Casein Micelle Dispersions Research Articles

Novel details on the dissociation of casein micelle suspensions as a function of pH and temperature

Membrane filtration is a widespread process for fractionation and recombination of milk components. Although the dissociation of micellar caseins has been studied in detail in skim milk, it is important to better understand the dissociation dynamics occurring between the colloidal and noncolloidal fractions in systems of modified composition. This research aimed at understanding the dissociation of casein proteins in micellar fractions depleted of whey proteins. Casein micelle dispersions were tested at neutral pH and pH 6 (using glucono-δ-lactone as acidulant), after incubation at 4°C or 22°C, and compared with skim milk. The ionic composition of the serum phase was measured using inductively coupled plasma-mass spectrometry, and the protein distribution analyzed using reversed phase-HPLC coupled with mass spectrometry. When incubated at 22°C, there were no differences in casein micelle dissociation between skim milk and whey protein-depleted micelles (∼2.6% dissociated casein). No additional dissociation occurred by lowering the pH from 6.8 to 6 at 22°C, albeit there were more soluble ions at low pH (71% Ca and 65% P). At 4°C, there was an increased amount of β-casein found in the serum phase (23-33% of total β-casein). In addition, there was an uneven dissociation behavior of the various genetic β-casein variants, whereof A2 was more readily released with cooling. In skim milk, approximately 22%, 18%, and 14% of κ-, αS2, and αS1-caseins, respectively, were dissociated from the micellar phase upon cooling and acidification to pH 6.0. This was in contrast to whey protein-depleted casein suspensions, in which only 6%, 5%, and 3% of κ-, αS2, and αS1-caseins, respectively, had dissociated. The results suggested that the whey proteins in the serum phase play a role in the equilibrium between colloidal and soluble caseins in milk. This is of great relevance in processes such as cold membrane fractionation, where more attention should be given to the protein composition in the serum phase, especially when concentration is combined with fractionation of the serum proteins.

Assessment of structures in phosphocaseinate dispersions by A4F, NMR and SAXS: The impact of demineralization and heat treatment on viscosity

The present work aims to better understand the relation between heat treatment (Ultra High Temperature - UHT), partial demineralization of casein micelles (CMs) dispersions and their corresponded rheological properties. A multiscale analysis (SAXS, solid state NMR and Asymmetrical Flow Field Flow Fractionation (A4F)), has been used. Different samples with different levels of calcium/phosphorous demineralization (up to −30% of calcium content) have been studied before and after UHT treatment. The present data show that CM dispersions, displaying a mild demineralization, display an important decrease in viscosity after UHT. NMR shows that, during UHT, mild demineralized samples display stronger calcium/phosphorus intake than both, the non-demineralized and the strong demineralized ones. SAXS and A4F show that UHT-dependent calcium intake, in mild demineralization dispersions, seems to be responsible for a transformation of large casein aggregates into small and denser ones. This decrease of the large aggregates is probably the main reason of the UHT-dependent decrease in viscosity. These data show that a sample with a rather small amount of demineralization can have a quite important susceptibility to UHT in terms of viscosity.

Are micelles actually at the interface in micellar casein stabilized foam and emulsions?

Different casein preparations are used for stabilizing emulsions and foams. For systems made with aqueous micellar casein dispersions, the molecular and colloidal mechanisms responsible for the stabilization of oil-water and air-water interfaces have not been conclusively ascertained. Whether the micelles themselves, small casein aggregates, or individual casein molecules are at the interface is still an open question. Understanding these mechanisms is important for food industries to improve product formulations. We investigated the nonlinear rheology and microstructure of oil-water and air-water interfaces stabilized with casein micelle dispersions and their fractions. Our results convincingly show that the micelles themselves are not adsorbed at the interfaces. For air-water interfaces, the behavior appears to be dominated by β-casein, whereas the properties of oil-water interfaces are dominated by small casein aggregates. These findings are important to understand the stabilization mechanisms of emulsions and foams prepared with caseins or milk.

Foaming properties of different forms of caseins in aqueous systems

Foaming properties of sodium caseinate (NaCas) particle dispersions, native NaCas solutions, and casein micelle dispersions were investigated by visual observations and by considering viscosity and structural differences. The effect of NaCl on the foaming properties of systems was also discussed. The presence of NaCl (50 mM, 100 mM, and 200 mM) changed the foam stability of NaCas particle dispersions and native NaCas solutions, whereas foam stability of casein micelle dispersions was not affected by the presence of salt. When there was no NaCl addition to the systems, NaCas particle dispersions showed the highest foam stability (~ 3 h), whereas when there was NaCl in the medium, native NaCas solutions showed the highest foam stability (~ 2 h). The presence of salt ions changed the repulsive forces between proteins, which could result in a partial disintegration of particles and aggregation of native proteins. The presence of both particles and their dissociated aggregates in NaCas particle dispersions was found to increase the foaming properties. The behavior of NaCas particles at the interface was similar to the colloidal particles, which was different from the behavior of native proteins. Therefore, the NaCas particles have the potential to be used as a food-grade Pickering stabilizer in aerated food products.

Build-up and relaxation of membrane fouling deposits produced during crossflow ultrafiltration of casein micelle dispersions at 12 °C and 42 °C probed by in situ SAXS

Skim milk filtration is performed either at low or high temperature. However, there is still a lack of knowledge concerning the influence of temperature on membrane fouling. We used in situ small-angle X-ray scattering (SAXS) to study external membrane fouling during crossflow ultrafiltration of milk protein (casein micelle) dispersions at 12 °C and 42 °C. Casein micelle concentration distribution was measured in the concentration polarization layer and the deposit in a three-step filtration experiment that consisted of (i) fouling development step (with applied pressure and average cross-flow velocity fixed at 110 kPa and 3.1 cm s−1, respectively), (ii) pressure relaxation step (with pressure reduced to 10 kPa), and (iii) deposit erosion step (with crossflow velocity increased to 15.6 cm s−1).Despite a higher average filtrate flux obtained at 42 °C, filtration at 42 °C resulted in the formation of a thicker and more concentrated deposit than filtration at 12 °C. At both temperatures studied, subsequent pressure relaxation resulted in deposit swelling, which was more intense in the less-compressed external part of the deposit. Deposit swelling rate was significantly higher at 42 °C than at 12 °C. The swelled deposit obtained at 42 °C was more eroded by the crossflow compared to the poorly swelled deposit obtained at 12°. This suggests that deposit formation and compression were more reversible at 42 °C than at 12 °C.

Casein core-polysaccharide shell nanocomplexes stable at pH 4.5 enabled by chelating and complexation properties of dextran sulfate

Casein micelle dispersions are turbid and unstable around the isoelectric point at pH 4.6. In this study, dextran sulfate (DS) was used to form stable and translucent 2% w/v casein dispersions at pH 4.5 through chelating calcium in micelles and forming nanocomplexes. Contrasting with turbid dispersions with large particles after brief mixing, thorough mixing micellar casein with DS stock dispersions resulted in stable and translucent dispersions after acidification to pH 4.5. The chelating capacity of DS was further confirmed by measuring ionic calcium. The dispersion turbidity was further reduced by changing pH from neutral to 11.30 to dissociate casein micelles followed by complexing with DS during acidification to pH 4.5. The formation of casein-DS nanocomplexes stable at pH 4.5 was mainly contributed to the electrostatic binding between basic amino acids of caseins and sulfate groups of DS, even when the two biopolymers were overall anionic at pH 7.6.

Stable casein micelle dispersions at pH 4.5 enabled by propylene glycol alginate following a pH-cycle treatment

Polysaccharides may form stable complexes with caseins to prevent precipitation near the isoelectric point of pH 4.6. In this study, dispersions of 1% w/v micellar caseins and propylene glycol alginate (PGA) were treated with a pH-cycle treatment from neutral to pH 11.30, to dissociate casein micelles, and then to pH 4.5 to re-associate caseins to form complexes with PGA. An increase in PGA concentration overall resulted in the reduced dispersion turbidity. The dispersion with equal masses of casein and PGA following the pH-cycle treatment had the lowest turbidity (260 NTU) and was absent of both precipitation and gelation during 30-day storage at 21 °C, contrasting with unstable and turbid (>4000 NTU) dispersions directly acidified to pH 4.5. The dispersion turbidity was determined by both the size and mass density of particles, and both covalent and non-covalent (mainly electrostatic and hydrophobic) interactions contributed to the complex formation and dispersion stability at pH 4.5.

Major Role of Voluminosity in the Compressibility and Sol-Gel Transition of Casein Micelle Dispersions Concentrated at 7 °C and 20 °C.

The objective of this work is to bring new information about the influence of temperatures (7 °C and 20 °C) on the equation of state and sol–gel transition behavior of casein micelle dispersions. Casein micelle dispersions have been concentrated and equilibrated at different osmotic pressures using equilibrium dialysis at 7 °C and 20 °C. The osmotic stress technique measured the osmotic pressures of the dispersions over a wide range of concentrations. Rheological properties of concentrated dispersions were then characterized, respectively at 7 °C and at 20 °C. The essential result is that casein micelle dispersions are less compressible at 7 °C than at 20 °C and that concentration of sol–gel transition is lower at 7 °C than at 20 °C, with compressibility defined as the inverse to the resistance to the compression, and that is proportional to the cost to remove water from structure. From our interpretations, these two features were fully consistent with a release of soluble β-casein and nanoclusters CaP and an increased casein micelle hydration and apparent voluminosity at 7 °C as compared with 20 °C.

Characterization of buttermilk serum fractions and their effect on rennet-induced coagulation of casein micelle dispersions

Upon centrifugation of milk and buttermilk (BM) the supernatant above the casein micelles pellet separates into three phases: a low-density opalescent fraction, a clear fraction, and a high-density opalescent (HDO) fraction. These fractions were collected and the concentrations of casein, whey and milk fat globule membrane (MFGM) proteins, fat and phospholipids determined. Reference casein micelles were pelleted from skim milk (SM) and re-suspended in the serum fractions. Rennet-induced aggregation, coagulation aptitude and gel syneresis were measured in these suspensions. Buttermilk serum fractions contained more MFGM components than SM fractions did. Overall, BM serum showed a negative impact on rennet-induced aggregation of casein micelles, gel formation and gel syneresis during cooking. This impact was greater for the HDO fraction, which contained much higher concentration of MFGM materials when isolated from buttermilk than from milk. The negative impact of buttermilk on rennet-induced coagulation of casein micelles was mainly attributed to the presence of tiny fat droplets and high density MFGM fragments.

Connection between dynamic rheometry and pair interactions of casein micelles in concentrated skim milk

The objective of this work was to relate dynamic rheological data to the pair interaction potential between casein micelles (CM) in concentrated systems. The elastic moduli at high frequencies (G∞′) were obtained by fitting experimental data from the literature for CM dispersions at different concentrations using a Maxwell model. G∞′ values were also obtained using the Buscall model and the pair interaction potential (U) obtained with DLVO-type theoretical predictions proposed for CM and for sterically stabilized colloidal particles. It was observed that moduli G∞′ obtained from experimental data are in agreement with theoretical predictions. Therefore, this methodology allows connecting the dynamic rheometric data with pair interaction potentials of CM in concentrated skim milk dispersions and it may help to infer about the stability of milk concentrates.

Interfacial properties, thin film stability and foam stability of casein micelle dispersions

Foam stability of casein micelle dispersions (CMDs) strongly depends on aggregate size. To elucidate the underlying mechanism, the role of interfacial and thin film properties was investigated. CMDs were prepared at 4°C and 20°C, designated as CMD4°C and CMD20°C. At equal protein concentrations, foam stability of CMD4°C (with casein micelle aggregates) was markedly higher than CMD20°C (without aggregates). Although the elastic modulus of CMD4°C was twice as that of CMD20°C at 0.005Hz, the protein adsorbed amount was slightly higher for CMD20°C than for CMD4°C, which indicated a slight difference in interfacial composition of the air/water interface. Non-linear surface dilatational rheology showed minor differences between mechanical properties of air/water interfaces stabilized by two CMDs. These differences in interfacial properties could not explain the large difference in foam stability between two CMDs. Thin film analysis showed that films made with CMD20°C drained to a more homogeneous film compared to films stabilized by CMD4°C. Large casein micelle aggregates trapped in the thin film of CMD4°C made the film more heterogeneous. The rupture time of thin films was significantly longer for CMD4°C (>1h) than for CMD20°C (<600s) at equal protein concentration. After homogenization, which broke down the aggregates, the thin films of CMD4°C became much more homogeneous, and both the rupture time of thin films and foam stability decreased significantly. In conclusion, the increased stability of foam prepared with CMD4°C appears to be the result of entrapment of casein micelle aggregates in the liquid films of the foam.

Particle size determines foam stability of casein micelle dispersions

The role of interfacial properties and size of casein micelles aggregates on foam stability of casein micelle dispersions (CMDs) was examined. CMDs were prepared by redispersing casein micelles pellets obtained by ultracentrifugation. The size of colloidal particles could be controlled by differences in redispersing temperature. CMDs redispersed at 20 °C (CMD20 °C) and 4 °C (CMD4 °C) had average particle sizes of around 200 nm (micelles) and 500 nm (micelles and aggregates), respectively. At 3% total protein, the foam half-life, t½, of CMD4 °C was significantly higher than that of CMD20 °C and skim milk. No correlation between foam stability and surface rheological properties or protein composition could be observed. Foam stability was strongly related to the size of colloidal particles present in CMD. This was confirmed by the observation that the foam stability of CMD4 °C decreased to that of CMD20 °C when the aggregates were broken down by homogenisation.

Rheological modelling of dispersions of casein micelles considered as microgel particles

Rheological behavior of dispersions of casein micelles (CM) was predicted through a viscosity model in which CM were considered as microgel particles. A simple structure for CM composed of a spherical core with a brush of κ-casein was considered. An explicit relationship between viscosity and shear stress allows obtaining key parameters such as the packing fractions corresponding to the shear stress limits and the critical shear stress closely related to the total interaction potential between two CM. The analysis of the model parameters fitted from experimental viscosity curves indicates that CM at concentrated regime behave as soft spheres. In addition, the model provides the pair interaction energy between CM directly from rheometric data. The values obtained agree with a DLVO-like theory previously proposed for CM. Indeed, using independent experimental data and parameters, it was possible to cross check both models. The results obtained are very promising for studying CM dispersions as colloidal systems and the stability of dairy products under different physicochemical environments.

Processing of phosphocasein dispersions by dynamic high pressure: Effects on the dispersion physico-chemical characteristics and the binding of α-tocopherol acetate to casein micelles

Dispersions of phosphocaseins (PCs) containing (w/w) 2.5% proteins at pH 6.7 were processed using a ~ 15 L/h homogeniser with a high-pressure valve immediately followed by cooling devices. The effect of dynamic high-pressure (or ultra-high pressure homogenisation, UHPH) at 100–300 MPa and two initial temperatures (Tin = 14 °C or 34 °C) was investigated on (i) casein micelle size distributions, (ii) the turbidity and viscosity of PC dispersions, and (iii) the binding efficiency of α-tocopherol acetate (α-TA) to casein micelles (α-TA:PC molar ratio ~ 1:1). A significant and gradual decrease of casein micelle sizes was observed after UHPH up to 300 MPa at Tin = 14 °C. The decrease in micelle sizes was less extensive after UHPH at Tin = 34 °C. Notably, the binding efficiency of α-TA significantly (p < 0.001) increased after processing, suggesting combined effects of temperature and dynamic high-pressure. Industrial operators in food, cosmetic and pharmaceutical areas are currently interested in developing encapsulating systems to delivery bioactive compounds, generally hydrophobic, unstable and sensitive to light, temperature or/and oxygen. The present study suggests that processing of casein micelle dispersions by dynamic high pressure at ≥ 200 MPa could modify casein micelle organisation with an enhancement of the binding of hydrophobic ligand (α-tocopherol acetate) to the newly UHPH-formed neo-micelles. Here is a potential application for UHPH, a physical technology that offers the added advantage of notably reducing the microbial load of processed samples.

Multiscale Characterization of Casein Micelles Under NaCl Range Conditions

Micellar casein (MC) dispersions were studied at a constant protein concentration of 5 wt % in high NaCl environment. The micellar edifices were characterized as to their morphology, size, and content of proteins in the supernatant after ultracentrifugation. Additionally, changes in secondary structures of the protein upon salt increase were followed by Fourier Transform Infrared Spectroscopy (FTIR). For the first time, the estimations of secondary structural elements (irregular, s-sheet, α-helix and turn) from Amide III assignments were correlated with results from Amide I. Casein micelles dispersions in water were characterized by Transmission Electron Microscopy (TEM) by a spherical shape and a size between 100 and 200 nm. A salt increase resulted to a destabilization of the micelle and the formation of mini-micelles more or less aggregated. The size of the new edifice was almost similar to the native micelle. These TEM observations were confirmed by a constant casein micelle hydrodynamic diameter determined by Dynamic Light Scattering (DLS) and ranging between 150 and 180 nm. Upon salt increase, FTIR revealed an increase in irregular structures and a concurrent decrease in s-sheet structures. Secondary structural elements percentages were almost similar from Amide I and Amide III. The use of these multiscale techniques led to a better understanding of the micellar edifice under high salt environment. Around 3% NaCl addition, a good correlation was observed between destabilization of the micellar edifice, modifications of the caseins secondary structure and repartition of caseins between supernatant and pellet after ultracentrifugation.

Rheology and phase behavior of dense casein micelle dispersions

Casein micelle dispersions have been concentrated through osmotic stress and examined through rheological experiments. In conditions where the casein micelles are separated from each other, i.e., below random-close packing, the dispersions have exactly the flow and dynamic properties of the polydisperse hard-sphere fluid, demonstrating that the micelles interact only through excluded volume effects in this regime. These interactions cause the viscosity and the elastic modulus to increase by three orders of magnitude approaching the concentration of random-close packing estimated at C(max) approximately 178 g/l. Above C(max), the dispersions progressively turn into "gels" (i.e., soft solids) as C increases, with elastic moduli G(') that are nearly frequency independent. In this second regime, the micelles deform and/or deswell as C increases, and the resistance to deformation results from the formation of bonds between micelles combined with the intrinsic mechanical resistance of the micelles. The variation in G(') with C is then very similar to that observed with concentrated emulsions where the resistance to deformation originates from a set of membranes that separate the droplets. As in the case of emulsions, the G(') values at high frequency are also nearly identical to the osmotic pressures required to compress the casein dispersions. The rheology of sodium caseinate dispersions in which the caseins are not structured into micelles is also reported. Such dispersions have the behavior of associative polymer solutions at all the concentrations investigated, further confirming the importance of structure in determining the rheological properties of casein micelle systems.

Casein Micelle Dispersions under Osmotic Stress

Casein micelles dispersions have been concentrated and equilibrated at different osmotic pressures using equilibrium dialysis. This technique measured an equation of state of the dispersions over a wide range of pressures and concentrations and at different ionic strengths. Three regimes were found. i), A dilute regime in which the osmotic pressure is proportional to the casein concentration. In this regime, the casein micelles are well separated and rarely interact, whereas the osmotic pressure is dominated by the contribution from small residual peptides that are dissolved in the aqueous phase. ii), A transition range that starts when the casein micelles begin to interact through their κ-casein brushes and ends when the micelles are forced to get into contact with each other. At the end of this regime, the dispersions behave as coherent solids that do not fully redisperse when osmotic stress is released. iii), A concentrated regime in which compression removes water from within the micelles, and increases the fraction of micelles that are irreversibly linked to each other. In this regime the osmotic pressure profile is a power law of the residual free volume. It is well described by a simple model that considers the micelle to be made of dense regions separated by a continuous phase. The amount of water in the dense regions matches the usual hydration of proteins.

Structure and rheological behavior of casein micelle suspensions during ultrafiltration process

The stability and mechanism underlying the formation of deposits of casein micelles during ultrafiltration process were investigated by small-angle and ultra small-angle x-ray scattering (SAXS and USAXS). The casein micelle dispersions consisted of phospho-caseinate model powders and the measurements probed length scales ranging from 1 to 2000 nm. Rheometric and frontal filtration measurements were combined with SAXS to establish the relationship between the rheological behavior of deposits (shear and/or compression) and the corresponding microstructure. The results revealed two characteristic length scales for the equilibrium structure with radius of gyrations R(g), about 100 and 5.6 nm pertaining to the globular micelles and their non-globular internal structure, respectively. The SAXS measurements further indicated that the increase of temperature from 20 to 70 degrees C or the decrease of pH from 6.6 to 6 lead to agglomeration of the globular micelles. In situ scattering measurements showed that the decrease of permeation flows is directly related to the deformation and compression of the micelles in the immediate vicinity of the membrane.

Determination of early stages of rennet-induced aggregation of casein micelles by diffusing wave spectroscopy and rheological measurements

Abstract Diffusing wave spectroscopy (DWS) and rheology have been used to study the aggregation of casein micelles dispersions induced by the addition of rennet, a proteolytic enzyme preparation. Aggregation times measured by DWS and by rheology were in good agreement. In addition, prior to the onset of aggregation DWS was able to detect the change in the casein micelle mobility due to the cleavage of κ-casein by the enzyme. We believe that, due to its the sensitivity to changes brought in particle size, in addition to being a non-invasive and a non-intrusive technique, DWS has a potential to be implemented and used in an industrial environment, such as manufacture of yogurts and cheeses.

Casein micelle dispersions into water, NaCl and CaCl2: physicochemical characteristics of micelles and rennet coagulation

We have studied the effect of ionic strength from about 0 to 0.12 m on physicochemical characteristics of micelles and on gelation by rennet. Native phosphocaseinate powder was used as it exhibits attractive technological properties. The powder was dissolved at 25 g l−1 casein concentration with 0.2 g l−1 sodium azide into deionised water, NaCl or CaCl2 solutions, at ionic strength from 0 to 0.12 m. The pH of micelle suspensions was corrected to pH 6.6 and, after overnight equilibration at 20°C, pH values were checked and eventually corrected. After re-equilibration, calcium and phosphorus partition, micelle size, centrifuged pellet hydration were determined and κ-casein hydrolysis and gelation of the suspensions were followed after rennet addition. The increase in NaCl concentration of casein micelle suspensions resulted in calcium and phosphorus solubilisation from micelles, and increase in pellet hydration. In the same way, the first-order rate constant of the κ-casein hydrolysis with rennet was reduced, while CMP at the clotting time determined by Formagraph was unchanged. Rennet gelation was consequently retarded by NaCl increase. Suspensions of casein micelles into CaCl2 solutions did not exhibit calcium binding onto the micelles, as in milk, but a small reduction of diffusible phosphorus from 1.55 to 0.24 mm was shown. Pellet hydrations were unchanged. The increase in ionic strength with CaCl2 retarded κ-casein hydrolysis, with an enhanced effect compared to NaCl study. As the CMP at the rennet clotting time was strongly reduced by CaCl2 addition, the effect of CaCl2 increase is typically biphasic, with an initial decrease and a following increase in rennet clotting times. From a technological point of view, reduction of ionic strength of milk led to reduced rennet clotting time. Results are discussed in terms of physicochemical modifications of milk micelles and the effect of ionic strength on enzymic reaction.