Assessing residual allergenicity in heat-treated red lentil flours through simulated digestion, in vitro and in silico assays and peptidome approach.

The increasing use of different legume flours (lentils, chickpeas, lupin, soybean) in food products, driven by their nutritional and technological properties, raises concerns for sensitised and allergic individuals. This study investigated the effect of thermal processing on the digestibility and immunoglobulin E (IgE)-binding capacity of red lentil proteins, using simulated gastrointestinal digestion models based on the INFOGEST 2.0 protocol. The protein profiles and the IgE-binding capacity before and after digestion were analysed using SDS-PAGE and immunoblotting with sera from lentil-allergic individuals. Both standard pancreatin digestion and a simplified trypsin/chymotrypsin model were applied. Heat treatment increased the susceptibility of proteins to pepsin during the gastric phase, particularly for vicilin subunits. During the intestinal phase, pancreatin digestion resulted in extensive protein degradation and induced non-specific IgE-reactivity in immunoblotting. In contrast, the trypsin/chymotrypsin model produced clearer IgE-binding profiles. High-resolution mass spectrometry revealed that thermal processing enhanced peptide release and increased proteolytic susceptibility, progressively reducing the number and length of digestion-resistant peptides. In silico epitope mapping showed a reduction in IgE-related epitopes from vicilin Len c 1 upon heating and digestion, particularly when using pancreatin. Overall, these results demonstrate that thermal processing improves lentil protein digestibility and reduces residual allergenic epitopes following digestion. Additionally, using a simplified digestion model improves the reliability of in vitro IgE-binding assessments. Together, these findings emphasise the importance of combining digestion models with immunochemical and proteomic tools for a comprehensive evaluation of the allergenic potential of processed legumes, thereby guiding the development of safer, hypoallergenic food ingredients.