Optimization of enzymatic hydrolysis for obtaining antioxidant hydrolysates from pumpkin seed protein: Improvement of the physicochemical, structural and functional properties
Mehvish Habib, Sakshi Singh, Entesar Hanan, Kulsum Jan, Khalid Bashir
Abstract
Plant-derived proteins are increasingly recognized as sustainable substitutes for animal-based proteins; however, their broader application in foods and nutraceuticals is often constrained by limited functional and bioactive properties. The present study aimed to optimize the enzymatic hydrolysis of pumpkin seed protein isolate (PSPI) to generate bioactive pumpkin seed protein hydrolysate (PSPH) with superior antioxidant activity and improved functional characteristics. Optimization was performed using Response Surface Methodology (RSM) based on a Central Composite Design (CCD) to evaluate the influence of enzyme concentration (1–3%) and hydrolysis time (30–90 min) on antioxidant capacity, determined through DPPH and FRAP assays. The optimal conditions—2.5% enzyme concentration and 60 min hydrolysis time—produced PSPH with significantly enhanced antioxidant activity and a high degree of hydrolysis (17.89%). Hydrolysis resulted in substantial reductions in particle size (436 nm), polydispersity index (0.69), turbidity (0.21), and zeta potential (–28.03 mV) compared to the native PSPI. Structural analyses confirmed protein degradation and conformational alterations, as indicated by SDS-PAGE (appearance of low-molecular-weight peptides), FTIR (amide I band shifts), and NMR (release of free amino acids). XRD analysis revealed decreased crystallinity, SEM micrographs showed a porous surface morphology, and DSC analysis indicated an increased denaturation temperature (99.5°C), reflecting improved thermal stability. This work presents a cost-effective, eco-friendly processing approach for upgrading underutilized pumpkin seed proteins into functional hydrolysates with enhanced bioactivity and physicochemical properties. The combination of multi-technique structural characterization (FTIR, XRD, DSC, SEM, NMR) with RSM-guided optimization underscores both the methodological innovation and industrial applicability of the study. The developed PSPH demonstrates strong potential as a natural antioxidant for incorporation into functional foods, dietary supplements, and clean-label nutraceutical formulations.