Changes in Particle Size, Sedimentation, and Protein Microstructure of Ultra-High-Temperature Skim Milk Considering Plasmin Concentration and Storage Temperature.

Age gelation is a major quality defect in ultra-high-temperature (UHT) pasteurized milk during extended storage. Changes in plasmin (PL)-induced sedimentation were investigated during storage (23 °C and 37 °C, four weeks) of UHT skim milk treated with PL (2.5, 10, and 15 U/L). The increase in particle size and broadening of the particle size distribution of samples during storage were dependent on the PL concentration, storage period, and storage temperature. Sediment analysis indicated that elevated storage temperature accelerated protein sedimentation. The initial PL concentration was positively correlated with the amount of protein sediment in samples stored at 23 °C for four weeks (r = 0.615; p < 0.01), whereas this correlation was negative in samples stored at 37 °C for the same time (r = -0.358; p < 0.01) due to extensive proteolysis. SDS-PAGE revealed that whey proteins remained soluble over storage at 23 °C for four weeks, but they mostly disappeared from the soluble phase of PL-added samples after two weeks' storage at 37 °C. Transmission electron micrographs of PL-containing UHT skim milk during storage at different temperatures supported the trend of sediment analysis well. Based on the Fourier transform infrared spectra of UHT skim milk stored at 23 °C for three weeks, PL-induced particle size enlargement was due to protein aggregation and the formation of intermolecular ß-sheet structures, which contributed to casein destabilization, leading to sediment formation.

Effect of gum tragacanth exuded by three Iranian Astragalus on mixed milk protein system during acid gelation.

The effects of various concentrations of three species of gum tragacanth on the gelation process, microstructure and viscoelastic properties of milk protein mixed gels acidified at 37°C by glucono-δ-lactone (GDL) were investigated using dynamic rheometry and microscopy. According to rheological measurements, the addition of gum tragacanth in the range of 0.05-0.2% (w/w) into milk protein dispersions led to a weaker structure for the milk protein network, compared to the control sample. This weakening effect could be eliminated by adding 0.3% (w/w) gum tragacanth exudates from A. gossypinus; the compositional features of gum tragacanth may have been responsible for the improved protein-protein interactions, greater structural strength and reduced gelation time onset. It was determined by scanning electron microscopy that the addition of gum tragacanth at a low concentration caused the density of the matrix to increase, while an open structure was observed in the presence of a higher gum concentration.

Starch addition in renneted milk gels: partitioning between curd and whey and effect on curd syneresis and gel microstructure.

Milk gels were made by renneting and acidifying skim milk containing 5 different starches, and then compressed by centrifugation to express whey and simulate curd syneresis during the manufacture of low-fat cheese. A series of 17 starches were examined, with 5 starches being selected for in-depth analysis: a modified waxy corn starch (WC), a waxy rice starch (WR), an instant tapioca starch (IT), a modified tapioca starch (MT), and dextrin (DX). Milks containing WC, WR, and DX were given a 72°C heat treatment, whereas those containing IT and MT had a 30-min treatment at 66°C that matched their optimum gelatinization treatments. Curd yields were calculated by weight, estimated starch content in whey was measured gravimetrically by alcohol precipitation, and starch retention in curd was calculated. Curd yields were 13.1% for the control milk (no added starch) and 18.4, 20.7, 21.5, 23.5, and 13.2% for the gels containing starches WC, WR, IT, MT, and DX, respectively. Estimated starch retentions in the curd were, respectively, 71, 90, 90, 21, and 1%. Laser scanning confocal microscopy was used to determine the location of the starches in the curd and their interaction with the protein matrix. Waxy corn, WR, and IT starches have potential to improve texture of low-fat cheese because they had high retention in the curd and they generated interruptions in the protein matrix network that may have helped limit extensive protein-protein interactions. Modified tapioca starch interfered with formation of the protein structure of the curd and produced a soft noncohesive gel, even though most (79%) of the MT starch was lost in the whey. Few distinct starch particles were present in the MT curd network. Dextrin was not retained in the curd and did not disrupt the protein network, making it unsuitable for use in low-fat cheese.

Whey protein nanofibrils: the environment-morphology-functionality relationship in lyophilization, rehydration, and seeding.

Amyloid-like fibrils from ß-lactoglobulin have potential as efficient thickening and gelling agents for food and biomedical applications, but the link between fibril morphology and bulk viscosity is poorly understood. We examined how lyophilization and rehydration affects the morphology and rheological properties of semiflexible (i.e., straight) and highly flexible (i.e., curly) fibrils, the latter made with 80 mM CaCl(2). Straight fibrils were fractured into short rods by lyophilization and rehydration, whereas curly fibrils sustained little damage. This was reflected in the viscosities of rehydrated fibril dispersions, which were much lower for straight fibrils than for curly fibrils. Lyophilized straight or curly fibrils seeded new fibril growth, but viscosity enhancement due to seeding was negligible. We believe that the increase in fibril concentration caused by seeding was counterbalanced by a decrease in fibril length, reducing the ability of fibrils to form physical entanglement networks.

Solid-state NMR reveals differences in the packing arrangements of peptide aggregates derived from the aortic amyloid polypeptide medin.

Several polypeptides aggregate into insoluble amyloid fibrils associated with pathologies such as Alzheimer's disease, Parkinson's disease and type 2 diabetes. Understanding the structural and sequential motifs that drive fibrillisation may assist in the discovery and refinement of effective therapies. Here we investigate the effects of three predicted amyloidogenic regions on the structure of aggregates formed by medin, a poorly characterised polypeptide associated with aortic medial amyloidosis. Solid-state NMR is used to compare the dynamics and sheet packing arrangement of the C-terminal region encompassing residues F(43) GSV within full-length medin (Med(1-50) ) and two shorter peptide fragments, Med(30-50) and Med(42-49) , lacking specific sequences predicted to be amyloidogenic.(.) Results show that all three peptides have different aggregate morphologies, and Med(30-50) and Med(1-50) have different sheet packing arrangements and dynamics to Med(42-49) . These results imply that at least two of the three predicted amyloidogenic regions are required for the formation and elongation of medin fibres observed in the disease state.

Storage induced changes to high protein powders: influence on surface properties and solubility.

BACKGROUND: MPC 80 is a high-protein (80%) milk powder commonly used in the food industry as a functional ingredient and valued for its nutritional quality. However, its rehydration properties decline during storage, causing more time to be required for rehydration of the powder by the end user. It is thought that changes at the surface of the powder particles contribute to this reduced solubility during storage. RESULTS: Surface composition and structural changes in milk protein concentrate (MPC) were observed during 90 days of storage at temperatures of 25 and 40 °C and relative humidities of 44, 66 and 84%. No significant changes to the surface composition (fat, protein and lactose) of the MPC powder samples occurred during storage; however, some changes in the microstructure of the powders were observed. Scanning electron microscopy analysis of the powder particles during dissolution showed the formation of a crust, consisting of a thin layer of fused casein micelles, on the surface of the stored powders. An increase in the hydrophobicity at the surface of the particles was evident by X-ray photoelectron spectroscopy analysis of the bonding state of the elements at or near the surface and by atomic force microscopy measurements of the adherence of particles to the surface of a material. CONCLUSION: The development of this 'crust' is thought to contribute to the decrease in the solubility of the powder particles during storage. The increase in the hydrophobicity at the surface and the casein micelle interactions resulting in the surface crust formation appear to contribute to the decrease in the solubility of MPC during storage.

How micro-phase separation alters the heating rate effects on globular protein gelation.

UNLABELLED: This study was conducted to determine how the combination of heating rate and pH can be used to alter viscoelastic properties and microstructure of egg white protein and whey protein isolate gels. Protein solutions (1% to 7% w/v protein, pH 3.0 to 8.5) were heated using a range of heating rates (0.2 to 60 °C/min) to achieve a final temperature of 80 °C. The gelation process and viscoelastic properties of formed gels were evaluated using small strain rheology. Single phase or micro-phase separated solution conditions were determined by confocal laser scanning microscopy. In the single phase region, gels prepared by the faster heating rates had the lowest rigidity at 80 °C; however, a common G' was achieved after holding for 4 h at 80 °C . On the other hand, under micro-phase separation conditions, faster heating rates allowed phase separated particles to be frozen in the network prior to precipitation. Thus, gels produced by slower heating rates had lower rigidities than gels produced by faster heating rates. The effect of heating rate appears to depend on if the solution is under single phase or micro-phase separated conditions. PRACTICAL APPLICATION: The effect of heating rate and/or time on protein gel firmness can be explained based on protein charge. When proteins have a high net negative charge and form soluble aggregates, there is no heating rate effect and gels with equal firmness will be formed if given enough time. In contrast, when electrostatic repulsion is low, there is a competition between protein precipitation and gel formation; thus, a faster heating rate produces a firmer gel.

Effects of ultrasound on the thermal and structural characteristics of proteins in reconstituted whey protein concentrate.

The sonication-induced changes in the structural and thermal properties of proteins in reconstituted whey protein concentrate (WPC) solutions were examined. Differential scanning calorimetry, UV-vis, fluorescence and circular dichroism spectroscopic techniques were used to determine the thermal properties of proteins, measure thiol groups and monitor changes to protein hydrophobicity and secondary structure, respectively. The enthalpy of denaturation decreased when WPC solutions were sonicated for up to 5 min. Prolonged sonication increased the enthalpy of denaturation due to protein aggregation. Sonication did not alter the thiol content but resulted in minor changes to the secondary structure and hydrophobicity of the protein. Overall, the sonication process had little effect on the structure of proteins in WPC solutions which is critical to preserving functional properties during the ultrasonic processing of whey protein based dairy products.

Transglutaminase catalysis of modified whey protein dispersions.

UNLABELLED: Transglutaminase (TGase) cross-linking reactions were accomplished using a heat-modified whey protein concentrate (mWPC) substrate after pH adjustment to 8. Based on earlier reports, the degree of lactosylation with respect to beta-lactoglobulin was lower in mWPC dispersions than measured in commercial whey concentrate (cWPC) protein solutions. In this study, a higher concentration of free sulfhydryl groups was detected in soluble supernatant fractions. Both factors potentially impact the availability of reactive lysine/glutaminyl residues required for TGase reactivity. The addition of 10 mM dithiothreitol (DTT) to the substrate mix, CBZ-glutaminyl glycine and hydroxylamine, revealed a 3.6-fold increase in TGase activity, likely due in part to maintenance of the catalytic cysteine residue in a reduced state. Furthermore, inclusion of DTT to mWPC dispersions significantly raised the apparent viscosity, independently of enzyme modification, while the rate of polymerization increased 2-fold based on OPA assay measurements. Limited cross-linking slightly increased the apparent viscosity, whereas extensive coupling lowered these values compared to equivalent nonenzyme-treated mWPC samples. Carbohydrate-staining revealed formation of glyco-polymers due to covalent linkages between glucosamine and mWPC proteins after TGase processing. Again, the apparent viscosity decreased after extensive enzymatic modification. Larger particles, sized 11.28 mum, were observed in the structural matrix of TGase-mWPC-fixed samples compared to 8 mum particles in control mWPC samples as viewed in scanning electron micrographs. Ultimately, the functional characteristics of TGase-mWPC ingredients may be custom-designed to deliver alternative functional attributes by adjusting the experimental reaction conditions under which catalysis is achieved. PRACTICAL APPLICATION: Taken together, these results suggest that unique TGase-mWPC and/or TGase-mWPC-glucosamine ingredients may be designed to provide novel, value-added, polymeric/glyco-polymeric protein products that afford added benefit for the milk industry.

Investigation of the microstructure of milk protein concentrate powders during rehydration: alterations during storage.

The aim of this work was to use scanning electron microscopy to investigate the microstructure of rehydrated milk protein concentrate powder (MPC) particles. A sample preparation method for scanning electron microscopy analysis of rehydrated MPC particles is described and used to characterize the time course of dissolution and the effects of prior storage on the dissolution process. The results show that a combination of different types of interactions (e.g., bridges, direct contact) between casein micelles results in a porous, gel-like structure that restrains the dispersion of individual micelles into the surrounding liquid phase without preventing water penetration and solubilization of nonmicellar components. During storage of the powder, increased interactions occur between and within micelles, leading to compaction of micelles and the formation of a monolayer skin of casein micelles packed close together, the combination of which are proposed to be responsible for the slow dissolution of stored MPC powders.

Efficacy of whey protein gel networks as potential viability-enhancing scaffolds for cell immobilization of Lactobacillus rhamnosus GG.

This study investigated cell immobilization of Lactobacillus rhamnosus GG in three separate protein products: native, denatured and hydrolysed whey protein isolate (WPI). Treatments were assessed for their ability to enhance probiotic survival during storage, heat stress and ex vivo gastric incubation. Spatial distribution of probiotic cells within immobilized treatments was evaluated by atomic force and confocal scanning laser microscopy, while cell viability was enumerated by plate count and flow cytometry (FACS). Microscopic analysis of denatured treatments revealed an oasis of immobilized cells, phase-separated from the surrounding protein matrix; an environmental characteristic analogous to hydrolysed networks. Cell immobilization in hydrolysed and denatured WPI enhanced survival by 6.1+/-0.1 and 5.8+/-0.1 log10 cycles, respectively, following 14 day storage at 37 degrees C and both treatments generated thermal protection at 57 degrees C (7.3+/-0.1 and 6.5+/-0.1 log(10) cfu/ml). Furthermore, denatured WPI enhanced probiotic protection (8.9+/-0.2 log(10) cfu/ml) following 3h gastric incubation at 37 degrees C. In conclusion, hydrolysed or denatured WPI were the most suitable matrices for cell immobilization, while native protein provided the weakest safeguard against thermal and acid stress, thus making it possible to envision whey protein gel networks as protective substrates for cell immobilization applications.

Anti-amyloid activity of the C-terminal domain of proSP-C against amyloid beta-peptide and medin.

Amyloid fibrils are found in approximately 25 different diseases, including Alzheimer's disease. Lung surfactant protein C (SP-C) forms fibrils in association with pulmonary disease. It was recently found that the C-terminal domain of proSP-C (CTC), which is localized to the endoplasmic reticulum (ER) lumen, protects the transmembrane (TM) part of (pro)SP-C from aggregation into amyloid until it has a folded into an alpha-helix. CTC appears to have a more general anti-amyloid effect by also acting on TM regions of other proteins. Here we investigate interactions of CTC with the amyloid beta-peptide (Abeta) associated with Alzheimer's disease and medin, a peptide that forms fibrils in the most common form of human amyloid. CTC prevents fibril formation in Abeta and medin and forms a complex with Abeta oligomers, as judged by size-exclusion chromatography and electrospray ionization mass spectrometry. These data suggest that CTC functions as a chaperone that acts preferentially against unfolded TM segments and structural motifs found during amyloid fibril formation, a mechanism that may be exploited in forming a basis for future anti-amyloid therapy.

Glycosylation and expanded utility of a modified whey protein ingredient via carbohydrate conjugation at low pH.

Whey protein, at one time considered a by-product of the cheese-making process, is now commonly used in foods for its thickening and emulsifying properties. Currently, approximately 30% of these proteinaceous resources remain under-utilized. Previously, an acidified, thermally treated whey protein concentrate (mWPC) was developed to produce a cold-set thickening ingredient. Mass spectroscopy revealed an approximate 2.5-fold decrease in the lactosylation of beta-lactoglobulin in mWPC starting materials compared with commercial whey protein concentrates, manufactured at a higher pH. Potentially, this should increase the number of reactive sites that remain available for carbohydrate attachment. With this study, the formation of glycoprotein complexes was demonstrated between the mWPC ingredient and lactose, naturally occurring in mWPC powders, or between mWPC protein components with dextran (35 to 45 and 100 to 200 kDa) materials at low pH. In fact, additional dry heating of mWPC powders showed a 3-fold increase in the amount of lactosylated beta-lactoglobulin. Evidence of Maillard reactivity was suggested using colorimetry, o-phthaldialdehyde assays, and sodium dodecyl sulfate PAGE followed by glycoprotein staining. Resultant glycoprotein dispersions exhibited altered functionality, in which case steady shear and small amplitude oscillatory rheology parameters were shown to be dependent on the specific reducing sugar present. Furthermore, the emulsion stability of mWPC-dextran fractions was 2 to 3 times greater than either mWPC or commercial WPC dispersions based on creaming index values. The water-holding capacity of all test samples decreased with additional heating steps; however, mWPC-dextran powders still retained nearly 6 times their weight of water. Scanning electron microscopy revealed that mWPC-dextran conjugates formed a porous network that differed significantly from the dense network observed with mWPC samples. This porosity likely affected both the rheological and water-binding properties of mWPC-dextran complexes. Taken together, these results suggest that the functionality of mWPC ingredients can be enhanced by conjugation with carbohydrate materials at low pH, especially with regard to improving the emulsifying attributes.

Disordered proteins: biological membranes as two-dimensional aggregation matrices.

Aberrant folded proteins and peptides are hallmarks of amyloidogenic diseases. However, the molecular processes that cause these proteins to adopt non-native structures in vivo and become cytotoxic are still largely unknown, despite intense efforts to establish a general molecular description of their behavior. Clearly, the fate of these proteins is ultimately linked to their immediate biochemical environment in vivo. In this review, we focus on the role of biological membranes, reactive interfaces that not only affect the conformational stability of amyloidogenic proteins, but also their aggregation rates and, probably, their toxicity. We first provide an overview of recent work, starting with findings regarding the amphiphatic amyloid-beta protein (Abeta), which give evidence that membranes can directly promote aggregation, and that the effectiveness in this process can be related to the presence of specific neuronal ganglioside lipids. In addition, we discuss the implications of recent research (medin as an detailed example) regarding putative roles of membranes in the misfolding behavior of soluble, non-amphiphatic proteins, which are attracting increasing interest. The potential role of membranes in exerting the toxic action of misfolded proteins will also be highlighted in a molecular context. In this review, we discuss novel NMR-based approaches for exploring membrane-protein interactions, and findings obtained using them, which we use to develop a molecular concept to describe membrane-mediated protein misfolding as a quasi-two-dimensional process rather than a three-dimensional event in a biochemical environment. The aim of the review is to provide researchers with a general understanding of the involvement of membranes in folding/misfolding processes in vivo, which might be quite universal and important for future research concerning amyloidogenic and misfolding proteins, and possible ways to prevent their toxic actions.

Whey protein concentrate and gum tragacanth as fat replacers in nonfat yogurt: chemical, physical, and microstructural properties.

The effect of whey protein concentrate (WPC) and gum tragacanth (GT) as fat replacers on the chemical, physical, and microstructural properties of nonfat yogurt was investigated. The WPC (7.5, 15, and 20 g/L) and GT (0.25, 0.5, 0.75, and 1 g/L) were incorporated into the skim milk slowly at 40 to 45 degrees C with agitation. The yogurt mixes were pasteurized at 90 degrees C for 10 min, inoculated with 0.1% starter culture, and incubated at 42 degrees C to pH 4.6, then refrigerated overnight at 5 degrees C. A control nonfat yogurt and control full fat yogurt were prepared as described, but without addition of WPC and GT. Increasing amount of WPC led to the increase in total solids, total protein, acidity, and ash content, whereas GT did not affect chemical parameters. Increasing WPC caused a more compact structure consisting of robust casein particles and large aggregates. Firmness was increased and susceptibility to syneresis was decreased as WPC increased. No significant difference was observed for firmness and syneresis of yogurt fortified with GT up to 0.5 g/L compared with control nonfat yogurt. Increasing the amount of gum above 0.5 g/L produced softer gels with a greater tendency for syneresis than the ones prepared without it. Addition of GT led to the coarser and more open structure compared with control yogurt.

Interactions between milk proteins and exopolysaccharides produced by Lactococcus lactis observed by scanning electron microscopy.

The interactions between exopolysaccharides produced by Lactococcus lactis ssp. cremoris JFR1 and dairy proteins (caseins and whey proteins) in fermented media (milk permeate and buttermilk) were observed using scanning electron microscopy. An immobilization technique by crosslinking was employed to bind the protein to the observation surface, so that a washing step could be performed to remove noninteracting material. The use of this novel technique allowed us, for the first time, to confirm that the exopolysaccharide molecules interact with dairy proteins. Exopolysaccharides appear as filament strands attached to the protein aggregates and to the bacterial cells. This new sample preparation technique proved to be very valuable for observing molecular interactions in fermented media.

Effects of moisture-induced whey protein aggregation on protein conformation, the state of water molecules, and the microstructure and texture of high-protein-containing matrix.

Moisture-induced protein aggregation through intermolecular interactions such as disulfide bonding can occur in a high-protein-containing food matrix during nonthermal processing and storage. The present study investigated the effect of moisture-induced whey protein aggregation on the structure and texture of such high-protein-containing matrices using a protein/buffer model system. Whey proteins in the protein/buffer model systems formed insoluble aggregates during 3 months' storage at temperatures varying from 4 to 45 degrees C, resulting in changes in microstructure and texture. The level of aggregation that began to cause significant texture change was an inverse function of storage temperature. The protein conformation and the state of water molecules in the model system also changed during storage, as measured by differential scanning calorimetry and Fourier transform infrared spectroscopy. During storage, the model system that had an initially smooth structure formed aggregated particles (100-200 nm) as measured by scanning electron microscopy, which lead to an aggregation network in the high-protein-containing matrix and caused a harder texture.

Effect of stirring and seeding on whey protein fibril formation.

The effect of stirring and seeding on the formation of fibrils in whey protein isolate (WPI) solutions was studied. More fibrils of a similar length are formed when WPI is stirred during heating at pH 2 and 80 degrees C compared to samples that were heated at rest. Addition of seeds did not show an additional effect compared to samples that were stirred. We propose a model for fibril formation, including an activation, nucleation, growth, and termination step. The activation and nucleation steps are the rate-determining steps. Fibril growth is relatively fast but terminates after prolonged heating. Two processes that possibly induce termination of fibril growth are hydrolysis of nonassembled monomers and inactivation of the growth ends of the fibrils. Stirring may break up immature fibrils, thus producing more active fibrils. Stirring also seems to accelerate the kinetics of fibril formation, resulting in an increase of the number of fibrils formed.

A novel technique for differentiation of proteins in the development of acid gel structure from control and heat treated milk using confocal scanning laser microscopy.

The incorporation of caseins and whey proteins into acid gels produced from unheated and heat treated skimmed milk was studied by confocal scanning laser microscopy (CSLM) using fluorescent labelled proteins. Bovine casein micelles were labelled using Alexa Fluor 594, while whey proteins were labelled using Alexa Fluor 488. Samples of the labelled protein solutions were introduced into aliquots of pasteurised skim milk, and skim milk heated to 90 degrees C for 2 min and 95 degrees C for 8 min. The milk was acidified at 40 degrees C to a final pH of 4.4 using 20 g glucono-delta-lactone/l (GDL). The formation of gels was observed with CSLM at two wavelengths (488 nm and 594 nm), and also by visual and rheological methods. In the control milk, as pH decreased distinct casein aggregates appeared, and as further pH reduction occurred, the whey proteins could be seen to coat the casein aggregates. With the heated milks, the gel structure was formed of continuous strands consisting of both casein and whey protein. The formation of the gel network was correlated with an increase in the elastic modulus for all three treatments, in relation to the severity of heat treatment. This model system allows the separate observation of the caseins and whey proteins, and the study of the interactions between the two protein fractions during the formation of the acid gel structure, on a real-time basis. The system could therefore be a valuable tool in the study of structure formation in yoghurt and other dairy protein systems.

Coalescence stability of emulsions containing globular milk proteins.

This review summarizes a large set of related experimental results about protein adsorption and drop coalescence in emulsions, stabilized by globular milk proteins, beta-lactoglobulin (BLG) or whey protein concentrate (WPC). First, we consider the effect of drop coalescence on the mean drop size, d32, during emulsification. Two regimes of emulsification, surfactant-rich (negligible drop coalescence) and surfactant-poor (significant drop coalescence) are observed in all systems studied. In the surfactant-rich regime, d32 does not depend on emulsifier concentration and is determined mainly by the interfacial tension and the power dissipation density in the emulsification chamber, epsilon. In the surfactant-poor regime and suppressed electrostatic repulsion, d32 is a linear function of the inverse initial emulsifier concentration, 1/C(INI), which allows one to determine the threshold emulsifier adsorption needed to stabilize the oil drops during emulsification, Gamma* (the latter depends neither on oil volume fraction nor on epsilon). Second, we study how the BLG adsorption on drop surface changes while varying the protein and electrolyte concentrations, and pH of the aqueous phase. At low electrolyte concentrations, the protein adsorbs in a monolayer. If the pH is away from the isoelectric point (IEP), the electrostatic repulsion keeps the adsorbed BLG molecules separated from each other, which precludes the formation of strong intermolecular bonds during shelf-storage as well as after heating of the emulsion. At higher electrolyte concentration, the adsorption Gamma increases, as a result of suppressed electrostatic repulsion between the protein molecules; monolayer or multilayer is formed, depending on protein concentration and pH. The adsorption passes through a maximum (around the protein IEP) as a function of pH. Third, the effect of various factors on the coalescence stability of "fresh" emulsions (up to several hours after preparation) was studied. Important conclusion from this part of the study is the establishment of three different cases of emulsion stabilization: (1) electrostatically-stabilized emulsions with monolayer adsorption, whose stability is described by the DLVO theory; (2) emulsions stabilized by steric repulsion, created by protein adsorption multilayers - a simple model was adapted to describe the stability of these emulsions; and (3) emulsions stabilized by steric repulsion, created by adsorption monolayers. Fourth, we studied how the emulsion stability changes with storage time and after heating. At high electrolyte concentrations, we find a significant decrease of the coalescence stability of BLG-emulsions after one day of shelf-storage (aging effect). The results suggest that aging is related to conformational changes in the protein adsorption layer, which lead to formation of extensive lateral non-covalent bonds (H-bonds and hydrophobic interactions) between the adsorbed molecules. The heating of BLG emulsions at high electrolyte concentration leads to strong increase of emulsion stability and to disappearance of the aging effect, which is explained by the formation of disulfide bonds between the adsorbed molecules. The emulsion heating at low electrolyte concentration does not affect emulsion stability - this result is explained with the electrostatic repulsion between the adsorbed molecules, which keeps them separated so that no intermolecular disulfide bonds are formed. Parallel experiments with WPC-stabilized emulsions show that these emulsions are less sensitive to variations of pH and thermal treatment; no aging effect is detected up to 30 days of storage. The observed differences between BLG and WPC are explained with the different procedures of preparation of these protein samples (freeze-drying and thermally enhanced spray-drying, respectively). Our data for emulsion coalescence stability are compared with literature results about the flocculation stability of BLG emulsions, and the observed similarities/differences are explained by considering the structure of the protein adsorption layers.