Caseins, the major proteins in milk, exist as colloidal particles called casein micelles. These polydisperse protein nanoparticles have diameters ranging from 20 to 600 nm. The size of the micelles can influence the fundamental and functional properties of milk. Therefore, this research work was designed with the assumption that various size fractions of casein micelles can produce products with desirable functional properties. This research focused on the size-fractionation of casein micelles from skim milk using two main techniques: differential ultracentrifugation and cross-flow filtration using ceramic membranes with pore sizes ranging from 0.05 µm to 0.2 µm. The fractionated casein micelles were characterized for their fundamental and functional properties.
Fractionation of casein micelles from skim milk by using differential consecutive ultracentrifugation could produce various classes of casein micelles with sizes in the range of 64 nm (the smallest) to 194 nm (the largest). The fraction of the smaller micelles had a narrower distribution compared to that of larger ones. A turbidity study of micelle pellets dispersed in SMUF buffer indicated that the turbidity of the casein micelle dispersion increased with size, from 11.6 to 80.6% corresponding to the smallest and largest. Zeta potential values showed no significant difference between micelles of different sizes but the surface tension of casein micelle solutions tended to be inversely correlated to micelle size. The voluminosity of the largest casein micelles was the highest (7.5 ml/g), compared to those of the intermediate (5.8 and 6.1 ml/g) and the smallest (5.8 ml/g) micelles. The proportion of individual caseins differed between various classes of casein micelles; smaller micelles had a higher percentage of κ-casein and a lower percentage of β-casein than larger micelles whereas the percentage of αs-caseins was size-independent.
The foaming and gelation properties of caseins as a function of casein micelle size were also investigated in this study. It was found that fractions containing the largest micelles had less foaming capacity (49%) than the intermediate (56% and 56.7%) and the smallest (55%) micelles; nonetheless these differences were insignificant between fractions of the intermediates and the smallest. The half-life of casein foams (expressed as the time taken for the foam to collapse to half its original volume at room temperature) increased with decreasing the micelle size; which was from 9 min (the largest) to 74 min (the smallest). This would lead to the conclusion that the foams generated from the smaller micelles were more stable than those from the larger ones. The rheological studies on acid- and rennet-induced gels of size-fractionated casein micelles at 25°C and 30°C, respectively, showed that the smaller the casein micelle size, the less time required for both rennet and acid casein gels to coagulate and the stronger the gels formed. The viscoelastic properties of the final rennet and acid gels were also studied. For both types of gels, the storage modulus (G’) was higher than loss modulus (G”) and these moduli increased with an increase in frequency in the linear viscoelastic region from 0.01 to 1 Hz. The results indicate that weak gel structures were formed. The stiffness of the final gels was enhanced by lowering the temperature from 25°C to 5°C (acid gels) or from 30°C to 5°C (rennet gels) after the gelation process. The rigidity of the smallest micelle gel increased more sharply with decreasing temperature than those of the intermediate and the largest micelles.
To investigate the feasibility of microfiltration to fractionate casein micelles according to size by cross-flow filtration using consecutive filtration with various porosities of ceramic membranes from 0.2 µm to 0.05 µm, the optimum operating conditions for microfiltration were determined. The pump frequency of 45 Hz, transmembrane pressures of 1.7 bar and temperature of 50°C were applied for the 0.2 µm membrane. In the case of 0.1 µm and 0.05 µm membranes, transmembrane pressures of 1.5 bar and temperature of 30°C were used. After filtration using 0.2, 0.1 and 0.05 µm membranes, three fractions of casein micelles with average sizes of 182, 166 and 161 nm, respectively, were collected from the retentates. The zeta potential of the fractions containing different sizes of casein micelles showed no significant differences.
In terms of commercial application of the centrifugation technique to partition the casein micelles by size, centrifugation at the maximum centrifugal force of 20,000 g, that could be scaled up for commercial fractionation of casein micelles in the dairy industry, was investigated. Whole casein micelles separated from skim milk were centrifuged at 20,000 g at 25°C for 70 min. Two fractions of casein micelles obtained had mean diameters of 200 nm (pellet) and 146 nm (supernatant). The gelation properties of acid and rennet induced gels made from these fractions showed that the smaller casein micelles formed a firmer gel than the larger ones. A study on large deformation properties of acid and rennet gels as a function of micelle size was also conducted. The breaking stress and the breaking strain values obtained from these types of gels increased as the micelle size decreased. Accordingly, the findings obtained from texture analysis of acid and rennet bulk gels confirm the influence of casein micelle size on the firmness of bulk gels, in which the smaller the micelles the harder the bulk gels formed.