The Study On Triterpenoids Biosynthesis By Inonotus Obliquus Submerged Fermentation

Inonotus obliquus is a rare edible and pharmaceutical fungus. The fruitbody andmycelium of Inonotus obliquus contains many kinds of bioactive substances, thus thefungus has a high value to be employed. Inonotus obliquus triterpenoids are importantbioactivity substances with antiviral, anti-inflammatory, immunomodulatory activites.Submerged fermentation is expected to be a suitable technology to product Inonotusobliquus triterpenoids, which has the advantage of short production cycle, norestriction of seasonal weather, high yield and precise control of fermentationparameter. In this paper, the kinetics feature of Inonotus obliquus fermentation wasinvestigated in basal medium and then the fermentation medium was optimized withstatistical method with the production of intracellular triterpenoids as an indicator.Trametenolic acid, betulin, inotodiol and lanosterol were isolated from the naturalsclerotia of Inonotus obliquus as standards, and the HPLC analytical method of thetriterpenoids compositions were established. On this basis, the total triterpenoidscontent and the compositions of four triterpenoids of the natural sclerotia, the myceliafrom the optimal medium and the basal medium were studied. Methyl jasmonate（MeJA） is signaling compound that modulates various physiological processes inplants, and induces or increases the biosynthesis of many secondary metabolites.MeJA at different concentrations of was added to the basal medium to study therelationship between the mycelia grown and the triterpenoids biosynthesis. AuxiliaryMeJA at optimal concentrations was further studied under different fermentationstages. A Inonotus obliquus fermentation system for triterpenoids production waseventually established.1. Inonotus obliquus CBS314.39was activated in the Mea plate, and themorphologies of colony, mycelium and conidium were observed, the pure culture wasalso successfully achieved at last. Based on the basal fermentation medium, we studied of the biomass, reducing sugar, total triterpenoids content, pH and otherfermentation parameters to determine the fermentation period10days.2. The statistical method was employed to optimize the fermentation medium.Glucose and mixed nitrogen sources （peptone+yeast powder） was determined to bethe best carbon and nitrogen sources by using the one-factor-one-time method. Withthe fractional factorial experiment design, the significant effect of glucose, peptone,CaCl₂and yeast powder on triterpenoids biosynthesis was screened. Ultimately,response surface methodology was used to optimize the screened factors. The optimalmedium contains （%w/v）: glucose,5.89; peptone,0.29; CaCl₂,0.05; yeast powder,0.12; KH₂PO₄,0.1; MgSO₄,0.02. Under optimal conditions, the triterpenoidsproduction and the mycelial biomass from I. obliquus were significantly enhanced andthe triterpenoids production reached57.1mg/g, representing an increase of1.4-foldand1.6-fold compared with that using the basal medium （41.8mg/g） and naturalsclerotia （36.3mg/g）, respectively. The content of four main triterpenoids compounds（trametenolic acid, betulin, inotodiol and lanosterol） analyzed by HPLC was improvedin the optimal medium as follows1.72±0.04,3.25±0.07,4.03±0.14,0.51±0.02mg/g,respectively.3. The four important triterpenoids were isolated from the natural sclerotia ofInonotus obliquus. Their structures were determined to be trametenolic acid, betulin,inotodiol and lanosterol by NMR. The HPLC method for the triterpenoidscompositions analysis was established with the four triterpenoids as standards.4. Effect of MeJA on triterpenoids biosynthesis in Inonotus obliquus submergedculture at various concentrations, inducing times and dissolve-helping solvents wereinvestigated detailedly. When tween-20was solvent,50μM MeJA had the significanteffect on triterpenoids biosynthesis （triterpenoids production reached57.4mg/g）, andthere were different results of triterpenoids biosynthesis in each fermentation stages.MeJA had a restrictive effect on mycelia grown in the initial stage of fermentation,and had no significant effect in the stationary phase. In the log phase, MeJA had asignificant effect on triterpenoids accumulation and the triterpenoids productionreached63.1mg/g on6day, representing an increase of1.5-fold compared with that on10day. But the inducing time for the four triterpenoids contents by MeJA wasDifferent.