抗金黄色葡萄球菌乳酸菌筛选及其抗菌物质特性研究

Discovery of Lactic Acid Bacteria Against Staphylococcus aureus and Characterization of Antibacterial Substances

为了利用乳酸菌(lactic acid bacteria,LAB)对金黄色葡萄球菌(Staphylococcus aureus,S.aureus)引起的奶牛乳房炎进行科学防治,本研究通过琼脂斑点法筛选了68株LAB,得到59株有抗S.aureus效果的LAB。根据抗菌效果从强到弱,选择L-12、L-30、L-31和L-37进行抗菌物质成分分析,结果表明,L-12、L-30、L-31无细胞上清液(cell-free culture supernatant,CFCS)的抗菌物质主要是酸性物质,而L-37 CFCS的抗菌物质主要是蛋白类物质和过氧化氢。因此选择L-37对其进行分子生物学鉴定并研究其抗菌物质的理化特性,结果表明,L-37是1株植物乳杆菌(Lactiplantibacillus plantarum,L.plantarum),其CFCS在pH值为2~6时,相对抗菌活性分别为115.34%、101.48%、55.06%、44.37%、20.23%,除pH值为3的处理组(pH值与初始CFCS相近),其余均与对照组呈显著性差异;将L-37 CFCS分别在65℃、85℃和100℃条件下处理,处理组与对照组的相对抗菌活性之间没有显著性差异,说明L-37 CFCS具有良好的热稳定性;分别使用10种不同金属离子处理L-37 CFCS,Na^(+)、K^(+)、Ca^(2+)、Mg^(2+)、Mn^(2+)、Cu^(2+)、Fe^(3+)和Al^(3+)组的相对抗菌活性与对照组呈显著性差异,前5种金属离子产生抑制作用,后3种产生促进作用,Zn^(2+)和Fe^(2+)组的相对抗菌活性与对照组相比没有显著性差异。本研究成功筛选到1株L.plantarum L-37对S.aureus有较强的抗菌效果,并对其抗菌物质特性进行了研究,为安全防治S.aureus引起的奶牛乳房炎提供实验依据。

In order to use lactic acid bacteria(LAB)to scientifi cally control bovine mastitis caused by Staphylococcus aureus(S.aureus),68 LAB strains were screened by agar spot method,of which 59 strains were found S.aureus resistant.According to the resistantl eff ect from strong to weak,L-12,L-30,L-31 and L-37 were selected for the composition analysis of antibacterial substances.The results showed that the antibacterial substances of L-12,L-30,L-31 cell-free culture supernatant(CFCS)were mainly acidic substances while L-37 CFCS were mainly protein substance and hydrogen peroxide.Therefore,L-37 was selected to conduct molecular biological identifi cation and study its properties.As a result,L-37 was Lactiplantibacillus plantarum and the relative antibacterial activities of its CFCS were respective 115.34%,101.48%,55.06%,44.37%and 20.23%when pH was 2,3,4,5 and 6.Except for the treatment group with pH value of 3(pH value was similar to the initial CFCS),the treatment groups were signifi cant diff erences from the control group.When L-37 CFCS was respectively treated at 65℃,85℃and 100℃conditions,the treatment groups were no signifi cant diff erences from the control group,indicating that L-37 CFCS had good thermal stability.Subsequently,10 diff erent metal ions were used to treat L-37 CFCS,Na^(+),K^(+),Ca^(2+),Mg^(2+),Mn^(2+),Cu^(2+),Fe^(3+)and Al^(3+) groups were signifi cant diff erences from the control group.The first five metal ions had inhibitory eff ect and the last three had promoting eff ect.The relative antibacterial activity of Zn^(2+)and Fe^(2+) groups was not signifi cantly diff erent from that of the control group.In this study,L.plantarum L-37 was found to have strong antibacterial eff ect on S.aureus,which provided experimental basis for safe prevention and treatment of bovine mastitis caused by S.aureus.