Release of beta-casomorphins during in-vitro gastrointestinal digestion of reconstituted milk after heat treatment

This study assessed the impact of heat treatment on beta-casomorphin 5 (β-CM5) and beta-casomorphin 7 (β-CM7) after in-vitro gastrointestinal digestion of milk representing beta-casein (β-CN) A1A1, A2A2 and A2I phenotype. After heat treatment at 73 °C/20 s, 85 °C/5 min and 121 °C/12 min, milk samples were subjected to in-vitro gastrointestinal digestion. β-CM5/7 were analysed using ultra high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) and further confirmed using ultra high-performance liquid chromatography-high resolution accurate mass Orbitrap™ mass spectrometry (UHPL-HRMS). β-CM5 was not released after in-vitro gastrointestinal digestion of all heated milk. Similarly, β-CM7 was not released in all milk with β-CN A2A2 phenotype. However, this peptide level ranged from 127.25 to 198.10 ng/mL (4.94–7.70 ng/mg protein) in heated milk with β-CN A1A1 phenotype, whereas it was released at much lower levels ranging from 19.35 to 24.50 ng/mL (0.71–0.91 ng/mg protein) in heated milk with β-CN A2I phenotype. •β-CM7 was not released after gastrointestinal digestion of heated A2A2 milk.•β-CM7 was released after gastrointestinal digestion of heated A1A1 and A2I milk.•β-CM7 ranges from 4.94 to 7.70 ng/mg protein in heated A1A1 milk.•β-CM7 ranges from 0.71 to 0.91 ng/mg protein in heated A2I milk.