Study On Fermentation、Extraction And Separation Of Triterpenoids From Inonotus Hispidus

Inonotus hispidus was known as a kind of very precious medicinal fungus belonging to Basidiomycota. As containing a variety of bioactive substances, it was used to cure many diseases in folklore such as gastrosia, rheumatism, diabetes mellitus, cancer and so on. There were many studies on classification, biological character and polysaccharide of Inonotus hispidus, but there were few studies on triterpenoids of Inonotus hispidus. In this study, firstly we chose Inonotus hispidus as the raw materials to optimize the submerged fermentation process for the yield of triterpenoids. Secondly we did some research for optimization of water and ultrasound-assisted extraction by Response Surface Methodology to improve triterpenoids extraction yield. Thirdly we purified triterpenoids by macro-porous resin adsorption. The main results are as follows:1. In order to improve the production of intracellular triterpenoid of inonotus hispidus, the submerged fermentation conditions of fermentation medium was optimized by the single factor experimental method and orthogonal experimental design, the best liquid fermentation medium was40g/Lglucose,5g/Lsoybean meal,1.5g/LMgSO4,3g/L KH2PO4,0.05g/LVB1. Then the shake-flask fermentation cultivation condition was optimized, and the result showed that under the condition of inoculation volume3ring, pH6, liquid volume100mL/250mL, rotate speed155r/min,27℃and11ds of cultivation, the mycelium dry weight could reached2.20g/100mL, and the yield of intracellular triterpenoid could reached100.83mg/100mL2. In order to improve the extraction yield of triterpenoids from inonotus hispidus, the process of water and ultrasound-assisted extraction were optimized by Response Surface Methodology. Based on the single-factor experiments, we chose solvent concentration, extraction temperature, solid-to-liquid ratio and extraction time as variables, and chose triterpenoids extraction rate as response to study the effect of the interaction of variables for triterpenoids extraction rate. We got a quadratic polynomial regression equation to predicted the optimum extraction process, and the optimum conditions were found as follows:73%ethanol, extraction temperature67℃, solid-to-liquid ratio (g/mL)1:53,extraction time5.25h. And triterpenoids extraction yield reached4.59%under such conditions. In the process of ultrasound-assisted extraction, based on the single-factor experiments, we chose solvent concentration, solid-to-liquid ratio, ultrasound time and ultrasound power as variables, and triterpenoids extraction rate was chose as response. The optimum extraction process were as follows:extraction temperature50℃,72%ethanol, solid-to-liquid ratio (g/mL)1:69, ultrasound time31min, ultrasound power21Ow.Under such conditions. triterpenoids extraction yield was5.61%. At the same time, the room temperature extraction and shaking extraction were studied, and their extraction yields were3.51%and4.23%. Compared with other methods, ultrasound-assisted extraction was the optimum methods. It not only saved some time, but also had simple operation. It was meaning for actual production.3. In order to obtain high purity triterpenoids, macro-porous resin adsorption was used in this study. Seven kinds of macro-porous resins was been selected by a static adsorption test, and the results showed that D101resin had the best adsorption capability on triterpenoids from inonotus hispidus compared with other resins, and its adsorption rate.was46.9%, desorption rate was89.71%, and its total recovery rate was42.07%. By dynamic adsorption and desorption test for D101resin, we got that the optimum purification conditions were as follows:concentration of the sample solution0.232mg/mL, sample solution pH7, flow velocity0.25mL/min,4BV eluotropic distilled water, concentration of ethanol desorption80%and the volume of80%ethanol2BV, and elution flow rate1mL/min. The recovery rate of triterpenoids reached74.92%after purified by D101resin, and the purify of triterpenoids was improved from5.05%to27.82%. It was meaning for industrial production by useing the conditions to purify triterpenoids from inonotus hispidus.