A Comparative Study on Novel-Assisted Extraction Techniques for Retrieving Protein from Moringa oleifera Seeds

<i>Moringa oleifera</i> seeds are rich in protein, yet their potential as plant-based protein in food remains underutilized. This study evaluated the extraction efficiency, composition, and techno-functional properties of moringa seed protein isolate (MSPI) using enzyme-assisted (EAE), ultrasonic-assisted (UAE), and microwave-assisted extraction (MAE) methods, compared to conventional alkaline extraction (CE). EAE was performed with viscozyme (2%, pH 8, 50 °C, 2 h) and papain (1%, pH 7, 50 °C, 1 h), UAE at 40% amplitude (20 kHz, 20 min), and MAE at 800 W (50 °C, 90 s). All methods significantly improved extraction yield (14.60-30.08%), protein content (80.47-86.61%), solubility (40.78-60.09% at pH 10), and techno-functional properties over CE. However, MAE slightly reduced solubility. Phytates (0.83-0.49 g/100 g) and trypsin inhibitor activity significantly decreased (4.48-1.92 U/mg). In vitro protein digestibility improved (<i>p</i> < 0.05) across all samples (88.11-93.81%), with hydrolysis patterns supporting the enhanced digestibility. Structural modifications were indicated by altered surface hydrophobicity and thermal properties. SDS-PAGE showed consistent major protein bands at 17, 25, and 48-63 kDa, with EAE showing reduced intensity at ~63 kDa. While UAE and MAE achieved high protein yield and purity, EAE offered the best balance of functionality and digestibility, making it the most promising method for producing high-quality MSPI. These findings are relevant for guiding the selection of extraction methods for MSPI recovery for food applications.