抑制肠出血性大肠杆菌感染的鸡源乳杆菌筛选及其抑菌机制  

Screening of chicken-derived Lactobacillus inhibiting enterohemorrhagic Escherichia coli infection and its antibacterial mechanism

作　　者：胡亚凡 张汝京 海丹 赵梦娜 陆兆新[1] 吕凤霞[1] 赵海珍[1] 别小妹[1] HU Yafan;ZHANG Rujing;HAI Dan;ZHAO Mengna;LU Zhaoxin;LÜ Fengxia;ZHAO Haizhen;BIE Xiaomei(College of Food Science and Technology,Nanjing Agricultural University,Nanjing 210095,China)

机构地区：[1]南京农业大学食品科学与技术学院,江苏南京210095

出　　处：《南京农业大学学报》2020年第6期1143-1150,共8页Journal of Nanjing Agricultural University

基　　金：国家重点研发计划项目(2018YFC1602500);国家自然科学基金项目(31972174)。

摘　　要：[目的]本文旨在筛选能够抑制肠出血性大肠杆菌黏附Caco-2细胞并抑制其诱导IL-8产生的乳杆菌。[方法]从健康鸡的肠道中分离菌株,通过双层琼脂法筛选出对大肠杆菌具有显著抑制能力的菌株,并进行16S rDNA鉴定。通过模拟胃肠液环境,评估分离菌株对胃肠液的耐受能力。通过FITC荧光标记大肠杆菌,对选定菌株抑制大肠杆菌黏附的能力进行测定,并利用ELISA试剂盒测定其对大肠杆菌诱导的IL-8过表达的抑制作用。[结果]10株乳杆菌对6株大肠杆菌的抑菌圈直径均大于10 mm,16S rDNA测序分析得到1株约氏乳杆菌、1株副植物乳杆菌和8株植物乳杆菌。其中Lactobacillus johnsonii NJ13对胃液耐受能力最佳,3 h的胃液存活率达73.19%;同时在肠液中能够生长,对肠出血性大肠杆菌ATCC35150黏附Caco-2细胞具有抑制作用,并能够显著降低由大肠杆菌诱导的IL-8表达量;可降低感染小鼠体内的大肠杆菌科比例(0.000205%),同时增加乳杆菌属的比例至3.11440%。[结论]L.johnsonii NJ13能够抑制大肠杆菌对细胞的黏附以及损伤,对有益乳杆菌资源的开发利用以及抑制肠出血性大肠杆菌的感染具有重要指导意义。[Objectives]This paper aimed to screen probiotics capable of inhibiting adhesion of enterohemorrhagic Escherichia coli(EHEC)and induction of IL-8 production by Caco-2 cells.[Methods]Strains were isolated from the intestines of healthy chickens and Lactobacillus with potential inhibition ability against E.coli were screened,and 16S rDNA sequencing was employed to identify the isolates.By formulating the environment of gastrointestinal fluid,the tolerance of Lactobacillus was evaluated.The inhibition of adhesion was detected by fluorescent labelling technique,and E.coli-induced IL-8 production was determined using an ELISA kit.[Results]The bacteriostatic circle diameter of ten Lactobacillus strains against tested E.coli isolates were all greater than 10 mm.16S rDNA sequencing analysis got one L.johnsonii,one L.paraplanta and eight L.plantarum.Among them,Lactobacillus johnsonii NJ13 had the best tolerance to formulated gastric fluid with a gastric fluid survival rate of 73.19%for 3 hours,and could grow in the formulated intestinal fluid.L.johnsonii NJ13 was proved to inhibit the adhesion of E.coli ATCC35150 on Caco-2 cells and production of IL-8 induced by it.Besides,it can reduce the proportion of Enterobacteriaceae in infected mice to 0.000205%and increase the proportion of Lactobacillus to 3.11440%.[Conclusions]L.johnsonii NJ13 can inhibit the adhesion and damage of E.coli ATCC35150 to Caco-2 cells,placing a clue for the development and utilization of beneficial Lactobacillus resources and inhibitory way of EHEC infection.

关 键 词：乳杆菌 肠出血性大肠杆菌(EHEC) 黏附 IL-8 抑制 

分 类 号：TS201.3[轻工技术与工程—食品科学]