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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-, α S2 , and α S1 -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-, α S2 , and α S1 -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., (© 2023, The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Casein micelles extracted from milk are 100-400 nm-sized particles, made up of proteins and calcium phosphates, with the latter as colloidal calcium phosphate particles (CCPs) in a size range of 2-4 nm embedded in a protein network. The hierarchical structures give rise to a variation of scattering intensity over many orders of magnitude, which can be measured by small-angle X-ray scattering and static light scattering. Expressions for the scattering intensity of a general simple model for composite particles with polydispersities of overall size and subparticles are derived, and some approximations are checked by generating scattering data for systems generated by Monte Carlo simulations. Based on the simpler models, a new model has been developed for casein micelles, where the scattering is expressed on an absolute scale and where the concentrations of, respectively, protein and CCPs are used as constraints, providing a consistent model. The CCPs are modelled as oblate ellipsoids and the protein as star structures. Correlations between the substructures of CCPs and protein structures are taken into account in terms of partial structure factors. The overall structure as well as some heterogeneities at intermediate length scale are modelled as polydisperse spheres. The model fits the data very well on all length scales and demonstrates that both the scattering from CCPs and protein is important. Thus, the model provides a detailed description of the casein structure, which is consistent with the information available in the literature.

This research focused on understanding physical and chemical changes occurring to concentrated milk protein suspensions as a function of time. Skim milk (untreated and heat treated at 90°C for 10 min) was concentrated at 6 times the original volume using osmotic stressing, a noninvasive concentration method, maintaining the serum composition as close as possible to that of native milk. A protease inhibitor cocktail, with broad specificity for the inhibition of serine, cysteine, aspartic proteases, and aminopeptidases, was added in selected samples. Within 9 d of storage at 4°C, the apparent viscosity increased markedly for both unheated and heated concentrated milk, but not for those in the presence of protease inhibitors. However, only unheated milk showed a significant increase in the apparent diameter of the casein micelles. Matrix-assisted laser desorption-ionization time-of-flight mass spectrometry measurements indicated a significantly lower extent of proteolysis in heated than in unheated samples. The microstructure of the aggregates was observed using field emission scanning electron microscopy, and unheated samples clearly showed aggregation of casein micelles with storage time. In heated samples, aggregation was instead triggered by heat-induced protein-protein interactions., (Copyright © 2021 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

The aggregation of a mixed suspension of native and pre-acidified casein micelles was observed using rheology. Pre-acidified micelles were prepared by addition of glucono-delta-lactone, once pH of 6 or 5.5 was reached, the suspension was concentrated and added to untreated skim milk. Gelation was followed by addition of chymosin, for mixes before or after re-equilibration to the original milk pH. When pre-concentrated micelles were acidified, an earlier gelation point was shown, as well as a higher elastic modulus, compared to control suspensions. When skim milk containing pre-acidified micelles was re-equilibrated to the original pH no gelation was observed after addition of chymosin, in spite of very similar levels of diffusible calcium and phosphate concentration to those of the original milk. It was concluded that it may be possible to fine tune the rheological properties of the final chymosin induced gel network, by pretreating only a portion of the casein micelles, and modifying their colloidal calcium phosphate and the ratio of soluble to micellar casein., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

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.

The colloidal properties of the casein micelles play a major role in the structural properties of milk protein concentrates. Because of their great technological importance, the structural-functional relationships of casein micelles have been studied for decades in skim milk; however, novel ingredients are now available with higher protein concentrations and varying in composition. The colloidal behavior of caseins in these systems is not fully understood. Concentrates prepared with membrane technologies, and subjected to pre- or post-modifications that affect their technological functionality, have become increasingly widespread. This has created large opportunities for innovation and generation of value-added ingredients. The manner in which caseins interact with themselves and the other components in these concentrates will affect the structure of the final matrix. During concentration by filtration, the interparticle distance between the micelles decreases considerably, increasing their spatial correlation and decreasing their diffusivity. Rearrangements occur due to changes in environmental conditions, such as ionic composition, osmotic stress, shear, pH, or heating temperature. This will have important consequences on bulk viscosity of the concentrates, as well as on the mode of formation of structures' building blocks. This paper aims at highlighting some of the important factors affecting the colloidal structure of casein micelles, their destabilization and network formation, namely, processing history, volume fraction, composition of the serum phase, and ionic equilibrium. Understanding these factors will lead to a better quality control of dairy ingredients and to the development of a new generation of ingredients with targeted functionality., (The Authors. Published by FASS Inc. and Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).)

Background: To understand the interactions between carriers and functional ingredients is crucial when designing delivery systems, to maximize bioefficacy and functionality. In this study, two different protein matrices were evaluated as means to protect the extract isolated from marjoram leaves (Origanum majorana), casein micelles from fresh skim milk and soy protein isolate (SPI)., Results: Marjoram extract was obtained from pressurization of ethanol and water solvent. Protein dispersions of casein and SPI (5 g L -1 each) with or without marjoram extract (0.1-3 mg mL -1 ) were prepared and homogenized. The physicochemical characterization of charge and entrapment efficiency were conducted. The results demonstrated that entrapment efficiency was highly dependent on the carrier itself where SPI formulations showed 20% higher affinity when compared to casein micelles. To investigate the physiological behaviour of the marjoram-protein dispersions, human macrophages were employed. A non-specific inflammatory response of macrophages stimulated with bacterial lipopolysaccharide was measured for TNF-α, IL-1β and IL-6 cytokine secretion., Conclusion: Casein and SPI protein formulations warranted high bioefficacy of marjoram extract, showing their potential as safe carriers. © 2018 Society of Chemical Industry., (© 2018 Society of Chemical Industry.)

The objective of this work was to investigate the properties of casein micelles after pH adjustment and their re-equilibration to the original pH and serum composition. Re-equilibration was carried out by dialyzing against skim milk at 2 different temperatures (4 or 22 °C). Turbidity, the average radius of the casein micelles, and the composition of the soluble phase were measured at different pH values, ranging between 5.5 and 8. Acidification led to the solubilization of colloidal calcium phosphate and decrease of the average radius of the micelles. With re-equilibration, casein dissociation occurred. In milk with pH values greater than 6.0, the average radius was recovered after re-equilibration. At pH values greater than neutral, an increase of the radius of casein micelles and increased dissociation of the casein were found. After re-equilibration, the radius of micelles and soluble protein in the serum decreased. The results were not affected by the temperature of re-equilibration. The changes to the calcium phosphate equilibrium and the dissociation of the micelles will have important consequences to the functionality of casein micelles., (Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Numerous studies have demonstrated that tea catechins form complexes with milk proteins, especially caseins. Much less work has been conducted to understand the metabolic conversions of tea-milk complexes during gastro-duodenal digestion. The objective of this study was to determine the significance of this association on the digestibility of the milk proteins and on the bioaccessibility of the tea polyphenol epigallocatechin gallate (EGCG). An in vitro digestion model mimicking the gastric and duodenal phases of the human gastrointestinal tract was employed to follow the fate of the milk proteins during digestion and determine the bioefficacy of EGCG isolated or encapsulated with the caseins. The samples, before and after digestion, were tested using two parallel colonic epithelial cell lines, a normal line (4D/WT) and its cancerous transformed counterpart (D/v-src). EGCG caused a decrease in proliferation of cancer cells, while in normal cells, neither isolated nor encapsulated EGCG affected cell proliferation, at concentrations <0.15 mg ml(-1). At higher concentrations, both isolated and encapsulated produced similar decreases in proliferation. On the other hand, the bioefficacy on the cancer cell line showed some differences at lower concentrations. The results demonstrated that regardless of the extent of digestion of the nanoencapsulated EGCG, the bioefficacy of EGCG was not diminished, confirming that casein micelles are an appropriate delivery system for polyphenols.

Many studies have shown that tea catechins bind to milk proteins. This research focused on the association of tea polyphenols with casein micelles, and the consequences of the interactions on the renneting behaviour of skim milk. It was hypothesized that epigallocatechin-gallate (EGCG), the main catechin present in green tea, forms complexes with the casein micelles and that the association modifies the processing functionality of casein micelles. The binding of EGCG to casein micelles was quantified using HPLC. The formation of catechin-casein micelles complexes affected the rennet induced gelation of milk, and the effect was concentration dependent. Both the primary as well as the secondary stage of gelation were affected. These experiments clearly identify the need for a better understanding of the effect of tea polyphenols on the processing functionality of casein micelles, before milk products can be used as an appropriate platform for delivery of bioactive compounds., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Background: Dietary selenium has the potential to reduce growth of mammary tumors. Increasing the Se content of cows' milk proteins is a potentially effective means to increase Se intake in humans. We investigate the effects of selenized milk protein on human mammary tumor progression in immunodeficient BALB/c nude mice., Methods: Four isonitrogenous diets with selenium levels of 0.16, 0.51, 0.85 and 1.15 ppm were formulated by mixing low- and high-selenium milk casein isolates with a rodent premix. MCF-7 cells were inoculated into the mammary fat pad of female BALB/c nude mice implanted with slow-release 17 β-estradiol pellets. Mice with palpable tumors were randomly assigned to one of the four diets for 10 weeks, during which time weekly tumor caliper measurements were conducted. Individual growth curves were fit with the Gompertz equation. Apoptotic cells and Bcl-2, Bax, and Cyclin D1 protein levels in tumors were determined., Results: There was a linear decrease in mean tumor volume at 70 days with increasing Se intake (P < 0.05), where final tumor volume decreased 35% between 0.16 and 1.15 ppm Se. There was a linear decrease in mean predicted tumor volume at 56, 63 and 70 days, and the number of tumors with a final volume above 500 mm3, with increasing Se intake (P < 0.05). This tumor volume effect was associated with a decrease in the proportion of tumors with a maximum growth rate above 0.03 day-1. The predicted maximum volume of tumors (Vmax) and the number of tumors with a large Vmax, were not affected by Se-casein. Final tumor mass, Bcl-2, Bax, and Cyclin D1 protein levels in tumors were not significantly affected by Se-casein. There was a significantly higher number of apoptotic cells in high-Se tumors as compared to low-Se tumors., Conclusions: Taken together, these results suggest that turnover of cells in the tumor, but not its nutrient supply, were affected by dairy Se. We have shown that 1.1 ppm dietary Se from selenized casein can effectively reduce tumor progression in an MCF-7 xenograft breast cancer model. These results show promise for selenized milk protein as an effective supplement during chemotherapy.

The majority of the protein in cow's milk is contained in the particles known as casein micelles. This review describes the main structural features of these particles and the different models that have been used to define the interior structures. The reactions of the micelles during processing operations are described in terms of the structural models.

The effects of sodium caprate on the gelation of β-lactoglobulin B and a β-lactoglobulin B/β-casein mixture at ambient temperature were investigated using ultrasonic spectroscopy and rheology. A 12% β-lactoglobulin B solution gelled in the presence of 3.6% sodium caprate. Conversely, sodium caprate did not induce the formation of a gel when β-casein was in isolation, regardless of the protein concentration. Although a 6% β-lactoglobulin B/1.8% sodium caprate solution did not form a gel, a gel was formed when 6% β-casein was added to a mixture containing 6% β-lactoglobulin B and 3.6% sodium caprate. This gel showed comparable rheological properties to that of a gel containing 12% β-lactoglobulin B. The results clearly indicated that β-casein aids in the gelation of a β-lactoglobulin B/sodium caprate mixture, when the concentration of β-lactoglobulin B is insufficient to allow for gelation. It appears that β-casein self-aggregation is also inhibited. Therefore, it could be concluded that β-casein can be used as a texture modifier for β-lactoglobulin gelation induced by sodium caprate., (Copyright © 2011 Elsevier B.V. All rights reserved.)

In colloidal systems physical-chemical changes are often a function of volume fraction and sample dilutions are critical. While most methods to characterize colloidal particles either require dilution or some disruption, acoustic spectroscopy can be performed in situ, without dilution. Objective of this work was to determine the effects of concentration, heating and acidification on the acoustic and electroacoustic properties of casein micelles in skim milk. The ultrasonic attenuation of skim milk increased with concentration of milk and frequency, and the average size of the colloidal particles calculated from the frequency dependence of attenuation was about 0.15 μm for both unheated and heated milk. When milk was concentrated by ultrafiltration, at 3× and 4× concentration (based on volume reduction), the calculated size deviated from that derived in undiluted or mildly concentrated milk, most likely because of increased particle-particle interactions. Electroacoustic measurements revealed a constant dynamic mobility of the particles in undiluted and concentrated milk, while lower mobilities were observed for milk diluted in permeate. The ζ-potential measured was significantly higher than the values measured using dynamic light scattering, with a value of -45.8 mV for casein micelles in unheated milk. With acidification, the ζ-potential decreased monotonically. Heating profoundly affected the change in charge with pH of the micelles, and it was concluded that the interaction of casein micelles with the whey proteins increased the surface charge of the casein micelles., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Aggregated particles of casein micelles and kappa-carrageenan were produced as a dried milk ingredient, then reconstituted and subjected to a heat treatment of 70 degrees C for 10 min. The reconstituted aggregates were found to be unstable when heated. Light scattering results showed that the aggregates dissociated partially into casein micelles. It was hypothesized that the removal of ions during ultrafiltration before spray-drying to produce the powdered ingredient significantly decreased stability upon reconstitution and heat treatment. When ions, either from whey permeate or calcium addition, were added to reconstituted aggregates, stability was greatly enhanced and the aggregates remained intact when subjected to heat. The effect of heat treatment on aggregates freshly produced with skim milk powder and kappa-carrageenan was also studied. These aggregates were found to be stable during heating due to the unchanged ionic environment. Therefore, incorporation of powdered aggregates of casein micelles and kappa-carrageenan into products would require the addition of whey permeate or calcium after reconstitution for stability during subsequent heating.

The rennet-induced aggregation of skim milk recombined with whey protein-stabilized emulsion droplets was studied using diffusing wave spectroscopy (DSW) and small deformation rheology. The effect of different volume fractions of casein micelles and fat globules was investigated by observing changes in turbidity (1/l*), apparent radius, elastic modulus and mean square displacement (MSD), in addition to confocal imaging of the gels. Skim milk containing different concentration of casein micelles showed comparable light-scattering profiles; a higher volume fraction of caseins led to the development of more elastic gels. By following the development of 1/l* in recombined milks, it was possible to describe the behaviour of the fat globules during the initial stages of rennet coagulation. Increasing the volume fraction of fat globules showed a significant increase in gel elasticity, caused by flocculation of the oil droplets. The presence of flocculated oil globules within the gel structure was confirmed by confocal microscopy observations. Moreover, a lower degree of kappa-casein hydrolysis was needed to initiate casein micelles aggregation in milk containing whey protein-stabilized oil droplets compared to skim milk. This study for the first time clearly describes the impact of a mixture of casein micelles and whey protein-stabilized fat globules on the pre-gelation stages of rennet coagulation, and further highlights the importance of the flocculation state of the emulsion droplets in affecting the structure formation of the gel.

Understanding the underlying processes that govern interparticle interactions in colloidal systems is fundamental to predicting changes in their bulk properties. In this paper we discuss the colloidal behavior of casein micelles and protein-stabilized fat globules individually and in a mixture. The colloidal interactions were observed by transmission diffusing wave spectroscopy. The turbidity parameter, l*, and the diffusion coefficients of the samples studied were measured experimentally and compared to the theoretically calculated parameters for a hard-sphere system. The light scattering properties of casein micelles (volume fraction phi = 0.1-0.2) dispersed in milk permeate showed no deviation from the theoretically predicted model. Whey protein isolate (WPI)-stabilized emulsions (phi = 0.025-0.1) prepared either in milk permeate or in 5 mM imidazole buffer at pH 6.8 showed a behavior identical to that of the hard-sphere model. Similarly to the WPI-stabilized fat globules, the sodium caseinate (NaCas)-stabilized emulsions (phi = 0.025-0.1) prepared in milk permeate also showed resemblance to the theory. In contrast, NaCas-stabilized emulsions prepared in 5 mM imidazole buffer exhibited some discrepancy from the theoretically calculated parameters. The deviation from theory is attributed to the enhanced steric stabilization properties of these droplets in a low ionic strength environment. When recombined milks made from concentrated milk and WPI- and NaCas-stabilized droplets prepared in permeate (phi = 0.125-0.2) were studied, the experimental data showed a significant deviation from the theoretical behavior of a hard-sphere model due to mixing of two different species.

A non-invasive technique, diffusing wave spectroscopy (DWS), and traditional dynamic light scattering (DLS) were used to study the interactions of high methoxyl pectin (HMP) with sodium caseinate-stabilized emulsion droplets. At pH 6.8, the droplet size measured by DLS did not change as a function of HMP concentration (up to 0.3%). However, the droplet diameter measured by DWS kept relatively constant up to 0.07% HMP after which it showed drastic increases. The turbidity parameter 1/l* decreased with HMP concentration and levelled off at 0.07% HMP, indicating that the system underwent reorganization and reached equilibrium at 0.07% HMP. During acidification at pH 5.4, right before the pH of aggregation of control emulsions, all emulsions containing 0.05-0.2% HMP showed an increase of 1/l*. This increase indicated the interaction of HMP with sodium caseinate at the interface. Emulsions containing 0.05 and 0.1% HMP also showed destabilization, and the pH of destabilization depended on the concentration of HMP. Sufficient amounts of HMP (0.2%) stabilized the caseinate-coated oil droplets, and the mean square displacement slope was close to 1 throughout, indicating free diffusion of emulsion droplets.

The interactions of high-methoxyl pectin (HMP) and soybean-soluble polysaccharide (SSPS) with sodium caseinate-stabilized emulsions were investigated using a multitechnique approach, including dynamic light scattering (DLS), electrophoretic mobility measurements, transmission diffusing wave spectroscopy (DWS), and ultrasonic spectroscopy (US). At pH 6.8, both polysaccharides are negatively charged and did not adsorb onto caseinate-coated droplets due to electrostatic repulsion; however, SSPS showed a different behavior compared to HMP in the turbidity parameter 1/l* and sound attenuation parameters measured by DWS and US, respectively. The present study brought the first evidence of the stabilization effect of SSPS in acidified sodium caseinate-emulsions. While destabilization occurred at low polysaccharide concentrations, probably via bridging flocculation, acid-induced aggregation of the oil droplet was completely prevented by 0.2% SSPS or HMP. However, the interaction behavior of SSPS during acidification was different from that of HMP. This was demonstrated by the different development of the parameter 1/l*, droplet sizes, sound attenuation, and velocity.

The objective of this research was to understand whether addition of soy protein to milk protein affects the properties of acid-induced casein gels. Different samples were prepared by suspending casein micelles pellets in milk serum containing soy proteins or whey proteins as well as mixtures of the two proteins. Glucono-delta-lactone was added, and the changes in apparent size (in diluted systems) as well as the viscoelastic properties of the mixtures were measured. Size exclusion chromatography was also carried out to characterize the soluble phase of the various mixtures before and after heating. Soy protein affected the gelation of the mixtures; however, not to the same extent as whey proteins, which dominated formation of the network in soy-whey-casein systems. It was concluded that, up to a critical ratio of soy/whey proteins, soy proteins can be incorporated in the mix without a significant change in structure of the casein gels.

Pectins are negatively charged polysaccharides employed as stabilizers in acidified milk dispersions, where caseins aggregate because of the low pH and serum separation needs to be prevented. The objective of this research was to study the effect of charge on the stabilizing functionality of the polysaccharide in acid milk drinks. Unstandardized pectins with various charges (as degree of esterification, DE) as well as soybean soluble polysaccharide (SSPS) were tested for their stabilizing behavior as a function of pH and concentration. Skim milk was acidified by glucono-delta-lactone and then homogenized in the presence of polysaccharide at different pH values (in the range from 4.2 to 3.0). Measurements of particle size distribution demonstrated that pectins with a DE of 71.4, 68.6, and 67.4 stabilized milk at pH > 4.0. Pectins with a lower DE (63.9%) needed a higher concentration (0.4%) at the same pH to show a monomodal distribution of particle sizes. Pectins with lower DE (<50%) did not stabilize the dispersions. Although this difference in behavior was attributed mainly to the pectin charge, the efficiency in stabilizing the casein dispersion decreased with decreasing pectin size. For example, the high methoxyl pectin (HMP) with 63.9 DE was smaller in size than the HMPs with a higher charge. Pectins showed a pH-dependent stabilization effect, as at pH < 4.0 the dispersions contained aggregates. When SSPS was used to stabilize acid milk, at pH < 4.0, it showed a better stabilization behavior than HMP. When SSPS and pectin were used in combination, the particle size distribution of the acid milk dispersion was pH-dependent, and results were similar to those for samples containing pectin alone. This suggested that in the mixture, pectin dominated the behavior over SSPS, even when an excess of SSPS was added to the dispersions before homogenization.

Polysaccharides are widely used in the food industry to modify the stability of protein-based drinks. However, an in depth knowledge of the interactions occurring in the system is still lacking. In this study, the interactions between sodium caseinate and high methoxyl pectin under acidification conditions were studied nondestructively and without dilution using transmission diffusing wave spectroscopy. Oil-in-water emulsions were prepared with 10% soybean oil and 0.5% sodium caseinate. Various concentrations of pectin (ranging from 0 to 0.2%) were added, and the emulsions were acidified with glucono-delta-lactone. With acidification, a "sol-gel" transition occurred and emulsions containing pectin were more stable at lower pH than those without pectin. Furthermore, the sol-gel transition of the mixtures was more sudden for control emulsions without pectin. While in control samples the final solidlike emulsion after gelation tended to be more inhomogeneous and more dissimilar to the starting emulsion, emulsions with pectin in solution gelled later under acidification. With a sufficient amount of pectin, the emulsions showed no aggregation and the destabilization pH varied depending on the amount of pectin present in the emulsions. At intermediate pH values (pH > 5.5), the emulsions displayed a decrease in particle size, more pronounced in samples containing pectin. The results collected using light scattering in concentrated systems, 10% (v/v) in our case, suggested that pectin stabilizes the emulsion oil droplets forming a network of oil droplets loosely connected by strands of pectin.

Membrane filtration, especially in combination with diafiltration, can affect the colloidal structure of casein micelles in milk and concentrated milks. The partial dissociation of casein proteins from the casein micelles into the serum phase has been shown to depend on diafiltration conditions. This dissociation can affect the technological functionality of the milk concentrates. The present study aimed at determining the contribution of the gel layer deposited onto the membrane during filtration in the colloidal equilibrium between soluble and micellar caseins. Skimmed milk was concentrated by microfiltration combined with diafiltration using a cross-flow spiral-wound membrane at two transmembrane pressure (TMP) levels, causing differences in the extent of the gel layer formed. Non-sedimentable casein aggregates were formed to a greater extent at a low TMP compared to a high operating TMP. This difference was attributed to the greater compression of the deposit layer during filtration at a high TMP. This study contributes new knowledge to the understanding of how to modulate the functionality of milk concentrates through the control of processing conditions. [ABSTRACT FROM AUTHOR]

The objective of this work was to evaluate the impact of changes during membrane filtration on the heat stability of milk protein concentrates. Dairy protein concentrates have been widely employed in high protein drinks formulations and their stability to heat treatment is critical to ensure quality of the final product. Pasteurized milk was concentrated three-fold by membrane filtration, and the ionic composition was modified by addition of water or permeate from filtration (diafiltration). Diafiltration with water did not affect the apparent diameter of the casein micelles, but had a positive effect on heat coagulation time (HCT), which was significantly longer (50 min), compared to the non diafiltered concentrates (about 30 min). UHT treatments increased the particle size of the casein micelles, as well as the turbidity of retentates. Differences between samples with and without diafiltration were confirmed throughout further analysis of the protein composition of the unsedimentable fraction, highlighting the importance of soluble protein composition on the processing functionality of milk concentrates. [ABSTRACT FROM AUTHOR]

The physico-chemical properties of casein micelles in milk adjusted to pH 6 and 5.6 were compared with native pH milk, as a function of concentration. Skim milk was acidified and concentrated using osmotic stressing. The apparent radius of the casein micelles, measured after redilution in their corresponding permeate, decreased with decreasing pH, but showed no differences with concentration. Furthermore, the casein micelles showed free diffusive behaviour at all three pH values; only suspensions at pH 5.6 showed hindered diffusion at 4× concentration. At protein concentrations >6% the extent of caseins dissociation increased in the acidified milk samples. The relative viscosity of the concentrated suspensions (up to 4×) was modeled according to the behaviour of colloidal hard spheres, taking into account hydrodynamic interactions. The voluminosity of the casein micelles was estimated to be 3.5 and 3.1 mg L −1 for pH 6 and 5.6, respectively. The rheological behaviour of casein suspensions at pH 6.0 could still be described as that of colloidal hard spheres. On the other hand, for casein suspensions adjusted at pH 5.6, very low maximum packing volumes were obtained using a hard sphere model. It was suggested that at this pH further rearrangements and aggregation need to occur during concentration >3×, because of dramatic changes in charge, dissociation of calcium and phosphate, a significant increase in soluble casein and the decrease in steric repulsion. Although much is known about the effect of pH on casein micelles in skim milk, very little data is available on the effect of concentration. This research contributed to a better understanding of the colloidal properties of casein suspensions as a function of pH and concentration, allowing better use of modified casein concentrates as functional ingredients. [ABSTRACT FROM AUTHOR]

The objective of this work was to investigate the properties of casein micelles after pH adjustment and their re-equilibration to the original pH and serum composition. Re-equilibration was carried out by dialyzing against skim milk at 2 different temperatures (4 or 22°C). Turbidity, the average radius of the casein micelles, and the composition of the soluble phase were measured at different pH values, ranging between 5.5 and 8. Acidification led to the solubilization of colloidal calcium phosphate and decrease of the average radius of the micelles. With re-equilibration, casein dissociation occurred. In milk with pH values greater than 6.0, the average radius was recovered after re-equilibration. At pH values greater than neutral, an increase of the radius of casein micelles and increased dissociation of the casein were found. After re-equilibration, the radius of micelles and soluble protein in the serum decreased. The results were not affected by the temperature of re-equilibration. The changes to the calcium phosphate equilibrium and the dissociation of the micelles will have important consequences to the functionality of casein micelles. [ABSTRACT FROM AUTHOR]

The addition of NaCl to milk is known to alter the mineral equilibrium and the structure of casein micelles. The objective of this study was to better understand the light scattering properties and viscosity of milk as a function of NaCl addition (0-500 mmol.L), for fresh skim milk and 2× concentrated milk protein. Suspensions (2×) were prepared using osmotic stressing. NaCl was added by dialyzing milk with milk serum (permeate) for 18 h. The presence of NaCl decreased the pH of milk and the zeta potential of casein micelles. When measured under diluted conditions, using dynamic light scattering, the average radius of the casein micelles showed a small increase, from 84 to 88 nm. Total calcium and phosphate in concentrated milk protein suspensions decreased with NaCl concentration, with a corresponding increase of the permeable calcium and phosphate, and of non-sedimentable caseins. In both untreated and concentrated milk protein samples, the viscosity increased with NaCl concentration. The characteristic decay time (τ) and turbidity measured under non-diluted conditions using diffusing wave spectroscopy showed a gradual decrease with NaCl addition. This work demonstrated that the changes to milk with addition of NaCl are mainly caused by the disruption of the internal structure of the casein micelles and changes in the composition of the serum phase, affecting the viscosity of the continuous phase as well as the refractive index contrast. [ABSTRACT FROM AUTHOR]

Cooling of milk causes the release of β-casein from the casein micelles. With subsequent washing steps, using milk UF permeate, it was possible to remove up to 60% of its initial concentration and study the interactions of β-casein depleted micelles with two model molecules, resveratrol and curcumin. With the release of β-casein there was an increase in the apparent diameter of the casein micelles, with no changes in the amount of colloidal calcium phosphate, or to the integrity of the protein particles when observed by Cryo-TEM. In addition, there was an increase in the affinity for both curcumin, a hydrophobic molecule and resveratrol, a hydrophilic molecule. Thus, with the removal of the β-casein, there appeared to be an overall loosening of the micellar structure, giving an increase in the accessibility to the inner hydrophobic regions. [ABSTRACT FROM AUTHOR]

Milk protein concentrates (MPC) are widespread food ingredients, due to their high protein-to-solids ratio, nutritional value, and technological properties, which can be tailored to various end uses. Interest has been given to modifying such ingredients through enzymatic cross-linking by microbial transglutaminase. However, no systematic studies on the cross-linking kinetics of casein micelles at different concentration factors are currently available, although the enzyme might act in a considerably different fashion at decreased inter-particle distances. In this study, cross-linking of casein micelles was studied in a 4x MPC obtained by ultrafiltration, and compared to that of the original skim milk. The cross-linking kinetics were evaluated by following the degree of casein polymerisation using size exclusion chromatography. The extent of polymerisation for both skim milk and 4x MPC could be scaled to a master curve by normalising the incubation time to concentration factor and enzyme level, indicating similar cross-linking kinetics. Likewise, the cross-linking of individual casein types followed the same trend regardless of treatment. However, the casein micelle size increased with cross-linking at 4x but decreased at 1x concentration, suggesting enhanced cross-linking on the surface of the casein micelles in MPC suspensions. These results are relevant for the design of novel milk protein ingredients. [Display omitted] • Casein micelles were cross-linked by transglutaminase at 1x and 4x concentration. • Cross-linking kinetics were followed using comparable enzyme:protein ratios. • Polymerisation kinetics are similar for 1x and 4x concentration. • Cross-linking increased casein micelles' size in concentrates compared to skim milk. [ABSTRACT FROM AUTHOR]

The colloidal stability of casein micelles suspensions prepared using ultrafiltration (UF) and microfiltration (MF) was studied by testing acid- and rennet-induced destabilization. Skim milk and 4× (based on volume reduction) concentrates were obtained by processing under similar conditions, at temperatures below 10°C. Concentrates were subjected to different levels of diafiltration (DF), resulting in samples with comparable casein volume fractions but different amounts of proteins and ions in the serum phase. The novelty of the work is the systematic comparison of MF and UF concentrates of similar history. More specifically, concentrates similar in ionic composition but with or without serum proteins were compared, to evaluate whether whey proteins and β-casein depletion from the micelles will play a role in the processing properties, or whether these are affected solely by the ionic balance. Microfiltered micelles' apparent diameter decreased by about 50 nm during the specific hydrolysis of κ-casein by chymosin, whereas those in skim milk control showed a decrease of about half that size. All concentrates subjected to extensive DF showed smaller hydrodynamic diameters, with reductions of ∼18 and 13 nm for MF and UF, respectively. Highly diafiltered UF retentates showed a delayed onset of rennet-induced gelation, due to low colloidal calcium, compared with other samples. Low-diafiltered samples showed weak storage modulus (∼1 Pa) after 60 min of onset of gelation. In addition, onset pH increased with diafiltration to ∼5.8 for UF and ∼6 for MF in high-diafiltered samples. These results clearly demonstrated that the functional properties of casein micelles change during membrane concentration, and this cannot be solely attributed to changes in ionic equilibrium. [ABSTRACT FROM AUTHOR]

Abstract: Protein–polysaccharide interactions are of great importance in the design of dairy formulations, as they play a key role in the formation of structure and texture in dairy products. With a detailed understanding of the factors affecting the interactions, the ability of charged polysaccharides to associate with the milk proteins is continuously exploited to create functional complexes, novel ingredients and delivery systems. In addition, formulations containing non-interacting polysaccharides also need to be carefully controlled, as these biopolymers may give rise to segregative phase separation, with important consequences to the stability and quality of the final matrix. As casein micelles play a major role in imparting structure to dairy products, emphasis in this review will be given to the molecular details of the interactions between polysaccharides with these protein particles. Some of the most researched polysaccharides will be highlighted in this context, and the progress made in understanding their role during structure formation of dairy matrices will be discussed. The opportunity of creating novel microstructures provided by association or/and incompatibility of milk proteins and different polysaccharides will be assessed. [Copyright &y& Elsevier]

Abstract: Pasteurized skim milk was subjected to membrane filtration using a molecular weight cut-off of 80kDa and a plate and frame pilot scale system at temperatures below 10°C. Via this process, transmission of whey proteins and β-casein through the membrane was achieved. The milk was concentrated to two times (based on volume reduction), and whey protein-free permeate was added to return to the original volume fraction of casein micelles in milk. This diafiltration process was carried out four times, and the retentate obtained was nearly free of whey proteins and with approximately 20% of β-casein removed. The same membrane filtration was also carried out at 25°C to achieve transmission of whey protein but not of β-casein, and to obtain whey protein-depleted milk without depletion of β-casein. The gelling behaviour of these samples, reconstituted to the original casein volume fraction, was examined using rheology and diffusing wave spectroscopy. When compared to the original skim milk it was found that there were no statistically significant differences in gelation behaviour during acidification, but differences were noted in gelation time and final stiffness modulus for samples undergoing renneting. These differences were attributed mostly to the changes in ionic composition, as when the serum composition of the retentates was re-equilibrated against the original skim milk by dialysis; the gelation behaviour of the samples was comparable to that of skim milk. The results clearly indicate the importance of the milk''s overall ionic balance in the early stages of aggregation of rennet-induced gelation of milk. [Copyright &y& Elsevier]

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Aggregated particles of casein micelles and κ-carrageenan were produced as a dried milk ingredient, then reconstituted and subjected to a heat treatment of 70 °C for 10 min. The reconstituted aggregates were found to be unstable when heated. Light scattering results showed that the aggregates dissociated partially into casein micelles. It was hypothesized that the removal of ions during ultrafiltration before spray-drying to produce the powdered ingredient significantly decreased stability upon reconstitution and heat treatment. When ions, either from whey permeate or calcium addition, were added to reconstituted aggregates, stability was greatly enhanced and the aggregates remained intact when subjected to heat. The effect of heat treatment on aggregates freshly produced with skim milk powder and κ-carrageenan was also studied. These aggregates were found to be stable during heating due to the unchanged ionic environment. Therefore, incorporation of powdered aggregates of casein micelles and κ-carrageenan into products would require the addition of whey permeate or calcium after reconstitution for stability during subsequent heating. Practical Application: Casein micelles and κ-carrageenan readily form aggregates when cooled together in solution with shearing. These aggregates have the potential to add functionality to skim milk powder; however, they have to be stable to subsequent applications. This research demonstrates that careful control of ionic balance is required to dry the aggregates, reconstitute them, and heat the reconstituted solution to pasteurization temperatures, but when controlled the intact aggregates can be maintained. [ABSTRACT FROM AUTHOR]

This review describes some of the effects of heat on several food systems containing milk proteins. Much of the recent research effort has been directed towards understanding the extensive effects of the thermal denaturation of the whey proteins (WPs) on the functional properties of heated systems (WP solutions and gels, emulsions and milks) that contain these proteins. Caseins play a more passive part during heating, apart from their specific interaction with denaturing WPs during the heat treatment of milk. [Copyright &y& Elsevier]

The impact of membrane concentration with different milieu exchange conditions on the structural changes of casein micelles was investigated by studying their dissociation as well as their internal structure using small-angle X-ray scattering (SAXS). Micellar casein concentrates were prepared using ultrafiltration of milk (< 10 °C) followed by microfiltration and diafiltration (DF) of the resulting 2 × milk protein concentrate (< 10 °C) using different DF media (demineralised water vs. ultrafiltration permeate) and DF modes (continuous at 2 × concentration vs. discontinuous at 4 × concentration). All concentrates showed a higher degree of casein micelle dissociation when DF was conducted with water compared to ultrafiltration permeate. SAXS data were evaluated using a model describing the scattering on an absolute scale, and using protein and mineral concentrations as constraints. The results showed changes in the internal structure of the casein micelles at intermediate length scales for the concentrates prepared using water as DF medium, linked to the partial dissociation of caseins from the colloidal phase to the dispersion phase. In contrast, the casein micelles, in spite of their concentrated state, retained their structural integrity better when DF was conducted with ultrafiltration permeate. These results clearly demonstrate that maintaining the ionic equilibrium during membrane filtration of skim milk is critical to preserve the original structure of the protein particles in skim milk. This has important consequences in developing processes aiming at tailoring the technological properties of micellar casein concentrates. [Display omitted] • Concentrated casein micelles were studied under different diafiltration treatments. • Casein micelle structure was assessed by fitting small-angle X-ray scattering data. • Diafiltration with water caused dissociation of caseins into the serum phase. • Diafiltration with water caused changes in the internal structure of the micelles. • Diafiltration with permeate preserved the structure of the casein micelles. [ABSTRACT FROM AUTHOR]

There is an increased interest in understanding the potential of modifying casein micelles composition, or even creating them de novo. In this study, soluble casein fractions were created by dissociation from native casein micelles, and their properties were studied in isolation, and after their addition to native casein micelles. The structure of these enriched casein micelles was investigated by small-angle X-ray scattering (SAXS) to evaluate if the enrichment caused changes in their internal structure. Acidification of native casein micelles from pH 6.8 to 5.6 caused colloidal calcium phosphate dissociation, and SAXS data showed a decrease in intensity at q = 0.08 Å−1, where q is the modulus of the scattering vector. Additionally, the acidification of native casein micelles (to pH 6.0) and incubation at 4 °C caused a decrease in scattering intensity at the intermediate q region (0.01–0.02 Å−1), signifying a loss of heterogeneity among the intermediate structure elements in the casein micelle. This change was caused by micellar dissolution of calcium phosphate and the release of β-casein at 4 °C. After the resuspension of casein micelles with a β-casein enriched serum, the absence of soluble β-casein suggested that most β-casein was re-associated in the colloidal fraction. These samples showed no changes in the structural features probed by SAXS. The work brings new evidence that, while a small extent of dissociation may affect the structure of the casein micelles, the enrichment with β-casein results in similar structures as those of the native micelle. [Display omitted] • CaseIn micelle structure is affected by β-casein dissociation, studied by SAXS. • CaseIn micelle internal structure becomes more homogeneous with acidification. • CaseIn micelle internal structure becomes more homogeneous with β-cas dissociation. • CaseIn micelle structure is not affected by β-casein enrichment. [ABSTRACT FROM AUTHOR]

Abstract: The objective of this study was to determine the effect of concentration of milk by ultrafiltration on the development of the gel structure induced by a combination of acidification (with mesophilic cultures) and rennet. As the initial aggregation was driven by rennet, all samples showed a similar initial gelation point and differences were observed after, during further acidification. All samples reached a maximum elastic modulus (G′) value, and then a breakdown of the structure occurred at a pH ranging from 6 to 5.6, depending on protein concentration. At pH ∼5.0 a maximum of tan δ (where δ is the phase angle) was observed, and the pH maximum depended on the concentration level. These differences were due to different rates of calcium release. This study clearly describes the molecular details occurring during mixed coagulation and allows for a better understanding of the processes occurring during making of quark-type cheeses. [Copyright &y& Elsevier]

Abstract: The changes in processing functionality of concentrated milk are caused by a number of factors, amongst the most important, the ionic equilibrium and the increase in the interactions between the casein micelles because of their increased volume fraction. The objective of this work was to characterize the physico-chemical properties of casein micelles as a function of their volume fraction, by using osmotic stressing as a non-invasive method to obtain concentrated milk, in the attempt to preserve the ionic balance during concentration. Osmotic concentration was carried out for 18 h at 4 °C, using different concentrations of polyethylene glycol dissolved in permeate as the stressing polymer. The viscosity of the concentrated milk could be predicted using established rheological models, when the changes occurring to the viscosity of the serum phase were taken into account. Both Eilers and Mendoza equations predicted a maximum packing volume fraction of 0.8 for the casein micelles. After concentration up to 20% protein, the casein micelles did not show a change in their size upon redilution. Light scattering measurements carried out using diffusing wave spectroscopy without dilution suggested that casein micelles behave as hard spheres with the characteristic of free diffusing Brownian particles up to a volume fraction of 0.3, and restricted motion at higher concentrations. Results of total and soluble calcium suggested release of colloidal calcium phosphate from the micelles at volume fractions >0.35. This research brings new insights on the changes occurring in skim milk during concentration. [Copyright &y& Elsevier]

In this study, milk protein isolate (MPI) and micellar casein concentrate (MCC) were obtained with ultrafiltration (UF) and microfiltration (MF), respectively. The objective of this work was to evaluate the effect of diafiltration (DF), the addition of water during filtration, on the colloidal dissociation of casein micelles. Two levels of DF were applied after an initial concentration step to 2x the volume fraction, at a total protein concentration of about 7% (w/v). Under these conditions, while permeation of protein was still measured in the permeates, calcium ion transmission reached a plateau. The molecular details of the serum phases were studied throughout the filtration process. A comparison of serum phases with and without whey protein (UF vs MF) was carried out by examining the two processes in parallel, applying similar filtration conditions. Final 4x concentrates (based on initial volume of milk) were also compared. Viscosity was higher for UF than the corresponding MF concentrates, is spite of the higher level of colloidal protein present in the MF concentrates, which would suggest a change in the hydration or the vol/wt ratio (voluminosity) of casein micelles in MCC retentates. Centrifugal supernatants contained casein proteins both in MPC and MCC retentates. There were no statistically significant changes in the apparent hydrodynamic diameter of the casein micelles when measured by dynamic light scattering, in the original milk serum. A two dimensional chromatography was carried out on the soluble aggregates to determine differences in the amount of individual caseins. The aggregates showed only traces of whey proteins. The composition of the final concentrates, albeit similar in total proteins, showed profound differences in ionic strength and soluble proteins, suggesting that these parameters may be used as an indication of ingredient quality and functionality. [Display omitted] • Diafiltration causes dissociation of the casein micelle. • More glycosylated к-casein releases compared to non-glycosylated. • Differences between ultrafiltration and microfiltration retentates show the importance of the serum phase. • In spite of the extensive diafiltration, colloidal calcium phosphate reaches a plateau. [ABSTRACT FROM AUTHOR]

Abstract: 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 1mM 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 αs-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. [Copyright &y& Elsevier]