Methodological approaches to assess protein digestibility with an emphasis on plant-derived foods.

The shift towards sustainable diets has increased interest in plant-based proteins and meat alternatives. The quality of protein depends on the content and digestibility of indispensable amino acids, which are shaped by molecular structure, food matrix interactions and processing. These factors influence amino acid bioavailability, digestion rates and metabolic outcomes. This work provides an overview of protein digestion, highlighting the influence of amino acid sequence, folding, β-sheet prevalence, disulfide crosslinking, aggregation and interactions with anti-nutritional factors on enzymatic accessibility and hydrolysis. Particular attention is given to plant-derived proteins and meat analogues, the digestibility of which is often modulated by intrinsic structural characteristics and complex matrix effects. Food processing has a dual effect, as mild treatments enhance proteolysis through unfolding and improved solubility, whereas harsh treatments cause aggregation, cross-linking, racemization and modifications that hinder enzyme access. This chapter covers in vitro digestion models, including the standardized static INFOGEST model, as well as semi-dynamic, and dynamic models. It details their uses, advantages and disadvantages for evaluating protein digestibility and nutritional value. Advanced tools such as high-resolution mass spectrometry and peptidomics help to characterized digestion products and offer a better understanding of how hydrolysis influences functionality and safety implications. The chapter also addresses protein quality metrics such as Digestible Indispensable Amino Acid Score, the challenges associated with them, and the need for a comprehensive framework to assess the nutritional and health impacts of alternative and novel plant proteins.