| Fertilized eggs,known as a kind of traditional and tonic food which can be eaten habitually in Xianfeng,Qing dynasty.Its rich nutrition and high utility value are best medicine of nutrition and supplements.With the emphasis on health,the development of food borne antioxidants has been paid more and more attention.Fertilized eggs protein is an important active substance in chicken embryo.It is not only the important material of chicken embryo life activity,and has the unique anti-wrinkle and antioxidant function,it is important to separate and purify the antioxidants protein from fertilized eggs.In this paper,the chicken embryos were incubated for 15 days.Then using ether reflux to remove fat in chicken embryo dry powder at low temperature and buffer method to extract protein in fertilized eggs.Selected on the basis of single factor,like material-liquid ratio,extraction temperature and extraction time.The mathematical model was established by the application of Box-Behnken center combination design.The optimum extraction conditions were determined by protein extraction rate,and then extracted by ion-exchange chromatography and Sephadex G-100 gel filtration chromatography.The protein with strong antioxidant ability was isolated and purified.Finally,the activity of antioxidant protein was verified by in vivo experiment.The denaturation onset temperature of the crude protein was first determined by differential scanning calorimetry?DSC?.Selected on the basis of single factor,like material-liquid ratio,extraction temperature and extraction time.The mathematical model was established by the application of Box-Behnken center combination design.The optimum extraction conditions were as follows: material-liquid ratio?1:16?,Temperature?50??and time?116 min?.Under the optimal conditions,processing three validation experiments.As a result,the extraction rate of chicken embryo protein was 73.87%.Moreover,fertilized eggs protein was isolated and purified.The chicken embryos extracted under the optimum conditions were separated by DEAE-cellulose-52 anion exchange chromatography and named as P1;P2;P3;P4.By using DPPH,superoxide anion?O2-·?and hydroxyl radical?·OH?evaluating the antioxidant activity of each component in vitro.The components with higher antioxidant activity were further purified by Sephadex G-100 gel filtration chromatography to obtain the P4-1 of chicken embryo antioxidant protein.The result of LC-MS analysis showed that the antioxidant protein P4-1 contained 386 amino acids and the isoelectric point was 5.07.The molecular weight of P4-1 was 45.0 kDa by SDS-PAGE.Through the determination of amino acid composition,the type of P4-1 amino acid was complete which rich in 17 kinds of amino acids?tryptophan in hydrochloric acid hydrolysis which destroyed was not detected?and 7 essential amino acids.The differential scanning calorimetry?DSC?analysis showed that the denaturation temperature of P4-1 was 46.59-67.00 ?,the melting point was 57.16 ?,the enthalpy??H?was 0.9183 J / g.Finally,we studied that temperature,pH,NaCl and metal ions had an influence on the activity of antioxidant protein in mice in vivo.The results showed that the ability of P4-1 to scavenge hydroxyl radicals?·OH?was stable at temperatures between 30-50 ?,and the ability of P4-1 to scavenge hydroxyl radicals?·OH?decreased with the temperature increasing and finally stabilized again.The ability of scavenging hydroxyl radicals?·OH?of P4-1 decreased with the increase of pH value.And the antioxidant protein remained relatively stable to hydroxyl radical scavenging rate when NaCl concentration was 0-4%.When the concentration was over 4%,the hydroxyl radical scavenging rate decreased significantly.Also,the metal ions had an inhibitory effect on the activity of antioxidant protein.It was found that antioxidant protein had a good antioxidant capacity in mice,which can decrease the content of MDA and increase the activity of SOD,GSH-Px and T-AOC.Significantly,the mice in the experimental group were higher in the low dose group. |
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