Study On The Characteristics And Mechanism Of Duck Gizzard Antioxidant Peptides

Bioactive peptides are biological,active,and safe biological products that have important research value and application prospects.Duck gizzard is a high-quality protein resource.High value of the development and utilization are the direction of industrial development.In this study,antioxidant peptides from duck gizzard protein were prepared by controlled enzymatic hydrolysis technology.The product was purified by ultrafiltration,gel filtration chromatography,and reversed-phase high performance liquid chromatography.The first-order amino acid sequence of the target peptide was identified by mass spectrometry.Quantitative structure-activity relationships of the antioxidant peptides based on molecular structure were established.The anti-oxidation mechanism of the antioxidant peptides was further elucidated from the molecular mechanisms of cellular activity,analysis of cell growth inhibition,apoptosis detection,growth cycle,intracellular antioxidant enzyme activity,and enzyme protein expression.In this study,the development and application of duck gizzard proteins were discussed,providing a theoretical basis and technical support for the development and utilization of duck gizzard protein resources.The main contents and results of this paper were as follows:(1)The amino acid content in the papain hydrolysate was 33594.73 mg/100 g,and the content of the main antioxidant amino acid was 9.54%.Based on the single factor experiment,the degree of hydrolysis and DPPH clearance were taken as the response values,and the optimal hydrolysis conditions were as follows: hydrolysis time 4 h,enzyme dosage 3000 U/g,hydrolysis temperature 55°C,and a material to liquid ratio of 1:3.Under these conditions,the degree of hydrolysis was 28.30%,and the DPPH removal rate was 75.22%.There was no significant correlation between the degree of hydrolysis and the DPPH free radical scavenging ability.(2)The antioxidant activities of ZT1(<3 kDa),ZT2(3-10 kDa),and ZT3(> 10 kDa)with different molecular weights were investigated.The DPPH scavenging rate and ORAC activity of the free radicals were significantly correlated with their molecular weight.The DPPH scavenging rate of the products with molecular weights less than 3 k Da was 77.59% and the ORAC activity was 885.25 ?mol TE/g.The antioxidant peptides with higher antioxidant activities were less than 3 kDa.The protective effect of the antioxidant peptides on scavenging the superoxide anions,hydroxyl radicals,hydrogen peroxide,and DNA damage showed an obvious dose-effect relationship.The IC50 values were 14.58,0.0163,19.80,and 1.93 mg/ml,respectively.Therefore,the duck gizzard antioxidant peptide had the strongest inhibitory effect on hydrogen peroxide radicals and the weakest inhibition on hydroxyl radical activity.(3)Separation and identification of antioxidant peptides from duck gizzard protein.The enzymolysis solution was first separated by hollow cellulose membrane ultrafiltration.The fraction with the highest ORAC activity(less than 3 kDa)was further separated by gel chromatography and semi-preparative RP-HPLC.Purified peptides with the highest ORAC activity were identified as Gly-Ile-Glu-Leu-Ile-Ile-Lys and Asn-Lys,respectively.These novel antioxidant peptides have never been identified before.(4)The chemical structure information of some known antioxidant peptides was described using the ACD/Labs software.Activity prediction of antioxidant peptides was performed by using multivariate linear regression(MLR),partial least squares regression(PLS),and artificial neural network(BPNN)analyses.The linear fitting value of MLR was 0.9915,the linear fitting value of PLS was 0.8313,and the linear fitting value of BPNN was 0.99239,indicating that the constructed neural network model had high accuracy and reproducibility.The F-test was performed on the model constructed by MLR,and showed that the equation was significant.The models of MLR,PLS,and BPNN were used to separate and identify two duck gizzard antioxidant peptides,DKFILK and SSYEGIELIIK.The ORAC activity was in agreement with the results of the amino acid structure analysis,further confirming the higher antioxidant activity of component DKFILK.The mechanism of the antioxidant peptide,DKFILK from the duck gizzard had a certain correlation with its amino acid composition and structure.(5)Anti-oxidative capacity and mechanism of duck gizzard antioxidant peptide in human benign prostatic hyperplasia cells.Different concentrations of duck gizzard peptide after treatment for 48 h could significantly inhibit B-cell proliferation by promoting apoptosis and preventing cell cycle progression.When the mass concentration of the duck gizzard active peptide was 200 × 10-3 g/L,the superoxide dismutase,catalase,and glutathione peroxidase activities increased by 26.16%,20.24%,and 69.41%,respectively.The expression of CAT and GSH-Px mRNA was increased and SOD mRNA expression was not significantly correlated with the concentration of the active peptide of duck gizzard as observed by qRT-PCR.Duck gizzard active peptides played an important role in antioxidant activity by upregulating the expression of CAT and GSH-Px mRNA in cells,and attenuating the oxidative damage in cells.