From By-Product to Bioactive: New Antioxidant and Bioavailable Peptides Derived from Milk Permeate Targeting the Nrf2/Keap1 Pathway in Intestinal Cell Models

This study investigates the antioxidant properties of several synthetic peptides derived from milk proteins previously identified in milk permeate, a by-product of the dairy industry. The aim of the research is to identify which peptides present in milk permeate are responsible for its antioxidant activity. A comprehensive experimental strategy was employed to evaluate their antioxidant potential, including in silico selection, in vitro free radical scavenging assays and cellular models using Caco-2 and HCT116 cell lines. The peptides were screened using a molecular docking approach for their potential interaction with the Kelch-like ECH-associated protein 1/nuclear factor erythroid 2-related factor 2 (Keap1/Nrf2) pathway, and eight out of twenty-eight were selected and synthesized for further analyses. In vitro, six of the selected peptides demonstrated significant direct antioxidant activity in the DPPH scavenging assay, and two in the ABTS scavenging test. In cellular environments, three peptides (LPAPELGPRQA, LPIIQKLEPQI and NGQVWEESLKRL) effectively protect cells from oxidative stress induced by tert-butyl hydroperoxide, reducing reactive oxygen species production and partially mitigating lipid peroxidation. Further investigation showed that two of them (LPAPELGPRQA and LPIIQKLEPQI) effectively induce the Keap1/Nrf2 pathway, as evidenced by a ∼1.5-fold increase in Nrf2 levels and overexpression of downstream proteins. Permeability studies revealed that these peptides can cross the intestinal monolayer (2–3% in 2 h), suggesting potential systemic effects. Overall, these findings highlight the multifunctional antioxidant properties of the investigated peptides and support their potential application as nutraceuticals or therapeutic agents for oxidative stress-related conditions.