Optimization of Moringa oleifera Lam. leaf hydrolysis for the release of bioactive di- and tripeptides with antioxidant and biostimulant potential

Bioactive peptides develop diverse functions and represent an underexplored biotechnological resource. Moringa oleifera Lam., due to its high protein content, is a promising raw material for generating these compounds. This study aimed to obtain di- and tripeptides-key molecules for rapid cellular absorption-by optimizing alkaline and enzymatic (Alcalase®) hydrolysis of dried M. oleifera leaves to maximize the release of small bioactive peptides. A full factorial response surface design was applied to evaluate two independent variables (M. oleifera mass and reagent concentration) and two response variables (protein content and degree of hydrolysis), using KOH, NaOH, and Alcalase®. The most promising hydrolysates were analyzed by liquid chromatography-tandem mass spectrometry for peptide identification, followed by DPPH assays to determine antioxidant activity and mineral analysis of the hydrolysis residues. A total of 66 dipeptides and 33 tripeptides were identified. Hydrolysis with 5% KOH and 21% M. oleifera yielded 4.54% total proteins, while the combination of 5% KOH and 11% Moringa produced the highest degree of hydrolysis (87.55%) and four tripeptides. Enzymatic hydrolysis with 10% Alcalase® and 11% M. oleifera resulted in a 63.41% degree of hydrolysis and the identification of 41 dipeptides and 10 tripeptides. These peptides demonstrated potential to inhibit ACE, dipeptidyl peptidase III and IV, renin, glycosidase, antioxidant action and the ability to chelate zinc, iron, and calcium. The solid residues generated during hydrolysis were rich in essential minerals. Application of the Alcalase® hydrolysate to Phaseolus vulgaris seedlings produced a 10% antioxidant response (compared to 0.19% in the control), supporting its potential as a biostimulant with antioxidant properties for vegetable crops.