Size Distribution Of Casein Micelles Research Articles

A quantitative calcium phosphate nanocluster model of the casein micelle: the average size, size distribution and surface properties.

Caseins (αS1, αS2, β and κ) are the main protein fraction of bovine milk. Together with nanoclusters of amorphous calcium phosphate (CaP) and divalent cations, they combine to form a polydisperse distribution of particles called casein micelles. A casein micelle model is proposed which is consistent with the way in which intrinsically disordered proteins interact through predominantly polar, short, linear, motifs. Using the model, an expression is derived for the size distribution of casein micelles formed when caseins bind to the CaP nanoclusters and the complexes further associate with each other and the remaining mixture of free caseins. The result is a refined coat-core model in which the core is formed mainly by the nanocluster complexes and the coat is formed exclusively by the free caseins. Example calculations of the size distribution and surface composition of an average bovine milk are compared with experiment. The average size, size distribution and surface composition of the micelles is shown to depend on the affinity of the nanocluster complexes for each other in competition with their affinity for free caseins, and on the concentrations of free caseins, calcium ions and other salts in the continuous phase.

Investigating the effect of powder manufacturing and reconstitution on casein micelles using asymmetric flow field-flow fractionation (AF4) and transmission electron microscopy

Milk powders are commonly used for a variety of food products in which among others the milk proteins add to the properties of the products. Processing of milk can, depending on the processing parameters, change the size and structure of the proteins. These changes can be difficult to measure due to the polydispersity of milk components, which makes it a challenge to obtain direct information about the individual proteins. In this paper, the results from an investigation of casein micelle size,size distribution, and structure in reconstituted skim milk and the comparison with raw and pasteurized skim milk are reported. The investigation used asymmetrical flow field-flow fractionation (AF4) in combination with online UV, multi-angle light scattering (MALS), and refractive index (RI) detection and the results were confirmed by transmission electron microscopy (TEM). The results show that there is a difference in casein micelle size distribution between the differently processed milk samples. The casein micelles of the reconstituted milk were found to have a z-average radius of gyration of 72 nm and the casein micelles in the raw and pasteurized skim milk were 58 and 62 nm respectively. The AF4 and TEM data suggest that the cause of the larger casein micelle size is a layer of aggregated whey proteins associated with the casein micelles surface. Moreover, the TEM investigation showed that a larger proportion of the casein micelles are aggregated in reconstituted milk compared to raw and fresh skim milk. Investigation of the effect of reconstitution time shows that the amount of aggregated casein micelles decreases during the first 20 min of reconstitution. The results show that the AF4-method can provide detailed insights into the reconstitution process and properties of different milk samples. Hence, it can be used as a reference or validation for more indirect methods to track the reconstitution of milk powders.

Composition, Structure, and Digestive Dynamics of Milk From Different Species-A Review.

Background: The traditional dairy-cattle-based industry is becoming increasingly diversified with milk and milk products from non-cattle dairy species. The interest in non-cattle milks has increased because there have been several anecdotal reports about the nutritional benefits of these milks and reports both of individuals tolerating and digesting some non-cattle milks better than cattle milk and of certain characteristics that non-cattle milks are thought to share in common with human milk. Thus, non-cattle milks are considered to have potential applications in infant, children, and elderly nutrition for the development of specialized products with better nutritional profiles. However, there is very little scientific information and understanding about the digestion behavior of non-cattle milks.Scope and Approach: The general properties of some non-cattle milks, in comparison with human and cattle milks, particularly focusing on their protein profile, fat composition, hypoallergenic potential, and digestibility, are reviewed. The coagulation behaviors of different milks in the stomach and their impact on the rates of protein and fat digestion are reviewed in detail.Key findings and Conclusions: Milk from different species vary in composition, structure, and physicochemical properties. This may be a key factor in their different digestion behaviors. The curds formed in the stomach during the gastric digestion of some non-cattle milks are considered to be relatively softer than those formed from cattle milk, which is thought to contribute to the degree to which non-cattle milks can be easily digested or tolerated. The rates of protein and fat delivery to the small intestine are likely to be a function of the macro- and micro-structure of the curd formed in the stomach, which in turn is affected by factors such as casein composition, fat globule and casein micelle size distribution, and protein-to-fat ratio. However, as no information on the coagulation behavior of non-cattle milks in the human stomach is available, in-depth scientific studies are needed in order to understand the impact of compositional and structural differences on the digestive dynamics of milk from different species.

The factors influencing rennet-induced coagulation properties of yak milk: The importance of micellar calcium during gelation

The objective of this study was to evaluate the importance and effects of age, hair color, lactation stage and number of yak, as well as physicochemical properties and composition of yak milk on rennet-induced coagulation characteristics. Results showed that casein micelle size distribution and total calcium content of milk from 68 yaks were the most important factors affecting the gelation characteristics, including gelling time and gel strength. The yak milk containing smaller casein micelles and higher calcium content formed stronger gels and had shorter incubation time. Moreover, casein micelle samples containing 20.6, 16.8, and 13.9 mmol/L micellar calcium were obtained through pH-induced dialysis method. The results clearly showed that although the removal of micellar calcium did not affect casein macropeptide (CMP) release, the gelation of samples containing lower micellar calcium value corresponded to lower aggregation rate of para-casein micelles and more CMP release. In addition, removal of micellar calcium may lead to longer incubation time and sparser gel structure. This study highlights that higher micellar calcium is indispensable to the aggregation of yak casein micelles and the formation of firm gel, because enough micellar calcium may promote the aggregation of para-casein and firmly links casein micelles.

Effect of cryoconcentration, reverse osmosis and vacuum evaporation as concentration step of skim milk prior to drying on the powder properties

Skim milk was concentrated by cryoconcentration (CC), reverse osmosis (RO) and vacuum evaporation (VE), and the concentrated milks were spray-dried. The powder morphology and shape were observed by electron microscopy and the mean size distribution by a sieve jet method. The resulting powders were reconstituted to 25% total dry matter (TDM) to carry out different measurements such as particle size distribution in solution, zeta-potential, heat stability, color, wettability, solubility and dispersibility. The surface of the particles was smooth with small amount of visible substructures on powder surface concentrated by VE and RO. The CC and RO corresponding powders have less particle fragments than VE powder. The mean particle size was larger for CC, followed by RO and VE powders. At the size of 250nm, the CC-powder occupied three times the volume (%) occupied by the RO-powder and the VE-powder. Analysis of reconstituted powders showed that the mean size distribution of casein micelles was found 190nm, 164 and 164nm for CC, RO and VE reconstituted powders, respectively. Moreover, it has been shown that RO and VE powder produced firm gels when heated whereas the CC powder presented flocs and weak gels. As it, all powders exhibited high solubility index (˃95%). However, depending on the size, the lowest solubility in water was observed at 75nm. Regarding the powder dispersibility, they presented a good dispersibility (<90%). The powders wettability was also size-dependent and was similar for all powders at a given particle size.

Effect of skim milk treated with high hydrostatic pressure on permeate flux and fouling during ultrafiltration.

Ultrafiltration (UF) is largely used in the dairy industry to generate milk and whey protein concentrate for standardization of milk or production of dairy ingredients. Recently, it was demonstrated that high hydrostatic pressure (HHP) extended the shelf life of milk and improved rennet coagulation and cheese yield. Pressurization also modified casein micelle size distribution and promoted aggregation of whey proteins. These changes are likely to affect UF performance. Consequently, this study determined the effect of skim milk pressurization (300 and 600 MPa, 5 min) on UF performance in terms of permeate flux decline and fouling. The effect of HHP on milk proteins was first studied and UF was performed in total recycle mode at different transmembrane pressures to determine optimal UF operational parameters and to evaluate the effect of pressurization on critical and limiting fluxes. Ultrafiltration was also performed in concentration mode at a transmembrane pressure of 345 kPa for 130 or 140 min to evaluate the decline of permeate flux and to determine fouling resistances. It was observed that average casein micelle size decreased by 32 and 38%, whereas β-lactoglobulin denaturation reached 30 and 70% at 300 and 600 MPa, respectively. These results were directly related to UF performance because initial permeate fluxes in total recycle mode decreased by 25% at 300 and 600 MPa compared with nonpressurized milk, critical flux, and limiting flux, which were lower during UF of milk treated with HHP. During UF in concentration mode, initial permeate fluxes were 30% lower at 300 and 600 MPa compared with the control, but the total flux decline was higher for nonpressurized milk (62%) compared with pressure-treated milk (30%). Fouling resistances were similar, whatever the treatment, except at 600 MPa where irreversible fouling was higher. Characterization of the fouling layer showed that caseins and β-lactoglobulin were mainly involved in membrane fouling after UF of pressure-treated milk. Our results demonstrate that HHP treatment of skim milk drastically decreased UF performance.

Impact of cryoconcentration on casein micelle size distribution, micelles inter-distance, and flow behavior of skim milk during refrigerated storage

Abstract Cryoconcentration combined with a cascade effect was used to concentrate skim milk up to 25.12% total dry matter. Size, shape, and inter-micellar distance of casein micelles were characterized by ZetasizerNano-ZS, transmission electron microscopy, and ImageJ analyses. Flow properties of the cryoconcentrated skim milk were evaluated during 5 weeks of storage under refrigerated condition at 4 °C. Milk color was also evaluated according to the L *, a *, and b * system. The cryoconcentrated skim milk obtained after three cryoconcentration cycles was characterized by a monomodal distribution of its micelles with a tendency to smaller casein micelles. Approximately 60% of the total micellar volume was occupied by the casein micelles with a size of 100–200 nm, less than 18% of the volume with a size of 50–100 nm and only less than 1% was occupied by micelles with a size > 350 nm. This result shows that cryoconcentration changed the distribution of the mean size of the casein micelles to smaller units. No significant difference was observed on the inter-micellar distance. Cryoconcentration significantly improved the color of skim milk by increasing the L * value up to 67 which was similar to that of whole milk. Transition from a Newtonian to a non-Newtonian behavior was observed from the fourth week storage with a slight increase of casein micelle size. Industrial relevance A concentration procedure of skim milk based on a complete block cryoconcentration technique was proposed. Application of this sub-zero technology permitted the concentration of skim milk total dry matter up to 25%. The casein micelle size was positively affected by moving the major part of the micelles toward the smaller size, whereas the inter-micellar distance was not affected. This new knowledge can be exploited in milk-based products to enhance the product stability. The cryoconcentrated skim milk color was positively affected since its L * value, which represents the milk whiteness, was significantly improved. The flow behavior of the cryoconcentrated milk was of Newtonian type up to 4 weeks of storage at 4 °C. The generated knowledge in this study can be easily used by the milk processing industry in order to make stable milk product with high dry matter content without adding milk powder, which negatively affects the product sensory properties (floury consistency).

Combination effect of sodium carboxymethyl cellulose and soybean soluble polysaccharides on stability of acidified skimmed milk drinks

Sodium carboxymethyl cellulose (CMC) and soybean soluble polysaccharide (SSPS) are negatively charged polysaccharides that are used to prevent casein aggregation in acidified skimmed milk drinks (ASMDs). The objective of this study was to examine the effect of CMC and SSPS combination on the stability of acidified skimmed milk drinks in comparison to their individual effects. Stability was evaluated based on the changes in sedimentation ratio, viscosity, zeta potential, and particle size distribution of casein micelles. Increased CMC or SSPS concentration resulted in decreased sedimentation ratio and size diameter, while viscosity and zeta potential increased. The use of CMC-SSPS blend at the ratio of 1:3 showed better stability of ASMDs compared to when the two stabilizers were used individually. Sedimentation ratio and size diameter obtained from the ratio of 1:3 (CMC-SSPS) had no clear significant difference (P < 0.05) with those stabilized by the ratio of 0:4, but CMC was crucial in controlling the blend viscosity and zeta potential, hence improving stability. Furthermore, SSPS showed a better stabilization behavior than CMC when they are used alone. It can therefore be suggested that a combination of CMC-SSPS is more effective than the individual use of the two stabilizers in stabilizing ASMDs, and thus, CMC can effectively supplement SSPS in the production of stable ASMDs.

Effect of solvent and temperature on the size distribution of casein micelles measured by dynamic light scattering

The objectives of this study were to investigate the effect of the solvent on the accuracy of casein micelle particle size determination by dynamic light scattering (DLS) at different temperatures and to establish a clear protocol for these measurements. Dynamic light scattering analyses were performed at 6, 20, and 50°C using a 90Plus Nanoparticle Size Analyzer (Brookhaven Instruments, Holtsville, NY). Raw and pasteurized skim milk were used as sources of casein micelles. Simulated milk ultrafiltrate, ultrafiltered water, and permeate obtained by ultrafiltration of skim milk using a 10-kDa cutoff membrane were used as solvents. The pH, ionic concentration, refractive index, and viscosity of all solvents were determined. The solvents were evaluated by DLS to ensure that they did not have a significant influence on the results of the particle size measurements. Experimental protocols were developed for accurate measurement of particle sizes in all solvents and experimental conditions. All measurements had good reproducibility, with coefficients of variation below 5%. Both the solvent and the temperature had a significant effect on the measured effective diameter of the casein micelles. When ultrafiltered permeate was used as a solvent, the particle size and polydispersity of casein micelles decreased as temperature increased. The effective diameter of casein micelles from raw skim milk diluted with ultrafiltered permeate was 176.4±5.3nm at 6°C, 177.4±1.9nm at 20°C, and 137.3±2.7nm at 50°C. This trend was justified by the increased strength of hydrophobic bonds with increasing temperature. Overall, the results of this study suggest that the most suitable solvent for the DLS analyses of casein micelles was casein-depleted ultrafiltered permeate. Dilution with water led to micelle dissociation, which significantly affected the DLS measurements, especially at 6 and 20°C. Simulated milk ultrafiltrate seemed to give accurate results only at 20°C. Results obtained in simulated milk ultrafiltrate at 6°C could not be explained based on the known effects of temperature on the casein micelle, whereas at 50°C, precipitation of amorphous calcium phosphate affected the DLS measurement.

Determination of heat-induced effects on the particle size distribution of casein micelles by dynamic light scattering and nanoparticle tracking analysis

The change in particle size distribution of casein micellar dispersions induced by heating both in the absence and presence of whey proteins was investigated by two techniques. Whereas dynamic light scattering (DLS) is an ensemble technique that tries to recover a particle size distribution from the combined signal of all particles present in the sample, nanoparticle tracking analysis (NTA) investigates the diffusion of individual particles. Both DLS and NTA indicated that heating of casein micellar dispersions in the presence of whey proteins gave rise to a bimodal distribution. Whereas the small diameter mode was not significantly affected by heating, an additional large diameter mode was obtained. This observation indicated the increase in average particle size to be primarily a consequence of casein micellar aggregation, which in itself is a further consequence of heat-induced whey protein deposition onto the casein micelles.

A novel application of neutron scattering on dairy products

The successful application of spin echo small angle neutron scattering (SESANS) has been demonstrated for studying concentrated, turbid colloid suspensions with respect to their structure without dilution. The structure could be studied in a distance range between 50 nm and 3 μm. Data on the size distribution of casein micelles in non-diluted fat-free milk was obtained. This distribution is similar to earlier observations. The structure of casein gels resulting from processes aimed to simulate curdling (early stage of cheese making) and yoghurt production (acidification) was investigated and found to be not very different, the yoghurt gel being somewhat less dense.

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.

Pressurisation of raw skim milk and of a dispersion of phosphocaseinate at 9°C or 20°C: effects on casein micelle size distribution

Abstract Raw skim milk (RSM) and a dispersion of industrial phosphocaseinate (PC) were pressurised for 15 min at 100–300 MPa and 9°C or 20°C. Treatment at 100–150 MPa and 9°C or 20°C induced an increase in the hydrodynamic volume of the main micelle population (30–70 nm) of RSM and PC and a slight dissociation of the largest micelles (300–600 nm), as determined by photon correlation spectroscopy (PCS). At 9°C, micelle dissociation increased progressively with pressure from 150 to 300 MPa, since turbidity and micelle diameters (PCS) of both RSM and PC decreased. At 20°C, pressurisation at ⩾200 MPa induced both micelle dissociation and aggregation phenomena. However, dissociation predominated over aggregation, for both RSM and PC, since small-sized micelles were mainly detected either by PCS (in particle number frequency) or by atomic force microscopy. Treatment at 300 MPa and 9°C or 20°C induced irreversible changes in casein micelles, with resulting diameters half the size of the original micelles.

The Two-Stage Coagulation of Milk Proteins in the Minimum of the Heat Coagulation Time-pH Profile of Milk: Effect of Casein Micelle Size

Milks with casein micelles larger or smaller than control milk were prepared by differential centrifugation. The heat stability of these modified milks increased markedly throughout the pH range 6.4 to 7.1 with decreasing casein micelle size. Within the region of the minimum in the heat coagulation time-pH profile, the control milk coagulated by a two-stage process, but the modified milks, because of their narrower casein micelle size distribution, coagulated by a single-stage process at the pH of minimum stability. The content of κ-CN and protein hydration increased as the size of the casein micelles decreased, and the level of glycosylation of κ-CN and protein surface hydrophobicity increased as a function of micelle size. The effect of casein micelle size on the heat stability of milk is likely to be related to changes in the above physico-chemical properties.

Size distribution and average size parameters of casein micelles determined by electron microscopy in bovine milk between pH 5·5 and 6·7. A comparison of several evaluation methods

SummaryThe conversion of the two-dimensional size distribution of casein micelles, observed by electron microscopy in a plane section, to the three dimensional distribution is discussed and the average size parameters evaluated by several methods are compared. It is shown that parameters containing the −1 moment of the two-dimensional distribution, i.e. Dn, the number of micelles per unit volume and the width of the size distribution, are sometimes uncertain. The occurrence of negative numbers in some of the classes of the distribution is discussed and remedies are suggested. Sections were made by freeze-fracturing skim milk samples; the pH of the milk was between 5·5 and 6·7.

Effect of Ultrafiltration of Skim Milk on Casein Micelle Size Distribution in Retentate

Casein micelles obtained from skim milk concentrated three or five times by UF were mixed with agar and embedded in resin, and their thin sections were examined by transmission electron microscopy. The examination revealed roughly spherical particles with a wide range of sizes similar to the range of sizes found in unaltered skim milk. However, there was a significant change in casein micelle size distribution when the milk was ultrafiltered to higher concentrations. The highest proportion of casein micelles was changed from the 80- to 100-nm range in skim milk to 60 to 80-nm in milk concentrated five times. At higher concentrations, the numbers of casein micelles with diameters less than 80nm increased, but those with diameters greater than 100nm decreased. Consequently, both the volume distribution and the average diameter of the casein micelles were altered. There was a decrease in casein micelle diameter from 118nm in normal skim milk to 92 and 87nm in milk concentrated three or five times, respectively. The change in the composition of casein and minerals, particularly Ca and P, as a result of UF may be responsible for the change in casein micelle size distribution, volume distribution, and average diameter of the casein micelles.

The disaggregation of calcium-depleted casein micelles.

The effect of depletion of Ca2+ on the composition and size distribution of casein micelles in milk has been examined using chemical analysis, size exclusion chromatography, fast protein liquid chromatography, turbidimetry and photon correlation spectroscopy. Partial removal of Ca2+ by EDTA and subsequent dialysis resulted in disaggregation of some of the casein micelles; as the EDTA concentration increased, the proportions of Ca2+ and phosphate relative to protein in the micelles remaining intact decreased. However, the composition of the intact micelles, with respect to the different caseins, and the number-frequency size distribution were essentially unchanged.

Size distribution of casein micelles in camels′ milk

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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.

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.