Addition of cholesterol esterase substantially enhances phytosterol ester bioaccessibility in emulsions with different droplet sizes using a standardized in vitro digestion model

In vitro digestion models are an important tool for the evaluation of foods and supplements, especially foods containing nutraceuticals and bioactive compounds. The INFOGEST network of researchers has published multiple iterations of a static in vitro digestion protocol for the study of food digestion. However, the protocol currently lacks cholesterol esterase (CE) activity during the intestinal phase, and few studies have attempted to evaluate its incorporation into the model. In the present study, the effect of additional CE on phytosterol ester (PSE) bioaccessibility in oil-in-water emulsions with different droplet sizes was evaluated. A range of droplet sizes was obtained by utilizing different homogenization techniques to produce large (9.9 um), medium (3.4 um), and fine (1.3 um) emulsions. Complete lipid digestion was observed for all emulsions. In agreement with previous studies on lipophilic bioactive compounds, emulsion droplet size and PSE bioaccessibility were inversely related to droplet size as smaller droplets led to increased bioaccessibility (12.7, 15.7, and 19.2% for large, medium, and fine emulsions, respectively). This relationship was significantly more pronounced following the addition of CE, with bioaccessibility of large (19.6%), medium (34.2%), and fine (40.8%) emulsions increasing 1.5, 2.2, and 2.1-fold, respectively. Additionally, Beta-sitosterol was significantly greater in bioaccessibility than stigmasterol in medium and fine emulsions with added cholesterol esterase. These findings provide compelling evidence for the inclusion of CE into the standardized INFOGEST protocol and have implications for the in vitro study of esterified micronutrients beyond PSEs. With an improved model, researchers can collect more accurate information about lipid bioaccessibility within different food matrices.