The aim of this research is to find the antimicrobial substances from Bacillus pumilus D5 and analysis its antibacterial activity and characteristics, and then we will use the results for field experiments of hard clams. In this study we find the growth of aquaculture pathogens, like S. agalactiae, S.iniae, V. anguillarum and V.vulnificus, were inhibited by cell-free supernatant of D5, and the supernatant of mid-stationary phase D5 could show the highest antimicrobial activity.
The second step we use the agar diffusion to test the growth inhibition of S. agalactiae, S. iniae, V. anguillarum and V. vulnificus incubated with B. pumilus D5 supernatant from various cultural time during 60 hours, V. anguillarum could keep the 99.9% antimicrobial activity after 60 hours, MICs of cell-free supernatant of D5 for five pathogens, ranged from 4 to 8 dilute times as the concentration of ampicillin ranged from 0.05 to 12.5µg/ml.
To obtain a preliminary characterization of the antimicrobial substance, we analyzed its stability in a wide range of pH and temperature conditions and after treatments with six kinds of enzymes. The purified substance was stable at pH 2-10, and treatment with 80°C and 100°C for at least 10 min showed 50% antimicrobial activity, Pepsin, proteinase K and lipase tested - 1 - 1082297 • • showed more effect on the antimicrobial activity of the B. pumilus D5 supernatant. To investigate the nature of the antimicrobial molecule, we performed a preliminary purification of the bioactive compound by using a reverse phase column and eluted with acetonitrile in different ratios. The obtained fractions were tested to evaluate the antimicrobial activity on Streptococcus spp. Fluorescence microscopy analyses showed that the major antimicrobial mechanism of the purified substance from D5 supernatant appeared to involve disruption of the pathogens cell membranes.