Screening Of Excellent Strains Of Lactic Acid Bacteria And Analysis Of CRISPR Diversity

Lactic acid bacteria(LAB)are widely used in many fields such as food fermentation industry,biomedicine and bioenergy.Some LAB strains have probiotic functions.The development of LAB strains with unique production performance and probiotic characteristics will be beneficial to enrich the diversity of LAB resources,provide excellent strains with potential application value for the fermentation industry,and provide good experimental materials for further study on the metabolic mechanism of LAB.It is especially important to accurately identify the genetic characteristics of LAB,which can lay a foundation for the screening of subsequent excellent strains and the analysis of CRISPR diversity.In this study,50 of laboratory isolated strains were identified by 16 S rRNA analysis.It was found that 36 strains belong to Lactobacillus casei group,four strains were Lactobacillus delbrueckii subsp.bulgaricus,three strains were Lactobacillus plantarum,one strain was Lactobacillus helveticus,one strain was Lactobacillus buchneri and five strains were Enterococcus faecalis.The experimental analysis of sugar fermentation found that the sugar metabolism ability of the strains was significantly different.Among the tested strains,the L.helveticus strain Y9-2 showed a strong ability to metabolize sugar,and all of the 14 tested sugars could be rapidly utilized in a short time.In this research,10 housekeeping genes(clpX,dnaA,groEL,murE,pyrG,pheS,recA,rpoC,uvrC and carB)were selected.The 38 L.casei were classified with Multilocus Sequence Typing(MLST)technology,based on allelic differences.The results showed that L.casei group were rich in genetic diversity and can be divided into two major categories,I and II,including 23 ST-types,especially the strain of E2 was special.The ability of LAB to grow,acid production capacity,acid and bile resistance,antibacterial ability,and ability to synthesize extracellular polysaccharides are important indicators for screening excellent strains.This study evaluated the production characteristics of 50 LAB strains such as growth ability,acid production capacity,acid and bile salt resistance,antibacterial ability,and extracellular polysaccharide synthesis ability.The results showed that the L.bulgaricus Y5,C2 and C10 of L.casei group,L.plantarum E1 had strong growth,acid production and bacteriostatic ability,and had potential application value.The LAB fermentation process is susceptible to infect with phage.The CRISPR-Cas system is an immune system formed by long-term competition between bacteria and viruses,and is effective against the invasion of host bacteria by foreign DNA.This study aims to reveal the diversity of CRISPR and provide a reference for the study of the use of CRISPR/Cas system to resist phage invasion and screening for excellent strains against phage.This study analyzed the CRISPR sequence of 36 LAB strains.Phylogenetic tree analysis indicated that the CRISPR sequence were diverse in LAB.The amplification and sequencing analysis of CRISPR sequence revealed that there were significant differences among the CRISPR sequence of L.bulgaricus strains.The Y1 only had the CRISPR2 sequence,while the Y5,CH3 and L6 only contained the CRISPR3 sequence,and the C1 did not invovle both of the CRISPR2 and the CRISPR3 sequences.L.helveticus Y9-2 detected a CRISPR sequence.The results showed that the CRISPR-Cas gene loci were distributed differently in different strains,and the CRISPR sequences were distributed in the upstream or downstream of the cas gene.There is a typical stem-loop structure in the secondary structure of the CRISPR repeats.The spacer BLAST alignment showed that the L.bulgaricus CRISPR spacers were homologous to multiple phages,a single spacer of L.casei and L.helveticus CRISPR were only homologous to 1-2 phages or plasmids.It is speculated that the strains were infected by phage or plasmid during evolution,and the CRISPR-Cas system formed acquired immunity against related foreign DNA such as phage.