Amylases are crucial enzymes for dietary starch hydrolysis and essential to human digestive physiology. To simulate gastrointestinal (GI) digestion in vitro, amylases may be included in a short oral phase to mimic human salivary amylase (HSA) activity. Due to the high procurement cost of HSA, alternate porcine or microbial sources of amylases have been used. The major objective of this study was to evaluate the suitability of porcine pancreatic amylase (PPA) as an alternative to HSA in the INFOGEST 2.0 static simulation of oro-gastric digestion. To start, a comprehensive literature review was conducted to categorize oral phase methodologies. We determined that 14.1 % and 5.2 % of 262 studies used PPA and microbial amylases, respectively, in the oral phase. Amylolytic activity was confirmed by HPLC analysis of free maltose after simulated potato digestion. PPA and HSA exhibited comparable starch hydrolysis, while a fungal amylase showed significantly higher activity (P <
0.0002). To investigate suspected proteolytic "side activity" of PPA, pea protein digestion was simulated with HSA, PPA, or no amylase in the oral phase. Proteolytic activity was evaluated in gastric digestas by measurement of free amino nitrogen (FAN) by UV spectroscopy and free amino acids by HPLC. Relative peptide hydrolysis patterns were evaluated by SDS-PAGE and size exclusion chromatography (SEC). PPA treatment showed significantly higher proteolytic activity based on FAN and free amino acid analysis compared to HSA (Both P <
0.0002, one-way ANOVA), and qualitatively greater protein hydrolysis by SDS-PAGE and SEC analyses. These data unanimously confirm the unintended proteolytic activity of PPA in the oral phase that carries over to the gastric phase, potentially confounding analyses of protein digestibility and nutrient bioavailability. These findings suggest PPA should be avoided in the oral phase and confirm that HSA remains the preferred amylase for simulating human oral digestion.