Joëlle Léonil

Identification of C-terminal peptides of bovine β-casein that enhance proliferation of rat lymphocytes

A casein polypeptidic fraction, obtained from a pepsin-chymosin digestion of caseins, showed a mitogenic effect on primed lymph node (LN) cells and unprimed spleen cells of rats. A biologically active C-terminal sequence of bovine beta-casein (residues 192-209) was characterized. The corresponding synthetic peptide had a stimulatory effect on primed LN cells but failed to enhance proliferation of spleen cells. We prepared two chymosin digests (PA and PB) of bovine beta-casein which contained, respectively, 80% and 95% of the sequence including residues 193-209. They induced a significant proliferative response in both LN and spleen cells. It is therefore possible that other active peptides in the PA preparation may be involved in mitogenic activity.

Food processing increases casein resistance to simulated infant digestion.

The objective of this study was to determine whether processing could modify the resistance of casein (CN) to digestion in infants. A range of different dairy matrices was manufactured from raw milk in a pilot plant and subjected to in vitro digestion using an infant gut model. Digestion products were identified using MS and immunochemical techniques. Results obtained showed that CNs were able to resist digestion, particularly κ- and αs(2)-CN. Resistant areas were identified and corresponded to fragments hydrophobic at pH 3.0 (gastric conditions) and/or carrying post-translational modifications (phosphorylation and glycosylation). Milk processing led to differences in peptide patterns and heat treatment of milk tended to increase the number of peptides found in digested samples. This highlights the likely impact of milk processing on the allergenic potential of CNs.

Peptides du lait à activité biologique

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Induction of New Physicochemical and Functional Properties by the Glycosylation of Whey Proteins

Nucleophilic primary amino groups of whey proteins (β-lactoglobulin and α-lactalbumin) were modified with reducing sugars in mild heat conditions. After 49 hr of heating (60°C) at pH 6.5, 20–30% of β-lactoglobulin amino groups were substituted with aldohexoses (galactose, mannose, glucose) and lactose, whereas up to 70% and 90% of β-lactoglobulin amino groups were modified with ribose and glyceraldehyde, respectively. Gel electrophoresis and reversed-phase HPLC coupled with electrospray ionization mass spectrometry of glycosylated proteins indicated that the substitution was random. Consequently, highly heterogeneous families of glycosylated proteins were generated. Proteins substituted with hexoses and lactose exhibited higher solubility and improved emulsifying properties as compared with nonglycosylated proteins, in the whole pH range studied. In contrast, proteins glycosylated with ribose and glyceraldehyde showed lower solubility close to their isoelectric points. β-Lactoglobulin modified with ribose and glyceraldehyde displayed substantial differences in denaturation behavior as compared with native protein. When compared with β-lactoglobulin, glycosylation of α-lactalbumin was quicker. There was no difference in glycosylation yields nor rates of α-lactalbumin in presence and absence of calcium.

Application of chromatography and mass spectrometry to the characterization of food proteins and derived peptides.

The following review describes the development of mass spectrometry off-line and on-line coupled with liquid chromatography to the analysis of food proteins. It includes the significant results recently obtained in the field of milk, egg and cereal proteins. This paper also outlines the research carried out in the area of food protein hydrolysates, which are important components in foodstuffs due to their functional properties. Liquid chromatography and mass spectrometry have been particularly used for the characterization of food peptides and especially in dairy products.

Heterogeneity of the bovine κ-casein caseinomacropeptide, resolved by liquid chromatography on-line with electrospray ionization mass spectrometry

Abstract Microheterogeneity occurs in the population of caseinomacropeptides (residues 106–169 of κ-casein) due to variation in the extent and type of oligosaccharide linked to this phosphoglycopeptide. Although caseinomacropeptide A variant (CMPA) was poorly resolved using reversed-phase high-performance liquid chromatography (RP-HPLC) with spectrophotometric detection, it could be analysed with on-line electrospray-ionization mass spectrometry (ESI-MS). From the already established O-linked glycan chains at least fourteen glycosylated forms of CMPA were identified, besides the non-glycosylated and multiphosphorylated (1, 2 or 3 phosphate groups) peptides, giving a maximum number of eighteen known forms. Major subcomponents in CMPA are disialylated species. A maximum of three out of the five potential glycosylation sites were found to be substituted with carbohydrate chains in the most highly glycosylated forms, which may contain up to six N-acetylneuraminic acid residues per molecule. A minor form, diphosphorylated with one disialylated chain, was also detected. From these results, it was shown that the on-line coupling of HPLC with ESI-MS offers a very promising alternative for the analysis of complex mixtures.

Lactolation of β-lactoglobulin monitored by electrospray ionisation mass spectrometry

Direct monitoring of the Amadori product formation between lactose and β-lactoglobulin was performed by electrospray ionisation mass spectrometry. As expected, the glycation reaction was faster at low water activity than in aqueous system. Heterogeneous β-lactoglobulin glycoforms were observed, with different number of lactose bound: populations of β-lactoglobulin with 1 to 7 and 2 to 11 linked lactose were detected, respectively, after 10 and 22 h of reaction under 65% relative humidity and 50°C. Trypsinolysis of glycated proteins, followed by reverse-phase HPLC coupled to tandem mass spectrometry in the neutral loss scanning mode, indicated that all potential reactive amino groups, except Lys101, were involved in sugar binding. The resulting order of reactivity, as a function of reaction time, was as follows: Lys47,91>α-amino-group, Lys15,70,100>Lys60,69,75,77,83,135,138>Lys8,141.

Phosphopeptides interacting with colloidal calcium phosphate isolated by tryptic hydrolysis of bovine casein micelles

After extended tryptic hydrolysis of large bovine casein micelles, a mineral-rich peptide fraction was recovered by ultracentrifugation. Its mineral part contained 72% of the colloidal Ca and 49% of the colloidal P1 originally present in the native micelle. Colloidal nitrogenous components were also present, amounting to 27% of the original N content. They contained most of the phosphopeptides and 82% of the micellar phosphoseryl residues. These tryptic peptides were characterized by reversed-phase HPLC on-line electrospray ion source-mass spectrometry analysis. Among the peptides produced 14 phosphopeptides were identified: alpha s2-CN(1-24), alpha s2-CN(1-21), alpha s1-CN(43-79), alpha s1-CN(35-79)7P, alpha s1-CN(35-79)8P, alpha s1-CN(37-79), alpha s1-CN(104-119), alpha s1-CN(104-124), beta-CN(1-25), beta-CN(1-28), beta-CN(1-29), beta-CN(30-97), beta-CN(33-97) and beta-CN(29-97). The proportion of the phosphopeptides interacting with colloidal calcium phosphate was correlated with their relative content of phosphoserine residues, since phosphopeptides containing more than four phosphoserine residues were consistently present within this fraction. It also appeared that other types of peptides, some of them hydrophobic in nature, were also partly or completely present within the colloidal fraction, including alpha s1-CN(91-100), alpha s1-CN(152-193), alpha s1-CN(23-34), alpha s1-CN(125-193), alpha s1-CN(125-199), beta-CN(177-209). beta-CN(184-209), beta-CN(114-169) and beta-CN(108-169). Their possible involvement in the micellar backbone is discussed.

A method for determination of macropeptide by cation-exchange fast protein liquid chromatography and its use for following the action of chymosin in milk

Cation-exchange chromatography on a Mono S column (Pharmacia) was used to separate macropeptide from whey proteins. Macropeptide was eluted by 0·1 M-NaCl in a 20 mM-KCl–HCl buffer, pH 2. This technique was suitable for quantitative determination of macropeptide in rennet whey and also for following the action of chymosin on κ-casein in skim milk. Precipitation at pH 4·6 was used to remove residual caseins and to keep macropeptide in solution. In comparison with other methods for determining macropeptide, the present one eliminates the need for pretreatment of samples with trichloroacetic acid (TCA) and allows the recovery of all the macropeptide. Quantitative determination of macropeptide in the 8% TCA-soluble fraction by cation-exchange chromatography showed that only 50–75% of the macropeptide was recovered. This chromatographic technique could also be applied for isolating and producing whole macropeptide on a preparative scale.

A novel bioactive peptide from yoghurts modulates expression of the gel-forming MUC2 mucin as well as population of goblet cells and Paneth cells along the small intestine

Several studies demonstrated that fermented milks may provide a large number of bioactive peptides into the gastrointestinal tract. We previously showed that beta-casomorphin-7, an opioid-like peptide produced from bovine β-casein, strongly stimulates intestinal mucin production in ex vivo and in vitro models, suggesting the potential benefit of milk bioactive peptides on intestinal protection. In the present study, we tested the hypothesis that the total peptide pool (TPP) from a fermented milk (yoghurt) may act on human intestinal mucus-producing cells (HT29-MTX) to induce mucin expression. Our aim was then to identify the peptide(s) carrying the biological activity and to study its impact in vivo on factors involved in gut protection after oral administration to rat pups (once a day, 9 consecutive days). TPP stimulated MUC2 and MUC4 gene expression as well as mucin secretion in HT29-MTX cells. Among the four peptide fractions that were separated by preparative reversed-phase high-performance liquid chromatography, only the C2 fraction was able to mimic the in vitro effect of TPP. Interestingly, the sequence [94-123] of β-casein, present only in C2 fraction, also regulated mucin production in HT29-MTX cells. Oral administration of this peptide to rat pups enhanced the number of goblet cells and Paneth cells along the small intestine. These effects were associated with a higher expression of intestinal mucins (Muc2 and Muc4) and of antibacterial factors (lysozyme, rdefa5). We conclude that the peptide β-CN(94-123) present in yoghurts may maintain or restore intestinal homeostasis and could play an important role in protection against damaging agents of the intestinal lumen.