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Cryo-TEM image of casein micelles from fraction 4 after 24 h at 4 ° C followed by separation from dissociated  -casein. Scale bar: 100 nm.
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Casein micelles are colloidal protein-calcium-transport complexes whose structure has not been unequivocally elucidated. This study used small-angle x-ray scattering (SAXS) and ultrasmall angle x-ray scattering (USAXS) as well as cryo transmission electron microscopy (cryo-TEM) to provide fine structural details on their structure. Cryo-TEM observa...
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... is released in the surrounding medium under free form or aggregate form with calcium present in serum phase. 16,44 It cannot be excluded that this type of aggregate is not dense enough to electrons. Hence, depleted casein mi- celles were separated from dissociated -casein by centrifu- gation and the micellar fraction was observed by cryo-TEM Fig. 6. They appeared similar to depleted micelles before separation of -casein Fig. 5 and also to native casein mi- celles Fig. 1b. Our results show that highly -casein-depleted casein micelles were able to maintain a micellar framework. These results were confirmed by the SAXS measurements Fig. 7 which shows no difference af- ter the ...
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Citations
... As a result of scattering and other non-perturbing structural methods (de Kruif, 2014;Marchin et al., 2007;Morris et al., 2000;Nogueira et al., 2021), a widely accepted medium-resolution structural model of the native casein micelle has been obtained. Casein micelles from cow milk are roughly spherical with a number average radius of about 75 nm. ...
... Notwithstanding the high hydration, a solute volume fraction of 0.2 is sufficiently high for the micelle to have viscoelastic properties (Bouchoux et al., 2009;Uricanu et al., 2004). Cryo-electron microscopy of raw milk micelles shows that the CaP nanocluster complexes are distributed more-or-less evenly through the core of the micelle (de Kruif et al., 2012;Hettiarachchi et al., 2020;Kamigaki et al., 2018;Marchin et al., 2007) but they are not found in the coat (Bouchoux et al., 2015;Shukla et al., 2009). The casein micelle is a dynamic structure: individual caseins are conformationally mobile and can exchange between the micelle and milk serum, particularly those that are not bound strongly to the CaP nanoclusters (Nogueira et al., 2021). ...
... Due to the presence of larger propyl residues casein lacks well organized secondary structures. Casein protein generally exists as micelle complex with α and β in the core of the complex and stabilized by κ fractions at the surface thus exhibiting a strong electrostatic repulsion which prevents collapse and flocculation of the milk matrix [146,147]. They are phosphorylated and possess a limited number of α helix and β pleated sheets. ...
... In addition, some darker particles with an estimated diameter of 2-3 nm were uniformly distributed inside the CMs, and the darker particles were deemed to be CCP clusters (Hettiarachchi et al., 2020). These observed unheated CM results are consistent with the results of previous studies (Marchin, Putaux, Pignon, & Léonil, 2007;Trejo et al., 2011). ...
... A calciumdependent shift of the features in the scattering curves to small q values was also observed in micellar systems in the study by Gebhardt et al. (2008). Besides, Marchin et al. (2007) reported the scattering curves of casein micelle fractions with different sizes, which were separated at different centrifugal forces. The scattering curves of these different casein fractions exhibited a similar variation as our results: a higher scattering intensity was also observed for larger casein micelles in the low q-region. ...
... Å -1 upon calcium addition; this peak was also clearly visible for the skim milk sample. According to the reported results on native casein micelles, this shoulder was indicative of protein inhomogeneities (which were recently modelled as star-like aggregates of rods by Pedersen et al. (2022)) reflecting localized variations in density arising from interactions of the caseins and calcium phosphate nanoclusters (Day et al., 2017;Marchin et al., 2007). Casein micelles can be regarded as a uniform protein matrix containing a disordered arrangement of calcium phosphate nanoclusters and protein aggregates Pedersen et al., 2022). ...
... Oppositely, the disappearance of this shoulder suggested the dissociation of colloidal calcium phosphate and/or protein aggregates, which was explored in acidification studies Marchin et al., 2007). ...
... The results verified that CaP is uniformly radial distributed in the core of micelles, and the shell is comprised of κ-casein but without any CaP nanoclusters. They believed that the core of micelles is made of a homogeneous network of caseins and CaP randomly distributed inside (Marchin, Putaux, Pignon, & Léonil, 2007). Analogously, it conforms to the recent models of sponge-like casein micelles with water-filled cavities present within the core (McMahon & Oommen, 2008;Trejo, Dokland, Jurat-Fuentes, & Harte, 2011). ...
Casein-based hydrogels (Casein Gels) possess advantageous properties, including mechanical strength, stability, biocompatibility, and even adhesion, conductivity, sensing capabilities, as well as controlled-releasing behavior of drugs. These features are attributed to their gelation methods and functionalization with various polymers. Casein Gels is an important protein-based material in the food industry, in terms of dairy and functional foods, biological and medicine, in terms of carrier for bioactive and sensitive drugs, wound healing, and flexible sensors and wearable devices. Herein, this review aims to highlight the importance of the features mentioned above via a comprehensive investigation of Casein Gels through multiple directions and dimensional applications. Firstly, the
composition, structure, and properties of casein, along with the gelation methods employed to create Casein Gels are elaborated, which serves as a foundation for further exploration. Then, the application progresses of Casein Gels in dairy products, functional foods, medicine, flexible sensors and wearable devices, are thoroughly dis�cussed to provide insights into the diverse fields where Casein Gels have shown promise and utility. Lastly, the existing challenges and future research trends are highlighted from an interdisciplinary perspective. We present the latest research advances of Casein Gels and provide references for the development of multifunctional biomass-based hydrogels.
... Inside the casein micelles used as raw material for fiber production, calcium neutralizes the negative charge of caseins, bridges colloidal calcium phosphate with the phosphoserines of caseins, and forms inter-protein or intra-protein linkages between two phosphoserines [12]. Drastic acidification leads to the complete dissociation of the calcium phosphate contacts, but the integrity of the casein micelle remains intact [20]. To investigate this effect on fiber stability, we studied the influence of acidic media on the swelling of the casein matrix. ...
Micellar casein fibers of defined size and internal structure can be produced by the extrusion of cold-renneted casein micelles into a warm, calcium-rich coagulation bath. Calcium phosphate contacts within the casein matrix are important for fiber stability and production but become less important under acidic pH conditions. We demonstrate this with swelling experiments in media with pH < 2, which we adjust with citric acid of different molarities. In contrast to the simple swelling of dried casein fibers in water, a two-phase process takes place in citric acid similar to swelling in 1 N HCl. However, instead of a second deswelling step, we observe in citric acid that the fiber swells further. The observation is explained by a pH-dependent transition from a rennet casein gel to an acidified rennet gel. This can be simulated with a kinetic model that couples two second-order rate equations via a time-varying ratio. The final swelling values decrease with increasing proton concentration via a scaling relation, which is also confirmed by swelling in other acids (HCl or acetic acid) in this pH range. We attribute this to a decrease in the molecular weights of the aggregated casein structures within the strands of the gel network.
... The plateau partly disappeared upon a decreasing ratio of native to dephosphorylated casein. Usually, an absence of this plateau is linked to an absence of nanoclusters, as for example upon dissolution of calcium phosphate upon acidification to pH 5.2 (Marchin et al., 2007). Alternatively, a polydisperse size distribution of the CaP nanoclusters and protein particles could have caused the gradual disappearance of the plateau. ...
Industrial-scale production of recombinant proteins for food products may become economically feasible but correct post-translational modification of proteins by microbial expression systems remains a challenge. For efficient production of hybrid products from bovine casein and recombinant casein, it is therefore of interest to evaluate the necessity of casein post-translational phosphorylation for the preparation of hybrid casein micelles and study their rennet-induced coagulation. Our results show that dephosphorylated casein was hardly incorporated into artificial casein micelles but was capable of stabilising calcium phosphate nanoclusters with an increased size through adsorption on their surface. Thereby, dephosphorylated casein formed larger colloidal particles with a decreased hydration. Furthermore, the presence of increasing amounts of dephosphorylated casein resulted in increasingly poor rennet coagulation behaviour, where dephosphorylated casein disrupted the formation of a coherent gel network by native casein. These results emphasise that post-translational phosphorylation of casein is crucial for their assembly into micelles and thereby for the production of dairy products for which the casein micelle structure is a prerequisite, such as many cheese varieties and yoghurt. Therefore, phosphorylation of future recombinant casein is essential to allow its use in the production of animal-free dairy products.
... Saffer et al. (2014) measured correlation length values of a similar scale to that reported herein, between 2 and 13 nm, ascribed to the pore size of a gel, which presumably relates to changes in the organization of protein-stranded clusters within the interior of the casein micelle. While changes in scattering patterns for the 0.08-0.1 Å − 1 region have previously been associated with colloidal calcium phosphate nanoclusters (Bouchoux et al., 2010;Gebhardt, Takeda, Kulozik, & Doster, 2011;Marchin, Putaux, Pignon, & Léonil, 2007;Mata et al., 2011), De Kruif (2014 attribute these shifts in scattering to changes in the size of the protein inhomogeneities, as described herein, and reinforced by the work of Ingham et al. (2016) and Singh, Hemar, Gilbert, Wu, and Yang (2020). Initial correlation lengths for all samples were approximately 1.5-2.5 nm and increased in size up to around 5.5 nm after 240 min of digestion, indicating either an increase in the aggregation of small protein fragments or agglomeration of adjacent clusters within the hard regions of the micelle to form larger entities, with a corresponding increase in the size of water channels within a cluster (Bouchoux et al., 2010). ...
... The surface-active behavior and hydrodynamic interactions between fat globules in emulsions can be altered by pH changes. The net charge of casein micelles was found to decrease as the pH decreased from 6.7 in milk to 4.6 at its isoelectric point [38][39][40]. At higher pH (7.0), Ca 2+ could bind more strongly to phosphate, thereby avoiding binding to the phosphoserine site of casein and reducing the instability of the emulsion. ...
The effects of different pH levels and ionic strength in calcium on the stability and aeration characteristics of dairy emulsions were investigated in this study. The results revealed that the stability and aeration characteristics of the emulsion were enhanced as the pH value increased from 6.5 to 7.0 and were optimal within the pH of 6.8~7.0, while the concentration of free calcium ions (Ca2+) was 2.94~3.22 mM. With the pH subsequently fixed at 6.8 and 7.0, when the addition of CaCl2 was increased to 2.00 mM (free Ca2+ strength > 4.11 mM), stability and aeration characteristics reduced significantly, including the flocculation of fat globules, an increase in particle size, and a decrease in the zeta potential and viscosity of the O/W emulsion, all leading to an increase in interfacial protein mass and decreased overrun and foam firmness. Overall, the results indicated that pH changes and CaCl2 addition significantly influenced the stability and aeration characteristics of dairy emulsions, by influencing free Ca2+ strength, which is an important factor in determining the quality of dairy emulsions.
... 1.5 nm [53]. Changes in this q-range were previously attributed to modifications of the colloidal calcium phosphate nanoclusters in the casein micelles, usually due to calcium dissociation [40,45]. The unaltered scattering pattern in this q-range is therefore in line with the minimal changes in colloidal calcium observed in the present study (see Fig. 5). ...
The impact of membrane concentration with different milieu exchange conditions on the structural changes of casein micelles was investigated by studying their dissociation as well as their internal structure using small-angle X-ray scattering (SAXS). Micellar casein concentrates were prepared using ultrafiltration of milk (< 10 °C) followed by microfiltration and diafiltration (DF) of the resulting 2 × milk protein concentrate (< 10 °C) using different DF media (demineralised water vs. ultrafiltration permeate) and DF modes (continuous at 2 × concentration vs. discontinuous at 4 × concentration). All concentrates showed a higher degree of casein micelle dissociation when DF was conducted with water compared to ultrafiltration permeate. SAXS data were evaluated using a model describing the scattering on an absolute scale, and using protein and mineral concentrations as constraints. The results showed changes in the internal structure of the casein micelles at intermediate length scales for the concentrates prepared using water as DF medium, linked to the partial dissociation of caseins from the colloidal phase to the dispersion phase. In contrast, the casein micelles, in spite of their concentrated state, retained their structural integrity better when DF was conducted with ultrafiltration permeate. These results clearly demonstrate that maintaining the ionic equilibrium during membrane filtration of skim milk is critical to preserve the original structure of the protein particles in skim milk. This has important consequences in developing processes aiming at tailoring the technological properties of micellar casein concentrates.
