| The bioactive compounds of the fungus Cordyceps militaris,such as cordycepin,polysaccharides and mannitol,make it exhibit a variety of pharmacological effects including anti-oxidant,anti-microbial,anti-virus,and anti-cancer activities.C.militaris has been increasingly and widely studied because of the similar chemical components and medicinal properties to Ophiocordyceps sinensis.Cordycepin(3'-deoxyadenosine),the main bioactive component of C.militaris,is the first nucleoside antibiotic isolated from the fungal culture,and possesses multiple physiological functions,such as anti-microbial,anti-cancer and anti-inflammatory.Cordycepin production using C.militaris with biotechnology strategies has important application values.In recent years,liquid cultivations of C.militaris for efficient production of cordycepin have been investigated extensively by researchers around the globe.In this study,C.militaris FFCC5111 was selected as the research subject,the effects of culture temperature and medium components(carbon sources,nitrogen sources,and metal elements)on cell growth and metabolism of C.militaris were investigated in submerged liquid cultures.The contents of this thesis mainly include the following aspects:1,The effects of different culture temperature on cell growth and cordycepin production were investigated,and the results showed that the C.militaris was able to metabolize glucose under 20 ?,25 ?,and 30 ? and produced cordycepin,and optimal culture temperature of either cell growth or cordycepin production for C.militaris was 25?,under which the cordycepin concentration reached 460.33 mg/L,more than twice of that at 20?(204.33 mg/L)and 30 ?(144 mg/L).2,The different effects of glucose and starch as carbon sources on cell growth and cordycepin production were investigated,and the results suggested that the biomass level could reach 11.75 g/L when using 20 g/L starch as carbon source,more than that of 20 g/L glucose(7.9 g/L).Additionally,both systems could obtain the cordycepin concentration of about 190 mg/L.These results proved that the fungus C.militaris could metabolize starch to cordycepin,thus reducing cost.3,Compared the growth and metabolism of C.militaris in both natural medium and synthetic medium(containing only inorganic nitrogen),results showed that cell growth and gluscose metabolism were severely inhibited when using synthetic medium,indicating that the synthetic medium may lack the essential growth factors for C.militaris.Amino acid was added to the original synthetic medium to obtain a new synthetic medium containing both inorganic and organic nitrogen source(Glucose 20 g/L,(NH4)2SO4 5 g/L,KH2PO4 0.5 g/L,K2HPO4 0.5 g/L,MgSO4·7H2O 0.5 g/L,Glycine 10 g/L,Aspartic acid 0.5 g/L,Glutamine 1 g/L,Tyrosine 0.1 g/L,Cysteine 0.1 g/L,Leucine 0.5 g/L,Lysine 1 g/L,and Phenylalanine 0.2 g/L)and a normal growth and metabolism activity was achieved in the C.militaris,thus ruling out the complex interference factors in natural medium for C.militaris.4,The effects of metal ions on cell growth and cordycepin production in submerged liquid cultivation in the established synthetic medium were investigated,and the results showed that the additions of either 5 mM Zn2+or 20mM Mg2+significantly improved the cordycepin production in C.militaris to 473.53 mg/L and 426.42 mg/L respectively,and at the same time facilitating cell growth and glucose metabolism,thus providing a new process engineering strategy for increased-yield cordycepin production.5,High performance liquid chromatography(HPLC)method was used to analyze the fermentation broth of C.militaris,methanol and water was chosen for the mobile phase,the separation of 12 nucleosides and nucleobases,including cordycepin,adenosine,adenine,uracil,thymidine,uridine,xanthine,hypoxanthine,guanosine cytidine,inosine and ergot,was achieved with the gradient elution technique on C18 column,thus providing technical support for the characterization and quantitation of the metabolite in the fermentation broth of C.militaris. |
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