Influence of peanut matrix on stability of allergens in gastric‐simulated digesta: 2S albumins are main contributors to the IgE reactivity of short digestion‐resistant peptides

I. Prodic, D. Stanic‐Vucinic, D. Apostolovic, J. Mihailovic, M. Radibratovic, J. Radosavljevic, L. Burazer, M. Milcic, K. Smiljanic, M. van Hage, T. Cirkovic Velickovic

Most food allergens sensitizing via the gastrointestinal tract are stable proteins that are resistant to pepsin digestion, in particular major peanut allergens, Ara h 2 and Ara h 6. Survival of their large fragments is essential for sensitizing capacity. However, the immunoreactive proteins/peptides to which the immune system of the gastrointestinal tract is exposed during digestion of peanut proteins is unknown. Particularly, the IgE-reactivity of short digestion-resistant peptides (<10 kDa) released by gastric digestion under standardized and physiologically relevant in vitro conditions has not been investigated. The aim of this study was to investigate and identify digestion products of major peanut allergens and in particular to examine IgE reactivity of short digestion-resistant peptides released by pepsin digestion of whole peanut grains. Two-dimension gel-based proteomics and shotgun peptidomics, immunoblotting with allergen-specific antibodies from peanut sensitised patients, enzyme-linked immunosorbent inhibition assay and ImmunoCAP tests, including far ultraviolet-circular dichroism spectroscopy were used to identify and characterize peanut digesta. Ara h 2 and Ara h 6 remained mostly intact, and short digestion-resistant peptides from Ara h 2 were more potent in inhibiting IgE binding than Ara h 1 and Ara 3. Ara h 1 and Ara h 3 exhibited sequential digestion into a series of digestion-resistant peptides with preserved allergenic capacity. A high number of identified short digestion-resistant peptides from Ara h 1, Ara h 2 and Ara h 3 were part of short continuous epitope sequences and possessed substantial allergenic potential. Peanut grain digestion by oral and gastric phase enzymes generates mixture of products, where the major peanut allergens remain intact and their digested peptides have preserved allergenic capacity highlighting their important roles in allergic reactions to peanut. This article is protected by copyright. All rights reserved.