| Triterpenoid acid is the key component of Ganoderma.In this paper,two new strains of Ganoderma were identified as Ganoderma lingzhi and their antitumor activities and main triterpenoid acid components were detected.On this basis,the strategy and technology of induction fermentation and the analysis of relevant induction mechanism were carried out.The main findings are as follows:1.Two new strains SCIM 1005 and 1006 were identified as Ganoderma lingzhi(East Asia)by its sequence.The mycelia the two strains were prepared by fermentation of them.The total amount of amino acids and the amount of essential amino acids in the mycelium of the two strains were higher than those of the reported Ganoderma samples.TAE-1 and TAE-2,triterpenoid acid extracts were prepared from fermented mycelia of SCIM 1005 and 1006,respectively.At least 6 kinds of active triterpenoid acids were presented in the two extracts,and the highest content was ganoderic acid T and Me.The inhibition of tumor cell growth in vitro showed that TAE-1 and TAE-2 had significant cytotoxicity on S-180,SGC-7901,SMMC-7721and SW620.The IC50 values of TAE-1 on the growth of these 4 kinds of tumor cells were 24.2,58.7,21.3 and 33.4?g/m L,respectively.The IC50 values of TAE-2 were28.1,45.2,28.6 and 32.1?g/m L,respectively.Both TAE-1 and TAE-2 have less cytotoxic effect on human normal hepatocyte L-02.In vivo,TAE-1 showed dose-dependent antitumor activity against sarcoma 180.The tumor size of mice in TAE-1(250 mg/kg)treatment group was significantly smaller than that in the control group,and the tumor growth inhibition rate was 46.6±4.6%;TAE-1 significantly improved the thymus index and spleen index of mice bearing tumor.2.Response surface methodology and box benhnken design were used to study the effect of the interaction among the initial p H,do and fermentation temperature on the yield and productivity of triterpenoid acid in G.lingshi.The results showed that in5 L fermentor,the optimal conditions for the fermentation of three terpene acids were initial p H 5.9,20%dissolved oxygen and 28.6?,and the yield of three terpene acids was 308.1 mg/L under these conditions.The optimal fermentation conditions of triterpenoid acid obtained in the study are also suitable for 200 L fermentation tank,and the maximum output and productivity of corresponding triterpenoid acid are295.3 mg/L and 49.2 mg/L respectively,which are 1.8 and 2.12 times of the yield and yield of triterpenoid acid before optimization,respectively.3.The induction strategies of exogenous substances,two-stage p H control,and two-stage p H control strategies of exogenous substances induction and integration were studied.The results showed that the ether extracts of Forsythia suspensa,dung beetle and honeysuckle can induce the synthesis of triterpenoid acid in G.lingzhi,of which EECM had the best inducing effect,and the yield of triterpenoid acid of G.lingzhi could reach 302.76 mg/L,which was 60%higher than that of the control group.The yield of triterpenoid acid reached 361.47 mg/L by two-stage p H control strategy.Then,the central composite design(CCD)method was used to integrate and optimize the strategy of exogenous material induction and two-stage p H control.Through response surface optimization,when the addition concentration of EECM was 185.89 mg/L,p H value was controlled to 5.07 within 72 hours of initial fermentation,and 4.37 after 72 hours of fermentation,the yield of triterpenoid acid was 449.37 mg/L,which was 2.37 times of that of the original control group.In addition,the strategy was successfully applied to the pilot test of 150 L fermentor.Under the pilot test conditions,the yield of triterpenoid acid was 431.83 mg/L.4.From the perspective of fermentation kinetics,the dynamic characteristics of triterpene acid production by submerged fermentation of G.lingzhi before and after adding 9,10-Methylene hexadecanoic acid(9,10-CMA)were studied by using Sigmoid model.The results showed that triterpene acids were mainly synthesized at4-9 days and reached the maximum at 9 days(268.62 mg/L)in control group of G.lingzhi,while the synthesis rate of triterpene acids was relatively fast,reaching the maximum at 8 d(343.52 mg/L)in 9,10-CMA treatment group.After adding9,10-CMA,the utilization rate of substrate glucose in G.lingzhi cells increased,and the cell specific growth rate(?)reached the maximum at 3.2 d(?max),0.94 d-1,which was higher than that of the control group(0.88 d-1 obtained at 3.4 d);the specific consumption rate of glucose reached the maximum at 1.7 d(q S,max),which was 8.34d-1,significantly higher than that of the control group,6.80 d-1(acquired on day 2.1 d).Intracellular triterpene acids were synthesized at a rate of 13.76 d-1(q IPS,max)at 6.2days,which was 1.42 times higher than that of the control group at 9.66 d-1.The triterpene acid synthesis of G.lingzhi in both groups showed a partial coupling relationship with cell growth,and after adding 9,10-CMA,there was no change in the relationship between cell growth and triterpene acid synthesis in the fermentation process.5.In 9,10-CMA-induced secondary metabolites of Ganoderma mycelium cells,no can be used as a signal molecule to mediate pal activation and CYP450 production in mycelium cells.No can be used as an effective trigger for PAL activation and CYP450 production in ganoderma mycelium cells.Although no is the upstream molecule of phenylalanine metabolism,it is not the only signal molecule.In addition to no,there are other signaling molecules or pathways in ganoderma mycelium cells that jointly mediate the promotion of 9,10-CMA on PAL activity and CYP450content.It can be seen that no can act as the upstream molecule of PAL activation and CYP450,the key enzyme of Ganoderma triterpene synthesis.It is a necessary but not the only signal molecule in 9,10-CMA-induced pal activation and CYP450 signal transduction pathway.6.The gene dynamic expression of eight key enzymes induced by 9,10-CMA was analyzed.The results showed that the expression of five genes,CYP5150L8,hmgr,mvd,fps and sqs were significantly up-regulated,suggesting that the five key enzyme might play more important roles in the induction of triterpenoid synthesis.In this work,the new strains were identified,the strategy of triterpenoid acid synthesis of G.lingzhi in submerged fermentation was innovated,and the induction mechanism of 9,10-CMA on the biosynthesis of triterpenoid acid of G.lingzhi was revealed,which provided an important theoretical reference for the synthesis and metabolism regulation of triterpenoid acid of G.lingzhi.At the same time,the induction and optimization strategy and technology can also provide practical guidance for the efficient and targeted production of triterpenoid acid of G.lingzhi. |
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