Screening Of Antimicrobial Microorganisms In The Soil Of Ji’an And Study On Antimicrobial Active Ingredients

In this experiment,we will select representative soil environment in Ji’an City,Jiangxi Province for sampling,screen and identify antimicrobial microorganisms in the soil,then select strains with strong antibacterial activity to carry out the separation of active metabolites,identify and study the structure and action mechanism of antibacterial active substances.It is hoped that it can provide assistance for the utilization of biocontrol strain resources and the development of new microbial drugs.1.Rhizosphere soil samples were collected from an ancient banyan tree planted in a Ming Dynasty in Ji’an City,Jiangxi Province.Collecting rhizosphere soil samples for antagonistic screening of actinomycetes in soil,after inoculation and purification,136 strains of actinomycetes were obtained.Finally,a broad-spectrum antagonistic strain AHF-20 which was passaged and stable was obtained.The inhibition zone of fermentation broth against E.coli reached 28.20 mm.According to the morphological observation,physiological and biochemical tests and molecular biological identification,the antagonistic strain was identified as Streptomyces lacticiproducens.2.The results showed that the fermentation products of Streptomyces AHF-20 had antagonistic effects on all 23 test indicator bacteria,and the antibacterial ability was stable,tolerated to temperature,light,ultraviolet,acid and alkali.Antibacterial activity still existed after heating at 121℃ for 20 min.The fermentation product was extracted with n-butanol according to the polarity of the active substance,and the obtained crude n-butanol extract was diluted to 1 μg/mL,and still had inhibitory effect for E.coli.The results indicated that it has well rutilization potential for biocontrol and the possibility of developing new microbial drug.3.TLC-Bioautography and TLC observations found that there should be multiple components in the fermentation metabolites of strain AHF-20 that have antibacterial activity and work together to produce antibacterial effects,and the polarities are very similar.Liquid-mass spectrometry and HPLC data analysis determined that the chemical formulas of the substances in the crude extract of the antibacterial active component after multiple purifications were C20H51N15O3、C20H51N15O4、C22H52N12O4、C20H51N15O4、C22H51N12O4、C22H51N12O3.The compound N-ethylpyridine was isolated from the fermentation product of SNB medium.Fluorescence microscopic observation combined with crude extracts of bacteriostatic active components on gram-negative(E.coli)and gram-positive(S.aureus)growth inhibition effect.4.Using the plate dilution method and the tube-dish method,with the help of Martin’s agar selection medium,226 fungi were isolated and purified from the soil at the accumulation site of Ji’an City in Jiangxi Province.Finally,a stable and broad-spectrum antagonistic fungus DAZ-2 was isolated,and the inhibition zone of fermentation broth against E.coli reached 24.15 mm.A passaging stable broad-spectrum antagonistic fungus DAZ-2 was isolated and identified as Aspergillus fumigatus by morphological observation,physiological and biochemical characteristics and ITS sequence analysis.5.Ethyl acetate was selected to extract the secondary metabolites of Aspergillus fumigatus DAZ-2.The TLC-Bioautography method showed that the antibacterial active substances in the two were not the same component,and the multi-component substances cooperated to produce bacteriostatic effect.The crude ethyl acetate extract of fermenting mycelium(200 μg/mL)has antibacterial effect on 11 indicator bacteria,and up to 32.75 mm inhibition zone against E.coli.The antibacterial ability was stable,tolerated to temperature,light,ultraviolet,acid and alkali.6.Microscopic observation and bacteriostatic mechanism experiment results show that the ethyl acetate extract of Aspergillus fumigatus DAZ-2 cell extract can significantly inhibit the growth of E.coli and Staphylococcus aureus,destroy the permeability of the cell membrane.The minimum bactericidal concentration for the two indicator bacteria is 1.5 mg/mL and 1mg/mL.It has the value of being developed as a new type of microbial medicine.Isolate the antimicrobial active substance of the secondary metabolite of Aspergillus fumigatus strain DAZ-2.After multiple purifications,the antibacterial activity of the crude extract disappeared,and the main point could not be found by TLC analysis.According to preliminary judgment,the metabolite of Aspergillus fumigatus DAZ-2 should be caused by the combined action of multi-component substances,and the single-component substance has no bacteriostatic effect or weak bacteriostatic effect.7.Three antagonistic strains were isolated from the soil of Qingyuan District Scenic Area and a vegetable garden in Ji’an County.They were named Streptomyces morookaensis strain F-19、Purpureocillium lilacinum strain TAZ-2 and Penicillium citrinum strain TAZ-8 based on molecular biology identification.