Background: Carotenoids are presumably health beneficial secondary plant compounds, as their consumption via the diet and their levels in body tissues (e.g. blood plasma) have been related to the reduced incidence of chronic diseases, including type 2 diabetes, and cardiovascular diseases. In addition, several carotenoids are provitamin-A constituents, and appear to be involved in age-related macular degeneration, the major cause of blindness in the elderly. However, owing to their rather low water solubility requiring emulsification prior to their potential absorption, carotenoids are of low and varying bioavailability (10-20% for e.g. beta-carotene), depending on host, but also on dietary factors including dietary fibre, lipids, and possibly other nutrients such as minerals or compounds competing for absorption (e.g. fat soluble vitamins). However, not much is known on the interaction between proteins and carotenoids during gastric and small intestinal digestion and solubilisation/micellization of carotenoids, a prerequisite for their bioavailability. Several proteins have been shown to have emulsifying properties during digestion, stabilizing oil-in-water emulsions, which can be expected to result in smaller sizes of digestible lipid droplets, possibly enhancing carotenoid micellization. While proteins have been employed in the production of encapsulated carotenoids (e.g. whey protein isolate (WPI) or sodium caseinate (SC)), the interaction between proteins with carotenoids in food matrices have, to our knowledge, never been studied systematically.Proposed Study and Aim: We propose the conduction of a human trial to investigate the effect of added proteins on carotenoid bioavailability from a test food, possibly a leafy vegetable (e.g. spinach). Preceding the human trial, in-vitro studies will be conducted, by means of simulated gastro-intestinal digestion coupled to Caco-2 cellular uptake models to select proteins most strongly interacting with carotenoid solubilisation, mixed micelle formation, absorption aspects as well as colonic fermentation.Materials and Methods: A consensus protocol (based on EU-Cost action FA-1005) will be employed to study the interaction of several proteins (including WPI, SC, soy-protein isolate, gelatin) with carotenoid (solubilized) standards (lutein, beta-carotene, lycopene, neoxanthin), as well as carotenoid containing solid and liquid matrices (spinach, apricot, carrot juice, tomato juice), on digestion (surface tension, protein digestion, lipid digestion, micelle size, zeta-potential etc.) and carotenoid availability (recovery, bioaccessibility, Caco-2 cellular uptake). The interaction with gastric lipases (e.g. Aspergillus niger) will be studied. Digestion parameters in the gastric phase, the small intestine, and the colon will be studied, and metabolites/degradation products scrutinized. The human trial will consist of a randomized, placebo-controlled, double blind postprandial cross-over trial with 24 subjects, receiving either test food (e.g. spinach) plus a drink (e.g. skimmed milk) with starch, and 2 promising proteins (given at approx. half the RDA amount), selected following in-vitro studies. Carotenoid bioavailability will be assessed by HPLC (LC-MS-MS if needed) in the triacyl-rich-lipoprotein fraction, representing newly absorbed carotenoids. Expected results: We hypothesize that proteins will enhance the solubilisation and digestion of lipid droplets, reduce their size, and foster the transition of lipid droplets to mixed micelles, at least for some proteins, and that this will result in improved carotenoid bioaccessibility, cellular uptake, and also enhanced bioavailability in vivo. This may have implications for dietary recommendations, e.g. for subjects consuming limited amounts of meat (vegetarians, developing countries), and for companies producing functional foods/food supplements, and could also have consequences for other liposoluble micronutrients.