrophila with the antibacterial area of(13.87±1.03)mm^(2),(11.78±0.46)mm^(2) and (7.93±0.21)mm^(2) respectively.[Conclusion]This study showed that B.subtilis RT-BS07 strain could secrete extracellular proteins with strong antibacterial properties,and the protein yield could be significantly increased by optimizing fermentation conditions.The results will provide practical data and theoretical reference for the biological green control of common conditional pathogens in rainbow trout culture and the development of antimicrobial agents.