Comparison Of Antioxidant Activities Of ?-Casein AA And BB Subtype Hydrolysates And Isolation Of Antioxidant Peptides From Buffalo Milk

Buffalo milk protein not only contains various amino acids necessary for human body,but also produces bioactive peptides after enzymatic hydrolysis,which is beneficial to human health.Although China has vigorously promoted the development of the milk buffalo industry,buffalo milk products have not been effectively developed and basic research is still weak.Studies on buffalo milk proteins were limited and less work was reported as regarding to buffalo casein.Therefore,more work is needed to further identify some new sequences of amino acids with higher bioactivity.In order to identify peptides with high antioxidant activities and develop functional buffalo dairy products,the antioxidant activites of ?-casein(?-CN)hydrolysates of different genotypes(AA and BB)of buffalo milk were compared in this study.Buffalo milk was collected from Guangxi Province of China.First,?-CN with genotypes AA and BB were separately isolated by selective precipitation method.The degree of hydrolysis(DH)was measured after ?-CN was hydrolyzed with alcalase,papain,trypsin and pepsin.Using ferric ion reducing antioxidant power(FRAP)as an indicator,the best enzymatic hydrolysis process was obtained through single factor test and orthogonal test.Then the antioxidant capacity was evaluated by six methods:ABTS radical scavenging ability,hydroxyl radical scavenging ability,FRAP,reducing ability,ferrous ion chelating ability and DPPH radical scavenging ability.The effects of temperature,pH and gastrointestinal digestion on the antioxidant activities of hydrolysates were further determined.Besides,ultrafiltration was used to purify the peptides to obtain three components of 0?3,3?5 and 5?10 kD,and their antioxidant activities were measured by FRAP method and cell test.Finally,the amino acid sequences of the component with the highest antioxidant activity were identified,its molecular weight distribution was verified by gel chromatography and it was further purified by cation chromatography.The main findings are as follows:(1)Among the enzymatic products of the four enzymes,the antioxidant activities and DH of the products hydrolyzed by alcalase were higher than those of the other three.When alcalase was used for hydrolysis,the temperature was 55?,pH 8.0,the enzymolysis time was 2.5 h,the substrate concentration was 3.0%and the total enzyme dose was 9 000 U/g protein,the product with the highest antioxidant activity was obtained.(2)After the hydrolysates were obtained through the optimal enzymatic hydrolysis,the antioxidant results showed that there was no significant difference between AA and BB genotypes in the determination of DPPH free radical scavenging ability(P>0.05).The antioxidant activities measured by the other five methods indicated that AA type was significantly higher than BB type(P<0.05)and all six assessment methods suggested that the ?-CN hydrolysates with alcalase had significantly improved antioxidant activities compared to the ?-CN(P<0.05).In addition,when the hydrolysates were treated at a temperature below 60? and a pH of 6 to 8,the antioxidant activities were stable and FRAP differences were less than 35 ?M FeSO4.During the gastric digestion stage,the antioxidant activity was not significantly reduced(P>0.05)and the antioxidant activity was significantly higher in gastric digestion than in gastric digestion(P<0.05).(3)Both FRAP and cell tests had verified that the 0-3 kD components in both AA and BB genotypes were significantly higher than other molecular weight components.Cell tests had shown that ?-CN enzymatic hydrolysates had better ROS scavenging activity and the 0?3 kD component of AA had the highest antioxidant activity.After purification of AA 0?3 kD component by cation chromatography,6 fractions were obtained for further screening of antioxidant peptides.The results of this study indicated that ?-CN alcalase hydrolysates could be used as an effective source of natural antioxidants.The hydrolysates showed good performance under various antioxidant evaluations and had good stability within 60?,pH 6?8 and through gastrointestinal digestion,which were suitable for food processing.Since the 0?3 kD component of the AA-type ?-CN hydrolysate had the best antioxidant activity,the purified peptide could be further separated.These results could provide food industry with beneficial technologies for a wider range of applications and provide superior quality and health benefits to products.