Development of a standardised human in vitro digestion protocol based on macronutrient digestion using response surface methodology

Bioaccessibility studies should be taken into account when evaluating the physiological effects of ingested compounds at the intestine level. Several in vitro digestion protocols have been described, with a wide range of experimental conditions but no optimised protocol exists. In order to fill in this gap, we evaluated the influence of three continuous factors (pH, incubation time, and enzyme concentrations), in the range of values found in literature, on the digestion of standard macronutrients (starch, albumin, triolein) alone or in mixture. Three central composite designs, using response surface methodology, were employed to model the three abiotic steps of pre-colonic digestion. A validated in vitro digestion was eventually set up for the salivary step (pH 6.9, 5 min, 3.9 units α-amylase/ml), the gastric step (pH 2, 90 min, 71.2 units pepsin/ml), and the abiotic duodenal step (pH 7, 150 min, 9.2 mg pancreatin and 55.2 mg bile extract/ml).