Cellular antioxidant activity and safety of gastrointestinal in vitro digests from gluten-free bread containing upcycled apple pomace and flaxseed

Apple pomace and flaxseed have antioxidant and anti-inflammatory potential, but evidence of their biological effects after incorporation into food matrices, in vitro digestion, and assessment in intestinal cell models relevant to celiac disease remains scarce. We evaluated the safety and bioactivity of digested samples from a gluten-free bread containing minimally processed apple pomace and local flaxseed, using Caco-2 cell models. Antioxidant and anti-inflammatory activities were assessed under basal and stimulated conditions, including celiac-like and bacterial inflammatory environment. Both the individual ingredients and the experimental bread exerted antioxidant effects at physiologically relevant concentrations. The bread digests reduced intracellular ROS under oxidative challenge (up to 65% at 0.5 h and 30% at 3 h; p <0.05) and in a recovery setting (20% at time 0 and 33% at 3 h; p<0.05 ), indicating antioxidant activity beyond prevention. In contrast, no significant anti-inflammatory effects were observed. However, digests from isolated apple pomace and flaxseed selectively modulated IFN-γ secretion. Under p31-43 model, with apple pomace and flaxseed digests (10 mg/dL) IFN- γ levels were reduced by 55% and 35% respectively, compared with the positive control. In the LPS model, treatment with apple pomace and flaxseed (10 mg/L) reduced IFN-γ levels by 90% and 81% respectively. Selected persistent organic pollutants were analysed in the digested samples with results below quantification levels. Overall, these findings provide a realistic assessment of the functional potential of agro-industrial byproducts when included in complex food matrices and subjected to digestion, supporting their use to improve nutritional quality of gluten-free foods.