Study On Genetic Stability Of Probiotic Lactobacillus Casei Zhang During Long-term Continuous Subculturing

As the project object, a probiotic strain of Lactobacillus casei Zhang was isolated from koumiss in the minority areas of China. This project was aimed at evaluating genetic stability of Lactobacillus casei Zhang systematically, thus during long-term continuous propagation, morphological characteristics from colony appearance, probiotic characteristics and whole genome re-sequencing technique were investigated inoder to unravel the molecular mechanism behind the variation by analyzing the evolution law. Normal MRS (De Man, Rogosa and Sharpe) and carbon source-limited MRS broth were applied as matrix for continuous subculturing.1%innoculum was subcultured at every24hours and each passage time with approximately at log2100≈6.64generations.The results showed that during2000generations subculturing, there is no changs for bacterial cell and colony appearance of L. casei Zhang was observed, as well as total biomass (OD6oo) and viable counts. Furthermore, there is no difference present for the metabolic ability of carbonhydrates and favorable bacterial activity with2000generations. Among2000generations, L. casei Zhang growed very well in two mediums with stable phenotypic characteristics and no microbial decay was observed. Additionally, the tolerance of L. casei Zhang for pH2.5simulated gastric fluid, pH8.0simulated intestinal fluid and bile salt remains unchanged. Moreover, the autoaggregating rate was not affected by2000generation subculturing in different medium.The probiotic properties of L. casei Zhang kept stable among2000generations. Through Miseq high-throughput sequencing system, the analysis of whole-genome re-sequencing for its1000th and2000th generations among two different mediums was performanced, and44single-nucleotide polymorphisms (SNPs) and23INsertions/DELetions (InDel) were found. There were less SNPs in its1000th generation in carbon resource-limited MRS broth, which is high-frequency presenters, once they are established, will be stably inherited. These perturbations are the classic signature of selection; while there were2SNPs increased in the2000th generation, the selection pressure has dropped and driven population differentiation. Meanwhile, among these two different mediums during2000generations, the genes rpoC (RNA polymerase, beta factor) along with high-frequency was found in the mutations of L. casei Zhang, which inferred that L. casei Zhang strains shows convergent evolution order in different culture conditions and the high-speed growth one owns a competitive priority.Overall, the high-frequency mutation occured less in the genome of the probiotic L. casei Zhang during the culturing in both medium, besides, the low-frequency mutation found in the1000th generation and then disappeared in the later2000th, which could be the sign of L. casei Zhang has been adapted in this similar environments for long-term survival. To sum up, the former statement indicates that L. casei Zhang is a probiotic with favorable genetic stability and could be used as safety probiotic for long-term period.