Enzymatically enhanced mustard (Brassica nigra) protein hydrolysates as a next-generation plant-based pragmatic alternative: Functional and hypotensive efficacy

• MPI was hydrolyzed with papain, pepsin, and alcalase to enhance functionality. • Hydrolysis enhanced solubility, emulsifying, and foaming properties significantly. • MPH showed strong antioxidant and antihypertensive activity with health benefits. • Supports mustard meal valorization, boosting sustainable protein from agro by-products. Mustard seed protein is a promising yet underutilized source of plant-based protein with potential health-promoting properties. However, its native form often lacks desired bioactivity due to poor solubility and the predominance of large, inactive protein molecules. This study aimed to enhance the functional, antioxidant, and anti-hypertensive properties of mustard protein through enzymatic hydrolysis using the enzymespapain, alcalase, and pepsin.The isolated mustard protein showed nutritionallysignificant BV and PER of 78.7% and 2.32, respectively, due to the presence of essential and non-essential amino acids in balanced proportions.Among the three enzymes, alcalase achieved the highest DH% after 210 minutes, thereby demonstrating the highest antioxidant activity, as evidenced by a vigorous DPPH radical scavenging activity of 57.6% at 75 mg/mL, followed by papain (51.8%) and pepsin (36.3%), respectively. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed that alcalase hydrolysates produced peptides with lower molecular weights. The MPH exhibited enhanced solubility at pH 7-9 compared to MPI. Papain hydrolysis resulted in a 113% increase in EAI (emulsifying activity index) and a 43% improvement in ESI (emulsifying stability index).Alcalase-derived hydrolysates showed that papain-treated hydrolysates after ultrafiltration using a 3 kDa membrane exhibited the highest ACE-inhibitory activity (IC₅₀: 95 µg/mL), suggesting a potential role in blood pressure regulation.Thus, mustard protein hydrolysates derived from the enzymatic hydrolysis of MPI emerge as a promising source for functional and nutraceutical applications.