The Identification And Biosynthesis Of Triterpenoids By Inonotus Obliquus Under Submerged Culture

Inonotus obliquus, a white rot fungi, has been regarded as one of the most promising medicinal fungi in China. Inonotus obliquus is paid attention by the scholars recently because of its biological activities, such as antitumor, antibacterial property, antioxidant, hypoglycemic and immune regulation etc.. Owing to scarce resources, high price and immaturity technology, Inonotus obliquus can not meet the people’s increasing needs. Besides, with the development of liquid submerged fermentation, Inonotus obliquus has been applied in it.Due to the limit of oxygen, nutrients, it is impossible to produce quantities of Inonotus obliquus, which leads to the low production of the triterpenoids. It has been reported that citrus peels have a stimulation on secondary metabolite production in A.cinnamomea. In this paper, two different forms of citrus peels were added to culture medium to improve the yield of triterpenoids.The results were as follows:1. The studies found that isopropano was the optimum solvent to extract triterpenoids, compared with 95% ethanol and chloroform. After extraction and column chromatographic separation, Triterpenoids were suited for the next separation and purification. Besides, the rate of extraction of lanosterol was 25.31%.2. The aim of this study was to evaluate the feasibility of adding citrus peel extract to enhance the content of crude triterpenoids in the submerged culture of Inonotus obliquus. Citrus peel extracts tested were proved to be beneficial to mycelial growth, Furthermore, Pomelo peel extract was the most effective for enhancing bioactive metabolite production and triterpenoids content. With an addition of 10 g/L, the mycelial biomass and triterpenoids concentrations reached the highest, and the corresponding maximum values were 11.68 g/L and 84.04 mg/g, respectively.3. The studies showed that four citrus peels tested were all demonstrated have no stimulation on the mycelium growth, however, it had different influence on the triterpenoids contents, in which pomelo peel powder was the most effective. The production of bioactive metabolites and the contents of crude triterpenoid with the addition of 1 g/L pomelo peel increased from 9.04 mg/g to 10.08 mg/g, 64.92 mg/g to 83.16 mg/g respectively, which was increased by 28.09%, compared with the control.4. In order to analyze the effect of citrus peels on Inonotus obliquus under submerged culture, Pectin and essential oils which extracted from citrus peels were added to the mediums. The results showed that adding pectin or mixture of pectin and essential oils, mycelium died. While 0.15 m L essential oil extract could enhance mycelia growth and crude triterpenoid production, When the amount was 0.1 m L, only the contents of crude triterpenoid were increased. So these impact on mycelia and triterpenoids by adding citrus peels might be associated with the presence of essential oils.5. Quantitative analysis of the contents of five individual triterpenoid in mycelium by HPLC. The studies had shown that the effects of different additives(10 g/L pomelo peel extract, 1 g/L pomelo peel powder, 0.1 m L and 0.15 m L essential oil) were different, the accelerator could effectively accelerate the formation of some triterpenoid, also it had an inhibitory effect. Compared to the control medium, the content of betulin, trametenolic acid, inotodiol, 3β-hydroxy-lanosta-8,24-dien-21-al and lanosterol in different medium was increased by 7.90-, 10.31-, 2.75-, 8.42- and 1.29- fold.In summary, from the view of the enhance of mycelial biomass and crude triterpenoid, Pomelo peel extract is the best accelerator to stimulate the triterpenoid production. When the concentration of pomelo peel extract is 1 g/L, the production of triterpenoids is up to 0.98 g/L.