枯草芽孢杆菌KC-WQ发酵液中抗菌脂肽的分离鉴定及发酵条件优化

Isolation and Identification of Antibacterial Lpopeptides from Bacillus subtilis KC-WQ Fermentation Broth and Optimization of Fermentation Conditions

本文研究了一株枯草芽孢杆菌KC-WQ的胞外分泌物的抑菌效果及功效成分。实验发现枯草芽孢杆菌KCWQ对大肠杆菌(Escherichia coli)、沙门氏菌(Salmonella enterica subsp.enterica)和嗜水气单胞菌(Aeromonas hydrophila)均有明显的抑菌效果,其中对沙门氏菌的抑制效果最为显著。在此基础上,采用酸沉淀法分离抗菌成分,通过薄层色谱法(TLC)、高效液相色谱法(HPLC)、基质辅助激光解吸电离飞行时间质谱(MALDI-TOFMS)分析结构发现,该菌株分泌抗菌物质为脂肽类,分子量分布在1030.5~1491.4 Da之间,主要由伊枯草菌素(Iturin)、表面活性素(Surfactin)和芬荠素(Fengycin)组成,最小抑菌浓度为0.8 mg/mL。该脂肽成分经热处理(105℃,20 min)后,仍保持良好的抗菌效果。采用响应面法对KC-WQ的发酵条件进行优化,得到最佳发酵条件为:初始pH为6.0,发酵温度为36℃,摇床转速为208 r/min,预测粗产物产量为1.311 g/L,后经实验验证,实际产量可达1.236 g/L。上述结果表明,枯草芽孢杆菌KC-WQ具有良好的抗菌性能和加工性能,可作为抑菌成分,在食品保藏和抗菌包装材料开发中具有应用前景。

This paper studied the antibacterial effect and functional components of the extracellular secretions of a strain of Bacillus subtilis KC-WQ.The experiment found that KC-WQ had obvious antibacterial effect on Escherichia coli,Salmonella enterica subsp.enterica and Aeromonas hydrophila,among which it had the most significant antibacterial effect on Salmonella.On this basis,the acid precipitation method was used to separate the antibacterial components,and the structure was found through thin layer chromatography(TLC),high performance liquid chromatography(HPLC)and matrix-assisted laser desorption ionization time-of-flight mass spectrometry(MALDI-TOF-MS).The antibacterial substances secreted by this strain were lipopeptides with a molecular weight distribution between 1030.5 and 1491.4 Da,mainly composed of Iturin,Surfactin and Fengycin,and the minimum inhibitory concentration was 0.8 mg/mL.The lipopeptide component still maintained a good antibacterial effect after heat treatment(105℃,20 min).The response surface method was used to optimize the culture conditions of KC-WQ,and the best fermentation conditions were obtained:The initial pH was 6.0,the fermentation temperature was 36℃,the shaker speed was 208 r/min,and the predicted crude product yield was 1.311 g/L.Later verified by experiments,the actual yield was 1.236 g/L.The above results indicated that Bacillus subtilis KC-WQ had good antibacterial and processing properties,could be used as a good antibacterial ingredient,and had a good application prospect in the development of antibacterial and preservation materials in food industry.