Effects Of Nutrient Factors On The Roduction Of Bioactive Metabolites In Ubmerged Cultivation Of Cord Yceps Militaris

Cordyceps militaris is a kind of Chinese traditional mushroom that is valuable, medicinaland edible. Due to its significant protective effects on human bodies, it can be used as analternative to C. sinensis. As the most two major ingredients of C. militaris, Cordycepspolysaccharide (CP) and cordycepin exhibit widely bioactive and pharmacological functions.Production of CP and cordycepin in large-scale submerged cultivation of C. militaris are thusvaluable in application. In this study, the influences of various nutrients (such as carbonsource, inorganic nitrogen and inorganic metal ions etc.) on the accumulations of CP,cordycepin and other nucleoside metabolites in fermentation of C. militaris were investigatedsimultaneously. Based on the above information, the regulation mechanism of differentnutrient factors on the CP and cordycepin biosynthesis were thus discussed in details. Finally,the orthogonal experimental design method was applied to optimize the culture medium forthe CP production. The results are shown as below:1. Slowly assimilated carbon source such as glycerol and soluble starch were beneficialto the cell growth. Soluble starch was found to be propitious to the biosynthesis ofintracellular polysaccharides (IPS) while glycerol was independent of the biosynthesis of CPor cordycepin. The biosynthesis of CP was uncorrelated to the cell growth. When maltoseregarded as the sole carbon source, the biosynthesis of CP was significantly promoted in thelogarithmic growing period. Galactose regarded as the sole carbon source resulted in thehighest cordycepin production (25.20mg/L on day9), which was significantly higher thanother carbon sources. Glucose was not the best for the cordycepin biosynthesis, however, itmarkedly stimulated the rapid accumulation of intermediate metabolites AMP and was alsobeneficial to production of IPS compared to the other carbon sources.2. The biosynthesis of CP and cordycepin were negatively correlated no matter which ofthe six carbon sources was used in fermentation process of C. militaris. The biosynthesis ofcordycepin was promoted while the biosynthesis of CP was then inhibited.3.(NH4)2SO4and (NH4)6Mo7O24·4H2O went against the cell growth of C. militaris butwere all beneficial to the biosynthesis of CP, especially the accumulation ofexopolysaccharides (EPS). The consumption of carbon source and ammonium ion were all relatively slow. The presence of ammonium ion was obviously conducive to the biosynthesisof IPS. The IPS content (160±12mg/g DCW) obtained from the case in the presence ofNH4NO3or (NH₄)₂HPO₄as the sole nitrogen source was about twice that of the one in thepresence of urea as the sole notrogen source. These results indicated that (NH₄)₂HPO₄was themost favorable to the biosynthesis of cordycepin, in which the highest prodution ofcordycepin reached to3.79mg/L on day9. Meanwhile the maximum accumulation of adeninewas also obtained. Different ammonium salts had different effects on the accumulation ofnucleoside metabolites. NH4NO3was the most beneficial to the accumulation of inosine and2'-deoxyadenosine while (NH4)2SO4was propitious to the accumulation of adenosine.4. Divalent metal ions significantly affected cell growth. Ca²⁺markedly inhibited the cellgrowth of C. militaris, the highest cordycepin production was obtained in the presence ofMg²⁺, however, the cell growth and the biosynthesis of CP were inhibited at the same time.Fe2+also remarkably promoted the biosynthesis of cordycepin, along with the correspondingadenosine production had the same trend. The influence of Fe2+on the cell growth, thebiosynthesis of CP and the accumulations of nucleosides were almost the same as that ofMg²⁺. Ca²⁺and Cu²⁺had no obvious effect on cordycepin biosynthesis while other metal ionsimproved the biosynthesis to a certain extent. The optimal concentration of Mg²⁺forhyperproduction of cordycepin was0.2mmol/L. All the investigated metal ions displayed aninhibitory effect on biosynthesis of cordycepin at high concentration (above5mmol/L) hadNo significant influence on the accumulation of cordycepin occurred in which the initialconcentration of metal ions ranged from0.2to2mmol/L.5. The optimal production of cell biomass and CP obtained from the cultivation in whichgalactose, peptone and (NH₄)₂HPO₄regarded as the carbon and nitrogen sources optimized bythe orthogonal experimental design method was all about1.2times higher than that obtainedfrom the one in which glucose, peptone and (NH₄)₂HPO₄regarded as the carbon and nitrogensources. Economically, the opital culture medium containing glucose of100g/L, peptone of20g/L and (NH₄)₂HPO₄of10mmol/L is more suitable for actual production of CP.