New insights into the bioaccessibility and metabolic fates of short-chain bioactive peptides in goat milk using the INFOGEST static digestion model and an improved data acquisition strategy

The metabolic fates of potentially bioactive short-chain peptides (SCPs; amino acid numbers between 2 and 4) in gastrointestinal digestion have received little attention due to their low concentration and broad suppression during high resolution mass spectrometry (HRMS) analysis. A tailored workflow integrating mesoporous magnetic solid phase extraction and a novel ion transmission strategy (data-dependent acquisition combined with both an inclusion list and an exclusion list followed by a data-independent acquisition) was used to profile the composition of SCPs during in vitro simulated digestion (LOQ 0.02 to 0.1 μg L-1). A total of 47 dipeptides, 59 tripeptides, and 21 tetrapeptides were identified and quantified from 0.01 to 27.84 mg L-1 (RSD ≤ 9.1%) based on parallel reaction monitoring and an internal standard method. The structural properties of stable SCPs resistant to intestinal digestion were determined by analysis of variance (p < 0.05), with a Pro residue at the C-terminal or penultimate position, a slightly greater negative charge at pH 7.0, and fewer C-terminal aliphatic and polar amino acids. SCPs' metabolic fates varied during digestion, but the overall trend of content change for either total or individual SCP increased as the digestion proceeded, and they were further assessed by a database-driven bioactivity search, which matched a wide variety of bioactivities with the predominance of dipeptidyl peptidase (DPP) IV and angiotensin-converting enzyme (ACE) inhibitors. This study facilitated the understanding of bioaccessibility of the food-derived SCPs and provided essential guidelines for the properties of conserved structure in vivo.