Disruption and reassociation of casein micelles during high pressure treatment: influence of whey proteins

In the study presented in this article, the influence of added alpha-lactalbumin and beta-lactoglobulin on the changes that occur in casein micelles at 250 and 300 MPa were investigated by in-situ measurement of light transmission. Light transmission of a serum protein-free casein micelle suspension initially increased with increasing treatment time, indicating disruption of micelles, but prolonged holding of micelles at high pressure partially reversed HP-induced increases in light transmission, suggesting reformation of micellar particles of colloidal dimensions. The presence of alpha-la and/or beta-lg did not influence the rate and extent of micellar disruption and the rate and extent of reformation of casein particles. These data indicate that reformation of casein particles during prolonged HP treatment occurs as a result of a solvent-mediated association of the micellar fragments. During the final stages of reformation, kappa-casein, with or without denatured whey proteins attached, associates on the surface of the reformed particle to provide steric stabilisation.     

Behaviour of partially cross-linked casein micelles under high pressure

High pressure (HP) treatment of a casein micelle suspension at 250 and 300 MPa leads to an initial rapid increase of its light transmission, as measured in situ , indicating micellar disruption. Subsequently, a much slower, partial reversal of the HP-induced increase in light transmission is observed, indicating re-association of micellar fragments. Partial internal cross-linking of the casein micelles by the enzyme transglutaminase prior to pressure treatment slows down both the disruption and the reassociation process considerably. It is proposed that covalent cross-linking provides the micelle with extra stability against pressure-induced disruption and also prevents a molecular reorganization process required to induce reassociation of micellar fragments during prolonged pressure treatment.     

Disruption and sedimentation of casein micelles and casein micelle isolates under high-pressure homogenization

Native casein micelles were isolated from raw skim milk by ultrafiltration (< 30 kDa) or microfiltration (< 0.2 μm) and subjected to high-pressure homogenization (HPH) at 100, 200, 250, 300, and 350 MPa. Of particular interest was the effect of HPH on casein micelle size in solutions varying in ionic strength (0, 5, 10, and 15 mM CaCl₂) and micelle size populations. Particle size distribution reflected an initial decrease in micelle diameter in all samples at 100 MPa. In samples containing 10 and 15 mM CaCl₂, there was an abrupt increase in particle size and subsequent casein precipitation followed by sedimentation upon centrifugation at elevated pressures (300 and 350 MPa). The amount of sedimentable casein protein increased as CaCl₂ concentration (10 and 15 mM) and pressure (300 and 350 MPa) increased as determined by UV absorbance of sample supernatant. SDS-PAGE indicated extensive micellar disruption at elevated pressures (300 and 350 MPa) and confirmed that the sedimented portion of the samples contained casein proteins and minimal amounts of whey proteins. Results indicated that through HPH treatment casein micelle size can be modified based on CaCl₂ concentration and pressure applied. Based on these findings, HPH in combination with an appropriate suspending medium has the ability to modify micelles to a desired size for a number of potential applications.Industrial relevanceThe modification of structure-function properties of the casein micelle from bovine milk by using high-pressure homogenization is relevant in (1) the development of new ingredients to change rheological/textural properties of dairy based foods, and (2) the discovery of new and/or improved functionalities for protein quaternary structures.     

Dynamics of casein micelles in skim milk during and after high pressure treatment

The effect of high hydrostatic pressure on turbidity of skim milk was measured in situ together with casein micelle size distribution. High pressure (HP) treatment reduced the turbidity of milk with a stronger pressure dependency between 50 and 300 MPa when the temperature was decreased from 20 to 5 °C, while at 30 °C (50–150 MPa) turbidity exceeded that of untreated milk. At 250 and 300 MPa turbidity decreased extremely. During pressurization of milk at 250 and 300 MPa, the turbidity initially decreased, but treatments longer than 10 min increased the turbidity progressively, indicating that re-association followed dissociation of casein micelles. Especially at 40 °C and at 250 and 300 MPa, the turbidity increased beyond untreated milk. Dynamic light scattering was used to investigate casein micelle sizes in milk immediately after long time (up to 4 h) pressurization at 250 and 300 MPa and casein micelle size distributions were bimodal with micelle sizes markedly smaller and markedly larger than those of untreated milk. Pressure modified casein micelles present after treatment of milk at 250 and 300 MPa were concluded to be highly unstable, since the larger micelles induced by pressure showed marked changes toward smaller particle sizes in milk left at ambient pressure.     

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

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

Reformation of casein particles from alkaline-disrupted casein micelles

In this study, the properties of casein particles reformed from alkaline disrupted casein micelles were studied. For this purpose, micelles were disrupted completely by increasing milk pH to 10.0, and subsequently reformed by decreasing milk pH to 6.6. Reformed casein particles were smaller than native micelles and had a slightly lower zeta-potential. Levels of ionic and serum calcium, as well as rennet coagulation time did not differ between milk containing native micelles or reformed casein particles. Ethanol stability and heat stability, >pH 7.0, were lower for reformed casein particles than native micelles. Differences in heat stability, ethanol stability and zeta-potential can be explained in terms of the influence of increased concentrations of sodium and chloride ions in milk containing reformed casein particles. Hence, these results indicate that, if performed in a controlled manner, casein particles with properties closely similar to those of native micelles can be reformed from alkaline disrupted casein micelles.     

High pressure treatment of bovine milk: effects on casein micelles and whey proteins

Effects of high pressure (HP) on average casein micelle size and denaturation of alpha-lactalbumin (alpha-la) and beta-lactoglobulin (beta-lg) in raw skim bovine milk were studied over a range of conditions. Micelle size was not influenced by treatment at pressures <200 MPa, but treatment at 250 MPa increased micelle size by approximately 25%, while treatment at > or = 300 MPa irreversibly reduced it to approximately 50% of that in untreated milk. The increase in micelle size after treatment at 250 MPa was greater with increasing treatment time and temperature and milk pH. Treatment times > or = 2 min at 400 MPa resulted in similar levels of micelle disruption, but increasing milk pH to 7.0 partially stabilised micelles against HP-induced disruption. Denaturation of alpha-la did not occur < or = 400 MPa, whereas beta-lg was denatured at pressures >100 MPa. Denaturation of alpha-la and beta-lg increased with increasing pressure, treatment time and temperature and milk pH. The majority of denatured beta-lg was apparently associated with casein micelles. These effects of HP on casein micelles and whey proteins in milk may have significant implications for properties of products made from HP-treated milk.     

Sub-structure of synthetic casein micelles.

Casein micelles of different composition were synthesized in various ways and their sub-structure was investigated with the electron microscope by means of thin sections. Earlier studies of Schmidt et al. (1974) using the freeze-fracturing technique had shown no differences in the sub-structure of natural micelles and artificial micelles containing Ca and casein only. Contrary to these results our present studies showed that for the production of synthetic casein micelles with the same sub-structure as the natural ones it is necessary to add at least 2 other ions to the casein solution besides Ca2+: these are phosphate and citrate. The citrate ions play an important role in forming the material of the dark framework of the micelles visible in electron micrographs of unstained thin sections. This supports the hypothesis of Pyne & McGann (1960) that casein micelles contain a citrate apatite.     

Stability of buffalo casein micelles.

Buffalo skim-milk is less heat stable than cow skim-milk. Interchanging ultracentrifugal whey (UCW) and milk diffusate with micellar casein caused significant changes in the heat stability of buffalo casein micelles (BCM) and cow casein micelles (CCM). Buffalo UCW dramatically destabilized CCM, whereas buffalo diffusate with CCM exhibited the highest heat stability. Cow kappa-casein stabilizes alphas-casein against precipitation by Ca better than buffalo kappa-casein. About 90% of alphas-casein could be stabilized by kappa:alphas ratios of 0.20 and 0.231 for cow and buffalo, respectively. Sialic acid release from micellar kappa-casein by rennet was higher than from acid kappa-casein in both buffalo and cow caseins, the release being slower in buffalo. The released macropeptide from buffalo kappa-casein was smaller than that from cow kappa-casein as revealed by Sephadex gel filtration. Sub-units of BCM have less sialic acid (1.57 mg/g) than whole micelles (2.70 mg/g). On rennet action, 47% of bound sialic acid was released from sub-units as against 85% from whole micelles. The sub-micelles are less heat stable than whole micelles. Among ions tested, added Ca reduced heat stability more dramatically in whole micelles, whereas added phosphate improved the stability of micelles and, more strikingly, of sub-micelles. Citrate also improved the heat stability of sub-micelles but not of whole micelles.     

Properties of artificial casein micelles.

A survey is given of the relationships between various properties of artificial casein micelle systems and their composition with respect to alphas1-, beta and kappa-casein, colloidal phosphate and citrate. Properties investigated were: the amount of colloidal phosphate, the micellar size, and the stability of the micelle towards dialysis, pressure, ethanol and heat.     

Hydration of casein micelles and caseinates: Implications for casein micelle structure

By studying the hydration of casein micelles using a variety of techniques, a distinction could be made between water that appeared bound by the protein (∼0.5 g g⁻¹ protein), water associated with the κ-casein brush (∼1.0 g g⁻¹ protein) and water entrapped in the casein micelles (∼1.8 g g⁻¹ protein), yielding a total micellar hydration of ∼3.3 g g⁻¹ protein, in line with casein micelle voluminosity derived from intrinsic viscosity measurements. For caseinate particles, however, the main contributor to intrinsic viscosity was not protein hydration but the non-spherical particle shape. These non-spherical particles in caseinate are likely to be naturally present as primary casein particles (PCP) in casein micelles. PCP could be used to build casein micelles by controlled introduction of micellar salts. Based on the findings of this study, casein micelles could be described as a porous network of non-spherical PCP linked by calcium phosphate nanoclusters.     

Kinetics of heat-induced interactions among whey proteins and casein micelles in sheep skim milk and aggregation of the casein micelles

The interactions among the proteins in sheep skim milk (SSM) during heat treatments (67.5–90°C for 0.5–30 min) were characterized by the kinetics of the denaturation of the whey proteins and of the association of the denatured whey proteins with casein micelles, and changes in the size and structure of casein micelles. The relationship between the size of the casein micelles and the association of whey proteins with the casein micelles is discussed. The level of denaturation and association with the casein micelles for β-lactoglobulin (β-LG) and α-lactalbumin (α-LA) increased with increasing heating temperature and time; the rates of denaturation and association with the casein micelles were markedly higher for β-LG than for α-LA in the temperature range 80 to 90°C; the Arrhenius critical temperature was 80°C for the denaturation of both β-LG and α-LA. The casein micelle size increased by 7 to 120 nm, depending on the heating temperature and the holding time. For instance, the micelle size (about 293 nm) of SSM heated at 90°C for 30 min increased by about 70% compared with that (about 174.6 nm) of unheated SSM. The casein micelle size increased slowly by a maximum of about 65 nm until the level of association of the denatured whey proteins with casein micelles reached 95%, and then increased markedly by a maximum of about 120 nm when the association level was greater than about 95%. The marked increases in casein micelle size in heated SSM were due to aggregation of the casein micelles. Aggregation of the casein micelles and association of whey protein with the micelles occurred simultaneously in SSM during heating.     

Stability of casein micelles cross-linked by transglutaminase

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

Properties of casein micelles cross-linked by transglutaminase

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

Rennet-induced coagulation of enzymatically cross-linked casein micelles

The enzymatic cross-linking of casein micelles with transglutaminase had an adverse influence on rennet-induced coagulation. Incubation with transglutaminase at 30 °C progressively reduced the levels of monomeric caseins and increased rennet flocculation time (RFT) in a Berridge test. For incubation up to 3 h at 30 °C, the reciprocal of the RFT was linearly correlated with the level of residual monomeric κ-casein, indicating that at complete cross-linking flocculation is absent. After treatment for 4–24 h at 30 °C, no residual monomeric κ-casein was detected and no rennet-induced flocculation of the casein micelle suspension was observed. Monitoring rennet-induced coagulation by diffusing wave spectroscopy revealed that transglutaminase-induced inhibition of rennet-induced coagulation of casein micelles is primarily due to an inhibition of the secondary phase of rennet coagulation, i.e., the gelation and gel-firming phase of the casein micelle coagulation. The gelation and fusion of κ-casein-depleted para-casein micelles as in normal milk appears to be absent if the casein macropeptide remains attached to the casein micelle.     

Size distribution of casein micelles during milk coagulation

Electron microscopy was employed to study the size distribution and the surface to volume ratios of casein micelles in coagulum from raw milk, heated milk and milk enriched with calcium chloride before the heating process. The particles size distribution of milk protein was changed by heating resulting in an increase in the free subunits of smaller size and the surface to volume ratios. On the other hand the clotting time was prolonged. Depression in the clotting time and surface to volume ratios was occurred by adding calcium chloride to the milk before the heating process to retard the formation of complex between the whey protein and milk casein.     

浓缩乳蛋白的离子交换脱钙及其对酪蛋白胶束的影响

研究了浓缩乳蛋白的离子脱钙技术,以及部分脱钙对截留液中酪蛋白存在形式及酪蛋白胶束水合率的影响。研究确定了离子交换树脂的平衡脱钙时间为2 h,并通过改变树脂添加量得到了0、5%.5%、10.5%、19.6%、29.6%、38.7%、49.9%、63.8%和83.6%系列脱钙程度的截留液。随着脱钙程度的增加,截留液超离心上清中游离酪蛋白的含量逐渐增加,而超离心沉淀的胶束酪蛋白减少,说明酪蛋白逐渐从酪蛋白胶束中游离出来。当脱钙程度为0~29.6%时,酪蛋白胶束的水合率从2.6 g/g(干基)增加到4.1 g/g(干基),而脱钙程度从29.6%进一步增加到83.6%时,酪蛋白胶束水合率则变小至3.3 g/g(干基)。浓缩乳蛋白的钙离子含量以及酪蛋白的存在状态决定了其在应用时的功能特性,研究对开发新型的浓缩乳蛋白配料具有重要的指导意义。