Casein micelles dissociation

The following factors must be considered when assessing the stability of the casein micelle The role of Ca++ is very significant in milk. More than 90% of the calcium content of skim milk is associated in some way or another with the casein micelle. The removal of Ca++ leads to reversible dissociation of P-casein without micellular disintegration. The addition of Ca++ leads to aggregation. The same reaction occurs between the individual caseins in the micelle, but not as much because there is no secondary structure in casein proteins. 

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). 

Creamer, L.K., Berry, G.P. (1975). A study of the properties of dissociated bovine casein micelles. Journal of Dairy Research, 42, 169-183. 

Dalgleish, D.G., Law, A.J.R. (1988). pH-Induced dissociation of casein micelles. 1. Analysis of liberated caseins. Journal of Dairy Research, 55, 529-538. 

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. 

Lipase associated with the casein micelles in skim milk is not fully active, but both dilution and the addition of sodium chloride stimulate or restore activity, presumably by dissociating the micelle-lipase complex. Sodium chloride is an inhibitor of lipolysis, but the proper dilution and addition of this salt can elicit maximal activity (Downey and Andrews 1966). 

The other major casein in cheese is /3-casein, but it is generally not hydrolyzed by rennet in low-pH cheeses. Alkaline milk protease (plas-min) plays the major role in the hydrolysis of /3-casein (Richardson and Pearce 1981). The plasmin level in cheese is related to the pH of the curd at whey drainage, since plasmin dissociates from casein micelles as the pH is decreased. Richardson and Pearce (1981) found two or three times more plasmin activity in Swiss cheese than in Cheddar cheese. Swiss cheese curds are drained at pH 6.4 or higher, while Cheddar cheese curds are drained at pH 6.3 or lower. Proteolysis of /3-casein is significantly inhibited by 5% sodium chloride. The inhibitory influence of sodium chloride is most likely due to alteration of /3-casein or a reduction in the attractive forces between enzyme and substrate (Fox and Walley 1971). 

Figure 12 Transmission electron microscopy study of protein desorption in icecream mix containing emulsifiers and hydrocolloids, (a) Immediately after homogenization the fat globules (0 are stabilized by adsorbed partially dissociated casein micelles (arrows), (b) During ageing the mix at 5°C, the previously adsorbed protein film is released in the form of coherent protein layers (arrows) into the water phase (w). (c) After mechanical treatment in the ice cream freezer, desorbed protein layers are seen more often in the water phase without association to fat giobules (arrows). From reference 48, courtesy of Dr. W.Buchheim, Kiel, Germany.

When casein micelles are dissociated, spherical particles are observed with a size similar to the scale of the substructure. Moreover, the number of spherical particles formed by dissociation appears to correspond roughly to the number of substructural elements in the micelle. In electron micrographs of mammary gland secretory cells, some of the Golgi vesicles contain particles of a size similar to that of the particles formed by dissociation of micelles, whereas others contain larger particles. Buchheim and Welsch (1973) proposed that the smaller particles are not small micelles but subunits that are to be assembled into full-sized micelles. The envisaged sequence of assembly is as follows ... 

As association polymers, the caseins can form aggregates in solution with a wide range of sizes, so it is difficult to idendfy any particular size as the units of bioassembly or dissociation of casein micelles. 

Griffin et al. (1988) reported that when the colloidal calcium phosphate was depleted, by addition of a EDTA solution to a micellar dispersion, there was essentially no selective dissociation of the individual caseins. This difference from the results of Holt et al. (1986) could reflect a difference of methodology. The method of Griffin et al. (1988) could bring about an almost complete and therefore non-selective disintegration of some micelles in the immediate vicinity of the added EDTA while leaving others virtually intact. In the dialysis method of Holt et al. (1986), the free Ca2+ concentrations is never depressed and hence micelles dissociate only because of the solubi-lazation of the colloidal calcium phosphate. 

The direct determination of some major elements (Ca, K, Mg, Na, and P) and Zn by ICP-AES was performed in powdered milk [14]. Samples were diluted with a 5 or 10 percent (v/v) water-soluble, mixed tertiary amine reagent at pH 8. This reagent mixture dissociated casein micelles and stabilized liquid phase cations. No decrease in analyte emission intensities was observed. Reference solutions were prepared in 10 percent (v/v) mixed amine solution, and no internal reference element was needed for ICP-AES. The direct technique is as fast as slurry approaches, without particle size effects or sensitivity losses. 

Avoidance of interference of other milk constituents with measurements is also of importance for example, dissociation of casein micelles by calcium-chelating agents, such as trisodium citrate or ethylenediamine tetra-acetic acid (EDTA), may used to avoid interference of the micelles in particle size measurement, while clusters of fat globules can be disrupted by adding a low level of sodium dodecyl sulphate (SDS). 

At 100 MPa the turbidity has nearly vanished, since the particle size has been reduced to 20 nm. A large fraction of the casein micelles is decomposed into smaller fragments dominating the number average up to 200 MPa suggesting the existence of stable mini-micelles. Above 250 MPa the micelles dissociate further forming particles with d = 3 nm, most likely the casein monomers. Releasing the pressure induces limited reassociation to particles with d = 5 nm, the native micelle is not recovered. 

Figure 19.20. In situ pressure dissociation-association kinetics of casein-micelles after a pressure jump monitored by the average back-scattered light intensity (a) dissociation upon pressure inerease (b) reassociation upon pressure release (Gebhardt et al. 2006).

Figure 19.21. Dissociation-association cycle of casein micelles. Pressure dissociation (verti-...

To elucidate the surface morphology and size distribution of pressure treated casein micelles and their irreversible fragments, AFM experiments were performed. The samples were pressure-treated for 30 min in discrete steps (0.1-400 MPa) across the dissociation transition (Figure 19.22). Instead of a continuous evolution of the structure with pressure three characteristic morphologies can be observed The native micelles, existent up to 50 MPa, appear to be composed of sub-elements, suggesting... 

Superior foaming properties of milk have been obtained by addition of calcium complexing agents. Kelly and Burgess (1978) demonstrated that addition of sodium hexametaphosphate to milk protein concentrate solutions prepared by ultrafiltration improved foam volume and stability on whipping. The addition of EDTA to milk, which causes dissociation of the casein micelle, improved the foaming properties of milk (Ward et al., 1997). 

Citrate salts have long been used in the processed cheese industry as "emulsifying salts," and there is still interest in the mechanism of their action. Shirashoji et al. (2006) examined the effects of trisodium citrate on the properties of processed cheese. Increasing concentration of sodium citrate decreased the size droplets of the cheese. This effect is typical when emulsifying properties of a system are improved. This is expected as the complexation of calcium by citrate causes dissociation of the casein micelle, making the casein more available for emulsifying fat droplets. This possibly contributed to the reinforcement of the structure of the processed cheese. 

Ward, B.R., Goddard, S.J., Augustin, M.A., and McKinnon, I.R. (1997). EDTA-induced dissociation of casein micelles and its effect on foaming properties of milk. /. Dairy Res. 64,495-504. 

The dissociation of a quaternary structure or denaturation of proteins is required prior to emulsification. Therefore, casein micelles are adsorbed at an interface in a semi-intact form (Oortwijn et al., 1977). The thermal denaturation of globular proteins prior to emulsification was reported to improve the emulsifying properties. The high level of the thermally denatured whey protein fraction in mixed proteins (of denatured and undenatured proteins) increased the emulsion viscosity and coalescence stability compared with the low-level denatured fraction (Britten et al., 1994). 

Casein dissociates from the micelles when milk or a dispersion of casein micelles at pH 5= 6.7 is heated to at least 90°C in the former, the dissociated K-casein is complexed with whey proteins. The functional properties of K-casein-)S-Ig complexes isolated by centrifugation of heated milk have been reported by Singh, Fox and Cuddigan (1993). 

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