| In the thesis, the strains of Cordyceps militaris, Isaria farinose and Paecilomyces hepiali and their artificial culture conditions were as main objects researched, and their process of artificial culture was studied. Culture and collection were carried out according to the culture time and growth stages. A batch of mycelia was collected every5days. The growth stages were divided into mycelia stage (named mycelia), primordia formation stage (named primordial)and stroma (or synnemata) stage. Besides, UV and HPLC methods were used to determine14chemical components of polysaccharide, cordycepic acid, total saponins, total flavonoids, uracil, uridine,2’-deoxyuridine, inosine, guanosine, adenine, thymidine, adenosine,2’-deoxyadenosine and cordycepin. In addition, the patterns distributed of the14chemical components of the three kinds of fungi strains in the process of growth were analyzed, and comparative analysis was conducted on the different growth phases. Comparative analysis was conducted on the different growth phases and the distribution patterns of the chemical composition metabolism of the three kinds of fungi strains. The results were as follows:The results showed that the biomass of C. militaris mycelia from0to35days was increased as cultivated days, after35days. The biomass of mycelia began to attenuation after35days, with growth of mycelia being decreased gradually. The biomass of mycelia of I. farinosa was increased from0to45days as being longer cultivated days, after45days mycelia began to be attenuation with its biomass being decreased gradually. The mycelia of P. hepiali of0to40days in the process of biomass fast growth, but no change of significant biomass of mycelia biomass after40days. The change of14chemical components was corresponded to change of mycelia biomass, the chemical components were positively correlated with accumulation of mycelia biomass. Except, the cordycepin of C. militaris had been rising since mycelia began to be attenuation gradually, with the biomass of mycelia being decreased. In view of the correlation between mycelia biomass and chemical components of these three kinds of Cordyceps, it was suggested the best culture time of C. militaris be from30to35days,I. farinosa should be from25to30days, and P. hepiali be from35to40days.At different development stage of C. militaris mycelia, the content of chemical component was not constant. For cordycepin, stroma (17266.15μg/g)> mycelia (13343.04μg/g)> primordia (6958.96μg/g); for polysaccharide, primordial (58890.49 μg/g)> mycelia (38846.46μg/g)> stroma (32236.17μg/g); for cordycepic acid, primordial (62911.50μg/g)> stroma (50274.47μg/g)> mycelia (49235.06μg/g); for total saponins content, primordial (28408.00μg/g)> stroma (18294.77μg/g)> mycelia (17142.16μg/g); for total flavonoids, mycelia (4281.08μg/g)> stroma (1797.35μg/g)> primordia (1592.11μg/g). Besides, the distribution pattern of nucleoside ingredients as follows:for uracil, mycelia (148.28μg/g)> primordia (118.μg/g)>stroma (80.22μg/g); for uridine, primordial (2527.41μg/g)> mycelia (1765.01μg/g)> stroma (1582.61μg/g); for2’-deoxyuridine, primordial (67.13μg/g)> mycelia (56.42μg/g)> stroma (31.82μg/g); for inosine, stroma (124.34μg/g)> mycelia (76.99μg/g)> primordia (54.24μg/g); for guanosine, primordial (1540.92μg/g)> stroma (1162.07μg/g)> mycelia (1037.01μg/g); for adenine, mycelia (122.55μg/g)> stroma (68.89μg/g)> primordia (39.70μg/g); for thymidine, primordial (96.28μg/g)> stroma (79.98μg/g)> mycelia (50.99μg/g); for adenosine, primordial (2071.93μg/g)> stroma (1582.77μg/g)> mycelia (1206.71μg/g); for2’-deoxyadenosine, primordial (88.01μg/g)> stroma (80.06μg/g)> mycelia (68.99μg/g). The strain CMSL had the highest content of cordycepin at the three stages, so it could be preserved and fully used to cultivational production.According to the Nested variance analysis of C. militaris, at the mycelia stage, it was indicated that about14.17%of the chemical component variability was attributable to the differentiation among populations, the rest85.83%of chemical component variability was derived from strains. At the primordia stage, about35.45%of the chemical component variability was resided among populations, and the rest64.55%of the chemical component variability was expressed between strains. At the synnemata stage, about20.28%of the chemical component variability was expressed between populations, while the rest79.92%of the chemical component variation was derived from individuals. It was indcate that the chemical component between populations had some differentiation at different stages.The "Chinese medicine chromatographic fingerprint similarity evaluation system (2004version)" was used to calculate and analyze the nucleosides data of the C. militaris at different growth stage, the samples of HPLC with the standard fingerprint similarity was0.98～0.99at the mycelia stage; the samples of HPLC with the standard fingerprint similarity was0.82～0.97at the primordia stage; and the samples of HPLC with the standard fingerprint similarity was0.90～0.99at the storma stage, except the fingerprint similarity of Pop CMYYH was0.66.As for I. farinosa at different growth stage, the chemical components were obviously different. At the mycelia stage, the minimum and the maximum values of total amount of the14chemical compoments of I. farinosa were55706.93μg/g and89228.48μg/g, respectively, with the average value being71896.26μg/g. At the primordia formation stage, the minimum and the maximum values of total amount of the14chemical components were109807.12μg/g and127164.07μg/g, respectively, with the average value being120808.12μg/g. At the synnemata stage, the minimum and the maximum values of total amount of the14chemical components were49763.14μg/g and128819.23μg/g, respectively, with the average value being73252.50μg/g. For polysaccharide, primordial (38415.47μg/g)> mycelia (19998.06μg/g)> synnemata (14859.59μg/g); for cordycepic acid, primordial (51020.91μg/g)> synnemata (41720.24μg/g)> mycelia (33966.94μg/g); for total saponins, primordial (24450.44μg/g)> mycelia (13267.66μg/g)> synnemata (9125.25μg/g); for total flavonoids, primordial (5498.85μg/g)> synnemata (2536.19μg/g)> mycelia (2367.84μg/g). Besides, the distribution pattern of nucleoside ingredients as follows:for uracil, synnemata (155.23μg/g)> mycelia (146.01μg/g)> primordia (44.83μg/g); for uridine, synnemata (1445.77μg/g)> mycelia (654.13μg/g)> primordia (511.75μg/g); for2’-deoxyuridine, mycelia (23.85μg/g)> synnemata (6.87μg/g)> primordia (5.40μg/g); for inosine, mycelia (22.30μg/g)> synnemata (6.06μg/g)> primordia (5.66μg/g); for guanosine, synnemata (1377.33μg/g)> mycelia (645.43μg/g)> primordia (341.34μg/g); for adenine, synnemata (47.08μg/g)> mycelia (18.56μg/g)> primordia (4.40μg/g); for thymidine, synnemata (31.25μg/g)> mycelia (13.17μg/g)> primordia (11.36μg/g); for adenosine, synnemata (1870.50μg/g)> mycelia (714.55μg/g)> primordia (489.12μg/g); for2’-deoxyadenosine, synnemata (51.87μg/g)> mycelia (20.52μg/g)> primordia (12.58μg/g). In addition, it was found that a small amount of cordycepin was detected in three strains of I. farinosa.The content of chemical components in P. hepiali mycelia was slightly lower than those in I. farinosa mycelia, and cordycepin was not detected in P.hepiali mycelia. Q-clustering showed that P. hepiali and I. farinosa were gathered in a large branch, showing that the chemical components of these two fungi were very similar.This investigation might provide a scientific basis for sustainable utilization of C. militaris, I. farinosa and P. hepiali. It was important to provide a specific guidance for their cultivational production. Meanwhile, according to the correlation between chemical component and mycelia biomass population at different growth stage, it is to select high-yield strain and optimize culture condition so as to improve production efficiency. |
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