Prestudy On Isolation And Identification Of Endophytic Fungi From Seeds Of Sinopodophyllum Hexandrum And Their Antimicrobial Activities

Sinopodophyllum hexandrum（Royle）Ying is a perennial herb of the genus Seven genus of the genus Berberidaceae.It is used as a premise of anticancer drugs with podophyllotoxin.Its demand is increasing day by day,coupled with the shortage of resources of Sinopodophyllum hexandrum.The rational development and utilization of Sinopodophyllum hexandrum has become a hot topic of current research.On the basis of previous research,this study selected the seeds of Sinopodophyllum hexandrum as the experimental object and carried out follow-up research:through the tissue culture of Sinopodophyllum hexandrum,endophytic fungi were obtained,and the endophytic fungi were isolated and purified to microscopic Morphological and molecular biology methods were used to identify endophytic fungi;S.aureus,Escherichia coli,Candida albicans,Fusarium graminearum,and Fusarium oxysporum f.sp.were used as indicators to screen for endophytic fungi with high bacteriostatic activity.The bacteria were used to optimize the fermentation process,and the active components in the secondary metabolites were initially identified.The main findings are as follows:1.Isolation and purification of endophytic fungi.A total of 9 endophytic fungi were isolated and purified from the seeds of Sinopodophyllum hexandrum by tissue separation.By identification,it was determined that seed 1 belongs to Mucor（Muscularus）;seed 2 belongs to Talaromyces（Cymbidium）;seed 3,seed 9 belongs to Penicillium（Pencilium）;seed 4 belongs to Rhizoctonia（Spirulina）;Seed 5 belongs to Aspergillus（Aspergillus）,seed 6,seed 7,and seed 8 belongs to Fusarium（Fusarium）.2.Screening of highly bacteriostatic strains.The antibacterial activities of endophytic fungi and secondary metabolites of 9 strains were tested.The 9endophytic fungi had different degrees of antibacterial activity against 5 indicator bacteria,but the antibacterial activity of Fusarium was the strongest.The secondary metabolites of this species are also more stable.3.Optimization of fermentation process of high antibacterial activity strains.Seeds 7 with high bacteriostatic activity were selected by single factor investigation.The Plackett-Burman test design was applied to the factors affecting fermentation.The influencing factors of the screening were designed by response surface methodology.The optimized process parameters were:inoculum size 6.5%,fermentation time 38h,rotation speed 185 r·min^-1,the bacteriostatic diameter was7.34mm under the optimized fermentation process.The inhibition diameter under this condition was 7.34 mm（RSD=16.56%）,and the deviation between the predicted value and the true value was 1.05%,indicating that the fermentation process was good,and the optimization process was accurate and reliable.4.Identification of secondary metabolite components.UPLC/Q-TOF-MS method was used to isolate 43 kinds of compounds from the endophytic fungus n-butanol fermentation broth with high antibacterial activity.Among them,37 kinds of chemical structures were initially identified,mainly including podophyllotoxin and other derivatives.Compounds such as lipoproteins,steroids,glycosides,and phenolic acids.Experiments have shown that the endophytic fungi isolated from the seeds of P.chinensis can produce the same bioactive substance as the host plant,podophyllotoxin,which provides ideas for future resource development.