Preliminary Study On The Types Of Bacteria In Wulingzhi And Their Biotransformation

Objective:Trogopterori faeces is the dry feces of Trogopterus xanthipes of Petauristidae,which has the effect of promoting blood circulation and removing blood stasis,dredging menstruation and relieving pain.Trogopterori faeces is mainly used for cardiovascular diseases and gynecological diseases,and its effect is remarkable.However,its hidden risk of security,unclear active ingredients and low compliance of patients which limits the development and application of medicinal materials.The purpose of this study is to explore the safety of clinical use of Trogopterori faeces by microbial limit test,clarify the structure and composition of natural microorganisms in Trogopterori faeces by sequencing,and isolate and identify its culturable bacteria and fungi.At the same time,the function of the fungi was studied by establishing co-fermentation system,to provide a partial basis for whether the flora is one of the effective components of Trogopterori faeces.Methods:In the first chapter,21 samples of Trogopterori faeces were collected after market investigation.According to the relevant methods under the microbial limit test of part IV of Chinese Pharmacopoeia(ChP)2015,the adaptability of the detection methods of total aerobic microbial count,total yeasts and molds count,Escherichia coli and Salmonella paratyphi B in Trogopterori faeces were investigated.Then the microbiological limit inspection of 21 batches of Trogopterori faeces medicinal materials and their decoction were examined with appropriate inspection methods.In the second chapter of this paper,ten samples were collected from the imitate wildness farmland in Shangluo City,Shaanxi Province.In this study,16S rDNA gene was used as molecular marker to explore the microbiome and the sequences were analyzed by Usearch analysis platform.The COG and KEGG database is used to predict and analyze the function of the flora.At the same time,the microorganisms in Trogopterori faeces were cultured,isolated and preserved by plate marking,and the 16S sequence of bacteria and the ITS1 sequence and ?-tubulin sequence of fungi were amplified.The sequences were Blast online,and the homologous sequences were analyzed by the software MEGA 5.0 to construct the N-J tree to identify the bacteria and fungi.In the third chapter,this study established the Microorganism-Typhae pollen bidirectional fermentation system and Microorganism-Ginsenoside transformation system,the changes of flavonoids and Ginsenoside in Typhae pollen before and after fermentation were detected to explore the function of some bacteria from Trogopterori faeces.Results:1.According to the microbiological limit inspection,it was found that aerobic microbial,yeasts and molds were detected in 21 batches of Trogopterori faeces,total aerobic microbial count ranged from 10 to 106 cfu/g,and total yeasts and molds count ranged from 10 to 106 cfu/g.Escherichia coli and Salmonella were positive in 11 dry Trogopterori faeces samples,Escherichia coli detection of 10 vinegar-processing samples were all negative,but Salmonella detection results were positive.Besides,Escherichia coli and Salmonella were not detected in 21 samples of Water decoction.The experimental results of the effects of processing and medication methods showed that vinegar and boiling could significantly reduce the total aerobic microbial count,total yeasts and molds count in Trogopterori faeces.Vinegar could eliminate the pollution effect of Escherichia coli in Trogopterori faeces..It had no effect on Salmonella,but boiling could.2.A great number of 285218 high quality clean reads with a length of 400-450 bp were obtained from 10 samples.Bacterial species detected in these samples covered 8 phyla,28 families,75 genera and 120 species.The dominant phylum microbial communities in these samples were Firmicutes(87.68%±2.68%)and the Bacteroidetes(7.62%±3.74%),all samples showed a high microbial diversity,the predicted functional metagenome was heavily involved in energy metabolism.This study provided that the beneficial bacteria in Trogopterori faeces may be one of its active ingredients,and no pathogens are detected in the sample.Through the microbial culture of Trogopterori faeces,22 strains of single bacterial colonies were isolated,including 1 strain of Lactobacillus,13 strains of Bacillus,4 strains of Enterococcus.and 4 strains of Escherichia.36 strains of fungi were isolated and identified as 5 genera and 7 species,including Aspergillus,Colletotrichum,Fusarium,Pestalotiopsis Clostridium and Nigrospora.3.The fingerprint similarity of Microorganism from Trogopterori faeces Typhae pollen co-fermentation system before and after fermentation is more than 99%,indicating that the bacteria and fungi have little effect on flavonoids in Typhae pollen.In Microorganism of Trogopterori faeces-ginseng saponins transformationsystem,it was found that Aspergillus fumigatus could transform ginsenoside Re,which has the highest content of total ginsenosides,into ginsenoside Rg1,and the conversion rate reached 35%after 5 d fermentation.Conclusion:1.This study found that there are a large number of culturable microorganisms in aerobic conditions in Trogopterori faeces.It is found that there are Escherichia coli and Salmonella paratyphi B contamination in Trogopterori faeces,but the potential harm can be reduced or even eliminated by clinical vinegar decoction,which provides a reference for the safety of Trogopterori faeces clinical drug use.2.High-throughput sequencing showed that the natural microorganisms carried by Trogopterori faeces were mainly Firmicutes and Bacteroidetes,and there were a large number of microorganisms related to the production of short-chain fatty acids,suggesting that there may be potential probiotics in Trogopterori faeces.In this study,22 strains of bacteria and 7 species of fungi were isolated from Trogopterori faeces,the bacteria are distributed in the genus Lactobacillus,Bacillus,Enterococcus,Escherichia;the fungi have been identified as Aspergillus,Colletotrichum,Fusarium,Pestalotiopsis Clostridium and Nigrospora.3.The fingerprints showed that the culturable microorganisms isolated from Trogopterori faeces had no effect on the flavonoids in Typhae pollen.Among which Aspergillus fumigatus could transform ginsenoside Re into ginsenoside Rg1 which can,be used as a strain for preparing special ginsenosides by biotransformation.