Preparation And Separation Of Cordyceps Militaris Polypeptides

Cordyceps militaris is a kind of food and medicine fungi which belong to the samespecies with Cordyceps sinensis. And the chemical composition of C. militaris is similar withC. sinensis. A lot of reports suggested that C. militaris can regulate immune, inhibite tumorgrowth, anti-aging and has other pharmacological effects. Now C. militaris is used in healthcare research. Polypeptide isolated from C. militaris stroma has anti-bacterial, anti-tumor andimmune enhancing functions. We took the HWM reminant of C. militaris as raw material tostudy preparation and separation techniques of C. militaris peptide, aiming at providingreference to the C. militaris protein polypeptide research.The research contents and results areshowed as follows:1. Identified enzyme hydrolysis process of the HWM reminant of C. militaris withoutprotein extraction. In protein extraction experiment of the HWM reminant of C. militaris, theprotein extraction rate was3.17%while the lye concentration was0.15mg/mL. The proteinextraction rate was low. Besides, the alkaline extraction process is cumbersome, increased thecost of inputs. So we omited protein extraction process and directed to enzymatic hydrolysisof C. militaris HWM residues.2. Using enzymatic hydrolysis to prepare the C. militaris peptides, TCA-SN%of C.militaris enzymatic hydrolysis as an index, we examined the enzymolysis effect of pepsin,alkaline protease, papain, snail enzyme and cellulose enzyme. The result showed that alkalineprotease is the best protease in hydrolysising the HWM reminant of C. militaris. Usingalkaline protease to enzymatic hydrolysis the HWM reminant of C. militaris, the enzymolysisliquid peptide index TCA-SN%was the highest,53.60%.3. By establishing the mathematical model, we studied the effects on the TCA-SN%ofthe substrate concentration, initial pH value, reaction temperature, enzyme amount, reactiontime, and meanwhile optimized the enzymatic hydrolysis process parameters of C. militarisHWM reminant. According to the result, the optimum enzymatic hydrolysis processparameters of C. militaris HWM reminant was pH12.0,55℃, enzyme amount5.12%,substrate concentration of70mg/mL, time of12h. In this condition, C. militaris enzymatichydrolysis liquid peptide index TCA-SN%reached90.32%.4. We adopted the Gel chromatography and the glucan Gel electrophoresis, aiming at separating the C. militaris polypeptide and analyzing molecular weight distribution of C.militaris peptide. The results of Tricine SDS-PAGE electrophoresis showed that the molecularweight of C. militaris peptide is mainly distributed in3180⁵620Da. The consequence of Gelchromatography displayed that the molecular weight of C. militaris peptide of6680¹580Daaccounted for31.00%and the molecular weight of1446³72Da taked over6.85%.5. At the vitro chemical simulation system, we admeasured the DPPH-scavenging ability,superoxide anion radical (O^2-) clearance ability and the hydroxyl radical （-OH） inbibitingability of C. militaris polypeptide. Experimental results showed that when the C. militarispeptide concentration was1.0mg/mL, the DPPH-scavenging rate, the superoxide anionradical clearance rate and the hydroxyl radical inbibiting rate were51.25%,38.35%and40.09%, respectively. It proved the C. militaris polypeptide by the mean of alkaline proteaseenzyme hydrolysis had certain ability of scavenging free radicals and lipid peroxidation.6. The plate filter method was used to examine bacteriostasis ability of the C. militarispolypeptide. The test results showed that the bacteriostasis results of C. militaris peptides onStaphylococcus aureus Rosenbach, Escherichia coli, Bacillus subtilis （Ehrenberg） Cohn,Bacillus Licheniformis, Salmonella Lignieres were negative. These indicated that C. militarispeptides had no antibacterial activity to the above five kinds of bacteria.