Casein micelle calcium phosphate

Skim milk can be considered as a two-phase system consisting of casein-colloidal calcium phosphate micelles in quasi-equilibrium with an aqueous solution of salts and proteins the phase boundary is ill-defined because of the intimate association between the calcium phosphate and the caseins (phosphoproteins). 

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. 

Casein monomers or small polymers — caseinate subunits + calcium phosphate — casein micelles... 

Milk contains about 40 gl of fat and 32 gl of protein. The milk fat occurs in droplets stabilizedby layers of phospholipid and protein [16]. Ofthe protein, about 25gl is casein and 7 gl is serum proteins (mostly lactoglobulins). The caseins form casein micelles , which involve both casein and calcium phosphate, may contain 500-10000 casein protein molecules, and are of the order of 200-300 nm in diameter [16, 18, 34, 36]. The casein micelles are quite complex. There are four types of casein molecules of which most (a j -, a 2 > P-caseins) complex with... 

Colloidal calcium phosphate (CCP) acts as a cement between the hundreds or even thousands of submicelles that form the casein micelle. Binding may be covalent or electrostatic. The casein micelles are not static there are three dynamic equilibria between the micelle and its surroundings ... 

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... 

Figure 6.1 The dual binding model of the structure of the casein micelle as built up from the individual caseins (0 1, o 2, P and k) in combination with calcium phosphate (CaP). Reproduced from Home (1998) with permission.

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). 

Because they occur as large aggregates, micelles, most (90-95%) of the casein in milk is sedimented by centrifugation at 100000 g for 1 h. Sedimentation is more complete at higher (30-37°C) than at low (2°C) temperature, at which some of the casein components dissociate from the micelles and are non-sedimentable. Casein prepared by centrifugation contains its original level of colloidal calcium phosphate and can be redispersed as micelles with properties essentially similar to the original micelles. 

Since the micelles are of colloidal dimensions, they are capable of scattering light and the white colour of milk is due largely to light scattering by the casein micelles the white colour is lost if the micelles are disrupted, e.g. by removing colloidal calcium phosphate (by citrate, ethylene... 

The K-casein content of casein micelles is inversely proportional to their size, while the content of colloidal calcium phosphate is directly related to size. 

Although the submicellar model of the casein micelle readily explains many of the principal features and physicochemical reactions undergone by the micelles and has been widely supported, it has never enjoyed unanimous support and two alternative models have been proposed recently. Visser (1992) proposed that the micelles are spherical conglomerates of individual casein molecules randomly aggregated and held together partly by salt bridges in the form of amorphous calcium phosphate and partly by other forces, e.g. hydrophobic bonds, with a surface layer of K-casein. Holt (1992, 1994) depicted the casein micelle as a tangled web of flexible casein... 

Although CCP represents only about 6% of the dry weight of the casein micelle, it plays an essential role in its structure and properties and hence has major effects on the properties of milk it is the integrating factor in the casein micelle without it, milk is not coagulable by rennet and its heat and calcium stability properties are significantly altered. In fact, milk would be a totally different fluid without colloidal calcium phosphate. 

Not all of the salt constituents are found in the dissolved state in milk. Calcium, magnesium, phosphate, and citrate are partitioned between the solution phase and the colloidal casein micelles (see Chapter 9 for the composition and structure of these micelles). For analytical purposes, partition of the salt constituents can be achieved by equilibrium dialysis or by pressure ultrafiltration. In the latter technique, pressures must be limited to about 1 atmosphere to avoid the so-called sieving effect (pushing water through the filter faster than the dissolved components (Davies and White 1960). 

Casein micelle proteins are primarily a8i-, as2-, /3-, and -caseins in approximate proportions 3 .8 3 1. asi-Casein has eight or nine phosphate groups, depending on the genetic variant. aS2-Casein is the most hydrophilic of the caseins. It has two disulfide bonds which, by severe heat treatment, can be caused to interact with those of /3-lactoglobulin. It also has 10 to 13 phosphate groups and is very sensitive to the calcium ion concentration (Kinsella 1984 Swaisgood 1982). 

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). 

Lowering the pH of milk to 4.6 solubilizes colloidal calcium phosphate. This removes its neutralizing effect, allowing electrostatic interactions between micelles. Under these conditions, micelles coagulate and precipitate from solution. Kudo (1980C) showed that release of whey proteins and K-casein from casein micelle surfaces as the pH is increased from 6.2 to 7.2 allows micelles to stick together and precipitate from solution. 

Stability of the complex protein system of milk or whey is decreased by concentration (Fox 1982 Muir and Sweetsur 1978 Sweetsur and Muir 1980B). In addition to closer packing of casein micelles and other proteins in concentrated milk, calcium phosphate is precipitated so that the pH decreases (Fox 1982). The pH effect causes protein which would be soluble at a normal solids concentration to precipitate. Casein in milk concentrated to three times its original solids level forms a flocculent after 1 to 3 weeks at -8°C (Lonergan 1978). 

---