Growth and structure of aggregates of heat-denatured β-Lactoglobulin

Summary The aggregation of the globular protein β-lactoglobulin after heat-denaturation was studied in aqueous solution at pH 7 using static and dynamic light scattering. The structure of the aggregates is self-similar with fractal dimension 2.0. Size exclusion chromatography and dynamic light scattering measurements show that the aggregates have a broad size distribution. Initially clusters of about 85 proteins and 15 nm radius are formed which are the elementary units of the larger fractal aggregates. At low ionic strength the formation of the larger aggregates is impeded by electrostatic interactions.
The structure of the aggregates is independent of the concentration and the temperature. The rate of aggregation has an Arrhenius temperature dependence with an activation energy of about 350 kJ/mol weakly dependent on the concentration. The apparent reaction order of the aggregation is 1.5. In the mixture both variants A and B have the same aggregation rate. The gel time increases with decreasing concentration and diverges at about 0.7g L⁻¹. At lower concentration the aggregate growth stagnates when all protein has aggregated.     

Caustic-induced gelation of β-lactoglobulin

The gelation kinetics of β-lactoglobulin (βLg) solutions has been determined in the alkaline regime over a wide range of protein concentrations, gelling temperatures and gelation pH (pH_gel), set using NaOH. The behaviour is compared with caustic-induced gelation of whey protein concentrate and the alkaline dissolution of heat-induced whey gels. The gelation time decreases significantly between neutral conditions and pH_gel 9, because of the activation of the free cysteine groups and displacement to the monomeric form, and between pH_gel 10 and 11, due to the base denaturation of βLg. Both transitions are associated with a significant decrease in the activation energy of gelation. At pH_gel >11.5 the gelation time is observed to increase with pH_gel, owing to destruction of interprotein crosslinks. These results are consistent with the recently reported observation that a minimum pH for the dissolution of βLg gels and aggregates exists around 11.6 [ Biomacromolecules 8 (2007) 1162]. This phenomenon has been assigned to the destruction of non-covalent interactions that would inhibit the final percolation of the gel.     

Adsorption of β-Lactoglobulin variants A and B to the air–water interface

Summary The adsorption of proteins to interfaces is a vital and complex process for the formation and stabilization of multiphase food systems (emulsions and foams). The process of protein adsorption is generally understood only at the phenomenological level, as the complexity of protein unfolding during adsorption is very difficult to predict and model. By comparing proteins with very similar structures, it is possible to attribute observed changes in adsorption behaviour. The A and B genetic variants of β-lactoglobulin (β-lg) differ by only two amino acids (Asp-64, Val-118 in A, and Gly-64, Ala-118 in B), thus making them ideal candidates for this type of comparison. In this study we monitored the surface behaviour of β-lg A and B, measuring the surface tension and surface dilational modulus of adsorbed protein, and the compression behaviour of spread protein films. At pH 7, variant B lowered the surface tension and increased the surface dilational modulus more rapidly than variant A. Raising the pH to 7.8 should increase the level of dissociation into monomers. Indeed, this was confirmed by the rate of adsorption, which increased in both cases. Also, the surface tension of both variants was much lower than at pH7. Variant B was less sensitive to the change of pH than A. Regardless of pH, after 3 h adsorption the difference between the variants in surface tension or surface dilational modulus was negligible. The differences in surface behaviour between the variants are discussed in terms of interactions between monomers at and with the interface, and the dimer : monomer equilibrium in the bulk solution.     

Interactions of (β-Lactoglobulin and High-methoxyl Pectins in Acidified Systems

The interactions that occur when β-lactoglobulin (β-lg) is mixed with a high-methoxyl pectin (HMP) and a modified pectin (mHMP, modified using plant pectinesterase) were examined at pH 3.8. Whereas soluble aggregates formed in β-lg-HMP, β-lg-mHMP precipitated upon mixing. β-lg-HMP mixtures showed soluble aggregates with larger hydrodynamic diameters when heated at 65°C than when heated at 90°C. β-lg-HMP mixtures adjusted to pH 6.0 after heating showed that the aggregates formed at 65°C could be dissociated, but the complexes were not reversible after heating at 90°C. A similar effect also was observed when resuspending the°-lg-mHMP precipitates at pH 6.0. The behavior of the 2 pectins was attributed to their differences in charge distribution.     

Functional Improvement of Alginic Acid by Conjugating with β-Lactoglobulin

Two bovine β-lactoglobulin-alginic acid (β-LG-ALG) conjugates were prepared to improve the function of ALG by using water-soluble carbodiimide and the Maillard reaction. Fluorescence studies suggested that the conformation around Trp had been changed in each conjugate and that the surface of each conjugate was covered with polysaccharide chain. Structural analyses with monoclonal antibodies indicated that the conformation around 15Val-29IIe (β -sheet) in each conjugate had changed, while the native structure was maintained around 125Thr-135Lys (α-helix). After conjugating with β -LG, ALG showed retinol-binding and high emulsifying ability. The aggregating property of ALG in acid and in the presence of Ca²⁺ was improved in each conjugate.     

β-Lactoglobulin Separation from Whey Protein Isolate on a Large Scale

A method for obtaining large quantities of β-lactoglobulin (β-Lg) from commercial whey protein isolate (WPI) was developed. β-Lg was separated from the rest of the whey proteins in a solution of 15% (W:W) WPI in distilled water adjusted to pH 2 and 7% NaCl. β-Lg was then separated from NaCl using diafiltration. The results indicate that more than 65% of the β-Lg originally in the WPI solution was recovered. The purity of the β-Lg was greater than 95%.     

Calcium-Induced Destabilization of Oil-in-Water Emulsions Stabilized by Caseinate or by β-Lactoglobulin

The stability to aggregation of 20% soya oil-in-water emulsions stabilized by 0.3 to 2% sodium caseinate or β-lactoglobulin in the presence of calcium chloride solutions was studied using light scattering and electron microscopy. Stability increased with the amount of protein in the emulsion, and decreased with the concentration of added calcium. Growth of particle size with concentration of Ca²⁺ was more in emulsions containing lower concentrations of protein. Sodium chloride at 50 and 100 mM stabilized both systems to the presence of calcium ions. Microstructure and light scattering showed caseinate emulsions formed clusters even at low concentrations of Ca²⁺ while β-lactoglobulin emulsions formed extensive strands.     

Relation of β-Lactoglobulin – Sodium Polypectate Aggregation to Bulk Macromolecular Concentration

Aggregation of β-Lactoglobulin β-Lg) solutions, with and without sodium polypectate (SPP), was investigated at pH 6.5 and 3.5 by turbidity measurements and gel permeation chromatography during heating at 1°C/min. The ratio of β-Lg:SPP was maintained at 10:1. At pH 6.5, the transition temperature of β-Lg aggregation decreased linearly with the logarithm of β-Lg concentration. Irrespective of β-Lg concentration, SPP did not affect the rate of β-Lg aggregation during heating at pH 6.5. However, SPP influenced the formation of high-molecular-weight (HMW) β-Lg aggregates during heating at pH 6.5 was related to bulk macromolecular concentration. No thermal aggregation transitions were detected for β-Lg solutions at pH 3.5. SPP interacted with β-Lg at pH 3.5 to form a complex that precipitated on heating.     

Cold Gelation of β-lactoglobulin in the Presence of Iron

ABSTRACT: To protect and transport iron, we investigated the trapping properties of a network formed from β-lactoglobulin. We studied the influence of different parameters—pH, iron, and protein concentrations—on gel properties (optical and mechanical properties, WHC, and microstructure). For all conditions tested, the results show the formation of a cold gel in the presence of iron. The mechanical properties reveal that the elastic behavior and the strength of rupture increase with higher protein concentrations and decrease with higher iron concentrations. The water-holding capacity is high for low iron concentrations. The microstructure shows that, at low iron/protein ratios, a homogeneous filamentous network is obtained whereas, at high iron/protein ratios, more random aggregated particles are present.     

Chemical changes in β-Lactoglobulin structure during ageing of protein-stabilized emulsions

Summary Commercial protein-stabilized emulsions are often stored for many months. Reversed phase high performance liquid chromatography (HPLC) analysis of protein displaced from β-lactoglobulin-stabilized oil-in-water emulsions by the competitive adsorption of Tween 20, showed that whereas the retention time of protein displaced from a tetradecane–water interface did not change greatly upon ageing, that of the protein displaced from a soya oil–water interface did. The changes in the retention time of the displaced protein were accompanied by an increase in the mass of the protein. When 2-mercaptoethanol was added prior to emulsification, the rate of modification was significantly slower than in its absence. Tryptic digests of the displaced, modified protein showed that the changes were specific. Analysis of volatile components present in the emulsions showed that emulsification induced the autoxidation of the polyunsaturated fatty acyl chains of the soya oil resulting in the time-dependent formation of low molecular weight compounds. 2-Mercaptoethanol inhibits the process probably by reacting with hydroperoxides and terminating the chain reaction. Aldehydes, particularly enals, are known to react with nucleophilic amino acid residues such as lysine via addition and/or condensation reactions, leading to the observed increase in the mass of the protein.     

Interactive Effects of Pressure, Temperature and Time on the Molecular Structure of β-Lactoglobulin

We studied the combined effects of several variables, using temperatures up to 79°C and pressures up to 105 MPa, on β-lactoglobulin at pH 7.0 and pH 5.6 by examination of circular dichroism spectra and application of experimental design methodology. The higher pressures were not sufficient to impart the energy necessary to disrupt the protein structure. When used in combination with temperature and holding time, the applied energy was sufficient to disrupt the tertiary structure of the protein. Processing conditions applied at specific pH therefore could act in combination to cause structural changes.     

Optimization of Thermal Pretreatment Conditions for the Separation of Native α-Lactalbumin from Whey Protein Concentrates by Means of Selective Denaturation of β-Lactoglobulin

ABSTRACT: In this study a method to obtain native α-lactalbumin with a high degree of purity of 98% (m/m) and recovery of 75% (m/m) by selective denaturation of β-lactoglobulin was developed. To achieve this goal, the thermal pretreatment of whey protein concentrate was optimized varying the composition of the liquid whey protein concentrate in terms of total protein, lactose and calcium content, and pH value. The kinetics of the thermal denaturation of α-la and β-lg were then investigated at predetermined optimal composition (protein content 5 to 20 g/L, lactose content 0.5 g/L, calcium content 0.55 g/L, and pH 7.5). Using the activation energies and reaction rate constants obtained, lines of equal effects for targeted denaturation degrees of α-la and β-lg were calculated. Depending on total protein content, an area of optimal heating temperature/time conditions was identified for each protein concentration level.     

β-Lactoglobulin – a three-dimensional perspective

Summary Bovine β-lactoglobulin has been the subject of intense study over the past 60 years by, effectively, every available physicochemical technique, of which one of the most powerful is X-ray crystallography. We present a short review of the X-ray crystallographic work on milk protein together with an overview of the properties as they are seen from the current state of crystallographic analyses. At the present time, structural analyses do not provide any further insights into the possible function of the protein.     

Involvement of Disulfide Bonds in Bovine β-Lactoglobulin B Gels Set Thermally at Various pH

To obtain information on forces important for maintenance of the structure of heat-set β-lactoglobulin gels, gels set from pure β-lactoglobulin at pH 3.0, 5.0, and 7.0 were solubilized in dissociating buffers, and solubilized material was characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and size exclusion high performance liquid chromatography. The gel formed at pH 7.0 was largely soluble in urea (or SDS), and this gel seemed to be built from covalently linked soluble aggregates, associating into a gel network mainly by strong noncovalent interactions. The gel set at pH 5.0 was solubilized only in the presence of a reducing agent, indicating that this gel structure was supported mainly by covalent disulfide bonds. The gel set at pH 3.0 was almost completely solubilized in SDS or urea without a reducing agent, showing the importance of noncovalent interactions in maintaining the gel structure.     

Heat-induced denaturation and aggregation of β-Lactoglobulin: kinetics of formation of hydrophobic and disulphide-linked aggregates

Summary Solutions of a whey protein mixture were subjected to various time/temperature treatments, at pH 6.7. Kinetic and thermodynamic activation parameters for the rates of irreversible denaturation/aggregation of the principal whey protein component—β-lactoglobulin (β-lg) were followed by gel permeation. Fast Protein Liquid Chromatography (non-dissociating, non-reducing conditions) and by SDS-PAGE (dissociating, non-reducing conditions). The rate of loss of native β-lg owing to the formation of disulphide linked protein aggregates (k_sds-page) and the rate of formation of aggregates via both covalent and non-covalent bonds (k_gp-fplc) showed similar biphasic Arrhenius plots. However, the break of the plot occurred at different points. The k_gp-fplc values were higher than values of k_sds-page for all the temperatures examined. There was a similar trend for the thermodynamic activation parameters implying that not all of the β-lg aggregates through thiol–disulphide interactions. Hydrophobically driven associations occur within the aggregates.     

Ligand and Flavor Binding Functional Properties of β-Lactoglobulin in the Molten Globule State Induced by High Pressure

ABSTRACT: β-lactoglobulin (β-LG) in the molten globule state induced by high hydrostatic pressure (HHP) at 500 MPa and 50 °C for 32 min exhibited a significant decrease in affinity for retinol and a significant increase in affinity for cis-parinaric acid (CPA) and 1-anilino-naphthalene-8-sulfonate (ANS) compared to native β-LG. The number of β-LG binding sites for retinol and CPA significantly decreased after HHP treatment. The HHP-induced molten globule state of β-LG exhibited less affinity for palmitic acid, capsaicin, or carvacrol ligands than native β-LG, and no detectable specific binding for α-ionone, β-ionone, cinnamaldehyde or vanillin flavors. HHP treatment resulted in changes in the hydrophobic calyx and surface hydrophobic sites of β-LG.     

Heating of β-Lactoglobulin A Solution in a Closed System at High Temperatures

ABSTRACT β-Lactoglobulin A solution at pH 6.4 was heated to 180 °C at the rate of 6 °C/min. By differential scanning calorimetry two independent endothermic peaks were observed. The first peak appeared below 100 °C is corresponded to the thermal denaturation of protein. This conformational change led to the aggregation and polymerization of molecules through disulfide linkage, particularly observed above 100 °C. The second endothermic peak appeared around 150 °C, which was brought by the decomposition of molecules as judged from electrophoresis. Up to 100 °C the viscosity of β-lactoglobulin A solution increased by heating, while the viscosity was reduced beyond 113 °C, due to change in the size of aggregate and decomposition of β-lactoglobulin A molecules.     

Core-Shell Biopolymer Nanoparticles Produced by Electrostatic Deposition of Beet Pectin onto Heat-Denatured β-Lactoglobulin Aggregates

ABSTRACT:  The purpose of this study was to produce and characterize core-shell biopolymer particles based on electrostatic deposition of an anionic polysaccharide (beet pectin) onto amphoteric protein aggregates (heat-denatured β-lactoglobulin [β-lg]). Initially, the optimum conditions for forming stable protein particles were established by thermal treatment (80 °C for 15 min) of 0.5 wt%β-lg solutions at different pH values (3 to 7). After heating, stable submicron-sized ( d = 100 to 300 nm) protein aggregates could be formed in the pH range from 5.6 to 6. Core-shell biopolymer particles were formed by mixing a suspension of protein aggregates (formed by heating at pH 5.8) with a beet pectin solution at pH 7 and then adjusting the pH to values where the beet pectin is adsorbed (< pH 6). The impact of pH (3 to 7) and salt concentration (0 to 250 mM NaCl) on the properties of the core-shell biopolymer particles formed was then established. The biopolymer particles were stable to aggregation from pH 4 to 6, but aggregated at lower pH values because they had a relatively small ζ-potential. The biopolymer particles remained intact and stable to aggregation up to 250 mM NaCl at pH 4, indicating that they had good salt stability. The core-shell biopolymer particles prepared in this study may be useful for encapsulation and delivery of bioactive food components or as substitutes for lipid droplets.     

Glycation of bovine β-Lactoglobulin: effect on the protein structure

Summary Since temperature and water activity are among the most important parameters that affect the Maillard reaction, the glycation sites in pure, native bovine β-lactoglobulin were determined after a mild heat treatment (60 °C) in an aqueous solution and after a treatment under a restricted water environment (50 °C, 65% relative humidity). In both systems, the results obtained underlined the structural heterogeneity of β-lactoglobulin (β-LG) glycoforms with respect to the number of lactose residues linked per protein molecule and to the binding sites involved. Subsequently, the effect of the glycation conditions on both the association behaviour and the conformational changes of the glycated β-LG were characterised by proteolytic susceptibility, binding of the fluorescent probe 8-anilino-1-naphtalene-sulfonic acid, SDS-PAGE and size exclusion chromatography. The results showed that dry-way glycation did not significantly alter the native-like behaviour of the protein while the treatment in solution led to important structural changes. These changes resulted in a specific denatured β-LG monomer, which covalently associated via the free thiol group. The homodimers thus formed and the expanded monomers underwent subsequent aggregation to form high molecular weight species, via non–covalent interactions. The use of monoclonal antibodies with defined epitopes, raised against native β-LG, confirmed that the protein conformation was much more modified when glycation was performed in a solution while the structural changes induced during dry-way treatment were limited to the AB loop region of the protein.     

Selective Solubilization of α- Lactalbumin and β-Lactoglobulin into Reversed Micelles from Their Mixtures

Phase transfer experiments were performed, involving contact between an aqueous 1:1 solution of α-lactalbumin and β-lactoglobulin and an AOT-in-isooctane reversed micellar phase. The resulting extraction and separation of the two proteins were analyzed as functions of pH, ionic strength and total protein concentration using SDS-PAGE, and compared with extractions from pure solutions. At low protein concentrations, the extent of reversed micellar solubilization of the two pure proteins predicted well the extraction from mixtures. However, at higher protein concentrations β-lactoglobulin appeared to be excluded from the micellar droplets. Because of the significantly different partitioning behavior of the two proteins, reversed micellar extraction from an initially equal weight mixture led to an effective separation of the proteins.