Isothermal Titration Calorimetry as an Innovative Method to Determine Pepsin Activity and Plant Protein Digestibility

Plant proteins are increasingly recognized as sustainable and ethical alternatives to animal‐derived proteins, especially in the light of environmental concerns and dietary restrictions [1]. Common industrial processing methods applied to plant‐based proteins, such as those derived from protein‐rich legumes, intentionally alter their structure to mimic meat products. This raises questions about unintentional modifications of amino acids side chains and their impact on digestibility by gastric and intestinal enzymes [2]. Understanding processing parameters influencing protein digestion is highly relevant for an accurate nutritional assessment of food processing innovations. The current standard to evaluate protein digestibility is the INFOGEST 2.0 in vitro digestion protocol [3]. It provides valuable specifications to simulate physiological conditions. The recommended assays to assess pepsin activity through peptide release are limited in sensitivity and require high amounts of specialized substrates. An innovative approach by isothermal titration calorimetry (ITC) is applied to determine pepsin activity during gastric digestion [4]. Unlike traditional assays, ITC measures the heat produced, monitoring hydrolyses in real time [5]. Validation of the ITC data was performed through comparison with UV‐based assays that quantify pepsin activity by hemoglobin degradation. Furthermore, several legume protein extracts were investigated according to their digestibility under in vitro gastric digestion conditions as a prerequisite for the subsequent intestinal digestion.