屎肠球菌FDT2的分离鉴定及抑菌特性研究

Isolation,Identification and Antimicrobial Properties of Enterococcus faecium FDT2

【目的】从牦牛粪便中分离和鉴定屎肠球菌并进行体外抑菌特性研究,以期筛选出具有优良抑菌特性的益生菌。【方法】用MRS培养基从青海省健康牦牛粪便中分离肠球菌,结合形态学和分子生物学鉴定后进行抑菌性研究。通过发酵过滤分离菌株上清液,采用打孔法观察其对应的抑菌效果,通过改变pH及加入过氧化氢酶、胃蛋白酶和胰蛋白酶等方法来分析其抑菌物质,并探究温度和pH对菌株发酵上清液的影响。【结果】结合形态学及系统进化树分析表明,分离的2株菌(FDT2和D2)均为屎肠球菌。抑菌试验结果表明,屎肠球菌FDT2对5株致病菌均有抑制作用,其中对产肠毒素性大肠杆菌K88和大肠杆菌ATCC 43888的抑菌性最强,而屎肠球菌D2对5株致病菌均无明显抑菌作用。通过排酸试验证明,屎肠球菌FDT2发酵上清液中起抑菌作用的不是酸性物质;通过加入过氧化氢酶试验证明了抑菌物质中有过氧化氢存在,且发挥部分的抑菌作用;加入胃蛋白酶和胰蛋白酶后其抑菌圈直径极显著低于对照组(P<0.01),表明抑菌物质中包含细菌素,且在抑菌过程中发挥重要作用。随着温度的逐渐升高,屎肠球菌FDT2的抑菌能力有所降低,在100℃下仍有抑菌性,表明其具有较强的热稳定性。随着pH的升高,屎肠球菌FDT2的抑菌活性逐渐下降,说明细菌素在酸性环境下活性较强。【结论】试验共分离到2株菌,其中屎肠球菌FDT2具有良好的抑菌特性,能分泌细菌素等抑菌物质,有必要对其深入研究。

【Objective】The purpose of this experiment was to isolate and identify Enterococcus faeciums isolates from yak faeces and conduct in vitro antibacterial studies,in order to screen probiotics with excellent antibacterial properties.【Method】In this experiment,Enterococcus spp.were isolated from faeces of healthy yaks in Qinghai province utilizing MRS medium,the strains were identified by combining morphology and molecular biology and conduct antibacterial research.The supernatants of the strains were isolated by fermentation filtration,and the corresponding antibacterial effect was observed using punching method.The antibacterial substances were analyzed by changing pH,adding catalase,pepsin and trypsin,and the effects of temperature and pH on the fermentation supernatants were explored.【Result】The results of morphology and phylogenetic tree analysis showed that the two new isolates(FDT2 and D2)belonged to Enterococcus faecium.The antibacterial experiment results showed that Enterococcus faecium FDT2 had inhibitory effect on all five pathogenic bacteria,and the inhibitory effects on enterotoxigenic Escherichia coli K88 and Escherichia coli ATCC 43888 were more stronger,while D2 had no inhibitory effect on all five pathogenic bacteria.The acid excretion test results proved that the acidic substance in the fermentation supernatant of Enterococcus faecium FDT2 did not have antibacterial effects.The experiment of adding catalase proved that hydrogen peroxide existed in the bacteriostatic substance and played partly bacteriostatic effect.After addition of pepsin and trypsin,it was found that the diameter of inhibition zone was extremely significantly lower than that of control group(P<0.01),indicating that antibacterial substances contained bacteriocin and played a major role in antibacterial effects.With the gradual increase of temperature,the antibacterial activity of Enterococcus faecium FDT2 decreased,and it remained antibacterial activity at 100℃,indicating its strong thermal stability.With the increase of pH,the bacteriostatic activity of Enterococcus faecium FDT2 decreased gradually,which also indicated that bacteriocin had strong activity in acidic environment.【Conclusion】Two strains of bacteria were isolated in the experiment,among which Enterococcus faecium FDT2 had good bacteriostatic properties and contained bacteriocin and other antibacterial substances,and it was necessary to further study on the bacteriocin of Enterococcus faecium FDT2.