Taxonomy Methods For Identification Of Milk Derived Bacillus Strains

The members of the genus Lactobacillus are industrially important lactic acid bacteria that areinvolved in the production of many fermented foods of both plant and animal origin. Because oftheir peculiar metabolic and technological properties, we need to control the safty and quality inLactobacillus fermented foods production. Rapid and reliable identification of Lactobacillus at thespecies and strain levels is of great interest for basic knowledge and also for industrial purposes.In this paper, polyphasic taxonomy identification of 9 milk derived lactic acid bacillus strains(2 Lb. delbrueckii subsp. Bulgaricu strains, 1 Lb. delbrueckii subsp. Delbrueckii strain, 3 Lb.acidophilus strains, 1 Lb. fermentum strain, 1 Lb. casei strain, 1 Lb. buchneri strains) were studied.The strains were isolated by MRS plate streaking cultivation and morphological observation,including colony observation, transmission electron microscope(TEM) examination basing on thenegative staining by PTA, and were identified by biochemical essays basing on the biochemicalmetabolism differences, specific PCR which primers were designed basing on the sequencedifferences among their genes coding certain biochemical metabolic key enzymes, and RAPD. Theresults of plate streaking cultivation were 9 strains according to the sizes of their colonies and ceils;The size, shape and schizogenesis styles of all strains were obtained by TEM observation; and theresults of biochemical identification had 6 distinct fermentation types which classified these strainsas 6 microbial populations.the PCR amplifications classified these strains as 9 strains, which wasconsistent with the RAPD result, one of which 20 random primers obtained 9 distinct genotypestoo.In addition, 5 species-specific PCR primers were acquired, as LbdbmanLF-R of Lb. delbrueckiisubsp. Bulgaricu strains, LbcmanNF-R of Lb. casei strain,LbarbsK1F-R of Lb. acidophilusstrains, LbpmelAF-R of Lb. fermentum strain, LbddlacZF-R of Lb. delbrueckii subsp.Delbrueckiistrain. Furthermore, one pair of strain-specific primers LbamanNF-R of Lb. acidophilus AS 1.2467was gained. It was assumed that AS 1.1878 had different nucleotides in its mannose-specific IIDcomponent gene (manN) from other Lb. acidophilus strains.Phenotypic traits of Lb'delbrueckii, Lb.acidophilus, Lb. casei and Lb.fermentum wereinvestigated at molecular level. The species-specific primers were designed by aligning sequencesof the genes coding metabolic enzymes to identify them at the level of strains, while a Lb. buchneristrain was used as control. We hope this method will offer theoretical and technical fundament forcorrelative research and industrialization, and provide reference for other lactic acid bacteria andeven other microorganism identification.