Lateral phase separation in adsorbed binary protein films at the air-water interface.

Lateral phase separation in two-dimensional mixed films of soy 11S/beta-casein, acidic subunits of soy 11 (AS11S)/beta-casein, and alpha-lactalbumin/beta-casein adsorbed at the air-water interface has been studied using an epifluorescence microscopy method. No distinct lateral phase separation was observed in the mixed protein films when they were examined after 24 h of adsorption from the bulk phase. However, when the soy 11S/beta-casein and AS11S/beta-casein films were aged at the air-water interface for 96 h, phase-separated regions of the constituent proteins were evident, indicating that the phase separation process was kinetically limited by a viscosity barrier against lateral diffusion. In these films, beta-casein always formed the continuous phase and the other globular protein the dispersed phase. The morphology of the dispersed patches was affected by the protein composition in the film. In contrast with soy 11S/beta-casein and AS11S/beta-casein films, no lateral phase separation was observed in the alpha-lactalbumin/beta-casein film at both low and high concentration ratios in the film. The results of these studies proved that proteins in adsorbed binary films exhibit limited miscibility, and the deviation of competitive adsorption behavior of proteins at the air-water interface from that predicted by the ideal Langmuir model (Razumovsky, L.; Damodaran, S. J. Agric. Food Chem. 2001, 49, 3080-3086) is in fact due to thermodynamic incompatibility of mixing of the proteins in the binary film. It is hypothesized that phase separation in adsorbed mixed protein films at the air-water and possibly oil-water interfaces of foams and emulsions might be a source of instability in these dispersed systems.     

Incompatibility of mixing of proteins in adsorbed binary protein films at the air-water interface.

Competitive adsorption of proteins from several binary protein solutions to the air-water interface has been studied. With a few exceptions, the equilibrium composition of the saturated monolayer of mixed protein films at various bulk concentration ratios did not follow a Langmuir-type competitive adsorption model. The deviation from ideal behavior results from incompatibility of mixing of proteins in the film at the air-water interface. This immiscibility alters the ratio of the binding affinity of the proteins in a protein 1/protein 2/water ternary film compared to that in a protein 1/water and protein 2/water binary film. A method to determine the extent of incompatibility between two proteins in a mixed protein film has been developed. It is shown that the incompatibility index derived for 19 protein 1/water and protein 2/water systems studied show a linear relationship with the absolute difference between Flory-Huggins protein-solvent interaction parameters, that is, /chi(1s) - chi(2)/, of the constituent proteins. On the basis of the evidence, it is theorized that, because of incompatibility, proteins in a mixed protein film at interfaces may undergo two-dimensional phase separation.     

Chemometrical analysis of capillary electrophoresis casein fractions for predicting ripening times of milk mixture cheese

The effect of the ripening time on the proteolytic process in cheeses manufactured from mixtures of cow's and ewe's milk during a 167-day ripening period was monitored by capillary electrophoresis of the pH 4.6-insoluble fraction. Totals of 21 and 16 peaks were recognized and matched in the electropherograms obtained with a fused-silica capillary and a neutral capillary (hydrophilically coated), respectively. These peaks corresponded to intact bovine and ovine caseins and their hydrolysis products (e.g., alpha(s1)-casein, gamma-caseins). In 167-day-old cheeses, bovine alpha(s0)-casein (alpha(s1)-casein 9P) had been completely degraded and 6% of the residual bovine alpha(s1)-casein remained intact. Breakdown of the beta-casein fraction was lower than that of the alpha(s)-casein fraction. Finally, partial least-squares regression and principal component regression were used to predict the ripening time in cheeses. The root-mean-square errors in prediction by cross-validation were <7.8 days in all cases.     

Competitive adsorption between beta-casein or beta-lactoglobulin and model milk membrane lipids at oil-water interfaces

This study investigated the competitive adsorption between milk proteins and model milk membrane lipids at the oil-water interface and its dependence on the state of the lipid dispersion and the formation of emulsions. Both protein and membrane lipid surface load were determined using a serum depletion technique. The membrane lipid mixture used was a model milk membrane lipid system, containing dioleoylphosphatidylcholine, dioleoylphosphatidylethanolamine, milk sphingomyelin, dioleoylphosphatidylserine, and soybean phosphatidylinositol. The model composition mimics the lipid composition of natural milk fat globule membranes. The interactions were studied for two proteins, beta-lactoglobulin and beta-casein. The mixing order was varied to allow for differentiation between equilibrium structures and nonequilibrium structures. The results showed more than monolayer adsorption for most combinations. Proteins dominated at the oil-water interface in the protein-emulsified emulsion even after 48 h of exposure to a vesicular dispersion of membrane lipids. The membrane lipids dominated the oil-water interface in the case of the membrane lipid emulsified emulsion even after equilibration with a protein solution. Protein displacement with time was observed only for emulsions in which both membrane lipids and beta-casein were included during the emulsification. This study shows that kinetics controls the structures rather than the thermodynamic equilibrium, possibly resulting in structures more complex than an adsorbed monolayer. Thus, it can be expected that procedures such as the mixing order during emulsion preparation are of crucial importance to the emulsification performance.     

Sugar-casein interaction in deuterated solutions of bovine and caprine casein as determined by oxygen-17 and carbon-13 nuclear magnetic resonance: a case of preferential interactions

17O NMR spectroscopy and (13)C NMR spectroscopy have been used to study the mechanism of interaction of sugars with bovine and caprine caseins in D(2)O. The (17)O NMR relaxation results showed in all cases an increase in water of hydration, as a result of added sugar; this was predominantly associated with "trapped" water in the caseins. Analysis of the vir al coefficients, obtained from the (17)O relaxation data, suggested that preferential interactions occur in the sugar-protein solutions. This could be the result of either sugar binding or a solute-solute thermodynamic effect, preferential hydration. The addition of sugars to deuterated solutions of bovine casein and caprine casein high in alpha(s1)-casein had little or no effect on either line width or chemical shifts of the (13)C NMR spectra of these milk proteins. (13)C NMR studies of sucrose, at various concentrations (100-300 mM) in the presence of caprine casein high in alpha(s1)-casein, showed no changes in either chemical shifts or T(1) values. This indicates that the sugar molecules tumble isotropically and therefore neither bind to the protein nor affect viscosity in the protein-sugar studies. All of these data suggest that the preferential exclusion of the sugar from the domain of the caseins results in preferential hydration of the caseins.     

Sensomics mapping and identification of the key bitter metabolites in Gouda cheese

Application of a sensomics approach on the water-soluble extract of a matured Gouda cheese including gel permeation chromatography, ultrafiltration, solid phase extraction, preparative RP-HPLC, and HILIC combined with analytical sensory tools enabled the comprehensive mapping of bitter-tasting metabolites. LC-MS-TOF and LC-MS/MS, independent synthesis, and sensory analysis revealed the identification of a total of 16 bitter peptides formed by proteolysis of caseins. Eleven previously unreported bitter peptides were aligned to beta-casein, among which 6 peptides were released from the sequence beta-CN(57-69) of the N terminus of beta-casein and 2 peptides originated from the C-terminal sequence beta-CN(198-206). The other peptides were liberated from miscellaneous regions of beta-casein, namely, beta-CN(22-28), beta-CN(74-86), beta-CN(74-77), and beta-CN(135-138), respectively. Six peptides were found to originate from alpha(s1)-casein and were shown to have the sequences alpha(s1)-CN(11-14), alpha(s1)-CN(56-60), alpha(s1)-CN(70/71-74), alpha(s1)-CN(110/111-114), and alpha(s1)-CN(135-136). Sensory evaluation of the purified, synthesized peptides revealed that 12 of these peptides showed pronounced bitter taste with recognition thresholds between 0.05 and 6.0 mmol/L. Among these peptides, the decapeptide YPFPGPIHNS exhibited a caffeine-like bitter taste quality at the lowest threshold concentration of 0.05 mmol/L.     

Interfacial shear rheology of aged and heat-treated beta-lactoglobulin films: displacement by nonionic surfactant

Interfacial shear rheology of adsorbed beta-lactoglobulin films (bulk protein concentration 10(-)(3) wt %) has been studied over the temperature range 20-90 degrees C using a two-dimensional Couette-type viscometer. Effects of the type of interface (air-water, triolein-water, and n-dodecane-water), the pH (2.0, 5.6, 6.0, 7.0, and 9.0), and the extent of the heat treatment have been assessed via measurements of changes in the apparent interfacial shear viscosity and elasticity before and after the addition of increasing amounts of nonionic surfactant Tween 20 (polyoxyethylene sorbitan monolaurate). The highest interfacial viscosities were obtained at the n-dodecane-water interface and the lowest at the air-water interface. Competitive displacement of protein from the interface by Tween 20 was easier at the air-water and n-dodecane-water interfaces as compared to the triolein-water interface. The surface shear viscosity was higher and the displacement by Tween 20 more difficult as the isoelectric point of the protein was approached, which is in agreement with the presence of a more strongly cross-linked protein network at the interface. The effect of heat treatment was dependent on the pH of the aqueous solution. No simple relationship between the surface rheological characteristics and the ease of displacement by Tween 20 could be inferred.     

Prediction of the ripening times of ewe's milk cheese by multivariate regression analysis of capillary electrophoresis casein fractions

The effect of the ripening time on the proteolytic process in cheeses made from ewe's milk during a 139-day ripening period was monitored by the use of capillary electrophoresis of pH 4.6 insoluble fraction. Totals of 18 and 21 peaks were recognized and matched in the electropherograms obtained with a fused-silica capillary and a neutral capillary (hydrophilically coated), respectively. These peaks correspond to intact ovine caseins and their hydrolysis products (alpha(s1)-casein I, alpha(s1)-casein II, alpha(s1)-casein III, alpha(s2)-casein, beta(1)-casein, beta(2)-casein, p-kappa-casein, alpha(s1)-I-casein, gamma(1)-casein, gamma(2)-casein, and gamma(3)-casein). The alpha(s)-caseins (alpha(s1)- and alpha(s2)-casein) displayed similar degradation pattern to one another, but different from those of beta-caseins (beta(1)- and beta(2)-casein). beta-Caseins were very much undergoing lesser degradation during the ripening time than alpha(s)-casein. Finally, partial least-squares regression and principal components regression were used to predict the ripening time in cheeses. The models obtained yielded good results since the root-mean-square error in prediction by cross validation was <8.6 days in all cases.     

Oleosins of Arabidopsis thaliana: expression in Escherichia coli, purification, and functional properties

The interfacial behavior of oleosins, the most abundant proteins from seeds oil bodies, was investigated using the pendant drop method at water/oil interfaces and compared to the behavior of beta-casein and lysozyme, proteins with contrasted emulsifying properties. Recombined high (rS3) and low (rS4) molecular weight oleosins comprising N-terminal histidine tags were purified to electrophoretic homogeneity. rS3 decreased the interfacial tension at the oil/water interface better than rS4, oleosins being more efficient than beta-casein. Oleosins formed aggregates when spread on noncompressed phospholipid (PL) films at the air/water interface as observed using a Langmuir-Blodgett balance equipped with a Brewster angle microscope. Oleosin spread at the surface of a compressed PL monolayer (5-20 mN/m) did not aggregate. Pressure increased immediately and proportionally to the amount of protein spread on the monolayer. The results stress the capacity of oleosins to be inserted in oil and in PL monolayers, which is of particular relevancy to their potential uses as water/oil emulsifiers.     

Analysis of beta-casein-monopalmitin mixed films at the air-water interface

The surface pressure (pi)-area (A) isotherms and Brewster angle microscopy (BAM) images of monopalmitin and beta-casein mixed films spread on buffered water at pHs 5 and 7 and at 20 degrees C were determined as a function of the mass fraction of monopalmitin in the mixture (X). The structural characteristics and morphology of monopalmitin-beta-casein mixed films are dependent on surface pressure, pH, and monolayer composition. The prevalence of monopalmitin in the interface increases with the amount of monopalmitin in the mixture and at higher pi. At the monopalmitin monolayer collapse the mixed film is practically dominated by the presence of monopalmitin. However, some degree of interactions exist between monopalmitin and beta-casein in the mixed film, and these interactions are more pronounced as the monolayer is compressed at the highest surface pressures.     

Structure,miscibility, and rheological characteristics of beta-casein-monoglyceride mixed films at the air-water interface

In this work we have used different and complementary interfacial techniques (surface film balance, Brewster angle microscopy, and interfacial dilatational rheology) to analyze the static (structure, morphology, reflectivity, miscibility, and interactions) and dynamic characteristics (surface dilatational properties) of beta-casein and monoglyceride (monopalmitin and monoolein) mixed films spread on the air-water interface. The static and dynamic characteristics of the mixed films depend on the interfacial composition and the surface pressure. At higher surface pressures, collapsed beta-casein residues may be displaced from the interface by monoglyceride molecules with important repercussions on the interfacial characteristics of the mixed films. From the frequency dependence of the surface dilatational properties, we have elucidated the relationships between interfacial dilatational rheology and changes in molecular structure, interactions, miscibility, and relaxation phenomena in protein-monoglyceride mixed films.     

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.     

Variability of hydrolysis of β-, αs1-, and αs2-caseins by 10 strains of Streptococcus thermophilus and resulting bioactive peptides

Milk proteins contain numerous potential bioactive peptides, which may be released by digestive proteases or by the proteolytic system of lactic acid bacteria during food processing. The capacity of Streptococcus thermophilus to generate peptides, especially bioactive peptides, from bovine caseins was investigated. Strains expressing various levels of the cell envelope proteinase, PrtS, were incubated with α(s1)-, α(s2)-, or β-casein. Analysis of the supernatants by LC-ESI-MS/MS showed that the β-casein was preferentially hydrolyzed, followed by α(s2)-casein and then α(s1)-casein. Numbers and types of peptides released were strain-dependent. Hydrolysis appeared to be linked with the accessibility of different casein regions by protease. Analysis of bonds hydrolyzed in the region 1-23 of α(s1)-casein suggests that PrtS is at least in part responsible for the peptide production. Finally, among the generated peptides, 13 peptides from β-casein, 5 from α(s2)-casein, and 2 from α(s1)-casein have been reported as bioactive, 15 of them being angiotensin-converting enzyme inhibitors.     

Overphosphorylation of milk caseins by a recombinant protein kinase CK2 catalytic subunit.

Milk caseins have been phosphorylated by a recombinant protein kinase CK2 catalytic subunit from Schizosaccharomyces pombe (rspCK2alpha). Phosphate incorporation stoechiometries into purified caseins and into native phosphocaseinate, a substrate exhibiting a micellar-like structure, were determined. We incorporated 2.01 mol of P/mol of alpha-casein, 6.46 mol of P/mol of beta-casein, up to 0. 29 mol of P/mol of kappa-casein in 4 h, and more than 1.36 mol of P/mol of casein into phosphocaseinate under optimized conditions. Phosphocaseinate was sequentially phosphorylated; beta-caseins being labeled at first; alpha-caseins being labeled later; and to a lower extend, kappa-caseins were the last to be phosphorylated. The solubility of phosphocaseinate micelles increased by 1.34 from 65 to 87%, and its rennetting time was increased 2.88 times. These results are discussed in terms of plausible structural organization of caseins micelles and the effect of phosphorylation on their structure.     

Polyphenol-beta-casein complexes at the air/water interface and in solution: effects of polyphenol structure

The interactions between proteins and plant polyphenols are responsible for astringency and haze formation in beverages and may participate in foam stabilization. The effect of phenolic compounds with different structures, namely, catechin (C), epicatechin (Ec), epigallocatechin (Egc), epicatechin gallate (EcG), and epigallocatechin gallate (EgcG), on the surface properties at the air/liquid interface of beta-casein, chosen as model protein, were monitored by tensiometry and ellipsometry. The formation of complexes in the bulk phase was measured by electrospray ionization mass spectrometry (ESI-MS). Adsorption of polyphenols from pure solution was not observed. Surface pressure, surface concentration, and dilational modulus of the protein adsorption layer were greatly modified in the presence of galloylated flavanol monomers (EcG and EgcG) but not of lower molecular weight polyphenols (<306 g/mol). The formation of polyphenol-protein aggregates in the bulk, as evidenced by ESI-MS and light scattering experiments, was related to the slowdown of protein adsorption.     

Proteomic analysis of temperature-dependent changes in stored UHT milk

Molecular changes in milk proteins during storage of UHT-treated milk have been investigated using two-dimensional electrophoresis (2-DE) coupled to MALDI-TOF mass spectrometry. UHT-treated samples were stored at three different temperatures, 4 °C, 28 °C, and 40 °C, for two months. Three main changes could be observed on 2-DE gels following storage. They were (1) the appearance of diffuse staining regions above the position of the monomeric caseins caused by nondisulfide cross-linking of α and β-caseins; (2) the appearance of additional acidic forms of proteins, predominantly of α(S1)-casein, caused by deamidation; and (3) the appearance of "stacked spots" caused by lactosylation of whey proteins. The extent of the changes increased with increased storage temperature. Mass spectrometric analysis of in-gel tryptic digests showed that the cross-linked proteins were dominated by α(S1)-casein, but a heterogeneous population of cross-linked forms with α(S2)-casein and β-casein was also observed. Tandem MS analysis was used to confirm deamidation of N(129) in α(S1)-casein. MS analysis of the stacked spots revealed lactosylation of 9/15 lysines in β-lactoglobulin and 8/12 lysines in α-lactalbumin. More extensive analysis will be required to confirm the nature of the cross-links and additional deamidation sites in α(S1)-casein as the highly phosphorylated nature of the caseins makes them challenging prospects for MS analysis.     

Binding of 2-nonanone and milk proteins in aqueous model systems

Interactions of the model flavor compound 2-nonanone with individual milk proteins, whey protein isolate (WPI), and sodium caseinate in aqueous solutions were investigated. A method to quantify the free 2-nonanone was developed using headspace solid-phase microextraction followed by gas chromatography with flame ionization detection. Binding constants (K) and numbers of binding sites (n) for 2-nonanone on the individual proteins were calculated. The 2-nonanone binding capacities decreased in the order bovine serum albumin > beta-lactoglobulin > alpha-lactalbumin > alpha s1-casein > beta-casein, and the binding to WPI was stronger than the binding to sodium caseinate. All proteins appeared to have one binding site for 2-nonanone per molecule of protein at the flavor concentrations investigated, except for bovine serum albumin, which possessed two classes of binding sites. The binding mechanism is believed to involve predominantly hydrophobic interactions.     

Rheology of mixed beta-casein/beta-lactoglobulin films at the air-water interface

The adsorption of dilute mixtures of beta-casein/beta-lactoglobulin to the air-water interface was investigated using surface dilatation and surface shear rheology. The data were fitted to simple rheological models to try to gain further information regarding the composition and nature of the interface. The dilatational measurements suggested that the composition of the interface could be determined using these models and that the surface concentration was dominated by the beta-casein in the early stages of adsorption but that high levels of beta-lactoglobulin were present in the final stages. Surface shear rheological measurements showed a similar trend. However, the shear measurements appeared to be more sensitive to the strength of the network than to the composition of the interface. Fluorescence microscopy supported the findings and demonstrated that any "phase separation" capable of affecting the surface rheological measurements occurred at the sub-micrometer scale. The results also demonstrated that the heterogeneity of the interface, once formed, is kinetically trapped, and no further phase separation occurs over the time span of the experiments.     

Effect of the pH of heating on the qualitative and quantitative compositions of the sera of reconstituted skim milks and on the mechanisms of formation of soluble aggregates

The effect of the pH of heating (6.3-7.3) on the composition of sera in reconstituted skimmed milks was investigated. A combination of SDS-PAGE analysis and size exclusion chromatography (SEC) combined with an original approach to the analysis of the SEC profiles was performed. The composition of the sera varied greatly when the pH of heating was adjusted below and above the natural pH of milk. The formation, composition, and concentration of heat-induced soluble complexes depended on the combination of the effect of adjusting the pH of the milk and the heat treatment. Two types of mechanism for the formation of soluble aggregates appeared to exist, depending on the pH of the milk. The first type results from the formation of WP/kappa-casein aggregates at the surface of the micelle, and these were detached partially into the serum in larger amount as the pH increased up to 6.7, where it reaches a maximum. The second type of complexes, whose amount increased as the pH of heating increased from 6.7 to 7.3, may be formed between caseins (kappa- but also perhaps some alpha(s)-casein) and aggregated WP resulting in complexes that are smaller in size and with a higher kappa-casein/whey protein ratio than the first type.     

Iron tissue storage and hemoglobin levels of deficient rats repleted with iron bound to the caseinophosphopeptide 1-25 of beta-casein.

Caseinophosphopeptides (CPP) issued from enzyme digestion of caseins bind cations and keep them soluble in the digestive tract. They could be used as ligands to improve iron (Fe) bioavailability. Fe-deficient young rats were repleted with Fe (40 or 200 mg/kg of diet) bound either to the beta-CN (1-25) of beta-casein or to whole beta-casein or as FeSO(4). A control pair-fed group was given 200 mg of Fe (FeSO(4))/kg of diet for 6 weeks. After repletion, hemoglobin concentration of the control group was reached only by the ) animals fed 200 mg of Fe/kg; beta-CN (1-25) bound Fe (40 and 200 mg) produced higher Fe liver and spleen stores than FeSO(4). Binding Fe to the whole, nonhydrolyzed beta-casein gave results intermediate between the other experimental groups. Binding Fe to phosphoserine residues of low molecular weight CPP improved its ability to cure anemia and to restore iron tissue stores, as compared to Fe bound to the whole casein and to inorganic salts.