Ca2+-mediated free radical grafting of whey protein with ferulic acid: Physicochemical characterization and antagonistic effects on lead toxicity.

Developing safe and effective dietary interventions to enhance lead (Pb) elimination and mitigate Pb-induced tissue damage is of considerable public health significance. This study prepared a calcium ion-mediated whey protein-ferulic acid (WP-FA) covalent complex via an ascorbic acid‑hydrogen peroxide radical-initiated system, aiming to improve FA stability and bioactivity and investigate its anti-Pb toxicity efficacy. Ca2+ significantly regulated the grafting rate and structural characteristics of the complex. Optimal WP-FA covalent complex with the highest grafting amount, smallest particle size, and best stability was obtained under 4 mmol/L Ca2+ supplementation. In vitro simulated digestion showed that the complex possesses satisfactory Pb-chelating capacity. Therefore, the optimized WP/FA-4 conjugate was chosen in vivo validations. Mice were exposed to Pb from day 1 to day 7, followed by a 14-day intervention period. The animals were euthanized on days 14 and 21 respectively for sampling. This covalent complex reduced Pb accumulation in blood, liver, kidneys, and brain, while mitigating Pb-induced weight loss, organ damage, and cognitive impairment. Mechanistically, these protective effects might be related to the alleviation of tissue oxidative stress and regulation of metal transporters expression by WP-FA. These findings highlight the important role that Ca2+ plays in augmenting the WP-FA covalently binding properties, providing theoretical support for modifying protein-polyphenol complexes and preventing food-derived Pb toxicity.