新疆维吾尔自治区呼图壁县驼乳源乳酸菌分离鉴定及其对金黄色葡萄球菌的抑制作用OACSTPCD

[Objective]The aims of the present study were to isolate and identify lactic acid bacteria from camel milk in Hutubi County of Xinjiang Uygur Autonomous Region,and to evaluate their inhibitory effects on Staphylococcus aureus(S.aureus).[Method]A total of 45 healthy camel milk samples were collected from a camel farm in Hutubi County.Lactic acid bacteria were isolated using MRS medium,and molecular biological identification was carried out using PCR amplification and sequencing targeting 16S rDNA sequence.Point inoculate method,agar spot method and Oxford cup double-layer agar diffusion method were used to screen the lactic acid bacteria with inhibitory effects on S.aureus ATCC 29213.The acid tolerance of the lactic acid bacteria was measured by adjusting pH value of MRS medium to 1-10.The bile salt tolerance of the lactic acid bacteria was assessed by adding 0-0.30%ox bile salt to MRS medium.The minimum inhibitory concentration(MIC)and subinhibitory concentration(SIC)of cell-free culture supernatant(CFCS)of the lactic acid bacteria against S.aureus ATCC 29213 were determined using micro-broth dilution method.Changes in inhibitory effect of CFCS on S.aureus ATCC 29213 under different pH value conditions and after enzymatic treatments were examined to preliminarily analyze the antibacterial active substances of CFCS.The inhibitory effect of CFCS on wild strains of S.aureus isolated from camel milk and dairy cow with mastitis were evaluated by Oxford cup double-layer agar diffusion method.[Result]A total of 20 lactic acid bacterial strains belonging to 5 species were isolated from the 45 camel milk samples,including Enterococcus faecalis(n=13),Lacticaseibcillus rhamnsus(n=1),Limosilactobacillus fermentum(n=3),Lactobacillus paracasei(n=1)and Weissella confusa(n=2).After screening with the above mentioned three methods,a strain of Lacticaseibcillus rhamnsus(L.rhamnsus,designated as KC4)exhibiting the strongest antibacterial effect on S.aureus ATCC 29213 was obtained.KC4 strain had good acid and bile salt tolerance,and showed normal growth in MRS medium with pH values adjusting to 4-7 and addition of ox bile salt at concentrations of 0-0.20%.The MIC and SIC of KC4 strain CFCS against S.aureus ATCC 29213 were 62.5 mg/mL and 7.812 5 mg/mL,respectively.Its antibacterial activity was still effective at pH values of 2-5,but reduced extremely significantly(P<0.01)after enzymatic treatments with pepsin,peroxidase,trypsin,peptidase,protease K,catalase and papain.It had good antibacterial effect on wild strains of camel and dairy cow derived S.aureus.[Conclusion]One strain of L.rhamnsus was obtained from the camel milk in Hutubi County,which had strong inhibitory effect on standard strain of S.aureus and wild strains of S.aureus isolated from camel milk and dairy cow with mastitis.It also exhibited high level tolerance to acid and bile salt,and the possible antibacterial active substances included acids,peroxides and proteins.The results obtained in this study provided potential bacterial resources for the development and utilization of lactic acid bacteria derived from camel milk.