Screening Of A Postbiotic Strain Against Aeromonas Hydrophila And Study On Its Bacteriocin

In this study,a pathogenic Aeromonas hydrophila strain Ah2 was isolated from Chinese giant salamander.Then a bacteriocin-producing Lactoplantibacillus paraplantarum M4L1 against Ah2 was obtained by screening.Subsequently,its antimicrobial physicochemical properties were explored and the whole genome sequence was characterized.Finally,the bacteriocin LP01 produced by the strain was isolated,purified and identified.Meanwhile,the antibacterial spectrum,antibacterial intensity and antibacterial effect of the bacteriocin were also evaluated here.Results are summarized as follows:1.Isolation and identification of pathogenic bacteria of diseased Chinese giant salamander:This chapter aimed to determine the main type of pathogen which caused a large number of deaths of giant salamander farms in Shaanxi.Firstly,viral pathogen were excluded by cytotoxicity test and gene amplification test of GSIV major capsid protein(MCP).10 strains of bacteria were then isolated from giant salamander by gradient dilution and plate coating.Hemolysis test showed that 1 of 10 bacteria from ascites hadβ-hemolytic activity and was identified as pathogenic Aeromonas hydrophila.Healthy giant salamanders were then tested for artificial infection of the suspected pathogen.Giant salamanders developed abnormal activity around a week and showed different degrees of surface ulcer after two weeks.It was finally determined that the main pathogen of infection with giant salamander was Aeromonas hydrophila and named it Ah2.Drug resistance test showed that the strain was not sensitive to 5 of 10 common antibiotics.Ah2 is highly drug resistant.2.Screening and whole-genome sequence characterization of an excellent bacteriocin-producing lactic acid bacterium(LAB):This chapter aimed to screen an excellent bacteriocin-producing LAB against the indicator Ah2,so that to evaluate physicochemical properties of the bacteriocin produced by the LAB and to analyze the whole genome sequence of the LAB.Through double-plate combined with agar punch method,a bacteriocin-producing Lactoplantibacillus paraplantarum M4L1 was screened from sufu sold in Qingdao.It was found that the strain was highly and moderately sensitive to 6 of the 10 tested common antibiotics.By analyzing the dynamic changes of M4L1 growth curve and bacteriocin production,results showed that when the biomass of the strain reached the maximum,its antibacterial activity also reached the peak,which indicated the biomass of M4L1 was positively correlated with its bacteriocin production.Then the bacteriocin produced by M4L1 was preliminarily purified by organic solvent extraction and named LP01.Physico-chemical analysis showed that,LP01 not only showed stable antibacterial activity at p H 2～10,-20～121 °C and storage period of 9 months,but also had good digestive enzyme and surfactant tolerance.LP01 also presented significantly characteristic peak at 220 nm corresponding to peptides,Bacteriocin LP01 belonged to peptide.In addition,the whole genome sequence of M4L1 was determined by Illumina Hi Seq2000 platform,and the virulence,drug resistance genes,pathogenic factors and potential bacteriocin gene clusters were excavated.It was verified that M4L1 didn’t contain any pathogenic factors and was predicted to be non-human pathogens.Moreover,genetic annotation predicted that M4L1 had two bacteriocin gene clusters(Ri PPs),and the corresponding products were Plantaricin K and Plantaricin E,respectively,which belonged to class II bacteriocin of Lactobacillus plantarum.As a bio-safety bacterium,L.paraplantarum M4L1 was equipped with good ability for bacteriocin production.3.Purification and identification of bacteriocin LP01 produced by L.paraplantarum and its antimicrobial characterization analysis:This chapter aimed to isolate and purify the bacteriocin LP01,and to explore its antibacterial characterization.Firstly,Tricine-SDS-PAGE showed that the molecular weight of LP01 was in the range of 3.3～4.0 k Da,LP01 was identified as small molecular bacteriocin.Highly purified bacteriocin LP01 was obtained by G-25 gel filtration chromatography,DEAE-52 anion exchange chromatography and reversedphase high performance liquid chromatography.Mass spectrometry analysis of highly purified LP01 were then carried out.MALDI-TOF / MS determined that the specific molecular weight was 3748.9643 k Da.Q-TOF-MS / MS predicted that the 34 amino acid sequences were HAEGTFTSDVSSYLEGQAAKKKEFLAWLVPVNV.The molecular mass of bacteriocin LP01 was different from that of other bacteriocins,and the sequence was not the same as the known bacteriocins in the NCBI protein BLAST analysis.Thus,LP01 was a novel bacteriocin.Moreover,LP01 presented a wide antimicrobial spectrum on 14 tested Gram positive and negative strains,including Acinetobacter baumannii,Listeria monocytogenes,Citrobacter freundii and etc.In addition,the MIC and MBC of LP01 against Ah2 were 12.94 μg/m L and 25.88 μg/m L,respectively.After treatment with LP01,the hemolytic activity and cytotoxicity caused by Ah2 were significantly relieved as confirmed.The microstructure of Ah2 treated with bacteriocin LP01 was observed by SEM and results showed that,LP01 destroyed the cell structure of Ah2,promoted the formation of cell cavities,depressions and content dissolution,resulting in cell rupture and death,which presented bacteriocin LP01’s excellent antibacterial effect on Ah2.To sum up,L.paraplantarum M4L1 and its novel bacteriocin LP01 were not only expected to become antibacterial agents for animal diseases in aquaculture,but also could be applied as biological preservatives to control multidrug-resistant strains in food,and evenmore provided a new direction for the screening of preventive and treament drugs in human disease.In all,bacteriocin-producing L.paraplantarum M4L1 possessed vast application prospects as a postbiotic candidate.