Uncovering the Protein Conversion Potential of Alfalfa (Medicago sativa L.) and Duckweed (Lemna minor L.) Through Enzymatic Hydrolysis and Digestibility Assessment

The growing demand for sustainable protein alternatives has increased interest in underutilized plant biomasses with high nutritional potential. This study investigated the conversion efficiency of alfalfa (<i>Medicago sativa</i> L.) and duckweed (<i>Lemna minor</i> L.) proteins through multienzyme hydrolysis, with the aim of evaluating how carbohydrate-protein matrix interactions influence enzymatic accessibility and apparent protein digestibility. Three biotechnological hydrolysis schemes were applied, involving combinations of α-amylase, amyloglucosidase, protease, pepsin, pancreatin, and bile salts, including an in vitro gastrointestinal digestion simulation. The first hydrolysis scheme demonstrated that starch-rich matrices formed a viscous medium that reduced protease mobility and limited protein cleavage. Improved substrate accessibility was achieved when plant material was pre-treated with amylolytic and proteolytic enzymes, which resulted in a noticeably higher release of free amino acids. Amino acid profiling revealed that this enzymatic sequence was the most effective for disrupting carbohydrate-associated protein fractions in both species. In vitro digestion assays indicated higher apparent protein conversion for duckweed compared to alfalfa under standardized laboratory conditions. Overall, the results confirm that appropriate multienzyme strategies can enhance amino acid liberation from complex plant matrices and highlight duckweed biomass as a promising candidate for sustainable protein valorization.