Study On Antioxidant Properties Of Hydrolysates Derived From Cordyceps Militaris With Protease

The fungus Cordyceps militaris was a congeneric and heterogeneic species to Cordyceps sinensis, and it belonged to Eumycota, Ascomycotina, Sphaeriales, Clavicipitaceae species. It had the same chemical composition as Cordyceps sinensis, their pharmacologic action, such as modulation of immune response, inhibition of tumor growth, delay of aging were the same too. In content of Cordycepin, Cordyceps militaris exceeds Cordyceps sinensis. As Cordyceps militaris had been artificial acclimated and planted extensively, its price cut down to be much lower than Cordyceps sinensis's.The thesis was about the protease enzymatic production of Cordyceps militaris hydrolysates (CMH) from residue after supercritical fluid extraction (SEE) and its antioxidant properties. The antioxidant properties evaluated by in vivo and in vitro antioxidant activity experiments.1. Cordyceps militaris hydrolysates (CMH) were directly hydrolyzed by protease without protein extraction. This scheme could make other antioxidant activities components in raw material multiple utilized, and it would be easy to implement in industrialization.2. The protease which could prepare CMH with antioxidant activities was screened. First, the sutable pH and temperature conditions for alcalase, flavorzyme, acid protease, neutrase, trypsin, papain and pepsin were determined through single factor experiment. Second, determined the enzymolysis scheme that using neutrase only, which was coming out of both single-enzyme and double-enzyme experiments with alcalase, flavourzyme, acid protease, neutrase, trypsin, papain and pepsin. This scheme prepared CMH with the strongest reducing power and scavenging ability on superoxide negative free radical (O₂^-·), and the process was comparatively simple.3. The effect of process conditions on antioxidant activity of CMH had been investigated using response surface methodology (RSM) in this paper. RSM was including three parts: Fractional factorial design(FFD), steepest ascent design, and central composite design(CCD). The optimum conditions of enzymatic hydrolysis were: 6.5 pH value, 45℃temperature, 7.03% ratio of enzyme and substrate, 25.41 ratio of water and raw material, 4h time, the predicted value at stationary point of reducing power(A₇₀₀) was 0.465.4. The hydrolysis characteristics of CMH were investigated. There was a positive correlation between DH (degree of hydrolysates) and time. When hydrolysis in progress at 4h, DH reached value 6.82% and reducing power reached the maximal value 0.463. After 4h, DH rose slowly and get mildly. Meanwhile, the content of peptide reached the maximal value when hydrolysis in progress at 2.5 h and broke down after 4h, this result was agreed with the result of DH.5. The antioxidant properties of hydrolysate was evaluated using a lipid peroxidation model, determination of scavenging ability on DPPH·free radical and reducing power. In a lipid peroxidation model, CHM showed higher antioxidant activities thanα-tocopherol. The order of scavenging abilities on DPPH·free radical was: BHT > CHM >α-tocopherol, this result agreed with result of lipid peroxidation model. The reducing power of antiscorbic acid and BHT were both higher than CHM, but CHM still owed comparative reducing power.6. In vivo experiments, 400mg/kg CMH could make index of antioxidase activities and coefficient of spleen and thymus gland significantly increased, delay the aging of senile mice induced by D-galactose and enhace their abilities of learning and memory. These results indicated 400mg/kg CMH had antioxidant abilities and established foundation of study on antioxidant activities of CMH. 200 mg/kg CMH also could delay the aging of senile mice induced by D-galactose, but the effects were not very good, it indicated there was a dose-effect relationship in the anti-aging effects of CMH.