Study On Phenotype And Genome Of Space Mutagenesis Isolates Of Lacticaseibacillus Rhamnosus Probio-M9

Lacticaseibacillus rhamnosus Probio-M9,isolated from breast milk of healthy women,is a probiotic with multiple physiological functions.As a new mutagenesis technique,space mutagenesis can induce strains to produce new phenotypic characteristics.Therefore,Probio-M9 sent into space aboard by Long March 5B carrier rocket in this study,was used as research project,and 100 mutagenic isolates were isolated and purified from the mutagenic isolates.Firstly,the growth characteristics of 100 mutagenesis isolates were preliminarily evaluated,and 3 mutagenesis isolates with significantly higher growth characteristics than Probio-M9 were obtained.Then the phenotype and genome were combined to comprehensively analyze the physiological and biochemical characteristics,probiotics and safety of the 3 mutagenesis isolates.The main research results are as follows:（1）The OD_600nm value of 3 mutagenesis isolates（HG-R7970-18,HG-R7970-29,HA-R7970-29）was significantly higher than Probio-M9 at each growth cycle point（P<0.05）by growth characteristics of 100 mutagenesis isolates.The viable count of HG-R7970-18,HG-R7970-29 and HA-R7970-29 was significantly higher than that of Probio-M9 after entering the logarithmic growth stage（P<0.05）.The maximum number of viable bacteria reached5×10⁹CFU/m L,6.2×10⁹CFU/m L and 7.38×10⁹CFU/m L,respectively.（2）The genome size of Probio-M9 and 3 mutagenesis isolates were 2.950±0.002 Mb,and GC content was 46.7%.A total of eight SNPs sites were identified in the 3 mutagenesis isolates,five of which were located in the intergenic region.There was one synonymous mutation site in HG-R7970-18,and one non-synonymous mutation site（ywq D）in HG-R7970-18 and HG-R7970-29 respectively.（3）Compared with Probio-M9,the morphological characteristics of HG-R7970-18 and HG-R7970-29 changed significantly,but HA-R7970-29 had no significant changes in morphological characteristics.API50 CHL results showed that carbohydrate utilization of the three mutagenic isolates did not change.TheΔOD_600nm values of the 3 mutagenesis isolates were not significantly different from those of Probio-M9 in different acid-alkaline environment and Na Cl concentrations（P>0.05）.Genome annotation results showed that the carbohydrate utilization genes of the 3 mutagenesis isolates were identical with Probio-M9,and genes encoding alkaline shock protein 23,acid shock protein,carnitine transporter penetrase protein and choline transporter penetrase protein were annotated.The results showed that the space environment did not change the utilization capacity of different carbohydrates and tolerance to different environments of the three mutagenesis isolates.The three mutagenesis isolates had the same physiological and biochemical characteristics as Probio-M9,which had certain application potential.（4）The probiotic properties and safety of the 3 mutagenesis isolates were further evaluated.The results showed that the 3 mutagenetic isolates showed the same bacteriostatic ability as Probio-M9.One identical bacteriotin gene cluster,encoding the key enzyme of acid production（ldh,atp）and H₂O₂ enzyme（pox,nox）genes was found in the genome.The auto aggregation ability of HA-R7970-29（76.5%）was significantly lower than that of Probio-M9（87.0%）（P<0.05）,but the cell surface hydrophobicity（43.0%）was not significantly different from that of Probio-M9（43.4%）（P>0.05）.The auto aggregation ability and cell surface hydrophobicity of HG-R7970-18 and HG-R7970-29 were significantly lower than Probio-M9（P<0.05）,the mutation of ywq D gene may be the reason why HG-R7970-18 and HG-R7970-29 showed different auto aggregation abilities and cell surface hydrophobicity.Glutathione synthesis（gsh AB）,NADH oxidase（nox）,NADH peroxidase（npr）and other antioxidant-related genes were annotated in the genome.The expression of antioxidant-related genes and the mutation of ywq D gene may be the main reasons for the difference in antioxidant capacity between the mutated isolates and Probio-M9.The safety evaluation results showed that the sensitivity of the three mutagenesis isolates to antibiotics was the same as that of Probio-M9,and no drug resistance genes and virulence genes were annotated,indicating that the three mutagenesis isolates had the potential for development and application.In conclusion,the phenotype and genome of Probio-M9 space mutagenesis isolates were analyzed in this study,and the genetic characteristics of phenotypic variation of 3mutagenesis isolates were expounded,and the causes of their changes were revealed.Space mutagenesis can not only obtain excellent strain resources,but also provide ideas for the breeding of lactic acid bacteria.The results of this study provide data support and theoretical guidance for the application of space mutagenesis isolates.