In vitro iron bioavailability studies

Garden peas were microwaved following the cooking instruction on the pack. In brief, 100 g frozen garden peas were placed in a beaker with 1 tablespoon (9.5 ml) MilliQ water (18.2 MΩ), and microwaved at 900 W for 2.5 min. Dried peas (100 g) were soaked in 500 ml Milli-Q water overnight, rinsed 3 times and boiled with 400 ml MilliQ water for 1 h. After cooking, samples were frozen at −80 °C, lyophilized, finely ground and stored at 4 °C until further use.

The simulated gastrointestinal digestion was performed as described¹⁹ with minor modifications. Pea samples were added to 10 ml of saline solution (140 mM NaCl, 5 mM KCl) at pH 2.0, followed by addition of pepsin (0.04 g ml⁻¹) and incubated for 90 min on a rolling platform at 37 °C to simulate gastric conditions. Ascorbic acid was added at a molar ratio of 10:1 ascorbate:iron. Subsequently, the pH of the samples was gradually adjusted to pH 5.5 with NaHCO₃. Bile extract (0.007 g ml⁻¹) and pancreatin (0.001 g ml⁻¹) were added, samples were readjusted to pH 7, and incubated for an additional hour on a rolling platform at 37 °C to mimic intestinal conditions. At the end of the simulated gastrointestinal digestion, samples were centrifuged at 3000 × g for 10 min and the supernatants were used for subsequent cell culture experiments. A volume of 1.5 ml supernatant was applied to an upper chamber consisting of a Transwell insert fitted with a 15 kDa molecular weight cut-off dialysis membrane (Spectra/Por 7 dialysis tubing, Spectrum Laboratories, Europe) suspended over Caco-2 cell monolayers grown in collagen-coated 6-well plates. After incubation for one hour at 37 °C in a humidified incubator containing 5% CO₂ and 95% air, inserts were removed and an additional 1 ml of low-iron Eagle’s minimal essential medium (MEM, GIBCO, Grand Island, NY) was added. Cells were incubated for a further 23 hours prior to harvesting for ferritin analysis. Details of Caco-2 cell culture and ferritin analysis by ELISA are described in ref.³³. To assess the effect of phytic acid on iron bioavailability, either 0.5 mM phytic acid was added to the digestate of microwaved garden peas, or 480 U phytase enzyme (Megazyme) was added to the digestate of boiled mature peas.