Formation of soluble and micelle-bound protein aggregates in heated milk

The formation of heat-induced aggregates of kappa-casein and denatured whey proteins was investigated in milk-based dairy mixtures containing casein micelles and serum proteins in different ratios. Both soluble and micelle-bound aggregates were isolated from the mixtures heated at 95 degrees C for 10 min, using size exclusion chromatography. Quantitative analysis of the protein composition of the aggregates by reverse phase high-performance liquid chromatography strongly suggested that primary aggregates of beta-lactoglobulin and alpha-lactalbumin in a 3 to 1 ratio were involved as well as kappa-casein, and alpha(s2)-casein in micellar aggregates. The results gave evidence that heat-induced dissociation of micellar kappa-casein was implicated in the formation of the soluble aggregates and indicated that a significant amount of kappa-casein was left unreacted after heating. The average size of the aggregates was 3.5-5.5 million Da, depending on the available kappa-casein or the casein:whey protein ratio in the mixtures. The size and density of these aggregates relative to those of casein micelles were discussed.     

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.     

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.     

Effect of the pH of heating on the qualitative and quantitative compositions of the sera of reconstituted skim milks and on the mechanisms of formation of soluble aggregates

The effect of the pH of heating (6.3-7.3) on the composition of sera in reconstituted skimmed milks was investigated. A combination of SDS-PAGE analysis and size exclusion chromatography (SEC) combined with an original approach to the analysis of the SEC profiles was performed. The composition of the sera varied greatly when the pH of heating was adjusted below and above the natural pH of milk. The formation, composition, and concentration of heat-induced soluble complexes depended on the combination of the effect of adjusting the pH of the milk and the heat treatment. Two types of mechanism for the formation of soluble aggregates appeared to exist, depending on the pH of the milk. The first type results from the formation of WP/kappa-casein aggregates at the surface of the micelle, and these were detached partially into the serum in larger amount as the pH increased up to 6.7, where it reaches a maximum. The second type of complexes, whose amount increased as the pH of heating increased from 6.7 to 7.3, may be formed between caseins (kappa- but also perhaps some alpha(s)-casein) and aggregated WP resulting in complexes that are smaller in size and with a higher kappa-casein/whey protein ratio than the first type.     

Role of the soluble and micelle-bound heat-induced protein aggregates on network formation in acid skim milk gels

Gel formation was monitored by low amplitude rheometry during acidification at 40 degrees C with 1.5% glucono-delta-lactone in combined milk systems containing soluble and/or micelle-bound heat-induced (95 degrees C/10 min) aggregates of denatured whey proteins and kappa-casein and in heated dairy mixes with varying micellar casein/whey protein ratio (CN/WP). Both soluble and micelle-bound aggregates increased gelation pH and gel strength. Micelle-bound aggregates seemed to modify the micelle surface so that micelles were destabilized at a pH of 5.1 (instead of 4.7), while soluble aggregates precipitated at their calculated pI of approximately 5.3, and initiated an early gelation by interacting with the micelles. Decreasing the CN/WP ratio produced larger aggregates with higher whey protein: kappa-casein ratio, which gave more elastic gels. The specific effects of the micellar and soluble aggregates on gel strength are discussed with respect to their relative proportions in the heated milk.     

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.     

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.     

Rennet-induced aggregation of heated pH-adjusted skim milk

Heated (20-100 °C/0-30 min) skim milks (pH 6.5-7.1) were diluted in buffer (pH 7.0). Rennet was added, and the particle size with time was measured. For all samples, the size initially decreased (lag phase) and then increased (aggregation phase). Milks heated at ≤60 °C had short lag phases and rapid aggregation phases regardless of pH. Milks heated at >60 °C at pH 6.5 had long lag phases and slow aggregation phases. As the pH increased, the lag phase shortened and the aggregation phase accelerated. The aggregation time was correlated with the level of whey protein associated with the casein micelles and with the level of κ-casein dissociated from the micelles. Heated milks formed weak gels when renneted. It is proposed that the milks heated at low pH have whey proteins associated with the casein micelles and that these denatured whey proteins stabilize the micelles to aggregation by rennet and therefore inhibit gelation. In the milks heated at higher pH, the whey proteins associate with κ-casein in the serum and, on rennet treatment, the κ-casein-depleted micelles and the serum-phase whey protein/κ-casein complexes aggregate; however, the denatured whey proteins stabilize the aggregates so that gelation is still inhibited.     

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.     

Interaction between β-casein and whey proteins as a function of pH and salt concentration

The effectiveness of β-casein as a chaperone in the aggregation of whey proteins was investigated. β-Casein altered heat-induced aggregation as shown by a reduction in turbidity of β-lactoglobulin, α-lactalbumin, and bovine serum albumin (BSA) solutions. The pH of the mixtures greatly affected how much β-casein reduced the turbidity of the solutions; the maximum reductions in turbidity were observed at pH 6.0. Reducing the pH decreased the effectiveness of β-casein as a chaperone. An increase in ionic strength by the addition of NaCl or CaCl(2) also decreased the effectiveness of the chaperone. The addition of CaCl(2) had a larger effect than the addition of NaCl. The chaperone effect was seen at temperatures up to 145 °C. Differential scanning calorimetry (DSC) showed that β-casein did not alter the denaturation temperature of β-lactoglobulin. The kinetics curves for loss of native protein and turbidity development showed that β-casein did not function by slowing the aggregation process. It was concluded that β-casein competes with whey protein in the aggregate process and the aggregates formed in the presence of β-casein are smaller in size than those formed during whey protein self-aggregation. The formation of smaller aggregates gives rise to less turbid, more soluble protein solutions.     

Effects of heat and high hydrostatic pressure treatments on disulfide bonding interchanges among the proteins in skim milk

Traditionally, milk has been heat treated to control microorganisms and to alter its functionality, for example, to increase its heat stability. Pressure treatment has been considered as a possible alternative for microorganism control, but some of the functionality-related milk protein interactions have not been explored. The present study used two novel two-dimensional polyacrylamide gel electrophoresis (2D PAGE) methods to explore the differences in the irreversible disulfide bond changes among the milk proteins after four common heat treatments and after 30-min pressure treatments of milk at 200, 400, 600, and 800 MPa at ambient temperature (22 degrees C). The pasteurizing heat treatment (72 degrees C for 15 s) denatured and aggregated only a few minor whey proteins, but the high heat treatments (100 degrees C for 120 s, 120 degrees C for 120 s, and 140 degrees C for 5 s) formed disulfide-bonded aggregates that included a high proportion of all of the whey proteins and kappa-casein (kappa-CN) and a proportion of the alpha(s2)-CN. Pressure treatment of milk at 200 MPa caused beta-lactoglobulin (beta-LG) to form disulfide-bonded dimers and incorporated beta-LG into aggregates, probably disulfide-bonded to kappa-CN. The other whey proteins appeared to be less affected at 200 MPa for 30 min. In contrast, pressure treatment at 800 MPa incorporated beta-LG and most of the minor whey proteins, as well as kappa-CN and much of the alpha(s2)-CN, into aggregates. The accessibility of alpha(s2)-CN and formation of complexes involving alpha(s2)-CN, kappa-CN, and whey proteins in the pressure treated milk is an important novel finding. However, only some of the alpha-lactalbumin was denatured or incorporated into the large aggregates. These and other results show that the differences between the stabilities of the proteins and the accessibilities of the disulfide bonds of the proteins at high temperature or pressure affect the formation pathways that give the differences among the resultant aggregates, the sizes of the aggregates, and the product functionalities.     

Limited enzymatic treatment of skim milk using chymosin affects the micelle/serum distribution of the heat-induced whey protein/kappa-casein aggregates

The effects of heat treatment and limited kappa-casein hydrolysis on the micelle/serum distribution of the heat-induced whey protein/kappa-casein aggregates were investigated as a possible explanation for the gelation properties of combined rennet and acid gels. Reconstituted skim milk was submitted to combinations of 0-67% hydrolysis of the kappa-casein at 5 degrees C and heat treatment at 90 degrees C for 10 min. The protein composition of the ultracentrifugal fractions was obtained by reverse-phase high-performance liquid chromatography (RP-HPLC). The aggregates contained in each phase were isolated by size-exclusion chromatography and analyzed by RP-HPLC and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Upon heating only, 20-30% of the total kappa-casein dissociated, while 20-30% of the total whey protein attached to the micelles. When heated milk was renneted, little changes were observed in the distribution and composition of the aggregates. Conversely, the heat treatment of partially renneted milk induced the formation of essentially micelle-bound aggregates. The results were discussed in terms of the preferred interaction between hydrophobic para-kappa-casein and denatured whey proteins.     

Rheological properties of acid gels prepared from heated pH-adjusted skim milk

Reconstituted skim milk was adjusted to pH values between 6.5 and 7.1 and heated (90 degrees C) for up to 30 min. The skim milk samples were then readjusted to pH 6.7. Acid gels prepared from heated milk had markedly higher G ' values, a reduced gelation time, and an increased gelation pH than those prepared from unheated milk. An increased pH at heating decreased the gelation time, increased the gelation pH, and increased the final G ' of acid set gels prepared from the heated milk samples. There were only small differences in the level of whey protein denaturation in the samples at different pH values, and these differences could not account for the differences in the G ' of the acid gels. The levels of denatured whey protein associated with the casein micelles decreased and the levels of soluble denatured whey proteins increased as the pH at heating was increased. The results indicated that the soluble denatured whey proteins had a greater effect on the final G ' of the acid gels than the denatured whey proteins associated with the casein micelles.     

Characterization of heat-induced changes in skim milk using asymmetrical flow field-flow fractionation coupled with multiangle laser light scattering

Separation and size measurement of protein particles are a relevant approach to monitor heat-induced changes in skim milk. Unfortunately, no method is currently available at low cost and without excessive preparation of the samples. Therefore, the present study aimed at evaluating the interest of asymmetrical flow field-flow fractionation (AFlFFF) coupled with multiangle laser light scattering (MALLS) for this purpose. Unheated and heated skim milk samples at pH 6.5 and 7.2 were prepared and comparatively analyzed using AFlFFF-MALLS, size exclusion chromatography (SEC-MALLS) and dynamic light scattering. The results showed that AFlFFF could evidence the conversion of the native whey proteins of unheated milk into heat-induced whey protein/κ-casein complexes in the serum phase of milk and possibly on the surface of the casein micelles. The pH-induced changes in the partition of the complexes between the serum and the micellar phases could also be observed. The results therefore showed the interest of AFlFFF-MALLS to monitor the heat-induced changes in particle sizes in skim milk and to separate the different protein components of unheated and heated skim milk.     

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.     

Controlled proteolysis and the properties of milk gels

Milk gels induced by partial proteolysis of the kappa-casein followed by acidification were studied, and their gelation behavior was compared to that of milk gels induced by simultaneous acidification and renneting, using dynamic rheology. There were generally two stages (at pH values below and above 5.0) in the gelation of the milk whose kappa-casein had been partially proteolyzed and acidified. The onset of gelation was at higher pH as the degree of kappa-casein proteolysis increased. The development of G' immediately after the onset of gelation was faster in the milk gels induced by simultaneous acidification and renneting, because of the continuing kappa-casein proteolysis. Preheat treatment caused the onset of gelation to occur at higher pH than for unheated milk. However, the maximum tan delta during gelation always occurred at the same pH (for a given concentration of acidulant), and its value and position were independent of the extent of renneting and whether the milks had been heat treated. The results are discussed in terms of the interactions between casein micelles occurring during gelation.     

Methionine oxidation enhances κ-casein amyloid fibril formation

The effects of protein oxidation, for example of methionine residues, are linked to many diseases, including those of protein misfolding, such as Alzheimer's disease. Protein misfolding diseases are characterized by the accumulation of insoluble proteinaceous aggregates comprised mainly of amyloid fibrils. Amyloid-containing bodies known as corpora amylacea (CA) are also found in mammary secretory tissue, where their presence slows milk flow. The major milk protein κ-casein readily forms amyloid fibrils under physiological conditions. Milk exists in an extracellular oxidizing environment. Accordingly, the two methionine residues in κ-casein (Met(95) and Met(106)) were selectively oxidized and the effects on the fibril-forming propensity, cellular toxicity, chaperone ability, and structure of κ-casein were determined. Oxidation resulted in an increase in the rate of fibril formation and a greater level of cellular toxicity. β-Casein, which inhibits κ-casein fibril formation in vitro, was less effective at suppressing fibril formation of oxidized κ-casein. The ability of κ-casein to prevent the amorphous aggregation of target proteins was slightly enhanced upon methionine oxidation, which may arise from the protein's greater exposed surface hydrophobicity. No significant changes to κ-casein's intrinsically disordered structure occurred upon oxidation. The enhanced rate of fibril formation of oxidized κ-casein, coupled with the reduced chaperone ability of β-casein to prevent this aggregation, may affect casein-casein interaction within the casein micelle and thereby promote κ-casein aggregation and contribute to the formation of CA.     

Effects of adding low levels of a disulfide reducing agent on the disulfide interactions of β-lactoglobulin and κ-casein in skim milk

Low concentrations of a disulfide reducing agent were added to unheated and heated (80 °C for 30 min) skim milk, with and without added whey protein. The reduction of the β-lactoglobulin and κ-casein disulfide bonds was monitored over time using electrophoresis. The distribution of the proteins between the colloidal and serum phases was also investigated. κ-Casein disulfide bonds were reduced in preference to those of β-lactoglobulin in both unheated and heated skim milk (with or without added whey protein). In addition, in heated skim milk, while the serum κ-casein was reduced more readily than the colloidal κ-casein, the distribution of κ-casein between the two phases was not affected.     

Proteomic analysis of temperature-dependent changes in stored UHT milk

Molecular changes in milk proteins during storage of UHT-treated milk have been investigated using two-dimensional electrophoresis (2-DE) coupled to MALDI-TOF mass spectrometry. UHT-treated samples were stored at three different temperatures, 4 °C, 28 °C, and 40 °C, for two months. Three main changes could be observed on 2-DE gels following storage. They were (1) the appearance of diffuse staining regions above the position of the monomeric caseins caused by nondisulfide cross-linking of α and β-caseins; (2) the appearance of additional acidic forms of proteins, predominantly of α(S1)-casein, caused by deamidation; and (3) the appearance of "stacked spots" caused by lactosylation of whey proteins. The extent of the changes increased with increased storage temperature. Mass spectrometric analysis of in-gel tryptic digests showed that the cross-linked proteins were dominated by α(S1)-casein, but a heterogeneous population of cross-linked forms with α(S2)-casein and β-casein was also observed. Tandem MS analysis was used to confirm deamidation of N(129) in α(S1)-casein. MS analysis of the stacked spots revealed lactosylation of 9/15 lysines in β-lactoglobulin and 8/12 lysines in α-lactalbumin. More extensive analysis will be required to confirm the nature of the cross-links and additional deamidation sites in α(S1)-casein as the highly phosphorylated nature of the caseins makes them challenging prospects for MS analysis.