| Ophiocordyceps sinensis (syn. Cordyceps sinensis) or'Dongchong Xiacao' in China is a special type of mushroom, which is formed on an insect larva infected by the fungus. It is a rare and precious medicinal mushroom with great medicinal value.Dendrolimus spp. is an important forest pest in our Country, however the body of Dendrolimus spp. is one kind of potential edible resources, for its rich in nutrition, such as:high quality protein, functional oil, carbohydrate materials and vitamin-like active substance and so on. And also the main nutrition constituent of Dendrolimus spp. and the hepialidae host of O. sinensis fungus are similar, it shows that the O. sinensis fungus has capacity to use the body of insect as growth matrix.Based on the previous studies in our lab, in this dissert, some research works as follows were done:1. Submerged fermentation of O. sinensis on a culture medium with insect, Dendrolimus punctantus as main nitrogen source and the toxicological safety evaluation of O. sinensis mycelium were all studied. The results showed that the freeze-dried powder of D. punctantus was suitable for cell growth of O. sinensis, and the supplementary carbon source, nitrogen source and mineral element were corn flour,(NH4)2SO4and KH2PO4, respectively. The fermentation medium was further optimized by Uniform Design, The fermentation medium was further optimized by Uniform Design was as follows:freeze-dried powder of D. punctantus13.0g/L, corn flour38.0g/L,(NH4)2SO43.0g/L, Glucose3.0g/L, KH2PO41.4g/L, Peptone1.Og/L, MgSO4·7H2O0.75g/L. In the optimized culture medium, the mycelial biomass of O. sinensis reached to8.678g/L. The results of acute test on mice through the mouth and skin stimulation test on rabbit were all indicated that Dendrolimus spp. fermented O. sinensis mycelium was low toxicity, belonged to actual non-toxic level.2. The effect of original pH, inculum quantity (%), temperature (℃), rotatory speed (r/min) factors on the production of polysaccharides was exploited. And the liquit culture of O. sinensis was optimized by means of orthogonal analysis. The results showed that the optimal fermentation conditions were:original pH7.0, inculum quantity9%, temperature24℃, rotatory speed170r/min. The production of polysaccharides reached0.294g/L. 3. The microwave-assisted extraction technology was used to extract polysaccharides from Dendrolimus spp. fermented O. sinensis mycelium (PDCM). The experiment factors and levels were firstly selected by one-factor test, then the method of response surface analysis with three factors and three levels was adopted and the factors influencing the technological parameters were determined by means of regression analysis. The results showed that the optimum conditions with the microwave-assisted extraction technique of Dendrolimus spp. fermented O. sinensis mycelium were:ratio of water and material was6.3:1, power of microwave was520W, extraction time was325S, and the extraction rate of PDCM was6.901%with three-times.4. Adopted the bath to extract D-mannitol from Dendrolimus spp. fermented O. sinensis mycelium in virtue of the difference of liquid-solid ratio, extracting temperature and extracting time were studied. The optimum parameters of extracting D-mannitol were obtained by response surface analysis, liquid-solid ratio (mL/g)54.2:1, extracting temperature40.3℃, extracting time40.5min, the yielding ratio of D-mannitol was6.771%at the condition of optimum parameters with three-times.5. A response surface analysis method was applied to optimize the conditions for extraction of adenosine from Dendrolimus spp. fermented O. sinensis mycelium. Based on the single factor experiments, a three-factor-three-level experiment was designed by Box-Behnken central composite design method. The influence of extraction time (S), extraction temperature (℃) and solvent-solid ratio (mL/g) on the yield of adenosine were studied. The response surface method was employed to analyze the results of experiments. The results indicated that the optimum extraction conditions of adenosine were as follows: extraction time175S, extraction temperature40.2℃and solvent-solid ratio37.6:1. The predictive maximum yield of adenosine was0.1375%, the average yield of adenosine in three validation experiments under the optimum process conditions was0.136%and the relative error was0.1091%.6. Compared the difference of crude fiber, crude fat, total sugar, polysaccharides, protein, D-mannitol, adenosine between cultivated mycelia and wild O. sinensis. The results showed that the contents of crude fiber, total sugar and D-mannitol in wild O. sinensis were higher than cultivated O. sinensis mycelia, but crude fat, polysaccharides, protein, and adenosine are lower. The submerged fermentation technology of Ophiocordyceps sinensis Fungus with Dendrolimus punctantus and the extraction predicting model of mycelium were obtained in this paper, and it also laid a foundation for the fermentation scale-up and industrial production of mycelium and polysaccharides of O. sinensis in the future. |
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