| This experiment took the Cerasus humilis Kernel removed oil by cold-pressed as a raw material and extracted protein from it using the alkaline extraction and acid precipitation method. Seven kinds of food grade protease were used to hydrolyze the Cerasus humilis Kernel respectively, and then selected the suitable enzymes for hydrolyzing the Cerasus humilis Kernep protein with the index of hydrolysis degree. The methods of single enzyme and mult-enzyme composite were applied to hydrolyze the protein using the selected enzymes. By comparing the effects of different enzyme solution on the DPPH free radical scavenging ability to determine the best process for preparation of Cerasus humilis Kernel peptide. Antioxidant activities and immunoregulating effects in vivo of the peptide which prepared by optimal hydrolysis process. Purified the peptide using Sephadex G-25 chromatography and collected all the components. Identificated the primary structure of the group which have the strongest scavenging ability of DPPH free radical. The results were shown as follows:1. Screening of enzymes for Cerasus humilis Kernel protein hydrolysisDetermined the optimum conditions and calculated the degrees of seven protease in hydrolysis time of 60 minutes using the single factor experiment. Selected the suitable enzymes for hydrolyzing the Cerasus humilis Kernel protein. The hydrolysis degrees of seven protease under the optimum conditions of neutral protease, alkaline protease, trypsin, acid protease, papain and flavor proteinase werel5.5%,14.9%, 13.7%,9.7%,7.8%,7.7% and 9.2%. The conclusion that the hydrolysis degree of alkaline protease, neutral protease and trypsin in the most suitable conditions for hydrolyzing the protein can reach more than 13%, So this three enzymes were selected to hydrolyze the Cerasus humilis Kernel protein.2. determining the method for hydrolying Cerasus humilis Kernel proteinSingle enzyme hydrolysis:alkaline protease, trypsin and neutral protease were used to hydrolyze the Cerasus humilis Kernel protein in their respective optimum conditions, and their enzyme solution were collected in the 30,60,90min and their DPPH radical scavenging rate were tested. The results showed that, with the time of hydrolysis prolonged in the 30 to 60min, the DPPH radical scavenging rate of the enzymatic solution gradually increased. The DPPH radical scavenging rate can reach 80.8% of the enzymatic solution by alkaline protease in 60min, the change of enzymatic solution on the DPPH radical scavenging rate was not obvious after 60min; The DPPH radical scavenging rate was up to 79.6% of the enzymatic solution hydrolyzed by trypsin in 60min; The scavenging rate of the enzymatic solution hydrolyzed by neutral proteinase was 79.1% in 60min. Double enzyme hydrolysis:the DPPH radical scavenging rate was 82.4% from enzymatic solution hydrolyzed by Alcalase for 30 min firstly and then hydrolyzed by trypsin for 30min. the DPPH radical scavenging rate was 84.2% of the enzyme solution hydrolyzed by alkaline protease for 30min and neutral protease for 30min. the DPPH radical scavenging rate was 81.1% of the enzyme solution hydrolyzed byTrypsin for 30min and neutral protease for 30min. Three enzymes composite hydrolysis:the DPPH radical scavenging rate was 93.4% of hydrolyzates after hydrolysis for 30 minutes respectively by alkaline protease, trypsin and neutral protease in succession.Therefore the three enzymes composite hydrolysis was the optimal enzymatic method for preparation of active peptide. Hydrolyzed the protein According to this method and collected the enzymatic solution. The Cerasus Humilis Kernep active peptide powder was obtained after inactivation, centrifugaland concentrated and dryed.3. The antioxidant experimentation of Cerasus humilis Kernel peptide in vivoCerasus humilis kernel active peptide will be divided into low, middle, high dose groups with the concentrations of 10,20,30mg/mL respectively, and gavaged mice for 30 days. Determined the antioxidant of the normal mice and model mice by analyzing the body weight, the SOD activity, the GSH-PX activity and the MDA concentration in serum, liver and kidney of mice.3.1 The effect of Cerasus humilis Kernel peptide on the antioxidant activity of normal miceThe Cerasus humilis Kernel active peptides could increase the body weight of mice, but the effects were not significant(P> 0.05); The high dose group could significantly increase the SOD activity in serum, liver and kidney(P< 0.05);The high, medium dose groups could significantly improve GSH-PX activity in serum and liver of mice, but the effects to improve GSH-PX activity in kidney were not significant(P> 0.05); The high, medium dose groups could significantly reduce the concentration of MDA in serum, liver and kidney(P< 0.05), the low dose group could significantly decrease the concentration of MDA in the kidney(P< 0.05), the MDA concentrations of low dose group had a trend of decrease in serum and liver, but the effect was not significant(P> 0.05).3.2 the effects of Cerasus humilis Kernel peptide on the antioxidant activity of model miceThe high, middle dose groups could significantly increase body weight of the model mice (P< 0.05);The low, middle and high dose groups could significantly improve SOD activity in the serum, liver of model mice(P< 0.05), the activity of SOD of low, middle dose groups in kidney had a trend of increase, but the effects were not significant(P> 0.05); The high, middle dose groups could significantly improve the GSH-PX activity in serum,liver and kidney of model mice; The high, middle dose groups could significantly reduce the concentration of MDA in serum, liver and kidneyof model mice, low dose group could significantly decrease the concentration of MDA in kidney (P< 0.05), but the effect of low dose group on MDA concentration was not significant(P> 0.05).4. Study on the immune regulative action of Cerasus humilis Kernel peptideThe immunoregulation function was determined depend on the effect of the Cerasus Humilis Kernel peptide on the development of immune organs, cellular immunity, humoral immunity and mononuclear macrophage function of the normal mice and immunodeficiency mice induced by injecting cyclophosphamide.4.1 the effects of Cerasus humilis Kernel peptide on immunitive function of normal miceThe high, middle dose groups could significantly increase the thymus and spleen index(P< 0.05), low dose group can significantly increase spleen index (P< 0.05), but the increase thymus index were not significant(P> 0.05); The high, middle, low dose groups could significantly improve the lymphocyte proliferation capacity(P< 0.05), high, middle dose groups could significantly enhance the delayed hypersensitivity in mice (P< 0.05); The high, middle dose groups could significantly increase the serum hemolysin and hemolytic plaque values (P< 0.05); The carbon clearance index in the high,low dose groups had a trend of increase of the normal mice, but the effects were not significant(P> 0.05).4.2 the effects of Cerasus humilis Kernel peptides on immunitive function of model miceThe high, middle,low dose groups could significantly increase the thymus and spleen index (P< 0.05); The low, middle, high dose groups could significantly enhance the delayed hypersensitivity of model mice(P< 0.05), but the effect of low dose on improving the lymphocyte proliferation capacity was not significant of model mice(P> 0.05); the serum hemolysin value of the high, middle, low dose groups had a trend of increase, but the effects were not significant(P> 0.05), the high dose group could significantly increase the hemolytic plaque values (P< 0.05); The middle dose group could also improve the the carbonclearance index of model mice (P< 0.05).5. Separation, purification and structure determination of Cerasus humilis Kernel peptideFour main components of Cerasus humilis Kernel active peptides were seperated and purificated by SephadexG-25 chromatography. The DPPH free radical scavenging rate of the fourth components was the strongest at the same concentration (2mg/ml), its scavenging rate could reach 95.6%, this group consists of two peptide fragments by the mass spectrometric identification. Their Amino acid sequence were Leu-Ala-Thr-Ala-Pro-Ser-Arg and Leu-Asp-Gln-Val-Pro-Arg. |
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