Studies On Metabolomics And Bioctive Compounds Of Mycelia Of Hirsutella Sinensis

High performance liquid chromatography mass spectrometry and metabolomics research methods were used to study mycelia metabolites of Hisutella sinensis strain RCEF5042which was the anamorph of Cordyceps sinensis collected in Qingshashan, Qinghai province. The research focused on secondary metabolites analysis and comparison of mycelia at different culture time, cultured in solid or liquid medium, treated with different concentration of oxidants, treated with different time of UV irradiation, subcultured strains with the ordinary light and dark conditions and long-term subculturing.The results showed that the metabolites of mycelia were significantly different in different incubation time especially the culture time of the15th day and21th fay, the24th day and27th day. According to metabolites changes the incubation time which were divided into early period (the day before19th days), medium period (19th-26th days) and later period (the day after26th days). In the process of cultivation, the kinds and contents of metabolites varied with cultural time. Metabolic Characteristics of many kinds of antibiotics and a variety of unique bioactive substances were revealed in this studies. Those could be guidlines for selection of culture time of Hisutella sinensis according to actual requirement.Different solid and liquid cultural conditions were studied, and the results showed that the metabolites were significantly difference between the mycelium of solid culture, the mycelium of liquid culture and the fermentation broth. This study was focused on metabolites in solid cultured mycelia, especially antibiotics and other bioactive substances. Some metabolites which existed only in liquid culture had different concentration in mycelia and fermentation broth. One can select proper cultural condition and time to get different useful substance.Influence on metabolites in submerged cultivation mycelium and fermentation broth of Hisutella sinensis RCEF5042treated with different concentrations of oxidant stress was analysed and the results showed that the metabolites in higher concentrations of oxidant were significantly different than lower one. The metabolites in the submerged cultivation mycelium of the control group were significant from that of the test group. Metabolites of the mycelia of the group treated with2μL oxidant were significant different from the others. In the presence of an oxidant, the concentration of some unique bioactive substances were decreased or disappeared, meanwhile, some antibiotics were detected. It indicated that Hisutella sinensis synthesized substances related with stress resistance.The results of the Hisutella sinensis RCEF5042treated with different time of UV irradiation showed that the more irradiation time the more different of metabolites. The difference between the control and the10seconds groups were more relatively significant than that of other groups. The analysis of metabolites with VIP scores greater than1.35revealed that the concentration of some compounds were gradually reduced or disappeared while the UV irradiation time increased. The UV irradiation stimulated the synthesis of the compounds which had cytotoxic activity and antibiotic. The concentrations of some others fluctuated with the change of the UV irradiation time.The metabolites were significantly difference between cultures under light and dark. The analysis showed that some of the active substances easily formed in the dark, at the same time, some were more biased in favor the light. The level of antibiotics was in the dark, while the level of cytotoxic compounds such as massarigenin A was more higher in the light.Comparison between metabolites of the five generation subcultures revealed that with the increase of the times of subculture, metabolites changes more significant. The changes of the metabolites of the subcultures were not a continuous process, there was a transition period. More anti-inflammatory, anti-cell cytotoxic and antimicrobial activity compounds were found in the5th generation, and the concentrations of some metabolites decreased or disappeared. This studies enriched the way to find special metabolites from the microorganisms, and also presented a new challenge to the traditional way of fungal subculture preservation.In short, through comparison of Hisutella sinensis metabolomics of different treatments, the studies revealed the metabolic pattern of some important metabolites, and revealed the relationship between metabolites and culture conditions, oxidative stress or ultraviolet and other factors. The results can be guidline for metabolites utilization, optimization of culture conditions, determination of the best harvest time, as well as metabolic theory studies on Hisutella sinensis.