羊源枯草芽孢杆菌分离鉴定及益生特性分析  

Isolation,Identification and Probiotic Characteristics Analysis of Bacillus subtilis from Sheep

作　　者：李永健 张润泽 吴忧 赵鹏飞 焦陇玲 周明 齐亚银[1] 任静静[1] LI Yongjian;ZHANG Runze;WU You;ZHAO Pengfei;JIAO Longling;ZHOU Ming;QI Yayin;REN Jingjing(College of Animal Science and Technology,Shihezi University,Shihezi 832003,China)

机构地区：[1]石河子大学动物科技学院,石河子832003

出　　处：《中国畜牧兽医》2025年第4期1590-1602,共13页China Animal Husbandry & Veterinary Medicine

基　　金：兵团农业关键核心技术攻关项目(2023AA310);天山英才“三农骨干人才”项目(2024-97号人才发展基金);石河子大学科技成果转化项目(CGZH202104);石河子大学高层次人才科研启动项目(RCZK202042)。

摘　　要：【目的】筛选益生特性好的枯草芽孢杆菌。【方法】从规模化羊场采集10份粪便和6份环境样品,通过LB固体培养基进行细菌分离纯化。利用形态学观察、生化鉴定和16S rRNA基因PCR扩增技术对菌株进行鉴定。然后通过溶血性测试、药敏试验、耐药与毒力基因分析、体外抑菌试验、酸碱及胆盐耐受性测试,以及小鼠安全性评估,全面评价菌株的益生特性。【结果】分离到的2株细菌在血平板上形成不规则白色突出菌落,表面粗糙呈山脉状,周围呈现明显的溶血环。生化鉴定结果显示,分离到的2株菌触酶、VP、葡萄糖、木糖、L-阿拉伯糖、甘露醇、蔗糖、柠檬酸盐、硝酸盐还原、淀粉水解、明胶液化、麦芽糖、蕈糖试验结果均呈阳性,硫化氢、枸橼酸盐、半乳糖试验均呈阴性,初步判定为枯草芽孢杆菌并分别命名为K1、K2。16S rRNA基因PCR扩增测序结果显示,分离株与枯草芽孢杆菌参考菌株的相似性为98%~100%,在系统进化树中处于同一分支,而与贝莱斯芽孢杆菌、沙门菌、屎肠球菌处于不同分支。抑菌试验结果表明,分离菌株K1、K2的上清对金黄色葡萄球菌有较好的抑菌效果,对大肠杆菌和沙门菌没有抑菌效果。药敏试验结果表明,分离菌株K1、K2对受试的青霉素、阿莫西林、头孢噻肟、恩诺沙星、氧氟沙星、复方新诺明、氟苯尼考、万古霉素具有较高的敏感性,对庆大霉素、大观霉素、罗红霉素中介,对四环素、多黏菌素B、林可霉素耐药。毒力基因检测结果表明,分离菌K1和K2不携带nheA、bceT、entFM、ces、cytK、hblA、hblC、hblD等毒力基因。耐药基因检测结果表明,分离菌K1携带aph(3′)-Ⅰa耐药基因,不携带MCR-1、bcr、floR、rpsl、strB、aadB、aac(6′)-Ⅰb、blaTEM、blaCTX、ermA、ermC、qnrA、qnrB、sul1、sul2、tetC、tetA、tetM、catA2耐药基因,而K2株携带ermA耐药基因,不携带其他验证的耐药基因【Objective】This study aimed to screen Bacillus subtilis with good probiotic properties.【Method】Ten fecal samples and six environmental samples were collected from large-scale sheep farms,and the bacteria were isolated and purified by LB solid medium.The strains were identified by morphological observation,biochemical identification and 16S rRNA gene PCR amplification.Then,the probiotic characteristics of the strains were comprehensively evaluated by hemolysis test,drug sensitivity test,drug resistance and virulence gene analysis,in vitro antibacterial test,acid-base and bile salt tolerance test,and mouse safety evaluation.【Result】The two isolated bacteria formed irregular white protruding colonies on the blood plate,the surface was rough and mountain-like,and there were obvious hemolytic rings around them.The results of biochemical identification showed that the isolated two strains were positive in catalase,VP,glucose,xylose,L-arabinose,mannitol,sucrose,citrate,nitrate reduction,starch hydrolysis,gelatin liquefaction,maltose and mushroom sugar tests,and negative in hydrogen sulfide,citrate and galactose tests.They were preliminarily identified as Bacillus subtilis and named K1 and K2,respectively.The results of 16S rRNA gene PCR amplification and sequencing showed that the similarity between the isolated strains and the reference strains of Bacillus subtilis was 98%-100%,which was in the same branch of the phylogenetic tree,but in different branches with Bacillus velezensis,Salmonella and Enterococcus faecium.The results of bacteriostatic test showed that the supernatant of isolated strains K1 and K2 had good bacteriostatic effect on Staphylococcus aureus,but had no bacteriostatic effect on Escherichia coli and Salmonella.The results of drug sensitivity test showed that the isolated strains K1 and K2 had high sensitivity to penicillin,amoxicillin,cefotaxime,enrofloxacin,ofloxacin,cotrimoxazole,florfenicol and vancomycin,and were intermediate to gentamicin,spectinomycin and roxithromycin,and resistant

关 键 词：枯草芽孢杆菌 分离鉴定 益生特性 

分 类 号：S816.7[农业科学—饲料科学]