Preparation Of Antioxidant And Immunomodulatory Peptides In Ovalbumin Of Duck Egg

Duck egg white protein is a full-price protein and contains essential amino acids,calcium,vitamins and trace minerals.However,in China’s industrial production,the use of egg white is less than egg yolk,resulting in waste of resources and environmental pollution.Ovalbumin accounts for 56%of duck egg white protein and is the most important protein in egg white.Studying its application value can help the high-value utilization of duck egg white.Bioactive peptides have shown great application potential in the fields of food and medicine.The separation and identification of active peptides from enzymatic hydrolysates has become one of the important ways of high-value utilization of various plant and animal proteins.Therefore,this article used duck egg white as raw material,extracted the egg white protein to prepare active polypeptide,and then isolated and purified it and peptide sequence was identified,and used in vitro antioxidant model and RAW 264.7 cell model to evaluate its antioxidant activity and immunomodulatory activity The results were as follows:This study compared p H 4.5,60%saturated ammonium sulfate precipitation method,p H9.0,60%saturated ammonium sulfate precipitation method,and PEG 4000 to extract egg white protein from duck eggs.It was found that p H 4.50,60%saturated ammonium sulfate precipitation method was optimal.The total nitrogen content was 86.75%,and the protein yield was 85.32%.Circular dichroism analysis were used to predict that the secondary structure was 15.06%α-helix,24.69%β-sheet,25.63%β-turn,and 33.83%random curl,respectively.In addition,under the stimulation of 1000μg/m L of ovalbumin,the secretion of NO,TNF-αand IL-6 in the supernatant of RAW 264.7 cells were 13.84μmol/L,1008.17pg/m L and 421 pg/m L,which was significantly higher than that of the blank group(p<0.05).Antioxidation experiments in vitro showed that the clearance rates of ABTS~+·,DPPH·for 2mg/m L samples were 57.05%,26.12%,and the highest ORAC value was 0.3148μmol TE/mg.The enzymatic hydrolysis conditions of duck egg ovalbumin were explored through orthogonal experiments.The results showed that using papain,the enzyme amount was4.5×10~4 U,the enzymolysis time was 5.5 h,the enzymolysis temperature was 55℃,and the enzymolysis p H was 6.40,DH reached 27.91%.HPLC results showed that the molecular weight of 83%of egg white proteolysis(HD)is less than 1450 Da.In addition,250μg/m L HD can significantly increase the secretion of NO,cytokines TNF-αand IL-6 in RAW 264.7cells(p<0.05),and inhibition rate of AAPH-induced red blood cell hemolysis was 53.9%,DPPH·,ABTS~+·free radical scavenging rates were 31.92%,86.46%,and ORAC value was0.3574μmol TE/mg,indicating that HD also had good in vitro antioxidant activity.After HD was purified by DEAE Sepharose F F anion column and Sephadex G-15dextran gel column,the component HD-1 was obtained.LC-MS/MS was used to analyze the amino acid sequence of the polypeptide in HD-1,and 8 peptides were selected for artificial synthesis.Among them,204,205 and 206 can significantly increase the secretion of NO,TNF-αand IL-6 in RAW 264.7 cells.The ABTS~+·clearance rates of 203,205,and 206 were72.89%,45.33%,and 81.50%,and the ORAC values were 0.997,0.4555,and 1.0918μmol TE/mg,respectively.Therefore,205 and 206 were polypeptides with both high immunomodulation activity and antioxidant activity,and their sequences are SEKMKILELPFASGMM and WTSSTMMEER,respectively.