The present study aimed to evaluate the effects of long-term storage on the carotenoid pigments present in whole-grain flours prepared from durum wheat and tritordeum. As expected, higher storage temperatures showed a catabolic effect, which was very marked for free carotenoid pigments. Surprisingly, for both cereal genotypes, the thermal conditions favoured the synthesis of lutein esters, leading to an enhanced stability, slower degradation, and, subsequently, a greater carotenoid retention. The putative involvement of lipase enzymes in lutein esterification in flours is discussed, particularly regarding the preferential esterification of the hydroxyl group with linoleic acid at the 3′ in the ε-ring of the lutein molecule. The negative effects of processing on carotenoid retention were less pronounced in durum wheat flours, which could be due to an increased esterifying activity (the de novo formation of diesterified xanthophylls was observed). Moreover, clear differences were observed for tritordeum depending on whether the lutein was in a free or esterified state. For instance, lutein-3′-O
-monolinoleate showed a three-fold lower degradation rate than free lutein at 37 °C. In view of our results, we advise that the biofortification research aimed at increasing the carotenoid contents in cereals should be based on the selection of varieties with an enhanced content of esterified xanthophylls.
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