Study On The Preparation,activity Identification And Hypoglycemic Study Of Antioxidant Peptide From Flaxseed

With the increasing demand for natural functional foods,natural antioxidant peptides,which have potential applications in food and pharmaceutical industry,are attracting more and more attention.As a kind of high quality protein resource,flaxseed protein has high research value.In this paper,the flaxseed meal(degummed and defatted)was used as the raw material to extract the protein and prepare antioxidant peptides.The antioxidant peptides were isolated and purified from the protein hydrolysates.The in vitro free radical scavenging ability,the protective activity of H2O2 damaged human endothelial cells(HAEC cells)and the hypoglycemic effect were also studied.The protein was extracted from flaxseed meal by alkali extraction and acid precipitation.With DPPH free radical scavenging rate as the index,the trypsin was chosen to hydrolyze flaxseed protein.The optimum hydrolysis conditions were obtained by response surface methodology.The optimum conditions are substrate concentration2%,pH value 8.50,the amount of enzyme 4000 U/g,temperature 37?,enzymolysis time3.0h,the DPPH radical of hydrolysis product was63.64±0.93%at 1mg/mL.Sephadex G-25 gel filtration chromatography and RP-HPLC were adopted for the purification of enzyme hydrolysate.Finally,three antioxidant peptides were obtained.Their sequences and molecular weights were determined as Glu-Gly-Leu-Ala-Arg(544.39 Da),Val-Phe-Ser-Val-Thr-Gly-Gly-Lys(793.59 Da)and Tyr-Ala-Pro-Phe-Tyr-Leu(772.49 Da)by LC-ESI-MS/MS.The antioxidative activies of peptides were evaluated by reducing power,superoxide anion radical scavenging activity,active oxygen free radical scavenging activity(ORAC)and ABTS radical scavenging activity.The results indicated that YAPFYL showed strongest the reducing power and can effectively remove active oxygen free radicals and ABTS radicals,among the three peptides.The aromatic amino acids(Y,F)in peptide YAPFYL were replaced by glycine.The results showed that three aromatic amino acids all contributed significantly to the antioxidant activity.The effect of Tyr on antioxidative ability was greater than that of Phe.The antioxidant activity was stronger when Tyr were at N terminus.To reveal the antioxidant activity in cell,HACE cell were treated with different concentrations of YAPFYL.The results suggested that the peptide with a concentration of 0.5 mg/mL did not have toxicity to cell.The final concentration of 0.25 mg/mL and 0.5 mg/mL of YAPFYL has obvious protective effect on HAEC cells.The DCFH-DA fluorescence staining showed that the YAPFYL can reduce intracellular ROS level,and reduce the degree of oxidative damage.In addition,we found that YAPFYL did not have the ability to inhibit the activity of alpha glucosidase,but also did not work in the treatment of insulin resistance.