益生芽孢杆菌脂肽类抑菌成分的高效液相色谱-电喷雾串联质谱分析

High-Performance Liquid Chromatography-Electrospray Ionization Tandem Mass Spectrometry Analysis of Lipopeptides Produced by Probiotic Bacillus spp.

本文旨在探明一株具有益生潜质的枯草芽孢杆菌N2-10对肠道菌的抗菌作用及其活性成分,为深入评价该菌株的益生功能和益生机理奠定基础。以常见的肠道致病菌和有益菌为指示菌,采用抑菌圈法对该菌株发酵液成分的抑菌活性进行测定,并通过高效液相色谱-电喷雾串联质谱(HPLC-ESI-MS/MS)对其中的活性成分进行分析。结果表明:枯草芽孢杆菌N2-10发酵液的氯仿和正丁醇提取液对供试菌无明显的抑制作用;脂肽类对大肠杆菌、痢疾杆菌和沙门氏菌等肠道致病菌表现出明显的抑菌作用,而对肠道有益菌保加利亚乳杆菌抑制作用很弱,对嗜热链球菌则无抑制作用。脂肽粗提物的HPLC-ESI-MS/MS分析结果显示,脂肽粗提物中的脂肽类抗生素为伊枯草素家族中的C14~C17抗霉枯草菌素(Mycosubtilin)的同系物。因此推断,枯草芽孢杆菌N_2-10对肠道致病菌产生抑制作用依赖于其产生的脂肽抗生素Mycosubtilin。

The aim of this paper was to study the antibacterial effect on the intestinal bacterial and active constituent of Bacillus subtilis N2-10 strain with a latent probiotic capability,to lay the foundation for the healthy function and mechanism of the in-depth evaluation of the probiotic strains.The antibacterial activity of the ingredients from culture broth was detected by the method of inhibition zone in this paper,using pathogens and probiotics commonly colonizing in the intestinal as indicators.The crude extract with antibacterial activity was further detected by high-performance liquid chromatography-electrospray ionization tandem mass spectrometry（HPLC-ESI-MS/MS）.The results of antibacterial assay displayed that the chloroform extract and n-butanol extract the corresponding culture media had not obvious inhibition activity against the tested strains.The crude lipopetide extact exhibited markedly antagonistic activity against Escherichia coli,Shigella flexneri and Salmonella enterica et cetera intestinal pathogenic bacteria and showed weak inhibitory effect on the probiotic Bulgarian lactobacillus,but had no effect on Streptococcus thermophilus.HPLC-ESI-MS/MS analysis data revealed that the active components in the crude lipopetide extact were C14 to C17 Mycosubtilin homologus.From all results,it can be concluded that Mycosubtilin is produced and is responsible for the antibacterial activity of Bacillus subtilis strain N2-10.