Rachel Boutrou

Sequential release of milk protein–derived bioactive peptides in the jejunum in healthy humans

BACKGROUNDnThe digestive hydrolysis of dietary proteins leads to the release of peptides in the intestinal tract, where they may exert a variety of functions, but their characterization and quantification are difficult.nnnOBJECTIVESnWe aimed to characterize and determine kinetics of the formation of peptides present in the jejunum of humans who ingested casein or whey proteins by using mass spectrometry and to look for and quantify bioactive peptides.nnnDESIGNnSubjects were equipped with a double-lumen nasogastric tube that migrated to the proximal jejunum. A sample collection was performed for 6 h after the ingestion of 30 g (15)N-labeled casein (n = 7) or whey proteins (WPs; n = 6). Nitrogen flow rates were measured, and peptides were identified by using mass spectrometry.nnnRESULTSnAfter casein ingestion, medium-size peptides (750-1050 kDa) were released during 6 h, whereas larger peptides (1050-1800 kDa) were released from WPs in the first 3 h. A total of 356 and 146 peptides were detected and sequenced in the jejunum after casein and WP ingestion, respectively. β-casein was the most important precursor of peptides, including bioactive peptides with various activities. The amounts of β-casomorphins (β-casein 57-, 58-, 59-, and 60-66) and β-casein 108-113 released on the postprandial window were sufficient to elicit the biological action of these peptides (ie, opioid and antihypertensive, respectively).nnnCONCLUSIONSnClear evidence is shown of the presence of bioactive peptides in the jejunum of healthy humans who ingested casein. Our findings raise the question about the physiologic conditions under which these peptides can express their bioactivity in humans. This trial was registered at clinicaltrials.gov as NCT00862329.

Glycosylations of κ-Casein-Derived Caseinomacropeptide Reduce Its Accessibility to Endo- but Not Exointestinal Brush Border Membrane Peptidases

Caseinomacropeptide (CMP) is a peptide obtained from kappa-casein hydrolysis by gastric proteinases and which exhibits various biological activities. The aim of this study was to analyze the intestinal processing of CMP at the brush border membrane (BBM) level. Intestinal BBM vesicles (BBMV) were used to digest glycosylated and unglycosylated CMP. Our results demonstrated that whatever was the glycosylated state of CMP, they were digested by BBMV intestinal enzymes, from macropeptides to free amino acids. The digestion of unglycosylated and glycosylated CMP throughout the action of exopeptidases was similar, but the activity of endopeptideases on glycosylated CMP was limited, certainly due to the attached O-glycosylations. Consequently, much more peptides were identified from the unglycosylated than from the glycosylated CMP. In addition, the glycosylation core as well as the number of the attached glycosylated chain modified the kinetic of digestion; the most heavily glycosylated forms being the slowest digested.

Phosphorylation and Coordination Bond of Mineral Inhibit the Hydrolysis of the β-Casein (1−25) Peptide by Intestinal Brush-Border Membrane Enzymes

Caseinophosphopeptides (CPP) are food mineral-rich components that may resist intestinal enzyme hydrolysis. We wondered whether phosphorylation and/or mineral binding induces resistance of CPP to intestinal hydrolysis. We used intestinal brush-border membrane vesicles to digest different forms of the beta-casein (1-25) peptide: unphosphorylated and phosphorylated carrier of varied cations. The results showed that the activity of alkaline phosphatase seems not to be specific to either the phosphorylation degree or the phosphorylation sites whereas phosphorylations limited the action of peptidases. Studying the mechanism and the kinetics of hydrolysis of the different peptides allows understanding how some cations prevent more CPP from hydrolysis than others. The action of both exo- and endopeptidases was limited for the beta-CN (1-25) peptide bound to zinc or copper. Actually the peptide bound to copper was almost not hydrolyzed during the digestion, suggesting that coordination bond of copper to CPP inhibits the action of both phosphatase and peptidases.

P059 Les peptides et les cinétiques de la digestion intestinale des protéines laitières chez l’homme

Milk proteins are of interest as they contain soluble (SP) and non-soluble (casein) fractions that behave differently during digestion. SP are rapidly evacuated from the stomach whereas gastric emptying of casein is delayed (Boirie et al. 1997). This difference that has been demonstrated in vitro or in vivo in animals and in humans induces differential appearance of derived peptides and amino acids in the blood and postprandial metabolic response. This study aims to assess in humans the intestinal digestion kinetics of both protein fractions. It has been performed on 16 in good health volunteers whose diets were standardized during 9 days. On the 9th day they ingested the meal composed among others of 30 g of either casein or SP. Ileal effluents were continuously collected every 30 min for 6 hours using a double lumen nasogastric tube that migrated to the jejunum. In order to verify whether casein and SP present differential digestion in jejunum, immunoreactive proteins were quantified and peptides were identified using proteomic tools (chromatography coupled on line with mass spectrometry). This study shows that i) immunoreactive proteins and peptides were present in a greater amount during the first hour of digestion for SP meal whereas a two-step digestion was observed for casein meal: a first one at 1.5-2.5 hours and a second one 4-5 hours after meal ingestion, ii) large protein fragments were present within the jejunum for both meals thus demonstrating that proteins digestion was incomplete in the jejunum, and iii) the peptides derived from casein were smaller than the ones from SP. The digestion of casein and soluble proteins differed in both the intestinal kinetics and the molecular weight of the peptides. These differences offer several applications in overweight and elderly people or in patients with wasting disorders