Gwénaële Henry

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.

Milk proteins and iron absorption: contrasting effects of different caseinophosphopeptides.

Clusters of phosphoserine residues in cow milk caseins bind iron (Fe) with high affinity. Casein inhibits Fe absorption in humans, but protein hydrolysis lessens this effect. Phosphopeptides from different caseins gave conflicting results on Fe absorption; release of phosphate residues by intestinal alkaline phosphatase could be a key point of that metabolism. The objectives of this study were to compare the absorption of Fe complexed to caseinophosphopeptides (CPP) of the main cow milk caseins β-casein (β-CPP) and αs-caseins (αs1-CPP) and to assess the role of alkaline phosphatase on this absorption. Two experimental models were used: an in vivo perfused rat intestinal loop and an in vitro Caco-2 cell culture model. In addition, we determined the effect of an intestinal phosphatase inhibitor on these various forms of Fe. Gluconate Fe was used as control. In both models, uptake and net absorption of Fe complexed to CPP from αS1-caseins were significantly lower than from Fe complexed to β-CPP. Inhibition of the intestinal phosphatase significantly increased the uptake and the absorption of Fe complexed to β-CPP without effect on the other forms of Fe. These results confirm the enhancing effect of β-casein and its CPP on Fe absorption. The differences between CPP could be explained by their structural and/or conformational features: binding Fe to αS1-CPP could impair access to digestive enzymes, whereas β-CPP–bound Fe is better absorbed than its free form. The differences in protein composition between cow and breast milk, which does not contain α-casein, could explain some of their differences in Fe bioavailability.

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.

Kinetics of Fibril Formation of Bovine κ-Casein Indicate a Conformational Rearrangement as a Critical Step in the Process

S-carboxymethylated (SCM) kappa-casein forms in vitro fibrils that display several characteristics of amyloid fibrils, although the protein is unrelated to amyloid diseases. In order to get insight into the processes that prevent the formation of amyloid fibrils made of kappa-caseins in milk, we have characterized in detail the reaction and the roles of its possible effectors: glycosylation and other caseins. Given that native kappa-casein occurs as a heterogeneous mixture of carbohydrate-free and carbohydrate-containing chains, kinetics of fibril formation were performed on purified glycosylated and unglycosylated SCM kappa-caseins using the fluorescent dye thioflavin T in conjunction with transmission electron microscopy and Fourier transform infrared spectroscopy for morphological and structural analyses. Both unglycosylated and glycosylated SCM kappa-caseins have the ability to fibrillate. Kinetic data indicate that the fibril formation rate increases with SCM kappa-casein concentration but reaches a plateau at high concentrations, for both the unglycosylated and glycosylated forms. Therefore, a conformational rearrangement is the rate-limiting step in fibril growth of SCM kappa-casein. Transmission electron microscopy images indicate the presence of 10- to 12-nm spherical particles prior to the appearance of amyloid structure. Fourier transform infrared spectroscopy spectra reveal a conformational change within these micellar aggregates during the fibrillation. Fibrils are helical ribbons with a pitch of about 120-130 nm and a width of 10-12 nm. Taken together, these findings suggest a model of aggregation during which the SCM kappa-casein monomer is in rapid equilibrium with a micellar aggregate that subsequently undergoes a conformational rearrangement into a more organized species. These micelles assemble and this leads to the growing of amyloid fibrils. Addition of alpha(s1)-and beta-caseins decreases the growth rate of fibrils. Their main effect was on the elongation rate, which became close to that of the limiting conformation change, leading to the appearance of a lag phase at the beginning of the kinetics.

Tryptic hydrolysis of κ-caseinomacropeptide: control of the enzymatic reaction in a continuous membrane reactor

Abstract The kinetics of the tryptic release of bioactive peptides from caseinomacropeptide was investigated in both batch and continuous mode in an enzymatic membrane reactor. The hydrolysis of the three susceptible bonds, Lys 111 –Lys 112 , Lys 112 –Asn 113 , and Lys 116 –Thr 117 , was monitored by quantitative determination of the released products. A kinetic study in the batch system showed that the overall catalytic process follows a sequential mechanism where the Lys 116 –Thr 117 bond was only cleaved on the intermediary products resulting from the cleavage of the Lys 111 –Lys 112 and Lys 112 –Asn 113 bonds. When the reaction was performed in the continuous enzymatic membrane reactor, it was found that the enzyme preference toward the Lys 116 –Thr 117 bond depended on the relative concentrations of both the caseinomacropeptide and the intermediary products accumulated at steady state. Such concentrations were controlled by the enzyme and substrate concentrations and the substrate feeding flow rate; hence, by control of the operating parameters and with the understanding of the reaction mechanism, the enzyme action toward various peptidic bonds can be oriented in the continuous mode, offering the possibility of better control of the type of product recovered in the reactor output.

Separation of small cationic bioactive peptides by strong ion-exchange chromatography

Abstract The high resolving power of a Hyper D cation-exchange column was evaluated to achieve the separation of small cationic bioactive peptides derived from tryptic digest of κ-casein: MAIPPKK (pI = 9.9), MAIPPKK (pI = 10.5), KNQDK (pI = 9.6) and NQDK (pI = 6.35). The influence of pH (1.5–6), gradient slope (2–10 mM sodium chloride/min) and elution of the mixture under isocratic conditions was investigated. Although their physico-chemical properties are very similar, these four peptides were readily resolved with an excellent selectivity and recovery. The selectivity of the exchanger was also expressed toward peptides of the same net positive charge; the most hydrophilic peptide always eluted last. It was also shown that the elution order of these molecules depends on pH. From the observed retention times and the elution order, we have established a simple approach to linearization of peptide retention behaviour on the S-Hyper D support.

Sensitivity of β-casein phosphopeptide-iron complex to digestive enzymes in ligated segment of rat duodenum

Binding iron to the phosphorylated beta(1-25) peptide derived from beta-casein improves iron bioavailability in the rat. The aim of the present work was to learn how injected beta(1-25) and iron-beta(1-25) complex behave in the duodenum of rats using the technique of intestinal ligation in situ and reversed-phase (RP)-high performance liquid chromatography-electrospray mass spectrometry analysis of the lumen contents. The results demonstrate that beta(1-25) is sensitive to digestive enzymes including proteases/peptidases and phosphatases during duodenal transit. The lumen contents of rats perfused with iron free beta(1-25) contained all peptidic sequences derived from beta(1-25). In contrast, the phosphorylated part of beta(1-25) [i.e., beta(15-24)] was not detected in lumen of rats perfused with iron-beta(1-25) complex.

Glucose slows down the heat-induced aggregation of β-lactoglobulin at neutral pH.

The behavior of β-lactoglobulin (β-Lg) during heat treatments depends on the environmental conditions. The influence of the presence or absence of a reducing sugar, namely, glucose, on the modification of the protein during heating has been studied using fluorescence, polyacrylamide gel electrophoresis (PAGE), size-exclusion chromatography (SEC), and transmission electron microscopy. Glycated products were formed during heating 24 h at 90 °C and pH 7. The fluorescence results revealed an accumulation of the advanced Maillard products and the formation of aggregates during heating. PAGE and SEC data suggested that the products in the control samples were essentially composed of covalently linked fibrillar aggregates and that their formation was faster than that for glycated samples. We showed that glucose affected the growing step of covalent aggregates but not the initial denaturation/aggregation step of native protein. Glucose-modified proteins formed a mixture of short fibrils and polydisperse aggregates. Our results revealed that β-Lg forms fibrils at neutral pH after heating and that glucose slows the formation of these fibrils.

Precipitation of hydrophobic peptides from tryptic casein hydrolysate by salt and pH

The precipitation of peptides derived from tryptic hydrolysis of caseins was studied as a function of the pH (3.5 to 7.5), ionic strength (from 0 to 1 m), and the chemical nature of the salt [NaCl (NH4)2 SO4] at 25°C. Precipitation occurred only in the acidic pH range. The precipitated fraction, analyzed by RP-HPLC, was constituted by a specific hydrophobic peptide group. The nine major peptides were identified as κ-CN (35–63), αs1-CN (91–100), αs1-CN (125–151), αs1-CN (152–193), β-CN (49–97), β-CN (108–169), β-CN (114–169), β-CN (184–202), and β-CN (184–209). The optimum precipitation was found to be at pH 3.5 and 0.25 m NaCl except for β-CN (184–202) and αs1-CN (125–151), which required a higher ionic strength. The results showed that pH had a greater effect on the precipitation of peptides than salt. However, neither pI nor hydrophobicity alone could have explained the salting-out behavior; probably a combination of the two properties is responsible.

Identification in milk of a serum amyloid A peptide chemoattractant for B lymphoblasts

BackgroundNormal mammary gland contains an extravascular population of B lymphoblasts, precursors of the immunoglobulin plasma cells that play a key role in the passive protection of neonates by secreting immunoglobulins to colostrum and milk. We investigated the presence of chemoattractants in the milk by analysing the chemoattractant activity of various fractions of this secretion. Milk chemoattractants are potentially involved in the recruitment of lymphocytes from the maternal bloodstream in lactating mammary glands.ResultsThe dilution-related lymphoid cell chemoattraction of whey was associated with a < 10 kDa ultrafiltrate. Active fractions were purified by reverse-phase high performance liquid chromatography. Two peptides of 2.7 kDa (DMREANYKNSDKYFHARGNYDAA) and 1 kDa (RPPGLPDKY) were identified as fragments of the SAA protein family, tentatively identified as SAA2. Only the 2.7 kDa synthetic peptide displayed chemotactic activity, at two different optimal concentrations. At the lower concentration (3.7 nM), it attracted B-cell lymphoblasts, whereas at the higher (3.7 μM), it attracted B lymphocytes. Then, the SAA mRNA expression was analysed and we observed more SAA transcripts during lactation than gestation.ConclusionThese data are consistent with the SAA23–45 fragment being involved in preplasma B-cell recruitment to the mammary gland and resultant benefit to the neonate.