Study On The Anti-vibrio Parahaemolyticus Activity And Mechanism Of Trypacidin From Marine Aspergillus Fumigatus

In the aquaculture industry,the economic loss caused by bacterial diseases of cultured animals has always been a difficult problem.For example,Vibrio parahaemolyticus can cause the death of fish,crab,shrimp,oyster and other aquatic animals,it has seriously affected the development of China’s aquaculture industry.At this time,it is particularly important to find green and efficient antibacterial compound from the rich marine resources,and marine fungi is a very good resource source.Aspergillus fumigatus HX-1 strain isolated from the symbiotic fungus of clam was cultured in our laboratory,and its antimicrobial activity was guided by ethyl acetate extraction,silica gel column chromatography,Sephadex LH-20 column chromatography and semi-preparative high performance liquid chromatography.One compound（trypacidin）with antibacterial activity against V.parahaemolyticus was obtained.In this study,the following results were obtained through the optimization of the fermentation conditions of HX-1 strain to produce trypacidin,the activity and mechanism of trypacidin against V.parahaemolyticus.（1）In order to improve the production of trypacidin from A.fumigatus HX-1 strain by fermentation,a single factor screening experiment was conducted on five factors including carbon source,nitrogen source,salinity,inorganic salt and p H in the culture conditions of HX-1strain for the production of trypacidin.Finally,the conditions of medium composition were determined as follows:3%maltose,1%beef extract,5%Na Cl,0.05%Mg SO₄,p H 7.（2）On the basis of determining the composition of culture medium,two-stage culture method was adopted to further improve the yield of trypacidin.The optimal fermentation conditions for synthesis of trypacidin by HX-1 strain were obtained by Box-Behnken experimental design and response surface analysis,with the main factors of shaking time,static time and inoculum size:shaking time for 5 days,static time for 6 days,inoculum size for 8%.Under the optimal fermentation conditions,the actual yield of trypacidin ether was 20.43±0.56mg/L,which was not significantly different from the predicted response value（21.54 mg/L）,indicating that the process of optimization was feasible.（3）When molasses was added into the fermentation medium of A.fumigatus HX-1 strain as substrate,more conconspores were produced by A.fumigatus.When molasses was added up to60 g/L,the production of trypacidin reached 24.06±0.57 mg/L,which was 17.7%higher than that of the group without molasses.The results showed that the addition of molasses could improve sporulation and biosynthesis of A.fumigatus HX-1 strain.（4）The Oxford cup method and test tube dilution method were used to evaluate the activity of trypacidin against aquatic pathogenic V.parahaemolyticus.The MIC and MBC of trypacidin against V.parahaemolyticus were 31.25μg/m L and 62.5μg/m L,respectively.The MIC and MBC of positive control streptomycin sulfate against V.parahaemolyticus were 62.5μg/m L and125μg/m L,respectively.Compared with streptomycin sulfate,trypacidin has a better inhibitory effect on V.parahaemolyticus.By reasearching the growth of bacteria,the integrity of the cell membrane and permeability,the permeability of the cell wall and cell morphology to find the mechanism of trypacidin against V.parahaemolyticus,the results showed that after acting on V.parahaemolyticus,trypacidin would destroy the cell wall and cell membrane of the bacteria,resulting in the leakage of intracellular biological macromolecules such as DNA,RNA,protein and other substances,changing the morphology of the bacteria,affecting the normal metabolism of nucleic acids,enzymes and ions in the cell,and ultimately leading to the death of V.parahaemolyticus.The effect of trypacidin on the ultrastructure of V.parahaemolyticus was observed by scanning electron microscopy.It was found that trypacidin had a destructive effect on V.parahaemolyticus cells.