Study On Endogenetic Fungi And Their Active Ingredients In Chinese Cordyceps

Chinese Cordyceps is a rare traditional Chinese medicine.It is a kind of complex composed of multiple fungi and the body of Hiepialus armoricanus.The isolation of the endogenetic fungi from Ophiocordyceps sinensis,determination of anamorphs and artificial fermentation have become a hot issue.Based on the isolation and identification of endogenetic fungi of Chinese Cordyceps,we studied the medicinal ingredients,similar to that of Chinese Cordyceps,generated by endogenetic fungi of Chinese Cordyceps and optimized the culture medium composition of Chinese Cordyceps.It laid a foundation for exploring new medicinal microbial resources,looking for the alternatives of Chinese Cordyceps and protecting the germplasm resources.Firstly,more than 30 isolates were isolated from Chinese Cordyceps with conventional isolation methods.Among them,6 isolates were identified based on their morphology,sporulation and molecular characteristics of clones.The results showed that 6 isolates belonged to three genera: Fusarium spp.,Mucor spp.,Mortierella spp.,respectively.Secondly,6 endogenetic fungi were submerged cultured in shaking flask.The nucleoside metabolites?uridine,guanosine,adenosine,cordycepin?related to Chinese Cordyceps in the fermentation broth of 6 endogenetic fungi were detected with HPLC?High performance liquid chromatography?.The results showed that all of the 6 endogenetic fungi can generate active ingredients similar to Chinese Cordyceps,such as Uridine,guanosine,adenosine and cordycepin.The uridine,adenosine and cordycepin that were generated by the strain NS2-B1 were relatively more than that of other strains.The guanosine that was generated by the strain NS2-Z?NS2-B1?NS1-B?NS2-B3 was relatively more than that of other strains.Thirdly,we optimized the composition of liquid medium based on the 2 strains submerged cultured in the shaking flask.The two carbon sources and two nitrogen sources were firstly selected based on the the wet weight of mycelia from five carbon sources?including sucrose,fructose,glucose,maltose and starch?and five nitrogen sources?including urea,beef extract,yeast extractand peptone?by one-factor-at-a-time experiments.The results showed that when carbon source was maltose or starch and nitrogen source was yeast extractor peptone,the wet weight of the 2 strains' mycelium reached the maxmium.Then,table L9?3⁴?was utilized to test four factors with three levels.The best carbon and nitrogen source combinations were screened based on the nucleosides?uridine,guanosine,adenosine,cordycepin?of Chinese Cordycepsin the fermentation broth detected by HPLC.The results showed that the suitable liquid medium for the NS2-B1?Fusarium spp.?consisted of 2% maltose,1.5% starch,1.5% yeast extract,1% peptone,plus to 0.8g/L KH₂PO₄ and 0.8g/L Mg SO₄?7H₂O.The maximum of total nucleosides was 267.67g/L.The suitable liquid medium for the NS2-Z?Mucor spp.?consisted of 1.5% maltose,1% starch,1.5% yeast extract,1% peptone,plus to 0.8g/L KH₂PO₄ and 0.8g/L Mg SO₄?7H₂O.The maximum of total nucleosides was 138.63 g/L.Finally,we attempted to analyze the microflora of Chinese Cordyceps in Nagqu Prefecture of the Tibet Autonomous Region and Chinese Cordyceps in Nyingchi City of the Tibet Autonomous Region with Illumina Hiseq3000.Differential expression genes were analyzed with GO?Gene ontology?function and KEGG?Kyoto encyclopedia of genes and genomes?Pathway.The results showed that a total of 382 genes were differentially expressed at two places,of which,there were 198 up-regulated genes and 184 down-regulated genes in samples in Nyingchi City compared to the samples in Nagqu Prefecture.In Go analysis,there were a total of 483 Go annotations.364 of them belonged to biological process,25 of them belonged to cellular component and 94 of them belonged to molecular function,which showed that genes were more related to biolobical process.The results of KEGG Pathway showed that there were few differences in the pathway of Chinese Cordyceps in two places.More differential expression genes were annotated to microbial metabolism in diverse environments and peroxisome,which showed that there were much more differences between Chinese Cordyceps in Nagqu Prefecture of the Tibet Autonomous Region and Chinese Cordyceps in Nyingchi City of the Tibet Autonomous Region in microbial metabolism in diverse environments and peroxisome.