Sylvie Marchesseau

Water-holding capacity and characterization of protein interactions in processed cheese

In order to predict the oozing susceptibility of heat-induced milk protein gels such as processed cheeses during storage, ultracentrifugation forces are required to accelerate the expulsion of water from the gel structure. Two predictive methods have been tested. Direct ultracentrifugation of processed cheese was used to study effects of centrifugal compression on the water-holding ability. The water release with optimal parameters (time, temperature and ultracentrifugation force) from a freshly manufactured processed cheese was correlated with visual assessment of oozing after 6 months storage. A second method was based on the susceptibility of gelled proteins to resist the dissociating action of solutions of chemical agents such as SDS, urea, EDTA and 2-mercaptoethanol. Most of the protein sedimented by ultracentrifugation in the presence of SDS represented that still in complexed form. This correlated with optimal water binding by the processed cheese. Response-surface methods, used to optimize the ultracentrifugation variables and the composition of the dissociation solutions, showed that the dissociation test was best with a dispersion of processed cheese in SDS solution (10 g/l) at a ratio of 1:6 (w/v) at 20°C, ultracentrifuged at 86000 g and 20°C for 25 min. Analysis of processed cheese cooked at 115°C and then dissociated in different solutions showed various protein–protein interactions in the gel network, whereas hydrophobic interactions were the most important stabilizers of the protein matrix of cheese cooked at higher temperature.

Epitope characterization of a supramolecular protein assembly with a collection of monoclonal antibodies: The case of casein micelle

In milk, kappa-, beta-, alphas(1)- and alphas(2)-casein (CN) are associated into a supramolecular assembly, the micelle. In this work, CN micelles contained in fresh skim milk were used to produce over 100 monoclonal antibodies. The specificity of these probes was determined using libraries of synthetic peptides and peptides fractionated from tryptic hydrolysis of purified CNs. Although kappa-CN and alphas(2)-CN are minor proteins in the micelle (ratio 1:1:4:4 for kappa, alphas(2), alphas(1), beta) a proportionally high number of clones were produced towards these two proteins (32 for each), compared to 9 and 29 for alphas(1)-CN and beta-CN, respectively. Most of the beta-CN and kappa-CN epitopes were identified, while about 50% of alphas(1)-CN and alphas(2)-CN antibodies were suspected to react to conformational linear or discontinuous epitopes, since no peptide binding could be identified. Antibody binding to the phosphoserine rich regions of the three calcium sensitive CNs was weak or non-existing, suggesting them to be hidden in the micelle structure together with alphas(1)-CN. The C-terminal glycomacropeptide of kappa-CN and the C-terminal moiety of beta-CN were well exposed generating the majority of the antibodies specific for these two proteins. The two major antigenic sites of alphas(2) were alphas(2)-CN (f96-114) and (f16-35). Cross-reaction between alphas(2)-CN specific antibodies with alphas(1)-CN illustrated the tangled structure between the two proteins. Immuno-dominant epitopes identified in the present study totally differ from those known for the purified caseins suggesting they were specific for the micelle supramolecular structure.

Topography of the Casein Micelle Surface by Surface Plasmon Resonance (SPR) Using a Selection of Specific Monoclonal Antibodies

Several theoretical models of the casein micelle structure have been proposed in the past, but the exact organization of the four individual caseins (α(s1), α(s2), β, and κ) within this supramolecular structure remains unknown. The present study aims at determining the topography of the casein micelle surface by following the interaction between 44 monoclonal antibodies specific for different epitopes of α(s1)-, α(s2)-, β-, and κ-casein and the casein micelle in real time and no labeling using a surface plasmon resonance (SPR)-based biosensor. Although the four individual caseins were found to be accessible for antibody binding, data confirmed that the C-terminal extremity of κ-casein was highly accessible and located at the periphery of the structure. When casein micelles were submitted to proteolysis, the C-terminal extremity of κ-casein was rapidly hydrolyzed. Disintegration of the micellar structure resulted in an increased access for antibodies to hydrophobic areas of α(s1)- and α(s2)-casein.

Influence of chemical agents on casein interactions in dairy products: chemical modification of milk proteins

Abstract Chemical modification of casein micelles by succinylation was investigated in order to assess the role of electrostatic charges in both acid and rennet coagulation, as well as to find a relationship between the chemical modification applied and the ability of milk proteins to coagulate. Succinylation of milk resulted in a delay of gelation and in a decrease in the final firmness of a rennet gel. The resistance of casein micelles to aggregation could be explained by the increase in electrostatic repulsion induced by the introduction of additional negative charges to the proteins. Results showed that the changes in the rheological properties upon rennet coagulation could be related to the microstructural differences observed between the succinylated and the reference milk: so changing the balance of protein charges upon decreasing the pHi increased the coagulation time of acid gels, but might also considerably alter the formation of hydrophobic interactions between succinylated casein micelles during the aggregation phenomena in rennet milk gels.

Foaming and adsorption behavior of bovine and camel proteins mixed layers at the air/water interface

The aim of this work was to examine foaming and interfacial behavior of three milk protein mixtures, bovine α-lactalbumin-β-casein (M1), camel α-lactalbumin-β-casein (M2) and β-lactoglobulin-β-casein (M3), alone and in binary mixtures, at the air/water interface in order to better understand the foaming properties of bovine and camel milks. Different mixture ratios (100:0; 75:25; 50:50; 25:75; 0:100) were used during foaming tests and interfacial protein interactions were studied with a pendant drop tensiometer. Experimental results evidenced that the greatest foam was obtained with a higher β-casein amount in all camel and bovine mixtures. Good correlation was observed with the adsorption and the interfacial rheological properties of camel and bovine protein mixtures. The proteins adsorbed layers are mainly affected by the presence of β-casein molecules, which are probably the most abundant protein at interface and the most efficient in reducing the interfacial properties. In contrast of, the globular proteins, α-lactalbumin and β-lactoglobulin that are involved in the protein layer composition, but could not compact well at the interface to ensure foams creation and stabilization because of their rigid molecular structure.

The foaming properties of camel and bovine whey: The impact of pH and heat treatment

The effect of heat treatment (70°C or 90°C for 30min) on the foaming and interfacial properties of acid and sweet whey obtained from bovine and camel fresh milk was examined. The maximum foamability and foam stability were observed for acid whey when compared to sweet whey for both milks, with higher values for the camel whey. This behavior for acid whey was explained by the proximity of the pI of whey protein (4.9-5.2), where proteins were found to carry the lowest negative charge as confirmed by the zeta potential measurements. Interfacial properties of acid camel whey and acid bovine whey were preserved at air water interface even after a heat treatment at 90°C. These results confirmed the pronounced foaming and interfacial properties of acid camel whey, even if acid and sweet bovine whey exhibited the highest viscoelastic modulus after heating.

The effect of pH and heat treatments on the foaming properties of purified α-lactalbumin from camel milk

The effect of pH (4.3 or 6.5) and heat treatment (70°C or 90°C for 30min) on the foaming and interfacial properties of α-lactalbumin extracted from camel milk were studied. The increased temperature treatment changed the foaming properties of camel α-lactalbumin solution and its ability to unfold at the air-water interface. At neutral pH, heat treatment was found to improve foamability, whereas at acid pH (4.3) this property decreased. Foams were more stable after a heat treatment at pH 4.3 than at 6.5, due to higher levels of protein aggregation at low pH. Heat treatment at 90°C for 30min affected the physicochemical properties of the camel α-lactalbumin by increasing free thiol group concentration at pH 6.5. Heat treatment also caused changes in α-lactalbumins surface charge. These results also confirm the pronounced aggregation of heated camel α-lactalbumin solution at acid pH.

X-ray diffraction investigation of amorphous calcium phosphate and hydroxyapatite under ultra-high hydrostatic pressure

The changes in the crystal structures of synthetically prepared amorphous calcium phosphate (ACP) and hydroxyapatite (HAP) in water (1:1 mass ratio) were studied by synchrotron X-ray diffraction (XRD) under ultra-high hydrostatic pressures as high as 2.34 GPa for ACP and 4 GPa for HAP. At ambient pressure, the XRD patterns of the ACP and HAP samples in capillary tubes and their environmental scanning electron micrographs indicated amorphous and crystalline characteristics for ACP and HAP, respectively. At pressures greater than 0.25 GPa, an additional broad peak was observed in the XRD pattern of the ACP phase, indicating a partial phase transition from an amorphous phase to a new high-pressure amorphous phase. The peak areas and positions of the ACP phase, as obtained through fitting of the experimental data, indicated that the ACP exhibited increased pseudo-crystalline behavior at pressures greater than 0.96 GPa. Conversely, no structural changes were observed for the HAP phase up to the highest applied pressure of 4 GPa. For HAP, a unit-cell reduction during compression was evidenced by a reduction in both refined lattice parameters a and c. Both ACP and HAP reverted to their original structures when the pressure was fully released to ambient pressure.

Performances of different protocols for exocellular polysaccharides extraction from milk acid gels: Application to yogurt

Dextran or xanthan were used as model exocellular polysaccharides (EPS) to compare the extraction efficiency of EPS from skim milk acid gels using three different protocols. Extraction yields, residual protein concentrations and the macromolecular properties of extracted EPS were determined. For both model EPS, the highest extraction yield (∼80%) was obtained when samples were heated in acidic conditions at the first step of extraction (Protocol 1). Protocols that contained steps of acid/ethanol precipitation without heating (Protocols 2 and 3) show lower extraction yields (∼55%) but allow a better preservation of the EPS macromolecular properties. Changing the pH of acid gels up to 7 before extraction (Protocol 3) improved the extraction yield of anionic EPS without effect on the macromolecular properties of EPS. Protocol 1 was then applied for the quantification of EPS produced during the yogurt fermentation, while Protocol 3 was dedicated to their macromolecular characterization.

Edible Wheat Gluten Films: Optimization of the Main Process Variables and Improvement of Water Vapor Barrier Properties by Combining Gluten Proteins with Lipids

An edible wheat gluten film was developed and relationships between film-formation conditions and properties were studied using Response Surface Methodology. pH and ethanol concentration of film-forming solution had strong interactive effects on film water solubility and moisture permeability. Mechanical properties were affected by gluten concentration and pH. Various formulations and methods of fabricating films consisting of gluten and lipids were investigated.