Preparation Of Peptides From Pufferfish Skin Collagen And Its Antioxidant Properties

The pufferfish Takifugu rubripes is a kind of economically important fish containing abundant nutrimental substance. As main byproduct in fish process, its skin could provide a valuable source of gelatin.In this study, the collagen was extracted by hot water from the pufferfish skin and was hydrolyzed by papain. To obtain the highest peptide yield and scavenging activity on hydroxyl radicals, the optimum hydrolysis conditions were ascertained by single-factor and orthogonal experiment. The resulting hydrolysate was fractionated through an ultrafiltration membrane system separately with three different molecular weight cut-offs (10,3and1kDa). The hydrolysate and its MW fractions (fraction1:3-10kDa; fraction2:1-3kDa; fraction3:<1kDa) were evaluated using hydroxyl radical scavenging assay, DPPH radical scavenging activity, reducing power and metal chelating activity. The fraction with highest antioxidant activities was separated using consecutive chromatographic methods on a DEAE Sepharose CL-6B anion exchange column, Sephadex G-25gel filtration column and TSK-G4000PWxL gel column.Experimental results indicated that the optimum hydrolysis enzyme was papain. Using papain, the optimum conditions were listed below:the papain vs collagen ratio was5000U/g; the hydrolytic time was2h; the hydrolytic temperature was45℃; the reactive pH was6.5; the concentration of collagen was33.6mg/100mL. Under the conditions, the yield of collagen peptides from hydrolysates could reach34.76%with hydroxyl radical scavenging effect of67.54%. The three MW fractions from pufferfish skin collagen exhibited high hydroxyl radical scavenging assay and DPPH radical scavenging activity and moderate reducing power and chelating potency. Meanwhile, there was a tendency that the lower the molecular weight of gelatin peptide, the stronger of antioxidant activity.The peptide fraction (1-3kDa) was initially separated into2fractions (A and B) on a DEAE Sepharose CL-6B anion exchange column. The fraction A showed stronger hydroxyl radical scavenging activity compared with fraction B. Fraction A and B were loaded onto a Sephadex G-25gel filtration column, respectively. The peptides in fraction A and B were then further separated into two fractions respectively (A-Ⅰ, A-Ⅱ, B-Ⅰ, B-Ⅱ) with fraction A-Ⅱ and B-II had higher hydroxyl radical scavenging activity. The fraction A-Ⅱ and B-Ⅱ were further separated by TSK-G4000PWXL gel column connected with HPLC on an and formed6and5visible peaks respectively. Experimental results indicated that the antioxidant activities decreased after the separation and purification, and the more lower the molecular weight of separated fractions, the stronger of antioxidant activity.