| The wide use of antibiotics has caused the serious drug resistance of Aeromonashydrophila, which in turn makes it difficult to effectively control fish aeromonasis. Thus,it is extremely urgent to develop the substitutes to antibiotics. Bacillus species arewidely distributed in nature and have high stress tolerance as a result of their endosporeproduction. They are reported to exhibit the ability to control fish diseases, promote fishgrowth, and have the merits of no side effects, no residues, no drug resistance and freeof pollution. Therefore, they are considered to be ideal substitutes for antimicrobials andhave tremendous applicable value. In this study, a potential strain G1of B.amyloliquefaciens against A. hydrophila was isolated, its inhibitory effect on A.hydrophila, as well as control efficacy on grass carp (Ctenopharyngodon idellus)aeromonasis, was determined. On this basis, its antibacterial mechanism was exploredusing comparative proteomics and antibiotics-related genes’ analysis. In addition, thelaboratory production process technique of its microcapsules was optimized, itsmicrocapsules’ acid resistance, alkali tolerance and safety to the aquacultureenvironment were further evaluated. The results of this study laid the solid foundationfor the future development of B. amyloliquefaciens microcapsules as a potentialsubstitute for antibiotic additives. The main results are as follows:1. Isolation of B. amyloliquefaciens against A. hydrophila and its antagonisticefficacy on A. hydrophilaA potential Bacillus strain G1against A. hydrophila was isolated from pondsediments, and identified as a Bacillus amyloliquefaciens isolate using API50CHidentification system and16S rDNA sequence analysis. Its16S rDNA sequence wassubmitted to the GenBank with an accession number of HM245965, and found to havethe high homology of99%100%with those of Bacillus species in GenBank, and exhibited most closely related to B. amyloliquefaciens strain Ba-74501(GenBankaccession no.: DQ422953). In addition, the G1isolate showed broad-spectrumantagonism against A. hydrophila pathogens, displayed good inhibitory effects on A.hydrophila, and exhibited good efficacy on the diseased grass carps infected with A.hydrophila.2. Comparative proteomic analysis of B. amyloliquefaciens against A. hydrophilaBased on the successful screening of the non-antagonistic mutant of B.amyloliquefaciens strain G1, two-dimensional gel electrophoresis (2-DE) maps of strainG1and its mutant were imaged by the2-DE technique, and the differentially expressedproteins were further identified using the mass spectrometry and database searching.The experimental results showed that the total protein spots in the G1isolate’s2-DEmap were1135with the matching scores of89.37%. In comparison with the2-DE mapof the G1isolate’s mutant,65different protein spots were detected, and36of them werehighly expressed and successfully identified to participate in physiological functionssuch as protein synthesis and modification, energy metabolism, stress responsetolerance, drug resistance, antibiosis. This revealed that besides the antimicrobialproteins, its self-protection in adverse environments and good growth performanceplayed important roles in the antibacterial action.3. Antibiotics-related gene analysis of B. amyloliquefaciens against A. hydrophilaThe antibiotics-related genes from B. amyloliquefaciens strain G1against A.hydrophila were amplified by PCR and sequenced, the amino acid sequence,transmembrane heices, domain and secondary structure of their coding products werealso analyzed. The experimental results showed that only iturin synthetase essentialgene was present in the G1isolate, naturally clustered with genes of iturin family fromBacillus sp. in GenBank, including iturin A, bacillomycin D, mycosubtilin, and showed98%similarity to the iturin A gene of B. subtilis strain MH25and strain RB14. Theamino acid sequence of its coding product exhibited high similarity to those of iturin,iturin A, bacillorin and bacillomycin D, and was close relative to the iturin A synthetaseB of B. subtilis strain MH25(GenBank accession no.: ABY89499). In addition, noobvious transmembrane structure was present in the coding product of iturin synthetaseessential gene of the G1strain. However, AMP-binding site, PP-binding site werepresent in its domain, and alpha helix, beta turn, random coil, extended strand were alsopresent in its secondary structure. 4. Production process technique and characteristics of microcapsules of B.amyloliquefaciens against A. hydrophilaOn the basis of gelatin as the wall materials, the influences of gelatin concentration,inlet temperature, feeding speed and air flow on the viable cells in the microcapsules ofB. amyloliquefaciens strain G1were assayed using single factor method, the spraydrying processing parameters of its microcapsules were optimized through orthogonalexperimental design, and the morphology and tolerance to artificial gastric and intestinaljuices were further examined. The experimental results showed that the optimum spraydrying processing parameters to prepare B. amyloliquefaciens microcapsules weregelatin concentration of3.0%, inlet temperature of155°C, feeding speed of8ml/minand air flow of700L/h, and the most leading influence factor on the production of B.amyloliquefaciens microcapsules was the gelatin concentration, followed by feedingspeed, air flow and inlet temperature. In addition, B. amyloliquefaciens microcapsuleswere spherical, featured dimpled surface without holes and cracks,uniformly-distributed with an average size of9.22μm. They were also found to havegood tolerance to artificial gastric and intestinal juices.5. Safety evaluation of the microcapsules of B. amyloliquefaciens against A.hydrophilaAccording to Chemicals—Alga growth inhibition test (GB/T21805-2008), Waterquality—Determination of the acute toxicity of substance to Daphnia (Daphnia magnastraus)(GB/T13266-91), Water quality—Determination of the acute toxicity ofsubstance to freshwater fish (Brachydanio rerio Hamilton-Buchanan)(GB/T13267-91)and Clinical experiment technical practice for fishery drugs, the growth inhibition ofthe B. amyloliquefaciens microcapsule on Chlorella sp. and its acute toxicity toDaphnia, zebra fish and grass carps were observed, and its influence on mainphysicochemical factors of aquaculture water was also assayed. The experimentalresults showed that the growth of Chlorella sp. was promoted with B. amyloliquefaciensmicrocapsules at the final concentrations of0.2mg/L~2000mg/L, its IC50to Chlorellasp. was2000mg/L, and its LD50to Daphnia, zebra fish and grass carps were all>2000mg/L (or mg/Kg body weight). In addition, in the period of14days after the input of theB. amyloliquefaciens microcapsule into the farming water at0.2mg/L~2000mg/L, thecontents of the ammonia and sulfide as well as the pH values were gradually reduced,only the nitrite nitrogen concentration first rose slightly and followed by a slow decrease, and the changes of the physiochemical factors related negatively to theconcentration of the B. amyloliquefaciens microcapsules. Thus, the B.amyloliquefaciens microcapsule was actually nontoxic, its effects on the ammonia,nitrite nitrogen, sulfide and pH in the fish faming water were under the control of theirsafe concentrations to aquatic animals, such as the goby fry, Pelteobagrus fulvidraco,Esox lucius, Procambarus clarkia. The present study provided important scientific basison the safe use of B. amyloliquefaciens microcapsules in aquaculture.In conclusion, this study confirmed B. amyloliquefaciens as potential probiotics inthe fish aeromonas control, enriched its antibacterial mechanism, and made up for thedeficiency in the new preparation and application safety evaluation of B.amyloliquefaciens probiotics, and played important significance in the development ofaquatic probiotics and establishment of their safe use systems in aquaculture. |
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