Investigation Of The Regulation Mechanism Of Free Radicals-induced Oxidation On The Structural And Functional Properties Of Egg White Protein

This research was funded by the scientific and technological research project of Jilin Provincial Department of Science and Technology(20190301015NY).Egg white protein(EWP),as a kind of valuable animal protein resource,was widely used in modern food industry due to its excellent nutritional value and processing properties.However,oxidation occurred during the production,processing and storage of egg products,thereby affected the processing characteristics and nutritional value of egg products.EWP was used as the research object.The effect of free radicals-induced oxidation(peroxy radical and hydroxyl radical)on protein structure transformation and functional properties was explored.The mechanism of free radicals-induced oxidation on foaming properties changes of EWP was investigated through structural,surface and rheological properties.In addition,oxidation sites were determined by HPLC-MS/MS to elucidate the molecular mechanism,which oxidation modulated the foaming properties of EWP.Furthermore,the in vitro digestion kinetics model was performed to monitor the effect of oxidation on the digestion rate of EWP.Structural changes of EWP before and after digestion were surveyed,which revealed the molecular mechanism of free radicals-induced oxidation on the digestion characteristics of EWP.This paper aimed to provide theoretical foundation for the improvement of protein functional properties and the design of nutritious foods.The main contents and results of this research were as follows:(1)2?-azobis(2-amidinopropane)dihydrochloride(AAPH)was used to generate peroxy radicals(ROO·)at 37?,in the dark,under aerobic conditions,which induced oxidation on EWP with different degrees(0,0.04,0.2,1.0 and 5.0 mM AAPH).Carbonyl and dityrosine contents were determined to verify oxidation degree.The size and morphology of EWP aggregates were reflected by atomic force microscope images,particle size distribution and viscosity,which contributed to investigate effect of lipid oxidation on EWP aggregation behavior.Additionally,surface properties were probed by surface hydrophobicity(H₀)and zeta potential.The results indicated that EWP treated with 0.2 mM AAPH and 5 mM AAPH acquired better foaming ability and foaming stability,respectively.This might result from the exposure enhancement of surface hydrophobic groups and negative charges induced by oxidation,which would accelerate protein adsorption to the air-water interface and strengthen protein-protein interactions and promote soluble aggregates formation.Thereby,the results above improved the apparent viscosity of the protein,which could promote the formation of thicker viscoelastic film to encapsulate gas,and meanwhile the increase of surface charge content helped to inhibit the coalescence and liquid drainage between the bubbles.(2)The structure of EWP was characterized by fluorescence spectroscopy,sulfhydryl(-SH)group content and fourier transform infrared spectroscopy.The results indicated that ROO·induced the unfolding of EWP,decreased the content of disulfide bonds and increased the flexibility of the protein.In addition,oxidation promoted the transformation of the ordered?-helix to the random coil and?-sheet.Furthermore,oxidation sites were determined by HPLC-MS/MS.5,10 and 19oxidation sites(in ovalbumin,ovotransferrin and lysozyme)were detected in EWP samples treated with 0 mM AAPH(control),0.2 mM AAPH(the highest foaming capacity)and 5 mM AAPH(the highest foaming stability),respectively.The results above suggested the oxidation sites in ovotransferrin and lysozyme might be related to the foaming properties of EWP.(3)Actually,ROO·was simulated lipid oxidation with limitations,taking long time.In contrast,the hydroxyl radical(·OH)decomposed by hydrogen peroxide(H₂O₂)can simulate the general oxidation state,and the preparation rate of oxidized protein is fast and the time is short,which is suitable for production and practice.Therefore,in the fourth and fifth chapters of this paper,the Fenton reaction model formed by hydrogen peroxide(H₂O₂)was used to generate·OH,simulating general oxidation state to induce oxidation on EWP.The final concentration of H₂O₂was 0,1,5,10 and 20 mM.Furthermore,the effect of·OH-induced oxidation on EWP foaming properties was explored by structural properties,surface properties,surface tension and rheological indicators.The results showed that moderate oxidation(5 mM H₂O₂)promoted EWP to unfold,reduced disulfide bonds,and increased structural flexibility.The ability of protein diffusion and adsorption to the air-water interface increased with lower surface tension and more internal groups exposure,and then promoted rapid film formation.Besides,more stable interface with smaller bubbles were formed by excellent rheological properties,which slowed coalescence,disproportionation and liquid drainage of foam.The above results significantly improved the stability of EWP foam.(4)The effect of·OH-induced oxidation on in vitro gastric digestibility of EWP was investigated by digestion kinetic model and multi-scale structural characterization including free amino groups,secondary structure and particle size distribution.The mechanism of oxidation effect on EWP digestion properties was explored by structural transformation before and after digestion.The results revealed that the exposure of internal active groups and hydrolysis sites increased with EWP unfolding,which enhanced the accessibility of pepsin and the degree of hydrolysis of EWP.Then,more disordered and smaller particles were formed with more free amino acids release at the same time.The curves of digestion kinetics and related indicators indicated that the oxidation-induced by·OH enhanced the digestion rate and extent of EWP.