Unlocking Alfalfa’s Potential as a Viable Source of Protein: Improving Protein Extraction and Characterizing the Isolated Protein

Alfalfa ( Medicago sativa ) stands out among other green biomass for its environmental advantages and potential as a novel plant protein source. However, the high extent of proteolysis post-harvest due to endogenous proteases limits the feasible production of functional alfalfa protein ingredients. This study aimed to provide a comprehensive evaluation of the structural and functional properties of alfalfa protein, as impacted by protease inactivation, extraction conditions, and inherent differences in plant parts across two harvests. Protein extraction from alfalfa total herbage was significantly enhanced ( P < 0.05) from 37 to 45% when double (15 min) solubilization at pH 7 was performed, where the second solubilization with fresh solvent (water) led to additional protein recovery. Protein polymerization, caused by the acid/thermal inactivation of proteases, hindered any further enhancement in protein extraction yield. Results demonstrated that protein extractability from alfalfa leaves was significantly higher than from stems. Regardless, the protease inactivation treatment resulted in protein denaturation and extensive polymerization that led to poor protein solubility across all alfalfa protein samples. In contrast, polymerization and intermolecular β-sheet contributed to strong gelation properties, namely of the protein isolated from leaf and total herbage, outperforming both commercial soy and pea protein isolates by forming strong gels at lower protein concentration (10% protein vs 15% and 20%, respectively). These results not only highlighted the potential of alfalfa as a source of functional protein but also emphasized the need to inactivate proteases while maintaining the integrity of the protein.