Skelte G. Anema

Association of denatured whey proteins with casein micelles in heated reconstituted skim milk and its effect on casein micelle size

When skim milk at pH 6.55 was heated (75 to 100 degrees C for up to 60 min), the casein micelle size, as monitored by photon correlation spectroscopy, was found to increase during the initial stages of heating and tended to plateau on prolonged heating. At any particular temperature, the casein micelle size increased with longer holding times, and, at any particular holding time, the casein micelle size increased with increasing temperature. The maximum increase in casein micelle size was about 30-35 nm. The changes in casein micelle size were poorly correlated with the level of whey protein denaturation. However, the changes in casein micelle size were highly correlated with the levels of denatured whey proteins that were associated with the casein micelles. The rate of association of the denatured whey proteins with the casein micelles was considerably slower than the rate of denaturation of the whey proteins. Removal of the whey proteins from the skim milk resulted in only small changes in casein micelle size during heating. Re-addition of beta-lactoglobulin to the whey-protein-depleted milk caused the casein micelle size to increase markedly on heat treatment. The changes in casein micelle size induced by the heat treatment of skim milk may be a consequence of the whey proteins associating with the casein micelles. However, these associated whey proteins would need to occlude a large amount of serum to account for the particle size changes. Separate experiments showed that the viscosity changes of heated milk and the estimated volume fraction changes were consistent with the particle size changes observed. Further studies are needed to determine whether the changes in size are due to the specific association of whey proteins with the micelles or whether a low level of aggregation of the casein micelles accompanies this association behaviour. Preliminary studies indicated lower levels of denatured whey proteins associated with the casein micelles and smaller changes in casein micelle size occurred as the pH of the milk was increased from pH 6.5 to pH 6.7.

Characterization of protein components of natural and heat-treated milk fat globule membranes

The proteins associated with the milk fat globule membrane (MFGM), isolated from early, mid and late season whole milks, were characterized using one- and two-dimensional SDS-PAGE under reducing and non-reducing conditions. In some experiments, MFGM separated from fresh whole milk was suspended in simulated milk ultrafiltrate (SMUF) and heated at various temperatures and times. SDS-PAGE under reducing conditions followed by staining with Coomassie blue showed the presence of about 37 protein bands, ranging in molecular weight from 15 to 200 kDa. SDS-PAGE under non-reducing conditions showed only about 25 distinct bands and the intensity of xanthine oxidase and butyrophilin bands was much less, while the intensity of PAS 6/7 band was similar compared with the reduced SDS-PAGE. Two-dimensional SDS-PAGE showed that the protein complexes that remained at the top of non-reducing gel were resolved into mostly xanthine oxidase and butyrophilin with a small proportion of PAS 6. These results indicate that xanthine oxidase and butyrophilin may be complexed via intermolecular disulfide bonds in the natural MFGM, although it is not possible to differentiate the individual protein distributions within these aggregates. It was found that the total protein content (mg/g fat) of MFGM and the percentage of xanthine oxidase and butyrophilin in early and late season MFGM were higher than that of mid season MFGM. In heated samples (above 60°C), xanthine oxidase and butyrophilin interacted further to form higher molecular weight protein complexes, while PAS 6/7 was relatively heat stable.

Effect of Calcium on the Morphology and Functionality of Whey Protein Nanofibrils

Self-assembly of amyloid-like nanofibrils during heating of bovine whey proteins at 80 °C and pH 2 is accelerated by the presence of NaCl and/or CaCl(2), but the rheological consequences of accelerated self-assembly are largely unknown. This investigation focused on the impact of CaCl(2) on the evolution of rheological properties and fibril morphology of heated whey protein isolate (WPI), both during self-assembly at high temperature and after cooling. Continuous rotational rheometry of heated 2% w/w WPI showed a nonlinear effect of CaCl(2) on the viscosity of fibril dispersions, which we attributed to effects on fibril flexibility and thus the balance between intrafibril and interfibril entanglements. Small-amplitude oscillatory measurements made in situ during heating of 10% w/w WPI at 80 °C suggest that CaCl(2) is not involved in either fibril structure or gel structure, and this was confirmed with dialysis experiments.

On heating milk, the dissociation of κ-casein from the casein micelles can precede interactions with the denatured whey proteins

When kappa-casein (kappa-CN) was added to milk, and the milk was subsequently pH adjusted (pH 6.5-6.9) and heated (90 degrees C/15 min), the serum contained considerably higher levels of denatured whey proteins than the milks without added kappa-CN. When milk at pH 6.5-6.9 was heated at 90 degrees C for different times, kappa-CN was found in the serum in the early stages of heating and before significant levels of whey proteins were denatured. kappa-CN reached its maximum level in the serum before the whey proteins were fully denatured. When milk at pH 6.5-6.9 was heated at 20-90 degrees C for 15 min, kappa-CN dissociated from the casein micelles at all temperatures, with the level in the serum increasing with the temperature and the pH at heating. kappa-CN dissociated from the micelles at temperatures below those at which significant levels of the whey proteins were denatured. When taken together, the results from these experiments strongly indicate that the dissociation of kappa-CN from the micelles can precede the interaction of the denatured whey proteins with kappa-CN, and that there is a preferential interaction of the denatured whey proteins with serum-phase kappa-CN.

Co-acervates of lactoferrin and caseins

On mixing positively charged lactoferrin (LF) with negatively charged caseins (*CN) it is observed that complexes are formed. The * stands for α, β, κ or Na. The size of the complex co-acervates appears to grow indefinitely and asymptotically near the point of charge equivalency. Away from the charge equivalent ratio it seems that build-up of (surface) charges limits complex size. We proposed a simple scaling law so as to predict the size of the complex. By assuming that surface charge density is constant or can reach only a maximum value, it follows that scattering intensity is proportional to |(1 − x/xcrit)|−3 where x is the mole (mass) fraction of the cationic protein and xcrit the value of the mole (mass) fraction at the charge equivalent ratio. Both scattering intensity and particle size obey this simple assumption. We investigated three different caseins, all of which formed co-acervate complexes with LF, but at different molar ratios. Critical composition varied inversely with pH, showing that charge neutrality is the determining factor. Sodium caseinate formed complexes as well but the growth was limited, presumably due to the intrinsic surfactant properties of whole casein. Adding NaCl diminishes the interaction and above 0.4 mol L−1 of NaCl no β-CN–LF complexes are formed. The charge neutral composition shifts to the LF side on adding NaCl, probably because the casein can wrap around the LF more effectively.

The effect of chymosin on κ-casein-coated polystyrene latex particles and bovine casein micelles

Abstract Bovine κ-casein was adsorbed onto negatively charged polystyrene latex particles of various sizes and the changes in the hydrodynamic diameters of the particles were monitored by photon correlation spectroscopy. The latex particle diameters were increased on addition of small amounts of κ-casein, and plateaued once a maximum adsorption was attained. The size increase was about 25 nm regardless of the initial size of the latex particle, indicating that the surface arrangement of κ-casein was similar on all latex sizes. Treatment of the casein micelles in skim milk and the κ-casein-coated latex particles with chymosin initially decreased the particle diameter by about 10nm after which the particle diameter rapidly increased as the aggregation reaction proceeded. The action of chymosin on both the casein micelles and the κ-casein-coated latex particles was retarded by increasing the NaCl concentration. The similarity between the κ-casein-coated latex particles and the casein micelles in milk, especially their behaviour towards chymosin, indicates that these latex particles may be a simple model system for studying some casein micelle properties under controlled conditions.

Complex coacervates of lactotransferrin and β-lactoglobulin

HYPOTHESIS Oppositely charged proteins should interact and form complex coacervates or precipitates at the correct mixing ratios and under defined pH conditions. EXPERIMENTS The cationic protein lactotransferrin (LF) was mixed with the anionic protein β-lactoglobulin (B-Lg) at a range of pH and mixing ratios. Complexation was monitored through turbidity and zeta potential measurements. FINDINGS Complexation between LF and B-Lg did occur and complex coacervates were formed. This behaviour for globular proteins is rare. The charge ratios of LF:B-Lg varies with pH due to changing (de) protonation of the proteins. Nevertheless we found that the complexes have a constant stoichiometry LF:B-Lg=1:3 at all pHs, due to charge regularization. At the turbidity maximum the zeta potential of complexes is close to zero, indicating charge neutrality; this is required when the complexes form a new concentrated liquid phase, as this must be electrically neutral. Complexes were formed in pH region 5-7.3. On addition of salt (NaCl) complexation is diminished and disappears at a salt concentration of about 100 mMol. The coacervate phase has a very viscous consistency. If we consider the proteins as colloidal particles then the formed complex coacervate phase may have a structure that resembles a molten salt comparable to, for example, AlCl3.

Interactions of fat globule surface proteins during concentration of whole milk in a pilot-scale multiple-effect evaporator.

The changes in milk fat globules and fat globule surface proteins during concentration of whole milk using a pilot-scale multiple-effect evaporator were examined. The effects of heat treatment of milk at 95 degrees C for 20 s, prior to evaporation, on fat globule size and the milk fat globule membrane (MFGM) proteins were also determined. In both non-preheated and preheated whole milk, the size of milk fat globules decreased while the amount of total surface proteins at the fat globules increased as the milk passed through each effect of the evaporator. In non-preheated samples, the amount of caseins at the surface of fat globules increased markedly during evaporation with a relatively small increase in whey proteins. In preheated samples, both caseins and whey proteins were observed at the surface of fat globules and the amounts of these proteins increased during subsequent steps of evaporation. The major original MFGM proteins, xanthine oxidase, butyrophilin, PAS 6 and PAS 7, did not change during evaporation, however, PAS 6 and PAS 7 decreased during preheating. These results indicate that the proteins from the skim milk were adsorbed onto the fat globule surface when the milk fat globules were disrupted during evaporation.

β-Lactoglobulin Self-Assembly: Structural Changes in Early Stages and Disulfide Bonding in Fibrils

Bovine β-lactoglobulin (β-Lg) self-assembles into long amyloid-like fibrils when heated at 80 °C, pH 2, and low ionic strength (<0.015 mM). Heating β-Lg under fibril-forming conditions shows a lag phase before fibrils start forming. We have investigated the structural characteristics of β-Lg during the lag phase and the composition of β-Lg fibrils after their separation using ultracentrifugation. During the lag phase, the circular dichroism spectra of heated β-Lg showed rapid unfolding, and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of samples showed increasing hydrolysis of β-Lg. The SDS-PAGE profiles of fibrils separated by ultra centrifugation showed that after six hours, the fibrils consisted of a few preferentially accumulated peptides. Two-dimensional SDS-PAGE under reducing and nonreducing conditions showed the presence of disulfide-bonded fragments in the fibrils. The sequences in these peptide bands were characterized by in-gel digestion electrospray ionization (ESI)-MS/MS. The composition of solubilized fibrils was also characterized by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS/MS. Both MS analyses showed that peptides in fibrils were primarily from the N-terminal region, although there was some evidence of peptides from the C-terminal part of the molecule present in the higher molecular weight gel bands. We suggest that although the N-terminal region of β-Lg is almost certainly involved in the formation of the fibrils, other peptide fragments linked through disulfide bonds may also be present in the fibrils during self-assembly.

Whey Protein Nanofibrils: The Environment–Morphology–Functionality Relationship in Lyophilization, Rehydration, and Seeding

Amyloid-like fibrils from β-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.