2.4. In Vitro-Simulated Digestion Coupled with the Caco-2 Cell Model

The impact of AA on the in vitro iron bioavailability from the ICCs was evaluated using the established in vitro-simulated digestion coupled with the Caco-2 cell model at USDA/ARS, Cornell University, Ithaca, NY, USA, as described previously [39,40]. In brief, the food matrix underwent a simulated gastric digestion with pepsin at pH = 2, 37 °C for 1 h to mimic the gastric phase of the digestion in individual above 2 years old. This step was followed by a simulated intestinal digestion with pancreatin and bile at pH = 7, 37 °C for 2 h (n = 3) to mimic the duodenal phase. This second step took place on a dialysis membrane placed above the Caco-2 cell monolayers. During the digestion process, iron was released from the food matrix. Solubilized iron can diffuse through the membrane and be taken up by the cells. Thus, in response to higher intracellular iron concentrations, Caco-2 cells will form ferritin. The formation of ferritin was quantified as an indicator of iron uptake by the cells. The Caco-2 cells (obtained at passage 21; American Type Culture Collection, Gaithersburg, MD, USA) were seeded in 6-well collagen-coated plates at passage 29–42. Cells were grown for 13 days before each bioassay at 37 °C in an incubator using Dulbecco’s modified Eagle’s medium supplemented with 25 mM HEPES (pH 7.2), 10% (v/v) fetal bovine serum and 1% antibiotic/antimycotic solution. The medium was changed every 2 days. Twenty-four hours prior to each bioassay, the culture medium was replaced with iron-free Minimum Essential Medium (MEM [pH 7]; GIBCO) supplemented with 10 mM PIPES (piperazine-N,N’-bis-[2-ethanesulfonic acid]), 1% antibiotic– antimycotic solution, hydrocortisone (4 mg/L), insulin (5 mg/L), selenium (5 μg/L), triiodothyronine (34 μg/L) and epidermal growth factor (20 μg/L). A fresh 1 mL aliquot of MEM (pH 7) covered the cells during each experiment. Eighteen hours after the start of the experiment, the cells were harvested, after rinsing and sonication of the plates for 15 min. Cells were then scraped from the plate surface and transferred into tubes for analysis. Ferritin was measured by enzyme-linked immunosorbent assay. The results were normalized to the total protein content of the Caco-2 cells and were expressed as ng ferritin/mg protein (n = 3). The complex was tested in a milk and water matrices, in comparison with FeSO₄, the reference compound for iron bioavailability, and with FePP, the main salt used for milk fortification. In water, only ICC (2.8%) was evaluated with and without AA at a molar ratio of AA to iron of 2:1, as a control. In milk, the two ICCs were evaluated with and without AA at molar ratios of AA to iron of 2:1 and 4:1. The milk was a full cream powder containing 26% fat and 875 mg calcium/100 g powder. The amount of iron corresponded to 3.3 mg Fe/serving of milk, i.e., 33 g of powder for 250 mL of milk ready to be consumed. An aliquot (2 mL) of each individual digest, corresponding to approximately 26.4 μg of iron, was loaded onto the dialysis membrane placed on the Caco-2 cell monolayers.