A guide for the evaluation of in vitro bioaccessibility of carotenoids

Ongoing efforts to improve the nutritional content of carotenoids in food sources to solve global health problems must also consider bioaccessibility and bioavailability of carotenoids. Carotenoid bioaccessibility encompasses carotenoid release from the food matrix, solubilization in lipid emulsion droplets, and transfer to mixed micelles for further absorption into the intestine. Bioavailability refers to the fraction of carotenoids which enters into circulation. To understand the mechanisms controlling bioaccessibility, in vitro digestion methods have been developed and are a useful approach to overcome some of the disadvantages associated with in vivo protocols. The INFOGEST protocol comprises three phases (oral, gastric and intestinal) to simulate adult human digestion of foods, as an international consensus for the conditions of static in vitro digestion. The last steps in any digestion protocol involve isolation of the target food compounds and subsequent analysis, steps not included in the INFOGEST protocol because they may differ depending on specific physical-chemical characteristics and quantification analysis of the target analyte. In this chapter, we describe the INFOGEST protocol which has been adapted for the analysis of carotenoid bioaccessibility, including determination and calculation of enzyme activities according to INFOGEST guidelines, removal of undigested material, collection of mixed micelles containing carotenoids, extraction, storage and subsequent identification and quantification by liquid chromatography analysis of both free and acylated carotenoids.