Colostrum protein digestion in newborn lambs.

The efficiency of colostral protein digestion was studied in nine newborn lambs fed one meal of bovine colostrum 3 h after birth. The results were compared with those obtained in two unfed lambs and four lambs fed bovine milk. The protein and peptide composition [immunoglobulins G1 and (IgG1), beta-lactoglobulin, alpha-lactalbumin, caseins and peptides resulting from casein hydrolysis] of digesta, gastrointestinal tissues, blood and urine were determined in samples taken 0.75 or 4 h after feeding. The amounts of ingested proteins in lambs fed colostrum were much higher than in those fed the milk diet, and their abomasal emptying was faster. alpha-Lactalbumin was highly degraded by abomasal and intestinal proteases, whereas beta-lactoglobulin and in particular the immunoglobulins were less sensitive. The gastric emptying of caseins was delayed in and the kinetics of appearance of peptides originating from casein hydrolysis was comparable to that observed in lambs fed milk and in 1-mo-old preruminant calves. Thirty-five percent of dietary amino acids ingested as colostrum were available within 4 h for amino acid metabolism; this percentage was 54% in the milk-fed lambs. In the lambs fed colostrum, these amino acids were provided by beta-lactoglobulin, casein and IgG1 (0.52, 0.43 and 0.30 g/kg body wt, respectively), whereas in milk-fed animals casein and beta-lactoglobulin were the most important sources of these amino acids (0.40 and 0.20 g/kg, respectively).

Characterization and kinetics of gastric emptying of peptides derived from milk proteins in the preruminant calf.

The gastric emptying kinetics of peptides derived from milk protein were studied in vivo in preruminant calves by collecting and characterizing the whole effluent leaving the stomach for 12 h after ingestion of crude skim milk. Peptides were isolated by reversed-phase HPLC and identified. Particular attention was paid to biologically active peptides and to peptides that could be precursors of biologically active sequences. A gastrin inhibitor, the caseinomacropeptide, was emptied from the stomach only during the first 0.5 h of digestion and rapidly hydrolysed. Precursors of immunostimulatory peptides from alpha s1- and beta-caseins were emptied throughout digestion in the gastric effluent. A precursor of beta-casomorphins (peptide 58-93 of beta-casein) was emptied from the stomach 3.5 h after the meal when it was taken on an empty stomach. From this precursor, peptides that may be resistant to hydrolysis by intestinal peptidase were obtained after in vitro hydrolysis by pancreatic enzymes. A phosphopeptide (fragment 110-142 of alpha s1-casein) was also found in digesta after a few hours of digestion. When the meal was not taken on an empty stomach, these peptides were emptied in the first digesta at a low concentration. The potential activity of these peptides is discussed. The results support the hypothesis that active sequences could still be present in the gut after the action of pancreatic enzymes.

Hydrolysis of beta-casein by gastric proteases. I. Comparison of proteolytic action of bovine chymosin and pepsin A.

Hydrolysis of beta A2-casein by bovine chymosin and pepsin A was performed in order to compare the hydrolysis of the two enzymes on this protein. Different conditions have been tested: pH 5.5 for 116h and pH 3.5 for 7 h [E/S = 1/100 (w/w)] for chymosin. pH 3.0 for 24 h [E/S = 1/1000 (w/w)] for pepsin A. Under these conditions 17 peptides were obtained after the action of chymosin and 23 after the action of pepsin A. They corresponded respectively to the cleavage of 14 and 15 peptide bonds for chymosin and pepsin A. However, six of the peptide bonds were only hydrolyzed by chymosin and seven other bonds only by pepsin A. Our results showed a preferential splitting at the Leu-X, Ser-X, and Trp-X bonds for chymosin and Leu-X, Met-X, and Thr-X, for pepsin A. Some of the identified peptides contained sequences with possible physiological roles.

Immobilized Fe3+ affinity chromatographic isolation of phosphopeptides.

Immobilized Fe3+ affinity chromatography is suggested as a means of concentrating phosphopeptides that are present in too low a proportion in a complex mixture to be purified by a single-step method. A high-performance liquid chromatographic system and a chelating Superose HR 10/2 column were used. The chromatographic conditions were optimized using a tryptic hydrolysate of whole casein. The unbound fractions did not contain any phosphorylated peptide. All caseinophosphopeptides were retained. Only four other strongly basic peptides were also retained. The quantitative accuracy of the method was evaluated. This method allowed the isolation of phosphopeptides in gastric effluents of calves fed with milk.

Solubility of peptides in trichloroacetic acid (TCA) solutions. Hypothesis on the precipitation mechanism.

The assessment of proteolysis levels is often achieved by global quantification of the peptides soluble at different TCA concentrations, but little information is available on the features of this precipitation mechanism. Peptic, tryptic and chymotryptic digests of alpha s1, beta, and kappa caseins have been prepared and fractionated by RP-HPLC and each isolated peptide was identified. Each digest was precipitated by adding TCA to different final concentrations (2, 4, 8, and 12%). The soluble fraction was analysed by RP-HPLC. Relationships have been searched between the properties of 75 peptides obtained in this way, and their solubilities in TCA. The best correlation was found with the peptide retention time in RP-HPLC, which can be regarded as the experimental measure of peptide hydrophobicity. We concluded that TCA, by interacting with peptides, induces an increase of the hydrophobicity of peptides which can lead to aggregation through hydrophobic interactions.

Digestion by pancreatic juice of a beta-casomorphin-containing fragment of buffalo beta-casein.

Degradation by pig pancreatic juice of a beta-casomorphin-containing fragment (tryptic peptide corresponding to residues 49-68 of buffalo beta-casein) was investigated. The FAB/MS (fast atom bombardment mass spectrometry) technique was used to identify the fragments produced by the concerted action of pancreatic proteases. Pancreatic juice, under our experimental conditions, is not able to release beta-casomorphins or morphiceptin from the tryptic peptide sequence. Furthermore, the present report shows that the rapid hydrolysis of a peptide bond by a single protease can prevent the cleavage of peptide bonds by a different protease. Therefore the formation of some peptides in the gastrointestinal tract can depend on the protease ratio.

[Synthesis of milk proteins]. / Synthèse des protéines du lait.

This review, written for non-specialists, describes briefly the steps of protein biosynthesis from their precursors in the blood to the excreted molecules, taking rat gamma-casein as an example. A schematic description is given of the procedures employed for preparing cDNAs which can provide the sequences of the corresponding mRNAs and proteins. Recent findings concerning milk proteins are briefly mentioned, and in particular those dealing with sequence determinations of mRNAs coding for milk proteins from several species.

[Digestion of milk proteins in the abomasum of the preruminant calf]. / Digestion des protéines du lait dans la caillette du veau préruminant.

Gastric digestion of milk proteins has been studied in the preruminant calf by analysing gastric effluents in the duodenum after the ingestion of 5 different diets: whole milk, skim milk, casein solution in water, casein solution with minerals, whey. The amino acid composition of the gastric effluents and HPLC analysis of the latter show that the major part of the whey proteins were evacuated rapidly without any proteolysis and after milk ingestion. On the contrary, all the caseins were largely proteolysed and arrived slowly in the gut. The major part of the peptides obtained came from alpha s1 casein. Only a few peptides, principally CMP, were quickly emptied from the stomach.

Inhibitory effect of a copper-dipeptide complex on the establishment of a Clostridium perenne strain in the intestinal tract of gnotobiotic mice.

A semisynthetic diet fed to axenic mice was found to prevent the establishment of a Clostridium perenne strain in their intestinal tract. This inhibitory effect did not occur when axenic mice were preinoculated with a strain of Clostridium difficile. The inhibitory effect was related to the presence in the intestinal contents of axenic mice of both dietary copper and a dipeptide, aspartic-epsilon-lysine. When C. difficile was inoculated into axenic mice, the dipeptide disappeared from the digesta, and C. perenne became established even in the presence of high concentrations of copper.

In vivo milk digestion in the calf abomasum. III. Amino acid compositions of the digesta leaving the abomasum.

Calves were fed five different test meals: whole milk, skim milk, 3% whole casein solution, 3% whole casein in simulated milk ultrafiltrate, and whey. The digesta leaving the abomasum before feeding and during the first 7 postprandial hours were collected by fractions. After precipitation with 12% TCA, the amino acid compositions of the sediments and the supernatants were determined and compared by multivariate analysis. The composition of prefeeding digesta was similar to that of gastric juice. When the calves were fed the two casein diets, the amino acid composition of the sediments changed little with time. In contrast, the changes observed in the composition of the supernatants suggested rapid abomasal emptying of caseino-macro peptide. With the whey diet, it was not possible to evidence more rapid hydrolysis or abomasal emptying of any particular whey protein. During the first 10 min following the ingestion of whole or skim milk, the amino acid composition of the sediment was close to that of milk protein. Immediately afterwards, the composition of the sediment was similar to that of whey protein. Thereafter, the composition of the sediment became more like that of casein and almost reached that of casein during the 7th hour. The amino acid composition of the supernatant was similar to that obtained with the casein diets; this fact suggests that the small peptides produced by proteolysis in the abomasum originated more from casein than from whey proteins.

In vivo milk digestion in the calf abomasum. I. Whole-casein digestion.

Peptide products insoluble in 12% TCA and obtained in the proximal duodenum of calves at different times after ingestion of casein solutions were characterized by polyacrylamide gel electrophoresis, electrofocusing and SDS pore gradient gel electrophoresis. With a 3% whole-casein solution in water the disappearance of electrophoretic bands corresponding to alpha s1 and beta caseins was observed after about 1 h 30. After 3 h and up to 7 h very acidic peptides appeared. With a 3% whole-casein solution in simulated milk ultrafiltrate, comparable patterns were obtained. Nevertheless, the acidic peptides appeared sooner, i.e. they were already detected in the first sample collected after meal ingestion. With the 2 diets, the importance of gastric proteolysis was demonstrated by the appearance of a great number of peptides of various sizes, charges and pHi. On the other hand, an effect of salts on casein proteolysis was detected.

Kinetic studies of in vivo digestion of bovine unheated skim-milk proteins in the rat stomach.

The in vivo action of gastric proteinases on bovine milk proteins was studied in rats fed with skim-milk, by analysing gastric contents after 30, 60 and 240 min of digestion. Gastric proteolysis was already marked after 30 min of digestion and liberated a large number of peptides with different molecular weights. alpha S1-Casein and beta-casein were still the main components of the stomach contents together with alpha-lactalbumin and beta-lactoglobulin, which were little degraded, even after 240 min of digestion. A statistical analysis (multivariate method), made on several parameters (such as pH, N, and NPN) showed changes in the stomach contents during the digestion. The amino acid composition of the protein fraction was close to that of the diet, whilst that of the non-protein fraction was very different, being between the amino acid composition of the endogenous proteins and that of the diet.

beta-Aspartyl-epsilon-lysine, a peptide of the fecal contents of axenic mice.

Small quantities of low-molecular weight peptides have been characterized in the feces of axenic mice. In fecal material of axenic mice fed an autoclaved synthetic (SN) diet, we isolated a dipeptide and characterized its structure as beta-aspartyl-epsilon-lysine. This product was also present in the feces of gnotobiotic mice harbouring Clostridium perenne. We could not detect the product in the fecal contents of holoxenic mice, Clostridium difficile-contaminated mice or axenic mice fed the irradiated SN diet. The peptide, beta-aspartyl-epsilon-lysine, was produced by heating proteins during sterilization, causing the formation of a pseudopeptide bond between the epsilon-amino group of lysine and the amide group of asparagine. The intestinal strains seemed to differ in their ability to split this pseudopeptide bond in vivo.

[Digestion of milk proteins in the abomasum of the preruminant calf. Gastric evacuation after a test meal]. / Digestion des protéines du lait dans la caillette du veau préruminant. Evacuation gastrique après un repas d'épreuve.

In this study, we used fasted, preruminant calves fitted with a double proximal cannula in the duodenum; gastric evacuation was investigated by recovering products leaving the abomasum after a test meal. Five types of meal were given: whole milk, skimmed milk, casein solution in water, casein solution in a mineral medium simulating the permeate of highly-filtered milk, and whey. Gastric emptying of nitrogen was relatively slow. The coagulation of "milk diets" slowed down this emptying. Precipitation of "casein diets" at acid pH also slowed down emptying, but to a lesser degree. The "casein diets", more proteolyzed than the "milk diets", released larger amounts of non-protein nitrogen (NPN) into the intestine. Calcium emptying was highly related to NPN emptying, demonstrating the important role of acid peptides (phosphopeptides) from casein hydrolysis.

[Search for phosphopeptides in the feces of axenic rats fed radioactive ovine casein]. / Recherche de phosphopeptides dans les fèces de rats axéniques alimentés avec de la caséine ovine radioactive.

Radioactive ovine casein was obtained by injecting 100 microCi of 14C-Ser into the jugular vein of an ewe. The milk collected 17 and 24 h after this injection contained 12 p. 100 of the radioactivity injected in protein form. The seryl residues were specificially labelled. This casein was used as the only protein source fed to axenic rats; 0.30 p. 100 of the tracer ingested was found in the feces of those rats. Since phosphoserine represented 25 p. 100 of the total casein seryl residues, the phosphopeptides may not be selectively unabsorbable.

Preparation and fractionation of goat kappa-casein: analysis of the glycan and peptide components.

Whole goat kappa-casein was prepared by chromatography of whole casein on hydroxyapatite. Chromatography of whole kappa-casein on DEAE-cellulose separated 5 fractions. All of them were sensitive to chymosin. Their amino acid and carbohydrate composition, phosphate content and molecular weight were determined. Galactose, N-acetylgalactosamine, N-acetyl and N-glycolyl neuraminic acids were identified in whole kappa-casein. It appears that goat kappa-casein, like cow, buffalo and ewe kappa-caseins, is composed of several fractions having identical peptide chains and differing in their carbohydrate contents. The main fraction, devoid of carbohydrate, was treated with chymosin. The para-kappa-casein and caseinomacropeptide were isolated. Their amino acid composition and phosphate content were determined.

[Primary structure of the casein macropeptide of caprine kappa casein]. / Structure primaire du caséinomacropeptide de la caseine K caprine

The amino acid sequence of caprine CMP, the negatively charged C-terminal fragment released by chymosin (rennin EC 3.4.23.4) from goat K-casein at the initial stage of the milk-clotting process, has been investigated. The complete sequence has been determined by analysing chymotryptic and "thermolysin" fragments of the CMP. Caprine CMP contains 66 amino acid residues, 2 being phosphorylated. Asp2, Asn5, Thr11, Ser6, SerP2, Glu7, Gln2, Pro6, Ala9 Val5, Met1, Ile6, Lys3, His1, and the carbohydrate-free polypeptide chain has a molecular weight of 6,998 daltons. The occurrence in caprine CMP of an additional phosphate group, linked to serine 168 in the C-terminal region Thr-Ser168-Thr-Glu170-Val.OH of the polypeptide chain, has given support to the phosphorylation code for caseins that we postulated earlier [28, 27]. According to this hypothesis, a specific phosphoryl kinase may recognize an anionic phosphorylation site corresponding to the tripeptide sequence Thr/Ser-X-Glu, X being any amino acid residue. Since the C-terminal sequence of bovine and caprine CMPs differ by the substitution Ala/Glu170 (caprine), phosphorylation of caprine serine 168 could be explained by the occurrence of the new phosphorylation site Ser168-Thr-Glu170.