Top-Down Glycopeptidomics Reveals Intact Glycomacropeptide Is Digested to a Wide Array of Peptides in Human Jejunum

Abstract

Background Bovine milk κ-casein-derived caseinomacropeptide (CMP) is produced in large quantities during cheesemaking and has various biological activities demonstrated via in vitro and in vivo experiments. Previous studies examined protein degradation and peptide release after casein or whey protein consumption. However, whether purified intact CMP which is partially glycosylated survives intact to its presumed site of bioactivity within the gut remains unknown. Objective The aim of this study was to determine the extent to which purified intact CMP (including glycosylated forms) is digested into peptide fragments within the jejunum of healthy human adults after consumption. Methods Jejunal fluids were collected from three adult participants (two men and one woman, age: 27 ± 7, BMI: 23 ± 1) for three hours after consuming 37.5 g of purified intact CMP. CMP and CMP-derived peptides were isolated from the collected jejunal fluids by ethanol precipitation and solid phase extraction and identified by mass spectrometry-based top-down glycopeptidomics. Relative abundances of CMP and CMP-derived peptides were compared qualitatively between the feed and the jejunal fluids. Results Intact CMP was dominant in feeding material, accounting for 90% of the total ion abundance of detected peptides, and in very low abundance (less than 2%) in the jejunal fluids. CMP-derived fragment peptides ranging from 11–20 amino acids in length were predominant (accounting for 68–88% of the total peptide ion abundance) in jejunal fluids during 1–3 h post consumption. Conclusions This study demonstrates that intact CMP (including glycosylated forms) is mostly digested in the human jejunum, releasing a wide array of CMP-derived peptide fragments. Some of the CMP-derived peptides with high homology to known bioactive peptides consistently survived across 3 h of digestion. Therefore, future research should examine the biological effects of the partially digested form—the CMP-derived fragments—rather than that of intact CMP.

Glycomacropeptide as an Efficient Agent to Fight Pathophysiological Mechanisms of Metabolic Syndrome

“…While the beneficial effects of GMP do not seem to involve mechanisms driven by the gut microbiota, we hypothesize that the peptide’s actions stem from direct interaction with various receptors within the gut or post-absorption. Although previous studies have suggested that GMP is predominantly found in circulation, recent research has revealed that a significant portion of GMP is degraded into smaller peptides of 11 to 20 amino acids within 1 to 3 h post-consumption [ 76 ]. This finding raises the possibility that the peptide’s effects may be primarily mediated by its interaction with various receptors directly within the gut rather than following absorption into circulation.…”

Macrophage-Immunomodulatory Actions of Bovine Whey Protein Isolate, Glycomacropeptide, and Their In Vitro and In Vivo Digests

“…When fed, GMP undergoes digestion within the gastrointestinal tract. Koh et al [26] demonstrated that GMP is broken down into many peptide fragments during human digestion, including many that are known bioactive peptides. We observed that in vitro-digested GMP strongly inhibited production of both TNF-α and IL-1β.…”

“…Peptides that are released from WPI and GMP during gastrointestinal digestion in the consumer would have the potential to affect immunoregulation by interacting with intestinal epithelial and immune cells. Koh et al [26] demonstrated that during human consumption, GMP is digested into smaller peptides, many of which share homology with known bioactive peptides. The digestion-released GMP-derived fragments thus have a higher likelihood of being able to exert immunomodulatory effects within the human gut than the intact form of GMP.…”

The Role of Bovine Kappa-Casein Glycomacropeptide in Modulating the Microbiome and Inflammatory Responses of Irritable Bowel Syndrome

“…More research is needed to determine the effects of GMP supplementation on the microbiome of IBS patients. Since GMP is known to be digested partially within the human gut [ 113 , 114 , 115 ], encapsulation and other delivery strategies may be needed to enhance its effectiveness.…”