| With the development of the mariculture industry,bacterial diseases have occurred widely in mariculture species.However,there are many safety problems in the use of chemical drugs and antibiotics.Therefore,more and more scholars have focused on finding antagonistic bacteria resistant to seawater pathogens.On the basis of previous studies,this experiment screened out strain with good inhibitory effect on Edwardsiella tarda,Vibrio harveyi and Streptococcus agalactiae.In order to analyze the way of its antibacterial activity,we verified the influence of physical and chemical factors on antibacterial substances of strain BA09.Furthermore,HPLC and ESI-Q-TOF were used to purify and identify the antibacterial substances of strain BA09.The effects of BA09 on growth and immunity of Scophthalmus maximus infected with Edwardsiella tarda were determined by adding BA09 into the diet.It is expected to provide a useful reference for the potential application of BA09 in aquaculture.The specific research contents are as follows:(1)Identification and antimicrobial activity analysis of BA09Seventeen probiotics were used as the basis,and Edwardsiella tarda,Vibrio harveyi and Streptococcus agalactiae were used as indicators.The strain BA09 was screened by double-plate method and stiletto method.Its metabolites had better antagonistic effect on the three Marine pathogens than other alternative strains,and it was identified as Bacillus cereus.The metabolites of BA09 treated by different physical and chemical factors did not change their antagonistic effect against the three pathogens.It showed that it was not sensitive to physical and chemical factors.Based on the experimental results and time cost,36 h fermentation and storage at-80 ℃were selected.(2)Separation and identification of metabolites of BA09The cell free supernatantwas extracted with methanol and tested by the stiletto method.The results showed that the antibacterial active substances were polar and soluble in methanol.The antifungal activity remained after desalting by dialysis.A total of 27 groups of fractions were collected by RP-HPLC.The results showed that the F19,F20 and F21 had antibacterial activity against Edwardsiella tarda.But the F16,F17,F19,F20,F21 and F26 showed antibacterial activity against Vibrio harveyi and Streptococcus agalactiae.Three lipopeptides were detected by LC-MS: Bacillomycin,Fengycin and Surfactin.(3)Study on the application of BA09 to the biocontrol of Edwardsiella tardaThe safety test was conducted by injecting and soaking,and the results showed that there was no significant difference in weight gain,specific growth rate and fatness between the test group and the control group.BA09 has no obvious adverse effects on Scophthalmus maximus and is safe to it.The protective test showed that there was no significant difference in growth indices and immune enzyme activities of Scophthalmus maximus fed with BA09.Therefore,feeding strain BA09 throughout the feeding process can reduce the death caused by Edwardsiella tarda infection in Scophthalmus maximus.In conclusion,a strain of Bacillus cereas was screened out to inhibit three Marine pathogens,and the antibacterial activity of its extracellular antibacterial substance was studied.The results showed that the antibacterial substances of strain BA09 were Bacillomycin,Fengycin and Surfactin.The death of Edwardsiella tarda infection could be reduced by feeding BA09 to Scophthalmus maximus. |
