Bacterial Diversity,Probiotic Characteristics And Whole Genome In Hurunge,Mongolia

With the continuous penetration of modern lifestyles into pastoral areas and the increasing variety of portable convenience foods,the types and processing of Hurunge are decreasing,and the valuable microbial resources contained therein are facing loss.Therefore,it is of great scientific research significance to increase the excavation and evaluation of lactic acid bacteria resources in related traditional dairy products.This study focused on 12 precious Hurunge samples collected from four regions of Mongolia.Firstly,the bacterial community diversity was evaluated by determining the sequences of the V3～V4 region of bacterial 16 S r RNA gene in Hurunge samples using highthroughput sequencing technology.Secondly,78 lactic acid bacteria were isolated from Hurunge and screened for potential probiotic properties.One potential probiotic strain was selected for further study on its probiotic properties and safety evaluation.Finally,the whole genome sequence of the selected strain was determined.By combining genotype and phenotype,the probiotic properties of the selected strain were explored in depth.The main findings are as follows:(1)The results showed that there were significant differences(P<0.05)in bacterial diversity and abundance in Hurunge samples from four regions,with the dominant phylum being Firmicutes and Proteobacteria,the dominant genus being Lactobacillus,samples from the former Ovorkhangay province(Q)region containing potential bacterial nanocellulose producing capacity of Komagataeibacter and the relative abundance of Lactococcus was significantly higher in Tov Province(Z)than in the other three areas,and traces of Enterobacter and Citrobacter associated with foodborne pathogenicity and environmental contamination were also found in this area.The prediction of gene functions by PICRUSt2 software showed that genes related to carbohydrate and amino acid metabolism among metabolism related to bacterial growth and reproduction dominated the microorganisms.(2)Twelve strains of lactic acid bacteria with good tolerance ability were initially screened by acid tolerance(p H 2.0 and p H 3.0)and bile salt tolerance(0.3% W/V)ability tests,and then one strain of Lacticaseibacillus rhamnosus Z23 with 90.19% survival rate in simulated artificial gastric fluid and 85.96% survival rate in simulated artificial intestinal fluid was finally screened by simulated artificial gastric fluid and simulated artificial intestinal fluid tests,indicating that this strain has good ability to tolerate the gastrointestinal environment.(3)The probiotic evaluation of strain Z23 revealed that its hydrophobicity to organic solvents xylene,ethyl acetate and hexadecane were 45.08%,19.33%,and 33.10%,respectively,while its adhesion rate to human colon cancer cells(Caco-2)was 5.20%.After5 h,the auto-aggregation rate of strain Z23 reached 74.63%,which was superior to the reference strain LGG.The co-aggregation rates of strain Z23 with Staphylococcus aureus,Escherichia coli,Listeria monocytogenes,and Salmonella typhimurium were all above 36%.Furthermore,strain Z23 also demonstrated inhibition effects against Bacillus subtilis,Bacillus cereus,Listeria monocytogenes,Pseudomonas fluorescens,Staphylococcus aureus,Escherichia coli and Salmonella typhimurium.The cell suspensions and fermentation supernatant of strain Z23 showed strong scavenging activities against DPPH radicals,hydroxyl radicals and superoxide anion radicals,while the antioxidant activity of cell lysate was weak and total antioxidant capacity(T-AOC)was lower than the reference strain LGG.(4)In vitro safety tests showed that strain Z23 has no hemolytic activity and no amino acid decarboxylase activity;it is hypersensitive to five antibiotics such as clindamycin,tetracycline,chloramphenicol,erythromycin and ampicillin;the oral acute toxicity test in mice is non-toxic,so it is determined that the strain is safe for consumption.(5)Whole genome sequencing of strain Z23 showed a whole genome sequence size of 3006999 bp with an average GC content of 46.65%,containing one 2960559 bp chromosome and one 46440 bp plasmid.The whole genome contained 2800 coding sequence(CDSs),15 r RNAs genes,59 t RNAs genes and 35 s RNAs genes,9 genomic islands,3 phages,4 CRISPR-Cas sequences,325 promoters and 126 repetitive sequences.A total of2 secondary metabolite gene clusters(T3PKS and Bacteriocin)were generated.The number of all genes in the whole genome of strain Z23 that could be annotated to COG information was 2147,the number of genes with GO information was 1748,and the number of genes that could be localized to specific Pathway level 2 by comparison with the KEGG database was 1862.