Preparation Of Hypoallergenic Ovalbumin By Glycation And Its Modification Mechanism

Hen Egg,as a common cooking ingredient in east and west eating cultures,is one of the best nutritional sources in foods.Egg is cheap,but abounding with a variety of nutrients(e.g.preotein,fat,lecithin and vitamins)beneficial to people’s health.However,it is also a worldwidely common allergenic food.In recent years,hen egg has been widely used as processing material in food industry,owning to its excellent functional properties,which has brought increasingly allergic potential to egg allergy(EA)patients.With the global EA rate being increased year by year,egg allergy has severely constricted the application of egg products and the expansion of their nutritional value.Therefore,it is urgent to find an effective protocol for desensitization.Maillard reaction(MR)is a common non-enzymatic browning in the processing,storage and transportation of high-protein foods.It has a wide range of applications in the food industry.Glycation occurs usually in the early stages of MR,decorating the primary sequence of proteins;moreover,it may destroy certain allergic linear epitopes by modifying amino acid residues containing free amino groups in the protein.At the same time,glycation could mediate intrinsic disorder in the 3D structure changes of proteins,destroying the integrity of some conformational epitopes.In addition,glycation can be carried out spontaneously under mild reaction conditions(>25℃)without introducing other chemical components,and is relatively safe and energy-saving desensitization methods for food processing.However,reports involving insight into mechanism of glycation desensitization at the molecular level are still limited.In this work,ovalbumin(OVA),the major protein allergen in eggs,was used as the research object and incubated under mild conditions(37℃)with D-ribose,D-xylose,D-galactose,D-glucose and D-fructose to change its potential allergenicity.SDS-PAGE was used to investigate the effect of monose glycation on the molecular weight of OVA;the glycation extent of different glycated samples were assessed with OPA method in combination with HRLC-HCD-MS;circular dichromatography was used to analyze the secondary structure changes in glycated OVA;UV absorption and fluorescence spectroscopy were used to study the effect of glycation on the conformation of OVA;An infrared spectroscopy was implied to study changes in molecular microenvironment of glycated OVA;ANS method was used to study the effect of glycation on the key molecular force in antigen-antibody interaction;the indirect competitive ELISA was used to detect the changes in IgG/IgE binding ability of glycated OVA,and finally monose with best desensitization activity was screened out for glycation optimization.The following conclusions are reached:(1)Monose glycation could increase the molecular weight of OVA,modifying its primary sequence and simultaneously inducing formation of OVA multimers.Moreover,it also resulted in a significant decrease in the free amino content of OVA.The order of monose glycation activity is as follows:D-fructose<D-Glucose<D-galactose<D-xylose≈D-ribose.(2)Monose glycation could gradually expand the tightly embedded globular structure of OVA,causing unfolding of the conformation and changes in the ultraviolet absorption spectrum and fluorescence spectrum,among which D-galactose,D-ribose and D-Xylose showed a stronger effect on the conformation of OVA.(3)Monose glycation could affect the secondary structure of OVA,and significantly increase the β-sheet content and molecular dynamics stability in OVA.This indicated that the overall structure of OVA is more orderly and stable with the unfolding of the conformation induced by glycation..(4)Monose glycation could significantly reduce the surface hydrophobicity of OVA and its ability to bind to IgG and IgE;the trend of IgE binding ability of OVA glycosylated samples is similar to the trend of its IgG binding ability.Among them,OVA-Rib exhibited the lowest surface hydrophobicity and the lowest antibody-binding capacity.(5)There were 15,17,15,11,and 12 glycation sites were determined in OVA samples glycated with D-ribose,D-xylose,D-galactose,D-glucose and D-fructose,respectively.Among them,6,6,6,4,and 4 glycosylation sites were found to be located on the large β-sheet coiled chains,which indicating that glycation may cause strong structural changes in OVA;moreover,different glycation samples showed different glycation extent.(6)The configuration,carbon chain length,and carbonyl type of monose had significant effects on its glycation activity.The difference in hydroxyl configuration at C-4 in the monose carbon chain may have significant effects on the glycation activity of the monose.This may cause differences in the degree of reaction with OVA,further affecting the ability of OVA to bind with antibodies.(7)Glycation may reduce the IgE binding capacity of OVA under certain reaction conditions,thereby inhibiting its potential allergenicity.However,the degree of inhibition obtained is limited.(8)The optimal glycosylation conditions for OVA desensitization were obtained through single factor experiments and orthogonal experiments:the sugar/protein addition ratio was 2:1,the initial pH of the mixture was 10,the reaction temperature was 50℃ and the reaction time was 2.4 h.The three repeated verifications were performed under this condition setting,and the OD450 value of native OVA and glycated OVA was 0.358±0.007 and 0.622±0.039,respectively,thus the relative inhibition rate was 73.74%.