Fabrication Mechanism And Structure Characterization Of Polyphenols And Gliadin Complexes

Gliadin is a type of allergenic protein that is beneficial to human health and harms humans.Due to its hydrophilic and lipophilic properties,gliadin is prone to non-covalent or covalent binding reactions with polyphenolic compounds during food processing,resulting in turbidity in beverages such as beer and fruit juices,affecting product perception and nutritional quality.In humans,proline residues in gliadin bind to intestinal mucosal epithelial cells,which cannot be completely digested,resulting in the residues of proteolytic peptides that ultimately trigger celiac disease and other allergic reactions,while the interaction of gliadin with specific polyphenol compounds can also reduce the immunogenicity and allergenicity of gliadin,which is related to the structure of polyphenolic compounds.Therefore,to investigate the non-covalent or covalent interaction and binding mechanism between gliadin and polyphenol compounds,to explore the binding ability of polyphenol structure and embedding the allergenic valine residues on gliadin,there is an important theoretical significance for the development of new multifunctional food materials and the reduction of the sensitizing properties of gliadin.The main research contents and conclusions of this article as followed:（1）Six polyphenolic compounds were studied in the pH 7.4 70%ethanol（V/V）simulation system:quercetin（Q）,isoquercetin（Q-3-β-D-G）,rutin（R）,interaction mechanism of ferulic acid（FA）,caffeic acid（CA）,p-coumaric acid（PCA）and gliadin（G）blend system.Soluble complexes can be formed between polyphenols and gliadin,when the molar ratio of gliadin with six polyphenol compounds is less than 1:8.The particle size of G+P complex can be significantly reduced via quercetin and its glycosylated derivatives Q,Q-3-β-D-G,R and phenolic compounds FA.（2）Basic information on the binding of Q,Q-3-β-D-G,R,FA,CA,PCA with G in the pH 7.4 70%ethanol（V/V）simulation system were obtained through fluorescence spectroscopy.All six polyphenols have strong fluorescence quenching effect on the gliadins,and quenching mode is the static quenching of the ground state complexes.Tryptophan is involved in the binding of polyphenols to gliadin,and the number of binding sites is 1,and the order of binding ability was G+R>G+PCA>G+Q-3-β-D-G>G+CA>G+Q>G+FA.（3）The distance of tryptophan in the combination of six polyphenols with gliadin were calculated in accordance with Forster’s non-radiative energy transfer theory,and G+Q,G+Q-3-β-D-G,G+R,G+FA,G+CA,G+PCA distances were 2.378 nm,2.189nm,2.418 nm,2.154 nm,2.146 nm,and 1.962 nm,respectively.The distances to tryptophan from the reaction between the six polyphenols and gliadin were:G+R>G+Q>G+Q-3-β-D-G>G+FA>G+CA>G+PCA.（4）In accordance with the Van’t Hoff equation,Q,R,PCA were the exothermic reactions of△H<0,△S<0,△G<0 in the binding reaction of six polyphenols to gliadin.In the thermal reaction,hydrogen bonding and van der Waals forces were the main driving forces;G+Q-3-β-D-G,FA,CA were exothermic reactions with△H<0,△S>0,△G<0,in which,hydrogen bonding and hydrophobic interactions were the main modes.The total order of△G for binding of six polyphenols to gliadin is:G+PCA>G+R>G+Q-3-β-D-G>G+CA>G+Q>G+FA.（5）G+Q,G+Q-3-β-D-G,G+R,G+FA,G+CA,G+PCA complexes were prepared via anti-solvent method under pH 7.4 conditions.Among them,quercetin and glycosylated derivatives have stronger binding activity than phenolic compounds with gliadin,and the final formed complex particle size is G+PCA<G+FA<G+R<G+Q<G+Q-3-β-DG<G+CA.（6）Six complexes were characterized through FTIR,UV-vis,SERS,CD,and FL.Results showed that the secondary structure of gliadin could be changed by Q,Q-3-β-D-G,R,FA,CA,PCA.The change is affected by the structure of six polyphenols,of which the glycosyl group of quercetin glycosylated derivatives Q-3-β-D-G and R contribute to the binding of G,while the phenolic acids The R₃hydroxyl group in CA inhibits the binding to G.In addition,Q-3-β-D-G and R promote the transition of G random coil structure toα-helix andβ-turn structure,resulting in G-sensitized proline residues being embedded,and CA decreasingβ-turn Structure,increased beta-sheet and glutamine structure,resulting in G-sensitized proline residue exposure,tryptophan and tyrosine are involved in the combination of polyphenols with gliadin.（7）The covalent complexes of G-R were prepared by the alkali treatment method and the conformational changes of G-R and non-covalent complexes prepared by anti-solvent method were analyzed and compared by means of FTIR,UV-vis,SERS,CD,and FL,and G-R covalent compound was found.The material has the characteristics of small particle size,strong stability,and regular shape of the sphere.Intermolecular formation of covalent bonds with G through C₂-O-C,C₃-O-C ether linkages in R and C-O-C ether linkages on pyranose,tyrosine,tryptophan,and amide I bands in G molecules(1600 cm^-11700 cm^-1)and amide III bands(1230 cm^-11340cm^-1)C=O,N-H,and C-N subunits all participate in the role of non-covalent interactions such as hydrogen bonding and hydrophobic interaction.