Submerged Fermentation Of Key Medicinal Components, Triterpene Acids From Ganoderma Lucidum

Triterpene acids are the key medicinal components from Ganoderma lucidum, which have various pharmacological effects. Because G. lucidum usually takes several months to form its fruiting body under culture conditions, and it is also difficult to control the triterpenoids quality during the cultivation, submerged fermentation of G. lucidum is viewed as a promising alternative for efficient production of triterpenoids. Based on the previous studies in our lab, in this dissert, the low-cost medium constituents and the fermentation conditions for triterpene acids fermentation by G. lucidum were optimized. Stimulators for triterpene acids formation from phytotherapic drugs as well as fermentation dynamic models of triterpene acids were preliminary systematically studied. The main results are as follows:1. The low-cost carbon and nitrogen sources selected from food were added to fermentation basal medium by single and combined way, results showed that low-cost carbon and nitrogen sources were ultilized well by G. lucidum mycelia cells, and the effect of combined add method was superior to the single way. Then the key fermentation medium constituents, glucose (A), corn flour (B), wheat bran flour (C) and peptone (D), were optimized by Box-Behnken design and response surface methodology, and the regression model which can predict intracellular triterpene acids (ITA) yield was obtained:Y1=233.40+13.15A+7.09B+7.34C+4.90D+2.95AB+5.30AC+1.93AD+2.33BC +6.83BD-0.81CD-36.36A2-19.38B2-25.94C2-23.76D2. The optimum medium for ITA production was glucose 26.0g/L, corn flour 11.2g/L, wheat bran flour 10.8g/L, peptone 5.9g/L. The validation tests showed that the result predicted by the model was similar to the actual fermentation results, so the model can be used in practical application.2. The results of one-factor experiments showed that the initial pH, rotation speed and culture temperature are key fermentation factors of triterpene acids from G. lucidum. Optimized by central composite rotatable design and response surface methodology, the regression models related to three key factors which can predict the production of intracellular triterpene acids (ITA) yield (Yi) and extracellular triterpene acids (ETA) yield (Y2) was obtained:Y1=282.00+4.41A+7.19B+3.02C+0.079AB-2.38AC-2.24BC-38.57A2-36.95B2-31.25C2; Y2=245.98-12.53A+8.99B+4.98C+0.20AB-0.71AC -1.24BC-29.00A2-28.50B2-31.83C2. The optimum fermentation condition for ITA was initial pH6.0, rotation speed 161.9r/min and culture temperature 30.1℃, while the optimum fermentation condition for ETA was initial pH 5.9, rotation speed 163.1r/min and culture temperature 30.2℃. The both products were not significantly different, then revised optimum conditions was initial pH-6.0, rotation speed 160r/min and culture temperature 30℃in order to apply conveniently in practice.3. The effects of water and ethanol extracts from ten kinds of phytomedicines on mycelial growth, ITA and ETA formation of G. lucidum in submerged fermentation were studied. The results showed that water and ethanol extracts from different phytomedicines had significantly different effects on mycelial growth and triterpene acids formation. Both water and ethanol extracts from Platycodon grandiflorum (Jacq.) had significantly stimulatory effects on the mycelial growth and triterpene acids production(P<0.05); extracts from Fructus lycii and Lonicera japonica (Thunb.) at low concentration showed positive effects on the mycelial growth and triterpene acids formation. However, both the water extracts and ethanol extracts from Isatis tinctoria L, Dendrobium loddigesii (Rolfe.) and Ginkgo biloba showed the inhibitory effects on the mycelial growth and triterpene acids production of G. lucidium.4. Analysing the submerged fermentation process of triterpene acids from G. lucidum, we discovered that triterpene acids formed in parallel with mycelium growth. Based on Sigmoid model, fermentation dynamics models of triterpene acids was founded, including the mycelium growth model, ITA and ETA formation models, and substrate consumption model, then the parameters of theses dynamic models were calculated by corresponding software. The values experimented matched well with the models, indicating these four models obtained from the investigation could reveal process characteristics of triterpene acids fermentation, the conclusion can provide reference for 30L fermentation and the fermentation scale-up in the future.