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.

When milk was preheat-treated after either evaporation or homogenization with fat present the degree of heat stability induced in the resulting evaporated milk (EM) was considerably less than with the normal process in which the preheat treatment was carried out first. When the preheat treatment was carried out after both evaporation and homogenization, no heat stability was induced by the preheat treatment. Removal of the whey protein fraction from the milk increased the heat stability of both the EM homogenized before preheat treatment and the EM made by the normal process.

Formation of covalent bonds at milk sterilization temperatures was studied using caseins and casein peptides. At 120°C lysinoalanyl residues produced even at pH 7·0 were derived from intra-molecular interactions between phospho-serine and lysine; the conditions of formation were determined. It was also found that the formation of isopeptidic cross-links was significant with conditions more severe than those used for milk sterilization.

Potentiometric acid-base titration is a simple method that can be used successfully in the investigation of the protein system of milk and other protein-containing products. Its applications include the examination of conventional heating procedures, cheese ripening, the investigation of processed cheese and the effect of melting salts. The principle depends on the performance of pH-dependent phase transitions which are recorded by a single titration cycle and evaluated quantitatively by means of hysteresis loops.

Buffalo skim-milk is less heat stable than cow skim-milk. Interchanging ultracentrifugal whey (UCW) and milk diffusate with micellar casein caused significant changes in the heat stability of buffalo casein micelles (BCM) and cow casein micelles (CCM). Buffalo UCW dramatically destabilized COM, whereas buffalo diffu-sate with CCM exhibited the highest heat stability.

Cow κ-casein stabilizes αs-casein against precipitation by Ca better than buffalo º-casein. About 90% of αs-casein could be stabilized by κ: αs ratios of 0·20 and 0·231 for cow and buffalo, respectively.

Sialic acid release from micellar κ-casein by rennet was higher than from acid κ-casein in both buffalo and cow caseins, the release being slower in buffalo. The released macropeptide from buffalo κ-casein was smaller than that from cow κ-casein as revealed by Sephadex gel filtration.

Sub-units of BCM have less sialic acid (1·57mg/g) than whole micelles (2·70mg/g). On rennet action, 47% of bound sialic acid was released from sub-units as against 85% from whole micelles. The sub-micelles are less heat stable than whole micelles. Among ions tested, added Ca reduced heat stability more dramatically in whole micelles, whereas added phosphate improved the stability of micelles and, more strikingly, of sub-micelles. Citrate also improved the heat stability of sub-micelles but not of whole micelles.