| Vibrio parahaemolyticus is not only the main pathogen of bacterial diseases in aquaculture animals,but also the leading foodborne pathogen causing bacterial food poisoning.Therefore,effective prevention and control of Vibrio parahaemolyticus is an urgent problem to be solved.The prevention and treatment of Vibrio parahaemolyticus in aquaculture is mainly by using antibiotics,but the abuse of antibiotics not only produces drug-resistant strains,but also damages the ecological balance of aquaculture environment.And the residues of antibiotics in aquatic products pose a threat to human health.Therefore,the ideal substitute for antibiotics is imminent.The antimicrobial peptides produced by probiotics can be used to control the disease of Vibrio parahaemolyticus.The antimicrobial peptides produced by probiotics can not only maintain the balance of intestinal flora of aquatic animals,improve the immunity of aquaculture animals,but also improve the aquatic environment.Therefore,antimicrobial peptides will play an important role in promoting the food safety of aquatic products and the development of aquaculture,It is an ideal substitute for antibiotics.At present,no matter in the laboratory or in the industrial stage,there are few reports on the use of antimicrobial peptides in the prevention and treatment of Vibrio parahaemolyticus,and the antimicrobial activity is not strong enough.Moreover,the bioinformatics analysis of probiotic strains using the third generation high-throughput sequencing technology is rare.In view of this,probiotics that inhibit the Vibrio parahaemolyticus will be explored from the aquatic environment and reused in the aquatic environment to reduce the decline or disappearance of antibacterial activity caused by changes in the growth environment of probiotics in this study.A strain of Bacillus subtilis with strong antibacterial activity and broad-spectrum antibacterial activity was screened from the East China Sea,and its taxonomic statuswas identified and identified.The physical and chemical properties of the antibacterial substances produced were explored.The antibacterial substance of Bacillus subtilis was purified.Finally,the bioinformatics analysis of the Bacillus subtilis strain was completed by the third generation high-throughput sequencing technology.1.Screening and identification of Bacillus subtilisA total of 50 bacteria were isolated from seawater samples from the East China Sea.The primary screening was carried out by the point method,and 7 strains of Bacillus having bacteriostatic activity were obtained.The re-screening was carried out by the perforation diffusion method,and only the strain H19 had the strongest antibacterial effect and the antibacterial effect was stable.Therefore,strain H19 was selected as the target strain.Strain H19 can inhibit 7 Gram-positive bacteria,9 Gram-negative bacteria,and 2 yeast strains,which can inhibit Gram-positive bacteria and inhibit most Gram-negative bacteria,and Gram-negative bacteria Positive bacteria have stronger bacteriostatic effect than Gram-negative bacteria.The strain H19 was identified as Bacillus subtilis by the morphological analysis and 16 S rDNA gene sequence homology analysis.The strain was named Bacillus subtilis H19,and the GenBank accession number was MG383451.2.Study on the properties of H19 antibacterial substancesThe growth curve of H19 strain was studied,and the pH stability,temperature stability and ultraviolet stability of H19 bacteriostatic substance were analyzed.The preliminary identification of H19 bacteriostatic substance was also carried out.The antimicrobial activity of H19 reached the maximum value of 640 AU/mL at 24 h,and maintained a balance of 640 AU/mL between 24 and 60 h.The growth logarithmic phase was 0-24 h and stable phase was 24-60 h.The antimicrobial activity declined after the bacteria entered the declining phase.The pH of fermentation broth increased continuously until about 8.0.The H19 bacteriostatic substance has good thermal stability,and retains 50% bacteriostatic activity after 10 minutes of treatment at 100 ℃.and maintains strong bacteriostatic activity at pH 2.0-9.0.The H19 bacteriostatic substance has good ultraviolet stability,and the bacteriostatic effect of the bacteriostatic substance is not affected by 10-60 minutes of irradiation under ultraviolet light.The crude extract of H19 bacteriostatic substance was crudely extracted and treated with five proteases and Tricine-SDS-PAGE electrophoresis in situ bacteriostasis test.The crude extract was preliminarily identified as a protein with molecular weight between 6.5-9.5kDa and 27.0-35.0 kDa.3.Isolation,Purification and Identification of Antimicrobial Peptides Produced by H19The isolation and purification of antimicrobial peptide H19 can be divided into 4steps: ammonium sulfate precipitation,Sephadex G-50 gel filtration chromatography,DEAE-52 ion exchange chromatography and HPLC chromatography.Among them,the protein precipitated by ammonium sulfate was dissolved in phosphoric acid buffer,and then dialyzed with dialysis bag with molecular weight of 1000 Da to obtain crude extract.Two elution peaks appeared in the 10-40 th tubes of the crude extract through Sephadex G-50 column.Component F1 of the first elution peak was obtained by merging the10-25 th tubes.Component F2 of the second elution peak was obtained by merging the40-70 th tubes.The antimicrobial activity of the two components was tested.The results showed that Component F1 had no antimicrobial activity and Component F2 had antimicrobial activity.Component F2 was dissolved by PBS buffer(pH7.0)after vacuum freeze-drying on DEAE-52 column.From the elution curve,three elution peaks appeared at 10 th,30th and 70 th tubes.Component F2-1 of the first elution peak was obtained by merging 10-20 th tubes,component F2-2 of the second elution peak was obtained by merging 30-60 th tubes,and component F2-3 of the third elution peak was obtained by merging 70-100 th tubes.The results showed that component F2-1 and component F2-3 had no bacteriostatic activity,while component F2-2 had bacteriostatic activity.Component F2-2 obtained by DEAE-52 ion exchange chromatography was further separated and purified by HPLC.Four elution peaks were obtained,and only component F2-2-3 of the third elution peak had bacteriostatic activity.Finally,MALDI-TOF-TOF/MS method was used to identify and analyze the isolated antimicrobial peptides.The results showed that there was an antimicrobial peptide.After amino acid analysis and comparison,the molecular weight was 1042 Da.4.H19 Bioinformatics AnalysisThe whole genome of Bacillus subtilis H19 was sequenced by the third generation single molecule sequencing technology,The GenBank accession number is CP039935.And then the bioinformatics analysis was carried out.Firstly,the gene prediction of Bacillus subtilis H19 was carried out by Glimmer 3.02 software.It was found that the genome length of Bacillus subtilis H19 was 3,595,203 bp,which contained 4217 predictive genes.The content of GC was 44.58%,and there was one Scafflod.The protein sequences of the predicted genes were blastp compared with COG,GO andKEGG databases respectively(BLAST 2.2.28+)to obtain annotation information of the predicted genes.The results showed that 3124 genes were successfully annotated with COG function.The most functional annotations involving genes were Amino acid transport and metabolism,Carbohydrate transport and metabolism,and Transcription.2711 genes could be annotated in GO database.According to the functional classification of each gene,it can be divided into three subclasses: Biological process(17 branches),Cellular component(14 branches),Molecular function(13 branches),totaling 44 branches.According to KEGG enrichment analysis,3431 predictive genes of Bacillus subtilis H19 gene participate in 178 KEGG metabolic pathways,of which 281 genes participate in secondary metabolites.The biosynthetic pathway includes six genes involved in the biosynthesis of non-ribosomal peptides in the iron carrier group and nine genes involved in the structure of non-ribosomal peptides.Then the Scaffold sequence in the whole genome of Bacillus subtilis H19 was predicted and analyzed by using antiSMASH software.27 gene clusters were obtained,including 6 antimicrobial peptide gene clusters,2 Sactipeptide gene clusters and 4 NRPS gene clusters.Finally,we compared Bacillus subtilis H19 gene with ARDB antibiotic resistance gene database and VFDB virulence factor database to evaluate the safety of Bacillus subtilis H19.We found that there were 0 antibiotic resistance genes and 18 virulence factors. |
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