Reconstituted whole milk was heated using pilot-scale heating equipment. Kinetic and thermodynamic parameters for the irreversible denaturation of β-lactoglobulins A and B and R-lactalbumin were determined. R-Lactalbumin denaturation was first order, whereas both β-lactoglobulin variants had a reaction order of 1.5. Arrhenius plots for all three proteins showed an abrupt change in temperature dependence. In the low-temperature range, the thermodynamic parameters were ascribed to typical denaturation processes in which the unfolding of the protein tertiary structure is the rate-determining step. At higher temperatures, these parameters were in the range expected for typical condensation reactions, suggesting that aggregation processes may be rate-determining in this temperature range. The rate constants for β-lactoglobulin denaturation were independent of the initial protein concentration at all temperatures. For R-lactalbumin at temperatures below 85 °C the rate constants may have been dependent on the initial R-lactalbumin concentration as higher rate constants were observed with decreasing protein concentrations.
When skim milk at pH 6·55 was heated (75 to 100 °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 β-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.
Skim milk was adjusted to pH values between 6.5 and 6.7 and heated (80, 90, and 100 degrees C) for up to 60 min. Changes in casein micelle size, level of whey protein denaturation, and level of whey protein association with the micelles were monitored for each milk sample. Changes in casein micelle size were markedly affected by the pH at heating. At low pH (6.5-6.55), the casein micelle size increased markedly during the early stages of heating, and the size plateaued on prolonged heating. The maximum increase in size was approximately 30-35 nm. In contrast, at high pH (6.7), much smaller changes in size were observed on heating and the maximum increase in size was only approximately 10 nm. An intermediate behavior was observed at pH values between these two extremes. The rate of denaturation of the major whey proteins, alpha-lactalbumin and beta-lactoglobulin, was essentially unaffected by the pH at heating for the small pH changes involved in this study, and the changes in casein micelle size were poorly related to the level of whey protein denaturation. In contrast, the level of denatured whey proteins associating with the micelles was markedly dependent on the pH at heating, with high levels of association at pH 6.5-6.55 and low levels of association at pH 6.7. Changes in casein micelle size were related to the levels of denatured whey proteins that were associated with the casein micelles, although there was a small deviation from linearity at low levels of association (<15%). Further studies on reconstituted and fresh milk samples at smaller pH steps confirmed that the association of whey proteins with the casein micelles was markedly affected by the pH at heating. These results indicate that the changes in casein micelle size induced by the heat treatment of skim milk were a consequence of the whey proteins associating with the casein micelles and that the level of association was markedly influenced by small pH changes of the milk. It was not possible to determine whether the association itself influenced the casein micelle size or whether parallel reactions involving micellar aggregation caused the increase in micelle size as whey protein association progressed.
The effect of pH (6.3−7.1) and temperature (20−90 °C)
on the dissociation of casein from the micelles
in reconstituted skim milk was investigated. These pH conditions
encompass those naturally found,
whereas heat treatments below 100 °C are commonly encountered during
the processing of milk
and milk products. Low levels of casein were rendered soluble at
pH below 6.7 regardless of heating
temperature, whereas increasing levels of casein were solubilized as
the pH was increased from 6.7
to 7.1. This pH-dependent dissociation of the casein micelles
showed an unusual dependence on
temperature. Low levels of casein were dissociated at 20 °C at
all pH values. The quantity of
casein solubilized increased with temperature to a maximum dissociation
at about 70 °C and then
decreased at higher temperatures. The dissociation behavior of
αs-casein and β-casein at pH ≥ 6.7
showed dependence on temperature similar to that of the total casein.
In contrast, above pH 6.5,
the dissociation of κ-casein increased essentially linearly with
increasing temperature over the entire
temperature range studied. The proportion of β-casein in the
soluble casein was essentially constant
regardless of the temperature and pH, whereas the proportions of
αs-casein and κ-casein varied
with both temperature and pH. The results of this study have
indicated that, at certain pH, a
marked dissociation of protein from the casein micelles occurs on
heating at temperatures below
100 °C; this phenomenom has not previously been reported to occur
under such mild heating
Keywords: Casein micelles; dissociation; heat treatment; reconstituted skim
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