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.     

High pressure effects on the structure of casein micelles in milk as studied by cryo-transmission electron microscopy

Milk was processed with high hydrostatic pressure in order to modify the casein micelles. Images, that in details showed the casein micelle structure in untreated and pressure-treated skim milk, were obtained by using cryo-transmission electron microscopy (cryo-TEM). Sizes and shapes adopted by casein micelles in pressurised milk are concluded to be a result of an equilibrium distribution between self-assembling casein molecules in the serum phase and caseins adsorbed to surfaces of casein micelles and are governed by an initial pressure-dependent displacement of caseins into the serum phase. Pressurisation of milk at moderately high pressure, in the range 150–300 MPa, favoured formation of a large number of small micelles that coexisted with a fraction of large micelles, and which appeared perfectly spherical with smooth and well-defined surfaces, features which are suggested to originate from secondary adsorption of caseins. Pressurisation of milk at 400 MPa favoured formation of smaller casein assemblies, with sizes between 30 nm and 100 nm. Measurements of free calcium concentration [Ca²⁺] showed that calcium was rebound to casein micelles after pressurisation of milk. Furthermore, the electron microscopy images indicated that the substructures were similar for pressure-modified casein micelles and casein micelles in untreated milk.     

Pressurisation of raw skim milk and of a dispersion of phosphocaseinate at 9 degrees C or 20 degrees C: effects on the distribution of minerals and proteins between colloidal and soluble phases

The effects of high pressure treatments (100-300 MPa; 15 min; 9 degrees C or 20 degrees C) on the distribution of minerals and proteins of raw skim milk (RSM) and of a dispersion of industrial phosphocaseinate (PC) were studied after separation of the micellar and soluble phases by ultracentrifugation (UCF). Whatever the temperature of high pressure treatments, the pressure-induced dissociation of the casein micelles was accompanied by calcium (Ca), phosphorus (P) and casein release from the micelles. The released Ca and P were or became bound to soluble proteins since progressive increases in Ca and P concentrations were observed in the UCF supernatants of RSM and of the PC dispersion but not in the ultrafiltrates from these UCF supernatants (free of soluble proteins). Simultaneously, alpha(S1-), alpha(S2-), beta- and kappa-caseins were progressively released from the micelles, as seen by electrophoretic analysis. The pressure-induced solubilisation of alpha(S1-) and alpha(S2-)caseins, essentially located in the core of the micelles, suggests that high pressure de-stabilized micelles including their internal structure.     

Aggregation and Dissociation of Milk Protein Complexes in Heated Reconstituted Concentrated Skim Milks

Heating milk at 120°C at pH 6.55 or pH 6.85 caused the denaturation of whey proteins and increased their association with the casein micelles. The dissociation of K -, β-, and α_s-caseins (in that order by extent) from the casein micelles increased with severity of heat treatment. The effect was greater at higher pH. Gel filtration chromatography followed by gel electrophoresis of fractions showed the dissociated protein was composed of disulfide-linked k -casein/β-lactoglobulin complexes of varying composition, casein aggregates of varying sizes and some monomeric protein. When reconstituted concentrate was prepared from NFDM made from heated milk the non-sedimentable (88,000 ± g for 90 min) caseins or whey proteins/heating time profiles were altered and the rate of aggregation, as measured by turbidity of heated milks, was significantly reduced.     

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-jet processing on the viscosity and foaming properties of pasteurized whole milk

The processing of milk using high-pressure technologies has been shown to dissociate casein micelles, denature whey proteins, and change the appearance and rheological properties of milk. A novel high-pressure processing technology called high-pressure-jet (HPJ) processing is currently being investigated for use in the food industry. Few studies have evaluated the effects of HPJ technology on dairy foods. The present study investigated the physicochemical and foaming properties of homogenized pasteurized whole milk processed at pressures from 0 to 500 MPa using HPJ processing. The apparent particle size exhibited a monomodal distribution in whole milk samples processed up to 125 MPa and a bimodal distribution for samples processed at 250, 375, and 500 MPa. The viscosity increased from approximately 2 to 5 mPa·s when whole milk was processed using HPJ at 375 MPa, and foam expansion increased from approximately 80 to 140% after processing at >125 MPa. Foam stability was limited to pressures in the 375 to 500 MPa range. We hypothesized that the increase in apparent particle size was due to the dissociation of casein micelles into surface-active casein protein monomers, and the formation of casein–casein and casein–fat particles. Ultracentrifugation of samples into 3 milk fractions (supernatant, serum, and precipitate), and subsequent fat and protein analysis on the 3 fractions, showed that a strong interaction between casein proteins and fat triglycerides occurred, evidenced by the increase in fat content associated with the precipitate fraction with increasing pressure. This suggests that stable casein–fat aggregates are formed when whole milk is processed using HPJ at pressure >125 MPa.     

Effects of pressurized thermal processing on native proteins of raw skim milk and its concentrate

Heating, pressurization, and shearing can modify native milk proteins. The effects of pressurized heating (0.5 vs. 10 MPa at 75 or 95°C) with shearing (1,000 s^?1) on proteins of raw bovine skim milk (SM, ～9% total solids) and concentrated raw skim milk (CSM, ～22% total solids) was investigated. The effects of evaporative concentration at 55°C and pressurized shearing (10 MPa, 1,000 s^?1) at 20°C were also examined. Evaporative concentration of SM resulted in destabilization of casein micelles and dissociation of α_S1- and β-casein, rendering CSM prone to further reactions. Treatment at 10 MPa and 1,000 s^?1 at 20°C caused substantial dissociation of α_S1- and β-casein in SM and CSM, with some dissociated caseins forming shear-induced soluble aggregates in CSM. The pressure applied at 10 MPa induced compression of the micelles and their dissociation in SM and CSM at 75 or 95°C, resulting in reduction of the micelle size. However, 10 MPa did not alter the mineral balance or whey proteins denaturation largely, except by reduction of some β-sheets and α-helices, due to heat-induced conformational changes at 75 and 95°C.     

Supramolecular structure of the casein micelle

The supramolecular structure of colloidal casein micelles in milk was investigated by using a sample preparation protocol based on adsorption of proteins onto a poly-l-lysine and parlodion-coated copper grid, staining of proteins and calcium phosphate by uranyl oxalate, instantaneous freezing, and drying under a high vacuum. High-resolution transmission electron microscopy stereo-images were obtained showing the interior structure of casein micelles. On the basis of our interpretation of these images, an interlocked lattice model was developed in which both casein-calcium phosphate aggregates and casein polymer chains act together to maintain casein micelle integrity. The caseins form linear and branched chains (2 to 5 proteins long) interlocked by the casein-stabilized calcium phosphate nanoclusters. This model suggests that stabilization of calcium phosphate nanoclusters by phosphoserine domains of α_s1-, α_s2-, or β-casein, or their combination, would orient their hydrophobic domains outward, allowing interaction and binding to other casein molecules. Other interactions between the caseins, such as calcium bridging, could also occur and further stabilize the supramolecule. The combination of having an interlocked lattice structure and multiple interactions results in an open, sponge-like colloidal supramolecule that is resistant to spatial changes and disintegration. Hydrophobic interactions between caseins surrounding a calcium phosphate nanocluster would prevent complete dissociation of casein micelles when the calcium phosphate nanoclusters are solubilized. Likewise, calcium bridging and other electrostatic interactions between caseins would prevent dissociation of the casein micelles into casein-calcium phosphate nanocluster aggregates when milk is cooled or urea is added to milk, and hydrophobic interactions are reduced. The appearance of both polymer chains and small aggregate particles during milk synthesis would also be expected based on this interlocked lattice model of casein micelles, and its supramolecule structure thus exhibits the principles of self-aggregation, interdependence, and diversity observed in nature.     

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.     

pH treatment as an effective tool to select the functional and structural properties of yak milk caseins

Qula is made from yak milk after defatting, acidifying, and drying. Yak milk caseins are purified from Qula by dissolving in alkali solution. The effects of different pH treatments on the functional and structural properties of yak milk caseins were investigated. Over a broad range of pH (from 6.0 to 12.0), functional properties of yak milk caseins, including solubility, emulsifying activities, and thermal characteristics, and the structural properties, including 1-anilino-8-naphthalene-sulfonate fluorescence, turbidity and particle diameter, were evaluated. The results showed that the yak milk casein yield increased as the pH increased from 6.0 to 12.0. The solubility dramatically increased as the pH increased from 6.0 to 8.0, and decreased as the pH increased from 9.0 to 12.0. The changes in emulsifying activity were not significant. Caseins were remarkably heat stable at pH 9.0. The turbidity of the casein solution decreased rapidly as the pH increased from 6.0 to 12.0, and the results suggested that reassembled casein micelles were more compact at low pH than high pH. At pH values higher than 8.0, the yield of yak milk caseins reached more than 80%. The highest solubility was at pH 8.0, the best emulsification was at pH 10.0 and the greatest thermal stability was at pH 9.0. According to the functional characteristics of yak milk caseins, alkali conditions (pH 8.0–10.0) should be selected for optimum production. These results suggested that pH-dependent treatment could be used to modify the properties of yak milk caseins by appropriate selection of the pH level.     

Influence of ethanol on the rennet-induced coagulation of milk

The influence of ethanol on the rennet-induced coagulation of milk was studied to investigate potential synergistic effects of these two mechanisms of destabilisation on the casein micelles. Addition of 5% (v/v) ethanol reduced the rennet coagulation time (RCT) of milk, whereas higher levels of ethanol (10-20%, v/v) progressively increased RCT. The temperature at which milk was coagulable by rennet decreased with increasing ethanol content of the milk. The primary stage of rennet coagulation, i.e., the enzymatic hydrolysis of kappa-casein, was progressively slowed with increasing ethanol content (5-20%, v/v), possibly due to ethanol-induced conformational changes in the enzyme molecule. The secondary stage of rennet coagulation, i.e., the aggregation of kappa-casein-depleted micelles, was enhanced in the presence of 5-15% ethanol, the effect being largest at 5% ethanol. Enhanced aggregation of micelles is probably due to an ethanol-induced decrease in inter-micellar steric repulsion. These results indicate an interrelationship between the effects of ethanol and chymosin on the casein micelles in milk, which may have interesting implications for properties of dairy products.     

Effect of heat and transglutaminase on solubility of goat milk protein‐based films

The effect of heat, transglutaminase and combination of heat and transglutaminase treatments on the solubility of films prepared from goat milk casein, goat milk whey proteins and whole goat milk proteins was investigated. Goat milk casein films were less soluble when treated with transglutaminase and combination of heat with transglutaminase compare with heat‐treated caseins alone. Heat treatment was more effective at decreasing the solubility of whey protein films. SDS‐PAGE patterns demonstrated that goat milk caseins were better cross‐linked by transglutaminase, whereas whey proteins were better cross‐linked by heat. The extent of cross‐linking was further enhanced when a combination of heat and transglutaminase was used.     

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.     

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.     

Casein micelle dissociation in skim milk during high-pressure treatment: Effects of pressure, pH, and temperature

The effect of pH (from 5.5 to 7.5) and temperature (from 5 to 40°C) on the turbidity of reconstituted skim milk powder was investigated at ambient pressure and in situ under pressure (up to 500 MPa) by measurement of light scattering. High-pressure treatment reduced the turbidity of milk for all combinations of pH and temperature due to micelle dissociation. The turbidity profiles had a characteristic sigmoidal shape in which almost no effect on turbidity was observed at low pressures (100 MPa), followed by a stronger pressure dependency over a pressure range of 150 MPa during which turbidity decreased extremely. From the turbidity profiles, the threshold pressure for disruption of micelle integrity was determined and ranged from 150 MPa at low pH to 350-400 MPa at high pH. The threshold pressure diagram clearly showed a relationship between the barostability of casein micelles and pH, whereas almost no effect of temperature was shown. This remarkable pH effect was a consequence of pressure-induced changes in the electrostatic interactions between colloidal calcium phosphate and the caseins responsible for maintaining micellar structure and was explained by a shift in the calcium phosphate balance in the micelle-serum system. Accordingly, a mechanism for high pressure-induced disruption of micelle integrity is suggested in which the state of calcium plays a crucial role in the micelle dissociation process.     

Acylation and Solubility of Casein Precipitated by Carbon Dioxide

Casein precipitated from milk using carbon dioxide (CO₂-casein) and calcium caseinate were acetylated and succinylated. Aqueous solubility of casein was measured at pH 3, 5, 7, and 9. Acetylation extent was lower for CO₂-casein while both succinylated to similar extents. Solubility between the unmodified caseins was similar except at pH 5 with CO₂-casein being less soluble. Succinylation enhanced solubility for both caseins at pH 5, 7, and 9 with similar solubility between the succinylated caseins at each pH. Acetylation enhanced solubility at pH 5, however CO₂-casein still possessed lower solubility than calcium caseinate. Acylation and solubility behavior indicated structural features in CO₂-casein which were not present in calcium caseinate.     

Skim milk protein distribution as a result of very high hydrostatic pressure

This work studies the micellar size and the distribution of caseins, major and minor whey proteins in different fractions of skim milk treated up to 900 MPa for 5 min. Transmission electron microscopy showed that the smallest casein micelles were formed around 450 MPa with no variations at higher pressures. The changes found in micellar size correlated with the concentration of soluble casein, because treatments at 250 MPa significantly enhanced the level of non-sedimentable casein while, between 700 and 900 MPa, there were no further increases with respect to lower pressures. There was a severe β-lactoglobulin (β-Lg) denaturation at pressures ≥ 700 MPa, which reached 77–87%. α-Lactalbumin (α-La) was stable up to 550 MPa, but it denatured at higher pressures. The content of soluble lactoferrin (Lf) decreased with pressure, particularly from 550 to 800 MPa, while that of secretory IgA (sIgA) progressively decreased from 250 up to 700 MPa. Our results indicated that treatment of milk at very high pressures, from 700 to 900 MPa, did not reduce micellar size nor released more soluble casein with respect to treatments at lower pressures (250–550 MPa). However, these treatments led to a severe denaturation of the whey proteins, in particular of β-Lg and the minor proteins Lf and sIgA. The possibility of using high hydrostatic pressure to obtain a soluble milk fraction with a casein and whey protein composition similar to that of human milk is discussed.     

Composition and Structure of Fat Globule Surface Layers in Recombined Milk

Protein coverage, composition and structure of surface layers of fat globules in recombined milk were determined. Average protein load was ～6 mg/m² fat surface. Both casein and whey proteins were present in the fat globule surface layer, with casein adsorbed in preference to whey proteins and α_s (α_sl+α_s2)-casein adsorbed in preference to β-casein. Transmission electron microscopy showed that the surface layer of fat globule was made up of casein micelles, fragments of casein micelles and a thin layer of protein, possibly whey proteins. Experiments with surface layers that had been dispersed in EDTA showed that the extent of dissociation of caseins followed the order: β-casein > α_s-casein ≦ K-casein, suggesting that most of the K-casein was probably associated directly with the fat surface.     

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.     

Characterization of ethanol-induced casein micelle dissociation using a continuous protein monitoring unit

The effect of ethanol on milk has been shown to be temperature-dependent, with higher ethanol concentrations and temperatures reversibly dissociating casein micelles. This work looked to expand on this knowledge, while also demonstrating the efficiency and precision of a custom-made continuous monitoring unit that combines solutions at defined concentrations and temperatures while measuring various parameters (i.e., absorbance, fluorescence, pressure). Caseins were found to self-associate at moderate ethanol concentrations (i.e., 12–36% vol/vol ethanol); however, they dissociated and remained in the serum at higher ethanol concentrations (≥48% vol/vol) and temperatures (24 and 34°C). Although serum casein content was found to be positively correlated with protein hydrophobicity, the addition of ethanol only increased protein hydrophobicity when the sample was held at high temperatures (34–64°C). Overall, the greatest dissociation of casein micelles was found between 40 and 60% (vol/vol) ethanol concentration at elevated temperatures (≥34°C). At these ethanol concentrations and temperatures, skim milk absorbance was minimized, serum casein content (including β-casein content) was maximized, and protein hydrophobicity reached a relative maximum.