Unlocking alfalfa’s potential as a viable source of protein: Improving protein extraction and characterizing the isolate protein

Authors: MOR, SHREEVATS - University Of Minnesota; Heuschele, Deborah; SAEIDY, SIMA - University Of Minnesota; ISMAIL, BARAEM - University Of Minnesota

Submitted to: Food Research International
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/10/2026
Publication Date: 7/17/2026
Citation: Mor, S., Heuschele, D.J., Saeidy, S., Ismail, B.P. 2026. Unlocking alfalfa’s potential as a viable source of protein: Improving protein extraction and characterizing the isolated protein. Food Research International. https://doi.org/10.1021/acsfoodscitech.6c00462.
DOI: https://doi.org/10.1021/acsfoodscitech.6c00462

Interpretive Summary: Alfalfa has a lot of potential as an environmentally friendly source of plant protein. However, after harvest, the natural enzymes in the plant break down its proteins very quickly making it difficult to produce high-quality, functional alfalfa protein ingredients. In this study, alfalfa was tested for protein extraction from whole plants, leaves, and stems, and how heating and acidity during extraction changed the protein’s structure and performance. Protein extraction was improved using two rounds of solubilzation, increasing production yields from 37% to 45%. Leaves were easier to extract protein than stems and the cutting late in the fall resulted in greater amounts of extracted protein than summer harvests. Despite lower solubility than soy, alfalfa proteins form very strong gels—stronger than commercial soy and pea protein isolates—even at lower protein concentrations. This study supports that alfalfa is a promising source of functional plant protein, especially for food products that rely on gel formation.

Technical Abstract: 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 minute) 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 ß-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.