Isolation And Mechanism Of Antibacterial Active Components From Bacillus Megaterium L2

Bacillus megaterium L2 was isolated from the rhizosphere of flue-cured tobacco and had strong inhibitory effects on various plant pathogenic bacteria.In this paper,the antibacterial active components of L2 strain were isolated and purified by bioactivity tracking method,and the antibacterial activity and antibacterial mechanism of the obtained monomer compounds were investigated.The main results are as follows:1.Active screening of crude extracts of Bacillus megaterium L2 different polar solvents petroleum ether,ethyl acetate,n-butanol and water.The results showed that the antibacterial effect of each crude metabolite on A.tumefaciens T-37 was water>ethyl acetate>n-butanol>petroleum ether;the antibacterial effect against R.solanacearum Q11-2 was ethyl acetate>Water=n-butanol>petroleum ether;the antibacterial effect against E.carotovora EC-1 is water>n-butanol=ethyl acetate>petroleum ether.Comprehensive analysis,the water and n-butanol layer crude extract solvent polarity is too large,it is not easy to separate,so choose the ethyl acetate layer with moderate polarity and strong antibacterial activity against the three bacteria as the L2 active part.814.8 g of the crude crude extract of the ethyl acetate layer was obtained,and the yield was 2.33%.2.Using biological activity tracking,the crude extract of the ethyl acetate layer was further separated and purified,and finally four kinds of monomer compounds were obtained,which were identified by ¹H-NMR,¹³C-NMR and EI-MS to determine the monomer compound J-1 is erucamide,preliminary identification confirmed that monomer compounds J-3,J-4 are the same substance,and the combined number is J-3,the specific structure has not been identified,monomer J-2,J-5 is still in the identification.3.The antibacterial activities of the isolated monomer compounds J-1,J-2,J-3,and J-5 were screened,and the results showed that J-1,J-2,and J-3 were at a final concentration of 1 mg/mL.The inhibition rates of J-5 against indicator strain T-37were 45.6±0.38%,66.55±0.58%,10.42±0.29%,and 80.00±0.62%,respectively.The inhibition rate against indicator strain Q11-2 was 50.05±0.49,respectively.%,86.03±0.36%,88.94±0.32%,81.01±0.41%;the inhibition rates for indicator indicator EC-1 were 48.40±0.52%,3.57±0.42%,76.39±0.63%,and 5.13±0.36%,respectively.Monomer compound J-3 has more than 75%antibacterial activity against all three pathogenic bacteria,which is used to study its antibacterial mechanism.4.The antibacterial mechanism of monomeric compound J-3 was investigated by using three pathogenic bacteria as indicator bacteria.The IC50 of J-3 for T-37,Q11-2,and EC-1 was 0.896±0.038 mg/mL,0.735±0.067 mg/mL,and 0.952±0.05 mg/mL,respectively.At the IC50 concentration,J-3 treated pathogenic bacteria showed that the cells of the cells showed different degrees of shrinkage,rupture and even dryness;J-3 increased the permeability of the pathogen’s cell membrane,leading to leakage of electrolyte;it would destroy the integrity of the cell membrane,resulting in Large amounts of macromolecular substances such as nucleic acids,proteins and soluble sugars are extravasated;with the prolongation of J-3 action time,the phosphorus metabolism of the three indicator bacteria is abnormal,and the normal metabolic activities of the cells are affected;T-37,Q11-2 total bacterial proteins The expression level decreased significantly,and the expression of total EC-1 protein was not significant,indicating that J-3 inhibited the synthesis of T-37 and Q11-2 proteins.