Production of Protein Hydrolysates and Bioactive Peptides From <i>Lablab purpureus</i> and <i>Macrotyloma uniflorum</i> via Optimized Extraction and Proteolysis Protocols

The rising global demand for sustainable, plant-based protein sources necessitates the exploration of underutilized legumes with high nutritional potential. This study systematically evaluates and compares five protein extraction techniques-ultrasound-assisted extraction (UAE), alkaline extraction-acid precipitation, phosphate buffer with TCA-acetone, TCA-acetone alone, and micellization-applied to Lablab purpureus and Macrotyloma uniflorum, two resilient and underexploited pulse crops. Protein recovery and yield were quantified for each method to identify the most efficient extraction approach. Among the tested methods, ultrasound-assisted extraction demonstrated superior performance in both legumes, with maximum protein yields of 84.02% for L. purpureus and 78.4% for M. uniflorum under optimized conditions (40% power, 10-15 min). Alkaline-acid precipitation resulted in the highest recovery (88.4%) but comparatively lower yields, indicating significant protein loss during isoelectric precipitation. Traditional precipitation techniques using TCA-acetone and phosphate buffer showed limited effectiveness, while micellization produced the lowest recovery and yield in both species. Importantly, the study reveals species-specific responses to extraction conditions, with L. purpureus consistently exhibiting higher extractability than M. uniflorum, likely due to differences in seed microstructure and protein distribution. These findings underscore the need for pulse-specific optimisation strategies rather than universal extraction protocols. The outcomes pave the way for their application in food, nutraceutical, and feed industries, contributing to sustainable protein innovation and improved utilisation of climate-resilient crops. Future work should focus on the structural, functional, and bioactive characterisation of the extracted proteins to enable targeted formulation in diverse food systems.