关键词: 枯草芽孢杆菌; 分离鉴定; 益生特性

Abstract: 【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 to tetracycline,polymyxin B and lincomycin.The results of virulence gene detection showed that the isolates K1 and K2 did not carry virulence genes such as nheA,bceT,entFM,ces,cytK,hblA,hblC and hblD.The results of drug resistance gene detection showed that the isolates K1 carried aph(3')-Ⅰa resistance gene and did not carry MCR-1,bcr,floR,rpsl,strB,aadB,aac(6')-Ⅰb,bla_TEM,bla_CTX,ermA,ermC,qnrA,qnrB,sul1,sul2,tetC,tetA,tetM and catA2 resistance genes,while K2 strain carried ermA resistance gene and did not carry other verified resistance genes.The two isolates had good growth activity in LB liquid medium with pH of 4.0,5.0,6.0,8.0,9.0 and 10.0.Among them,strain K2 still had good growth activity in LB liquid medium with pH of 3.0.In LB liquid medium containing 0.3%,0.5%,0.7% and 1.0% bovine bile salts,the two isolates could still survive and maintain relatively good growth activity.The safety test in mice showed that there was no obvious pathological change in mice.【Conclusion】 The two strains isolated in this study were Bacillus subtilis, which had hemolytic properties, antibacterial activity against Staphylococcus aureus and excellent growth activity.In the simulated gastrointestinal environment,it showed diversity in acid,alkali and bile salt tolerance,and remained sensitive to most antibiotics.No major virulence genes were detected.It had good safety to mice and had the potential as a probiotic.which could further explore its application in animal husbandry and human health.

Key words: Bacillus subtilis; isolation and identification; probiotic characteristics