Studies On The Liquid Fermentation And Chemical Constituents Of Cordyceps Gunnii

The liquid fermentation and partical chemical constituents of Cordyceps gunnii fruitbodies collected from Liuyang Hunan and its cultured mycelium were studied by means of tissue isolation, liquid fermentation, PCR, column chromatography, TLC and spectrum technology, which including riddling of culture condition of C. gunnii, determining the growth survey of the spawn, analyzing the ITS sequence of C.gunnii and Paecilomyces gunnii, separating, purification and structural analyzing of polysaccharide mycelia of C. gunnii, extracting of alkali soluble polysaccharide and mannitol and their content. The result showed as below:1. The optimum liquid fermentation medium of Cordycep gunnii was potato juice 20%, peptone 1%, glucose 2%, magnesium sulfate 0.1%, potassium dihydrogen phosphate 0.05%, dipotassium hydrogen phosphate 0.1%. The optimum culture conditions of C. gunnii was investigated by the orthogonal experiment in three factors and four levels, temperature was 25 ~26 C, rotation rate 150 r/min, pH 6.5 and culture period 5 days.2. Four typical growth periods in the spawn of liquid fermentation were studied: the period of growth hesitation, the rapid growth period, the growth steady period and the old period. We got a best fermentation period of 66 hours.3. The internal transcribed spacer (including ITS1, ITS2 and 5.8S rDNA gene) of nuclear ribosomal DNA from the stroma and mycelium of C. gunnii were amplified by means of PCR technology. The PCR products electrophoretic patterns and nucleotide sequence were complete concordant, the sequences of ITS1 was 207 bp in size, 5.8S was 157 bp and ITS2 was 202 bp. The result indicated that the asexual stage of C. gunnii was the isolated mycelium, namely Paecilomyces gunnii. The sequences of comparison ITS of stroma and mycelium of C. gunnii and Cordyceps sinensis, showed that they had a close predestined relationship.4. Crude polysaccharide was obtained from the cultured mycelium of C. gunnii by extraction with hot water and precipitated by alcohol, The proteins in it was removed by Sevage method, individual constituent CG-1 was isolated and purified by DEAE-52 chromatography. Its homogeneity was verified by UV scan and chromatography, TLC shows that CG-1 was composed by glucose only. The reaction of periodic acid suggested that the sugar residue be interlinked by 1- 6 bonds. The result of IR spectrum shows that there was typical characteristic absorption peaks of polysaccharides and a a - pyranoglucan in CG-1.5. The optimal extractive technology of alkali soluble polysaccharide from mycelium was determined by orthogonal experiment in four factors and three levels, the temperature was 35 C, marinated in 5 multiple volume 0.75 mol/L NaOH solution for 5 hours. A maximum extraction of 41.92 mg / g has been obtained.6. There was plenty of mannitol in the cultured mycelium of C. gunnii,the sum of mannitol was 60 mg / g, the stroma was 61 mg /g, the sclerotium was 105mg/g.