Studies On Protoplast Mutagenesis And Technology Of Active Component Extraction Of Cordyceps Militaris

Cordyceps militaris, also named North Aweto, is a type of famous traditional Chinese medicine with high economic value, being the ideal remedy of strengthening physical health, invigorating the lung and the kidney, suppressing cough and relieving dyspnea. The nutritional physiology of cultured Cordyceps militaris, the conditions of separation and regeneration mutagenesis of Cordyceps militaris protoplasts were studied in this paper. The extracting conditions of two kinds of active substances (cordycepin and cordyceps polysaccharides) were studied at last.1. For the purpose of studying the nutritional physiology of Cordyceps militaris, the effects of carbonhydrates, nitrogens, minerals, vitamins and phytohonnones on the mycelial growth were determined. The results showed that cane sugar and glucose were suitable and there was no significant difference between them. The organic nitrogen sources, amino acids and inorganic nitrogen sources investigated different effects on the mycelial growth, with complex organic sources being the best, followed in order by amino acids and inorganic nitrogen sources, pupa powder was the best organic nitrogen source, followed by beef peptone and malt extract; it was also found that mineral nutrition calcium, kalium, iron and nicotinic acid, could greatly stimulate the growth. Among the tested phytohormones, gibberellin (GA3) and 2,4-D had significant influences on the mycelial growth whereas naphthaleneacetic acid (NAA) had inhibitory action.2. The conditions of protoplast formation and regeneration (Enzymolysis temperature, enzymolysis time, osmotic pressure stabilizer, pH and spawn age) from the mycelium of Cordyceps militaris were studied. The orthogonal test results indicated that the highest production rate of protoplast was obtained by using spawn age of 6 d, 0.6 mol/L KCl, digesting for 1 hour at the condition of 24℃. The highest regeneration rate was obtained using spawn age of 6 d, 0.6 mol/L KCl, digesting for 3 hours at the condition of 24℃. Spawn age was the most influence factor on the protoplast formation and regeneration of Cordyceps militaris.3. The mutagenesis of protoplast was preliminary studied and it was concluded that the optimal ultraviolet mutation dosage was 20.63 s~27.61 s, and the nitrous acid mutation dosage was 14.84 min~19.89 min.4. The extraction conditions of cordycepin and cordycepin polysaccharide of Cordyceps militaris stroma and the residual culture after Cordyceps militaris cultivation were studied in this paper. The optimal extraction conditions of cordycepin of Cordyceps militaris stroma and the residual culture after Cordyceps militaris cultivation were showed as followed respectively: the extraction temperature 100℃, liquid-to-solid ratio 25:1, extraction time 330 min; the extraction temperature 40℃, liquid-to-solid ratio 25:1, extraction time 330 min, and in such conditions the yields of cordycepin were 0.27 %, 0.23% respectively. Further proven experiments showed the yields of cordycepin of Cordyceps militaris stroma and the residual culture after Cordyceps militaris cultivation were 0.25 %, 0.29 %, the results were closed to the predictions.5. The best extraction conditions of cordycepin polysaccharide of Cordyceps militaris stroma and the residual culture after Cordyceps militaris cultivation were showed as followed respectively: the extraction temperature 100℃, liquid-to-solid ratio 25:1, extraction time 329.99 min; the extraction temperature 69.98℃, liquid-to-solid ratio 17:1, extraction time 112.18 min, and in such conditions the yields of cordycepin polysaccharide were 4.13 %, 3.71 % respectively. Further proven experiments showed the yields of cordycepin polysaccharide of Cordyceps militaris stroma and the residual culture after Cordyceps militaris cultivation were 4.26 %, 3.55 %, the results were closed to the predictions.