Study On Purification And Separation Of Casein Antioxidant Peptides

In this paper,rennin was used to hydrolyze casein for getting peptide. Single factor examinations were design to study the influence of various factors,including enzyme dose,temperature, pH and hydrolysis time, on the antioxidant activity of peptide in the hydrolyzate with DPPH free radical scavenging activity as indicator. The optimum condition of hydrolyzation was get through orthogonal test. Then,the peptides were purified by ultrafiltration,gel chromatography, ion exchange chromatography and RP-HPLC to observe the relationship between antioxidant activity of peptides and their properties,which include their molecule weights and polarities.The optimum condition of hydrolysis was obtained as follows:pH7.0,40℃,1.5h, the enzyme amount200U/g. In this condition, the scavenging activity of hydrolysate on the DPPH·,O2-· and·H were79.41%,70.63%,and64.59%respectively. The influence of peptide concentration on DPPH free radical scavenging activity were also observed as the concentration of peptides at2,4,6,8,10,12mg/mL,the DPPH free radical scavenging rate was43.74%,70.32%,82.13%,85.46%,90.82%,87.14%respectively.The maximum scavenging activity on DPPH free radical was90.82%at10mg/mLThe results of purification and separation indicated:The peptides in the hydrolyzate were divided into four groups with different molecular weights, which were less than3kDa,between3～5kDa, between5～10kDa,greater than lOkDa. The scavenging rate on DPPH free radicals was maximum as the molecular weights were less than3kDa. Then the peptides with molecular weights less than3kDa was separated by Sephadex G-15using water as mobile phase,0.3mL/min,40mg.Four peaks were observed and the peak2had the strongest antioxidant activities,with the scavenging rate on DPPH free radical as82.65%. Then the peak2were isolated by ion exchange chromatography, three peaks were observed, the peakl had strongest antioxidant activities, with the DPPH free radical scavenging rate at82.71%. A kind of peptide which had strong antioxidant activity as DPPH free radical scavenging rate at85.37%(0.1mg/mL) was isolated by using RP-HPLC. In conclusion, the antioxidant activities of casein hydrolysate had a relationship with their molecular weights and polarities.