Release of wheat gluten exorphins A5 and C5 during in vitro gastrointestinal digestion of bread and pasta and their absorption through an in vitro model of intestinal epithelium

• Gluten exorphins (GEs) A5 and C5 were present in digestates of bread and pasta.
• For the 1st time, release of GEs during digestion of wheat-derived foods was shown.
• A5 and C5 were studied for transport across monolayers of Caco-2/HT-29 co-culture.
• Less than 3% of GEs A5 and C5 crossed intact the monolayer after 120 min.
• For the 1st time, transepithelial transport of intact GEs A5 and C5 was shown.

The release of wheat gluten exorphins (GEs) A5, A4, B5, B4, C5, Gd-7 and dGd-7 during in vitro gastrointestinal digestion (GID) of bread and pasta samples has been investigated by UPLC/ESI–HRMS. The GEs that survived in vitro GID were studied for transport studies across monolayers of 70% Caco-2/30% HT-29 co-culture grown on semi-permeable membranes, used as a model of the human intestinal epithelium. Among the GEs, only the peptides A5 and C5 were detected at the end of in vitro GID of bread and pasta samples. Levels of GEs A5 and C5 were in the range 0.747–2.192 mg kg− 1 and 3.201–6.689 mg kg− 1, respectively. The absorption behavior of A5 and C5 was studied in detail using synthetic peptides. The two peptides were susceptible to the action of Caco-2/HT-29 co-culture peptidases, and 3% and 1% of A5 and C5, respectively, added to the apical chamber were transported to the basolateral chamber after 120 min of incubation, without being hydrolysed. Several hydrolytic fragments of both GEs crossed the co-culture layer and were found in the basolateral chamber. For the first time, these results demonstrate that GEs are released during in vitro GID of real wheat-derived foods and highlight the possibility of transport of the GEs A5 and C5 across the human intestinal mucosa.