Study On The Preparation And Purification Of Antioxidant Peptides From Sturgeon Intestine Protein And Its Antioxidant Activities

Antioxidant peptides were prepared from sturgeon intestine protein through controlled enzymatic technology.Separation and isolation of the activity peptides from the sturgeon intestine protein hydrolysate?LPH?by a series of purification technology,including ultrafil-tration,gel-filtration chromatograghy and anion-exchange chromatography and reversed-phase high performance chromatography.Tricine-SDS-PAGE and RP-HPLC-MS were used to identify the purity,the molecular weight and the amino acid sequence of the peptide Finally chemical models in vitro and animal models in vivo were used to evaluate their antioxidant activities.The preparation of peptide with enzymolysis method was studied and it was also tested in vitro experiment.The results showed that four enzymes were respectively used to preparation of sturgeon intestine anti-oxidation peptide for screening the best one.Pepsase was detected as the most enzyme.Single factor experiment and Orthogonal test were used for optimizing the enzymatic hydrolysis conditions in this study.The optimal values of temperature,pepsase enzyme dosage,solid-to-water ratio and reaction time were 35?,1:20?g/mL?,3200U/g and 1.5h.The polypeptide content's scavenging rates against hydroxyl free radicals was 85.80%with concentration of 1.6 mg/mL under the optimal conditions.Sturgeon intestine peptide was isolated and purified by ultrafiltration and consecutive chromatographic methods including gel filtration chromatography Sephadex G-25,ion-exchange chromatography DEAE-52 cellulose and reverse high-performance liquid chromatography?RP-HPLC?.The purified peptide showed a 1.08-fold higher scavenging activity for DPPH radical compared with the crude sturgeon intestine peptide,and it is indicated that the separation and purification can improve the antioxidant activity of the sturgeon of sturgeon intestine protein peptide.The target peptide was a single component with a molecular weight of about 4.2KD.Finally six antioxidant peptides sequence,FDKPVSP?GVDNPGHPF?IEEELEEL?TGVDNPGHPF?NLKGGDDLDPN?NDHFVKL,were got from target peptide through RPLC-MS and were matched and searched in the database Actinopteri.The molecular weight of those peptides were 788.4069?938.4246?1002.4757?1039.4723?1156.536?871.4552 Da.Preliminary analysis of the antioxidant activity was mainly manifested in strong hydrophobic properties?Leu?Phe?Pro?Ile?Val?,ability of chelating metal from His and Glu and its structure stability.And Gly can improve the ability of resistance to oxidative stress,etc.Three different chemical models in vitro were used to evaluate the antioxidant activities of sturgeon intestine peptide.In a certain concentration range?0.3-1.5 mg/mL?,the results showed that the antioxidant capacity decreased with the increasing of peptide mass concentration.When the concentrate-ion was 1.5 mg/mL,scale antioxidant peptide on DPPH radical scavenging capacity reached 83.64%of Vc and its scavenging rates against hydroxyl free radicals was 78.06%and its reducing activity was about 1/3 of V_C.Generally speaking,the sturgeon intestine showed certain antioxidant activities with three different analytical methods in vitro.