Purification Of Antioxdative Peptides Derived From Buffalo Milk Whey Protein And Developmention Of Product

Buffalo milk as the "King of milk", is rich of the protein and essential amino acids, and researches of antioxidant activity peptides of buffalo milk are still in the preparation of the study, the products of buffalo milk containing antioxidant activity peptides are not yet. To fill the market and increasing the added value of buffalo milk, study aims to develop products of buffalo milk to have antioxidant activity,also research how to purify antioxidant peptide of whey protein to obtain high purity peptide.To fix neutral protease (Solarbio company) which was selected by preliminary studies, it contrasts activity recovery of neutral protease enzyme fixed by agar, sodium alginate and chitosan in its optimum conditions. Activity recovery of immobilized enzyme fixed by agar is the highest,reached37.80%, while the recovery of immobilized enzyme fixed by sodium alginate up to19.89%, while the highest chitosan immobilized enzyme activity recovery is only16.74%. The optimal conditions for immobilization is that agar concentration of3%,per gram agar adds160mg neutral protease,optimal immobilization time is4h. Through comparing the relative activity of Free enzyme and immobilized protease, it is found immobilized pH stability, thermal stability, storage stability, and stability of operation are better than the free enzyme.and the immobilized protease used after10cycles, maintains the viability of more than50%of the original.Skim buffalo milk is hydrolyzed by immobilized enzyme which neutral protease is fixed to Agar for production of antioxidant hydrolyzate. The antioxidant hydrolyzate is deployed to develop antioxidant activity beverages. Optimal parameters that Immobilized enzyme hydrolyzed skim buffalo milk is drawn as PH7.5, temperature55℃, enzyme dosage of2500U/g and hydrolysis time of5h. The main factors second order is that A (pH)> C (enzyme dosage)> B (temperature)> D (hydrolysis time). Validation experiments show that, under optimum conditions, DPPH radical scavenging rate reachs65.24%. Then diluted enzyme solution into the antioxidant activity of the peptide content of50ug/mL allocate the production of liquor beverage antioxidant activity, the best recipe for the buffalo milk:enzymatic liquid=1:3, white sugar3%(w/v),(3-cyclodextrin0.3%(w/v), coffee2%(w/v).Validation experiments show that this recipe is feasible, color is to coordinate, shape is uniform, the smell is fragrant, sweet and sour is moderate, and DPPH radical scavenging of drink is still reached54.34%.Using ultrafiltration to separate buffalo milk whey protein hydrolyzate, the best ultrafiltration pressure is0.20MPa. After ultrafiltration,three components are obtained, which molecular weight less than5000Da component is the highest antioxidant activity, DPPH radical scavenging rate reached60.18%Using DA201-C macroporous resin for desalination of component of the whey protein hydrolyzate molecular weight less than5000Da, the optimal conditions is that lh per100mL sample on the sample, and then with70%ethanol to50ml/h of flow rate6hours. DA201-C macroporous resin operate according to the optimal conditions for desalination of buffalo milk whey protein antioxidant activity peptide, desalination final measured rate is97.33%, recovery rate peptides is80.10%. The whey protein hydrolysates after desalination treated are separated by Sephadex G-15column chromatography to obtain the optimum separation conditions,which is that measure the wavelength of280nm, the sample volume was200ug, eluted at a flow rate4mL/12.5min. The hydrolyzate samples were isolated and purified by Sephadex G-15chromatography under optimal conditions, four peaks appeared and concentrated which were measured DPPH radical scavenging, found that2component peaks was the strongest antioxidant activity,1component peaks was second, but its content is greater. Finally, using RP-HPLC to separate F1and F2, F1isolated three peaks, the first two peaks maximum purity of70.15%; F2got three distinct peaks and four obvious peaks of seven peaks, the largest of the first six peak purity reached81.45%.