Β-lactoglobulin Gels Research Articles

Globular protein gelation—theory and experiment

Heat-set globular protein gel networks are discussed in relation to protein charge and screening. Homogeneous ‘fine-stranded’ gels form when electrostatic repulsion is high, with a transition to microphase-separated structures as repulsion falls. Fine-stranded networks showing density fluctuations occur close to this transition and probably arise from a combination of kinetic trapping and a drive towards only limited phase separation. For the most uniform structures (pH far from pI, and low salt content) network building appears to involve three main stages: initial protein unfolding, linear fibrillar aggregation, and random cross-linking of the fibrils. A mean field model is described which incorporates these features and includes the possibility of cooperative linear aggregation (nucleation and growth). Application of this to cure data for acid β-lactoglobulin gels was only partially successful, however, a higher order ( n=4) nucleation process being required to explain gel time—concentration data, while only a lower second order process could reproduce the shapes of the cure curves. As uniform gels give way to phase-separated structures network building becomes still more complex. Here solution demixing of unfolded monomers, and/or the initial aggregates, must be included in the model. This seems beyond the current mean-field approach, and simulation is likely to be required. This is true, even for the more homogeneous structures, when gel properties of interest extend from the linear elastic, to the time-dependent, and non-linear.

Heat-induced gelation of globular proteins: part 3. Molecular studies on low pH β-lactoglobulin gels

Heat-set gels and aggregates from β-lactoglobulin (β-Lg), one of the major globular proteins from milk, have been studied on a molecular distance scale using negative-staining transmission electron microscopy (TEM), wide-angle X-ray diffraction (WAXD), and Fourier transform infrared spectroscopy (FTIR). The microscopy showed long linear aggregates forming in solutions at pH 2 (and sometimes 2.5) after prolonged heating. While there appeared to be no differences in aggregates formed under these conditions in H 2O as compared with D 2O, at all other pH and pD values, and in the presence of added salt, much shorter linear aggregates were formed. These became slightly more extended the further the pH was removed from pI. Wide-angle X-ray diffraction (WAXD) showed a diffuse β-sheet halo at 2 θ=19° in patterns for both dried native and aggregated protein (irrespective of pH) with only a small change (sharpening) of this feature on heat treatment. Solution FTIR spectra, measured at pD=2, 2.5, 3, and 7, during heating, indicated shoulder development at 1612 cm −1 in the carbonyl-stretching Amide I region diagnostic of a modest increase in intermolecular β-sheet. In terms of the shoulder size, no distinctions could be made between acid and neutral aggregate structures. At all pHs, β-lactoglobulin showed only limited secondary and tertiary structural changes in aggregation, in contrast to previous studies of insulin aggregation, where highly ordered crystalline fibrils were indicated. The current work has implications both in structural studies of food biopolymers and in ongoing studies of pathological protein self-assembly in disease states, such as spongiform encephalopathies.

THE SENSORY PERCEPTION OF FLAVOR RELEASE AS A FUNCTION OF TEXTURE AND TIME: A TIME INTENSITY STUDY USING FLAVORED GELS

The focus in this study was to study time intensity (TI) methodology and procedures of getting the sensory panel acquainted with this technique. By means of a descriptive profiling exercise effects of altering the structure of a β-lactoglobulin gel, laced with banana aroma on the perception of banana flavor were obtained and a suitable attribute for TI was selected. Samples made up by protein, without any fat are not ideal for a TI study of flavor release because of the fast release of aroma. However, although the concentration of banana aroma was identical in the four groups of gels the intensity was perceived as different. This was reflected in the descriptive profiling as well as in the dynamic study. The TI study showed that it is important to minimize distractive, noncrucial information during the exercise. The assessors had different abilities to connect with the dynamic data acquisition procedure and training made the assessments more congruent. The results also indicate a relationship between the appearance of the TI-curve and the score of the total banana aftertaste.

Rheological influence of non-gelling amylopectins on β-lactoglobulin gel structures

The effect of non-gelling potato amylopectin on the gel properties of a particulate β-lactoglobulin gel was studied by small and large deformation rheology and by light microscopy. Two different techniques using small deformations, one measuring the modulus in shear and the other measuring the modulus in compression, have been compared. The fracture tests of the gels were performed in tension. The concentration of β-lactoglobulin was kept constant at 6 wt%, and the amount of potato amylopectin was varied from 0 to 2 wt%. Two preparations of potato amylopectin, with different rheological behaviour, were used. The results illustrate the importance of a full rheological characterisation, since small and large deformation tests responded different to the structure. Both the viscoelastic and the failure properties of β-lactoglobulin gels changed on addition of potato amylopectin even at low concentrations. The effect of the potato amylopectin concentration on the rheological properties of the mixed system varied with the properties of the potato amylopectin. The higher viscosity (HV) potato amylopectin had a shear-thinning behaviour and a small yield stress, while the lower viscosity (LV) potato amylopectin had a lower viscosity with a Newtonian behaviour. The gels containing the LV potato amylopectin increased in the modulus of small deformations with increasing potato amylopectin concentration, while the stress at fracture was constant up to a concentration of 0.5 wt% potato amylopectin and then increased with increasing potato amylopectin concentration. For the gels containing the HV potato amylopectin the modulus of small deformations reached a maximum at 0.25 wt% potato amylopectin and then decreased with increasing potato amylopectin concentration. The stress at fracture was constant up to 0.5 wt% potato amylopectin and decreased at higher concentrations. The state of aggregation of β-lactoglobulin was influenced both by concentration and properties of potato amylopectin. The higher the potato amylopectin concentration the larger the pores in the β-lactoglobulin gel.

The effects of pH on the rheology of β-lactoglobulin/κ-carrageenan mixed gels

Gelation and melting of single and mixed systems of 10% β-lactoglobulin and 1% κ-carrageenan have been investigated by small-deformation oscillatory measurements over pH 4–7. For single β-lactoglobulin, it was found that the gelation temperature and the storage modulus of the gels were strongly pH-dependent. However, the rheological behaviour was almost the same; the β-lactoglobulin gels were formed upon first heating from 45 to 90°C, reinforced on cooling to 20°C, and then weakened during reheating steps to 70°C. Single κ-carrageenan systems showed also the same behaviour over pH 4–7; gelation occurred at temperatures below 25°C and the melting took place on reheating above 40°C. In contrast, over this pH range, mixed systems showed two distinct types of behaviour: one over pH 5–7 and the other at pH 4.0. Over pH 5–7, the behaviour of β-lactoglobulin and κ-carrageenan, upon gel formation and melting, can easily be identified in that of the mixed systems, thus suggesting that phase-separated gels were formed. While at pH 4.0, the mixed gel showed a peculiar behaviour, similar to that of the pure protein gel. The presence of κ-carrageenan is only evidenced by the significant enhancement of the protein gel strengths. At this pH, below the protein p I, the electrostatic attractive forces between β-lactoglobulin and κ-carrageenan are important. Therefore, these two biopolymers associated and then formed a mixed gel, with one continuous network structures, similar to that of complex coacervate networks.

Effect of addition of κ-carrageenan on the mechanical and structural properties of β-lactoglobulin gels

The effect of the addition of a polysaccharide, κ-carrageenan (KCG), on the properties of heat induced gels of a globular protein, β-lactoglobulin (BLG), was studied at pH 7 and 0.1M NaCl The shear modulus was measured as a function of heating time. At a given heating time and BLG concentration, the shear modulus has a maximum as a function of the KCG concentration. The initial increase can be attributed to the increase of the kinetics of the aggregation of BLG induced by KCG. The subsequent decrease can be attributed to the effect of micro phase separation. Macroscopic phase separation is not observed, but can be easily induced by centrifugation. Transmission electron microscopy of gels with and without the addition of KCG demonstrates the increased micro phase separation induced by KCG.

Heat induced aggregation and gelation of β-lactoglobulin in the presence of κ-carrageenan

The effect of addition of κ-carrageenan on heat induced aggregation of β-lactoglobulin is investigated. The gel time of β-lactoglobulin at pH 7 and 0.1 M salt is strongly reduced if κ-carrageenan is added. The structure of the aggregates was studied using scattering techniques and is shown not to be modified by the presence of κ-carrageenan. The kinetics of the early stage of the aggregation is unaffected by κ-carrageenan, but the larger scale aggregation leading to gel formation is accelerated if κ-carrageenan is present.

Rheological properties of mixed gels made of microparticulated whey proteins and β-lactoglobulin

Microparticulated whey proteins (MWP) in suspension or included in a globular protein network revealed that the degree of flocculation was important in the final structure of the material. The MWP dispersions developed an antithixotropic character when low shear rate was applied to the system even at low temperature. Similar results were obtained on globular protein solutions and could be interpreted in terms of an orthokinetic aggregation. MWP participated to the formation and reinforcement of heat-induced β-lactoglobulin (BLG) gels. The mechanical properties of mixed gels demonstrated synergestic effects at low MWP massic fraction (BLG:MWP ratio higher than 2:1). A possible interpretation would be that both interaction of MWP with the network and flocculation of the MWP inside the pores could occur. However, increasing the fraction of MWP induced heterogeneities in the protein network. In particular, micro-phase separation due to steric hindrance effects or thermodynamic incompatibility between the two components could occur and decrease viscoelastic properties of mixed gels.

Fractal Analysis of the Elasticity of BSA and β-Lactoglobulin Gels

The fractal structure of aggregates in heat-induced BSA and β-lactoglobulin gels prepared with and without CaCl2 was examined. From the concentration dependence of the gel elasticity determined from a uniaxial compression test of the gel, the fractal dimensions Df of the aggregates in the gels were evaluated, using the theory of Shih et al. It was confirmed that the gels with and without CaCl2 showed weak- and strong-link behavior, as defined in the theory of Shih et al., respectively. The obtained values of Df were about 2 for strong-link gels and about 2.7 for weak-link gels. In addition, for the β-lactoglobulin gels containing CaCl2 (weak-link type), from the analysis of the gel image obtained with confocal scanning laser microscopy of the gels, the fractal dimensions were also evaluated, the value being close to that found from the gel elasticity measurements. These results indicate that the elastic behavior of the aggregate gels is a reflection of the fractal structure of the aggregates in the gels. ...

Analysis of Aggregate Structure in Food Protein Gels with the Concept of Fractal.

The fractal structure of the aggregates in food protein gels was analyzed. Three kinds of food protein gels were prepared: (1) β-lactoglobulin (β-LG) gel; (2) 11S soybean globulin gel; and (3) caseinate gel. From the concentration dependence of the gel elasticity, the fractal dimensions Df of the aggregates in the gels were evaluated, according to the theory of Shih et al. These gels showed the weak-link behavior described in the theory of Shih et al The values obtained for Df were 2.6-2.7, which were larger than those predicted by the cluster-cluster aggregation model for a dilute system. In addition, for the β-LG gels, the fractal dimension was also evaluated from the analysis of the gel image obtained with a confocal scanning laser microscopy, the value being close to that evaluated from the concentration dependence of the gel elasticity. These results indicate that the elastic behavior of the aggregate gels is a reflection of fractal structure of the aggregates in the gels.

Pressure-induced aggregation of β-lactoglobulin in ph 7.0 buffers

Solutions of a β-lactoglobulin isolate in water, in potassium phosphate buffer (20 or 50 mmol/L), and in pressure-resistant buffers, at a protein concentration of 25 g/kg and at pH 7.0, were processed at 150, 250, 350 or 450 MPa and 25 °C, for 15 min, then stored at 4 °C, usually for 24 h, before analysis. Bis-Tris (20 or 50 mmol/L) and bis-Tris-propane (10, 20 or 50 mmol/L) were selected as pressure-resistant buffers, while the pH of water or phosphate buffer is known to decrease reversibly by 0.2–0.3 pH unit per 100 MPa. After pressurization, nitrogen solubility at pH 4.7 and 7.0, and aggregation patterns by polyacrylamide gel electrophoresis were determined. Both indicated that aggregation of β-lactoglobulin was more extensive in pressure-resistant buffers than in phosphate buffer or in water. Electrophoretic patterns also revealed the progressive formation of dimers to hexamers and of higher polymers of β-lactoglobulin as a function of the type and molarity of buffer and of the pressure level. All high molecular weight aggregates and most oligomers disappeared when pressurized solutions were treated with β-mercaptoethanol (15 mL/L) prior to electrophoresis. Thus pressure induced the formation of intermolecular S-S bonds, especially when the pH was maintained close to neutral. Previous studies with thermally-induced β-lactoglobulin gels had shown that SH/ S-S interchange reactions were enhanced at or above neutrality.

NMR Studies of Thermal Denaturation and Cation-Mediated Aggregation of .beta.-Lactoglobulin

Thermal denaturation, aggregation, and gelation of β-lactoglobulin in solutions containing either no additional salt, 100 mM NaCl 2 or 20 mM CaCl 2 were investigated using 1 H and 111 Cd NMR spectroscopies. 1 H NMR temperature dependence experiments suggest that an oligomeric protein aggregate (octamer/differ) undergoes dissociation to form a dimer/monomer as the temperature is increased from 10 to 25/30 o C, and at temperatures above 25/30 o C, the protein undergoes a conformational change that leads to denaturation and aggregation/gelation

Thermal aggregation and gelation of bovine β-lactoglobulin

Heat-induced aggregation and gelation of β-lactoglobulin, dispersed in imidazole-HCl buffer containing 0.1 mol/dm 3 NaCl at a concentration of 10% (w/v), was studied at different temperatures and pHs. Gel electrophoresis under non-dissociating (PAGE, in the absence of dissociating and reducing agents) and dissociating but not reducing conditions (SDS-PAGE) was used to determine the extents of aggregation. The rate of loss of β-lactoglobulin from non-dissociating-PAGE was faster than from SDS-PAGE and the rates could be described by second-order kinetics. The loss of protein on heat treatment at 75°C increased as the pH increased from 6.0 to 9.0, under both non-dissociating and dissociating conditions. Dynamic shear measurements were used to determine the development of gel structure by measuring the changes in storage modulus ( G′) during heating. No detectable changes in G′ were observed during heating at 70°C. At 75 and 80°C, G′ increased with heating time, the rate of increase being greater at 80°C. The increase in G′ on heat treatment at 75°C was faster at pH 7.0 than at pH 6.0, 8.0 and 9.0. Comparison of the loss of native β-lactoglobulin structure due to the formation of non-covalently linked protein aggregates and covalently cross-linked aggregates with the changes in G′ showed that no measurable changes in G′ occurred until most of the protein had formed covalently cross-linked aggregates. This suggested that protein aggregates are formed as an intermediate prior to the formation of self-supporting macroscopic gel networks.

Microstructure and rheological behaviour of particulate β-lactoglobulin gels

The microstructure of the network as well as the strands of particulate β-lactoglobulin gels formed at pH 5.3 have been characterized by microscopy. The microstructural influence on the rheological properties both at small and large deformations has been measured. It was shown that the microstructure depends on the heating rate used. Gels formed at a fast heating rate (5–10°C/min) consisted of a homogeneous network with pore sizes of 20–30 μm. The strands were formed by evenly sized spherical particles linked like a flexible string of beads. At a slow heating rate (0.1–1°C/ min) the network had larger pores, ~100–150 μm. The network formed at 0.1°C/min was inhomogeneous, with regions of small and large pores. The particle size distribution was broader at a slow heating rate and the strands, formed by several particles fused together, were thicker. Tensile measurements of fracture properties showed that the gels formed at a fast heating rate had higher stress and strain at fracture due to the network structure. The size of the weakest element of the network was deduced from notch sensitivity measurements and correlated well with the pore size, i.e. the fracture starts at the largest pores. Viscoelastic measurements showed that the gels formed at a slow heating rate had a higher storage modulus, G′, which was explained by the microstructure of the strands. The thick strands of particles fused together were stiffer, thus causing a higher storage modulus than the flexible strands formed at a fast heating rate. The concentration dependence of G′ was measured, and a model assuming clustering of clusters was applied to the results. The model shows that the particulate gels are self-similar within the region of concentration measured, with a fractal dimension of ~2.5.

Inhomogeneous fine-stranded β-lactoglobulin gels

Inhomogeneities occur in the network of fine-stranded β-lactoglobulin gels. They have been characterized by electron microscopy and their influence on the rheological properties at small deformations measured. The inhomogeneities occurred as dense and loose regions in the network and their formation depended on the heating rate during gel formation (0.017–12°C/min) at pH 7.5 or on pH if close to where the network structure shifts from fine-stranded to particulate (pH 5.8–6.5). An inhomogeneous microstructure was formed at pH 7.5 with a slow heating rate, whereas the network was homogeneous after fast heating. These homogeneous gels had higher G′ than the inhomogeneous gels. In addition the character, as expressed by the frequency dependence, changed from strong homogeneous gels to weaker, more frequency-dependent inhomogeneous gels. The formation of the inhomogeneities was monitored at the slowest heating rate by measuring G′ during gelation. G′ showed a maximum around 70°C, indicating a separation into polymer-rich and polymer-poor regions in the network. Electron microscopy showed a transient, homogeneous network before the separation. Both types of inhomogeneous gels had broken frequency curves, i.e. G′ f had two slopes, which were caused by different relaxation times in the dense and loose regions. Relaxation measurements of the inhomogeneous gels showed relaxation time spectra with two peaks. A model was used to confirm that a broken frequency curve may be caused by two peaks in the relaxation time spectrum.

Fine-stranded and particulate gels of β-lactoglobulin and whey protein at varying pH

The effects of pH on the gel network structure have been characterized by means of different microscopy techniques. β-Lactoglobulin forms white particulate gels in the region of pH 4–6, and transparent fine-stranded gels below and above this region. Microstructural analysis showed differences in the regularity and particle size of the particulate network structures. The fine-stranded gels formed at low pH were composed of stiff short strands, whereas the fine-stranded gels formed at high pH had longer, more flexible strands. A mixture of particulate and fine-stranded structures was found at the lower shift, at pH 4. At the higher shift pH 6 no mixtures of structures were found and either a particulate or a fine-stranded structure was formed. The microstructure of whey protein gels at different pH showed similarities with the microstructure of β-lactoglobulin gels at comparable pH. The results of the microstructure correlated with previously published data on fracture properties.

Large deformation properties of β-lactoglobulin gel structures

Different gel structures formed by β-lactoglobulin dissolved in distilled water (12% w/w) at pH 3.0–7.5 have been investigated using tensile measurements at large deformations. Gels formed at pH 4–6 were opaque, whereas at pH values below or above this range they were transparent. The fracture properties showed large variations over the pH range studied. Gels formed at low pH were brittle with low strain and stress at fracture, as opposed to those formed at high pH, which were rubber-like with high strain and stress at fracture. Gels formed at intermediate pH (pH 4–6) had an intermediate, near-constant, strain at fracture. The fracture stress was, however, higher at pH 5.5–6.0 than at pH 4.0–5.2. The specific fracture energy resembled the stress at fracture, with a maximum of 6 J/m 2 at pH 6.0. Gels formed at pH 4.5, 5.5, 6.5 and 7.5 were all notch-sensitive. The opaque gels were defined as aggregate gels and the transparent gels were defined as fine-stranded gels. The fracture properties clearly showed there are differences between the fine-stranded gels formed at high pH and those formed at low pH. The fracture stress demonstrated that there are structural differences within the pH range in which the aggregate gels are formed. The non-linearity of the stress-strain curve was the same for all fine-stranded gels, which had r-shaped stress-strain curves. The stress-strain curves of the aggregate gels were almost linear. The non-linearity did not influence the fracture properties. Young's modulus showed two peaks, at pH 3.5 and 6.0, coinciding with the range where the structure changes between aggregated and fine-stranded. The stress at fracture also has a maximum at pH 6.0. The high elasticity and fracture stress may depend on strong, elastic areas in the network structure. Apart from the two peaks, Young's modulus shows the same behaviour as the storage modulus, G′, measured at small deformations, but Young's modulus is slightly larger than 3 G′.

Effects of structuring and destructuring anionic ions on the rheological properties of thermally induced β-lactoglobulin gels

The effects of increasing levels (0–1.0 mol/dm 3) of protein-structuring (chloride, NaCl; sulfate, Na 2SO 4) and -destructuring anionic ions (thiocyanate, NaSCN) on rheological properties and electron microstructures of protein gels were examined. The effects of the ions varied with concentration and species. At concentrations above 100 mM they enhanced gel strength in the order SCN − > Cl − > SO 4 2−. The hardness of gels increased with increasing concentrations up to 0.1, 0.2 and 1.0 mol/dm 3 with SO 4 2−, Cl − and SCN − respectively. Electron microstructures of the gels showed that distribution of the protein in the gel matrix became less ordered with increasing ion concentration.

Viscoelastic behaviour of β-lactoglobulin gel structures

The viscoelastic properties of β-lactoglobulin gels, made by dissolution of β-lactoglobulin in distilled water, have been investigated by dynamic rheological measurements at pH 2.5–9.0. Gels formed at pH 4–6 were opaque and have been defined as aggregate or particle gels; at pH below or above this range gels were transparent and have been defined as fine-stranded gels. The aggregate gels were more strain sensitive during gelation and more frequency dependent than the fine-stranded gels. They also had a higher storage modulus than the fine-stranded gels at a constant β-lactoglobulin concentration of 12% (w/w). The critical concentration for gel formation was lower for the aggregate gels than for the fine-stranded gels, and as low as 1% in the pH range 4.5–5.5. This indicates a very open structure. The onset of gelation of 12% β-lactoglobulin gels at intermediate pH, when measured as G ′ = G ″ at 1 Hz or as the development of a measurable G′ takes place at temperatures far below the temperature of denaturation. This is not the case at higher or lower pH, where the onset of gelation is above the temperature of denaturation. The heating rate during gel formation was varied between 0.008 and 1°C/min. The gelation temperature of the fine-stranded gels was found to increase with an increasing heating rate.

Rheological Properties of Heat-Induced β-Lactoglobulin Gels

Formation of heat-induced β-lactoglobulin gels was studied by oscillation rheometry for up to 16h at pH 4.5, 5, and 7 and a concentration of 3, 4, and 5% (wt/vol) protein. The absolute shear moduli (a measure of gel stiffness) continued to increase even after 16h of aging. During aging, the character of the gels became progressively more elastic. Despite a large variation in the dynamic moduli of the gels studied, their relative increase and the changes in loss tangent were very similar as a function of aging. The gels at pH greater than 6 were, over short time scales, relatively more elastic and less viscous. The influence of temperature was nearly the same for all gels. The shear moduli were generally proportional to the concentration to the power of 2.5.