Enzymatic Production, Fractionation, and Multi-Assay Bioactivity Profiling of Dual-Function Antimicrobial and Antioxidant Peptides from Moringa oleifera Leaf Protein

Abstract

The growing prevalence of antimicrobial resistance, coupled with consumer demand for clean-label ingredients, has driven research into plant-derived bioactive peptides as natural food preservatives. This study reports the systematic optimization of enzymatic hydrolysis of Moringa oleifera leaf protein using three food-grade proteases (Alcalase, trypsin, and papain), followed by molecular weight-based fractionation, multi-assay bioactivity profiling, and safety evaluation including hemolytic activity. Alcalase at pH 9.0 and 53 °C yielded the highest degree of hydrolysis (59.9 ± 1.2%). Sequential ultrafiltration through 10, 3, and 1 kDa membranes produced defined peptide pools; Tricine-SDS-PAGE confirmed progressive molecular weight reduction, and ATR-FTIR verified peptide bond integrity. Bioactivity assays revealed a clear size–activity relationship: the 1–3 kDa fraction (F3) exhibited maximal antimicrobial activity against Staphylococcus aureus (70.4% inhibition; MIC 62.5 µg/mL) and Escherichia coli (61.9%; MIC 125 µg/mL), alongside multi-assay validated antioxidant capacity (DPPH IC50 = 0.62 mg/mL; ABTS IC50 = 0.89 mg/mL; FRAP = 142.5 µmol TE/g). Hemolysis assays demonstrated low erythrocyte lysis at bioactive concentrations (5.2% at the MIC against S. aureus), yielding a Hemolytic Index of 8.0 (HC50 ≈ 500 µg/mL). MTT cytotoxicity assays on MCF-7 and HepG2 cells confirmed acceptable safety (Selectivity Index 5.6). Exploratory AlphaFold2 modeling of representative peptide sequences suggested conformational flexibility and potential amphipathic character consistent with the observed bioactivity pattern. These findings establish M. oleifera leaf-derived peptide fractions as promising dual-function natural preservatives for food applications.