Optimization Of Enzymatic Hydrolysis And Purification Of Goose Bone Antioxidant Peptides

Goose bone was used to product antioxidant peptides.Neutral protease, alkaline protease, the flavourzyme, papain, pepsin and trypsin protease were used to enzymatic hydrolyze goose bone protein respectively.Three kinds of enzymes were selected as better enzyme at the target of superoxide anion radical scavenging activity. Enzymatic hydrolysis conditions were optimized by single factor experiment and response surface method. Three different types of proteases were utilized to hydrolyze goose bone protein in different ways, i.e., double-enzyme hydrolysis,three-enzyme hydrolysis,under the optimum conditions of every single enzyme. The optimum hydrolysis conditions of goose bone protein for production of antioxidant peptides was determined by comparing the hydrolysis effect of different ways. The antioxygenic property of goose bone protein hydrolysis was studied by four analytical methods..Peptides derived from goose bone protein hydrolysates were separated by ultrafiltration(UF),ion exchange chromatography(IEC),and gel filtration chromatography(GFC).Alkaline protease, the flavourzyme, papain were selected as the better enzyme. The optimal enzymolysis conditions of goose ptotein using flavourzyme were determined as follows:enzyme dosage8800U/g, hydrolyzing temperature50℃, pH6.0, hydrolysis time8h,concentration of substrate12g/100mL. The superoxide anion radical scavenging activity and degree of hydrolysis were64.14%,17.76%respectively at the optimum hydrolysis conditions. The optimal enzymolysis conditions of goose ptotein using alkaline protease were determined as follows:enzyme dosage12000U/g, hydrolyzing temperature55℃, pH9.0, hydrolysis time6h,concentration of substrate12g/100mL. The superoxide anion radical scavenging activity and degree of hydrolysis were63.98%,8.15%respectively at the optimum hydrolysis conditions. The optimal enzymolysis conditions of goose ptotein using papain were determined as follows:enzyme dosage12000U/g, hydrolyzing temperature69℃, pH5.5, hydrolysis time4h,concentration of substrate8g/100mL. The superoxide anion radical scavenging activity and degree of hydrolysis were63.12%,11.27%respectively at the optimum hydrolysis conditions.The optimum hydrolysis conditions of goose bone protein for production of antioxidant peptides was determined by comparing the hydrolysis effect of different ways. Sequential hydrolysis with flavourzyme protease followed by papain was the best way under the optimum conditions of every single enzyme. The superoxide anion radical scavenging activity and degree of hydrolysis were67.94%,22.86%respectively.The antioxidant properties of goose bone protein hydrolysates which was pruducted under the optimum conditions were determined in vitro. In a certain concentration range(10-50mg/mL),the results showed that the enzymic hydrolysates of collagen had scavenging ability on superoxide anion radical,hydroxyl radical and DPPH free radical, the maximum scavenging rates to superoxide anion radical was60.80%,IC50was28.02mg/mL;the maximum scavenging rates to hydroxyl radical was99.58%,IC50was9.45mg/mL;the maximum scavenging rates to DPPH free radical were98.16%, and inacertein concentration(30-50mg/mL), the scavenging rates of the enzymic hydrolysates was higher than Vc;the goose bone protein hydrolysates also had certain reductive ability.Generally speaking,the goose bone protein hydrolysate showed certain antioxidatative activities with four different analytical methods in vitro.The peptides with superoxide radical scavenging activity were isolated and purified from goose bone protein hydrolysates.Two different UF centrifuge tubes having a range of molecular weight cut-offs(MWCO)of10KDa,5KDa were used to fractionated the enzymic hydrolysates.The portion<5KDa showed the highest superoxide radical scavenging ability, its IC50value was7.93mg/mL. The portion<5KDa were isolated and purified by ion exchange chromatography and gel filtration chromatography in order. The results showed that, the portion<5KDa were load onto ion exchange column for further purification and five peaks(S1-S5) were eluted.The second peak S2exhibited higher superoxide radical scavenging ability, IC50value was2.93mg/mL.The S2portion was loaded onto Sephadex G-25gel filtration column for further isolation.Three peaks were eluted and the second peak S2B showed the highest superoxide radical scavenging ability, IC50value was1.21mg/mL. The portion<5kDa were isolated and purified by gel filtration chromatography and ion exchange chromatography in order. The results showed that, the portion<5KDa were loaded onto Sephadex G-25gel filtration column for further isolation.Four peaks (N1、N2、N3、N4)were eluted and the third peak N3showed the highest superoxide radical scavenging ability,IC50value was2.55mg/mL. The third peak N3was load onto ion exchange column for further purification and six peaks were eluted. second peak N3B exhibited higher superoxide radical scavenging ability, IC50value was0.6mg/mL.Comparing the separation effect of goose bone protein hydrolysates with the two ways,the best way was the hydrolysates were isolated and purified by ultrafiltration,gel filtration chromatography and ion exchange chromatography in order.The peptides showed highest superoxide radical scavenging ability,IC50value was0.6mg/mL.