Synergy of Analytical Characterization and Biocompatible Extractions for the Enhancement of High-Quality Biorefinery Products from <i>Medicago sativa</i>

This study presents the development of an analytical characterization strategy tailored to end products derived from an alfalfa (<i>Medicago sativa</i>)-based biorefinery with particular emphasis on protein concentrates and phenolic-enriched fractions. Our approach began with a comprehensive full-factorial experimental design aimed at optimizing the extraction process, taking care to design a biocompatible extraction protocol. Liquid chromatography with tandem mass spectrometry (LC-MS/MS) techniques were used to characterize the molecular profile of the extracts. In particular, the extracts showed a significant relative abundance of flavonoids and isoflavonoids in both their aglycone and glycosylated forms, in which antioxidant activity was evaluated. In addition, we elucidated the proteomic profiles of the protein concentrates. This proteomic characterization served as a valuable resource for understanding the differences between these end products, providing insights that can guide informed decisions about their potential applications.