Effects Of Sugar Group On Hen-Egg Ovomucoid Allergenicity Alleviation By Protease Digestion And Ultrahigh Pressure Treatments

Up to date,hen-egg ovomucoid（HOVM）is the strongest allergenic protein among four main allergen proteins from egg-white,and its strong allergenicity may be attributed to high content of sugar group,which make HOVM to owe high structural stability,anti-heat ability,and anti-digestion from preteases,however,research on the relationship between sugar group and HOVM allergenicity is so limited.In this study,phenolsulfuric method and ninhydrin method was applied to analyze total sugar content and proteolysis degree,respectively,circular dichroism spectroscopy and fluorescence spectroscopy was used to reveal the the secondary structure content surface hydrophobicity of protein,respectively;and indirect enzyme-linked immunosorbent assay（ELISA）to characterize changes in allergenicity by analyzing the ability of allergen proteins to bind to IgG.First,effects of heat treatment on the variation of HOVM’s structure and its binding ability to IgG were explored;and then four specific glycosidases（such asβ-galactosidase,β-mannosidase,β-N-acetylglucosaminidase and Peptide-N-glycosidase F）were utilized to cause different degree of depleting sugar group from HOVM and deglycosylated hen-egg ovomucoid（dHOVM）with different sugar group content were obtained,and allergenicity or structural variation of dHOVM,which were further digested by proteases or treated by ultrahigh-pressure treatment,were investigated.And the effects of sugar group content on its structure（including secondary structure and surface hydrophobicity）and allergenicity were analyzed.Results are listed as following.Firstly,heat treatment can affect the surface hydrophobicity index of HOVM and its binding ability to IgG:with the heat treatment strength（temperature and time）increasing the binding capacity of HOVM to IgG was decreased.Especially at 90°C and 100°C,the binding capacity of HOVM to IgG decreased significantly and reached the lowest value when heated for 110 min;as the heat treatment strength increases,the surface hydrophobicity index showed a tendency to rise first and then decrease.Secondly,Sugar-based content affects HOVM structure under ultrahigh pressure（UPT）,which affects its ability to bind to IgG.When the sugar group content decreased,the content ofβ-sheet also decreased.When pressure of UPT arranged from 100 to 400 MPa,the surface hydrophobicity index（S₀）of HOVM and its binding ability to IgG shared the same trend,which indicates that the alteration of tertiary structure of HOVM affects its binding ability to IgG.When pressure of UPT arranged from 500 to 800 MPa,the sugar group content has little effect on the secondary structure variation,and there is no connection between hydrophobicity index S₀ and its binding ability to IgG,which indicated that HOVM’s allergenicity variation is no longer directly related to the tertiary structure in this treating pressure range.Moreover,the sugar group content can affect pretease-catalyzed hydrolysis degree of HOVM,which further affects HOVM’s binding ability to IgG:with the sugar group content decreasing,the hydrolysis degree of HOVM by three proteases（trypsin,papain,bromelain）were increased.The extent（from the lowest to the highest）of dHOVM’s hydrolysis by these three proteases were listed as following:trypsin（hydrolysis degree being3.78%-5.09%）,papain（6.63%-19.40%）and bromelain（7.36%-24.91%）.The digestion products’（these dHOVMs degested by protease）binding ability to IgG changed:binding ability of reaction products（gotten by trypsin-digestion,papain-digestion and bromelain-digestion）to IgG decreased by 1.8%-29.61%,37.35%-46.73%,and41.89%-47.06%,repectively.Afterβ-galactosidase deglycosylation（HOVM’s sugar group content is 15.21%）,dHOVM hydrolysate（gotten by bromelain digestion）binding ability to IgG is the weakest one.