HIGH-PRESSURE HOMOGENIZATION ENHANCES ANTIOXIDANT AND CYTOPROTECTIVE PROPERTIES OF PEQUI ALMOND PROTEIN PEPTIDES

The growing interest in plant proteins has stimulated the search for novel processing methods capable of overcoming the limitations of their industrial application. Among emerging approaches, non-thermal processes such as high-pressure homogenization (HPH) show potential to modify protein structures, enhance enzymatic accessibility, and promote bioactive peptides. In this context, the pequi almond (Caryocar brasiliense Camb.), a by-product of oil extraction, represents an underexplored source of proteins with functional properties. This study evaluated the effect of HPH at 100 MPa and 150 MPa on pequi almond protein (PAP) proteolysis and the release of bioactive peptides with antioxidant properties, as well as their protective effects against oxidative damage in RAW264.7 macrophages. PAP was obtained by alkaline extraction followed by isoelectric precipitation (pH 4.5). The dispersions (1% w/v in phosphate buffer, pH 7.5) were subjected to HPH in a single cycle, followed by enzymatic hydrolysis with Alcalase® (1%, 180 min, 55 °C). Proteolysis was assessed by quantifying trichloroacetic acid (TCA)-soluble proteins. Antioxidant activity of PAP peptides at 1000 µg/mL was evaluated using ABTS and FRAP assays. Cell viability was determined by the MTT assay after 24 h of treatment with hydrolysates, with or without H₂O₂ exposure for 3 h. Results demonstrated that PAP enzymolysis increased TCA-soluble proteins after 180 min for the 100 MPa and 150 MPa samples (35% and 53%, respectively) (p < 0.05), compared to control. Moreover, HPH significantly enhanced antioxidant peptides. In the ABTS assay, PAP peptides at 1000 µg/mL showed radical inhibition of 21% in control (0 MPa), 34% at 100 MPa, and 41% at 150 MPa (p < 0.05). Similarly, in FRAP assay, PAP peptides exhibited reducing capacity equivalent of 53 µM ferrous sulfate in control, increasing to 154 µM at 1000 µg/mL under both 100 and 150 MPa. Cell viability assays indicated no significant cytotoxic effects, with values consistently exceeding 80%. Under H₂O₂ exposure, PAP peptides exerted a protective effect, with ~9% increase in cell viability at 1000 µg/mL (150 MPa) compared to the untreated H2O2-exposed control. In conclusion, HPH is an effective strategy to enhance PAP bioactivity, improving both chemical antioxidant capacity and cytoprotective effects. These findings highlight its potential as a sustainable and functional ingredient for food and nutraceutical applications.