Fractionation of casein

As discussed in section 4.4, individual caseins may also be isolated on a laboratory scale by methods based on differences in solubility in urea solutions at around pH 4.6, by selective precipitation with CaCl2 or by various forms of chromatography, especially ion-exchange or reverse-phase high performance liquid chromatography (RP-HPLC). Obviously, these methods are not amenable to scale-up for industrial application. 

There is considerable interest in developing techniques for the fractionation of caseins on an industrial scale for special applications. For example  

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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Although less susceptible than / - and as2-caseins, isolated xsl -casein in solution is also readily hydrolysed by plasmin. It has been suggested that a minor ill-defined fraction of casein, called A-casein, consists of plasmin-produced fragments of asl-casein, but the situation is unclear. 

The viscosity of milk and creams tends to increase slightly with age, due in part to changes in ionic equilibria. Heating skim milk to an extent that denatures most of the whey proteins increases its viscosity by about 10%. Homogenization of whole milk has little effect on its viscosity. The increase in the volume fraction of fat on homogenization is compensated by a decrease in the volume fractions of casein and whey proteins because some skim milk proteins are adsorbed at the fat-oil interface. Pasteurization has no significant effect on the rheology of whole milk. 

Differential Solubility Methods. Numerous methods have been developed to obtain one or more of the various caseins from whole casein or directly from skim milk based on their differential solubility (Thompson 1971 Mackinlay and Wake 1971 Whitney 1977). While some early procedures indicated the possibility of fractionating whole casein into different components, it was not until the 1950s that systematic procedures were proposed for the fractionation of casein into Warner s a-, 0-, and y-caseins. Hipp et al. (1952) developed two procedures which have been used extensively or partially incorporated into other methods. The first is based upon the differential solubilities of the caseins in 50% alcohol in the presence of ammonium acetate by varying the pH, temperature, and ionic strength. The second procedure involves the dispersion of whole casein in 6.6 M urea and the separa-... 

Partition Methods. Walter (1952) devised a countercurrent distribution procedure for the fractionation of casein in a two-phase system containing water-ethanol-phenol at pH 8.2. The concentration of /3-casein increased in the phenol phase. Ellfolk (1957) employed a two-phase system consisting of collidine, ethanol, and distilled water at 20 °C. The a-caseins were concentrated in the water-rich phase, while the /3-caseins were concentrated in the collidine-rich phase. 

Another technique employed to facilitate the chromatographic fractionation of casein involves the reduction and subsequent alkylation of the casein prior to chromatography (Rose et al. 1969 Yaguchi and Rose 1971 Davies and Law 1977). Rose et al. (1969) reduced whole casein with mercaptoethanol, alkylated the product with iodoacetamide, and separated the components on a DEAE-cellulose column (Figure 3.18). Davies and Law (1977) modified this procedure and achieved a quantitative estimation of the major caseins. 

Cation-exchange columns have been used effectively by some investigators for the fractionation of casein (Annan and Manson 1969 Kim et al 1969 Kopfler et al. 1969 Snoeren et al. 1977 Saito et al 1979). Sulfoethyl-Sephadex was used by Annan and Manson (1969) with formate buffer to fractionate the as-casein complex. Cellulose phosphate, carboxyl-methyl-cellulose (CMC), potassium-K-carrageenan, and sodium Amberlite CG50 columns have also been used to fractionate the caseins (Kim et al. 1969 Kopfler et al. 1969 Snoeren et al 1977). A batch method for the preparation of para-K-casein from rennin-treated whole casein has been developed with CMC Sephadex (Saito et al. 1979). 

Agarose gels have also been used as columns for the fractionation of caseins (Yamashita et al. 1976 Pepper and Farrell 1977). Nijhuis and... 

Klostermeyer (1975) used an activated thiol-Sepharose 4B column with Tris-HCl buffer containing dithiothreitol to separate the K-and aa2-caseins from the aai-and /3-caseins in whole casein. More recently, Creamer and Matheson (1981) studied the fractionation of casein by hydrophobic interaction chromatography on octyl- or phenyl-Sephar-ose CL-4B columns. The whole casein was adsorbed onto the column from dilute phosphate buffers. A gradient of 0 to 40% ethylene glycol followed by 6 M urea was employed to desorb the protein. Optimum separation was obtained with an increasing urea gradient. Under all conditions, the major /3-casein component was eluted more readily than the asi-casein in spite of its higher hydrophobicity. 

Ellfolk, N. 1957. Fractionation of casein by distribution in a liquid two-phase system. Acta Chem. Scand. 11, 1317-1322. 

El-Negoumy, A. M. 1976. Two rapid and improved techniques for chromatographic fractionation of casein. J. Dairy Sci. 59, 153-156. 

Nakahori, C. and Nakai, S. 1972. Fractionation of caseins directly from skim milk by gel chromatography. I. Elution with sodium dodecylsulphate. J. Dairy Sci. 55, 25-29. 

Vujicic, I. F. 1973. Fractionation of casein complex by ion-exchange chromatography on DEAE-Sephadex. Milchwissenschaft 28, 175-176 (German). 

Wei, T.-M. 1982. Batch fractionation of casein with diethylaminoethyl cellulose. M. S. thesis, University of Illinois, Urbana. 

Microfiltration processing for clarification and defatting of cheese whey, for selective separation and concentration of micellar caseins from milk for various purposes, for fractionation of caseins and their peptides, for recovery of native whey proteins from milk, for gentle sterilization of milk to produce extended shelf fife liquid milk and cheese milk, for fractionation of globular milk fat and its components, for the reduction of microorganisms in cheese brine, and for the removal of colloidal particles in membrane cleaning solutions. 

Fat Membrane Structure, Figure 3 shows the isotherms for different ratios of NaCas/GMS at pH 6.8. The curves present two distinct regions. The first one yccurs below the collapse pressure of pure caseinate (0 to 20 mNm ). In that region, we observed an increase in area as the fraction of caseinate in the film increased. We used the additivity law to calculate the interaction between the two components at the interface. We have observed that mixed films do not follow ideality. In fact, there is a condensation of the film (30). 

Turhan, K. N., D. N. Barbano, and M. R. Etzel. 2003. Fractionation of caseins by anion-exchange chromatography using food-grade buffers. J. Food Sci. 68 (5) 1578-1583. 

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