Preparation of casein and whey proteins

Skim milk prepared by mechanical separation (see Chapter 3) is used as the starting material for the preparation of casein and whey proteins. 

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Depending on the preparative method used, the membrane may or may not contain skim-milk proteins (i.e. caseins and whey proteins) if the membrane has been damaged prior to isolation, it may contain considerable amounts of these proteins. The membrane contains unique proteins which do not occur in the skim-milk phase. Many of the proteins are glycoproteins and contain a considerable amount of carbohydrate (hexose, 2.8-4.15% hexosamine, 2.5-4.2% and sialic acid, 1.3—1.8%). 

The applications of colloid solutions are not restricted to paints and clay. They are also to be found in inks, mineral suspensions, pulp and paper making, pharmaceuticals, cosmetic preparations, photographic films, foams, soaps, micelles, polymer solutions and in many biological systems, for example within the cell. Many food products can be considered colloidal systems. For example, milk is an interesting mixture containing over 100 proteins, mainly large casein and whey proteins [6,7]. 

Milk protein films are widely used in food and non-food sectors such as textile, paper, leather etc. These films and composites are used in different scopes of food science such as coating of meat and fresh-cut fruits, bread wrapping and preparation of antimicrobial packaging. Cow s milk proteins are divided into 2 parts caseins and whey proteins. The major fraction of milk proteins (approximately 80 %) belongs to casein group which consists of si-, s2-, ) -, and jc-casein and their portions are nearly 38, 10, 36 and 12, respectively with the corresponding molecular masses of 23,164, 25,388, 23,983 and 19,038 Da [78]. 

In contrast to the caseins, the whey proteins do not precipitate from solution when the pH of milk is adjusted to 4.6. This characteristic is used as the usual operational definition of casein. This difference in the properties of the two milk protein groups is exploited in the preparation of industrial casein and certain varieties of cheese (e.g. cottage, quarg and cream cheese). Only the casein fraction of milk protein is normally incorporated into these products, the whey proteins being lost in the whey. 

CE, with its high resolving power, rapid method development, easy sample preparation, and low operational cost, is reported to be an excellent technique for resolving caseins (including different genetic variants), peptides derived from them, and whey proteins (16-19). Peptide profiles obtained by CE supplement the information obtained by reversed-phase high performance liquid chromatography (RP-HPLC) (17, 20). The application of CE to the assessment of proteolysis in milk and different cheese types has acquired an enormous importance in recent years. Reviews on the application of CE to this field can be found in papers by Otte et al. (21) and Redo et al. (22). 

The most abundant milk protein is casein, of which there are several different kinds, usually designated a-, (1-, and K-casein. The different caseins relate to small differences in their amino acid sequences. Casein micelles in milk have diameters less than 300 nm. Disruption of the casein micelles occurs during the preparation of cheese. Lactic acid increases the acidity of the milk until the micelles crosslink and a curd develops. The liquid portion, known as whey, containing water, lactose and some protein, is removed. Addition of the enzyme rennet (chymosin) speeds up the process by hydrolysing a specific peptide bond in K-casein. This opens up the casein and encourages further cross-linking. 

Other Protein Components. Other protein components In complex food systems and In protein Ingredient preparations may Interfere with or modify gelation reactions. Protein Interaction between whey protein and casein upon heating has a profound Influence on the characteristics of the casein gel structure In cheesemaking. Similarly protein Interactions are Important to meat structures. Protein-protein Interaction between soy and meat proteins has also been demonstrated with heat treatment (28). While concrete Interaction data have not been collected on protein gels formed from protein combinations, gelation properties of whey proteln/peanut flour blends have been Investigated GU) ... 

Whey protein concentrates (WPC), which are relatively new forms of milk protein products available for emulsification uses, have also been studied (4,28,29). WPC products prepared by gel filtration, ultrafiltration, metaphosphate precipitation and carboxymethyl cellulose precipitation all exhibited inferior emulsification properties compared to caseinate, both in model systems and in a simulated whipped topping formulation (2. However, additional work is proceeding on this topic and it is expected that WPC will be found to be capable of providing reasonable functionality in the emulsification area, especially if proper processing conditions are followed to minimize protein denaturation during their production. Such adverse effects on the functionality of WPC are undoubtedly due to their Irreversible interaction during heating processes which impair their ability to dissociate and unfold at the emulsion interface in order to function as an emulsifier (22). 

The whey produced during cheese and casein manufacturing contains approximately 20% of all milk proteins. It represents a rich and varied mixture of secreted proteins with wide-ranging chemical, physical and functional properties (Smithers et al., 1996). Due to their beneficial functional properties, whey proteins are used as ingredients in many industrial food products (Cheftel and Lorient, 1982). According to Kinsella and Whitehead (1989), functional properties of foods can be explained by the relation of the intrinsic properties of the proteins (amino acid composition and disposition, flexibility, net charge, molecular size, conformation, hydrophobicity, etc.), and various extrinsic factors (method of preparation and storage, temperature, pH, modification process, etc.). 

Enzymatic gelation of partially heat-denatured whey proteins by trypsin, papain, pronase, pepsin, and a preparation of Streptomyces griseus has been studied (Sato et al., 1995). Only peptic hydrolysate did not form a gel. The strength of the gel depended on the enzyme used and increased with increasing DH. Hydrolysis of whey protein concentrate with a glutamic acid specific protease from Bacillus licheniformis at pH 8 and 8% protein concentration has been shown to produce plastein aggregates (Budtz and Nielsen, 1992). The viscosity of the solution increased dramatically during hydrolysis and reached a maximum at 6% DH. Incubation of sodium caseinate with pepsin or papain resulted in a 55-77% reduction in the apparent viscosity (Hooker et al., 1982). 

We chose to prepare 14C-methyl-K-casein (M-k-C) as a tracer because of the important role of K-casein in stabilizing casein micelles (8) and because K-casein is known to participate in heat-induced interactions with whey proteins, thereby influencing the heat stability of milk (9). The reductive methylation radiolabeling procedure used low concentrations of reagents (10) and resulted in M-k-C containing approximately 1 fiinol of 14C-methyl groups for every micromole of protein monomer (about 3 /xCi/mg). When tracer M-k-C was added to skim milk, and trichloroacetic acid was added to a concentration of 2%, about 1% of the radioactivity remained soluble. After clotting of the milk with excess... 

FIGURE 7.14 Solubility of protein preparations as a function of pH. (a) Turbidity (expressed as absorbancy) of solutions of a whey protein isolate, heated at 70°C for various times (indicated, minutes). (From results by S. Damodaran, see Bibliography), (b) Solubility (percentage of protein in supernatant after centrifuging) of various protein products sodium caseinate (C), peanut (P), and soya (S) proteins. (Approximate results after various sources.) (c) Solubility (as in b) of the protein in a potato juice extract (pH = 7.0, 7=0.2 molar) as a function of solvent volume (v, in ml). See text for lines 1 and 2. (After results by G. A. van Koningsveld. Ph.D. thesis, Wageningen University, 2001.)... 

The development of large-pore membranes facilitates the separation of whey proteins from casein micelles by microfiltration (MF). Membranes used in MF have cut-offs in the range 0.01-10/im, and therefore casein micelles may be in the permeate or retentate streams, depending on the pore size of the MF membranes chosen. MF with large-pore membranes very effectively removes bacteria and somatic cells from milk and may also be used to remove lipoprotein complexes from whey prior to the production of WPCs with improved functionality. The preparation of micellar casein by MF is still at the exploratory stage. 

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