Studies On The Screening Of Fungal Polysaccharide Elicitor And Improving The Accumulation Of Bioactive Substance From Cordyceps Militaris

Cordyceps militaris is considered to be the suitable alternative of Cordyceps sinensiswhich was used as a valuable herb in Chinese for centuries. The excellentimmunomodulation, antitumor, and anti-oxidant effect of the bioactive metabolin obtainedfrom Cordyceps militaris had been recognized by researchers. In recent years, the fungalpolysaccharide elicitor had been successfully applied to increase the contents of bioactivemetabolin for cell and microbe. The objective of this study was to screen the fungalpolysaccharide elicitors from medicinal fungi, and to increase the contents of the bioactivemetabolin from Cordyceps militaris, optimal conditions and mechanism for the predominantinfluences in the stimulation of the fungal polysaccharide elicitor were studied.In the study, six kinds of fungal hyphae （Ganoderma, Antrodia cinnamomea,Ganoderma applanatum, Phellinus igniarius, Schizophyllum commune and Cordycepssinensis） were selected to prepare the fungal polysaccharide elicitors. In addition, five typesof fruiting bodies of CM-js-D （from rice medium）, CM-js-Y （from pupa medium）, CM-yf-D（from rice medium）, CM-yf-Y （from pupa medium）, CM-jd-D （from rice medium） wereselected to prepare the fungal polysaccharide elicitors. The results showed that thepolysaccharide from fruiting bodies of CM-js-D, and hyphae of Antrodia cinnamomea andGanoderma stimulate the bioactive metabolin expression from Cordyceps militarissignificantly. Under the stimulation of fungal polysaccharide elicitors from CM-js-D, theamount of polysaccharide and cordycepin from Cordyceps militaris increased54.16%and5.49%, respectively. The cordycepic acid of Cordyceps militaris increased21.96%after thestimulation of Antrodia cinnamomea polysaccharide. The fungal polysaccharide elicitorsfrom Ganoderma caused3.5%increase for the adenoside content of Cordyceps militaris.Optimal conditions for the predominant influences in the stimulation of the fungalpolysaccharide elicitor were studied by using monofactorial and response surfacemethodology testing. The effects of the concentration of the fungal polysaccharide elicitor,addition time and stimulation time were investigated by single factor test. On the basis ofthese, optimization of stimulation conditions of fungal polysaccharide elicitor was studiedusing response surface methodology. By the study, the regression model for the optimalcondition of the stimulation was expressed as: y=320.39+2.48x₁+0.4x2-0.19x₃-7.43x₁x₂-2.27x₁x₃-4.48x₂x₃-35.55x₁²-16.94x22-16.9x₃²（x₁, x₂, x₃were the concentration of the fungal polysaccharide elicitor, addition time and stimulation time, respectively）. The resultsshowed that, the importance of three factors affecting activities for the stimulation from bigto small was the concentration of the fungal polysaccharide elicitor, addition time andstimulation time. The R-Squared of this model was0.9996, suggesting the model wasreliable and feasible. The optimal stimulation conditions were defined as: concentration ofthe fungal polysaccharide elicitor70.328μg/ml, addition time48h and stimulation time72h.Under the optimal conditions, the stimulation of the fungal polysaccharide elicitorapproached to get the highest.The mechanism of fungal polysaccharide elicitor was discussed in the paper.Enzymatic hydrolysis on the fungal polysaccharide elicitor was researched by selectingthree kinds of glycosidases （alpha-amylase, pectinase, beta-galactosidase）, obtained thedifferent fragments by controlling hydrolysis time, determination of the relative molecularweight of different fragments by viscosimeter and compared the stimulation which werearoused by different fragments. The results showed that stimulation aroused by fungalpolysaccharide elicitor fragment （relative molecular weight from10000to15000） was thestrongest.