The size and number average molecular weight have been determined for β-casein monomers and polymers from electron micrographs using the freeze-etching procedure with spray-frozen specimens. For the spherical β-casein monomers we found a mol. wt of 22600 and a diam. about 10 nm, which compared quite well with data obtained from ultracentrifugation, light scattering and viscosity measurements. Polymer sizes were in agreement with molecular weight determinations from ultracentrifugation and light scattering, assuming that the volume and weight of the particles are proportional.

The temperature and concentration dependent association of β-casein was studied by means of viscometry, gel filtration chromatography, electron microscopy, analytical ultracentrifugation and UV difference spectrophotometry. Degrees of polymerization of 12, 22 and 49 and free energies of association of –21, –23 and – 25 kJ/mole monomer were found at temperatures of 10, 15 and 20 °C respectively in 0·2 M Na phosphate buffer pH 6·7.

Monomeric β-casein was not a completely random coil but became more compact with increasing temperature, due to hydrophobic interactions.

Multiple regression analysis was applied to sedimentation equilibrium data for determination of the molecular weight distribution (MWD) of model systems consisting of up to 3 components. Negative weight fractions which were frequently encountered during multiple regression analysis were forced to zero by sequentially eliminating from the regression matrix the corresponding molecular weights in order of the magnitude of negative t-values. The simplex optimization of Morgan & Deming (1974), modified by incorporating a prohibit–range– trespassing routine, was used to search for the best fit values for weight average molecular weights and relative concentrations of the components. This method almost quantitatively reproduced the molecular weights and concentrations of the original model systems. This quantitative information supplemented the multiple regression matrix to improve the resolution of MWD.

A direct comparison using model systems revealed that the multiple regression method in conjunction with the simplex optimization routine was more quantitative than the linear programming method of Scholte (1969). When applied to mixtures of standard proteins (ribonuclease A; ovalbumin; γ-globulin and ovalbumin; γ-globulin; apoferritin), the simplex optimization routine yielded values for average molecular weights and relative concentrations of the component proteins which were in good agreement with the known values in the original mixtures.

The MWD of αs1-k-casein mixtures at an ionic strength of 0.1 suggested that k-casein was readily dissociated to the monomer (or the dimer) and interacted with the monomer (or the dimer) of αs1-casein forming a complex of approximately 400000.

The colloidal properties of micellar casein are reviewed. It is shown that the behaviour of intact micelles is much at variance with the predictions from the Schulze–Hardy rule, and that therefore their stability cannot be explained by the principles of the DLVO theory. Towards electrolyte, micelles behave as a protein rather than a lyophobic colloid.

Casein is a strong protective colloid. In the micelle, however, it does not completely cover the inorganic constituent which remains sensitive to changes in the ionic environment.

The rate theory of the enzyme-induced clotting of casein micelles is summarized. It is shown that the lag phase in the clotting is due to the second order of the coagulation reaction. Flocculation rate constants of micelles have been deduced from clotting times. Their relatively low values can be attributed to an orientational constraint. Practical consequences of the theory with respect to clot structure, gelation of sterilized products and cheese manufacture are discussed.

Skim-milk was fractionated by permeation chromatography on CPG-10 (300 nm) at 20 °C with synthetic milk serum. The elution profile, which was highly reproducible, showed 3 partly resolved peaks. On treatment with 0.1% glutaralde-hyde before chromatography, a similar profile was obtained indicating no appreciable micellar dissociation during chromatography of the milk. The casein composition of the eluent fractions, determined by quantitative chromatography on hydroxyapatite, showed that 12.1% of total casein was associated with the low molecular weight peak. The micelle content of ²- and κ-casein increased with increasing elution volume, and the ±s/κ ratio showed a progressive diminution from 4·3 to 2·3. Large differences were found in the colloidal phosphate contents and the weight average diameters of the micelle fractions which clearly show that CPG-10 fractionates on the basis of molecular size.

Casein micelles fractionated on controlled pore glass (CPG-10/3000) were shown to be stable by recycling experiments. Only minor effects on the size distribution of the casein micelles were found after heating skim-milk to 100 °C for 10 min, or freeze-drying skim-milk at – 70 °C followed by resuspension in the synthetic milk serum of Jenness & Koops (1962). The heating caused some whey proteins (β-lactoglobulin) to enter the micelle fractions while the freeze-drying caused some of the largest micelles to disrupt. In colloidal calcium phosphate-free skim-milk prepared according to Pyne & McGann (1960) all the micelles appeared to dissociate into monomeric caseins.

A comparison of literature results on the voluminosity of casein micelles yields large differences, largely related to the method of determination. After applying some corrections, methods based on hydrodynamic radius yield a value of roughly 3·9ml/g dry casein, other methods (microscopy, sediment volume) about 2·2. Roughly half of the discrepancy can be explained by the micelles being not perfectly spherical. To explain the remaining difference, it is assumed that the micelles are hairy, i.e. they have molecular chains protruding into the milk serum. The hairyness would increase with decreasing temperature and be largely removed by the action of rennet. It would cause some entropic repulsion between micelles at close approach.

Water vapour sorption isotherms of casein micelles prepared from raw milk and various heat-treated milks were determined. The equilibrium water contents of the heated preparations were markedly lower than that of the raw-milk casein over the whole range of vapour pressures studied. An analysis of the sorption isotherms in the relative vapour pressure range 0·1–0.45, according to the Brunauer, Emmett & Teller (1938) equation, showed that there were significant differences between preparations in the computed monolayer contents. Differences in the rates of water sorption were also observed between the different preparations.

As judged from the amount of absorbed water, the influence of the heating methods could be ranked in the order: HTST (92 °C) ≃ UHT (direct) <UHT (indirect) < HTST (72 °C).

The principle of moving boundary electrophoresis has been employed for the measurement of the electrophoretic mobility and subsequent calculation of zeta potential of bovine casein micelles. The zeta potential of casein micelles was observed to increase with increase in temperature (from 10 to 50 °C) and to decrease with decrease in pH. Heat treatment of milk between 90 °C/30 min and 135 °C/50 min had no significant effect on zeta potential. The zeta potential of casein micelles during rennet action decreases until all the κ-casein has been cleaved by the enzyme.

The results of this study indicate that electrostatic interactions alone are not sufficient for an understanding of the absolute stability of casein micelles.

To obtain greater insight into the interaction of Ca, citrate and phosphate ions with casein, 31P NMR measurements were performed on combinations of these ions with αs- and κ-caseins. It was found that addition of αs-casein to a Ca phosphate solution in D2O at 27 °C and pD 6·4 resulted in a downfield shift of the 31P singlet. An almost identical shift was observed with κ-casein, but no shift was found when only phosphate ions were present or when Ca2+ were added to phosphate ions in the absence of casein. Separate experiments with poly-l-lysine, mol. wt approx. 35000, resulted in similar downfield 31P chemical shifts of Ca phosphate as with both caseins, whereas no shift was observed when poly-glycine was added. From these results it can be concluded that Ca and phosphate ions associate with casein in a co-operative manner, probably in the way described by ter Horst (1963) as a complex with the NH3+-groups of lysine or arginine in a structure such as: (casein—NH3+)—P043-—Ca2+. The formation of this complex may be enhanced by citrate ions, since preliminary results have shown that addition of Na citrate to a solution of αs-casein with added Ca phosphate produces a broadening of the 31P signal as well as a chemical shift.

The elution behaviour of β-casein from columns of hydroxyapatite has been studied and the effect examined of the enzymic addition of an extra phosphate residue. When the additional phosphate is located near pre-existing phosphate residues stronger binding to hydroxyapatite is observed. When the additional phosphate is remote from the pre-existing phosphates no increase in strength of binding is observed. It is suggested that the clustering of phosphate residues which characterizes αs- and β-caseins can be rationalized on the basis that it facilitates cooperative interactions between these phosphates and the Ca phosphate of the casein micelle and/or basic residues within casein polypeptide chains.

Calorimetric measurements of the heat of solution of micellar Ca phosphate suggest that this material more closely resembles OH-apatite than amorphous Ca phosphate or any other crystalline form. Preliminary computer calculations of the ionic equilibria of milk salt solutions indicate that, over a wide range of pH and temperature, such solutions are supersaturated with respect to OH-apatite and to various other Ca salts under certain conditions.

Casein micelles of different composition were synthesized in various ways and their sub-structure was investigated with the electron microscope by means of thin sections. Earlier studies of Schmidt et al. (1974) using the freeze-fracturing technique had shown no differences in the sub-structure of natural micelles and artificial micelles containing Ca and casein only. Contrary to these results our present studies showed that for the production of synthetic casein micelles with the same sub-structure as the natural ones it is necessary to add at least 2 other ions to the casein solution besides Ca2+: these are phosphate and citrate. The citrate ions play an important role in forming the material of the dark framework of the micelles visible in electron micrographs of unstained thin sections. This supports the hypothesis of Pyne & McGann (1960) that casein micelles contain a citrate apatite.

A survey is given of the relationships between various properties of artificial casein micelle systems and their composition with respect to αs1-, β and κ-casein, colloidal phosphate and citrate. Properties investigated were: the amount of colloidal phosphate, the micellar size, and the stability of the micelle towards dialysis, pressure, ethanol and heat.

The heat stability and rennet coagulation time (second stage) of milk were reduced by brief dialysis against water. Destabilization appears to arise from a developed imbalance between Ca and phosphate plus citrate due to the very slow diffusion of Ca on dialysis. Average micelle size as indicated by permeation chromatography in porous glass CPG 10 was slightly reduced by dialysis for 24 h. Direct addition of low levels (10–100 mM) of NaCl to milk markedly reduced heat stability at pH > 7·0 (normal minimum) possibly due to dissociation of κ-casein, but increased rennet coagulation times; higher levels of NaCl decreased heat stability throughout the pH range 6·4–7·4.

A suspension of casein micelles in a milk-salts solution bound highly cationic polypeptides and charged surfactants extensively, and globular proteins and low molecular weight amines less extensively. All additives were bound equally to native micelles and to a rennet coagulum. The distribution of each additive between the micellar and soluble phases was constant over a wide range of concentration, indicating that the micelle contained a large number of equivalent binding sites. Additives that did not bind Ca2+ caused a decrease in the rennet coagulation time which was related to the charge on the additive and the amount adsorbed. Salmine affected the time course of the viscosity of milk treated with rennet in a similar way to increase in temperature, accelerating the aggregation phase. Extensively-bound additives protected milk against loss of coagulability by rennet on heating, but did not affect the extent of whey protein denaturation. They did not cause aggregation of micelles or produce significant changes in their size distribution, but did cause a decrease in micellar hydration. It was concluded that the additives were bound to the apatite and casein components of the micelle, causing a change in the environment of the κ-casein and para-κ-casein, which affected their properties.

The modern food-processing industry is placing more and more emphasis upon the utilization of protein ingredients to provide specific functional properties to a wide range of formulated foods. Isolated milk protein products represent an important and valuable source of protein ingredients due to their recognized superior nutritional, organoleptic and functional properties. This paper provides up-to-date information on the quantities, production processes, composition, general properties, and specific functional properties of the major milk protein products, e.g. caseinates, co-precipitates, lactalbumin, whey protein concentrates and milk blends. The subject of chemical and enzymic modification to improve certain functional properties of milk proteins is considered briefly.

The theory of branching processes is used to derive a simple kinetic model for the heat-induced coagulation of a type A milk. Coagulation time-pH profiles and N depletion curves for the overall reaction can be fitted with the model, and most of the major properties for a milk exhibiting a type-A behaviour can be accounted for. Possible reactions for the heat-induced coagulation are also discussed in relation to the model.

When urea is added to concentrated skim-milk (22·5% total solids) no increase in heat stability is observed. However, a similar addition of urea to concentrate stabilized with aldehyde brought about a significant further increase in heat stability. Urea can further stabilize concentrated skim-milk only when aldehyde addition has brought about a change in coagulation mechanism from the normal 2-stage process to a single-stage coagulation. These results can be related to the 2 coagulation mechanisms observed in unconcentrated milks.

Heat stability as a function of pH has been studied in concentrated and non-concentrated milk systems. The influence of preheating has also been examined. The concentration of β-lactoglobulin has been shown to affect markedly the heat stability behaviour in both systems but with different characteristics. Increasing the level of urea resulted in increased heat stability in non-concentrated milks, but corresponding concentrated milks showed a small decrease in maximum heat stability. It has not been possible to extrapolate heat stability data from nonconcentrated to concentrated milks.