Using membrane filtration it is possible to selectively concentrate proteins and, in the case of microfiltration, concentrate casein micelles. During filtration, water is often added and this practice, called diafiltration, causes further release of permeable components and maintains filtration efficiency. Filtration causes changes in composition of the protein as well as the soluble phase, including soluble calcium, which is a critical factor controlling the gelation properties of the casein micelles in milk. It was hypothesized that concentrates obtained using membrane filtration with or without diafiltration would have different gelation behavior. To test this hypothesis, two concentrates of similar casein micelle volume fraction were prepared, using spiral wound polymeric microfiltration membranes with a 800 kDa molecular weight cutoff, with or without diafiltration. The concentrates showed a gelation behavior comparable to that of skim milk, with a similar gelation time and with a higher firmness, due to the higher number of protein linkages in the network. In contrast, the hydrolysis of κ-casein by chymosin and casein aggregation were inhibited in diafiltered casein micelle suspensions. When the concentrates were recombined with the original skim milk to a final concentration of 5% protein, which re-established a similar soluble phase composition, differences in gelation behavior were no longer observed: both treatments showed similar gelation time and gel firmness. These results confirmed that membrane filtration can result in concentrates with different functionality, and that ionic environmental conditions are critical to the aggregation behavior of casein micelles. This is of particular significance in industrial settings where these fractions are used as a way to standardize proteins in cheese making.

In this study, the relationship between the dissociation of κ-casein from casein micelles due to heat-induced denaturation and the strength of acid milk gel was investigated. The κ-casein-dissociated micelles were fractionated by gel filtration chromatography and two-dimensional polyacrylamide gel electrophoresis, and their zeta potential and surface hydrophobicity were measured. The negative charge of the κ-casein-dissociated micelles was lower than that of native micelles, and micellar surface hydrophobicity was higher. For confirmation, the isoelectric point of the casein micelles was measured. The κ-casein-dissociated micelles were found to cohere at an earlier stage of acidification than the native micelles. These results demonstrated that the heat-induced increase in the strength of acid milk gel was partly due to the decrease in micellar surface charge and partly to the increase in surface hydrophobicity caused by the dissociation of κ-casein.

Gelation is a significant operation in dairy processing. Protein gelation can be affected by several factors such as temperature, pH, or enzyme addition. Recently, the use of ultrasonication has been shown to have a significant impact on the formation of whey protein gels. In this work, the effect of ultrasonication on the gelation of casein systems was investigated. Gels were formed by the addition of 7·6 mm Tetra Sodium Pyro Phosphate (TSPP) to 5 wt% micellar casein (MC) solutions. Sonication at 20 KHz and 31 W for up to 30 min changed the surface hydrophobicity of the proteins, whereas surface charge was unaltered. Sonication before the addition of TSPP formed a firm gel with a fine protein network and low syneresis. Conversely, sonication after TSPP addition led to an inconsistent weak-gel-like structure with high syneresis. Gel strength in both cases increased significantly after short sonication times, while the viscoelastic properties were less affected. Overall, the results showed that ultrasonication can have a significant effect on the final gel properties of casein systems.

The susceptibility of total casein and the individual caseins in reconstituted skim milk to transglutaminase (TGase)-induced cross-linking was studied as a function of incubation temperature (5–40 °C), pH (5·0–7·0) and mineral addition. Within the ranges studied, the level of total casein cross-linked increased with increasing temperature, pH and concentration of added trisodium citrate, whereas adding calcium chloride had the opposite effect. These effects can be largely related to the effects of these parameters on TGase activity. In addition, the parameters were also found to influence the susceptibility of κ-casein, and to a lesser extent β-casein, to cross-linking, whereas the susceptibility of αs1-casein was not affected. The susceptibility of κ-casein to cross-linking increased with increasing temperature and calcium chloride addition, but decreased with increasing pH and citrate content, whereas the susceptibility of β-casein to TGase-induced cross-linking decreased with increasing temperature, but was not affected by other parameters. These findings highlight the fact that selection of environmental conditions during cross-linking can be applied to tailor the surface, and hence possibly colloidal stability, of casein micelles in TGase-treated milk.

Standard method for the determination of the activity of milk coagulants is the rotating bottle method, where clotting time is defined as the time when visually observable flocculation starts. Aim of this study was to verify whether it is possible to determine milk clotting time by rotational viscometry. Using three different coagulants and reconstituted milk of different pH and temperature, flocculation time and viscosity in steady shear was determined, and milk gelation was monitored by small amplitude oscillating shear rheometry. The results show that, independent of pH and temperature, milk clotting time is related to an apparent viscosity of 7·24±0·45 mPa.s, indicating that rotational viscometry can be used for the screening of flocculation time with an accuracy of approximately 6%.

The effects of high-pressure homogenisation (HPH) in the pressure range 100–300 MPa on the gel formation and rheological properties of rennet-induced skim milk (0·08%, fat, w/w) and standardised milk (3·60% fat, w/w) gels at pH 6·60 were studied. The average casein micelle size in skim milk was significantly reduced and the gel formation time decreased when skim milk was subjected to the pressures of 200 and 300 MPa. The storage modulus of rennet-induced skim milk gels at 2700 s after rennet addition was higher for samples homogenised at higher pressures, which contained smaller casein particles. HPH had little effect on the large deformation properties of rennet-induced skim milk gels. The gel formation time of renneted standardised milk was significantly reduced as a result of HPH, while the storage modulus of rennet-induced milk gels 2700 s after rennet addition increased with increasing homogenising pressure. The apparent fracture stress was slightly higher for standardised milk gels formed from HPH-treated milk, whereas the apparent strain at fracture was lower, than that of unhomogenised milk. In conclusion, HPH treatment influenced gel formation processes of skim milk and its small-deformation rheological properties, mainly through modification of casein micelles. HPH also significantly affected the gel formation process of standardised milk gels and its rheological properties as a result of an increase in volume fraction of aggregating particles, while the particle size was of lesser importance.

When κ-casein (κ-CN) was added to milk, and the milk was subsequently pH adjusted (pH 6·5−6·9) and heated (90°C/15 min), the serum contained considerably higher levels of denatured whey proteins than the milks without added κ-CN. When milk at pH 6·5−6·9 was heated at 90°C for different times, κ-CN was found in the serum in the early stages of heating and before significant levels of whey proteins were denatured. κ-CN reached its maximum level in the serum before the whey proteins were fully denatured. When milk at pH 6·5−6·9 was heated at 20−90°C for 15 min, κ-CN dissociated from the casein micelles at all temperatures, with the level in the serum increasing with the temperature and the pH at heating. κ-CN dissociated from the micelles at temperatures below those at which significant levels of the whey proteins were denatured. When taken together, the results from these experiments strongly indicate that the dissociation of κ-CN from the micelles can precede the interaction of the denatured whey proteins with κ-CN, and that there is a preferential interaction of the denatured whey proteins with serum-phase κ-CN.

The formation of pressure-induced casein structures (600 MPa for 30 min at 30°C) was investigated for different pressure release rates (20 to 600 MPa min−1) and casein contents (1 to 15 g/100 ml). Structures from liquid (sol) to solid (gel) were observed. The higher the protein content and the pressure release rate, the higher was the dynamic viscosity. A firm gel was built up at a casein content of 7 g/100 ml for a pressure release rate of 600 MPa min−1, while lower release rates resulted in less firm gels (200 MPa min−1) or liquid structures (20 MPa min−1). In a 5 g/100 ml casein solution and at a pressure release rate of 600 MPa min−1, casein aggregates were generated which were built from smaller casein particles with a larger hydrodynamic diameter and higher voluminosity than in the untreated solution. After a slow release rate casein micelles had a smaller hydrodynamic diameter and a lower voluminosity, but were similar in shape and diameter as compared with the micelles in solution before high pressure treatment.

The influence of ethanol on the rennet-induced coagulation of milk was studied to investigate potential synergistic effects of these two mechanisms of destabilisation on the casein micelles. Addition of 5% (v/v) ethanol reduced the rennet coagulation time (RCT) of milk, whereas higher levels of ethanol (10–20%, v/v) progressively increased RCT. The temperature at which milk was coagulable by rennet decreased with increasing ethanol content of the milk. The primary stage of rennet coagulation, i.e., the enzymatic hydrolysis of κ-casein, was progressively slowed with increasing ethanol content (5–20%, v/v), possibly due to ethanol-induced conformational changes in the enzyme molecule. The secondary stage of rennet coagulation, i.e., the aggregation of κ-casein-depleted micelles, was enhanced in the presence of 5–15% ethanol, the effect being largest at 5% ethanol. Enhanced aggregation of micelles is probably due to an ethanol-induced decrease in inter-micellar steric repulsion. These results indicate an interrelationship between the effects of ethanol and chymosin on the casein micelles in milk, which may have interesting implications for properties of dairy products.

Dynamics of micro-structural changes in milk during the renneting process were analysed using high-resolution ultrasonic spectroscopy in combination with dynamic rheology and NIR transmission measurements. Two independent ultrasonic parameters, velocity and attenuation were measured in the frequency range 2 to 15 MHz, as a function of time after addition of rennet to milk. The results show an initial decrease of 20 nm for the average diameter of micelles caused by hydrolysis of the κ-casein ‘hairy’ layer followed by an aggregation of the micelles into small clusters (effective aggregation number of 3) and then formation of the gel structure. It was found that evolution of ultrasonic attenuation in the renneting process could well be described by the scattering of the ultrasonic waves on aggregates. The evolution of ultrasonic velocity is well described by the scattering theory but deviates from the predicted curve at the gelation stage of the process, which shows the difference in propagation of ultrasonic waves in a gel structure compared with dispersions. Overall, we found high-resolution ultrasonic spectroscopy to be a powerful tool for analysis of microscopic processes in the formation of milk gel. It allows the characterisation of the pre-gelation processes, such as hydrolysis and aggregation, and the initial stages in the formation of the gel network as well as monitoring of the microscopic evolution in the gel at the post-gelation stage.

In this study, effects of high pressure (HP) on some constituents and properties of buffalo milk were examined. HP treatment at 100–600 MPa for 30 min affected casein micelle size only slightly, whereas treatment at 800 MPa increased it by ~35%. Levels of non-micellar αs1- and β-caseins were increased by treatment [ges ]250 MPa, and were highest after treatment at 400–800 MPa. The level of non-micellar calcium increased with increasing pressure up to 600 MPa. The L*-value of the milk decreased gradually with increasing pressure, from ~82 for untreated milk to ~65 for milk treated at 800 MPa. Milk pH was increased by ~0·07 units after treatment at 100–800 MPa, with no significant difference between treatment pressures. Denaturation of α-lactalbumin occurred at pressures [ges ]400 MPa, and reached >90% after treatment at 800 MPa, whereas β-lactoglobulin (β-lg) was denatured >100 MPa, reaching ~100% after treatment at 400 MPa; after treatment [ges ]400 MPa, all β-lg was associated with the casein micelles. The rennet coagulation time of buffalo milk increased with increasing pressure, whereas the strength of the coagulum formed decreased after treatment at 250–800 MPa. Overall, HP treatment affected many constituents and properties of buffalo milk; some of these effects have also been observed in the milk from other species, but the extent of the effects, and the pressure at which they occurred, differed considerably.

Effects of high pressure (HP) on average casein micelle size and denaturation of α-lactalbumin (α-la) and β-lactoglobulin (β-lg) in raw skim bovine milk were studied over a range of conditions. Micelle size was not influenced by treatment at pressures <200 MPa, but treatment at 250 MPa increased micelle size by ∼25%, while treatment at [ges ]300 MPa irreversibly reduced it to ∼50% of that in untreated milk. The increase in micelle size after treatment at 250 MPa was greater with increasing treatment time and temperature and milk pH. Treatment times [ges ]2 min at 400 MPa resulted in similar levels of micelle disruption, but increasing milk pH to 7·0 partially stabilised micelles against HP-induced disruption. Denaturation of α-la did not occur [les ]400 MPa, whereas β-lg was denatured at pressures >100 MPa. Denaturation of α-la and β-lg increased with increasing pressure, treatment time and temperature and milk pH. The majority of denatured β-lg was apparently associated with casein micelles. These effects of HP on casein micelles and whey proteins in milk may have significant implications for properties of products made from HP-treated milk.

Sedimentation Field Flow Fractionation (SdFFF) was combined with Photon Correlation Spectroscopy (PCS), to characterize changes in the structure of the colloidal particles of reconstituted skim milk of diameter >50 nm (aggregates of casein and calcium phosphate known as casein micelles) with the changes in partitioning (with the addition of salt) of calcium (Ca), inorganic phosphate (Pi) and casein between the serum and colloidal phases of the milk. The number weighted particle size distributions are determined. These are well represented by a log-normal distribution. Methods are presented for estimating the relative contributions of scattering and absorbance to the SdFFF detector signal and for taking both into account when analysing SdFFF data. The values found for the effective density of the casein micelles were in good agreement with the literature and ranged from (1·06–1·08 g cm−3) according to the composition of micelles. The changes in the scattering intensity as determined by PCS correlated with the changes in the particle composition. Although the concentrations of colloidal calcium phosphate (CCP) (1·1–3·5 g/kg milk) and micellar casein (18·1–27·2 g/kg milk) varied considerably only small changes in the size distribution of particles >50 nm diameter were observed except for milk to which 30 mmol Pi+10 mmol Ca/kg milk had been added where the particle size distribution shows a swelling of the particles consistent with a lower than expected value for the particle density. These observations suggest that the micelles have the ability to both lose (depleted micelles) and accommodate (enriched micelles) more casein, calcium and inorganic phosphate in their interior, thus confirming the model of the micelles which postulates an open structure allowing freedom of movement of casein and small ions.

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.