Digestibility of peanut and hazelnut allergens investigated by a simple invitro procedure

Stability under digestion is thought to be an important prerequisite determ ining the allergenicity of food proteins. To test this hypothesis, two impo rtant allergenic plant-derived foods were selected for this investigation. Protein extracts from roasted peanuts and unprocessed (native) hazelnuts we re digested by a static, two-step in vitro procedure with commercial enzyme tablets containing peptic and pancreatic enzymes, respectively. The food e xtracts were subjected to gastric digestion for 2 h followed by a 45-min tr eatment under duodenal conditions. Undigested control samples and the two d igests were investigated by analytical sodium dodecyl sulphate-polyacrylami de gel electrophoresis (SDS-PAGE), by SDS-PAGE immunoblotting, by an enzyme allergosorbent test (EAST) with human IgE, and by a rat basophil leukaemia (RBL) cell mediator release assay that depends on specific IgE raised in m ice. Peanut proteins appeared to be more stable under digestion than hazeln ut proteins, as shown by electrophoresis. These results were also underline d by immunblot data. The gastric digest from peanut contained various prote in fragments that were detected by antibodies from a peanut-specific rabbit antiserum and by IgE from patients allergic to peanuts. These immunoblot r eactivities decreased strongly after subsequent pancreatic digestion. In th e gastric digest from hazelnuts, a rabbit antiserum with a broad reactivity against native hazelnut proteins exclusively recognized small protein frag ments of <15 kDa. This serum showed no binding to blots of the pancreatic d igest. Sera from hazelnut-allergic patients presented IgE reactivities to a n 18-kDa major allergen with homology to major tree-pollen allergens, to a minor allergen of 12 kDa, and to multiple bands >30 kDa in native hazelnut extract. No binding was observed with these sera on blot strips of the two digests prepared from hazelnut extract. Under the native conditions of EAST , both digests from peanuts strongly reacted with human IgE. Their IgE bind ing capacity persisted at a level of about 50% when compared to undigested peanut. In the case of hazelnuts the IgE reactivity of the untreated sample s was reduced to <10% by both gastric and combined gastric/duodenal digesti on for a serum pool prepared from four patients and sera from three additio nal participants. By contrast, a constantly high immunoreactivity of the ha zelnut digests was detected with serum from one patient. The results of the EAST were confirmed by dose-related mediator release experiments performed with RBL cells passively sensitized with allergen-specific murine IgE. As a whole, our results indicated that the EAST and the RBL cell assay are sup erior-to immunoblotting for the immunologic testing of digests. In accordan ce with clinical observations, the allergenicity of peanut proteins was ver y persistent during digestion, whereas the native birch-pollen-related haze lnut allergens appeared to be relatively labile under identical conditions.