| In view of the limitations of the existing control technologies for cow milk allergy（CMA）,dietary polyphenols such as anthocyanins and flavonoids can interact with allergen proteins to change protein conformational structure and allergenicity.Given this,this study carried out the construction and mechanism study of the hypoallergenic milk protein interaction system based on the polyphenol-protein interaction.Through the construction of a characteristic polyphenol-milk typical allergen hypoallergenic system,from the perspectives of molecular interactome formation,allergenicity evaluation,and structure-activity correlation changes,the molecular and molecular mechanisms of polyphenol-protein interaction to reduce milk allergens were systematically elucidated.The study aims to provide a reference for the application of the hypoallergenic system of dairy products.The main contents and results of the research are as follows:Firstly,the interaction of C3G and two typical milk allergens（α-CN andβ-LG）under covalent and non-covalent conditions was studied.The fluorescence quenching data showed that C3G quenched the fluorescence of both proteins through a static process under non-covalent conditions and revealed the dominant interaction force in the non-covalent system.For covalent complexes,C3G fluoresced the proteins,the intensity decreases andλmax is red-shifted.CD spectroscopy indicated that the addition of C3G induced structural unfolding of the protein through transitions between random coils and ordered secondary structural components.In addition,covalent protein-C3G complexes showed lower levels of Ig E-binding compared to native controls.Considering that non-covalent binding is more common in nature and daily life,and the adverse effects of this interaction on the properties of milk protein are relatively low,and its significant effects on the structure and physical and chemical properties of milk protein suggest that other polyphenols may affect the conformation of milk protein similarly,thus affecting its antigenic properties.At the same time,considering thatβ-LG is the Bos d 5 allergen named by the WHO/IUIS Allergen Nomenclature Subcommittee,another study has shown that 82%of cow’s milk allergy patients are allergic to cow’s milkβ-LG[1,2].Therefore,the non-covalent interaction system ofβ-LG and polyphenols was selected as the further research object to further compare the allergenicity changes of differentβ-LG-plant extracts complexes.The Ig E-and Ig G-binding abilities of the complexes were determined by ELISA,and LRE,BE,SBE,BBE,and BSE were confirmed as characteristic hypoallergenic polyphenols.The physicochemical properties and structural characteristics of different complexes were characterized by the multispectral method,and it was found that the combination of characteristic polyphenol extracts andβ-LG would increase the particle size distribution and stability of the system.The hydrophilic group of polyphenol extracts and binding with the hydrophobic group on the surface of the protein lead to the decrease of the H0 and the increase of the soluble protein content.After binding with the characteristic polyphenol extract,the secondary structure ofβ-LG becomes more compact（the proportion ofα-helix increased and the proportion of random coils decreased）,resulting in the masking of the original epitopes ofβ-LG,thus showing a decrease in the in vitro allergenicity ofβ-LG.It was elucidated that specific polyphenolic extracts could alter the in vitro allergenicity by altering the structural properties ofβ-LG.Finally,LC-MS/MS technology was used to identify the dominant ligand polyphenols in the low-allergenicity complexes.Starting from the molecular structures of different polyphenols,dynamic light scattering,fluorescence spectroscopy,and CD spectroscopy were used to analyze the physicochemical and structural properties of various complexes.The study found that,polyphenols with large MW and polyhydroxy structure（represented by EGCG,MC,and ET）tend to have higher affinity and binding efficiency toβ-LG,and thus could reduce the H0 ofβ-LG and increase its soluble protein content.Using indirect ELISA and LAD2 mast cell degranulation assays,it was demonstrated that the dominant polyphenols have the potential to inhibitβ-LG Ig G-binding capacity andβ-LG-induced mast cellβ-hexosaminidase release rate.An important factor affecting the Ig G-binding ability ofβ-LG is the change ofα-helix andβ-sheets ratio in the secondary structure.Whether theβ-LG structure becomes more compact,or the unhelix and refolding occurs,it may lead to the covering of the Ig G-binding site or the change of conformational epitopes.β-hexosaminase release rate may be correlated withβ-turns and random coil changes.It was verified that the complex of polyphenols andβ-LG induced structural changes,which led to the covering or destruction of the epitope,and finally led to the change ofβ-LG allergenicity.In conclusion,this study systematically explored the physicochemical properties,structural properties,and in vitro allergenicity changes of the polyphenol-milk protein complex system,and clarified“structure-activity”relationship of allergenicity between complex.Provide basis and support for the control of dairy allergy risk and the formation of a hypoallergenic/allergic food processing system. |
