健康奶牛子宫颈乳酸菌筛选方法的建立及其抑菌活性分析

Establishment of Selected Method for Lactic Acid Bacteria in Healthy Cows and Analysis of Its Antibacterial Activity

为筛选用于防制奶牛子宫炎的乳酸菌菌株,本试验选取产后30~50d的健康奶牛,采集子宫颈口处分泌物,利用MRS选择性培养基进行乳酸菌初步筛选。纯化后的菌株进行革兰氏染色镜检,将具有乳酸球菌或乳酸杆菌典型形态的疑似菌株进行针对性生化鉴定。提取分离菌株的基因组DNA,扩增其16SrDNA全序列并测序,将所得序列与NCBI数据库中的相应序列进行同源性比对分析。采用琼脂扩散法检测分离菌株对致病性金黄色葡萄球菌和大肠杆菌的抑菌活性,并检测分离菌株对山羊上皮细胞的黏附活性。结果显示,经染色镜检、生化鉴定和16SrDNA测序分析,得到13株乳酸菌,其中5株具有较强抑菌活性,6株具有较强黏附上皮细胞的能力,7、12和23号3株兼有良好的抑菌活性和黏附上皮细胞能力。综上,本试验建立了健康奶牛子宫颈中乳酸菌的筛选方法,得到3株具有良好益生特性的乳酸菌,为制备微生态制剂提供了基础材料,对防制奶牛子宫炎具有重要意义。

To select the lactic acid bacteria strains that could be used to prevent and control the metritis in dairy cows,healthy dairy cows 30 to 50 dpostpartum were selected to collect the secretions of the cervix,and the lactic acid bacteria were screened by MRS selective medium.The purified strains were subjected to microscopic examination,and the suspected strains with the typical morphology of Lactococcus lactis or Lactobacillus were biochemically identified.The genomic DNA of the isolated strains was extracted,and the 16 SrDNA sequence was amplified and sequenced.The sequence of the 16 SrDNA sequences was aligned with the NCBI nucleic acid data-base to identify the genus of the isolates.Antibacterial activity of isolated strains against pathogenic Staphylococcus aureus and Escherichia coli was evaluated using agar diffusion method.The adhesion activity of the isolated strains to goat epithelial cells was also tested.As a result,13 strains of lactic acid bacteria were obtained by staining microscopic examination,biochemical identification and 16 SrDNA sequencing.Among them,5 strains had strong antibacterial activity,6 strains had strong ability of adhering epithelial cells,strains of No.7,No.12 and No.23 both had good antibacterial activity and adhesion of epithelial cells.In summary,the experiment established a selected method for probiotic lactic acid bacteria in healthy cow＇s cervix.3 strains of lactic acid bacteria with good probiotic properties were obtained,which provided the basic materials for the preparation of probiotics and was of great significance for the prevention and treatment of dairy cows.