Purification And Macrophage Activation By Polysaccride From The Culture Medium Of C.Militaris

Cordyceps militaris L is a famous fungus which widely used in Chinese herb.Considering the scarce yield of natural Cordyceps militaris L, it's very important to proceedthe studies on producing Cordyceps militaris mycelium and some functional ingredients bysubmerged culture. The submerged culture technology and the macrophage activation bypolysaccharide of Cordyceps militaris was detailed studied in this paper.The optimum fermentation condition:Cordyceps militaris was inoculated in at 24℃ for4-6d, the primary pH is 7.0, glucose is as carbon source, soya peptone is the source ofnitride.Coarse polysaccharide isolated from mycelia of culture medium of C.militaris. wasserved as available material.The pure polysaccharide(CPS) was obtained through alcoholprecipitation and Cellulose CM-52 gel filtration purification.The purity of C.militaris wasidentified by Sephadex G-150 column chromatography and infrared(IR).The results showedthat the CPS was an unique and homogeneous component.The average molecular weight ofCPS wase estimated to be 34,938.The component of CPS is manna.No characteristicabsorption peaks of nucleic acid and protein were shown in CPS spectrum. However, typicalabsorption peak of Polysaccharide was shown in its IR spectrum. The IR results indicatedthat CPS was linked by β-glucoside bond.To investigate the effect of CPS on NO production, Nitrite accumulation was used as anindicator of NO production in the culture media using Griess reagent.The treatment of cellswith CPS(50mg/L, 100mg/L, 200mg/L, 400mg/L) caused a significant increase in theproduction of nitrite by macrophage cell line RAW 264.7(). Potent macrophage activatorLPS increased the nitrite synthesis compared to the control group.The pretreatment of CPS with polymyxin B does not affect nitrite generation, whilepolymyxin B abolished LPS stimulation of macrophages.This results indicated that CPSdoes not contain lipid A, the active moiety of LPS. Collectively, CPS is assumed to usedifferent membrane receptors for its biological activities.