具有拮抗空肠弯曲杆菌能力鸡源乳酸菌的筛选及特性

Screening and characterization of lactic acid bacteria with antagonistic activities against Campylobacter jejuni from chicken manure

摘要:

【目的】从鸡粪中筛选具有拮抗空肠弯曲杆菌能力的乳酸菌，研究其肠道益生特性，探讨其对空肠弯曲杆菌鞭毛毒力因子的影响。【方法】利用牛津杯法测定40株鸡粪源乳酸菌菌株的抑菌活性以确定抑菌性能好的菌株，利用16S rRNA基因分析进行菌株鉴定，采用HT-29细胞测定菌株的细胞粘附能力，通过模拟胃肠液实验分析菌株对胃肠道环境的耐受性，利用扫描电镜分析乳酸菌无细胞提取物对空肠弯曲杆菌鞭毛毒力因子的影响。【结果】从鸡粪中分离得到40株菌株，进一步筛选得到X13、X14和G20等3株拮抗空肠弯曲杆菌能力较强的菌株，经16S rRNA基因序列分析分别鉴定为罗伊氏乳杆菌、唾液乳杆菌和鸡乳杆菌；HT-29细胞粘附实验表明X13、X14及G20的粘附指数分别为11.5、20.3和14.3个/细胞，均具有良好的粘附能力；3株乳酸菌对人工胃肠液均具有良好的耐受性；扫描电镜观察表明，与对照组相比，3株纯培养乳酸菌无细胞提取物均能抑制空肠弯曲杆菌鞭毛毒力因子的合成。【结论】从鸡粪中筛选得到了3株能有效抑制空肠弯曲杆菌生长并能抑制其鞭毛合成的乳酸菌，有望作为拮抗性饲用益生菌用于控制禽畜的空肠弯曲杆菌感染。

Abstract:

[Objective] This study is aimed to screen and identify new lactic acid bacteria (LAB) with antagonistic activities against Campylobacter jejuni from chicken manure, to understand their intestinal probiotic properties, and to probe the effects of LAB’s cell-free extracts on the virulence factor, flagella formation of C. jejuni. [Methods] The inhibition activities of 40 strains isolated from chicken manure were analyzed based on an oxford cup method to screen the strains with excellent antagonistic inhibitory effects on the indicator bacteria C. jejuni. The 16S rRNA gene-based technique was used to identify the selected strains. The adherence abilities of LAB strains to human intestinal epithelial cell (HT-29) were evaluated. The tolerance of LAB strains to artificial gastrointestinal juices was tested. Scanning electron microscopy was used to observe the effects of LAB’s cell-free extract on the flagella formation of C. jejuni. [Results] Forty LAB strains were isolated from chicken manure, among which X13, X14 and G20 strain showed the strongest antagonistic activities against C. jejuni. The 16S rRNA gene sequence analysis showed that X13, X14, and G20 strain were respectively identified as Lactobacillus reuteri, Lactobacillus salivarius and Lactobacillus gallinarum. HT-29 cell adhesion tests showed that the adhesion index of X13, X14, and G20 were respectively 11.5, 20.3, and 14.3 cells per HT-29 cell, such results suggests that all these three strains showed strong adhesion abilities to HT-29 cell. Tolerance tests to artificial gastrointestinal juices showed that all these three strains showed good capabilities. SEM observation showed that the cell-free extracts of these three strains were able to inhibit the flagella formation of C. jejuni. [Conclusion] Three selected LAB strains from chicken manure have inhibit the growth and the flagella formation of C. jejuni in vitro effectively, which suggested that such three LAB strains could be used as potential animal protective probiotics for the control of C. jejuni infection in vivo.