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Schematic representation of two casein micelles; the interaction is modeled by a square well potential.
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![Figure 2. Log-normal size distribution [c(d)] of casein micelles in...](profile/Cg-kees-De-Kruif/publication/239919296/figure/fig2/AS:669961227878412@1536742612590/Log-normal-size-distribution-cd-of-casein-micelles-in-NILAC-experimental-results_Q320.jpg)
![Figure 3. Log-normal size distribution [c(d)] of casein micelles in...](profile/Cg-kees-De-Kruif/publication/239919296/figure/fig3/AS:669961227870226@1536742612606/Log-normal-size-distribution-cd-of-casein-micelles-in-NILAC-which-is-heated-for-15_Q320.jpg)
Native casein micelles (i.e., the micelles in fresh milk) can be treated as a collection of polydisperse hard spheres. This follows from small angle neutron scattering, viscosity, diffusivity, and other measure- ments. Therefore, the equilibrium and transport properties of native casein micelles in an ultrafiltra- tion permeate solvent can be descr...
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... the periphery of the parti- cles is the glycomacropeptide ( GMP) portion, which is cleaved by the enzyme chymosin. In Figure 1, two casein micelles are depicted according to the ideas and model of Holt (18,19). The GMP part of the k- CN is about 7 nm long and represents only a fraction of the micellar size. ...
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... behavior can be described by the adhe- sive hard sphere ( AHS) model. The model is referred to as the AHS model or sticky sphere model because the range of the attraction is very short compared with the radius of the particles (see Figure 1). ...
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... the AHS model, the interaction potential is characterized by a short-ranged attraction preceding a steep repulsion (Figure 1). Although the model was developed for molecular systems, its applicability to colloidal systems is far better because, in colloidal systems, the particle size is much larger than the range of the interaction. ...
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... a physically realistic potential, three parameters are needed: the hard core diameter ( s) , the depth ( _) , and the range ( D) of the attraction well. A commonly used model is the square well potential described by V(r)/kT = ∞; r < s = -_; s _r _s + D = 0; s + D < r and schematically depicted in Figure 1. To solve the statistical mechanics equations, Bax- ter ( 3 ) used a limiting case of this square well poten- tial: ...
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... amount of renneting or, better, the brush density, and the interaction between two casein micelles can be modeled as is shown in Figure 1. In this AHS model, the well depth is related to the brush density, given by [14] in which ( [15] in which h = adjustable parameter of order 2. The width of the well is chosen as D = a/(6f) where f = hydrodynamic length of the GMP ( f = 7 nm), and a = radius of the particles ( a = 105 nm). ...
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... this AHS model, the well depth is related to the brush density, given by [14] in which ( [15] in which h = adjustable parameter of order 2. The width of the well is chosen as D = a/(6f) where f = hydrodynamic length of the GMP ( f = 7 nm), and a = radius of the particles ( a = 105 nm). Details are given by De Kruif et al. (11). Now, given the well depth and at not too high volume fractions, the radial distribution function g(r) can be calculated as well as the second osmotic virial coefficient B 2 from Equations [5], [6], [7], and [8]. ...
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Abstract
Milk is a complex liquid, which contains many different species, for example proteins, fat, minerals etc. It is the primary source of nutrition for young mammals before they are able to digest other types of food. The proteins can be divided into two groups: caseins and whey. Whey proteins are about 20 wt % of the total protein amount in m...
The aim of this work was to study the effect of pressure (50; 90; 160; 250; 350 MPa) on a physical property of casein micelle: hydrodynamic radius, tyrosine and tryptophan fluorescence and IR spectra characteristics. According to photon-correlation spectroscopy, the average hydrodynamic radius of the casein micelle was 128 nm, increasing at 50 MPa...
In this work, we present a computer simulation study of the acid coagulation of casein micelles. For different conditions, we predict the acidity (pH value) at which aggregation transition occurs. The interaction potential implemented consists of three contributions, the electrostatic repulsion, the steric repulsion between the polyelectrolyte brus...
![Fig. 3. a. Model of casein micelles proposed by Waugh 1958 [44]. b....](profile/Cg-kees-De-Kruif/publication/266323236/figure/fig3/AS:671513795637266@1537112773957/a-Model-of-casein-micelles-proposed-by-Waugh-1958-44-b-Representations-of-the-model_Q320.jpg)
Casein micelles are association colloids found in mammalian milk. Small-angle scattering data on casein micelles have been collected and are reviewed, including contrast variation. The scattering spectra are quite consistent at medium and high scattering wavevectors [Q = 4πnsin(θ/2)/λ, where n is the refractive index, λ is the wavelength and θ is t...
![SEM and AFM images of casein capsules taken from a), [66, 76] b) and c)...](publication/353642600/figure/fig2/AS:11431281256754098@1719563618732/SEM-and-AFM-images-of-casein-capsules-taken-from-a-66-76-b-and-c-11-59-and-TEM_Q320.jpg)
Casein is an abundant, cheap, and easy to modify milk protein, which is useful as a wall material to encapsulate organic and inorganic substances that can be applied to protect, load, and deliver flavorings, phenolic compounds, drugs, or inorganic substances. There are many reviews that have explored casein itself as well as the configuration of mi...
Citations
... The electron microscopic studies conducted on the gelation process show similar findings to previous works [20,21], which used different analytical methods. These findings also highlight the existence of an aggregation process of casein micelles during the enzymatic stage prior to coagulation [32]. ...
... This can be explained by the higher proportion of κ-casein in the sodium caseinate used for the preparation of ACM in this study (20% as opposed to 17%) and the gradual pH decrease from 7.25 to 6.70 during ACM preparation. Nonetheless, the S-ACM were similar in diameter to natural bovine casein micelles, which generally range from 50 to 600 nm with an average size of about 150 nm (de Kruif, 1998) and were measured at 167.0 ± 0.8 nm with an almost identical method as in this work (Antuma et al., 2024). When an equal preparation time of 60 min was applied, the size of S-ACM (156.3 ± 3.5 nm) and VE-ACM (161.0 ± 7.6 nm) was not significantly different (p = 0.73). ...
Industrial-scale production of artificial casein micelles (ACM) is required to produce dairy alternatives from recombinant casein. However, the currently common micelle preparation method of dropwise mixing casein and salt solutions is inefficient and may prove difficult to scale up. Here, we view casein micelle formation as a process driven by calcium phosphate phase separation in the presence of casein. On this basis, we developed novel routes to prepare ACM through vacuum evaporation, forward osmosis, or reverse osmosis. ACM prepared through these methods have similar properties and improved coagulation behaviour compared to those prepared through the currently common method and natural bovine casein micelles. The properties and functionality of the micelles depend on the preparation time and surface area available for micelle formation, with longer times and larger surfaces (i.e. lower fluxes) yielding smaller ACM that form firmer curds. These novel processes enable fast, efficient, and continuous production of ACM for application in future dairy alternatives.
... The electron microscopic studies of the gelation process at this stage yield results that are consistent with earlier findings in [30,31] obtained through different methods and indicate the occurrence of a clustering process of casein micelles as a preliminary step prior to coagulation. Regarding the timeframe, the formation of large clusters of casein micelles occurs around the "gel point", which refers to the moment when the viscosity of the dispersed system rapidly increases. ...
The aim of this study is to enhance the comprehension of the mechanism of enzymatic gelation in milk by visualizing the evolution of its microstructure through transmission electron microscopy. In order to minimize the potential for artifacts during the preparation process and eliminate any possible difficulties in interpreting the resulting images, three distinct methods were employed in the research: shading the surface topography with vacuum deposition of heavy metal, negative staining of the specimen with a heavy metal solution and replicating a cleavage of a quick-frozen sample. The selection of time intervals for sampling the gel during its evolution is determined by the most probable significant modifications in the resulting gel. Based on the research, it has been shown that natural milk is a nonequilibrium system from the perspective of statistical thermodynamics. A notable observation is that the glycomacropeptides forming the hair layer on the surface of casein micelles are unevenly distributed, leading to the formation of micelle dimers and trimers. It has been determind that during the initial stage of enzymatic gelation in milk, clusters of loosely bound micelles are formed in areas with the highest concentration. The formation of micelle chains is absent at this stage due to the non-anisometric nature of micelles and the energetic disadvantage of their formation. It has been found that under the influence of enzymatic gelation near the gel point, a hierarchical process involving the transformation of the milk’s protein component is activated. The trigger mechanism for this process is a cooperative conformational transition in clusters of casein micelles, which initiates a chain of more energy-intensive reactions in the following sequence: hydrophobic interactions → hydrogen bridges → electrostatic interactions → calcium bridges. The result is the conversion of loosely bound micelle clusters into denser aggregates, predominantly contributing to the formation of milk curd. It is worth noting that gelation in milk can be regarded as a process that reduces the free energy of the dispersed system. Understanding the correlation between the decrease in the free energy value during gelation and the physical properties of the finished cheese and other dairy products continues to be a relevant area of research.
... The average diameter of casein micelles from skim milk was 167.0 ± 0.8 nm and the diameter of ACM linearly increased (R 2 = 0.921) with the preparation rate from 189.0 ± 3.3 to 229.6 ± 14.8 nm for ACM prepared at 100 and 600 mL h − 1 , respectively ( Fig. 2A). This corresponds well to the average size distribution of casein micelles in bovine milk, which has been found to range from 50 to 600 nm with an average of 150 nm (De Kruif, 1998). ...
Artificial casein micelles (ACM) could play a key role in producing animal-free dairy products from recombinant casein, although their coagulation and curd functionality are currently unexplored. Moreover, efficient processes are required to cost-effectively engineer ACM for food applications. In this study, ACM were prepared from bovine casein at various preparation rates and their structure and functionality were compared to natural bovine casein micelles. Overall, the micelles were comparable, although ACM were larger, more polydisperse, and more mineralised. ACM showed similar coagulation behaviour as bovine micelles and their curd grains could be plasticised. ACM prepared at increased preparation rates showed an increased size, polydispersity, and mineralisation. These micelles required longer coagulation times and showed no plasticisation behaviour. Therefore, casein micelle self-assembly is a time-sensitive process, for which we found a characteristic time of about 18 min. Our findings highlight that ACM prove a feasible route to produce future cheese alternatives.
... The elevated viscosity greatly reduced flowability, compared with the liquid formed by untreated VCF5 under similar conditions (Fig. S9). At a low to moderate protein concentration of 0-13%, the casein micelles are considered as hard-sphere colloids, where the attractive interactions between the particles are insignificant, due to the presence of electrostatic repulsion (Dahbi et al., 2010;De Kruif, 1998). When the protein concentration reaches a certain level (~13%, w/w) or the volume fraction is above 0.55, the casein proteins interact more, deviating from hard-sphere behavior (Bouchoux, Cayemitte, et al., 2009;Dahbi et al., 2010). ...
... 20 The micelle voluminosity was 4.4 mL g À1 . 48 The densities of serum, casein protein, and CCP were 1.023, 1 1.359, 49 and 2.31 g mL À1 , 20 respectively. Micelles contained 7% CCP in dry casein micelle. ...
Bovine milk is the complex colloidal system containing nano to micrometer scale components. Earlier, our research group reported the structural changes in bovine casein micelles in the temperature range of 10-40 °C by in situ small-angle X-ray scattering (SAXS) [H. Takagi, T. Nakano, T. Aoki and M. Tanimoto, Food Chem., 2022, 393, 133389]. In this study, we extend our previous research by investigating the temperature-associated structural alterations in casein micelles over a wide spatial scale using in situ SAXS and ultra-SAXS (USAXS). Furthermore, the temperature dependences of various physical properties of the casein micelles were investigated by analyzing the SAXS intensities. The USAXS results showed that micelles formed 1-dimensional aggregates and that these aggregate structures did not change in the temperature range of 10-40 °C. Changes in electron densities calculated from SAXS intensities showed that the voluminosity reduced and the weight fraction of protein inside the micelles increased during the heating process. The number of water domains in a micelle decreased when the temperature increased from 10 to 40 °C, but did not substantially change in the cooling run at a rate of 1 °C min-1. The number of colloidal calcium phosphate (NCCP) in a micelle can also be calculated from the SAXS intensities; NCCP increases upon heating. This study on the behavior of casein micelles with respect to temperature change in milk over a wide spatial scale showed that the casein micelle structure was sensitive to temperature and can change dramatically with temperature variations.
... 90% w/w) consists of six major milk proteins identified as α s1casein (α s1 -CN), α s2 -casein (α s2 -CN), β-casein (β-CN), κ-CN, α-lactalbumin, and β-lactoglobulin. Caseins form a supramolecular structure termed a 'casein micelle' to transport calcium and phosphate to the neonate (Holt et al., 2013), and κ-CN is widely recognised as the protein which creates the outer layer of the micelle and provides steric and electrostatic repulsion between micelles to prevent aggregation (De Kruif, 1998;Horne, 2003;Walstra, 1999;Waugh, 1958). κ-CN is 169 amino acids in length, containing one tryptophan residue and belongs to the intrinsically disordered protein family that lacks an ordered tertiary structure (Farrell et al., 2004;Farrell et al., 2003). ...
In order to explore the functions of glycosylation of κ-Casein (κ-CN) in bovine milk, unglycosylated (UG) and twice glycosylated (2G) forms of κ-CN B were purified by selective precipitation followed by anion exchange chromatography from κ-CN BB milk and tested for their amyloid fibril formation and morphology, oligomerisation states and protein structure. The diameter of self-assembled κ-CN B aggregates of both glyco-form were shown for the first time to be in the same 26.0–28.7 nm range for a 1 mg mL⁻¹ solution. The presence of two bound glycans in the protein structure of 2G κ-CN B led to a greater increase in the maximum amyloid fibril formation rate with increasing protein concentration and a difference in both length (82.0 ± 29.9 vs 50.3 ± 13.7 nm) and width (8.6 ± 2.1 vs 13.9 ± 2.5 nm) for fibril morphology compared to UG κ-CN B. The present results suggest that amyloid fibril formation proceeds at a slow but steady rate via the self-assembly of dissociated, monomeric κ-CN B proteins at concentrations of 0.22–0.44 mg mL⁻¹. However amyloid fibril formation proceeds more rapidly via the assembly of either aggregated κ-CN present in a micelle-like form or dissociated monomeric κ-CN, packed into reorganised formational structures above the critical micellar concentration to form fibrils of differing width. The degree of glycosylation has no effect on the polarity of the adjacent environment, nor non-covalent and disulphide interactions between protein molecules when in the native form. Yet glycosylation can influence protein folding patterns of κ-CN B leading to a reduced tryptophan intrinsic fluorescence intensity for 2G compared to UG κ-CN B. These results demonstrate that glycosylation plays an important role in the modulation of aggregation states of κ-CN and contributes to a better understanding of the role of glycosylation in the formation of amyloid fibrils from intrinsically disordered proteins.
... Casein is considered to be a highly hydrated spherical protein with a diameter distribution between 20 and 300 nm. The average diameter is about 200 nm and the average isoelectric points (pI) range from 4.6 to 4.8 (De Kruif, 1998). The four phosphoproteins are self-assembled by calcium phosphate to form casein micelles and rely on casein-calcium phosphate association and hydrophobic interactions together to maintain casein integrity and stability (Elzoghby et al., 2011). ...
Chitosan can be used as a stabilizer in dairy products to help suspend and stabilize casein micelles, but the molecular weight (MW) effect of chitosan has not been much explored. This work aimed to construct interaction models with casein using chitosan with MW of 30 kDa, 150 kDa, 250 kDa and 300 kDa to reveal the effect of MW on the structure and stability of chitosan-casein complexes. These models were based on the ordered porous layer interferometry (OPLI) system that can generate time-resolved signals to monitor the chitosan-casein interaction process dynamically in real time. The zeta potential, size distribution, binding mechanism and binding constants of chitosan-casein complexes with different MW were also analyzed. It was found that the chitosan and casein at pH 5.5 with the strongest interaction. The zeta potential and size analysis of chitosan-casein complexes showed opposite trends with MW before and after the saturation concentration, and MW(150 kDa) chitosan produced the largest size change in the bridging flocculation stage. The order of binding constants for chitosan-casein interactions is MW(300 kDa) < MW(250 kDa) < MW(30 kDa) < MW(150 kDa). In addition to the attraction between chitosan and casein by electrostatic driving forces, the dependence of the hydrophobic interaction between chitosan molecules on MW all influenced the interaction with casein. The dynamic monitoring and accuracy of the OPLI technique open up more possibilities to obtain the interactions of chitosan/casein and other biopolymers in different condition variables.
... Caseins are flexible and unfolded proteins that consist of both hydrophobic and hydrophilic amino acid segments (Holt, Carver, Ecroyd, & Thorn, 2013;Huppertz, 2013). Due to proteineprotein interactions, these individual caseins associate together and form nano-scale amorphous aggregates together with colloidal calcium phosphate, referred to as a casein micelle (De Kruif, 1998;Holt et al., 2013). The basic biological function of the casein micelle is believed to be the transportation of protein and mineral calcium phosphate from the mammary gland of the mother to the stomach of the neonate, while preventing alternative aggregation pathways and ensuring efficient releasing of calcium phosphate during digestion (Holt et al., 2013). ...
Whilst there are only four genes that encode caseins found in bovine milk, there is a vast array of naturally occurring variants of each protein arising from both genetic polymorphisms and post-translational modification. Effects of the different casein genetic variants on production traits, composition and functional properties of milk have been discovered over the last two decades, and their potential use as an active ingredient in products for improved health outcomes has formed the basis for consumer marketing. Research focused on casein post-translational modifications is an emerging area, and the influence of phosphorylation and glycosylation modifications on the compositional and functional properties of milk is not yet fully elucidated. This review article outlines the influence of casein genetic variants, phosphorylation and glycosylation on the structure of casein and how the properties of certain casein variants associate with human nutrition and the physicochemical and sensory characteristics of dairy products.
... However, when casein is acidified to a pH near the isoelectric point (at about 4.6), the κ-casein on the surface of casein micelles collapses. The lack of electrostatic and steric repulsions leads to the aggregation and sedimentation of casein [11,12]. For a long time, pectin, propylene glycol alginate, and carboxyl methyl cellulose (CMC) have been used to stabilize proteins in acidified milk drinks [13][14][15]. ...
The effects of molecular weight (MW) and degree of esterification (DE) of soluble soybean polysaccharide (SSPS) on the stability of casein under acidic conditions were investigated. The ability of SSPS to stabilize casein was characterized by the content of SSPS–casein complex, the LUMiSizer instability index, average particle size, zeta potential, and storage experiments. The long-term storage stability of the mixtures was related to their ability to combine casein and the stability of the complexes. At the same DE, SSPSs with medium MW formed more complexes with casein than SSPSs with high or low MW; and at the same MW, SSPSs with medium or low DE formed more complexes than SPSSs with high DE. In addition, SSPSs with higher MW had a better stabilizing behavior due to the large steric repulsion between complexes. SSPSs with high MW and low DE showed the best ability to stabilize casein under acid conditions.
