Study On Liquid Fermentation Of Paecilomyces Cicadae (Miquel.) Samson

Paecilomyces cicadae is the asexual stage of traditional Chinese medicine Cordyeps cicadae. Cordyeps cicadae and Cordyceps sinensis both belong to Cordyceps (Clavicipitales:Claviceptaceae). In recent years, scholars did a lot of research on the chemical composition, physical and pharmacological action, and clinical application etc of Paecilomyces cicadae. Results showed that the components of Cordyeps cicadae and the artificial cultivation of Paecilomyces cicadae were similar to that of Cordyceps sinensis. As a kind of Cordyceps sinensis excellent replacement, the research and utilization of them were carried out. With the advantages of the sources of the raw materials stability, low cost, short fermentation period, the target product easy to control and improve, product easy to development, using microorganism fermentation technology to further develop Cordyeps cicadae is the effective way to look for, extract and prepare the biological activity material, and also is the development direction of the depth development and utilization of fungi.In this paper, by preliminary study, a set of P. cicadae (asexual stage of C. cicadae) liquid fermentation technology at pilot level was obtained. It was as follwing:(1)The optimum shaker-flask fermentation conditions were:medium initial pH 4.5-5.5, culture medium amount 40-60%(V/V) of flask, inoculum 2×107spores/100ml culture medium, culture temperture 22℃, shake rotation 140 r/min, fermentation period 4-6 days; (2)Through determining the contents of adenosine, N6-(2-hydroxyethyl) adenosine and cordycepin by HPLC for the first time, a optimization liquid medium of P. cicadae was gained using the carbon and nitrogen source single factor screening, carbon and nitrogen sources interactive test, inorganic salts influences test and stability test. It was:the sucrose (1%), glycerin (0.5%), soybean meal (6%), yeast extract (1%), KH2PO4 (0.05%), MgSO4·7H2O(0.075%), FeSO4·7H2O (0.001%), ZnSO4·7H2O(0.001%)and CaCl2(0.01%). Among them, soybean meal had the most influence; (3) Using peptone-glucose medium, P.cicadae was cultured according to the above optimized shaker-flask fermentation conditions. Then it was cultured in 10L fermenter. Finally,it was further cultured in 70L fermenter using the optimized medium formula. Three nucleosides (adenosine, N6-(2-hydroxyethyl) adenosine and cordycepin) growth curves were analyzed. Results showed that N6-(2-hydroxyethyl) adenosine was mainly existed inside the cell (mycelia), and cordycepin was mainly existed in exocellular (filtrate). Adenosine may be the premise substances of N6-(2-hydroxyethyl) adenosine and cordycepin. Therefore, using adenosine, N6-(2-hydroxyethyl) adenosine and cordycepin as the objective products, collecting fermentation total product (including mycelia and filtrate) has good economy. The best time for fermentation was 132h. At this time, three nucleosides contents were 0.80mg/100ml (adenosine),3.59 mg/100ml [N6-(2-hydroxyethyl) adenosine]and 5.50mg/100ml (cordycepin) respectively.