Native casein

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). 

Nature itself gives us a spectacular example of a biopolymer-based delivery system in the form of the native casein micelle of mammalian milk (Lemay et al, 2007). This is primarily a colloidal delivery system for calcium, where the micronutrient is in the form of calcium phosphate, which does not give a bitter taste, and which provides good bioavailability owing to its colloidal size, amorphous state and quick dissolution in gastric conditions (pH 1-2). Nevertheless, the casein micelle structure is unique there are no other readily available natural delivery systems for most nutraceuticals. Therefore some new designs are clearly required (Velikov and Pelan, 2008 McClements et al, 2008, 2009). 

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... 

The self-assembly of caseins may be readily manipulated by processing methods that affect the integrity of native casein micelles and the character of the casein interactions in aqueous media. Examples of such procedures are (Dickinson, 2006) (i) acidification toward the isoelectric point (p/) (pH 4.6-4.8), leading to a neutralization of the net protein charge (ii) enzyme action, as exploited in the production of cheeses and fermented milks (iii) addition of divalent ions, especially, Ca2+ ions (iv) addition of sucrose or ethanol (v) temperature treatment and (vi) high-pressure treatment. 

These calculations also demonstrate the general theoretical principle, which has been confirmed in practice for various dairy-type emulsions, that the depletion interaction is of insufficient magnitude to induce flocculation when the non-adsorbed protein species are too small (e.g., individual protein molecules) or too large (e.g., native casein micelles). 

We have seen earlier in this chapter how the self-assembly of casein systems is sensitively affected by temperature. Another thermodynamic variable that can affect protein-protein interactions in aqueous media is the hydrostatic pressure. Static high-pressure treatment causes the disintegration of casein micelles due to the dismption of internal hydro-phobic interactions and the dissociation of colloidal calcium phosphate. This phenomenon has been used to modify the gelation ability of casein without acidification as a consequence of exposure of hydrophobic parts of the casein molecules into the aqueous medium from the interior of the native casein micelles (Dickinson, 2006). High-pressure treatment leads to a reduction in the casein concentration required for gelation under neutral conditions, especially in the presence of cosolutes such as sucrose (Abbasi and Dickinson, 2001, 2002, 2004 Keenan et al., 2001). 

It is difficult to obtain meaningful results on colloidal interactions unless the samples have low polydispersity. Studies of colloidal interactions between whole casein micelles can be affected by the polydispersity of native casein micelles. (Stothart,1987b). To circumvent the problem of polydispersity, the food system can be deposited on monodisperse silica spheres (Rouw and de Kruif,1989). 

The subject matter of this section has been treated, for the most part, in some earlier reviews (Swaisgood, 1982 Schmidt, 1982 Pay-ens and Vreeman, 1982 Farrell and Thompson, 1988). The coverage here is highly selective, reviewing mainly the more recent findings. However, because of their relevance to our understanding of the structure and stability of native casein micelles, studies dealing with the interactions of the caseins with calcium phosphate are considered more fully. 

C-Methyl-Labeled c-Casein. The effect of reductive methylatior on the properties of asi-, / -, and c-caseins was investigated in this labora tory (7). The purified proteins were treated to obtain either 20% oi 60% modification of lysine residues. Electrophoretic mobilities at pE 8.5 or 6.5 of the modified proteins were indistinguishable from those oi the native caseins. 

Thomsen, J.K., Jakobsen, H.J., Nielsen, N.C., Petersen, T.E., and Rasmussen, L.K. (1995). Solid-state magic-angle spinning P-NMR studies of native casein micelles. Eur. J. Bio-chem. 230,454-459. 

As can be seen from Figure 19.27 skim milk can be fractionated by means of UTP MF (0.1 pm), in combination with an UF diafiltration step, in its two main protein fractions, i.e. native casein micelles and native whey proteins since no heat treatment step was required to separate the proteins. The MF permeate can be considered a sort of sweet whey, however, without containing the caseinomacropeptide (CMP). This MF permeate can be further concentrated to obtain a WPC product. The... 

Figure 19.30. Process to obtain GMP from native casein micelles (Thoma and Kulozik 2004).

Bulca, S., and Kulozik, U. (2004). Eleat-induced changes in native casein micelles obtained by microfiltration. In Advances in Fractionation and Separation Processes for Novel Dairy Applications. Bull. 389, International Dairy federation (IDE), Brussels, pp. 36-39. 

Besides its in vivo activity, mammary gland casein kinase can phosphorylate proteins other than casein, as demonstrated by the in vitro phosphorylation of several food proteins as shown in Table 4 [90], As expected, there is a marked preference of the enzyme for dephosphorylated caseins over native casein, and for native caseins over other food proteins. So far, no protein kinases have been evaluated for the phosphorylation of plant proteins. An examination of known primary structural sequences of soy glycinin and /3-conglyci-nin revealed several potential phosphorylation sites for the mammary gland kinase but not for the CK-2 (unpublished observations). Based on this information, mammary gland kinase appears most likely to be active toward soy proteins. 

Native casein. An exciting new development is the production of native casein. Few details on the process are available at present but it involves electrodialysis of skim milk at 10°C against acidified whey to reduce the pH to about 5 the acidified milk is centrifuged and the sedimented casein dispersed in water, concentrated by UF and dried. The product disperses readily in water and is claimed to have properties approaching those of native casein micelles. 

K-casein on the other (31). Moreover, these simple systems show appreciable differences from native casein micelles in their response to Ca ". In casein micelles, the binding sites for Ca appear to be some distance from the surface of the hairy layer (13) and the same argument can be presumably used for the individual caseins, and show that the calcium binding sites in the synthetic particles are within the surface of shear. On the other hand, the binding of Ca may cause conformational changes in the interfacial layer. 

Reported studies on native casein micelles have indicated that P NMR spectroscopy was useful to determine the nature of phosphate molecules as phosphoserins and inorganic calcium phosphate" . Quantitative assessment of the various micellar components was also probed by Rasmussen et al (1997). Nevertheless, in the case of cheeses which are highly-hydrated and heterogeneous samples, investigations by high-field NMR appear as a true challenge. The NMR technique implies some evident technical limitations as for instance, the sample preparation (introduction in NMR tubes or rotor), but NMR is also dependant on the intrinsic sample properties such as heterogeneity and multi-phase liquid/solid nature which induce some susceptibility effects and the presence of anisotropic interactions. The feasibility of the P NMR spectroscopy to study cheeses has been first probed on the milk powder, main component of these dairy products. 

Casein is the other, larger, protein fraction of milk, accounting for approximately 80% of the milk proteins. Casein is the protein source of cheese and forms curds during processing because it exists as a micelle in milk. The clotting properties of casein cause it to be digested and released into the intestine slowly. -" This slow release into the intestine and ultimately circulation leads to a muted peak in plasma amino acid content compared to whey and soy proteins, which will be explained in Section 8.3. In supplemental form, casein is often made into caseinates because the native casein does not dissolve well in solution and forms clumps or curds. It is most often combined with calcium for dietary supplements, resulting in calcium caseinates. Casein is made up of three protein fractions a-casein, P-casein, and K-casein. 

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