Isolation And Identification Of Bacillus Velezensis DH82 From The Yap Trench In The Western Pacific And Preliminary Study On Its Antimicrobial Protein

At present,there are many diseases in aquaculture in China,especially those caused by aquatic pathogenic bacteria,which are explosive and rapid.How to effectively prevent and control aquatic diseases is an important task facing aquaculture industry.Traditionally,chemical antibiotics or chemical drug are the important means to prevent and control aquatic bacterial diseases.Chemical antibiotics or chemical drug do solve the problem of bacterial diseases to a certain extent,but using chemical antibiotics or chemical drug often brings problems such as drug resistance of pathogenic bacteria,drug residues in aquatic products and environmental pollution.Therefore,non-pollution,non-drug resistance and pollution-free chemical antibiotic or chemical drug substitutes become the key to solve the problem.The diversity of metabolic pathways and tenacious vitality of deep-sea Bacillus endow it with the ability to secrete new antimicrobial substances.Isolation of proteins from deep-sea Bacillus spp.that can prevent and control aquatic pathogenic bacteria without pollution and drug resistance is a research hotspot in the prevention and treatment of aquatic diseases.This thesis aims to solve the serious problem of aquatic bacterial diseases and aquatic product safety in our country.A strain which has strong antimicrobial effects on aquatic pathogenic bacteria were screened from deep water samples in the Yap Trench in the Western Pacific,and bioactive proteins were obtained through separation and purification by ammonium sulfate fractional precipitation and Sephadex chromatography from fermentation and culture of the strain,this protein shows good ability to prevent and control aquatic pathogenic bacteria and can be regard as an alternatives to replace chemical antibiotics or chemical drug in solving the problems of aquatic bacterial diseases.The main results are as follows:1.Screening,isolation and identification of DH82 strain against aquatic bacterial diseasesIn this paper,106 strains of Bacillus spp.were isolated from 6000 m deep water samples in the Yap Trench of the Western Pacific by heating treatment,gradient dilution and morphological observation.79 strains of antagonistic bacteria were screened for the preliminary screening test of antagonistic bacteria against aquatic pathogens.Then,DH82 strain which has high activity of antibacterial protein was found by rescreening requirement.The rescreening requirement whether the product of antibacterial activity was a protein.DH82 strain was identified as Bacillus velezensis by combining morphological observation,physiological and biochemical characteristics and molecular biological analysis.2.Study on preparation and physicochemical properties of crude antimicrobial proteins from Bacillus velezensis DH82In order to further study the properties of antimicrobial active substances,B.velezensis DH82 was cultured in modified LB medium,and the growth characteristics were studied.The result showed the optimum fermentation time for producing antimicrobial protein was determined to be 24 h.Then the protein was concentrated by ammonium sulfate fractional precipitation method,and we could find that the optimum ammonium sulfate precipitation saturation was 70%.The physicochemical properties of crude antimicrobial proteins were studied.Edwardsiella tarda was used as the indicator of pathogenic bacteria.It was found that the crude antimicrobial proteins from B.velezensis DH82 had a good thermostability,and performed no significant difference with the treatment of the protease including protease K,papain,pepsin and trypsin.The optimum p H was 7.0.The crude antimicrobial protein activity could be enhanced by metal ions K+ and Na+,whilst be inhibited by metal ions Mg2+,Ca2+ and Fe3+.3.Preliminary isolation and purification of crude antimicrobial protein and detection of its inhibiting spectrumThe crude protein from the fermentation supernatant of B.velezensis DH82 was separated by chromatography using Sephadex G-75 column.Two peak proteins Pr I and Pr II were obtained.The result of inhibition spectrum analysis showed that the proteins had good antimicrobial activity against the aquatic pathogens such as E.tarda,Aeromonas hydrophila,Vibrio campbellii,V.alginolyticus,V.harveyi,Streptococcus agalactiae and Staphylococcus aureus.It showed that the antimicrobial active substance of B.velezensis DH82 had a broad inhibition spectrum for some aquatic pathogenic bacteria.The protein Pr II showed extra antimicrobial activity against the plant pathogen fungi of Fusarium equisetum.The Minimal Inhibitory Concentration(MIC)of the proteins were determined.The MIC of the proteins to E.tarda and V.alginolyticus were lower than 7.16 ?g/m L.The findings in this study indicated that B.velezensis DH82 was valued on the potential application on the prevention and control of common bacterial diseases in aquaculture and some case of plant fungal diseases on agriculture.