Casein micelle cross-linking

Bovine casein is listed by the International Union of Immunological Societies as a single allergen, Bos d 8. However, it contains four main protein components, a-sl-, a-s2-, 3-, and K-Casein, in approximate proportions of 40% 10% 40% 10%, respectively (Bernard et al. 1998). The fractions obtained after acid coagulation of individual caseins are cross-linked to aggregates called nanoclusters, which combine into micelles. Their central part is hydrophobic and peripheral hydrophilic parts contain sites of phosphorylation. 

The most abundant milk protein is casein, of which there are several different kinds, usually designated a-, (1-, and K-casein. The different caseins relate to small differences in their amino acid sequences. Casein micelles in milk have diameters less than 300 nm. Disruption of the casein micelles occurs during the preparation of cheese. Lactic acid increases the acidity of the milk until the micelles crosslink and a curd develops. The liquid portion, known as whey, containing water, lactose and some protein, is removed. Addition of the enzyme rennet (chymosin) speeds up the process by hydrolysing a specific peptide bond in K-casein. This opens up the casein and encourages further cross-linking. 

A) Caserns cross-linked by die enzyme transglutaminase within the casein micelle ... 

The weak physical forces that hold together self-assembled nanoparticles are, of course, susceptible to disruption under the influence of thermodynamic and/or mechanical stresses. Hence some workers have investigated ways to reinforce nanoscale structures via covalent bonding. For instance, improved stability of protein nanoparticles, in particular, casein micelles, can be achieved by enzymatic cross-linking with the enzyme transglutaminase, which forms bonds between protein-bound glutamine and lysine residues. By this means native casein micelles can be converted from semi-reversible association colloids into permanent nanogel particles (Huppertz and de Kruif, 2008). 

Huppertz, T., de Kruif C.G. (2008). Structure and stability of nanogel particles prepared by internal cross-linking of casein micelles. International Dairy Journal, 18, 556-565. 

These different casein monomers combine with calcium phosphate to form discrete particles on the nano-size scale. The phosphoserines of the caseins are seemingly clustered for the purpose of linking within the micelle to putative calcium phosphate microcrystallites, also known as nanoclusters (Holt, 1992 Home, 1998, 2002, 2003, 2006 Holt et al., 2003 Home et al., 2007). Structural evidence for the existence of such nanoclusters has come from neutron and X-ray scattering (de Kruif and Holt, 2003 Holt et al., 2003 Pignon et al., 2004 Marchin et al., 2007). The presence of nanoclusters allows native casein micelles to be effective natural suppliers of essential calcium salts in the human diet in a readily assimilated functional form. Protein-nanocluster interactions are the central concept of the cross-linking mechanism in Holt s model of casein micellar assembly (Holt et al., 2003 de Kruif and Holt, 2003). Any analogy with conventional soap-like micelles is considered to be... 

Like as2-casein, /e-casein has two disulfide bonds which can form cross-links with /3-lactoglobulin. The N-terminal two-thirds of the molecule is hydrophobic and contains the two disulfide bonds. The C-termi-nal end is hydrophilic, polar, and charged. It varies in the number of attached carbohydrate moieties and has only one phosphate group. These characteristics make /c-casein ideal for the surface of casein micelles, where it is most often found. It is not susceptible to calcium ion binding, as the other caseins are, and when present on the surface of micelles, it protects the other caseins from calcium (McMahon and Brown 1984A Swaisgood 1982). 

In principle, the proportion of K-casein on the external surface can be studied by using cross-linked or immobilized enzymes, antibodies, lectins, or other specific functionalities. This approach is not without its problems, however. The rate of exchange of K-casein between the micelle and milk serum has not been established but may have a relaxation time of several hours, similar to that for p-casein (Creamer et al., 1977). If this rate is of the order of the time of the experiment, or faster, the proportion apparendy in the surface will be overestimated. Likewise, solubilization of micellar K-casein, e.g., by dissociation during the experiment, would lead to a similar error. 

Figure 19.24. Degree of polymerisation in Tg cross-linked casein micelles and caseinate (Bonisch et al. 2007).

Bonisch, M.P., Lauber, S., and Kulozik, U. (2007). Improvement of enzymatic cross-linking of casein micelles with transglutaminase by glutathion addition. Int. Dairy J. 17, 3-11. 

Transglutaminase treatment of milk offers a novel way to improve the heat stability of milks without the use of chemical additives. Transglutaminase-treated milk had markedly improved heat stability at pH > 6.5 compared to untreated milk. This may be related to the effect of intramolecular cross-links formed in transglutaminase-treated milk, which prevents the dissociation of caseins from the micelles imder conditions where it would have otherwise occurred (e.g., when colloidal calcium phosphate is removed). This was considered to be the mechanism by which the enzyme-treated milk was stabilized to heat treatment (O Sullivan et al., 2002a,b). 

The treatment of micellar casein dispersions altered pH-heat stability profiles. At a pH up to 6.45, there was a negligible effect on heat stability, but stability to heat treatment at 140 °C was markedly improved when pH was increased to 7.1. In these systems, there was minimal intermolecular cross-linking between micelles (Mounsey et al., 2005). 

Schorsch, C., Carrie, H., Clark, A.H., and Norton, l.T. (2000a). Cross-linking casein micelles by a microbial transglutaminase conditions for formation of transglutaminase-induced gels. Int. Dairy J. 10, 519-528. 

Huppertz and Smiddy (2008) demonstrated that application of high pressure (250-300 MPa) led to an initial rapid micellar disruption this was followed by a partial reversal of the high-pressure-induced reassociation of micellar fragments. Partial internal cross-linking of casein micelles by transglutaminase prior to pressure treatment considerably slowed down both the disruption and reassociation processes. 

Huppertz, T. and Smiddy, M.A. 2008. Behaviour of partially cross linked casein micelles under high pressure. International Journal of Dairy Technology 61 51-55. 

Gel-like structure is another model that states casein molecules are located in micelle in a tangle manner and comphcated situation, forming a sphere. In addition, /c-caseins stretch out of the micelle and form a hairy layer. In this model, which was proposed by Holt [35], calcium phosphate nanoclusters act as linkers of thread-like casein monomers by cross-linking [2, 29, 36, 37]. In this model the calcium phosphate nanoclusters are responsible for linking the caseins molecules to each other whereas in previous proposed model nanoclusters are the joiners for submicelles. 

Based on a new proposed model, each CCP nanocluster is assumed as a core and si-> o s2- ]S-caseins are linked to this core. Since / -casein just contains 1 hydrophobic site, it links to only 1 CCP nanocluster in essence, as soon as ]S-casein links to CCP (core) the growth of micelle in that direction ceases. Contrary, si- and as2-caseins are multi-functional (bi-functional) caseins and own 2 hydrophobic sites. These caseins develop the network via cross-linking since each as-casein that is linked to a CCP is able to interact with the next CCP and in this way nanoclusters link to each other. The tendency of Ca-sensitive caseins to interact with CCP is directly related to their phosphoseryl residues. The multifunctional caseins continually link CCP nanoclusters to each other and this process continues till the end nanocluster links to the first one, and a loop is formed. Bacause multi-functional caseins link randomly to each other a variety range of micelle size is obtained. The location of K-casein and its role in the stability of micelle appear to be unclear in this model [15]. 

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