Antioxidation And Isolation Of Collagen Hydrolysis From Pig Skin

Collagen was extracted from pig skin, the process of preparing of collagen hydrolysis from pig skin by Bromelain, Papain,Alcalase were studied in the present paper.The factors of effecting process of hydrolysis were investigated by determining the hydrolysis degree of collagen hydrolysis and the scavenging capacities of the hydrolysates on superoxide free radical.Enzymatic hydrolysis conditions were optimized through single factors and orthogonal experiment.The antioxygenic property of collagen hydrolysis was studied by five analytical methods.The stability of antioxygenic property was also evaluated.Peptides derived from porcine collagen hydrolysates were separated by ultrafiltration(UF) and consecutive chromatographic methods including ion exchange chromatography(IEC),and gel filtration chromatography(GFC).The optimum hydrolysis conditions of Bromelain were determined:pH3.5, hydrolyzing temperature 45℃,ratio of Bromelain 4000 U/g, concentration of substrate 6% and total hydrolysis time 5 h.Under these conditions the hydrolysis degree of collagen protein could reach 5.25%,while the scavenging capacities of the hydrolysates on superoxide free radical could reach 44.49%; the optimum hydrolysis conditions of Papain were determined:pH5.5,hydrolyzing temperature 70℃,ratio of Papain 6250 U/g, concentration of substrate 6% and total hydrolysis time 6 h.Under these conditions the hydrolysis degree of collagen protein could reach 13.28%,while the scavenging capacities of the hydrolysates on superoxide free radical could reach 41.97%; the optimum hydrolysis conditions of Alcalase were determined:pH7.5, hydrolyzing temperature 55℃,ratio of Alcalase 6000 U/g, concentration of substrate 4% and total hydrolysis time 4 h,under these conditions the hydrolysis degree of collagen protein could reach 9.55%,while the scavenging capacities of the hydrolysates on superoxide free radical could reach 60.11%. The hydrolysates hydrolyzed by Alcalase showed the highest superoxide radical scavenging ability.The antioxidant properties of collagen hydrolysates were determined in vitro. In a certain concentration range(10 mg/mL-50 mg/mL),the results showed that the enzymic hydrolysates of collagen had scavenging ability on hydroxyl radical, the maximum scavenging rates to·OH free radical were 56.38%,IC5o=40.99mg/mL; the maximum scavenging rates to DPPH·free radical were 95.06%, IC50=3.89mg/mL;the maximum scavenging rates to O2-·free radical was 65.89%,IC50 was 16.43mg/mL;the collagen hydrolysates also had certain reductive ability; moreover, the collagen hydrolysates could inhibit the lipid peroxidation.Generally speaking,the collagen hydrolysate showed certain antioxidatative activities with five different analytical methods in vitro.The antioxygenic property of collagen hydrolysates were stabile to heat and freezing,furthermore,the antioxygenic property in acidic condition was better than in alkaline condition.The peptides with superoxide radical scavenging activity were isolated and purified from collagen hydrolysates.Four different UF centrifuge tubes having a range of molecular weight cut-offs(MWCO)of 10,5,3 and 2kDa were used to fractionated the enzymic hydrolysates.The portion<2 kDa showed the highest superoxide radical scavenging ability, the maximum scavenging rates to O2-·free radical was 41.11% when the peptide concentration was 2.24 mg/mL, its IC50 value was2.85mg/mL.The collagen hydrolysates were load onto SP-Sephadex C-25 ion exchange column for further purification and seven peaks were eluted.The first peak P1 exhibited higher superoxide radical scavenging ability, the maximum scavenging rates to O2-·free radical was 46.30% when the peptide concentration was 0.85 mg/mL,IC50 value was 1.24 mg/mL.The P1 portion was loaded onto Sephadex G-25 gel filtration column for further isolation.Two peaks were eluted and the second peak P1-B showed the highest superoxide radical scavenging ability, the maximum scavenging rates to O2-·free radical was 49.43% when the peptide concentration was 0.9 mg/mL,IC50 value was 0.98mg/mL...