Proteolysis Preparation And Purification Of Antioxidant Peptides Derived From Porcine Scapula Protein

Porcine scapula was used as raw material to prepare antioxidant peptides by controlled enzymatic hydrolysis.Based on the results of single factor experiment and the response surface,antioxidant capacity was selected as the index to analyze the antioxidant activity.In addition,the antioxidant peptides were separated and purified by ultrafiltration,Gel chromatography,RP-HPLC.Molecular weight identification and sequence were identified by LC-MS/MS.The enzymatic hydrolysis effect of 6 different proteases from the skapular bone powder of the skimmed pig was analyzed and compared on the evaluation index of DPPH clearance rate and the polypeptide yield.Besides the optimal enzyme was screened out.Based on the results of single factor experiment,selecting the best enzyme for hydrolysis as the index to analyze response surface.The optimal technological conditions was that bone powder was hydrolyzated with 6200 U/g of enzyme/substrate,3%of substrate concentration in pH 6.0 for 4 h.The free radical scavenging rate of DPPH was 75.90%,and the yield of peptides obtained was 62.80%.The antioxidant activity of the obtained hydrolysate was analyzed using four vitro antioxidant assays.The IC₅₀ value of DPPH was 4.59 mg/mL,the IC₅₀ of·OH value was2.363 mg/mL,the IC₅₀ value of·O^2-was 13.031 mg/mL.When the concentration is 10mg/mL,the A₇₀₀00 of Fe²⁺was 0.3526,equaled to 15.11%of Vc.The hydrolysates of pig scapula were filtered by 0.45?m and 0.22?m filter membranes.Then ultrafiltration filtrate was filtrated by ultrafiltration centrifuge with a cut-off molecular weight of 10 kDa and 5 kDa.The IC₅₀0 value of DPPH scavenging rate,color value of filtrate,clarity and protein yield were compared.The results showed that the color,clarity and protein yield of the filtrates obtained from the 0.45?m and 0.22?m filters were not significantly different?p>0.05?.After comprehensive consideration,0.45?m was used to filtration of the pork shoulder hydrolyzate.When the molecular weight was less than 5 kDa,the antioxidant activity was the strongest and it was suitable for the next step of separation and purification.After ultrafiltration,the filtrate was successively passed through dextran gel Sephadex G-25.The effects of eluent,flow rate and sample size on the isolation and purification of porcine scapular antioxidant peptides were studied in three factors.The optimum condition of Sephadex G-25 were hyperpure waterthe of eluent,0.3 mL/min of the flow velocity and sample volume of 1 mL.Three fraction A,B,and C were observed,and the fraction C with the highest DPPH clearance rate(IC₅₀ was 0.6528 mg/mL).The optimum condition of Sephadex G-15 were hyperpure waterthe of eluent,0.15mL/min of the flow velocity and sample volume of 3 mL.Four fraction C₁,C₂,C₃,C₄ were separated from the fraction C.The IC₅₀ value of the active fraction C₂ with the highest DPPH clearance rate is 0.1797 mg/mL.The results shows that the mobile phase A:B=5:95?V:V?,0.75 mL/min of the control velocity,and 25?of the column temperature were the optimum condition.Three fraction C₂₁,C₂₂,C₂₃ were separated from the fraction C₂,and the fraction C₂₃ with the highest DPPH clearance rate(IC₅₀ was 0.0710 mg/mL).The sequence of the antioxidant peptide was identified by ESI-TOF-MS/MS as ARGPDGG?700.4 Da?.In general,this study established a process to increase the function added value of the porcine scapula andget the better of protein product for the industrial production and applications.