奶牛源乳酸菌的分离、鉴定及其微生态制剂的研究

Isolation and Characterization of Lacto-Bacterial Strain and Its Micro-Ecological Preparation from Milk Cow

为筛选出能用于防治奶牛子宫内膜炎的益生性乳酸菌菌株并以其为原材料制作微生态制剂,采集产后30～50 d健康奶牛的子宫颈口处分泌物,利用MRS选择性培养基厌氧培养方式进行乳酸菌初步筛选。纯化得到的单株菌株进行染色和镜检,将典型的乳酸菌进行生化鉴定。提取其DNA,扩增16S rDNA全序列并测序,将所得序列与NCBI核酸数据库进行同源性比对,明确各分离菌株种属归属。将鉴定得到的单株乳酸菌过滤以及离心获取上清,得到的上清进行抑菌实验。采用琼脂扩散法检测对大肠埃希菌的抑菌活性。以得到的良好抑菌能力的菌株为原材料进行混合抑菌,进行微生态制剂最优比例的探究,并研究这些菌株的生长曲线和产酸能力。结果显示,经染色镜检、生化鉴定和16S rDNA测序分析和单株抑菌实验得到6株较好的抑菌能力的乳酸菌,其中鼠李糖乳杆菌、魏斯式细菌和粪肠球菌的混合上清抑菌效果最好,具有较强的生长和产酸能力。研究得到了可用于防治奶牛子宫内膜炎益生菌株,可进一步用于临床试验探究。

In order to screen probiotic lacto-bacteria strains to be used to control cow endometritis and use them as raw materials to make probiotics. Excretion from the cervix exit of healthy cows 30-50 days postpartum were used as material, and MRS selective medium was used to carry out initial lacto-bacteria screening. The purified strains were stained and microscopically examined for biochemical identification of typical lacto-bacteria. The DNA was extracted and the full sequence of 16S rDNA was amplified and sequenced. The homology comparisons between the obtained sequences and the NCBI nucleic acid database were carried out to clarify the species and genus attribution of each isolate. The identified lacto-bacteria were filtered and centrifuged to obtain its supernatant, and the resulting supernatant was subjected to a bacteriostasis experiment. Agar diffusion method was adopted to test the antibacterial activity against E.coli . In order to obtain strains with good antibacterial ability as raw materials and carry out mixed bacteriostatic, and find out the optimal proportion of microecological preparations, as well as the growth curve and acid production ability of these strains. The results showed that six strains of lactic acid bacteria with good antibacterial activity were obtained by staining microscopy, biochemical identification, 16S rDNA sequencing analysis and antibacterial test of each single strain. Among them, the antibacterial effects of supernatant of mixed Lactobacillus mucosae, Weissella cibaria and Enterococcus faecalis were the best, and possessing comparative growth and acid production ability. It can be concluded that the probiotic strains can be used to control cow endometritis were obtained, and can be used for further exploration in clinical experiments.