实验方法 Experimental Methods

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

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  • 河北农业大学生命科学学院, 保定 071000
李红亚(1977-),女,河北蠡县人,副教授,博士,主要从事农业微生物学研究。E-mail:lihy77@sina.com

收稿日期: 2016-10-01

  网络出版日期: 2017-04-14

基金资助

河北省科技支撑计划项目(12226605);河北省高等学校科学技术研究项目(2011232)

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

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  • College of Life Science, Agriculture University of Hebei, Baoding 071000, China

Received date: 2016-10-01

  Online published: 2017-04-14

摘要

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N2-10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

本文引用格式

李红亚, 李术娜, 王树香, 王全, 李文, 朱宝成 . 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[J]. 动物营养学报, 2017 , 29(4) : 1191 -1197 . DOI: 10.3969/j.issn.1006-267x.2017.04.014

Abstract

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.

参考文献

[1] 张娟,杨彩梅,曹广添,等.解淀粉芽孢杆菌及其作为益生菌的应用[J].动物营养学报,2014,26(4):863-867.

[2] SOROKULOVA I B,PINCHUK I V,DENAYROLLES M,et al.The safety of two Bacillus probiotic strains for human use[J].Digestive Diseases and Sciences,2008,53(4):954-963.  

[3] HONG H A,DUC L H,CUTTING S M.The use of bacterial spore formers as probiotics[J].FEMS Microbiology Reviews,2005,29(4):813-835.  

[4] JOHNSON B A,ANKER H,MELENEY F L.Bacitracin:a new antibiotic produced by a member of the B. subtilis group[J].Science,1945,102(2650):376-377.  

[5] KOUMOUTSI A,CHEN X H,HENNE A,et al.Structural and functional characterization of gene clusters directing nonribosomal synthesis of bioactive cyclic lipopeptides in Bacillus amyloliquefaciens strain FZB42[J].Journal of Bacteriology,2004,186(4):1084-1096.  

[6] SOUTO G I,CORREA O S,MONTECCHIA M S,et al.Genetic and functional characterization of a Bacillus sp. strain excreting surfactin and antifungal metabolites partially identified as iturin-like compounds[J].Journal of Applied Microbiology,2004,97(6):1247-1256.  

[7] KIM P Ⅱ,CHUNG K C.Production of an antifungal protein for control of colletotrichum lagenarium by Bacillus amyloliquefaciens MET0908[J].FEMS Microbiology Letters,2004,234(1):177-183.  

[8] WANG J,LIU J,WANG X,et al.Application of electrospray ionization mass spectrometry in rapid Typing of fengycin homologues produced by Bacillus subtilis[J].Letters in Applied Microbiology,2004,39(1):98-102.  

[9] 李冠楠,夏雪娟,隆耀航,等.抗菌肽的研究进展及其应用[J].动物营养学报,2014,26(1):17-25.

[10] 曹小红,廖振宇,王春玲,等.Bacillus natto TK-1产脂肽的纯化,抑菌活性及其表面活性剂特性[J].中国生物工程杂志,2008,28(1):44-48.

[11] DELEU M,PAQUOT M,NYLANDER T.Effect of fengycin,a lipopeptide produced by Bacillus subtilis,on model biomembranes[J].Biophysical Journal,2008,94(7):2667-2679.  

[12] KIM P I,BAI H,BAI D,et al.Purification and characterization of a lipopeptide produced by Bacillus thuringiensis CMB26[J].Journal of Applied Microbiology,2004,97(5):942-949.  

[13] YU G Y,SINCLAIR J B,HARTMAN G L,et al.Production of iturin A by Bacillus amyloliquefaciens suppressing Rhizoctonia solani[J].Soil Biology and Biochemistry,2002,34(7):955-963.  

[14] SEYDLOVÁ G,SVOBODOVA J.Review of surfactin chemical properties and the potential biomedical applications[J].Central European Journal of Medicine,2008,3(2):123-133.

[15] CAZORLA F M,ROMERO D,PÉREZ-GARCÍA A,et al.Isolation and characterization of antagonistic Bacillus subtilis strains from the avocado rhizoplane displaying biocontrol activity[J].Journal of Applied Microbiology,2007,103(5):1950-1959.  

[16] ONGENA M,JACQUES P.Bacillus lipopeptides:versatile weapons for plant disease biocontrol[J].Trends in Microbiology,2008,16(3):115-125.  

[17] 张宝.解淀粉芽孢杆菌抗菌脂肽Bacillomycin L的纯化鉴定及抑菌机理研究[D].博士学位论文.北京:中国农业大学,2014:34-39.

[18] 翟少伟,李剑,史庆超.抗菌脂肽Surfactin的抗菌活性及应用[J].动物营养学报,2015,27(5):1333-1340.

[19] FUKUSHIMA M,NAKANO M.The effect of a probiotic on faecal and liver lipid classes in rats[J].British Journal of Nutrition,1995,73(5):701-710.  

[20] MIKKOLA R,KOLARI M,ANDERSSON M A,et al.Toxic lactonic lipopeptide from food poisoning isolates of Bacillus licheniformis[J].European Journal of Biochemistry,2000,267(13):4068-4074.  

[21] 侯红漫,靳艳,金美芳,等.环脂肽类生物表面活性剂结构、功能及生物合成[J].微生物学通报,2006,33(5):122-128.
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