Screening Of Gut Antibacterial Strains Of Periplaneta Americana And Studies On The Active Components Of Symbiotic Streptomyces Sp.PAS8

Objectives: In this study,the wild Periplaneta americana which originated from a harsh environment was used as the research material to isolate and purify the gut symbiotic microorganisms.The obtained strains were detected the potential antibacterial activity.A strain with significant antibacterial activity was selected,the antibacterial active parts of its secondary metabolites were isolated and purified.In order to find the natural compounds with antibacterial activity,the minimum inhibitory concentration of the isolated monomer compounds was detected.Methods:1.The gut symbiotic microorganism of Periplaneta americana were isolated and purified by the dilution coating method and plate marking method.2.The antibacterial activities of symbiotic bacteria,fungi,and actinomycetes were detected by the cross-fringe method,confrontation culture method,and Oxford cup method,respectively.The strains with significant antibacterial activity were identified by molecular biology technology.3.A strain Streptomyces sp.PAS8 with significant antibacterial activity was selected for large-scale solid fermentation.The antibacterial active part of the secondary metabolites of the strain was further determined by the Oxford cup method.The active extract was isolated and purified by silica gel column chromatography and Sephadex LH-20 gel column chromatography.The structure of the compound was analyzed combined with nuclear magnetic resonance spectroscopy and HRESI-MS,as well as the minimum inhibitory concentration of the compound was determined by the double dilution method.Results:1.A total of 185 strains of symbiotic bacteria were isolated from the gut of 10 Periplaneta americana,including 68 strains of bacteria,76 strains of fungi,and 41 strains of actinomycetes.2.The antibacterial activity result of secondary metabolites from the gut symbiotic microorganism showed that 28 strains（41.2%）of bacteria had weak antibacterial activity,but mainly had weak antibacterial activity,and 23 strains（30.3%）of fungi and 34 strains（82.9%）of actinomycetes held varying degrees of inhibitory activity human pathogenic bacteria,and the antibacterial spectrum is extensive.The actinomycetes with significant antimicrobial activity and broad-spectrum antibacterial activity were classified by 16 S r RNA sequencing.The results showed that all the actinomycetes belonged to Streptomyces.Among them,Streptomyces sp.PAS8 with obvious antibacterial activity,and its 16 S r RNA sequence is99.86% similar to Streptomyces violaceoruber（KJ676477）.3.The crude secondary metabolic extract of Streptomyces sp PAS8 was extracted with water and ethyl acetate respectively,and the results of Oxford cup method showed that the ethyl acetate part of Streptomyces PAS8 had antibacterial activity.The antib-acterial active part was isolated and purified by normal phase silica gel column chro-matography and Sephadex LH-20 gel column chromatography,and three compounds wereobtained.They are respectively aloesaponarin II（1）,5-（2′-oxo-4′R-hydroxy-hexanoic acid）-2-methyl-chromone（2）,and13-methyltetradecanoic acid（3）.The minimum inhibitory concentration of compound 1against Enterococcus faecalis was 128 μg/m L.Conclusions: In this study,a total of 185 strains were isolated from the gut of wild Periplaneta americana,and 6 kinds of pathogenic bacteria were used as indicator strains.85strains（45.9%）of gut symbiotic strains of Periplaneta americana with antibacterial activity were screened,including 28 strains of bacteria,23 strains of fungi,and 34 strains of actinomycetes.The active strain bank of gut symbiotic microorganisms of Periplaneta americana was established.Streptomyces sp.PAS8,which has a wide antibacterial spectrum and obvious antibacterial activity against MRSA,was selected for large-scale fermentation,and three compounds were isolated.Compound aloesaponarin II showed certain inhibitory activity against Enterococcus faecalis and Pseudomonas aeruginasa.The research showed that the potential of discovering antibacterial active compounds from the gut symbiotic microorganisms of Periplaneta americana.