Caseins with calcium

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 is used to fortify flour, bread, and cereals. Casein also is used for glues and microbiological media. Calcium caseinate is made from a pressed casein, by rinsing, treating with calcium hydroxide [1305-62-0], heating, and mixing foUowed by spray drying. A product of 2—4% moisture is obtained. 

Phosphates, which react with calcium to reduce the calcium ion activity, assist in stabilizing calcium-sensitive proteins, eg caseinate and soy proteinate, during processing. Phosphates also react with milk proteins. The extent of the reaction depends upon chain length. Casein precipitates upon addition of pyrophosphates, whereas whey proteins do not. Longer-chain polyphosphates cause the precipitation of both casein and whey proteins. These reactions are complex and not fully understood. Functions of phosphates in different types of dairy substitutes are summarized in Table 9 (see also Food additives). 

The butter fat is a coarse dispersion readily removable on standing or by a centrifuging operation. The casein will be present in the skimmed milk as colloidally dispersed micelles of diameter of the order of 10 cm, and is associated with calcium and phosphate ions. 

Casein may be considered to be a conjugated protein, that is the protein is associated in nature with certain non-protein matter known as prosthetic groups. In the case of casein the prosthetic group is phosphoric acid. The protein molecule is also associated in some way with calcium. The presence of these inorganic materials has an important bearing on the processability and subsequent use of casein polymers. 

Although acid caseins are employed for a number of purposes, rennet caseins in which the protein remains associated with calcium and phosphate are preferred for plastics applications. Rennet is the dried extract of rennin, obtained from the inner lining of the fourth stomach of calves, and is a very powerful coagulant. As little as 0.2 parts per million are said to be sufficient to coagulate slightly acidic milk. Its coagulating power is destroyed at 100°C. 

Milk from cows contains 3.2% protein, about 80% of which is casein. Casein is isolated by a precipitation process from milk, involving heating, rinsing to remove whey, and drying to a powder. The yield is about 3 kg/ 100 kg skim milk. Rennet casein is obtained when the casein is precipitated by chymosin enzyme, also known as rennet, and acid casein is produced when precipitation is accomplished by acidification. Acid casein is usually found in the form of sodium caseinate or calcium caseinate, which are water-soluble salts. Caseinates are made by reacting NaOH or CaOH with a slurry of casein curd or powder and then spray drying (Southward, 2010). 

The casein, which is contaminated with calcium phosphate and fats, is filtered off to as small a volume as possible (about 500 cc.) and transferred to a 2-1. beaker. It is then treated with 0.1 M sodium hydroxide, the alkali being added slowly and with stirring through a capillary extending to the bottom of the beaker (Note 4). The addition of alkali is continued until the / ll of the mixture reaches 6.3 (Note 5) 100-150 cc. of the alkali is required. The end-poinl is determined by matching against... 

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.

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). 

Decrease in pH. After heating at 140°C for 20 min, the pH of milk has decreased to about 5.8 due to acid production from pyrolysis of lactose, precipitation of soluble calcium phosphate as Ca3(P04)2, with the release of H+, and dephosphorylation of casein with subsequent precipitation of the liberated phosphate as Ca3(P04)2 with the release of H+. The heat-induced precipitation of Ca3(P04)2 is partially reversible on cooling so that the actual pH of milk at 140°C at the point of coagulation is much lower than the measured value and is probably below 5.0. 

SOFTEN 4 g CASEIN WITH 4 ml WATER. SHAKE UP 1 g CALCIUM OXIDE IN 4 ml WATER. POUR THE CALCIUM OXIDE MIXTURE INTO THE CASEIN WHILE STIRRING. THE RESULTING SMOOTH PASTE IS AN EXCELLENT GLUE FOR PAPER AND FOR WOOD. 

The proteins of milk fall into several classes of polypeptide chains. These have been delineated most completely in bovine milk, and a system of nonmenclature has been developed for them (Chapter 3 Eigel et al. 1984). One group, called caseins, consists of four kinds of polypeptides asr, as2-. and 3-, and k- with some genetic variants, post translational modifications, and products of proteolysis. Almost all of the caseins are associated with calcium and phosphate in micelles 20-300 fim in diameter (see Chapter 9). The other milk proteins, called whey proteins, are a diverse group including /3-lactoglobulin, a-lactalbumin, blood serum albumin, and immunoglobulins (Chapter 3). Almost all... 

Eigel, W. N. and Randolph, H. E. 1974. Preparation of whole 7-casein by treatment with calcium phosphate gel. J. Dairy Sci. 57, 1444-1447. 

Clarification by removal of casein with such agents as calcium chloride, acetic acid, cooper sulfate, or rennin has often been employed to obtain a serum more suitable for refractometric measurements. Obviously the composition, and hence the refractive index, of such sera will depend on the method of preparation. Furthermore, some of the serum proteins may be precipitated with the casein by some of the agents used, particularly if the milk has been heated. Refractive index measurements of such sera are not generally considered as satisfactory as freezing point measurements for detection of added water (David and MacDonald 1953 Munchberg and Narbutas 1937 Schuler 1938 Tell-mann 1933 Vleeschauwer and Waeyenberge 1941). Menefee and Overman (1939) reported a close relation between total solids in evaporated and condensed products and the refractive index of serum prepared therefrom by the copper sulfate method. Of course, a different proportionality constant would hold for each type of product. 

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). 

Micelles in bovine milk contain physiologically significant amounts of calcium and phosphorous because of phosphorylated seryl residues in asl-, aS2- and (3-casein. These casein phosphopeptide (CPP) fragments help to create thermodynamically stable casein micelles super-saturated with calcium and phosphate, thus contributing to the stability of milk during... 

The temperature-sensitive precipitation of unmodified and methylated /3-caseins in the presence of calcium was measured also (see Figure 3). Methylation caused an increase of up to 3°C in the precipitation temperature of calcium /3-caseinate. Results from rennet clotting of an asi-K casein micelle system indicated that replacing native asi-casein with the reductively methylated protein had little influence on clotting time, while replacing K-casein with its reductively methylated derivative re-... 

Protein Composition of Milk. Skim milk is a colloidal suspension of extreme complexity. The particulate phase, the casein micelles, consists primarily of a mixture of asi, as2, / , and x-caseins combined with calcium ions and an amorphous calcium-phosphate-citrate complex. The soluble phase contains lactose, a fraction of the caseins and calcium, and, in raw milk, the whey proteins, which are predominantly /3-lacto-globulin and a-lactalbumin. When milk is centrifuged at high speed (in our experiments, 30 min at 110,000 X gravity), the casein micelles sediment. This permits one to separate the two physical phases of skim milk and to measure changes in composition of the phases resulting from... 

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