牦牛肠道与粪便乳酸菌的分离鉴定及PCR-16 S rDNA鉴定

Isolation and identification of lactic acid bacteria from yak and sequence analysis of its 16 S rDNA

以取自四川省不同地区的牦牛粪便、肠道内容物为材料,用MRS琼脂双层培养基进行厌氧培养,分离到50株乳酸菌,经生化鉴定为嗜热链球菌(2株)、乳酸乳球菌(1株)、保加利亚乳杆菌(5株)、嗜粪乳杆菌(10株)、嗜酸乳杆菌(8株)、乳酸乳杆菌(9株)、肠乳杆菌(10株)、弯曲乳杆菌(5株)。采用乳酸菌16 S rDNA通用引物,对分离的8种菌的16 S rDNA一段可变区序列进行扩增,均得到大小约470 bp的产物;扩增产物经纯化、测序后与GenBank中标准菌株的核甘酸序列比较,同源性均大于97．5％,同源性分析与生化试验的结果是一致的。证实,牦牛肠道和粪便的乳酸菌较为丰富,且乳杆菌的数量较多,这可能与牦牛复杂的生长环境有关。

50 strains of lactic acid bacteria were isolated from stools and intestinal tracts of yak （Bos grunniens） and eight species were identified based on their biochemical characteristics：Streptococcus therrnophilus （2 strains） ,Lactococcus lactis （1 strains）, Lactobacillus bulgaricus （5 strains）, L. coprophilus（ 10 strains）, L. acidophilus（ 8 strains） ,L. lactis（ 9 strains） ,L. intestinalis（ 10 strains） ,L. curvatus（ 5 strains）. A fragment of 470 bp in the variable region of 16 S rDNA was amplified from the 8 species by using the universal primers for lactic acid bacteria. Nucleotide sequences of the fragments for the 8 species shared over 97.5% homology with that of the standard sequence available in GenBank. The results of homology analysis was in accordance with that of biochemical identification. It was concluded that abundant lactic acid bacteria exist in the stool and intestinal tract of yak,in particular lactobacilli,probably due to the complex environment.