Abstract In vitro food digestion methods are increasingly used due to standardised protocols and validation with in vivo data. Porcine pepsin is the major gastric enzyme used to replace human pepsin during the gastric phase of in vitro digestion models. Until now, limited information exists on the combined effect of the temperature and pH variations that occur during digestion on the activity of these enzymes. This study addresses gaps in the understanding of how pH (1–7) and temperature (4–60 °C) affect pepsin activity. Human pepsin exhibited broader activity retention than porcine pepsin, maintaining 80% and 46% activity at pH 3 and 4, respectively, versus 47% and 13% for porcine pepsin. Predictive models for both enzymes were developed to quantify activity under varied conditions. Porcine pepsin required heating to 75 °C for ≥ 5 min for complete inactivation, while 65 °C for 15 min proved insufficient. These findings allow direct comparison of standardised in vitro digestion results across studies, improving the translation of in vitro outcomes to in vivo human digestion. Additionally, this study clarifies optimal inactivation protocols for sample preparation. This work therefore enhances the accuracy of in vitro models, supporting their use in food research.
J. et al. (Sun,) studied this question.