Study On Breeding With Protoplast Of Cordyceps Sinensis And Zn, Se, Ge Accumulation In Liquid Medium

Cordyceps sinensis, a traditional Chinese herb, which contains many biological active substances, such as cordycepin, cordycepic polysaccharides, proteins, vitamins, trace elements, etc, is a kind of ideal mild pharmaceutical agent. Therefore, it has notable therapeutic and precautionary function to the diseases, such as chronic bronchitis, chronic hepatitis, high blood pressure, heart diseases, nephritis, kidney decline, tinnitus, cancer, etc. Rare natural resources and unreasonable excavation cause serious shortage of C. sinensis. Accordingly, liquid culture of mycelia is becoming one of the most important means to solve this problem. In this paper, we studied the preparation and regeneration of protoplast of C. sinensis and hydrolytic conditions of the enzymes. A C. sinensis strain with high polysaccharides'production was obtained through protoplast mutagenesis. The characters about the bioaccumulation of Zn, Se and Ge were investigated by single element and triple elements bioaccumulation of mutant strain. Finally, the antioxidation effects of C. sinensis accumulated Zn, Se, Ge were studied. The results are as follows:1. The result of optimizing liquid cultural conditions of Cordyceps sinensis was: potato 20%, glucose 2%, yeast extract 0.4%. The biomass reached 1.72g/100mL at the condition of 25℃and 160r/min.2. The highest yield of protoplast can be obtained by using 1.5% lywallzyme made by 0.6mol/l KCl, digesting for 3 hours at the condition of 30℃and pH value 6.0. The production of protoplast reached 4.35×107 /mL.3. The optimal medium for regeneration of C. sinensis protoplast were: maltose 2%, glucose 2%, peptone 0.2%, yeast extract 0.40%, KH2PO4 0.1%, MgSO4·7H2O 0.25%, VB1 0.001%, VB6 0.001%. The highest regeneration rate was 38.07%.4. The medium lethal dose of UV irradiation on C. sinensis protoplast was 20s. According to antagonistic effect and state of growth, we screened 100 strains which polysaccharides'yield were higher than control. Finally, we screened strain 035 for its'high polysaccharides'yield and genetic stability.