Assessment of Iron Bioavailability in Biofortified Bell Pepper Using a Mucin-Enhanced In Vitro Digestion/Caco-2 Cell Model

Iron deficiency is a major global health concern, primarily attributable to inadequate dietary intake and limited absorption of iron. Foliar fertilization of vegetables like bell pepper (Capsicum annuum) may increase their iron content. When especially rich in ascorbic acid, intestinal iron absorption might be improved even further. However, iron content in foods and/or bioaccessibility after in vitro digestion alone would be unreliable predictors of iron bioavailability. Consequently, it seems to be necessary to reconsider digestion models and bioavailability evaluation. The objective of this study was to establish a mucin-enhanced in vitro digestion model to assess the bioavailability of non-heme iron from food matrices in combination with the widely utilized Caco-2 model of intestinal iron absorption. Compared with Fe(III), incubation with 20 µM and 200 µM Fe(II) sulfate increased ferritin formation (normalized to total protein (TP)) in differentiated Caco-2 cells by 80% and 130%, respectively. Furthermore, no loss of cellular viability was observed across the tested Fe(II) and Fe(III) concentrations (20–2000 µM). Three in vitro digestion models (DIN, DIN-lite, and DIN+G), differing mainly in digestive enzyme content, were evaluated for iron bioaccessibility and bioavailability. Only DIN-lite and DIN+G were compatible with Caco-2 cells. Although DIN-lite yielded 25% higher bioaccessible iron than DIN+G, both models resulted in comparable ferritin formation in Caco-2 cells. The DIN+G/Caco-2 model was applied to bell pepper cultivars (‘Ferrari’, ‘Morbidelli’, and ‘Jack Miller’), treated with foliar Fe(II) sprays during cultivation, achieving up to 3.9-fold increased iron content. However, this increase did not translate into enhanced in vitro iron bioavailability in the bell pepper pericarp. Consistent with previous studies, these findings indicate that iron content and bioaccessibility alone are insufficient predictors of iron bioavailability in plant-based foods. At the same time, the mucin-enhanced DIN+G/Caco-2 model proved to be a suitable approach for investigating iron bioavailability in plants.