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Caseinophosphopeptides (CPPs) are bioactive peptides originating from proteolysis of caseins, the main proteins of milk. In this study, the generation of CCPs during Beaufort cheese making and by in vitro simulated gastro-intestinal hydrolysis using pepsin and pancreatin were assessed using selective precipitation and liquid chromatography–electrospray ionisation tandem mass spectrometry. Seventy-two water-soluble CPPs, mainly originating from β-casein, were identified in Beaufort cheese, while 79 CPPs, mainly generated from α s1 -caseins, were obtained from enzymatic hydrolysates. Most of the peptides generated by the action of digestive enzymes were monophosphorylated; however, 17 out of the 23 polyphosphorylated CPPs identified from digested Beaufort cheese still contained the characteristic cluster sequence Ser(P)-Ser(P)-Ser(P)-Glu-Glu, providing calcium-binding properties to CPPs. The content of CPPs in Beaufort cheese before and after the action of digestive enzymes was estimated to be 230 and 275 mg per 100 g of fresh cheese, respectively. The action of proteolytic digestive enzymes on CPPs is discussed.

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.

Digestion of proteins leads to the release of numerous peptides in the gastrointestinal tract, among them several bioactive peptides. This review compiles, organises and critically analyses the information available on bioactive peptides present in vivo in the digestive tract of adult humans and animals. It focuses on milk proteins, which are recognised as one of the main sources of bioactive peptides with different biological functions. To date, few studies have been performed on the presence of milk-derived peptides in the intestinal tract in vivo. The diversity of these studies is remarkable in terms of the milk products that have been tested, the animal models selected and the segment of the digestive tract considered. Altogether, 69 bioactive peptides identified in vivo are reviewed. Most of them are released from casein, and especially from β-casein, to which 38 bioactive peptides are attributed. These sequences can be classified mainly as angiotensin-converting enzyme (ACE) inhibitors, opioids and antimicrobial peptides. Particular attention is paid to β-casomorphins and caseinomacropeptide-derived peptides. The function of caseinophosphopeptides as a mineral carrier is also discussed. Few bioactive peptides derive from whey proteins., The authors are involved in the Food and Agriculture COST (European Cooperation in Science and Technology) Action FA1005 “Improving health properties of food by sharing our knowledge on the digestive process” (INFOGEST)

By studying the hydrolysis of -casein during soft cheese manufacture, we demonstrated that this casein was hydrolysed before renneting during milk maturation. The quantitative time course of the peptides -CN (f161-169) and -CN (f162-169) was determined throughout the drainage, and the CMP was sought out within the milk before inoculation and before renneting. In this way, we showed that before renneting, the lactococcal cell envelope proteinase produced the peptide -CN (f161-169) mainly from -casein, if not all, whereas the peptide -CN (f162-169) originated from CMP through the action of chymosin. -casein / lactococcal starter / milk maturation / soft cheese

Background: The 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. Objectives: We 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. Design: Subjects 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. Results: After 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. b-casein was the most important precursor of peptides, including bioactive peptides with various activities. The amounts of b-casomorphins (b-casein 57‐, 58‐, 59‐, and 60‐66) and b-casein 108‐113 released on the postprandial window were sufficient to elicit the biological action of these peptides (ie, opioid and antihypertensive, respectively). Conclusions: Clear evidence is shown of the presence of bioactive peptides in the jejunum of healthy humans fed 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. Am J Clin Nutr doi: 10.3945/ajcn.112.055202.

Infant formulas (IFs) are major sources of proteins for the neonate when breast-feeding is not possible. Heat treatments used during IF manufacture affect the structure of milk proteins but the impact of these structural modifications on the digestion of milk proteins and the identity of the bioactive peptides released in the gut have never been studied so far. The objective of this work was to determine the impact of two technological parameters (percentage of dry matter of the milk concentrate prior to heat treatment and intensity of the heat treatment applied to it) considered as essential in IF manufacture on the resistance of milk caseins (CNs) to digestion. Six skim milk powders were manufactured by spray-drying after having submitted 25% or 35% dry matter milk concentrates to three types of heat-treatment traditionally used in industry (80°C/20 s, 85°C/180 s, 105°C/60 s). Those six samples and a reference unheated one were characterised (particle size, composition…) and submitted to an in vitro digestion model mimicking the gastrointestinal digestion of an infant. Digested samples were characterised by SDSPAGE and western-blotting and by ELISA using a collection of 23 αs1, αs2, β and κ-CN-specific monoclonal antibodies. Kinetics of β-CN hydrolysis was studied by antibody arrays. Peptides resisting to digestion were identified using mass spectrometry. Heat-treatment of milk prior to spray-drying was shown to significantly increase the residual CNs immunoreactivity monitored after digestion. Areas showing either post-translational modifications or high hydrophobicities were identified as being the most resistant to digestion whatever the process studied was. Ke

International audience; Caseinphosphopeptides (CPPs) are phosphorylated fragments of caseins, which are found in dairy products. One of their unique features is their ability to bind calcium. There are several published methods to extract CPPs from cheese, but none allows their quantitative evaluation. A chromatographic procedure allowing a semi-quantitative evaluation of cheese CPPs was adapted from a previously published method and tested on Beaufort cheese. Water-soluble peptides were extracted from cheese and purified using cation-exchange chromatography followed by immobilised metal-ion affinity chromatography (IMAC). CPPs were identified using mass spectrometry (LC-ESI-MS/MS). Fifty-five peptides, including 48 CPPs carrying one to three phosphorylations, were identified in Beaufort cheese. It can thus be considered that the selectivity of the chromatographic procedure proposed is sufficient for a semi-quantification of CPPs with a molecular mass ranging from 600 to 10 000 g*mol−1 in cheese. As IMAC fractions contain molecules with a molecular mass

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.

The structure development of a soft cheese curd model has been studied in relationship to its rheological properties and its biochemical characteristics (pH, amount and partition of minerals, casein proteolysis) at different technical steps including cutting, drawing, three turns and demoulding. Scanning electron microscopy was used to observe structural changes during the drainage of a fat-free soft cheese. The micrographs provided visual evidence of changes in the casein matrix from casein particles aggregated in clusters to uniform strands observed at the demoulding. The initial increase of loss tangent and of the exponent of the power law between G′ and G′′ and frequency (that were maximal at the second turn) was related to the solubilization of micellar calcium phosphate, while intact caseins and large casein fragments accumulated in the curd. After the second turn, the strength, Youngs' and loss moduli of the curd increased greatly. The hydrolysis of αs1-casein into αs1-I-CN f(24–199) may facilitate the rearrangement of casein particles within the curd. The pH-induced solubilization of calcium phosphate continued throughout the manufacture process but was unexpectedly incomplete at the end of the drainage. Combination of electron microscopic observations with dynamic rheological measurements and chemical and biochemical assessments provided increased knowledge about the structure of soft cheese during drainage, an important but poorly understood cheese making stage.

We emphasise the role of the lactococcal cell envelope proteinase (lactocepin) on the proteolysis in a curd of soft cheese. During cheese manufacture, proteolysis occurred as soon as the renneting stage with the action of the chymosin, and the level of proteolysis intensified rapidly. The peptides, present within the whey collected throughout the drainage, were identified using tandem mass spectrometry. From the sixteen peptides identified at the first turn, we showed the early predominant action of the lactocepin type III. From the forty eight peptides identified at the end of the drainage, we demonstrated, in addition to the predominant action of lactocepin, other activities that could result from the action of lactococcal peptidases. In addition, this study of proteolysis confirmed that the action of proteinases on casein in solution differed from their action in situ: some sites cleaved in solution were not cleaved in cheese and vice versa.

Proteolysis was considered as a typical biochemical parameter which allows cheese ripening and maturity to be characterised. Owing to methods for peptide sequencing, identification of peptides from cheese has been established as an efficient means to determine which proteolytic enzymes are active within cheese, their specificity, and casein molecular structure accessibility via cleavage site identification. This review is an attempt to determine whether these peptides produced in cheese can give information on the structure of paracasein matrix through the action of the inherent proteolysis (added coagulant, milk endogenous proteinases (plasmin, cathepsin) and proteinases and peptidases of bacterial origin). Some answers will be given with special reference to Emmental cheese of which some data on peptide characterisation are presented here.

The variations of the autolytic and proteolytic potential of lactococci need to be taken into account because these variations probably influence the development of organoleptic properties during cheese ripening. To predict lytic capacity, proteolytic potential, and specificity of cell surface-associated proteinase, we developed simple tests that were applied to 26 industrial strains and a few reference strains of Lactococcus lactis . The tests allowed us to measure the autolytic capacity of lactococci in a buffer or in a pseudo curd under conditions that were close to those of cheese ripening and to evaluate global peptidase activity, proteinase activity, and specificity using casein or casein hydrolysate as a substrate. We confirmed the variability of the autolytic capacity of lactococci and classified the strains into three groups by low, moderate, or high lytic capacity according to their behavior in the buffer and in pseudo curd tests. Validation of the latter was obtained by the observation of similar lytic behavior in the two reference strains of Lactococcus lactis , AM2 and NCDO763, which were highly autolytic and poorly autolytic, respectively, both in our tests and in previously reported cheese experiments. The global activities of peptidases and proteinase varied from 1 to 6 among the strains. Most of the proteinases that were isolated from the highly proteolytic industrial strains hydrolyzed β -casein preferentially and, consequently, are more like the PI-type proteinase than the PIII-type. We used simple, rapid methods to test a large number of strains to predict their lytic behavior and proteolytic activity in cheese.

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

International audience; The casein micelle is a highly aggregated colloid consisting of phosphoproteins and minerals, in particular calcium and phosphate. Its properties are affected by physico-chemical changes which provide possibilities for the development of new casein aggregates (CAs) with novel functionalities. The aim of this study was to investigate the emulsifying and emulsion-stabilizing capacity of gradually demineralized CAs in model dairy emulsions. Tri sodium citrate (TSC) was used to remove calcium and inorganic phosphate from pure casein micelles in order to produce four suspensions of differently demineralized CAs. Two types of milkfat-in-suspension (30:70 v/v) emulsions were then prepared to study the emulsifying and emulsion-stabilizing capacity of these CAs separately. Casein micelles were progressively demineralized (from 24 to 81% calcium reduction) and dissociated with the increase in TSC concentration. Three distinct populations of particles (micelle-like aggregates, sodium caseinate-like aggregates and casein monomers) were present in every suspension in different proportions. The smaller CAs had better emulsifying capacity and similar surface activity according to interfacial studies. The state of aggregation of the CAs was thus the main factor that controlled their emulsifying capacity. However, the emulsions formed with these smaller aggregates were less stable against creaming and flocculation, but still resisted coalescence under our storage conditions (21 days at 50 °C). The properties of the interfacial casein layers did not depend on the aggregation state of the CAs used to form the emulsions. The differences in instability were attributed to the nature of the non-adsorbed CAs and storage conditions.