A Preliminary Study On Isolation, Identification And Antibacterial Activity Of Wild Strains Produced High Optical Purity D-lactic Acid

Objectives1To separate and screen wild strains which produced high optical purity D-lactic acid from a fermented food.2To identify these wild strains.3To observe the antibacterial activity of these isolates’ fermentation broth.Methods1Wild strains from a fermented food were separated and purified by streak plate method, and then the strains which produced high optical purity D-lactic acid were screened from these bacteria.2According to the phenotypic characteristics and16S rRNA gene sequence analysis, the isolated wild strains were identified to the species level, and then these isolates were identified to the subspecies level via the multilocus sequence analysis technology based on hsp60, pheS, rpoA and tuf genes.3The antibacterial activity of these isolates’fermentation broth was observed by Oxford Cup method.Results1Nine wild strains have been isolated and screened from a fermented food. These wild strains which were named SZD produced D-lactic acid of100%optical purity.2The SZD strains which stained Gram-positive were non-spore forming. According to the physiological and biochemical characteristics, these strains were identified as genus Lactobacillus. According to the similarity rates of more than99.9%of16S rRNA gene sequence among the SZD strains, Lactobacillus coryniformis subsp. coryniformis and Lactobacillus coryniformis subsp. torquens, these strains were identified as Lactobacillus coryniformis, but partial biochemical characterizations of the SZD strains were not entirely consistent with two known subspecies of Lactobacillus coryniformis. The similarity rates of the corresponding sequence of pheS gene among the SZD strains, Lactobacillus coryniformis subsp. coryniformis and Lactobacillus coryniformis subsp. torquens were100%and97.87%, respectively, while the similarity rates of the corresponding sequence of rpoA gene among these strains were98.65%and100%, respectively. The similarity rates of the corresponding sequence of tuf gene among the SZD strains and two known subspecies of Lactobacillus coryniformis were more than99.00%; in contrast, the similarity rates of the corresponding sequence of hsp60gene among these strains were less than99.00%. The phylogenetic trees constructed by unweighted pair-group method using arithmetic average based on16S rRNA, hsp60, tuf and pheS genes showed that the SZD strains were independent of the two known subspecies of Lactobacillus coryniformis, and formed a unique branch. According to the phenotypic characteristics and genotypic characteristics, the SZD strains were identified as a novel subspecies of Lactobacillus coryniformis.3The fermentation broth of all SZD strains had antimicrobial activity against4indicator bacteria (Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Staphylococcus saprophyticus) when the pH value was3.0, but with the pH value increasing, the antibacterial activity weakened gradually, and completely disappeared when the pH value was increased to5.0.Conclusions1Nine wild strains which produced high optical purity D-lactic acid have been isolated and screened from a fermented food.2According to the physiological and biochemical characteristics, these strains were identified as genus Lactobacillus. Combined the phenotypic characteristics with analysis of the16S rRNA gene sequence, these strains were identified as Lactobacillus coryniformis. These isolates were identified as a novel subspecies of Lactobacillus coryniformis via the MLS A technology based on hsp60, pheS, rpoA and tuf genes finally.3The fermentation broth of these strains had antimicrobial activity against4indicator bacteria when the pH value was3.0～4.5. The antibacterial activity depended mainly on the lactic acid of fermentation broth, and might be associated with lactobacillin and other antibacterial substances.