Casein Micelles In Skim Milk Research Articles

Effect of temperature and protein concentration on the protein types within the ultracentrifugation supernatant of liquid micellar casein concentrate

Liquid micellar casein concentrate (MCC) is an ideal milk-based protein ingredient for neutral-pH ready-to-drink beverages. The texture and mouthfeel of liquid MCC-based beverages depend on the beverage protein content, as well as the composition of soluble proteins in the aqueous phase around the casein micelle. The objective of this study was to determine the composition of soluble proteins in the aqueous phase around the casein micelles in skim milk and liquid MCC containing 7.0% and 11.6% protein content. Skim milk was pasteurized and concentrated to 7% protein content by microfiltration and then to 18% protein content by ultrafiltration. The 18% MCC was then serially diluted with distilled water to produce 11.6% and 7.0% protein MCC. Skim milk, 7.0% MCC, and 11.6% MCC representing starting materials with different protein concentrations were each ultracentrifuged at 100,605 × g for 2 h. The ultracentrifugation for each of the starting materials was performed at 3 different temperatures: 4°C, 20°C, and 37°C. The ultracentrifugation supernatants were collected to represent the aqueous phase around the casein micelle in MCC solutions. The supernatants were analyzed by Kjeldahl to determine the crude protein, casein, and casein as a percentage of crude protein content, and by sodium dodecyl sulfate PAGE to determine the composition of the individual proteins. Most of the proteins in MCC supernatant (about 45%) were casein proteolysis products. The remaining proteins in the MCC supernatant consisted of a combination of intact αS-, β-, and κ-caseins (about 40%) and serum proteins (14-18%). Concentrations of αS-casein and β-casein in the supernatant increased with decreasing temperature, especially at higher protein concentrations. Temperature and interaction between temperature and protein explained about 80% of the variation in concentration of supernatant αS- and β-caseins. Concentration of supernatant κ-casein, casein proteolysis products, and serum protein increased with increasing MCC protein concentration, and MCC protein concentration explained most of the variation in supernatant κ-casein, casein proteolysis products, and serum protein concentrations. Predicted MCC apparent viscosity was positively associated with the dissociation of αS- and β-caseins. Optimal beverage viscosity could be achieved by controlling the dissociation of these proteins in MCC.

Influence of Chymosin on Physicochemical and Hydrolysis Characteristics of Casein Micelles and Individual Caseins.

The effects of chymosin on the physicochemical and hydrolysis characteristics of casein micelles and individual caseins were investigated. Adding 0.03 units of chymosin/mL led to the casein micelles in skim milk coagulating after a 3 h incubation period at 30 °C. SDS–PAGE investigation showed that β-CN, κ-CN, αs-CN, and a portion of β-lactoglobulin (β-LG) in the milk supernatant fraction (MSF) were precipitated into the milk pellet fraction (MPF). The mean particle size of the MSF with chymosin decreased from 254.4 nm to 179.2 nm after a 3 h incubation period. Mass spectrometry and SDS–PAGE analysis suggested that chymosin hydrolyzed individual β-CN, κ-CN, and αs-CN, but not β-LG. Chymosin hydrolysis led to a decrease in the molecular weights of the hydrolyzed β-CN, κ-CN, and αs-CN. Particle size analysis indicated that there was no difference in the particle size distribution of hydrolyzed β-CN and αs-CN. Moreover, our outcomes demonstrated that the hydrolysis of κ-CN by chymosin occurs before that of β-CN and αs-CN.

The relationship between ultra-small-angle X-ray scattering and viscosity measurements of casein micelles in skim milk concentrates

Skim milk concentrates have important applications in the dairy industry, often as intermediate ingredients. Concentration of skim milk by reverse osmosis membrane filtration induces water removal, which reduces the free volume between the colloidal components, in particular the casein micelles. Thermal treatment before or after concentration impacts the morphology of casein micelles. These changes affect the flow behavior and viscosity, but the consequences for supermicellar structure have not been elucidated. In the present study, skim milk concentrates with different total solid contents from 8.7% (control) up to 22.8% (w/w), prepared by reverse osmosis membrane filtration of non-heated and pasteurized skim milk, were heat treated at 75 °C for 18 s, and compared with non-heated concentrates. The structure of the concentrates was studied using Ultra Small Angle X-ray Scattering (USAXS), and the viscosity of concentrates was measured. The USAXS intensity I(q) was fitted at small and intermediate q-regions (0.0005 < q < 0.003 Å−1 and 0.0035 < q < 0.03 Å−1, respectively) with a power law. The value of the power law exponent was used to assess the heat- and concentration-induced aggregation of the milk solids and correlate it with the apparent viscosity. The results showed that increased viscosity of skim milk concentrates, due to water removal and heat-load, can be explained by increased aggregation of the casein micelles into elongated aggregates and increased smoothening of the casein micelle surface.

A novel approach for characterisation of stabilising bonds in milk protein deposit layers on microfiltration membranes

The gel formation mechanism of low-pressure casein gels during the filtration of skim milk remains unknown this far. We investigated the prevailing bond types in this gel and correlated these findings with deposit layer properties and filtration performance. At high pH and low transmembrane pressure, calcium bridges predominated among the bond types stabilising the deposit layer gel. However, there were less calcium bridges present than within or between casein micelles in skim milk. With a decreasing amount of calcium bridges at low pH and higher transmembrane pressures, the deposit layer turned into a dense and mushy gel, which impaired solvent and solute permeability. Additionally, ionic and hydrogen bonds, as well as disulphide bonds, were formed between the highly concentrated deposited proteins during gelation. Hydrophobic interactions played a negligible role. We concluded that intramicellar calcium bridges are associated with a voluminous micelle and a stable gel backbone, which benefits filtration efficiency.

The association of lipophilic phospholipids with native bovine casein micelles in skim milk: Effect of lactation stage and casein micelle size

A known biological role of casein micelles is to transport calcium from mother to young and provide amino acids for growth and development. Previous reports demonstrated that modified casein micelles can be used to transport and deliver hydrophobic probes. In this study, the distribution of lipid-soluble phospholipids, including sphingomyelins (SM) and phosphatidylcholines (PC), was quantified in whole raw milk, skim raw milk, and casein micelles of various sizes during early, mid, and late lactation stages. Low-pressure size exclusion chromatography was used to separate casein micelles by size, followed by hydrophobic extraction and liquid chromatography-mass spectrometry for the quantification of PC and SM. Results showed that the SM d18:1/23:0, d18:1/22:0, d18:1/16:0, d16:1/22:0, d16:1/23:0, and d18:1/24:0 and the PC 16:0/18:1, 18:0/18:2, and 16:0/16:0 were dominating candidates appearing in maximum concentration in whole raw milk obtained from late lactation, with 21 to 50% of total SM and 16 to 35% of total PC appearing in skim milk. Of the total SM and PC found in skim milk, 35 to 46% of SM and 22 to 29% of PC were associated with the casein micelle fraction. The highest concentrations of SM d18:1/22:0 (341 ± 17 µg/g of casein protein) and PC 16:0/18:1 (180 ± 20 µg/g of casein protein) were found to be associated with the largest casein micelles (diameter = 149 nm) isolated in milk from late lactation, followed by a decrease in concentration as the casein micelle size decreased.

Colloidal properties of casein micelles suspensions as a function of pH during concentration by osmotic stressing

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

Heat stability of yak micellar casein as affected by heat treatment temperature and duration

Heat treatment is one of the essential operations widely used in most dairy processes, and heat stability is one of the essential properties of milk. Casein micelles are the major component in milk responsible for the heat stability of milk during processing. This study assessed the effects of heat treatment temperature and duration on the average size, turbidity, polydispersity index and heat stability of casein micelles in yak skim milk and distilled water. The results showed that whey protein had an important role in influencing the heat stability of casein micelles. The average size, polydispersity index and turbidity of micelles in skim milk were higher than those of micelles in distilled water in all cases while the heat stability of casein micelles in skim milk was lower than those in distilled water. As a result of the heat treatment, the size of micelles in skim milk increased due to complex of casein/whey protein formed via covalent bonds, whereas it decreased in distilled water attributed to the change of hydrophobicity in micelles. The size distribution of particles broadened with increasing heating temperature, resulting in the increase in turbidity and polydispersity index of casein micelles both in skim milk and distilled water. The micelles in skim milk combined with whey protein during heating. These findings will help processors design appropriate heating conditions for yak milk and yak casein products and help identify new opportunities for product development.

Temperature-dependent dynamics of bovine casein micelles in the range 10–40 °C

Milk is a complex colloidal system that responds to changes in temperature imposed during processing. Whilst much has been learned about the effects of temperature on milk, little is known about the dynamic response of casein micelles to changes in temperature. In this study, a comprehensive physico-chemical study of casein micelles in skim milk was performed between 10 and 40°C. When fully equilibrated, the amount of soluble casein, soluble calcium and the pH of skim milk all decreased as a function of increasing temperature, whilst the hydration and volume fraction of the casein micelles decreased. The effect of temperature on casein micelle size, as determined by dynamic light scattering and differential centrifugation, was less straightforward. Real-time measurements of turbidity and pH were used to investigate the dynamics of the system during warming and cooling of milk in the range 10–40°C. Changes in pH are indicative of changes to the mineral system and the turbidity is a measure of alterations to the casein micelles. The pH and turbidity showed that alterations to both the casein micelles and the mineral system occurred very rapidly on warming. However, whilst mineral re-equilibration occurred very rapidly on cooling, changes to the casein micelle structure continued after 40min of measurement, returning to equilibrium after 16h equilibration. Casein micelle structure and the mineral system of milk were both dependent on temperature in the range 10–40°C. The dynamic response of the mineral system to changes in temperature appeared almost instantaneous whereas equilibration of casein was considerably slower, particularly upon cooling.

Binding of vitamin A by casein micelles in commercial skim milk

Recent studies have shown that reassembled micelles formed by caseinates and purified casein fractions (αs- and β-casein) bind to hydrophobic compounds, including curcumin, docosahexaenoic acid, and vitamin D. However, limited research has been done on the binding of hydrophobic compounds by unmodified casein micelles in skim milk. In the present study, we investigated the ability of casein micelles in commercial skim milk to associate with vitamin A (retinyl palmitate), a fat-soluble vitamin commonly used to fortify milk. Milk protein fractions from different commercially available skim milk samples subjected to different processing treatments, including pasteurized, ultrapasteurized, organic pasteurized, and organic ultrapasteurized milks, were separated by fast protein liquid chromatography. The fractions within each peak were combined and freeze-dried. Sodium dodecyl sulfate-PAGE with silver staining was used to identify the proteins present in each of the fractions. The skim milk samples and fractions were extracted for retinyl palmitate and quantified against a standard using normal phase-HPLC. Retinyl palmitate was found to associate with the fraction of skim milk containing caseins, whereas the other proteins (BSA, β-lactoglobulin, α-lactalbumin) did not show any binding. The retinyl palmitate content in the various samples ranged from 1.59 to 2.48µg of retinyl palmitate per mL of milk. The casein fractions contained between 14 and 40% of total retinyl palmitate in the various milks tested. The variation in the retention of vitamin A by caseins was probably explained by differences in the processing of different milk samples, including thermal treatment, the form of vitamin A emulsion used for fortification, and the point of fortification during processing. Unmodified casein micelles have a strong intrinsic affinity toward the binding of vitamin A used to fortify commercially available skim milks.

Lactoferrin binding to transglutaminase cross-linked casein micelles

Casein micelles in skim milk were either untreated (untreated milk) or were cross-linked using transglutaminase (TGA-milk). Added lactoferrin (LF) bound to the casein micelles and followed Langmuir adsorption isotherms. The adsorption level was the same in both milks and decreased the micellar zeta potential, indicating charge neutralization and the formation of complex coacervate-type interactions. For the untreated milks, the adsorption of LF was initially accompanied by an increase in turbidity of the milk and in the size of the casein micelles; however, after several hours, the turbidity and the casein micelle size of these milks decreased markedly. For the TGA-milks, no change in casein micelle size was observed on adsorption of LF, but the turbidity increased due to the increased mass of the casein micelles, and remained constant on holding. These results indicate that the cross-linking of the casein micelles prior to adding LF prevents disintegration of the casein micelles.

Probing the colloidal properties of skim milk using acoustic and electroacoustic spectroscopy. Effect of concentration, heating and acidification

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

Observations of casein micelles in skim milk concentrate by transmission electron microscopy

The microstructure of casein micelles in ultrafiltrated (UF) skim milk concentrate at pH 6.5 and 5.8 was investigated by transmission electron microscopy using three different preparation methods. The volume fraction of the casein micelles in the UF concentrate was 62.8% (v/v) at pH 6.5 and fixation by glutaraldehyde revealed the close packing of micelles in the UF concentrate as well as a higher degree of micelle aggregation at pH 5.8. No details of the microstructure of the micellar surface or core could, however, be observed. Freeze-fracture of cryoprotected, i.e. glycerol, UF concentrate on the other hand, exposed the finer structures of the micellar core but no pH dependent differences were observed. As cryoprotection includes a dilution of the sample with glycerol, the packing of the micelles in the UF concentrate could not be observed. Undiluted UF concentrate exposed to rapid freezing using a propane jet followed by freeze-fracture exhibited development of ice crystals but rough areas on the micrographs were identified as fractured casein micelles. The micellar core appeared rougher and differences in the micellar core microstructure due to changing pH could be observed when this preparation method was used.

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.

Molecular Mechanism of the Renneting Process of Casein Micelles in Skim Milk, Examined by Viscosity and Light-Scattering Experiments and Simulated by Model SCF Calculations

The properties of the outer hairy layer of casein micelles have been studied using several methods. Viscosity and light-scattering measurements have been performed on rennet-destabilized skim milk at different concentrations of calcium. We have reproduced the known result that calcium promotes destabilization at relatively high κ-casein surface coverages (even at only 50% of the κ-casein molecules cut off). This suggests that calcium bridges contribute to the casein micelle attraction. The effect may be direct by calcium-mediated bridging of β-casein or αs1-casein (phosphate/carboxylate) or κ-casein (carboxylate) or indirect by altering the interplay between several types of caseins in the micelle. In the analysis of the experiments, we make use of an adhesive hard sphere approximation. The casein hairy layer has further been modeled by a self-consistent field (SCF) theory in which coarse-grained molecular details were included. In these calculations the effect of calcium bridges cannot directly be accounted for. It is shown that a κ-casein layer induces repulsive interactions of mainly steric origin. Cutting the N-terminus part results in reduced (shorter-ranged) repulsive pair potentials, which still dominate over the van der Waals attraction. As a result of the altered interplay between the several types of casein molecules during the renneting process, the αs1-casein concentration in the outer hairy layer may increase. Again using the SCF model, but now using a less-detailed description of the system, we examine a possible result of this. We confirm that diblock copolymers (simple model for κ-casein) and diblock copolymer with shorter soluble block (simple model for para-κ-casein) give repulsion and triblock copolymers (simple model for αs1-casein) give attraction by means of bridging (adsorption). The renneting process is simulated by increasing the ratio of model para-κ-casein to κ-casein at a fixed αs1-casein concentration. As a result, the potential shifts from repulsive to attractive. We show that by reducing the range of repulsive interactions of the diblock copolymers, we can make room available to express the attractive contributions of the model αs1-casein. This comprises a novel molecular-based mechanism for the renneting process.

Association of β-Lactoglobulin and β-Lactalbumin with the Casein Micelles in Skim Milk Heated in an Ultra-high Temperature Plant

Abstract Skim milk was subjected to various time–temperature treatments using a direct steam injection (DSI) system in a pilot-scale ultra-high temperature (UHT) plant. The heated samples were ultracentrifuged and the supernatants analysed using quantitative polyacrylamide gel electrophoresis to determine the extent of denaturation of β-lactoglobulin (β-lg) and α-lactalbumin (α-la) and their association with the casein micelles. The extent of β-lg and α-la denaturation and association increased with an increase in both heating time and temperature; the rate of association was markedly less than that of denaturation. The association behaviour was affected by heating temperature; during the initial stages of heating in the range 80–130°C, mainly β-lg appeared to associate with the casein micelles, but after prolonged heating, α-la began to associate with the micelle. In contrast, below 80°C both β-lg and α-la appeared to associate simultaneously with the micelles. The maximum level of association of α-la varied with heating temperature, ∼40% of total in the range 95–130°C and ∼55% below 90°C. For β-lg, the maximum level of association was ∼55% of total regardless of the temperature. A pseudo-first-order model was used to calculate the reaction kinetics of the association of β-lg with the casein micelle.

The effect of chymosin on κ-casein-coated polystyrene latex particles and bovine casein micelles

Abstract Bovine κ-casein was adsorbed onto negatively charged polystyrene latex particles of various sizes and the changes in the hydrodynamic diameters of the particles were monitored by photon correlation spectroscopy. The latex particle diameters were increased on addition of small amounts of κ-casein, and plateaued once a maximum adsorption was attained. The size increase was about 25 nm regardless of the initial size of the latex particle, indicating that the surface arrangement of κ-casein was similar on all latex sizes. Treatment of the casein micelles in skim milk and the κ-casein-coated latex particles with chymosin initially decreased the particle diameter by about 10nm after which the particle diameter rapidly increased as the aggregation reaction proceeded. The action of chymosin on both the casein micelles and the κ-casein-coated latex particles was retarded by increasing the NaCl concentration. The similarity between the κ-casein-coated latex particles and the casein micelles in milk, especially their behaviour towards chymosin, indicates that these latex particles may be a simple model system for studying some casein micelle properties under controlled conditions.

Electroacoustic determination of size and zeta potential of casein micelles in skim milk

SummaryMeasurements of the zeta potential and particle size of casein micelles in skim milk suspensions at natural and lower pH have been made using the technique of electroacoustics. This technique requires no dilution or change of environment of the casein micelles. The zeta potential obtained at natural pH for a commercial skim milk suspension was −18 mV; it became less negative with decreasing pH. The median particle size observed at natural pH for a commercial skim milk suspension was 0·2 εm, in good agreement with previously reported values. The particle size increased as the pH was decreased.

Effect of temperature and pH on the interactions of whey proteins with casein micelles in skim milk

Skim milk was heated at temperatures in the range 75–90 °C, at pH values of 6.8, 6.2 and 5.8, The amounts of α-lactalbumin and β-lactoglobulin which interacted with the casein micelles during heat treatment were quantified by SDS-polyacrylamide gel electrophoresis of the micellar fractions isolated by ultra-centrifugation. Both α-lactalbumin and β-lactoglobulin appeared to interact similarly with casein micelles at temperatures up to 85 °C. The amount of whey protein complexed with micelles increased with time, reaching plateau values that, at the highest temperatures, were comparable with the quantity present in the original skim milk. In general, faster reaction of the whey proteins with the micelles was found at lower pH and higher temperatures. The rates and extent of the reaction changed also when additional α-lactalbumin and β-lactoglobulin isolates were added to milk before heating. The reaction between α-lactalbumin and casein micelles depended to a relatively small extent upon environmental variations (pH and temperature), while β-lactoglobulin interactions were more affected, so that a more complex behaviour may be attributed to the latter protein.

Fixation of bovine casein micelles for chromatography on controlled pore glass

SummaryGlutaraldehyde was used to fix the size distribution of casein micelles in skim milk before their fractionation by permeation chromatography on controlled pore glass. The effect of fixation was assessed by comparing the size and absorption profile for column fractions obtained from samples which were fixed before fractionation with those of unfixed samples and of samples that were fixed only after completion of chromatography. Micelle size was determined by photon correlation spectroscopy. Column profiles were obtained from absorption measurements at 340 nm and after pronase digestion at 280 nm to determine relative protein concentration. For comparative purposes the elution profile was divided into 4 peak areas, of which I-III contained most of the casein micelles, and IV consisted of the smallest micelles, soluble casein and whey protein. Average micelle size was unaltered by fixation but was larger in fractions from prefixed skim milk than those from unfixed samples. Fixation increased the absorbance readings but the elution profile at 340 nm for peak areas I-III was essentially the same for unfixed fractions and those obtained when fixation was applied after chromatography. However, areas I-III accounted for a much larger proportion of the total profile area when the latter procedure was followed. Total profile area increased with fixation time and temperature but this did not affect the elution profile. The results indicate that fixation with glutaraldehyde does not induce artefactual changes in casein micelle size.

Molecular Weight of Bovine Milk Casein Micelles From Diffusion and Viscosity Measurements

We have measured the average molecular weight of casein micelles in skimmilk by combining diffusion and intrinsic viscosity results. The diffusion coefficients and hydrodynamic radius were measured by inelastic laser light scattering spectroscopy. The results in simulated milk ultrafiltrate buffer at 25C were diffusion coefficient D = 2.18×10-8 cm2/sec, hydrodynamic radius Rh = 941 A, intrinsic viscosity [ђ] = .1011 100 ml/g, and molecular weight M = 5.20×108. This value for the molecular weight, obtained entirely by measurements in solution and not depending on assumptions about the voluminosity of the micelles, is about half that obtained by Nitschmann from electron microscopy. The average micelle molecular weight by these measurements is approximately weight average.