Elimination of β-Lactoglobulin from Whey to Simulate Human Milk Protein

When the pH of cottage cheese whey was adjusted to 4.5 in the presence of 6.7 mM FeCI³, β-lactoglobulin was eliminated from the whey as a precipitate. However, the majority of immunoglobuhns were also coprecipitated. To recover immunoglobulins together with α-lactalbumin, the whey pH was adjusted to 3.0 in the presence of 4.0 mM FeCI³. After centrifugation of the whey, the supernatant contained exclusively β-lactoglobulin; other whey proteins were found in the precipitate. Excess Fe₊₊₊ in the precipitate was removed by ion exchange or by ultrafiltration. This protein concentrate had a protein composition much more similar to that of human milk whey than that of ultrafiltered whey protein concentrate.     

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%.     

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.     

Changes in Gelling Behavior of Whey Protein Isolate and β-Lactoglobulin During Storage: Possible Mechanism(s)

Dry-heat treatment of a dialyzed whey protein isolate at 80°C for 7 days resulted in a decrease in hardness (from 1.55N to 0.49N) of gels formed from a 12% solution. Partial denaturation and progressive polymerization of protein was observed. The monomeric β-lactoglobulin concentration of the whey decreased from 60.64% to 33.33%after 7 days at 80°C. The rate constants determined at 40 to 80°C were used to calculate an Arrhenius relationship for the polymerization. After one year at 25^oC, 18% of monomeric β-lactoglobulin was projected to be converted to higher-molecular-weight material. The polymerization apparently did not involve disulfide cross-links.     

Effects of Succinylation on β-Lactoglobulin Foaming Properties

The extent of succinylation of β-lactoglobulin (β-Lg) increased logarithmically with increasing concentrations of succinic anhydride. The surface pressure of 27.5% succinylated β-Lg was higher than native (β-Lg but higher levels of succinylation (50% and 100%) reduced the surface pressure. Overrun and foam stability were reduced following succinylation. The electrostatic interaction caused by the addition of 100% succinylated β-lactoglobulin (0.5g/100 mL) to a solution (2.5%) of native β-lactoglobulin at pH 4.0 improved overrun (47%) and foam stability (61%).     

Preparation of β-Lactoglobulin and p-Lactoglobulin-Free Proteins from Whey Retentate by NaCI Salting Out at Low pH

Solubility of the main proteins in 10 x acid and rennet whey retentates was studied in the pH range 2.0 to 4.0 in the presence of NaCI from 2 to 15% (w/v) final concentration, at 20°C to find fractionation conditions suitable for preparing pure β-lactoglobulin and β-lactoglobu-lin-free whey proteins and scaling up. At pH 2.0, 7% NaCI, 20 min holding time, nearly all (3-lactoglobulin remained soluble while a precipitate (PI) containing all other proteins was formed. Pure p-lacto-globulin was quantitatively recovered by salting-out the centrifugation supernatant at 30% NaCI (w/v) final concentration. PI, insoluble at pHs lower than 4.0, was made soluble at any pH by dissolving at pH 9.0, dialyzing against 50 mM formic acid (pH 3.0) and freeze-drying.     

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.     

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.     

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.     

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.     

Effects of Lysozyme, Clupeine, and Sucrose on the Foaming Properties of Whey Protein Isolate and β-Lactoglobulin

Lysozyme and clupeine interacted with β-lactoglobulin to form aggregates. Sucrose reduced the aggregation. The addition of lysozyme (0.5%) to β-lactoglobulin (2.5%) reduced the time required to reach an overrun maximum and increased foam stability and heat stability by 124% and 377%, respectively. Lysozyme (0.5%) also improved overrun (98%), foam stability (114%) and heat stability of the foams (12%) made with whey protein isolate (WPI, 5%). Lysozyme and sucrose further improved the foaming properties of β-lactoglobulin and WPI. The addition of clupeine and sucrose gave similar results. The foaming properties of β-lactoglobulin and WPI with the inclusion of sucrose and lysozyme were superior to those of egg white.     

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.     

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.     

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.     

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.     

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 Gelation and Modification: Effect of Selected Acidulants and Heating Conditions

ABSTRACT: The effect of acidulant selection, heating temperature, and heating rate on the properties of low-pH β-lactoglobulin (β-Lg) gels and powders derived from these gels was investigated by rheological and microscopic techniques. As isothermal gelation temperature was increased from 75 to 85 °C, gels made with hydrochloric and lactic acid showed more rapid gel formation and increased stress at gel fracture. Thickening and water-holding properties of powders derived from these gels also increased with temperature. Increases in gel strength and derivatized powder functionality appeared to plateau above 85 °C. Gels and derivatized powders prepared with phosphoric acid exhibited attributes similar to samples prepared with HCl and lactic acid at lower temperatures. The ion-specific ability of phosphate to increase denaturation temperature was responsible for the shift in properties of gels made with phosphoric acid. Microscopy revealed temperature effects on network building block size, but variations in rheological properties could not be linked to changes in gel micrographs. Alteration of heating rates from 2.0 to 0.2 °C/min during gelation affected the observed gelation temperature, but had little effect on final gel mechanical properties. Acid selection and gelation temperature offer alternatives to control β-Lg gel strength and the functional properties of instant thickening protein ingredients.     

Factors Affecting Rheological Characteristics of Fibril Gels: The Case of β-Lactoglobulin and α-Lactalbumin

ABSTRACT:  Some of the factors that affect the rheological characteristics of fibril gels are discussed. Fibrils with nanoscale diameters from β-lactoglobulin (β-lg) and α-lactalbumin (α-la) have been used to create gels with different rheological characteristics. Values of the gelation time, t_c , the critical gel concentration, c ₀, and the equilibrium value of the storage modulus, G , such as     at long gelation times, derived from experimental rheological data, are discussed. Fibrils created from β-lg using solvent incubation and heating result in gels with different rheological properties, probably because of different microstructures and fibril densities. Partial hydrolysis of α-la with a serine proteinase from Bacillus licheniformis results in fibrils that are tubes about 20 nm in diameter. Such a fibril gel from a 10% (w/v) α-la solution has a higher modulus than a heat-set gel from a 10% (w/w) β-lg, pH 2.5 solution; it is suggested that one reason for the higher modulus might be the greater stiffness of α-la fibrils. However, the gelation times of α-la fibrils are longer than those of β-lg fibrils.     

Foaming properties of β-Lactoglobulin: impact of pre-heating and addition of isoamyl acetate

Summary Foaming properties of solutions of β-lactoglobulin alone or in a mixture with isoamyl acetate were investigated through a conductimetric method. It was observed that addition of isoamyl acetate to β-lactoglobulin at molar ratios, R = 1 and 2 led to enhanced foaming properties in comparison with β-lactoglobulin alone. In regards to the observed changes in tryptophan fluorescence properties, it is suggested that β-lactoglobulin + isoamyl acetate might form a complex with a specific surface activity.