Interaction of a cationic acrylate polymer with caseins: biphasic effect of Eudragit E100 on the stability of casein micelles

When whole or skim milk was incubated with the cationic acrylate polymer Eudragit E100, a biphasic effect on the stability of casein micelles was observed. A precipitation phase was observed at low polymer/casein ratios. Strikingly, a solubilization phase of the aggregates was observed when the ratios of polymer/casein were increased. Purified alpha(s)-, beta-, and kappa-caseins or dephosphorylated caseins were equally precipitated and resolubilized by the cationic polymer, indicating no special selectivity for a particular protein or phosphate residue for these events. An increase in the size of the aggregates as the optimum precipitating amount of Eudragit E100 was reached suggests a crossbridging of the micelles by the polymer. The inhibition of the precipitation phase by high ionic strength indicates that electrostatic interactions play a critical role in complex formation. Furthermore, a dramatic reduction in size of the protein colloidal particles upon solubilization of the aggregates was observed by dynamic light scattering, indicating a dissociation of the micellar structure. Taken together, the results indicate that at low concentration Eudragit E100 may act as a precipitant of casein micelles, mainly by ionic interaction and at high concentration as an amphipathic agent, solubilizing casein micelles with a disruption of their internal structure.     

Acid gelation properties of heated skim milk as a result of enzymatically induced changes in the micelle/serum distribution of the whey protein/kappa-casein aggregates

Changes in the acid gelation properties of skim milk as a result of variations in the micelle/serum distribution of the heat-induced whey protein/kappa-casein aggregates, induced by the combination of heat treatment and limited renneting, were investigated. No dramatic change in the zeta potential or the isoelectric point of the casein micelles was suggested, whether the aggregates were all attached to the casein micelle or not. Fluorescence intensity measurement using 8-anilino-1-naphthalenesulfonic acid (ANS) showed that the heat-induced aggregates were highly hydrophobic. Dynamic oscillation viscosimetry showed that acid gelation using glucono-delta-lactone (GDL) started at a higher pH value in prerenneted milk. However, no change in the gelation profile of skim milk could be related to the proportion of aggregates bound to the surface of the casein micelles. The results support the idea of an early interaction between the serum aggregates and the casein micelles on acidification.     

Aggregation of rennet-altered casein micelles at low temperatures

The rennet-induced coagulation of bovine milk at 10 degrees C was investigated. The rate of change of absorbance at 600 nm was higher in milk renneted at 30 degrees C than that at 10 degrees C. The amount of casein sedimented on centrifuging skim milk at 5000g for 1 h at 10 degrees C increased with time after renneting. The viscosity of milk at 10 degrees C at low shear rates did not change significantly until 10 h after rennet addition, but it increased markedly after 20 h. Smaller particles in milk at 10 degrees C disappeared slowly over 36 h after rennet addition and aggregated into larger particles. These results suggested that casein micelles in milk aggregate at low temperatures. Reasons for the slow aggregation of milk renneted at 10 degrees C were investigated by inhibiting chymosin activity by pepstatin A. It is likely that beta-casein, or its hydrolysis, plays a role in aggregation of rennet-altered casein micelles at low temperatures.     

Reversible precipitation of casein micelles with a cationic hydroxyethylcellulose

The cationic hydroxyethylcellulose Polyquaternium 10 (PQ10) was found to produce a dose-dependent destabilization of casein micelles from whole or skim milk without affecting the stability of most of the whey proteins. The anionic phosphate residues on caseins were not determinant in the observed interaction since the destabilization was also observed with dephosphorylated caseins to the same extent. However, the precipitation process was completely inhibited by rising NaCl concentration, indicating an important role of electrostatic interactions. Furthermore, the addition of 150 mM NaCl solubilized preformed PQ10-casein complexes, rendering a stable casein suspension without a disruption of the internal micellar structure as determined by dynamic light scattering. This casein preparation was found to contain most of the Ca2+ and only 10% of the lactose originally present in milk and remained as a stable suspension for at least 4 months at 4 degrees C. The final concentration of PQ10 determined both the size of the casein-polymer aggregates and the amount of milkfat that coprecipitates. The presence of PQ10 in the aggregates did not inhibit the activity of rennet or gastrointestinal proteases and lipases, nor did it affect the growth of several fermentative bacteria. The cationic cellulose PQ10 may cause a reversible electrostatic precipitation of casein micelles without disrupting their internal structure. The reversibility of the interaction described opens the possibility of using this cationic polysaccharide to concentrate and resuspend casein micelles from whole or skim milk in the production of new fiber-enriched lactose-reduced calcium-caseinate dairy products.     

Effects of casein and fat content on water self-diffusion coefficients in casein systems: a pulsed field gradient nuclear magnetic resonance study

The water self-diffusion coefficients in casein matrixes were measured using a pulsed field gradient spin-echo nuclear magnetic resonance technique (PFG-SE NMR). The dependence of the water self-diffusion coefficient on the casein concentration and the aqueous phase composition is reported in both a rehydrated native phosphocaseinate dispersion and a concentrated casein retentate. A model has been proposed to explain the different behavior of the water self-diffusion coefficient in the two casein systems. This model demonstrates that the water self-diffusion cannot be simply explained by the water content only. So, taking into account the specific effect of each constituent of the aqueous dispersing phase, the water self-diffusion reduction induced by the casein micelle can be modeled. The effect of fat on the water self-diffusion coefficients was investigated. Anhydrous milk fat-reconstituted retentate samples were used in order to estimate the obstruction effect of fat globules in the modeling process. The dependence of the self-diffusion coefficient of water on the fat and casein content is reported. A general model included the effect of the aqueous phase composition, and the obstruction effects of casein micelles and fat globules were proposed. This model was validated for water self-diffusion coefficients in industrial fatty retentates.     

Heat-induced covalent complex between casein micelles and beta-lactoglobulin from goat's milk: identification of an involved disulfide bond.

Goat milk is characterized by a very low heat stability that could be attributed, in part, to the covalent interaction between whey proteins and casein micelles. However, the formation of such a complex in goat milk has never been evidenced. This study was designed to assess whether heat-induced covalent interaction occurs between purified casein micelles and beta-lactoglobulin. We used a multiple approach of ultracentrifugation of heated mixture, chromatographic fractionation of resuspended pellets, sequential enzyme digestion of disulfide-linked oligomers, and identification of disulfide-linked peptides by on-line liquid chromatography-electrospray ionization mass spectrometry (LC-ESI/MS), and tandem MS. We identified three different types of disulfide links: (1) expected intermolecular bridges between beta-Lg molecules; (2) disulfide bond involving two kappa-casein molecules; and (3) a disulfide bond between two peptides, one from beta-Lg and the other from kappa-casein. The involved sites in this last bond were Cys(160) of beta-Lg and Cys(88) of kappa-casein. Although the identified heterolinkage is possibly only one of several different types, the results of this study constitute the first direct evidence of the formation of a covalent complex between casein micelles and beta-lactoglobulin derived from goat milk.     

Role of kappa-casein in the association of denatured whey proteins with casein micelles in heated reconstituted skim milk

Reconstituted skim milk at pH from 6.5 to 7.1 was unheated, preheated (68 degrees C/20 min), or heated at 90 degrees C for 20-30 min. On preheating, the size of the casein micelles decreased by about 5-20 nm, with a greater effect at higher pH. The casein micelle size of the heated milk at pH 6.5 increased by about 30 nm when compared to that of the unheated or preheated milk. As the pH was increased before heating, the particle size gradually decreased so that, at pH 7.1, the size was markedly smaller than that for the unheated milk and slightly smaller than that for the preheated milk. High levels (about 85%) of denatured whey protein associated with the casein micelles at pH 6.5, and this level decreased as the pH increased so that, at pH 7.1, low levels (about 15%) were associated with the micelles. Low levels of alphaS-casein and beta-casein were found in the serum regardless of the heat treatment or the pH of the milk. At pH 6.5, low levels (about 10%) of kappa-casein were also found in the milk serum. In the unheated milk, the level of serum kappa-casein increased slightly with increasing pH; in the heated samples, the level of serum kappa-casein increased markedly and linearly with increasing pH so that, at pH 7.1, about 70% of the kappa-casein was in the serum phase. The results of this study indicate that the pH dependence of the levels of serum phase kappa-casein may be responsible for the change in distribution of the whey proteins between the colloidal and serum phases. This is the first report to demonstrate significant levels of dissociation of kappa-casein from the micelles at pH between 6.5 and 6.7, although this dissociation phenomenon is well known on heating milk at high temperatures at pH above 6.7.     

Effect of pH on the association of denatured whey proteins with casein micelles in heated reconstituted skim milk

Skim milk was adjusted to pH values between 6.5 and 6.7 and heated (80, 90, and 100 degrees C) for up to 60 min. Changes in casein micelle size, level of whey protein denaturation, and level of whey protein association with the micelles were monitored for each milk sample. Changes in casein micelle size were markedly affected by the pH at heating. At low pH (6.5-6.55), the casein micelle size increased markedly during the early stages of heating, and the size plateaued on prolonged heating. The maximum increase in size was approximately 30-35 nm. In contrast, at high pH (6.7), much smaller changes in size were observed on heating and the maximum increase in size was only approximately 10 nm. An intermediate behavior was observed at pH values between these two extremes. The rate of denaturation of the major whey proteins, alpha-lactalbumin and beta-lactoglobulin, was essentially unaffected by the pH at heating for the small pH changes involved in this study, and the changes in casein micelle size were poorly related to the level of whey protein denaturation. In contrast, the level of denatured whey proteins associating with the micelles was markedly dependent on the pH at heating, with high levels of association at pH 6.5-6.55 and low levels of association at pH 6.7. Changes in casein micelle size were related to the levels of denatured whey proteins that were associated with the casein micelles, although there was a small deviation from linearity at low levels of association (<15%). Further studies on reconstituted and fresh milk samples at smaller pH steps confirmed that the association of whey proteins with the casein micelles was markedly affected by the pH at heating. These results indicate that the changes in casein micelle size induced by the heat treatment of skim milk were a consequence of the whey proteins associating with the casein micelles and that the level of association was markedly influenced by small pH changes of the milk. It was not possible to determine whether the association itself influenced the casein micelle size or whether parallel reactions involving micellar aggregation caused the increase in micelle size as whey protein association progressed.     

Mechanism for the ethanol-dependent heat-induced dissociation of casein micelles

An explanation as to how casein micelles dissociate when heated in the presence of ethanol is presented. Dissociation of casein micelles in milk-ethanol mixtures was studied using (1)H NMR, and the effects of addition of CaCl(2), NaCl, or EDTA or alteration of milk pH on this dissociation were studied. It is proposed that at low temperatures, ethanol reduces the solvent quality of milk serum, but above a critical temperature (approximately 30 degrees C in a 35% ethanol solution), ethanol enhances solvent quality and dissociates the casein micelles. Ethanol reduced protein hydrophobicity and increased the pK(a) value of phosphoserine, effects that are likely to be significant in the dissociating effect of ethanol at elevated temperatures.     

Disruption and reassociation of casein micelles under high pressure: influence of milk serum composition and casein micelle concentration

In this study, factors influencing the disruption and aggregation of casein micelles during high-pressure (HP) treatment at 250 MPa for 40 min were studied in situ in serum protein-free casein micelle suspensions. In control milk, light transmission increased with treatment time for approximately 15 min, after which a progressive partial reversal of the HP-induced increase in light transmission occurred, indicating initial HP-induced disruption of casein micelles, followed by reformation of casein aggregates from micellar fragments. The extent of HP-induced micellar disruption was negatively correlated with the concentration of casein micelles, milk pH, and levels of added ethanol, calcium chloride, or sodium chloride and positively correlated with the level of added sodium phosphate. The reformation of casein aggregates during prolonged HP treatment did not occur when HP-induced disruption of casein micelles was limited (<60%) or very extensive (>95%) and was promoted by a low initial milk pH or added sodium phosphate, sodium chloride, or ethanol. On the basis of these findings, a mechanism for HP-induced disruption of casein micelles and subsequent aggregation of micellar fragments is proposed, in which the main element appears to be HP-induced solubilization of micellar calcium phosphate.     

Study of the effect of soy proteins on the acid-induced gelation of casein micelles

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.     

Ethanol-dependent heat-induced dissociation of casein micelles

The dissociation of casein micelles when heated to approximately 65 degrees C in the presence of ethanol [1:1 mixture (v/v) of milk and 65% (w/w) aqueous ethanol] was investigated using L* values and transmission measurements. Mixtures of milk and ethanol became transparent on heating, which suggests dissociation of casein micelles. Results of experiments using confocal laser scanning microscopy, light scattering (static and dynamic), and dialysis to examine the changes of milk during heating in the presence of ethanol supported the assertion that such treatments result in dissociation of casein micelles, as did studies of model beta-casein micellar systems.     

Solvent-mediated disruption of bovine casein micelles at alkaline pH

The disruption of casein micelles at alkaline pH was investigated using turbidity measurements. The rate and extent of disruption of casein micelles at alkaline pH (8.0-11.0) increased with pH. Furthermore, the extent of alkaline disruption increased with increasing temperature (5-40 degrees C). Preheating milk for 10 min at 90 degrees C did not influence the extent of alkaline disruption of casein micelles, suggesting that whey proteins do not influence the alkaline disruption process. Levels of ionic calcium and serum calcium and phosphate decreased in a logarithmic fashion with increasing pH, indicating precipitation of calcium phosphate onto the casein micelles. A mechanism for alkaline disruption of casein micelles is proposed, in which increasing the milk pH improves the solvent quality for the caseins, thereby leading to the disruption of casein micelles into their constituent nanoclusters; increases in the net-negative charge on the caseins on increasing pH may contribute to micellar dissociation.     

Noncovalent cross-linking of casein by epigallocatechin gallate characterized by single molecule force microscopy

Interaction of the tea polyphenol epigallocatechin gallate (EGCG) with beta-casein in milk affects the taste of tea and also affects the stability of the tea and the antioxidant ability of the EGCG. In addition, interaction of polyphenols with the chemically similar salivary proline-rich proteins is largely responsible for the astringency of tea and red wine. With the use of single molecule force microscopy, we demonstrate that the interaction of EGCG with a single casein molecule is multivalent and leads to reduction in the persistence length of casein as calculated using the wormlike chain model and a reduction in its radius of gyration. The extra force required to stretch casein in the presence of EGCG is largely entropic, suggesting that multivalent hydrophobic interactions cause a compaction of the casein micelle.     

Casein micelles: size distribution in milks from individual cows

The size distribution and protein composition of casein micelles in the milk of Holstein-Friesian cows was determined as a function of stage and number of lactations. Protein composition did not vary significantly between the milks of different cows or as a function of lactation stage. Differences in the size and polydispersity of the casein micelles were observed between the milks of different cows, but not as a function of stage of milking or stage of lactation and not even over successive lactations periods. Modal radii varied from 55 to 70 nm, whereas hydrodynamic radii at a scattering angle of 73° (Q2 = 350 μm?2) varied from 77 to 115 nm and polydispersity varied from 0.27 to 0.41, in a log-normal distribution. Casein micelle size in the milks of individual cows was not correlated with age, milk production, or lactation stage of the cows or fat or protein content of the milk.     

Effects of added sodium caseinate on the formation of particles in heated milk

The effects of heat at temperatures in the range of 80-90 degrees C on mixtures of reconstituted skim milk powder (RSMP) and sodium caseinate have been determined. In the absence of caseinate, the action of heat on RSMP produces soluble complexes of whey proteins and kappa-casein, as well as complexes of whey protein with the casein micelles. When sodium caseinate was added to RSMP at levels of 0.5 and 1.0%, the denaturation of the whey protein and the production of the soluble complexes in the serum were hardly affected, either in rate or in amount. However, during the heating, the caseinate disappeared from the serum. Further studies on model mixtures of the different components showed that it was probable that the bulk of the caseinate associated with the casein micelles during heating, probably by binding inside the surface layer of kappa-casein, because no increase in the diameters of the casein micelles could be observed.     

Water sensitivity, antimicrobial, and physicochemical analyses of edible films based on HPMC and/or chitosan

Several properties of chitosan films associated or not with hydroxypropylmethylcellulose polymer (HPMC) and HPMC films incorporating or not nisin and/or milk fat were studied. Nisin addition at a level of 250 microg mL-1 and likewise chitosan at 1% (w/v) concentration were efficient for total inhibiting Aspergillus niger and Kocuria rhizophila food deterioration microorganisms. HPMC and chitosan films were transparent, whereas nisin and/or fat incorporation induced a 2-fold lightness parameter increase and, consequently, involved more white films. Measurements of tensile strength, as well as ultimate elongation, showed that chitosan and HPMC initial films were elastic and flexible. High thermal treatments and additive incorporation induced less elastic and more plastic films. Water vapor transmission as far as total water desorption rates suggested that chitosan films were slightly sensitive to water. Water transfer was decreased by <60% as compared with other biopolymer films. Regarding its hydrophobic property, the capacity of fat to improve film water barrier was very limited.     

Mechanism of pressure-induced gelation of milk.

The pressure-induced gelation of concentrated skimmed milk and milk-sugar mixtures was studied to discover the main components responsible for gelation. The major protein component responsible for gelation is micellar casein. Gelation occurs at similar pressures to casein micelle disintegration in dilute milk, and both can be prevented by inclusion of excess calcium chloride. Transmission electron micrographs show that the protein network is formed from particles with diameters approximately an order of magnitude smaller than those of intact casein micelles. Gelation occurs on decompression and is found to be baroreversible. Concentrations of sugar up to 30% reduce the critical concentration of casein required for gelation, but higher sugar concentrations inhibit gelation. A mechanism of gelation based on the aggregation of casein submicelles formed by pressure-induced disintegration of casein micelles is proposed. Observations on the effect of sucrose on gelation are discussed in terms of the influence of sugars on the solvent quality in aqueous casein systems.     

Interaction between casein micelles and whey protein/κ-casein complexes during renneting of heat-treated reconstituted skim milk powder and casein micelle/serum mixtures

Casein micelles were separated from unheated reconstituted skim milk powder (RSMP) and were resuspended in the serum of RSMP that had been heated, with and without dialysis of this serum against unheated RSMP. Using size-exclusion chromatography, it was found that the soluble complexes of whey protein (WP) with κ-casein in the serum of the heated milk bind progressively to unheated casein micelles during renneting, even prior to the onset of clotting. Similar trends were noted when casein micelles from RSMP heated at pH values of 6.7, 7.1, or 6.3, each with different amounts of WP coating the micelles, were renneted in the presence of soluble WP/κ-casein complexes. No matter what was the initial load of micelle-bound WP complexes, all micelle types were capable of binding additional serum protein complexes during renneting. However, it is not clear that this binding of WP/κ-casein complexes to the micellar surface is a direct cause of the impaired rennet clotting of the RSMP.