Study On The Production Of Bioactive Substance Inotodiol By The Fermentation Of Inonotus Obliquus

Inonotus obliquus is a kind of precious edible and medicinal mushroom,which possesses a wide range of pharmacological activities,such as anticancer and immunomodulatory.Inotodiol is a specific lanostane triterpenoid compound of Inonotus obliquus,which is also a characteristic metabolic compound with significant physiological activity in the fermentation mycelium of Inonotus obliquus.Recent studies have shown that inotodiol has notable anticancer activity with extremely high research value.Therefore,this research mainly focuses on the production of inotodiol by solid-state fermentation and submerged fermentation as the research object,aiming at solve the existing some problems in the solid-state fermentation and the submerged fermentation to carry out relevant research.On the one hand,in order to improve the production of inotodiol by solid-state fermentation,eight different substrates were selected for solid-state fermentation,and the differences of fermentation results were preliminarily explained by combining the grain size and its own characteristics.Then,the single factor optimization experiments were carried out on the inoculation,initial moisture content,the type and amount of nitrogen source.Based on the experimental results,a response surface experiment on the production of inotodiol was designed and analysed.The results showed that under the condition of 25.5%inoculation,54.5%initial moisture content,0.28g·100 g^-1 nitrogen source and 25 days fermentation time,the production of inotodiol reached995.32 mg·kg^-1,which was about 2.78 times as high as that of control(358.62 mg·kg^-1).On the other hand,in order to solve the problem of low production of inotodiol in the process of submerged fermentation,this research first carried out single factor optimization experiments on the composition of inotodiol fermentation medium and fermentation conditions,and determined the best medium as follows:glucose 30 g·L^-1,sodium nitrate 1.5 g·L^-1,corn steep power 1.5 g·L^-1,KH₂PO₄ 1.5 g·L^-1,MgSO₄·7H₂O 0.3 g·L^-1,CaCl₂ 0.1 g·L^-1,the best fermentation conditions were culture temperature 27°C,incubate time 4 days,inoculum density 100 m L·500 m L^-1,initial pH 7,inoculation 15%.At this time,the optimized production reached 12.52 mg·L^-1,which was 2.28times of the maximum production(5.50 mg·L^-1)before optimization.However,the production of inotodiol was still low only after the optimization of culture medium and fermentation conditions.Therefore,in order to improve the production of inotodiol,the appropriate elicitors?extractant,microparticle,precursors?were selected.The results showed that the optimal elicitor was geraniol,the appropriate addition amount and time were 0.02%?v/v?and 144 h respectively.On this basis,subsequently,the mechanism of high production of inotodiol induced by geraniol was preliminarily studied.Compared with the control group,the process curve of submerged fermentation with inotodiol,the production changes of squalene and lanosterol,and the relationship between the consumption of geraniol and the production of inotodiol in the fermentation process were studied.It is suggested that geraniol can stimulate the synthesis of inotodiol by increasing the production of squalene and lanosterol.Finally,the enzymatic activities of farnesyl pyrophosphate synthase,squalene synthase,squalene epoxidase and lanosterol synthase in the synthesis pathway of inotodiol were detected.The results showed that the activities of the four enzymes were significantly higher than those of the control group,and the corresponding production of inotodiol was also significantly increased,indicating that the four enzymes played a positive role in the pathway of inotodiol synthesis,which indirectly verified the right pathway of inotodiol synthesis.The experimental results lay a theoretical foundation for further elucidating the synthesis mechanism of inotodiol.