Interactions and mechanisms in iron-fortified skim milk: Physicochemical properties, calcium bioavailability, and cellular responses.

Iron fortification of skim milk offers a promising strategy to address global micronutrient deficiencies, however, its effects on the physicochemical properties, mineral distribution, and bioavailability of milk remains poorly understood. The present study provides a comprehensive analysis of the interactions and mechanisms involved in iron-fortified skim milk systems. Iron salts (ferrous chloride, iron(II) lactate, and iron(II) gluconate) induced a concentration-dependent pH reduction, with ferrous chloride causing the most significant acidification. Iron distribution varied by salt type, with iron(II) gluconate showing higher serum iron levels. Calcium redistribution revealed a 22-28 % aqueous-phase depletion due to competitive iron-phosphate binding. Particle size remained stable, while ζ-potential decreased, reflecting iron-induced charge neutralization. Viscosity increased with iron concentration, indicating enhanced casein cross-linking. In vitro digestion and cellular uptake studies demonstrated that skim milk protein hydrolysates (MH) improved calcium bioavailability by forming soluble peptide‑calcium complexes, upregulating calcium transporters (TRPV6 and Claudin-2) and mitigating iron-induced oxidative stress. MH also enhanced antioxidant activity, reducing ROS and lipid peroxidation while upregulating ferroportin (FPN1) via Nrf2 activation. These findings highlight skim milk as an effective carrier for iron fortification, enabling simultaneous calcium and iron supplementation while maintaining stability and bioavailability.