Whole casein

Helstad, K., Rayner, M., van Vliet, T., Paulsson, M., and Dejmek, P. (2007). Liquid dropletlike behaviour of whole casein aggregates adsorbed on graphite studied by nanoindentation with AFM. Food Hydrocolloids 21, 726-738. 

In 1956, Waugh and von Hippel showed that the a-casein fraction of Hipp et al. contained two proteins, one of which was precipitated by low concentrations of Ca2 + and was called as-casein (s = sensitive) while the other, which was insensitive to Ca2+, was called /c-casein. as-Casein was later shown to contain two proteins which are now called a8l- and as2-caseins. Thus, bovine casein contains four distinct gene products, designated a5l-, x,2-, / - and /c-caseins which represent approximately 37, 10, 35 and 12% of whole casein, respectively. 

In the fractionation of the milk proteins, usually the first step in the process is to separate the so-called whole casein from the whey in a skim milk. A number of procedures are available (McKenzie 1971C), but the most commonly used method is based upon classical acid precipitation at the pH of minimum solubility. Several different temperatures have been employed 2, 20, and 30°C. Except for precipitation at 2°C, where minimum solubility occurs at pH 4.3, the skim milk is adjusted to pH 4.5-4.6 with hydrochloric acid (1 M). A more recent investigation of the relationship of temperature and pH to the completeness of casein precipitation indicated that optimum yield was obtained at pH 4.3 and 35°C (Helesicova and Podrazky 1980). 

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-... 

Electrophoretic Methods. Several electrophoretic procedures have been developed to fractionate or purify the various caseins (McKenzie 1971C Thompson 1971 Whitney 1977). Wake and Baldwin (1961) fractionated whole casein by zone electrophoresis on cellulose powder in 7 M urea and 0.02 ionic strength sodium phosphate buffer at pH 7 and 5°C. Payens and co-workers employed several somewhat different electrophoretic conditions for the fractionation and purification of the caseins on cellulose columns (Payens 1961 Schmidt and Payens 1963 Schmidt 1967). Three fractions, as-, k-, and /3-caseins, were separated at pH 7.5 and 30°C with 4.6 M urea-barbiturate buffer. The purification of asi-casein and the separation of the genetic variants of K-casein were accomplished by altering the electrophoretic conditions. Manson (1965) fractionated acid casein on a starch gel column stabilized by a density gradient at 25 °C. 

Creamer (1974) to separate asl-casein-A from whole casein containing both the A and B variants. The B variant was degraded with pepsin or rennet, and the A variant was isolated from the degradation products on a DEAE-cellulose column with an NaCl gradient (0.0 to 0.5 M) in 4.5 M urea buffered at pH 5.5 containing 0.1% mercaptoethanol. 

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). 

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. 

Addeo, F., Chobert, J.-M. and Ribadeau-Dumas, B. 1977. Fractionation of whole casein on hydroxyapatite. Application of a study of buffalo kappa-casein. J. Dairy Res. 44, 63-68. 

McKenzie, H. A. 1971C. Whole casein Isolation properties and zone electrophoresis. In Milk Proteins, Vol. II. H.A. McKenzie (Editor). Academic Press, New York. 

Nijhuis, H. and Klostermeyer, H. 1975. Partial fractionation of whole casein by affinity chromatography. Milchwissenschaft 30, 530-531 (German). 

Saito, T., Itoh, T. and Adachi, S. 1979. Rapid preparation method of para-kappa-casein from whole casein. Jpn. J. Dairy Food Sci. 28, A183-A188. 

M. pusillus var. Lindt protease has given satisfactory results as a chymosin substitute in the manufacture of a number of cheese varieties, but not all varieties of M. pusillus var. Lindt are capable of producing acceptable cheese (Babel and Somkuti 1968). The clotting activity of M. pusillus var. Lindt protease is more sensitive to pH changes between 6.4 and 6.8 than chymosin, but is much less sensitive than that of porcine pepsin (Richardson et al 1967). The same authors reported that CaCL added to milk affected the clotting activity of M. pusillus var. Lindt rennet more than it did that of chymosin rennet. They also reported that this rennet was more stable than chymosin between pH 4.75 and 6.25. M. pusillus var. Lindt rennet is not destroyed during the manufacture of Cheddar cheese, although less than 2% of the enzyme added to the milk remains in the curd. Nearly all of it is found in the whey (Holmes et al. 1977). Mickelsen and Fish (1970) found M. pusillus var. Lindt rennet to be much less proteolytic than E. parasitica rennet but more proteolytic than chymosin rennet on whole casein, a8-casein and /3-casein at pH 6.65. 

Velcheva, P., Kolev, D. A. and Chipileva, R,. 1975B. Bacterial enzyme complex with milk-coagulating activity. IV. Action of its components on whole casein. Prilozh. Mikro-biol. 5, 44-51. 

Stothart and Cebula (1982) and Stothart (1989) obtained linear Guinier plots of sub-micelles of whole casein, giving a radius of gyration of 64 A (Figure 1). On a uniform sphere model, this would indicate sphere radius of 84 A. This sphere size was input to a model fitting calculation (below). Thum et al (1987) found similar sizes for sub-micelles of kappa-casein. 

Figure 3. SANS intensity for wet pellets of whole casein micelles made with (a) 96% D20,4% H20 (b) 74% D20, 26% H20 (c) 41% D20, 59% H20. Casein concn. approx. 250 mg/ml. Calculated intensities for models with subunits in close packing are (—) for 74% D20 and Nl=2, N2=l, N3=2, D=168 A.

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). 

Fig. 6. (a) Ion-exchange chromatogram of whole casein according to the method of Davies and Law (1977a) and (b) alkaline PAGE of the fractions. WC, Whole casein. Reproduced by permission of Cambridge University Press from Davies and Law (1977b). 

Casein micelles are remarkably stable structures. Milk may be boiled, sometimes for several hours, without coagulating the micelles. Also, the addition of CaCU to milk does not precipitate the micelles up to concentrations greatly in excess of that required to precipitate purified whole casein. On the other hand, micelles rapidly flocculate after treatment with chymosin, at or above room temperature, and casein... 

The emulsifying activity index (EAI) curves for phosphorylated whole casein solution versus pH showed shift of their minima towards the acid... 

FIG. 3. Emulsifying activity index of native ( ) and phosphorylated whole caseins [4 (O) 7 ( ) and 11 (V) mol P/mol protein]. (Source From Haertle and Chobert (1999), by courtesy of Food Nutrition Press, Inc.)... 

Table I shows that the foaming properties of whole casein improved by slight phosphorylation. The lowest phosphorylated form of casein (4 mol P/mol protein) showed higher foam hydration and stability than the native whole casein. However, the highly phosphorylated whole casein (11 mol P/mol protein) showed poor foaming properties. The foam hydration of as-casein deteriorated while that of K-casein improved by phosphorylation. This discrepancy seemed to be caused by a different initial hydrophobic/ hydrophilic balance of the proteins in their native states. However, foam stabilities of all casein fractions were reduced by phosphorylation, with K-casein being only slightly affected.

Whole casein and individual casein fractions and their derivatives have been shown to modulate lymphocyte proliferation in vitro. Carr et al. (1990) found that asl-casein can enhance the mitogen-stimulated proliferation of murine splenic T-lymphocytes, when induced in in vitro cell culture at a concentration of 10 6 M. Wong et al. (1996b) showed that (3-casein significantly enhances the mitogen-induced proliferation of ovine T- and B-lymphocytes in a dose-dependent manner, when added to in vitro cell culture. With K-casein, on the other hand, the opposite effect was found, as K-casein was suppressive for murine... 

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