Screening And Characterization Of Beneficial Bacterial Strains Isolated From Pericarpium Citri Reticulatae ‘Chachiensis'

Pericarpium Citri Reticulatae ‘Chachiensis',the pericarps of Citri Reticulatae Blanco cv.Chachiensis in Xinhui,Guangdong Province,was a superior food condiment and traditional medicine in southeast and eastern Asia,which belongs to the ‘genuine chenpi' according to the pharmacopoeia.As a product of natural aging,its rich and various bacterial community awaits exploration.In this study,a large number of bacterial strains were isolated,purified and identified from Pericarpium Citri Reticulatae ‘Chachiensis' purchased by several manufacturers for different years.Then,the isolated bacteria were screened and evaluated from the perspectives of probiotic characteristics and antimicrobial activity,respectively,in order to excavate the potential beneficial strains in Pericarpium Citri Reticulatae‘Chachiensis'.64 strains of bacteria were identified by 16 S r RNA sequencing.At order level,they were divided into two groups,bacillales and lactobacillales.They were further divided into four families,namely bacillaceae,paenibacillaceae,planococcaceae and lactobacillaceae.Eventually,they were clustered into ten genera.Among them,Bacillus accounted for the largest proportion,49 strains of which covered 33 species.The identification range of all strains was 95% to 100%.Based on the safety and gastrointestinal tolerance test,8 strains were screened out to have great survival in the simulated gastrointestinal stressful condition,the characteristics of non-hemolytic and non-biogenic amines.Among them,3 strains belong to Bacillus,3 strains belong to Lactobacillus,and the rest belong to bacillales.The basic probiotic properties,cholesterol-lowering potential and carbohydrate utilization capability were further investigated.These strains all had the basic characteristics of probiotics,including gastrointestinal tolerance,self-aggregation and hydrophobic interpretation of adhesion,and antibiotic resistance safety.Compared with other strains,Bacillus licheniformis N17-02 showed both remarkable cholesterol removal capability and presence of bile salt hydrolase gene.Thus,it could be a good probiotic candidate with hypocholesterolaemic potential.While Bacillus megaterium N17-12 displayed widest carbohydrate utilization profile.Hence,it was promising to be probiotics in host and as fermentation starter in fermented food or feed.Further based on antibacterial activity,64 bacterial strains were screened,among which Bacillus pumilus X14-67 had the best antibacterial activity against Staphylococcus aureus.After eliminating the interference of acidic substances and hydrogen peroxide,it was found that the crude extract of supernatant was sensitive to pepsin and protease K,and could not tolerate high temperature above 80 degree C.The complete genome map of 3714404 bp with GC content of 41.82% was assembled by the whole-genome de novo sequencing.The sequence similarity of 16 S r RNA with B.pumilus(NZ CP016784.1)was 100%.GO,COG and KEGG annotation showed that there were a large number of genes related to translation,transcription and transmembrane transport proteins in the strains,and the most of the pathways related to metabolism,which were speculated to be closely related to the synthesis,transport and secretion of antimicrobial substances.According to functional gene annotation analysis,a large number of glycoside hydrolase,carbohydrate esterase and glycosyltransferase are existed in the carbohydrate metabolism pathway.The existence of chloramphenicol and penicillane resistance genes as well as invasiveness virulence factor and weak toxic endotoxin genes reflects the self-protection,adaptability and survival ability of the strain.From the perspective of metabolic pathway,the strain has relatively complete biosynthesis pathways of lycopene,kanosamine and bacilysin,which may contribute to antibacterial activity.Anti SMASH software was used to predict the gene cluster of secondary metabolite synthesis.It also found that there were several gene clusters of secondary metabolites,which synthesis lichenysin-like cyclopeptide,antibacterial iron carrier bacillibactin and bacteriocin with uberolysin or circularin A as the core.Combined with the phenotype and genotype,it was suggested that there might be two mechanisms,competitive inhibition of iron ion and inhibition of bacteriostatin.The bacteriostatin secreted might be a mixture of protein and non-protein substances.In conclusion,the present study is the first report on beneficial strains screening and characterization of bacteria from Pericarpium Citri Reticulatae ‘Chachiensis'.which proved it was an important source of beneficial microbes.It also provides a new perspective for the subsequent research on the functional characteristics of Pericarpium Citri Reticulatae‘Chachiensis'.