Search

Self-assembly of α-lactalbumin after partial hydrolysis by a protease from Bacillus licheniformis can result in nanotubular structures, which show many similarities to microtubules. Calcium plays a crucial role in this process. The objective of this investigation was to study the role of calcium in more detail. The kinetics of the hydrolysis step and the self-assembly step were monitored by respectively liquid chromatography−mass spectrometry and dynamic light scattering. The microstructure of the gels finally formed was investigated by transmission electron microscopy. This investigation demonstrates that calcium accelerated the kinetics of the self-assembly, but it had no effect on the hydrolysis kinetics. As a result of the accelerated self-assembly kinetics at a high calcium concentration, the time of gelation decreased as well. A minimum concentration of calcium needed to obtain the tubular α-lactalbumin structures was determined. Below R = 1.5 (mole calcium/mole α-lactalbumin), turbid gels with randomlike structure were obtained. Between R = 1.5 and R = 6, translucent gels with a fine stranded network of tubules were formed, while higher calcium concentrations had a negative effect on the tubule formation, resulting in amorphous structures. The optimum calcium concentration for α-lactalbumin nanotube formation seemed to be around R = 3.

Structural properties of whey protein (WP)/gum arabic (GA) coacervates were investigated by measuring the diffusivity of WP and GA in their coacervate phase as a function of pH by means of three different complementary techniques. The combination of these measurements revealed new insights into the structure of coacervates. Nuclear magnetic resonance (NMR) measured the self-diffusion coefficient of the GA in the coacervate phase prepared at various pH values. Fluorescence recovery after photobleaching (FRAP) was measured using a confocal scanning laser microscope. The WP and GA were covalently labeled with two different dyes. The time of fluorescence recovery, related to the inverse of the diffusion coefficient, was evaluated from the measurements, and the diffusivity of the WP and GA on a long time scale could be individually estimated at each pH value. Diffusing wave spectroscopy (DWS) combined with transmission measurement was carried out in the coacervate phase, and the diffusion coefficient, corresponding to the averaged diffusion of all particles that scattered in the system, was calculated as a function of pH. Independently of the technique used, the results showed that the diffusion of the WP and GA within the coacervate phase was reduced as compared to a diluted biopolymer mixture. NMR, DWS, and FRAP measurements gave similar results, indicating that the biopolymers moved the slowest in the coacervate matrix at pH 4.0−4.2. It is assumed that the diffusion of the WP and GA is reduced because of a higher electrostatic interaction between the biopolymers. Furthermore, FRAP results showed that in the coacervate phase WP molecules diffused 10 times faster than GA molecules. This result is very relevant because it shows that WP and GA move independently in the liquid coacervate phase. Finally, DWS measurements revealed that the coacervate phase rearranged with time, as evidenced by a decrease of the diffusion coefficient and a loss of the turbidity of the sample. A more homogeneous transparent coacervate phase was obtained after a few days/weeks. Faster rearrangement was obtained at pH 3.0 and 3.5 than at higher pH values.

Complex coacervation in whey protein/gum arabic (WP/GA) mixtures was studied as a function of three main key parameters:  pH, initial protein to polysaccharide mixing ratio (Pr:Ps)ini, and ionic strength. Previous studies had already revealed under which conditions a coacervate phase was obtained. This study is aimed at understanding how these parameters influence the phase separation kinetics, the coacervate composition, and the internal coacervate structure. At a defined (Pr:Ps)ini, an optimum pH of complex coacervation was found (pHopt), at which the strength of electrostatic interaction was maximum. For (Pr:Ps)ini = 2:1, the phase separation occurred the fastest and the final coacervate volume was the largest at pHopt = 4.0. The composition of the coacervate phase was determined after 48 h of phase separation and revealed that, at pHopt, the coacervate phase was the most concentrated. Varying the (Pr:Ps)ini shifted the pHopt to higher values when (Pr:Ps)ini was increased and to lower values when (Pr:Ps)ini was decreased. This phenomenon was due to the level of charge compensation of the WP/GA complexes. Finally, the structure of the coacervate phase was studied with small-angle X-ray scattering (SAXS). SAXS data confirmed that at pHopt the coacervate phase was dense and structured. Model calculations revealed that the structure factor of WP induced a peak at Q = 0.7 nm^-1, illustrating that the coacervate phase was more structured, inducing the stronger correlation length of WP molecules. When the pH was changed to more acidic values, the correlation peak faded away, due to a more open structure of the coacervate. A shoulder in the scattering pattern of the coacervates was visible at small Q. This peak was attributed to the presence of residual charges on the GA. The peak intensity was reduced when the strength of interaction was increased, highlighting a greater charge compensation of the polyelectrolyte. Finally, increasing the ionic strength led to a less concentrated, a more heterogeneous, and a less structured coacervate phase, induced by the screening of the electrostatic interactions.

Whey proteins (WPs) and the exopolysaccharide B40 (EPS B40) form electrostatic complexes under specific conditions. EPS B40 is a natural thickener in yogurt-like products. It is a phosphated polysaccharide and thus has a strong polyelectrolyte character. When the WP and the EPS B40 were mixed at pH values near or below the isoelectric point (pI) of the protein, soluble complexes were formed at pHc and phase separation took place below pHφ. The formation and the structure of those complexes were studied by various methods, including turbidity, dynamic and static light scattering (DLS and SLS), and viscosity measurements. The results showed that the strength of the interaction was strongly pH- and salt-dependent. The ζ-potential of the protein at pHc and pHφ was linearly dependent on the square root of the ionic strength (√I), showing the electrostatic nature of the interaction. Light scattering and viscosity measurements provided new results on the behavior of the complexes at the molecular level. In the region where the complexes were still soluble and at low ionic strength, the DLS radius measured in the WP/EPS B40 mixture was smaller than the coil size in the EPS B40 solution but the apparent molar mass was increased. The increase of the molecular mass was attributed to the complexation of WP on the EPS B40 chain, which, at low salt, induced a reduction of the intramolecular repulsion and led to the compaction of the polysaccharide. Also, the ratio of protein to polysaccharide was varied in order to get more insight into the dynamics, the structure, and the apparent stoichiometry of the EPS B40/WP complexes. The results illustrated that phase separation was a consequence of charge neutralization of the complexes and that the apparent stoichiometry of the complexes depends on the order of mixing of the compounds. In time, the complexes rearranged to form neutralized complexes and free EPS B40. The concept of cooperative binding was highlighted in the case studied.

High-sensitivity differential scanning calorimetry was applied for the first time to study the micellization of bovine β-casein in aqueous solutions, complemented by analytical ultracentrifugation data. The micellization was described as a single endothermic heat capacity peak located in the temperature range of 0−50 °C. The transition originated at temperatures very close to 0 °C. The position of the heat capacity peak was strongly dependent on the protein concentration and the solvent composition. Thermodynamic parameters of the micellization, the transition temperature and enthalpy, were determined over a wide range of protein concentration. The observed thermal behavior of β-casein was well fitted by the thermodynamic shell model of micellization proposed by Kegeles (J. Phys. Chem. 1979, 83, 1728). The effects of different cosolutes (inorganic salts, urea, Tris, and ethanol) on the thermodynamic parameters of micellization of β-casein underline the hydrophobic character of the transition.

Mixtures of gum arabic and whey protein (whey protein isolate, WP) form an electrostatic complex in a specific pH range. Three phase boundaries (pHc, pHφ1, pHφ2) have been determined using an original titration method, newly applied to complex coacervation. It consists of monitoring the turbidity and light scattering intensity under slow acidification in situ with glucono-δ-lactone. Furthermore, the particle size could also be measured in parallel by dynamic light scattering. When the pH is lowered, whey proteins and gum arabic first form soluble complexes. This boundary is designated as pHc. When the interaction is stronger (at lower pH), phase separation takes place (at pHφ1). Finally, at pHφ2 complexation was suppressed by the charge reduction of the gum arabic. The major constituent of the whey protein preparation used was β-lactoglobulin (β-lg), and it was shown that β-lg was indeed the main complex-forming protein. Moreover, an increase of the ionic strength shifted the pH boundaries to lower pH values, which was summarized in a state diagram. The experimental pHc values were compared to a newly developed theory for polyelectrolyte adsorption on heterogeneous surfaces. Finally, the influence of the total biopolymer concentration (0−20% w/w) was represented in a phase diagram. For concentrations below 12%, the results are consistent with the theory on complex coacervation developed by Overbeek and Voorn. However, for concentrations above 12%, phase diagrams surprisingly revealed a “metastable” region delimited by a percolation line. Overall, a strong similarity is seen between the behavior of this system and a colloidal gas−liquid phase separation.

The milk proteins β- and κ-casein are in a monomeric state below their critical micelle concentration (cmc) and spontaneously self-associate above the cmc, at or above room temperature. Small-angle neutron scattering (SANS) measurements at 5 g L^-1 confirmed the high specific volume of these micelles, and ¹H-NMR and calorimetric measurements showed that the polypeptide chains remain open and their side chains largely flexible in the associated state. The experimental values of parameters used to describe both types of micelle were radius of gyration of 8 nm, scattering radius (in ²H2O) of 11 nm, and interaction radius close to 15 nm. The scattering radius is obtained by assuming a homogeneous sphere, and the interaction radius by assuming hard-sphere-like behavior. The micelle size, structure, and interaction radius were independent of concentration. The interparticle structure factor of the micelles was determined using a polydisperse hard-sphere model, which showed that the wave vector position of the peak of the interparticle structure factor was independent of concentration over the range 5−20 g L^-1, consistent with effective hard-sphere behavior. The SANS and ¹H-NMR experiments indicated that the β-casein micelles were less compact and more dynamic than the κ-casein micelles. The micelle dispersions were treated with the cross-linking enzyme transglutaminase. Scattering results showed that the cross-linking hardly influenced the structure or interaction of the micelles, indicating that there was little or no interparticle cross-linking. Addition of 6 M urea did not change the structure of the cross-linked micelles, whereas addition of the urea to the non-cross-linked micelles caused extensive dissociation.

Pectin, a polysaccharide derived from plant cells of fruit, is commonly used as stabilizer in acidified milk drinks. To gain a better understanding of the way that pectin stabilizes these drinks, we studied the adsorption and layer thickness of pectin on casein micelles in skim milk dispersions. Dynamic light scattering was used to measure the layer thickness of adsorbed pectin onto casein micelles in situ during acidification. The results indicate that the adsorption of pectin onto casein micelles is multilayered and takes place at and below pH 5.0. Renneting, i.e., cleaving-off κ-casein from the casein micelles, did not alter the adsorption pH. It did, however, show that pectin arrests the rennet-induced flocculation of casein micelles below pH 5.0. From the findings we concluded the attachment of pectin onto casein micelles is driven by electrosorption. Adsorption measurements confirmed the multilayered nature of the adsorption of pectin onto casein micelles. Both the adsorbed amount and the layer thickness increased with decreasing pH in the relevant range 3.5−5.0. The phase behavior of a casein micelles/pectin mixture was determined and could be explained in terms of thermodynamic incompatibility being relevant above pH 5.0 and adsorption, leading to either stabilization and bridging, being relevant below pH 5.0. The results confirm that electrosorption is the driving force for the adsorption of pectin onto casein micelles.

The viscoelastic properties of a well-characterized exocellular polysaccharide (EPS) produced by the lactic acid bacterium Lactococcus lactis subsp. cremoris strain B40 were investigated. Dynamic rheological measurements were made as a function of frequency and EPS concentration. The bead−spring model of Rouse could reasonably describe the dynamic properties. Concentrated EPS solutions have a significant elasticity (G‘ > G‘ ‘) at high frequencies. The relatively high G‘ values at high concentrations and high frequencies are indicative of significant normal stress differences, and we put forward a hypothesis that suggests that these normal stresses may explain the contribution of EPSs to the ropy behavior of yogurts.

An attractive interaction, commonly referred to as depletion interaction, is induced between aggregated whey protein colloid (AWC) particles when they are mixed with exocellular polysaccharides (EPSs) from a lactic acid bacterium. This interaction originates from a loss of conformational entropy of the EPSs near the surfaces of neighboring AWC particles and leads to a phase separation at high enough EPS and AWC concentrations. The effect of the depletion interaction on the properties of the mixtures of EPS and AWC particles is first studied in the stable, that is, one-phase, region. Small-angle neutron scattering (SANS) and dynamic light scattering (DLS) were used to characterize the strength of attractions. The SANS results can be described quantitatively by a model for depletion interaction. From Ornstein−Zernike plots, we derive the position of the spinodal. The DLS results can be described qualitatively quite well by using a recently derived expression for the wavevector (Q)-dependent diffusion coefficient as a function of the correlation length. Further, the experimental phase boundary is determined and compared with a mean-field theory, which evaluates the free energy of a mixture of colloids and large nonadsorbing polymers. The independently calculated spinodal was found to be consistent with the experimentally determined position of the phase boundary. Spinodal phase separation kinetics is investigated by small-angle light scattering (SALS). At low Q, a scattering peak was detected, which shifted to lower Qs with time, in agreement with other experimental data and theoretical predictions for spinodal decomposition. Both the scaling of the scattered intensity with Q and the scaling of the Q-position of the peak with time agree with theoretical predictions.

The physical properties of a polysaccharide produced by the lactic acid bacterium Lactococcus lactis subsp. cremoris strain NIZO B40 were investigated. Separation of the polysaccharide from most low molar mass compounds in the culture broth was performed by filtration processes. Residual proteins and peptides were removed by washing with a mixture of formic acid, ethanol, and water. Gel permeation chromatography (GPC) was used to size fractionate the polysaccharide. Fractions were analyzed by multiangle static light scattering in aqueous 0.10M NaNO3 solutions from which a number- (Mn) and weight-averaged (Mw) molar mass of (1.47 ± 0.06) · 10³ and (1.62 ± 0.07) · 10³ kg/mol, respectively, were calculated so that Mw/Mn ≈ 1.13. The number-averaged radius of gyration was found to be 86 ± 2 nm. From dynamic light scattering an apparent z-averaged diffusion coefficient was obtained. Upon correcting for the contributions from intramolecular modes by extrapolating to zero wave vector a hydrodynamic radius of 86 ± 4 nm was calculated. Theoretical models for random coil polymers show that this z-averaged hydrodynamic radius is consistent with the z-averaged radius of gyration, 97 ± 3 nm, as found with GPC. © 1999 John Wiley & Sons, Inc. Biopoly 49: 1–9, 1999

The properties of the outer hairy layer of casein micelles have been studied using several methods. Viscosity and light-scattering measurements have been performed on rennet-destabilized skim milk at different concentrations of calcium. We have reproduced the known result that calcium promotes destabilization at relatively high κ-casein surface coverages (even at only 50% of the κ-casein molecules cut off). This suggests that calcium bridges contribute to the casein micelle attraction. The effect may be direct by calcium-mediated bridging of β-casein or αs1-casein (phosphate/carboxylate) or κ-casein (carboxylate) or indirect by altering the interplay between several types of caseins in the micelle. In the analysis of the experiments, we make use of an adhesive hard sphere approximation. The casein hairy layer has further been modeled by a self-consistent field (SCF) theory in which coarse-grained molecular details were included. In these calculations the effect of calcium bridges cannot directly be accounted for. It is shown that a κ-casein layer induces repulsive interactions of mainly steric origin. Cutting the N-terminus part results in reduced (shorter-ranged) repulsive pair potentials, which still dominate over the van der Waals attraction. As a result of the altered interplay between the several types of casein molecules during the renneting process, the αs1-casein concentration in the outer hairy layer may increase. Again using the SCF model, but now using a less-detailed description of the system, we examine a possible result of this. We confirm that diblock copolymers (simple model for κ-casein) and diblock copolymer with shorter soluble block (simple model for para-κ-casein) give repulsion and triblock copolymers (simple model for αs1-casein) give attraction by means of bridging (adsorption). The renneting process is simulated by increasing the ratio of model para-κ-casein to κ-casein at a fixed αs1-casein concentration. As a result, the potential shifts from repulsive to attractive. We show that by reducing the range of repulsive interactions of the diblock copolymers, we can make room available to express the attractive contributions of the model αs1-casein. This comprises a novel molecular-based mechanism for the renneting process.

The denaturation and aggregation of β-lactoglobulin was studied by isothermal calorimetry. Experiments with several β-lactoglobulin concentrations (15−100 g/L) were performed at temperatures in the range 62−68.5 °C. Even a small change in temperature had a tremendous effect on the shape of the thermograms, depending also very strongly on the β-lactoglobulin concentration used. The measured thermograms were modeled using the kinetic model for the denaturation and aggregation of β-lactoglobulin recently developed by Roefs and De Kruif. In this kinetic model we recognize four consecutive steps:  dissociation, unfolding, exchange of disulfide bonds, and aggregation. The numerical calculations yielded results that were in qualitative and quantitative agreement with typical experimental isothermal calorimetry curves. DSC curves and the decrease in concentration of native β-lactoglobulin as a function of time could also be reproduced well. We think that isothermal calorimetry, in combination with this kinetic model, provides a valuable and useful approach to the study of the denaturation and aggregation of β-lactoglobulin and other proteins.

The turbidity of skim milk dispersions was measured in a spectrophotometer at four different wavelengths and at various dilutions. After adding rennet to milk the transmission (~exp(-turbidity)) of the samples decreases gradually. This decrease in transmission, which depends on the volume fraction of the micelles and the wavelength of the light used, is caused by the interactions between the micelles. During the renneting the interactions change from repulsive to attractive and can be characterized by the second osmotic virial coefficient. Before the rennet is added to the milk the casein micelles can be characterized as hard spheres for which B'₂ = 4V_HS, where V_HS is the (excluded) volume of a hard sphere. Renneting causes the casein micelles to lose their colloidal stability and B'₂ decreases strongly. The micelle interaction can be described by the so-called adhesive hard sphere model, also called the "sticky" sphere model. A theory is presented which quantitatively describes the changes in transmission on renneting in the initial stages. Copyright 1993, 1999 Academic Press