Polyphasic Taxonomy Methods For Identification Of Milk Derived Coccus Strains

In this paper, polyphasic taxonomy identification of 9 milk derived lactic acid coccus strains (5 lactococcus lactis strains, 3 Streptococcus thermophilus strains and 1 Streptococcus agalactiae strain) were studied. The strains were isolated by MRS plate streaking cultivation and morphological observation, including colony observation, light microscope examination basing on the Gram staining of the strains cell wall, and transmission electron microscope(TEM) examination basing on the negative staining by PTA, and were identified by biochemical essays basing on the biochemical metabolism differences between Lc. lactis and St. thermophilus, specific PCR which primers were designed basing on the sequence differences among their genes coding these biochemical metabolic key enzymes, and RAPD. The results of plate streaking cultivation were 11 strains according to the sizes of their colonies and cells; The size, shape and schizogenesis styles of every strain were obtained by TEM observation; And the result of biochemical identification had 2 distinct fermentation types which classified these strains as 2 microbial population, one was Lc. lactis subsp. lactis, and the other was St. thermophilus; the PCR amplifications classified these strains as 9 strains, which was consistent with the RAPD result, one of which 20 random primers obtained 9 distinct genotypes too, the 9 stains were 6 Lc. lactis strains, 2 St. thermophilus strains and 1 St. agalactiae strain. In addition, 2 species-specific PCR primers of Lc. lactis which named LclamyLF-R and LclmapA2F-R respectively were acquired, and St. thermophilus too, SttglgPF-R and SttamyLF-R respectively, and even one strain-specific primers named LclamyYF-R of L. lactis subsp. lactis AS 1.1936 which might loss nucleotides in itsα-amylase gene(amyY) were gained. Phenotypic traits of Lc. lactis and St. thermophilus strains were investigated at molecular level.The species-specific primers were designed first time by aligning sequences of the genes coding metabolic enzymes to identify them at the level of strains, and a St. agalactiae strain which has been applicated to yoghort was as control. We hope this method will offer theoretical and technical fundament for correlative research and industrialization of Lc. lactis and St. thermophilus,and provide reference for other lactic acid bacteria and even other microorganism identification.