Preparation,Purification And Identification Of Antioxidant Collagen Peptides From Yak Bone

In this study,yak bone powder was used as raw material to prepare antioxidant peptide through enzymatic hydrolysis.At the same time,the physical properties and stability of yak bone peptides were studied.The yak bone antioxidant peptides were isolated and purified by prepacked ceramic hydroxyapatite column chromatography,gel chromatography and C18 reversed-phase high performance liquid chromatography.The sequence of yak bone peptides was determined by reversed-phase high performance liquid chromatography-mass spectrometry.Through computer simulation of gastrointestinal digestion experiments,the stability of these peptides'sequences was predicted.The results of this study were as follows:The oil absorption of freeze-dried yak bone peptides was 0.2 mL/g.With the increase of the concentration of yak bone peptides,emulsification,emulsion stability,foam ability and foam stability were all increased.The low-temperature stability and heat resistance of the yak-bone-peptide solution were low.There was no effect on the antioxidant activity of yak bone peptides with the addition of low concentration of NaCl.The antioxidant capacity of yak bone peptides was significantly increased with the addition of 6%NaCl.Yak bone peptides were sensitive to acid and alkaline environment,and the antioxidant activity of these peptides was stronger in alkaline environment than in others.The optimal irradiation time of UV for yak bone peptides was 6 h.The antioxidant activity of yak bone peptides was significantly increased with the addition of 0.1g glucose and fructose,and there was no protective effect on yak bone peptides with the addition of0.1g sucrose.In vitro gastrointestinal simulation experiments,the antioxidant activity of yak bone peptides was high.The optimal elution conditions for prepacked ceramic hydroxyapatite column chromatography were as follows:the concentration of samples was 15 mg/mL.The volume of loading samples was 60 mL.The Na₂HPO₄ solution?pH 6.8,5 mmol/L?was used for equilibrating prepacked ceramic hydroxyapatite column,and the equilibrium volume was 50 mL.Following that,1 mol/L NaCl solution was used as eluent,and the volume of it was 30 mL.Then,100 mmol/L Na₂HPO₄ solution?pH8.0?with the volume of 35 mL was used to elute samples.The yak bone peptides were isolated by ceramic hydroxyapatite column,and two peptide groups,F1 and F2,were obtained.And group F2 had higher antioxidant activity than group F1.G15 gel was used to separate peptides,group F2,with the following conditions:the concentration of samples was 1 mg/mL.The volume of samples was 4 mL,and the flow rate was 0.4 mL/min.The peptide group F2 was isolated by G15 gel,and three peptide groups,F21,F22 and F23,were obtained.And group F23 had higher antioxidant activity than other groups.The peptide group F23 was separated by C18 reversed-phase high-performance liquid chromatography,and only one peptide peak,F231,was detected.When the concentration of the samples was 1 mg/mL,the·OH radical scavenging rate,DPPH·radical scavenging rate,and reducing power were 84.27%,24.55%and 0.07,respectively.Through reversed-phase high performance liquid chromatography-tandem mass spectrometry,129 kinds of peptides were detected from the isolated and purified peptides,and the range of theoretical molecular weight of these peptides was from699.36 to 1228.58 Da.Gastrointestinal digestive experiments were simulated by a software named PeptideCutter.The peptides were not stable,most peptide sequences were experienced enzymatic reactions,resulting in 197 peptide sequences.And most of the peptide sequences contained less than 10 amino acids.