Phosphate, colloidal calcium

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 ... 

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). 

The inorganic colloidal calcium phosphate associated with casein in normal milk dissolves on acidification of milk to pH 4.6 so that if sufficient time is allowed for solution, isoelectric casein is essentially free of calcium phosphate. In the laboratory, best results are obtained by acidifying skim milk to pH 4.6 at 2°C, holding for about 30 min and then warming to 30-35°C. The fine precipitate formed at 2°C allows time for the colloidal calcium phosphate to dissolve (Chapter 5). A moderately dilute acid (1 M) is preferred, since concentrated acid may cause localized coagulation. Acid production by a bacterial culture occurs slowly and allows time for colloidal calcium phosphate to dissolve. The casein is recovered by filtration or centrifugation and washed repeatedly with water to free the casein of lactose and salts. Thorough removal of lactose is essential since even traces of... 

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. 

Casein may be coagulated and recovered as rennet casein by treatment of milk with selected proteinases (rennets). However, one of the caseins, K-casein, is hydrolysed during renneting and therefore the properties of rennet casein differ fundamentally from those of acid casein. Rennet casein, which contains the colloidal calcium phosphate of milk, is insoluble in water at pH 7 but can be dissolved by adding calcium sequestering agents, usually citrates or polyphosphates. It has desirable functional properties for certain food applications, e.g. in the production of cheese analogues. 

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... 

As the pH of milk is reduced, the colloidal calcium phosphate (CCP) dissolves and is completely soluble at pH 4.9 (Chapter 5). pH adjustment, followed by dialysis against bulk milk, is a convenient and widely used technique for varying the CCP content of milk. As the concentration of CCP is reduced, the properties of the micelles are altered but they retain some of their structure even after removing 70% of the CCP. Removal of more than 70% of the CCP results in disintegration of the micelles into smaller particles (aggregates). 

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. 

Removal of colloidal calcium phosphate (CCP) results in disintegration of the micelles into particles of mass 3 x 106 Da. The properties of the CCP-free system are very different from those of the normal milk system, e.g. it is sensitive to and precipitated by relatively low concentrations of Ca2 +, it is more stable to high temperatures, e.g. 140°C, and is not coagulable by rennets. Many of these properties can be restored, at least partially, by increased concentrations of calcium. 

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). 

Figure 5.10 Association of colloidal calcium phosphate (Ca3(P04)2) with the serine phosphate groups of casein (from Schmidt, 1982).

Association with casein. The colloidal calcium phosphate is closely associated with the casein it does not precipitate out of solution and is considered to be protected against precipitation by the casein. Two possible forms of protection are suggested ... 

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. 

Acidification of milk is accompanied by a progressive solubilization of colloidal calcium phosphate and other colloidal salts from casein. Solubilization is complete below about pH 4.9 (Figure 5.11). 

Divalent cations. Addition of calcium to milk causes precipitation of soluble phosphate as colloidal calcium phosphate, an increase in ionized calcium, a decrease in the concentration of soluble phosphate and a decrease in pH. 

Phosphate. Addition of secondary Na or K phosphate (i.e. Na2HP04 or K2HP04) causes the precipitation of colloidal calcium phosphate, with concomitant decreases in the concentration of soluble calcium and calcium ion. Polyphosphates, e.g. Na-hexametaphosphate, chelate Ca2+ strongly and dissolve CCP. 

Citrate. Addition of citrate reduces the concentrations of calcium ions and colloidal calcium phosphate and increases the soluble calcium, soluble phosphate and pH. 

Since milk is saturated with respect to calcium and phosphate, dilution reduces the concentration of Ca2+ and HPO - and causes solution of some colloidal calcium phosphate, making the milk more alkaline. Concentration... 

Reducing the level of colloidal calcium phosphate increases stability in the region of the HCT maximum. 

Solubilizes of colloidal calcium phosphate and thereby affects cheese texture rapid acid production leads to a low level of calcium in the cheese and a crumbly texture (e.g. Cheshire) and vice versa (e.g. Emmental). 

The bioavailability of calcium from dairy foods is considered to be excellent (Schaafsma 1983). Evidence from animal studies suggests that the form of calcium in dairy foods may influence the bioavailability of this mineral (Wong and LaCroix 1980). For example, dairy foods that contain colloidal calcium phosphate or calcium caseinate (e.g., as in Cheddar cheese) appear to be somewhat better sources of calcium than foods that contain ionic calcium (e.g., yogurt, buttermilk). However, calcium in milk and other milk products is of greater bioavailability to humans than calcium found in other food sources. According to Renner (1983), calcium utilization from skim milk powder is 85% compared with 22-74% from vegetables. Dietary fiber in plant cell... 

Ling, E. R. 1936. The titration of milk and whey as a means of estimating the colloidal calcium phosphate of milk. J. Dairy Res. 7, 145-155. 

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. 

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