Studies On Screening Of Antimicrobial Active Fungi And Secondary Metabolites From The Gut Of Blaps Rynchopetera

Objective:In this study,the metabolites of Blaps rynchopetera gut fungi were screened by Oxford cup agar diffusion method.In order to obtain antimicrobial active substances,the fungi with strong inhibitory activity and broad antibacterial spectrum were fermented on a large scale under the dominant culture conditions.Methods:1.The B.rynchopetera gut fungi were fermented on a small scale and the metabolites were extracted.9 strains of pathogenic microbes including methicillin-resistant Staphylococcus aureus(MRSA),Staphylococcus aureus,Escherichia coli,Enterococcus faecalis,Salmonella typhimurium,Pseudomonas aeruginosa,Aspergillus Niger,Penicillium expansum and Candida albicans were selected as indicator strains.The antibacterial activity of secondary metabolites of the gut fungi was detected by Oxford cup agar diffusion method.The strains with significant antimicrobial activity were identified by morphological and molecular biological methods,and the phylogenetic tree was constructed.And the antitumor activity of the identified strains was tested by using5 fine tumor cells.2.The culture conditions of 7 strains of gut fungi with significant antimicrobial activity were screened.The metabolites under different culture conditions were extracted and 6 strains of pathogenic bacteria were used as indicator strains.The antibacterial activity of the metabolites was detected by Oxford cup agar diffusion method.The fungi with strong inhibitory activity and wide antibacterial spectrum were screened.3.The secondary metabolites of fungi with significant activity were obtained by large-scale fermentation.The metabolites of fungi were analyzed by Ultra-performance Liquid Chromatography-Q-TOF-Mass(UPLC-Q-TOF/MS),and the antibacterial active parts were determined by activity tracking method.The monomer compounds were separated and purified from the active parts by thin layer chromatography(TLC),silica gel column chromatography and gel column chromatography,and the structure of the compounds were analyzed by nuclear magnetic resonance spectroscopy(NMR).Results:1.17 fungi with different levels of antibacterial activity were screened out from 108 strains of intestinal fungi of B.rynchopetera by Oxford cup agar diffusion method.7strains of BPF6,BPF33,BPF47,BPF55,BPF56,BPF100 and BPF106 were identified.According to ITS sequencing and Blast comparison,7 strains of fungi with significant activity were classified into 5 genera.The sequence similarity of fungi BPF6 and BPF33 with Trichoderma longibrachiatum reached 100%.The sequence similarity of BPF47 with Trichoderma atroviride reached 100%,and that of BPF55 with Fusarium oxysporum reached 100%.BPF56 and BPF100 showed 100% similarity to Penicillium citrinum,and BPF106 showed 100% similarity to Cladosporium pseudocladosporioides.The results also showed that the secondary metabolites of Trichoderma longibrachiatum BPF6,Trichoderma longibrachiatum BPF33 and Fusarium oxysporum BPF55 showed broadspectrum antitumor activity.2.After screening of culture conditions,the metabolites of Trichoderma longibrachiatum BPF6 obtained after 14 days of PDA culture had a wide antibacterial spectrum and significant antibacterial activity compared with the metabolites of other strains,and had a strong inhibitory effect on all the 6 pathogenic strains tested,and 4 of them had a strong inhibitory effect.The metabolites of intestinal fungus Penicillium citrinum BPF100 obtained after 14 days of SDA culture had significant antibacterial activity,and had strong inhibitory effect on the 4 tested pathogens.3.Large-scale fermentation of B.rynchopetera gut fungi Trichoderma longibrachiatum BPF6 and Penicillium citrinum BPF100 was carried out,and the total extracts were analyzed by UPLC-Q-TOF/MS.10 and 23 compounds were inferred from the metabolites of Trichoderma longibrachiatum BPF6 and Penicillium citrinum BPF100,respectively.The ethyl acetate extraction active parts of Trichoderma longibrachiatum BPF6 were isolated and purified,and one monomer compounds were identified,Compound 1 is(22E)-5α,8α-Epidioxyergosta-6,22-Dien-3β-ol,ergosterol compounds,which has been reported to have certain antibacterial,antitumor and antiplasmodium activities.Conclusion:In this study,the antibacterial activity of B.rynchopetera gut fungi was screened systematically.The results showed that there were abundant fungal resources with antibacterial activity in the gut of B.rynchopetera.Two strains of fungi with broad antibacterial spectrum and significant antibacterial activity were selected to study the active components,and one antimicrobial active components were obtained from fungal Trichoderma longibrachiatum BPF6.