Deamidization Modification Of Duck Egg White Protein And Plastein Reaction Of Egg White Peptide To Improve The Calcium Binding Capacity

In order to improve the added value of the food industry waste(salted duck egg white),bioactive desalted duck egg white peptides with high calcium carrying capacity were prepared.In the present study,salted duck egg white was used and the modification research of duck egg white protein or protein hydrolysate through two methods were conducted.Firstly,the effects of organic acid(citric acid)and inorganic acid(hydrochloric acid)on the deamidation of duck egg white protein were compared by DSC,CD and AFM.Then,the deamidated egg white protein was decomposed into peptides through enzymolysis which product called DDPs.Finally,the DDPs were subjected to plastein reaction by adding an exogenous amino acid(glutamic acid).The sequences of peptides before and after the plastein reaction were carried out by amino acid analyzer,LC-ESI/MS/MS and so on.The main findings of this paper were as follows:1.Deamidation modification of duck egg white proteinThe optimum reaction conditions for the deamidation of hydrochloric acid were as follows: deamidation temperature of 95℃,reaction time of 4h,hydrochloric acid concentration of 0.5mol/L.In this condition,the deamidation degree of the deamidated modified product can reach to 72.67%.The optimal reaction conditions for the deamidation of citric acid were as follows: deamidation temperature of 50℃,reaction time of 1h,citric acid concentration of 0.04 mol/L.Under these conditions,the deamidation degree of duck egg white protein was 49.69%.2.Functional properties and structural characterization of deamidated egg white proteinCompared with unmodified egg white protein,some properties as the solubility,foaming,foaming stability and water holding capacity of deamidated egg white protein were significantly enhanced(P﹤0.05),while the emulsifying ability decreased.The whiteness of deamidated protein were increased,especially the hydrochloric acid deamidated protein.The deamidation treatment increased the polarity and decreased the surface hydrophobicity.The Zeta potential showed that the deamidated protein system were more stable,especially the deamidated hydrochloric acid protein.The denatured heat absorption of the deamidated protein were increased,and the thermal denaturation temperature also changed significantly.The results of CD showed that the molecular structure of the deamidated hydrochloric acid protein was more disordered,and the structure of the citric acid deamidated protein was more stretched.The results of scanning electron microscopy showed that both unmodified egg white protein and citric acid deamidated protein were in the form of flakes,but the overall structure of citric acid deamidated protein was more loose.The morphology of hydrochloric acid deamidated protein was changed from flake structure to spherical shape,and its molecular size became smaller.The diameter of the sphere increased as the degree of deamidation increased.The results of AFM showed that the molecular morphology of unmodified egg white protein and citric acid deamidated protein were similar,but the molecular height of citric acid deamidated protein decreased slightly compared with the unmodified egg white protein.And the citric acid deamidated protein is more loose and more distributed evenly.The molecular morphology of hydrochloric acid deamidated protein changed greatly,and the three-dimensional map showed that its height was significantly higher than egg white protein and citric acid deamidated protein,showing dense convex and concave shape,indicating its surface roughness increased.It was apparent from the plan view that it is a nearly uniform spherical structure.3.Preparation of deamidated duck egg white peptidePapain was screened from the five commercial proteases as papain used for this experiment could obtain the deamidated duck egg white peptide with higher calcium carrying capacity.The optimum conditions for the preparation of deamidated duck egg white peptide were as follows: temperature of 50℃,p H of 6.5,material to liquid ratio of5%,enzyme mass fraction of 0.3%,and reaction time of 3h.Under these conditions,the soluble calcium binding amount of DDPs was 14.42 mg Ca/g peptide.After ultrafiltration fractionation,it was found that fractions with smaller molecular weight had stronger calcium binding ability,especially Mw<3k D and Mw<1k D fractions contributed to the high calcium carrying capacity of DDPs.4.Plastein reaction modification of DDPsThe optimum conditions for the plastein reaction were as follows: reaction temperature of 45℃,enzyme addition of 4%,material to liquid ratio of 45%,reaction time of 4h,and the amount of glutamic acid added was 0.3 times amount of the total free amino acids.The modified product obtained under these conditions and through dialysis treatment was called P-D,and its calcium binding ability was up to 19.57 mg Ca/g peptide,which was significantly improved compared with DDPs.Compared with DDPs,the particle size of P-D increased significantly.The results of amino acid analysis showed that the content of Glu in P-D was significantly improved.The peptide structure was analyzed by LC-ESI/MS/MS,and 28 Glu-containing peptides were analyzed from DDPs map,43 Glu-containing peptides were identified from the P-D map,which indicating that Glu and peptide were efficiently bound.We found there were six same peptides before and after the plastein reaction.In addition,compared with the peptide sequences involved Glu before and after the plastein reaction,we found that the products mainly included two ways: Glu substituted 1-2 amino acids at the carbon terminal and 1-2 amino acids at the nitrogen terminal.