Simulated gastrointestinal digestion generates a peptide-rich macauba kernel hydrolysate that modulates cellular redox homeostasis in HepG2 cells

Macauba (Acrocomia aculeata), a native Brazilian palm, contains protein-rich fractions that remain underexplored as sources of bioactive peptides. During gastrointestinal digestion, food proteins can release peptide sequences capable of interacting with cellular pathways involved in oxidative balance and health-related responses. This study aimed to characterize peptides generated from macauba kernel protein isolate after simulated gastrointestinal digestion (HD) and evaluate their effects on cellular oxidative responses in HepG2 cells. Macauba kernel protein isolate obtained from defatted kernel flour was subjected to simulated gastrointestinal digestion using pepsin and pancreatin. The resulting hydrolysate was characterized by SDS-PAGE, nanoLC-ESI-MS/MS, and bioinformatic prediction of peptide bioactivity. Antioxidant capacity was determined by the ABTS radical scavenging assay (1%, w/v). Cell viability was evaluated in A549, HepG2, CCD-18Co, and HCT-8 cell lines to establish non-cytotoxic concentrations prior to intracellular reactive oxygen species (ROS) determination in HepG2 cells using the DCFH-DA fluorescent probe under basal conditions and following H₂O₂-induced oxidative stress. SDS-PAGE revealed peptide fractions predominantly below 15 kDa, indicating extensive proteolysis. NanoLC-ESI-MS/MS identified peptides ranging from 571 and 2391 Da (6–22 amino acids) with high sequencing confidence (10lgP > 20 and confident amino acid probabilities predominantly at 100%). Approximately half (51.1%) of the identified peptide sequences were predicted to possess redox-related bioactivity, supporting the functional potential of the hydrolysate. Consistent with these findings, HD showed strong ABTS radical scavenging activity (84.67 ± 0.09% inhibition at 1%, w/v). Cell viability remained above 75% in all evaluated cell lines, with IC₅₀ values exceeding 3200 µg/mL, confirming low cytotoxicity. Although HD slightly increased intracellular ROS under basal conditions at the highest concentration evaluated, pretreatment significantly attenuated H₂O₂-induced ROS accumulation at all tested concentrations. Treatment with 500 µg/mL restored intracellular ROS to levels statistically indistinguishable from untreated cells, indicating effective modulation of the oxidative response under stress conditions. Together, peptide characterization, chemical antioxidant capacity, cellular compatibility, and intracellular redox modulation support the biological potential of the simulated gastrointestinal hydrolysate. These findings support further investigation of macauba-derived protein hydrolysates as food-derived bioactives and encourage future studies to elucidate the molecular mechanisms underlying their effects on cellular oxidative stress.

Acknowledgments: CAPES. S.Oleum.