S. Bouhallab

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.

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.

Mechanisms of absorption of caseinophosphopeptide bound iron.

Binding iron (Fe) to the 1-25 caseinophosphopeptide obtained from enzyme hydrolysis of beta casein (beta CPP) improves Fe bioavailability in the rat. To assess the mechanisms involved in its absorption, a perfused, vascularized duodenal rat loop model was used in controls and in Fe-deficient (bleeding of 25% blood volume) rats. Inhibitors of oxidative phosphorylation [2-4 dinitrophenol (DNP)] and/or of endocytosis [phenylarsine oxide (PAO)] were added to the perfusion solution containing 50 microM Fe as beta CPP bound Fe (Fe-beta CPP) or gluconate (Fe Gluc). Fe-beta CPP enhanced Fe uptake, reduced mucosal storage, and improved net absorption both in controls and in deficient animals. DNP reduced uptake, mucosal storage, and net absorption by the same percentage in Fe-beta CPP and Fe Gluc perfused rats in both control and Fe-deficient animals. PAO decreased uptake, mucosal storage, and net absorption of Fe-beta CPP but not of Fe Gluc. At the end of the experiment Fe serum levels were increased only in Fe Gluc animals. These results confirm the improved bioavailability of beta CPP bound Fe. They suggest that at least part of its absorption can occur by a different pathway than usual Fe salts. Fe-beta CPP can be taken up by endocytosis and absorbed bound to amino acids or peptides.

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.

Continuous hydrolysis ofβ-casein in a membrane reactor: Preparation of a bioactive peptide

SummaryContinuous Stirred Tank Membrane Reactor was used to investigate the continuous and selective extraction of a bioactive peptideβ-CN (193–209) from bovineβ-casein/chymosin hydrolysate. It was shown that the feasibility of the process depends on the nature and the area of ultrafiltration membrane used. With an inorganic (carbon-zirconia) membrane, high retention of the peptide constitutes a limit to the operation. However, when the reactor was equipped with a cellulosic type membrane, satisfactory transmission of the peptide was obtained. As shown by RP-HPLC and mass spectrometry analysis, onlyβ-CN (193–209) permeates through the membrane.

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.

Modification of bovine β-lactoglobulin by glycation in a powdered state or in aqueous solution: adsorption at the air–water interface

Abstract The adsorption at the air–water interface of native and various glycated forms of β-Lactoglobulin B (β-LG), prepared under two different experimental conditions, was investigated by ellipsometry, surface tension and shear elastic constant measurements. The measurements were performed in 0.1 M phosphate buffer, 0.1 M NaCl, pH 6.8. It was found that the interfacial properties of β-LG were more affected when the glycation was performed in solution than in the dry-way system. Dry-way glycated β-LG, despite a higher glycation extent, affected slightly its interfacial behaviour. Solution glycated β-LG exhibited a higher adsorption and more rigid interface as expressed by shear elastic constant measurement at saturation (16.5 mN/m against 8.7 and 11.5 mN/m for native and control treated β-LG, respectively). These results were attributed to the specific molecular species induced during glycation in solution, which includes monomers and unfolded covalent homodimers of β-LG molecules with a high tendency to self-association via non-covalent interactions.

Resistance of β-lactoglobulin-bound lactose to the hydrolysis by β-galactosidase

An in vitro study was conducted to investigate the sensitivity of lactose-β-lactoglobulin conjugates to β-galactosidase from Kluyveromyces lactis. The hydrolysis was monitored by ion exchange chromatography. Compared to free lactose or lactulose, which were rapidly hydrolysed, protein-bound lactose was not hydrolysed by β-galactosidase even after an extended hydrolysis time. Tryptic digestion of the conjugates before addition of β-galactosidase improved the substrate accessibility and led to the release of about 50% of the linked lactose. The results strongly suggest that the resistance of protein bound lactose is linked to the globular and compact structure of lactose-β-lactoglobulin conjugates.