Protein-Polyphenol Complexation to Reduce Food Protein Allergenicit

Food allergy is a worldwide health concern. Peanuts are among the "big eight" foods that can cause food allergy, and has been cited as one of the most serious and prevalent. Peanut allergy is an immunoglobulin (Ig) E-mediated hypersensitivity disorder towards certain proteins (allergens) in peanut. Upon initial exposure to peanut allergens, the immune system of a susceptible individual produces peanut-specific IgE that bind to IgE receptors on mast cell and basophil surfaces (sensitization). Upon re-exposure, allergens bind and crosslink with mast and basophil cell-bound peanut-specific IgE and trigger those cells to degranulate and release inflammatory mediators (allergic reaction). Several promising intervention strategies to reduce peanut allergenicity or modulate human immune response have been investigated, but none of these techniques have led to a cure.;Plant polyphenols are known for their natural ability to bind proteins. In previous studies, we have described a new approach to bind fruit juice- and herbal extract-derived polyphenols to peanut proteins (in the form of peanut flour), and demonstrated that some of the resulting protein-polyphenol aggregate particles were hypoallergenic in vitro, ex vivo and in vivo. In this work, we aimed to better understand the molecular mechanisms by which such peanut protein-polyphenol aggregate particles are rendered less allergenic.;In the first study, we created peanut protein-polyphenol aggregate particles containing different concentrations of polyphenols derived from cranberry (Vaccinium macrocarpon Ait.) and lowbush blueberry ( Vaccinium angustifolium Ait.) pomaces. Immunoblotting revealed that peanut protein-bound cranberry and blueberry polyphenols significantly decreased IgE binding to peanut proteins in a polyphenol concentration-dependent manner. In an allergen model system, anti DNP-sensitized RBL-2H3 mast cells challenged with DNPBSA (antigen) and ionomycin in the presence of aggregate particles showed a significant reduction in histamine and beta-hexosaminidase release, also in a polyphenol concentrationdependent fashion. We demonstrated that polyphenolic compounds bound to peanut proteins (reversibly and irreversibly). Quercetin, -- in aglycone or glycosidic form- was the main phytochemical identified to be covalently bound to peanut proteins (determined by alkaline hydrolysis and subsequent HPLC analysis). The modification of peanut proteins with cranberry or blueberry polyphenols led to the formation of peanut protein-polyphenol aggregate particles with significantly reduced allergenic potential.;One of the main peanut allergens bound by blueberry and cranberry polyphenols was Ara h 2. While our previous investigations have shown the potential of using polyphenol binding as a way to mediate allergenicity, the activities of individual polyphenols was not investigated. In the second study, we used in silico molecular modeling techniques to prioritize polyphenols from cranberry and lowbush blueberry predicted to bind strongly to Ara h 2 in general, and to bind to specific IgE epitopes. Predicted binders delphinidin-3- glucoside, cyanidin-3-glucoside, procyanidin C1, and chlorogenic acid, as well as benzoic acid, a low predicted binder, were selected. UV-Vis spectroscopy suggested that procyanidin C1 and chlorogenic acid did interact with Ara h 2 and circular dichroism tests further showed that those compounds also induced changes in the Ara h 2 secondary structure upon binding. Previous studies using ATR-FTIR (Attenuated Total Reflectance-Fourier Transform Infrared spectroscopy) had suggested, but did not confirm changes in secondary protein structure when peanut proteins were bound to polyphenols. Immunoblotting showed that procyanidin C1, chlorogenic acid, and benzoic acid binding to Ara h 2 significantly inhibited IgE epitope binding capacity.;Collectively, these findings suggest that different classes of polyphenols can both non-covalently and covalently bind to peanut allergens and consequently inhibit epitope binding by epitope-specific IgE, presenting a potential strategy for alleviating peanut allergenicity symptoms in susceptible individuals.