| Antrodia cinnamomea is a rare and precious medicinal and edible fungus.At present,solid-state fermentation and submerge fermentation are the main methods for artificial culture of Antrodia cinnamomea.Antroquinonol?a ubiquinone derivative?and Antrodin C?a maleic acid succinic acid derivative?are the characteristic bioactive metabolites in A.cinnamomea.However,the related research is still at a preliminary stage.Therefore,in this thesis,further research is carried out to solve the existing problems in the solid-state fermentation and the submerged fermentation for producing Antroquinonol and Antrodin C.For solid-state fermentation,most of the studies are concentrate on the production of Antroquinonol via optimizing the solid substrate and fermentation conditions.However,the yield of Antroquinonol is still low,and the reasons for the influence of different substrates on the production of Antroquinonol are rarely discussed.In this thesis,various kinds of grains were used as substrates,among which millet was found to be the best substrate.Furthermore,the underlying reasons were preliminary investigated by comparing the amylose content and grain size of different cereal substrates.Afterwards,the appropriate fermentation time,initial moisture content,inoculation amount and addition nitrogen source were studied by single factor experiments.Based on these results,the response surface design was further used for the process optimization.The results show that the best antroquinonol-producing conditions are as follows:the solid substrate was millet,the inoculation amount was 21%,the culture period were 25 days,the concentration of soybean hydrolysate was 82.4mL·L-1,and the initial moisture content was 41%.Under the optimized conditions,the yield of antroquinonol was 1340.7 mg·kg-1,which was about 4.1 times as high as that of control?325.0 mg·kg-1?.For submerged fermentation,the yield of Antrodin C can be enhanced by establishing a biphasic fermentation system coupling use of surfactant and in situ extractant.However,there are still some remaining problems,such as the low yield of Antrodin C,difficulty in separation and purification of Antrodin C and the high cost of recycling the extractant.Therefore,it is particularly important to seek a regulatory method that can directly stimulate the synthesis of Antrodin C.At first,Antrodin C was found to have good antioxidant activity,which may be involved in the maintenance of intracellular ROS balance.To prove this hypothesis,oxidative stress was applied in submerged fermentation process to promote ROS production in mycelia,and its effect on Antrodin C synthesis was also studied.The results showed that all the three oxidative stress agents could enhance the production of Antrodin C and among which hydrogen peroxide was the best oxidative stress agent.Under the optimal concentration and addition time,the yield of Antrodin C was 375.20 mg·L-1,which was about5 times as high as that of control.Moreover,the intracellular CAT activity increased by 1.37 times due to the addition of hydrogen peroxide,and the production of Antrodin C decreased when supplemented with ROS scavenger,indicating that the production of Antrodin C was related to the level of intracellular ROS.Subsequently,the submerged fermentation conditions of A.cinnamomea were optimized under the stimulation of ROS.After screening the suitable carbon and nitrogen sources,culture temperature and initial pH,a three-factor and three-level response surface experiment was further designed for optimization.When the hydrogen peroxide concentration was 25 mmol·L-1,the culture temperature was 24?,and the initial pH was 7,the maximal production of Antrodin C was 605.12 mg·L-1,which was 8.06 times as high as that of the control.Finally,in order to further explore the mechanism of ROS-induced Antrodin C production,transcriptomics was used for preliminary analysis.The results showed that the expression level of the regulatory genes in mitochondrial electron transport chain were significantly lower than that of the control group after addition of hydrogen peroxide.Therefore,it was speculated that hydrogen peroxide may induce the decrease of the efficiency of the electron transport chain,break the balance of intracellular ROS in mycelia,and promote the synthesis of Antrodin C.To verify the result,one type of mitochondrial electron transport chain complex inhibitor rotenone was added during the fermentation process.It was found that the inhibition of the mitochondrial electron transport chain did stimulate the synthesis of Antrodin C,which laid a theoretical foundation for further investigation on the mechanism of Antrodin C biosynthesis. |
PDF Full Text Request