| Intensive breeding mode has been widely used in aquaculture in China,but the establishment of this breeding mode has produced a series of problems,among which the most prominent is the frequent occurrence of diseases caused by aquatic pathogens.Vibrio parahaemolyticus is not only a foodborne pathogen but also one of the main culprits of aquatic product diseases.Seafood infected with vibrio parahaemolyticus and other aquatic pathogens will produce a variety of symptoms,if people eat these seafood will pose a threat to human health to a certain extent.Vibrio parahaemolyticus diseases not only destroy the green and healthy development mode of aquaculture industry in China,but also threaten the economy of China to a certain extent.The use of antibiotics can play a good effect in the short term,but in the long run,the use of antibiotics causes a series of problems: reducing the immunity of water and aquatic animals,increasing the number and types of drug-resistant strains,killing harmful bacteria while destroying beneficial bacteria,etc.Therefore,the research on antibiotic substitutes is not only a problem that people are most concerned about,but also a research hotspot for researchers.In this context,in this study,a strain with resistance to Vibrio parahaemolyticus was isolated from the symbiotic microorganisms of mariculture animals and its activity was preliminarily studied.The results of the study are as follows:(1)From 11 kinds of seafood such as conch,prawn and yellow croaker,76 strains of microorganisms were isolated by dilution coating plate method and plate scribe method,including 50 strains of bacteria and 26 strains of fungi.After screening,two strains with strong anti-vibrio parahaemolyticus activity and stable inhibitory effect were selected.HL-3 strain from conch and XHY-1 strain from yellow croaker,respectively.(2)HL-3 was identified as Aspergillus flavus based on the cultural characteristics and ITS sequence analysis.The optimum culture conditions for producing active substances against Vibrio parahemolyticus in Aspergillus flavus HL-3 strain were studied.The results showed that the best antibacterial activity was obtained by using the modified Sabouraud’s medium,2% Na Cl,28 ℃,160 r/min for 7 d.The optimal medium was used for large fermentation.After culture,200 m L ethyl acetate was added into every 500 m L triangular flask to extract secondary metabolites.The extract was extracted with ethyl acetate twice and finally combined with organic phase and steamed by rotary evaporator.Using various chromatographic method for separation of secondary metabolites with active compounds,the first is using silica gel column elution,active tracking by Oxford cup method is pair of hemolysis activity of vibrio components,the active component gel column separation again,again by Oxford cup method for active tracking gel column chromatography to get active component,Finally,the active components were prepared by HPLC.An active compound was prepared by separation of silica gel column,gel column and high performance liquid chromatography,and was finally determined as kojic acid by carbon,hydrogen and mass spectrometry.The MIC of kojic acid for Vibrio parahaemolyticus was 4 mg/m L,for Vibrio harveyi was 4 mg/m L,and for Aeromonas hydrophila was 6 mg/m L.(3)By observing the morphological characteristics of XHY-1 active strain combined with ITS sequence analysis,the active strain was finally identified as Neurospora sp.The results of condition optimization showed that the optimum culture conditions of the active strain were as follows: the content of Na Cl was 3% in the czapek yeast extract medium,and static culture for30 d.After the fermentation of the active strain XHY-1,The fermentation products were extracted with organic solvent,and the compounds with anti-Vibrio parahaemolyticus activity were isolated by silica gel column chromatography,gel column separation and HPLC.The thesis has 14 figures,9 tables and 114 references. |
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