Assessing the impact of food-derived bioactives on digestive proteases by <i>in vitro</i> and <i>in silico</i> approaches

The interactions between food components and digestive tract enzymes can affect nutrient absorption and impact an individual's health. Certain components, particularly polyphenols, are reported to inhibit digestive enzymes and are commonly referred to as anti-nutritional factors. Reports on this subject often contradict each other, highlighting the need for consistent methodologies to assess the potential impact of bioactive compounds. This study evaluated the "<i>in vitro</i>" activity of pepsin, trypsin, and chymotrypsin using ovalbumin, gluten, and haemoglobin as substrates in the presence or absence of twenty-five bioactive natural compounds belonging to different chemical classes at gastro-intestinal physiological concentrations (0.1 mM). The results indicate that bioactives may have opposite effects on proteolytic activity depending on the substrate/enzyme combination and bioactives structure. With ovalbumin as substrate, piceid and resveratrol were described as strong chymotrypsin activators (+1.46- and 1.17-fold change, respectively), phloridzin dihydrate as a weaker activator (+0.41-fold change), while phloretin was a strong inhibitor (-0.65-fold change). A computational approach based on molecular docking and dynamics simulations was used to investigate the interactions between selected bioactives, chymotrypsin and ovalbumin. The "<i>in silico</i>" study included piceid and phloridzin dihydrate, as well as their respective aglycones (resveratrol and phloretin). The results obtained through computational modelling indicate that all four bioactives can interact with chymotrypsin. However, only those bioactives that enhance <i>in vitro</i> proteolytic activity induce a partial unfolding of ovalbumin's structure. This suggests that the effect of bioactive compounds on protein digestion may be substrate-dependent, and may vary depending on the specific protein being digested.