2.9.1. Carotenoids Extraction

Carotenoids were extracted following the method described by Sadler et al. [30], with slight modifications. An extraction solution (50 mL) composed of hexane:acetone:ethanol (50:25:25) and 1 g·L⁻¹ BHT was added to carrot puree (2 g) or juice (1 g) and was stirred for 20 min. Then, 15 mL of NaCl (10% (w/v)) solution was added, and the samples were stirred for 10 additional minutes. The samples were left to stand for ≥3 minutes, and the upper organic phase was microfiltered across a nylon filter (0.45 μm, ø 13 mm, Labbox Labware S.L., Barcelona, Spain) and analyzed using High-Performance Liquid Chromatography with Diode Array Detection (HPLC-DAD).

Recovery of the carotenes from the micellar digested fraction was performed by adding 5 mL of the extraction solution to 0.2 g of freeze-dried digesta. After that, the samples were vortexed for 20 s, and 1 mL of NaCl solution (10% (w/v)) was added. The samples were vortexed for another 20 s and centrifuged at 4000× g for 5 min [29]. An aliquot of the upper organic phase was microfiltered across a nylon filter (0.45 μm, ø 13 mm, Labbox Labware S.L., Barcelona, Spain) and analyzed by HPLC using the same method as for non-digested fractions.

All extractions were performed in duplicate, and the samples were protected from light throughout extraction and analysis to avoid carotenoid degradation and isomerization.