Baking results in impaired detection of clinically relevant food allergens.

Quantifying food allergens in complex matrices is hindered by thermal processing, particularly in baked products used for oral food challenges (OFCs), where limited data exists on effects on clinically relevant allergens. To address this, an incurred biscuit was prepared, and 12 allergens were quantified using a multiplex-immunoassay array or ELISA. Biscuit dough was incurred with allergenic materials from egg, milk, peanut, almond, cashew, hazelnut, walnut, sesame, and soy at 1,000 ppm total protein. Dough portions (40 g) were baked at 185°C or 210°C for 15 min. Raw dough, whole biscuits, and biscuit middle and edge fractions were extracted using an optimised buffer at 60°C for 15 min. Baking reduced measurable allergen levels compared with raw dough, with the greatest reductions observed for Gal d 1, Gal d 2 (egg) and Bos d 5 (milk) (86%-98%). Ara h 3 (peanut), Jug r 1 (walnut), and Bos d 11 (milk) were least affected, showing 22%-26% reductions. All allergens showed significant intra-biscuit variation, with lower levels at the biscuit edge than the middle. Gal d 1, Gal d 2, and Bos d 5 were most affected, with up to 450-fold higher levels measured in the middle. These findings show heat processing differentially affects allergen quantification within complex matrices. The mechanism of reduction may be attributed to native conformational epitope loss, epitope masking or cleavage, or heat induced loss of solubility, including aggregation, which may impact