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.     

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.     

Dynamics of competitive adsorption of alphas-casein and beta-casein at planar triolein-water interface: evidence for incompatibility of mixing in the interfacial film

Competitive adsorption of alpha(s)-casein and beta-casein from a bulk solution mixture to the triolein-water interface has been studied. Although the binding affinity of alpha(s)-casein to the triolein-water interface was lower than that of beta-casein in single-component systems, in a 1:1 mixture of alpha(s)-casein and beta-casein in the bulk solution the ratio of interfacial concentrations of alpha(s)-casein to beta-casein at equilibrium was about 2:1, indicating that alpha(s)-casein was preferentially adsorbed to the triolein-water interface. Furthermore, the equilibrium composition of alpha(s)-casein and beta-casein in the interfacial film at various bulk concentration ratios did not follow a simple Langmuir adsorption model. This deviation from ideal behavior was mainly due to thermodynamic incompatibility of mixing of these caseins in the interfacial region. The value of the incompatibility parameter, X(12), for these caseins at the triolein-water interface was much greater than that at the air-water interface. Displacement experiments showed that while alpha(s)-casein could dynamically displace beta-casein when the latter was in an unsaturated monolayer state at the interface, it could not do so when beta-casein was in a saturated monolayer film state. It is hypothesized that, because of thermodynamic incompatibility of mixing, the alpha(s)-casein and beta-casein mixed film at the oil-water interface may undergo two-dimensional phase separation.     

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.     

Adsorption of whey protein isolate at the oil-water interface as a function of processing conditions: a rheokinetic study

In this paper we present surface dynamic properties (interfacial tension and surface dilational properties) of a whey protein isolate with a high content of beta-lactoglobulin (WPI) adsorbed on the oil-water interface as a function of adsorption time. The experiments were performed at constant temperature (20 degrees C), pH (5), and ionic strength (0.05 M). The surface rheological parameters and the interfacial tension were measured as a function of WPI concentration (ranging from 1 x 10(-)(1) to 1 x 10(-)(5)% w/w) and different processing factors (effect of convection and heat treatment). We found that the interfacial pressure, pi, and surface dilational modulus, E, increase and the phase angle, phi, decreases with time, theta, which should be associated with WPI adsorption. These phenomena have been related to diffusion of the protein toward the interface (at short adsorption time) and to the protein unfolding and/or protein-protein interactions (at long-term adsorption) as a function of protein concentration in solution and processing conditions.     

Influence of competitive adsorption on flocculation and rheology of high-pressure-treated milk protein-stabilized emulsions

The effect of high-pressure treatment (HPT) on the droplet-size distribution and small-deformation rheology of oil-in-water emulsions containing beta-lactoglobulin and a nonionic surfactant or sodium caseinate has been investigated at neutral pH. Addition of Tween 20 (polyoxyethylenesorbitan monolaurate) to a beta-lactoglobulin-stabilized emulsion results in competitive displacement of the adsorbed globular protein film and, following HPT, the formation of a less flocculated emulsion. The age of the beta-lactoglobulin-stabilized emulsion prior to addition of sodium caseinate influences the competitive adsorption behavior. The strengthening of the beta-lactoglobulin layer with time makes it more resistant to disruption by sodium caseinate. The level of pressure-induced flocculation of beta-lactoglobulin-coated oil droplets depends on the intensity of processing conditions and on the degree of interfacial displacement. In contrast, beta-lactoglobulin added after emulsification appears to show little evidence of competitive adsorption behavior at the caseinate oil-water interface. Changes in the rheological properties of these latter systems following HPT can be attributed to pressure-induced denaturation and gelation of beta-lactoglobulin in the continuous phase of the emulsion.     

Interactions of vitamin D3 with bovine beta-lactoglobulin A and beta-casein

It is of nutritional significance to fortify processed dairy products (e.g., cheese, yogurt, and ice cream) with vitamin D3; however, the inherent complexity of these foods may influence the stability and bioavailability of this nutrient. In the present study, the interactions of vitamin D3 with beta-lactoglobulin A and beta-casein were investigated under various environmental conditions (i.e., pH and ionic strength) using fluorescence and circular dichroism spectroscopic techniques. The results indicated that vitamin D3 was bound to both beta-lactoglobulin A and beta-casein depending on the solution conditions. The apparent dissociation constants ranged from 0.02 to 0.29 microM for beta-lactoglobulin A, whereas the beta-casein apparent dissociation constants ranged from 0.06 to 0.26 microM. The apparent mole ratios were also comparable, i.e., 0.51-2.04 and 1.16-2.05 mol of vitamin D3 were bound per mole of beta-lactoglobulin A and beta-casein, respectively. It was concluded that these interactions may strongly influence vitamin D3 stability and, hence, bioavailability in processed dairy products.     

Transfer of aroma compounds in water-lipid systems: binding tendency of beta-lactoglobulin

Interactions of volatile aroma compounds with protein in aqueous solutions, especially whey proteins, have received significant attention in recent years. This work attempts to improve our understanding of the mass transfer in multiphasic systems, such as emulsions at the lipid-water interface, and to reveal the role of beta-lactoglobulin in the release rate of solutes. For this purpose the rotating diffusion cell has been used. From a practical point of view it enables evaluation of the transfer through the aqueous phase, through the oil and the interfacial transfer. The effect of beta-lactoglobulin, medium pH, and solute concentration has been investigated. Benzaldehyde and 2-nonanone have been studied, and miglyol has been chosen as an oil phase. It has been demonstrated that mass transfer has a rate-limiting step, which depends on physicochemical parameters such as hydrophobicity of the volatile, diffusion and partition coefficients, and rheological properties of the aqueous phase.     

Effect of processing on the displacement of whey proteins: applying the orogenic model to a real system

Atomic force microscopy (AFM) has been used to investigate the displacement of a commercial whey protein system and the behavior as compared to that of beta-lactoglobulin (Mackie, A. R.; Gunning, A. P.; Wilde, P. J.; Morris, V. J. Orogenic displacement of protein from the air-water interface by competitive adsorption. J. Colloid Interface Sci. 1999, 210, 157-166). The whey protein isolate (WPI) was displaced from an air-water interface by the surfactants Tween 20 and Tween 60. Displacement data obtained were compared with data obtained for pure beta-lactoglobulin and have shown that WPI was more resistant to displacement from the air-water interface than native beta-lactoglobulin. This was related to the greater surface elasticity of WPI at higher surface stresses. In the presence of Tween 20, WPI was observed to remain on the interface at surface pressures up to 8 mN/m greater than the surface pressure at which complete displacement of beta-lactoglobulin was observed. Displacement of WPI and beta-lactoglobulin films by the surfactant Tween 60 showed similar results. However, because of the lower surface activity of Tween 60, it was not possible to reach surface tension values similar to those obtained for Tween 20. Despite the lower surface activity of Tween 60, WPI was still observed to be present at the interface at surface pressure values greater than those by which beta-lactoglobulin had been completely displaced.     

Changes in structures of milk proteins upon photo-oxidation

Changes in protein structures as a result of riboflavin-induced photo-oxidation were studied for six milk proteins: alpha-casein, beta-casein, kappa-casein, lactoferrin, alpha-lactalbumin, and beta-lactoglobulin. The milk proteins showed significant variability in sensitivity to photo-oxidation. After photo-oxidation, an increase in carbonyl content because of oxidation of tryptophan, histidine, and methionine, as well as formation of dityrosine, was observed for all proteins studied, although at very different levels. Generally, the increment was highest for alpha- and beta-casein and was lowest for lactoferrin. Loss of tryptophan because of photo-oxidation was well-correlated with the formation of the tryptophan oxidation products, N-formylkynurenine and kynurenine. Changes at the tertiary protein structure level were observed after photo-oxidation of the globular proteins, where tryptophan fluorescence emission indicated unfolding of alpha-lactalbumin and beta-lactoglobulin, whereas lactoferrin achieved a more compact tertiary structure. Changes in secondary structure were observed for alpha-lactalbumin and beta-lactoglobulin, whereas the secondary structure of lactoferrin did not change. Polymerization of alpha- and beta-casein and of lactoferrin was observed, whereas kappa-casein, alpha-lactalbumin, and beta-lactoglobulin showed little tendency to polymerize after photo-oxidation. Lability toward photo-oxidation is discussed according to the structural stabilities of the globular proteins.     

Identification of the wheat seed protein CM3 as a highly active emulsifier using a novel functional screen

Lyophilized albumin protein fractions were prepared from flour of four varieties of wheat: Triticum aestivum cvs. Mercia and Riband, Triticum aestivum var. spelta, and Triticum turgidum var. durum (Kamut). The dry powders were redissolved in sodium phosphate buffers at pH 3.0, 6.5, or 8.0 and at ionic strengths of 0.1 or 1.0 M to a concentration of 0.1% (w/v). Emulsions formed by sonication of protein solutions with n-hexadecane were aged at room temperature and separated into aqueous, interstitial, and interfacial phases. The distinct emulsion components were lyophilized and analyzed by RP-HPLC. A protein was observed to be preferentially located in the interfacial component and subsequently purified from a total albumin fraction and identified by N-terminal sequencing as CM3, an alpha-amylase inhibitor subunit. Measurement of the equilibrium surface tension of CM3 as a function of protein concentration demonstrated that it was at least as active as bovine beta-lactoglobulin, an established protein emulsifier. Furthermore, measurement of the surface dilational elastic modulus at an air/water interface demonstrated the formation of a viscoelastic film, while fluorescence and FT-IR spectroscopic measurements on adsorbed and nonadsorbed CM3 suggest that the secondary structure is essentially unchanged upon adsorption to an oil/water interface. It is concluded that functional screening is a valid approach to identify novel protein emulsifiers in complex mixtures.     

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.     

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.     

Rheological and proteolytic properties of Monterey Jack goat's milk cheese during aging

To enhance the understanding of the quality traits of goat's milk cheeses, rheological and proteolytic properties of Monterey Jack goat's milk cheese were evaluated during 26 weeks of 4 degrees C storage. As expected with aging, beta-casein levels decreased with concomitant increases in peptide levels and were correlated with changes in rheological properties of the cheese. Hydrolysis of the protein matrix resulted in more flexible (increased viscoelastic properties) and softer (decreased hardness, shear stress, and shear rigidity) cheeses. During the first 4-8 weeks of storage, cheese texture changed significantly (P < 0.05) and then stabilized. Characterization of rheological and proteolytic properties of the goat's milk semihard cheese during aging provided insight into the changes occurring in the protein matrix, the relationship to structure, and a shift in cheese quality.     

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.     

Producibility and digestibility of antihypertensive beta-casein tripeptides, Val-Pro-Pro and Ile-Pro-Pro, in the gastrointestinal tract: analyses using an in vitro model of mammalian gastrointestinal digestion

Val-Pro-Pro (VPP) and Ile-Pro-Pro (IPP) are antihypertensive tripeptides isolated from milk fermented with Lactobacillus helveticus and inhibit angiotensin-converting enzyme (ACE). We investigated whether these peptides were generated from beta-casein by digestive enzymes and whether they were resistant to enzymatic hydrolysis, using an in vitro model. VPP and IPP were not generated from beta-casein by gastrointestinal enzymes; instead, a number of longer peptides including VPP and IPP sequences were detected. The fermentation step would therefore be necessary to produce these antihypertensive tripeptides. VPP and IPP themselves were hardly digested by digestive enzymes, suggesting that orally administered VPP and IPP remain intact in the intestine, retaining their activity until adsorption. The present study also demonstrated that various functional peptide sequences in beta-casein were resistant to gastrointestinal enzymes. There may be a strong correlation between the resistance of peptides to gastrointestinal digestion and their real physiological effects after oral administration.     

Transfer of aroma compounds through the lipidic-aqueous interface in a complex system

The transfer kinetics of aroma compounds from the aqueous phase to the lipidic phase (miglyol) and from miglyol to the aqueous phase have been studied in the presence or absence of a protein, beta-lactoglobulin, and at different pH values. In the presence of beta-lactoglobulin, the transfer at the interface from the aqueous to the lipidic phase decreases, with a greater effect of the presence of the protein at pH 3 than at pH 6. This barrier effect of the protein plays a role in the transfer of the aroma compounds between the different phases of the matrix.     

果品静载流变特性的研究进展

本文通过对国内外各类果品的静载压缩流变特性研究现状的分析，指出了在果品的静载流变学研究领域内存在的主要问题，提出了今后果品流变学研究中需要加强和进一步研究的内容：1)加强对采后各阶段受压时果品的微观组织结构、生理生化过程的变化以及储运环境温度等因素与流变损伤效应的交叉性影响的研究； 2)加强对实际储运中较多出现的多果接触、受压问题的系统性研究；3)重视研究储运受压后流变损伤的发展与果品储藏品质关系的研究；4)根据国内外生产实际，应加强特色果品的流变学研究,像西瓜、甜瓜等，既具有较高的营养价值和经济价值，又在世界范围内广泛种植。同时，提出了以果品本身的粘弹性为依据，在ADAMS软件平台上利用计算机虚拟样机技术建立流变学模型并获得相应参数的新方法。     

Effect of denaturants on the emulsifying activity of proteins

The relationship between protein flexibility and emulsifying activity was investigated by disrupting disulfide bonds and/or noncovalent interactions of the protein. Oil-in-water emulsions using model proteins (apomyoglobin, beta-casein, alpha-casein, lysozyme, bovine serum albumin, kappa-casein, and beta-lactoglobulin) were made in the presence of chemical denaturants (dithiothreitol and/or urea). In most cases, the presence of denaturants enhanced emulsifying activity. The effect was protein-specific and depended on the relative importance of disulfide bonds and noncovalent interactions in stabilizing the native conformation of each protein. Implications for the design of novel protein emulsifiers are discussed.     

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.