Concentrates from skim milk heated at pH 6.5 to 7.1 had viscosities that followed power law behaviour and could be described by consistency coefficients (K) and flow behaviour indices (n). On evaporation to 40% total solids, milk heated at pH 6.5 produced concentrates with higher K than those from unheated milk. Milk samples heated at pH 7.1 produced concentrates with lower K than those from unheated milk. Above 40% totals solids, the K values of concentrates from heated milk were similar regardless of the pH at heating, and were higher than those from unheated milk. It is proposed that denatured whey proteins associated with the casein micelles increase the voluminosity of the micelles leading to an increased viscosity and K at all milk concentrations. In contrast, denatured whey proteins and κ-casein in the serum only make a small contribution to the viscosity and K until the milk is at high concentrations.

The kinetics of beta-lactoglobulin (beta-LG) denaturation in pressure-treated reconstituted skim milk samples over a wide pressurization range (100-600 MPa) and at various temperatures (10-40 degrees C) was studied. Denaturation was extremely dependent on the pressure and duration of treatment. At 100 MPa, no denaturation was observed regardless of the temperature or the holding time. At higher pressures, the level of denaturation increased with an increasing holding time at a constant pressure or with increasing pressure at a constant holding time. At 200 MPa, there was only a small effect of changing the temperature at pressurization. However, at higher pressures, increasing the temperature from 10 to 40 degrees C markedly increased the rate of denaturation. The two major genetic variants of beta-LG (A and B) behaved similarly to pressure treatment, although the B variant appeared to denature slightly faster than the A variant at low pressures (<= 400 MPa). The denaturation could be described as a second-order process for both beta-LG variants. There was a marked change in pressure dependence at about 300 MPa, which resulted in markedly different activation volumes in the two pressure ranges. Evaluation of the kinetic and thermodynamic parameters suggested that there may have been a transition from an aggregation-limited reaction to an unfolding-limited reaction as the pressure was increased.

Changes in the structure and chemistry of beta-lactoglobulin (beta-LG) play an important role in the processing and functionality of milk products. In model beta-LG systems, there is evidence that the aggregates of heated beta-LG are held together by a mixture of intermolecular non-covalent association and heat-induced non-native disulfide bonds. Although a number of non-native disulfide bonds have been identified, little is known about the initial inter- and intramolecular disulfide bond rearrangements that occur as a result of heating. These interchange reactions were explored by examining the products of heat treatment to determine the novel disulfide bonds that form in the heated beta-LG aggregates. The native protein and heat-induced aggregates were hydrolyzed by trypsin, and the resulting peptides, before and after reduction with dithiothreitol, were separated by high-performance liquid chromatography and their identities confirmed by electrospray ionization mass spectrometry. Comparisons of these peptide patterns showed that some of the Cys160 was in the reduced form in heated beta-LG aggregates, indicating that the Cys160-Cys66 disulfide bond had been broken during heating. This finding suggests that disulfide bond interchange reactions between beta-LG non-native monomers, or polymers, and other proteins could occur largely via Cys160.

Reconstituted skim milk samples were pressure treated at 100–600MPa from 0 to 60min at 10–40°C. The changes in casein micelle size, the size distribution and the level of casein dissociated from the casein micelles were monitored after pressure treatment. Small decreases in size were observed at 100MPa, with slightly greater effects at higher temperatures or longer pressure treatments. At pressure ≥400MPa, the casein micelles disintegrated; the effect was more rapid at higher temperatures although the final size was similar in all samples regardless of the pressure or temperature. At 200MPa and 10°C, the casein micelle size decreased slightly on heating, whereas, at higher temperatures, the size increased as a result of aggregation. Whey proteins were not involved in the aggregation reaction. At 300MPa, there was an intermediate behaviour, with micelle aggregation and disintegration observed. Aggregation was observed at higher temperatures and shorter pressurisation times, whereas disintegration was observed in all samples at lower temperatures or at the longer pressurisation times at the higher temperatures. Considerable casein was rendered non-sedimentable on treatment at pressures above 100MPa, even in the samples that had aggregated. There appeared to be a relationship between the size reduction and the level of non-sedimentable casein, although the aggregated samples deviated from this relationship.

Reconstituted skim milk was adjusted to pH values between 6.5 and 6.7 and heated (90°C) for up to 30 min. The change in the viscosity, as well as the casein micelle size and the level of whey protein association with the micelles, was monitored for each milk sample. The change in the viscosity was dependent on the pH of the milk at heating. At pH 6.5, the viscosity increased markedly during the early stages of heating, and plateaued on prolonged heating. The magnitude of the change in viscosity decreased as the pH of the milk was increased, so that only small changes in viscosity were observed on heating at pH 6.7. The change in viscosity was linearly correlated with the change in particle volume (calculated from the particle size). This indicates that the viscosity changes were related to a change in the volume fraction of the casein micelles as a consequence of heating. The level of denatured whey proteins associating with the micelles was also markedly dependent on the pH at heating. High levels associated at pH 6.5, and the level associated was lower as the pH was increased to pH 6.7. The changes in viscosity and casein micelle volume were correlated with the levels of denatured whey proteins that were associated with the casein micelles, although there was some deviation from linearity at low levels of association (<15%).

Heat treatment of milk causes the heat-denaturable whey proteins to aggregate with kappa-casein (kappa-CN) via thiol-disulfide bond interchange reactions. The particular disulfide bonds that are important in the aggregates are uncertain, although Cys(121) of beta-lactoglobulin (beta-LG) has been implicated. The reaction at 60 degrees C between beta-LG A and an activated kappa-CN formed small disulfide-bonded aggregates. The tryptic peptides from this model system included a peptide with a disulfide bond between a Cys residue in the triple-Cys peptide [beta-LG(102-124)] and kappa-CN Cys(88) and others between kappa-CN Cys(88) or kappa-CN Cys(11) and beta-LG Cys(160). Only the latter two novel disulfide bonds were identified in heated (90 degrees C/20 min) milk. Application of computational search tools, notably MS2Assign and SearchXLinks, to the mass spectrometry (MS) and collision-induced dissociation (CID)-MS data was very valuable for identifying possible disulfide-bonded peptides. In two instances, peptides with measured masses of 4275.07 and 2312.07 were tentatively assigned to beta-LG(102-135):kappa-CN(11-13) and beta-LG A(61-69):kappa-CN(87-97), respectively. However, sequencing using the CID-MS data demonstrated that they were, in fact, beta-LG(1-40) and beta-LG(41-60), respectively. This study supports the notion that reversible intramolecular disulfide-bond interchange precedes the intermolecular interchange reactions.

Reconstituted skim milk samples at pH between 6.5 and 7.1 (heating pH) were heated at 80°C, 90°C or 100°C for 30min (heating temperature). The particle size of the casein micelles was measured at pH 4.75–7.1 (measurement pH) and at temperatures of 10°C, 20°C and 30°C (measurement temperature) using photon correlation spectroscopy. The particle size of the casein micelles, at a measurement pH of 6.7 and a measurement temperature of 20°C, was dependent on the heating pH and heating temperature to which the milk was subjected. The casein micelle size in unheated milk was about 215nm. At a heating pH of 6.5, the casein micelle size increased by about 15, 30 and 40nm when the milk was heated at 80°C, 90°C or 100°C, respectively. As the heating pH of the milk was increased, the size of the casein micelles decreased so that, at pH 7.1, the casein micelles were ∼20nm smaller than those from unheated milk. Larger effects were observed as the heating temperature was increased from 80°C to 100°C. The size differences as a consequence of the heating pH were maintained at all measurement temperatures and at all measurement pH down to the pH at which aggregation of the micelles was observed. For all samples, size measurements at 10°C showed no aggregation at all measurement pH. Aggregation occurred at progressively higher pH as the measurement temperature was increased. Aggregation also occurred at a progressively higher measurement pH as the heating pH was increased. The particle size changes on heating and the aggregation on subsequent acidification may be related to the pH dependence of the association of whey proteins with, and the dissociation of κ-casein from the casein micelles as milk is heated.

Reconstituted skim milk was adjusted to pH values between 6.5 and 7.1 and heated (90 degrees C) for up to 30 min. The skim milk samples were then readjusted to pH 6.7. Acid gels prepared from heated milk had markedly higher G ' values, a reduced gelation time, and an increased gelation pH than those prepared from unheated milk. An increased pH at heating decreased the gelation time, increased the gelation pH, and increased the final G ' of acid set gels prepared from the heated milk samples. There were only small differences in the level of whey protein denaturation in the samples at different pH values, and these differences could not account for the differences in the G ' of the acid gels. The levels of denatured whey protein associated with the casein micelles decreased and the levels of soluble denatured whey proteins increased as the pH at heating was increased. The results indicated that the soluble denatured whey proteins had a greater effect on the final G ' of the acid gels than the denatured whey proteins associated with the casein micelles.