Sodium caseinate-stabilized fat globules inhibition of the rennet-induced gelation of casein micelles studied by Diffusing Wave Spectroscopy

Sodium caseinate (NaCas)-stabilized oil-in-water emulsions were added to skim milk and the rennet-induced aggregation was observed in situ using light scattering and dynamic oscillatory rheology. The gelation of the recombined milk was greatly inhibited by the addition of the oil droplets, at volume fractions >0.025. The development of the turbidity parameter, 1/l*, and the apparent hydrodynamic radius during renneting were determined using diffusing wave spectroscopy. Although the recombined milk samples contained two scattering particles, namely, casein micelles and fat globules, the latter overwhelmingly contributed to the overall light-scattering signal. This made possible to follow the behaviour of NaCas-stabilized fat globules during the gelation process. The enzymatic reaction associated with the hydrolysis of micellar κ-casein was not significantly affected by the presence of the NaCas-stabilized fat globules. However, the emulsion droplets impeded the aggregation of rennet-altered casein micelles preventing the formation of a gel network. The inability of renneted casein micelles to develop a gel network can be attributed in part to an altered equilibrium between soluble and micellar calcium phosphate, caused by the association of soluble Ca²⁺ with casein molecules, but mostly can be attributed to the effect of non-adsorbed caseins on the surface of the casein micelles.     

Effect of Ultra-high Temperature Treatment on the Enzymatic Cross-linking of Micellar Casein and Sodium Caseinate by Transglutaminase

ABSTRACT: It was found that ultra-high temperature (UHT) treatment of sodium caseinate and native whey protein-depleted micellar casein drastically increases the protein polymerization effect of an enzymatic treatment by microbial transglutaminase (TG). As a result the concentration of the isopeptide ε-(γ-glutamyl)lysine was increased significantly in UHT-treated micellar casein solutions after TG incubation compared with the unheated casein solution. Sodium caseinate was more susceptible to the cross-linking reaction as compared with the native casein micelles. The results demonstrate that the protein structure significantly affects the TG cross-linking reaction. The effect of an UHT treatment was considered to be related to a better TG accessibility due to a more open casein micelle structure and to the inactivation of a TG inhibitor substance. The results demonstrate that an unidentified component in the natural milk serum inhibits the TG reaction. The thermal inactivation of a TG inhibitor is the dominant effect explaining the improved cross-linking of UHT-treated casein micelles as well as sodium caseinate.     

Inactivation of an indigenous transglutaminase inhibitor in milk serum by means of UHT-treatment and membrane separation techniques

An indigenous inhibitor in raw milk inhibits cross-linking by transglutaminase (TG). The enzymatic cross-linking of micellar casein, compared with sodium caseinate, taking thermal inactivation of the TG inhibitor in the milk serum into consideration, was investigated. Inhibitor-free micellar casein was prepared by membrane separation combined with heat treatment of the UF permeate. The inhibitor permeated through MF (nominal pore size 0.1 μm) and UF (cutoff 25 kDa) membranes. TG-catalyzed cross-linking of casein micelles was clearly enhanced by UHT-treatment of UF permeate. Variation of the enzyme concentration showed that the inhibitory effect could not be compensated by higher enzyme concentrations when the casein micelles were suspended in unheated milk serum. Sodium caseinate, however, underwent high degrees of cross-linking even in unheated milk serum. By mixing an unheated milk serum and a UHT-treated milk serum at different ratios, the relative TG inhibitor activity was analysed. High inactivation (>80%) of the TG inhibitor is necessary to achieve high degrees of protein cross-linking.     

Effects of depleting ionic strength on 31P nuclear magnetic resonance spectra of micellar casein during membrane separation and diafiltration of skim milk

Membrane separation processes used in the concentration and isolation of micellar casein-based milk proteins from skim milk rely on extensive permeation of its soluble serum constituents, especially lactose and minerals. Whereas extensive literature exists on how these processes influence the gross composition of milk proteins, we have little understanding of the effects of such ionic depletion on the core structural unit of micellar casein [i.e., the casein phosphate nanocluster (CPN)]. The ³¹P nuclear magnetic resonance (NMR) is an analytical technique that is capable of identifying soluble and organic forms of phosphate in milk. Thus, our objective was to investigate changes to the ³¹P NMR spectra of skim milk during microfiltration (MF) and diafiltration (DF) by tracking movements in different species of phosphate. In particular, we examined the peak at 1.11 ppm corresponding to inorganic phosphate in the serum, as well as the low-intensity broad signal between 1.5 and 3.0 ppm attributed to casein-associated phosphate in the retentate. The MF concentration and DF using water caused a shift in the relevant ³¹P NMR peak that could be minimized if orthophosphate was added to the DF water. However, this did not resolve the simultaneous change in retentate pH and increased solubilization of micellar casein protein. The addition of calcium in combination with orthophosphate prevented micellar casein solubilization and simultaneously contributed to preservation of the CPN structure, except for overcorrection of retentate pH in the acidic direction. A more complex DF solution, involving a combination of phosphate, calcium, and citrate, succeeded in both CPN and micellar casein structure preservation while maintaining retentate pH in the region of the original milk pH. The combination of ³¹P NMR as an analytical technique and experimental probe during MF/DF processes provided useful insights into changes occurring to CPN while retaining the micellar state of casein.     

Properties of casein micelles cross-linked by transglutaminase

Factors affecting the cross-linking of milk proteins by transglutaminase (TGase) were studied. Cross-linking of caseins in bovine skim milk was optimal over a very wide pH range. The role of micellar calcium phosphate (MCP) in maintaining the integrity of TGase-treated casein micelles was studied by incubating skim milk with 0.01% (w/v) TGase at 30°C for 1–24 h, followed by removal of MCP from untreated or TGase-treated milk by acidification and dialysis. The protein content and profile of the samples were determined by Kjeldahl and SDS-PAGE, respectively. Whey proteins in unheated milk were not susceptible to TGase-induced cross-linking. The higher level of sedimentable protein in MCP-free TGase-treated milk than in MCP-free control milk indicated that TGase treatment partially prevented disintegration of the micelle on removal of MCP, probably due to extensive intramicellar TGase-induced cross-linking of casein molecules which led to the formation of sedimentable covalently bonded casein aggregates.     

Stability of casein micelles cross-linked by transglutaminase

In this study, caseins micelles were internally cross-linked using the enzyme transglutaminase (TGase). The integrity of the micelles was examined on solubilization of micellar calcium phosphate (MCP) or on disruption of hydrophobic interactions and breakage of hydrogen bonds. The level of monomeric caseins, determined electrophoretically, decreased with increasing time of incubation with TGase at 30°C; after incubation for 24 h, no monomeric β- or κ-caseins were detected, whereas only a small level of monomeric α_S1-casein remained, suggesting near complete intramicellar cross-linking. The ability of casein micelles to maintain structural integrity on disruption of hydrophobic interactions (using urea, sodium dodecyl sulfate, or heating in the presence of ethanol), solubilization of MCP (using the calcium-chelating agent trisodium citrate) or high-pressure treatment was estimated by measurement of the L*-value of milk; i.e., the amount of back-scattered light. The amount of light scattered by casein micelles in noncross-linked milk was reduced by >95% on complete disruption of hydrophobic interactions or complete solubilization of MCP; treatment of milk with TGase increased the stability of casein micelles against disruption by all methods studied and stability increased progressively with incubation time. After 24 h of cross-linking, reductions in the extent of light scattering were still apparent in the presence of high levels of dissociating agents, possibly through citrate-induced removal of MCP nanoclusters from the micelles, or urea- or sodium dodecyl sulfate-induced increases in solvent refractive index, which reduce the extent of light-scattering.     

The liquid-state 31P-nuclear magnetic resonance study on microfiltrated milk

To gain further insight into diversiform phosphorus in bovine milk, we separated skim milk into casein micelle and serum fractions by microfiltration and subjected them to liquid-state 31P-nuclear magnetic resonance (NMR) spectroscopy. As previously reported, the skim milk spectrum showed a broad and indistinct peak from phosphoserine residue (SerP) of casein. In the casein micelle spectrum, however, the SerP peak was more clearly observed with a phosphate peak that may be from micellar calcium phosphate (MCP). The serum spectrum was the same as skim milk spectrum, except for SerP peak. Furthermore, two types of casein micelle fractions, with 0.90 and 1.04 of [beta-casein + kappa-casein]/[alpha(s1)-casein + alpha(s2)-casein] ratios were generated by different temperature microfiltrations, occurring because beta-casein is released from the micelle at a low temperature. The shape of SerP peaks changed dramatically in both the casein micelle spectra, when the temperature dropped from 35 to 5 degrees C. Deconvolution analysis indicated that each SerP peak comprised the same set of four peaks. Half-width and composition discriminated between the two types of casein micelle fractions. As a consequence, there was significant interaction between casein micelle and milk serum, causing cloudiness of SerP in the liquid-state 31P-NMR spectrum of milk. Casein composition influenced the SerP-MCP interaction in micellar structure. Shape changing of the SerP peak was discussed in connection with beta-casein-release phenomenon.     

Addition of sodium caseinate to skim milk inhibits rennet-induced aggregation of casein micelles

The objective of this paper was to observe the rennet-induced aggregation behaviour of casein micelles in milk in the presence of additional sodium caseinate. Analysis of the centrifugal supernatants by size exclusion chromatography confirmed an increase in the soluble protein in the milk serum phase after addition of sodium caseinate. Although the total amount of κ-casein hydrolyzed over time was not affected, there was a significant effect of soluble casein on milk gelation, with a dose-dependent decrease of the gelation time as measured by rheology. Light scattering experiments also confirmed that the addition of soluble caseins inhibited the aggregation of casein micelles. Addition of 1 mM CaCl₂ prior to renneting increased the extent of rennet aggregation in samples containing additional sodium caseinate, but the inhibiting effect was still evident. The amount of soluble casein (as measured by chroma tography) significantly decreased after renneting, suggesting its association with the micellar fraction. Supporting experiments carried out with purified fractions of soluble caseins demonstrated that both α_s-casein and β-casein played a role as protective colloids (increasing steric repulsion) during renneting. It was concluded that the inhibiting effect observed during gelation was caused by the adsorption of soluble casein molecules on the surface of rennet-altered casein micelles.     

Supramolecular structure of the casein micelle

The supramolecular structure of colloidal casein micelles in milk was investigated by using a sample preparation protocol based on adsorption of proteins onto a poly-l-lysine and parlodion-coated copper grid, staining of proteins and calcium phosphate by uranyl oxalate, instantaneous freezing, and drying under a high vacuum. High-resolution transmission electron microscopy stereo-images were obtained showing the interior structure of casein micelles. On the basis of our interpretation of these images, an interlocked lattice model was developed in which both casein-calcium phosphate aggregates and casein polymer chains act together to maintain casein micelle integrity. The caseins form linear and branched chains (2 to 5 proteins long) interlocked by the casein-stabilized calcium phosphate nanoclusters. This model suggests that stabilization of calcium phosphate nanoclusters by phosphoserine domains of α_s1-, α_s2-, or β-casein, or their combination, would orient their hydrophobic domains outward, allowing interaction and binding to other casein molecules. Other interactions between the caseins, such as calcium bridging, could also occur and further stabilize the supramolecule. The combination of having an interlocked lattice structure and multiple interactions results in an open, sponge-like colloidal supramolecule that is resistant to spatial changes and disintegration. Hydrophobic interactions between caseins surrounding a calcium phosphate nanocluster would prevent complete dissociation of casein micelles when the calcium phosphate nanoclusters are solubilized. Likewise, calcium bridging and other electrostatic interactions between caseins would prevent dissociation of the casein micelles into casein-calcium phosphate nanocluster aggregates when milk is cooled or urea is added to milk, and hydrophobic interactions are reduced. The appearance of both polymer chains and small aggregate particles during milk synthesis would also be expected based on this interlocked lattice model of casein micelles, and its supramolecule structure thus exhibits the principles of self-aggregation, interdependence, and diversity observed in nature.     

High pressure effects on the structure of casein micelles in milk as studied by cryo-transmission electron microscopy

Milk was processed with high hydrostatic pressure in order to modify the casein micelles. Images, that in details showed the casein micelle structure in untreated and pressure-treated skim milk, were obtained by using cryo-transmission electron microscopy (cryo-TEM). Sizes and shapes adopted by casein micelles in pressurised milk are concluded to be a result of an equilibrium distribution between self-assembling casein molecules in the serum phase and caseins adsorbed to surfaces of casein micelles and are governed by an initial pressure-dependent displacement of caseins into the serum phase. Pressurisation of milk at moderately high pressure, in the range 150–300 MPa, favoured formation of a large number of small micelles that coexisted with a fraction of large micelles, and which appeared perfectly spherical with smooth and well-defined surfaces, features which are suggested to originate from secondary adsorption of caseins. Pressurisation of milk at 400 MPa favoured formation of smaller casein assemblies, with sizes between 30 nm and 100 nm. Measurements of free calcium concentration [Ca²⁺] showed that calcium was rebound to casein micelles after pressurisation of milk. Furthermore, the electron microscopy images indicated that the substructures were similar for pressure-modified casein micelles and casein micelles in untreated milk.     

Ethanol stability of casein micelles cross-linked with transglutaminase

The influence of transglutaminase (TGase)-induced cross-linking on the ethanol stability of skimmed milk was investigated. The stability of milk against ethanol-induced coagulation increased in sigmoidal fashion with milk pH (5.0–7.5) for all samples; ethanol stability also increased upon incubation (0–24 h) with 0.05 g L⁻¹ TGase at 30 °C. In untreated milk, addition of ethanol induced a collapse of the polyelectrolyte brush of κ-casein on the micelle surface, thereby facilitating micellar aggregation. Dynamic and static light scattering measurements indicated that in TGase-treated milk, the ethanol-induced collapse of the polyelectrolyte brush was far less than in untreated milk, suggesting that the increased ethanol stability of TGase-treated casein micelles is caused by the cross-linking of the polyelectrolyte brush on the micellar surface.     

Cross‐linking of bovine and caprine caseins by microbial transglutaminase and their use as microencapsulating agents for n‐3 fatty acids

Bovine and caprine caseins were cross‐linked with microbial transglutaminase (mTG). The mTG‐cross‐linked bovine or caprine casein dispersion, mixed with 14.5% maltodextrin (DE = 40), was used to prepare emulsions with 10.5% algae oil. Oxidative stability of emulsions was evaluated by peroxide values (PVs) and anisidine values. Adding liposoluble rosemary extract rich in carnosic acid and δ‐tocopherol lowered the formation of hydroperoxides and their subsequent decomposition products in emulsions. Emulsions stabilised with liposoluble rosemary extract rich in carnosic acid and δ‐tocopherol were spray‐dried at 180/95 °C. Algae oil microencapsulated with mTG‐cross‐linked bovine casein reduced PV by ≈ 34%, while the algae oil microencapsulated with mTG‐cross‐linked caprine casein with low levels of α_s1‐casein reduced PV by ≈ 42% at 4 weeks of storage at 30 °C. The investigation suggests that liposoluble rosemary extract rich in carnosic acid and δ‐tocopherol effectively protected algae oil during the coating process with mTG‐cross‐linked bovine and caprine caseins. The above results clearly indicated that the choice of milk caseins (bovine vs. caprine) cross‐linked with mTG impacts the oxidative stability of spray‐dried algae oil emulsions (microcapsules) enriched with n‐3 fatty acids.     

Technical note: Fourier transform infrared spectral analysis in tandem with 31P nuclear magnetic resonance spectroscopy elaborates detailed insights into phosphate partitioning during skimmed milk microfiltration and diafiltration

Our previous study identified peaks in the ³¹P nuclear magnetic resonance (³¹P NMR) spectra of skim milk, denoting the interaction of different phosphate species such as inorganic and casein-associated phosphate during the separation of colloidal and serum phases of skim milk by microfiltration (MF) and diafiltration (DF). In the current study, we investigated the same samples generated by the aforementioned separation using attenuated total reflectance (ATR) Fourier transform infrared (FTIR) spectroscopy analysis. The results confirmed that the technique was not only capable of differentiating between the mineral equilibrium of the casein phosphate nanocluster (CPN) and milk serum, but also complemented the application of ³¹P NMR. An ATR-FTIR broad band in the region of 1,055 to 1,036 cm^?1 and a specific band at 1,076 cm^?1 were identified as sensitive to the repartitioning of different phosphate species in milk in accordance with the ³¹P NMR signals representing casein-associated phosphate and inorganic phosphate in the serum. A third ATR-FTIR signal at 1,034 cm^?1 in milk, representing precipitated inorganic calcium phosphate, had not previously been detected by ³¹P NMR. Thus, the results indicate that a combination of ATR-FTIR and ³¹P NMR spectroscopies may be optimally used to follow mineral and protein phase changes in milk during membrane processing.     

Size of native and heated casein micelles,content of protein and minerals in milk from Norwegian Red Cattle—effect of milk protein polymorphism and different feeding regimes

Milk samples of 59 cows of the Norwegian Red Cattle breed receiving three different supplementary concentrates, were analysed for genotypes of caseins and whey proteins, the content of different milk salts (Ca²⁺, Ca, Mg and citrate), the content of total protein, casein and whey protein and the mean micellar size of native and heated casein micelles. The genotype of α_s1-casein had a statistically significant effect on the content of protein and casein, and the content of whey protein and the casein number were significantly influenced by different feeding regimes, and the content of citrate. The mean size of native and heated casein micelles was significantly influenced by the feeding regimes, genotype of α_s1-casein (native mean size only) and κ-casein, pH and the content of casein, whey protein and casein number. The heat-induced changes in mean micellar size were significantly affected by the calcium ion activity which accounted for approximately 40% of the total variation.     

Structural changes induced by high-pressure processing in micellar casein and milk protein concentrates

Reconstituted micellar casein concentrates and milk protein concentrates of 2.5 and 10% (wt/vol) protein concentration were subjected to high-pressure processing at pressures from 150 to 450 MPa, for 15 min, at ambient temperature. The structural changes induced in milk proteins by high-pressure processing were investigated using a range of physical, physicochemical, and chemical methods, including dynamic light scattering, rheology, mid-infrared spectroscopy, scanning electron microscopy, proteomics, and soluble mineral analyses. The experimental data clearly indicate pressure-induced changes of casein micelles, as well as denaturation of serum proteins. Calcium-binding α_S1- and α_S2-casein levels increased in the soluble phase after all pressure treatments. Pressurization up to 350 MPa also increased levels of soluble calcium and phosphorus, in all samples and concentrations, whereas treatment at 450 MPa reduced the levels of soluble Ca and P. Experimental data suggest dissociation of calcium phosphate and subsequent casein micelle destabilization as a result of pressure treatment. Treatment of 10% micellar casein concentrate and 10% milk protein concentrate samples at 450 MPa resulted in weak, physical gels, which featured aggregates of uniformly distributed, casein substructures of 15 to 20 nm in diameter. Serum proteins were significantly denatured by pressures above 250 MPa. These results provide information on pressure-induced changes in high-concentration protein systems, and may inform the development on new milk protein-based foods with novel textures and potentially high nutritional quality, of particular interest being the soft gel structures formed at high pressure levels.     

pH-dependent sedimentation and protein interactions in ultra-high-temperature-treated sheep skim milk

Sheep milk is considered unstable to UHT processing, but the instability mechanism has not been investigated. This study assessed the effect of UHT treatment (140°C/5 s) and milk pH values from 6.6 to 7.0 on the physical properties of sheep skim milk (SSM), including heat coagulation time, particle size, sedimentation, ionic calcium level, and changes in protein composition. Significant amounts of sediment were found in UHT-treated SSM at the natural pH (～6.6) and pH 7.0, whereas lower amounts of sediment were observed at pH values of 6.7 to 6.9. The proteins in the sediment were mainly κ-casein (CN)–depleted casein micelles with low levels of whey proteins regardless of the pH. Both the pH and the ionic calcium level of the SSM at all pH values decreased after UHT treatment. The dissociation levels of κ-, β-, and α_S2-CN increased with increasing pH of the SSM before and after heating. The protein content, ionic calcium level, and dissociation level of κ-CN were higher in the SSM than values reported previously in cow skim milk. These differences may contribute to the high amounts of sediment in the UHT-treated SSM at natural pH (～6.6). Significantly higher levels of κ-, β-, and α_S2-CN were detected in the serum phase after heating the SSM at pH 7.0, suggesting that less κ-CN was attached to the casein micelles and that more internal structures of the casein micelles may have been exposed during heating. This could, in turn, have destabilized the casein micelles, resulting in the formation of protein aggregates and high amounts of sediment after UHT treatment of the SSM at pH 7.0.     

Fluorescent labeling study of plasminogen concentration and location in simulated bovine milk systems

A fluorescent labeling method was developed to study plasminogen (PG) concentration and location in simulated bovine milk. Activity and stability of PG labeled with Alexa Fluor 594 (PG-594) were comparable to those of native PG. The fluorescent signal of PG-594 exhibited pH, temperature, and storage stability, and remained stable throughout typical sample treatments (stirring, heating, and ultracentrifugation). These characteristics indicate broad applicability of the fluorescent labeling technique for milk protease characterization. In an example application, PG-594 was added to simulated milk samples to study effects of heat and β-lactoglobulin (β-LG) on the distribution of PG. Before heating, about one-third of the PG-594 remained soluble in the whey fraction (supernatant) whereas the rest became associated with the casein micelle. Addition of β-LG to the system slightly shifted PG-594 distribution toward the whey fraction. Heat-induced PG-594 binding to micelles in whey-protein-free systems was evidenced by a decrease of PG-594 from 31 to 15% in the whey fraction accompanied by an increase of PG-594 from 69 to 85% in casein micelle fractions. When β-LG was present during heating, more than 95% of PG-594 became associated with the micelle. A comparison with the distribution pattern of PG-derived activities revealed that heat-induced PG binding to micelles accompanies heat-induced PG inactivation in the micelle fraction. Incubation of the casein micelles with the reducing agent β-mercaptoethanol revealed that disulfide bonds formed between PG and casein or between PG and casein-bound β-LG are the mechanisms for heat-induced PG binding to casein micelles. Western blotting and zymography results correlated well with fluorescent labeling studies and activity studies, respectively. Theoretically important findings are: 1) when heated, serum PG is capable of covalently binding to micellar casein or complexing with β-LG in whey and then coadhering to micelles, and 2) PG that associated with micellar casein through lysine binding sites before heating is capable of developing heat-induced disulfide bonds with casein. The overall results are PG covalently binding to micelles and inactivation thereafter. Our results suggest that, instead of thermal denaturation through irreversible unfolding, covalent bond formation between PG and other milk proteins is the mechanism of PG inhibition during thermal processing.     

Effect of high pressure - low temperature processing on composition and colloidal stability of casein micelles and whey proteins

The aim of this study was to identify the impact of high pressure treatments at sub-zero temperatures (high pressure - low temperature; HPLT) on milk proteins. Whey protein solutions, micellar casein dispersions and two mixtures (micellar caseins:whey proteins, 80:20 and 20:80, w/w) were pressure treated (100–600 MPa) at pH 7.0 or 5.8 at −15 °C, −35 °C and ambient temperature. Solubility data showed that whey proteins could only be affected by HPLT treatments at pH 7.0 if caseins were present, while effects could be induced at pH 5.8 without the presence of caseins. The caseins formed on the one hand large aggregates (flocs) and on the other hand the solubility was increased by the creation of smaller micelles. The formation of flocs could only be observed for HPLT treated samples, which indicates the formation of different protein interactions in milk protein based samples compared with common HP treatments.     

Effect of hydrolysis of casein by plasmin on the heat stability of milk

This study examined the effect of hydrolysis of casein by added plasmin (6 mg L⁻¹) on the heat stability of raw, pre-heated, serum protein-free or concentrated skim milk. Plasmin activity markedly affected the heat stability–pH profile of skim milk and serum protein-free milk, apparently by altering the properties of the casein micelles. It is probable that changes in the surface charge of the micelles, as a result of the hydrolysis of caseins, contributed to this effect. Hydrolysis by plasmin reduced the zeta-potential of the casein micelles from ∼−19 to ∼−16 mV. The effect of hydrolysis of casein by plasmin on the heat stability of pre-heated milk was less pronounced, shifting the heat stability–pH profile to more alkaline values; the heat stability of concentrated milk was unaffected by plasmin. A very high (50 mg L⁻¹) level of added plasmin resulted in clearing of the skim milk; the L^* value decreased from ∼75 to ∼35 after 24 h incubation at 37 °C. Clearing was correlated with a change in casein micelle diameter from an initial value of ∼175 to ∼250 nm. It is suggested that plasmin-induced changes in zeta-potential may promote micellar aggregation or changes in micelle stucture.     

Hydration of Native and Rennin-Treated,Cold-Solubilized Caprine Caseins as Determined by Oxygen-17 Nuclear Magnetic Resonance

The hydration of native and rennin-coagulated bovine and caprine caseins was investigated by oxygen-17 nuclear magnetic resonance (NMR) and fitted by nonlinear regression analysis. A charge-charge interaction model (B₀) was employed to analyze the transverse relaxation (1/T₂) data. Relaxation differences between reconstituted native micelles at 10°C and rennin-coagulated, cold-solubilized micelles of caseins strongly suggest that important structural dissimilarities exist between these milk proteins that are due to differences in the ratios of α_s1- to β-CN. Variants of α_s1-CN had significant effects on caprine casein hydration. The differences were more pronounced in rennin-coagulated than in native casein micelles. All rennin-coagulated, cold-solubilized casein micelles demonstrated significant decreases in hydration. Micelles containing low α_s1-CN retain an open, highly hydrated structure, in comparison with similarly treated bovine and caprine casein micelles containing high α_s1-CN. Second virial coefficients (B₀ values) derived from oxygen-17 NMR data suggest that reconstituted bovine and caprine casein micelles containing high α_s1-CN exhibit strong interactions, which at first decrease then increase during clotting. In contrast, the native caprine casein micelles containing low α_s1-CN exhibit strong charge repulsions, which increase with clotting. The compositional differences are reflected in differences in the extent of protein-protein aggregation during casein clotting by rennin. Our results demonstrate that alteration in casein composition can dramatically alter cheese-making properties.