Indigestible proteins and peptide-bound AGEs in ultra-processed foods: Dulce de leche as a model of severely transformed protein food systems

Caramel milk confectioneries, such as dulce de leche, are produced by simmering whole or reconstituted milk with sugar and alkali at temperatures ranging from 115 to 130 ◦C for several hours. This process triggers extensive non-enzymatic browning through the Maillard reaction, leading to significant modifications in milk proteins. Our research focused on the effects of such severe processing conditions on the structural integrity and digestibility of milk proteins, using dulce de leche as a model for severely transformed food protein systems. Due to structural modifications, covalent interchain cross-links, and sugar-derived bridging moieties, milk proteins in dulce de leche form high molecular weight aggregates that are not resolvable by SDS-PAGE. Proteomic analysis revealed that all major milk proteins are involved in the formation of these covalent molecular complexes. When subjected to static gastro-duodenal digestion using both adult and infant models, these macroaggregates showed resistance to pepsin and marginal susceptibility to duodenal proteases. High pressure liquid chromatography-tandem mass spectrometry (HPLC-MS/MS)-based peptidomic analysis of dulce de leche digests allowed identifying variously sized polypeptides bearing the hallmarks of the Maillard reaction. However, many MS signals could not be assigned due to unknown modifications. The prevalence of free and peptide-bound advanced glycated end products (AGEs) in caramel milk products and other ultra-processed foods poses significant health concerns, stressing the need for a comprehensive dedicated evaluation.