Casein precipitation

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). 

For cheese sample Ultraturrax homogenization (two cycles) with H2O, centrifugation (for skimming), acidification at pH 4.2. 6 (for casein precipitation), centrifugation, filtration For cheese sample extraction with H2O (sonication) for milk centrifugation, acidification (pH 4.6) to precipitate caseins, centrifugation, filtration... 

Any factor that accelerates the crystallization of lactose shortens the storage life of the product. At very low temperatures (below — 23°C), neither lactose crystallization nor casein flocculation occurs, even after long periods. Enzymatic hydrolysis of lactose by /S-galactosidase before freezing retards or prevents lactose crystallization and casein precipitation in proportion to the extent of the hydrolysis (Figure 2.14). 

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). 

Explain why casein precipitates when acetic acid is added to it. 

Note Rapid and continuous agitation during the addition prevents casein precipitation. 

Renders casein precipitable by calcium Cleaves fats... 

Figure 15.14 Separation of milk proteins [reproduced with permission from B.B. Gupta, J. Chromatogr., 282, 463 (1983)]. Conditions sample, 100 pi of whey from raw skimmed milk (casein precipitated at pH 4.6) column, 60 cm x 7.5 mm i.d. stationary phase, TSK 3000 SW (silica, 10pm) mobile phase, 0.5 ml min buffer containing 0.1 M NaH2PO4, 0.05 M NaCl and 0.02% NaNa (pH 6.8) UV detector, 280 nm. Peaks (with molecular masses) 1 = high molecular weight proteins 2 = -y-globulin (150000) 3 — bovine serum albumin (69000) 4 = /3-lactoglobulin (35000) 5 = a-lactalbumin (16500) other components not identified.

The caseins exist in milk as polydisperse aggregates ranging in size from ca. 40 to 220nm (3), but the size distribution of micelles depends upon the method of measurement. These casein micelles scatter light and are responsible for the whitish, opaque nature of skim milk. The casein micelles are also associated with a colloidal apatite comprised of calcium-phosphate-citrate (CPC) which has a stabilizing influence on the micelle structure. The colloidal CPC is in equilibrium with soluble CPC in the milk serum phase and is solubilized as the pH is reduced. Thus, as the pH is reduced to the isoelectric point of the caseins (4.6), the colloidal CPC solubilizes, and the caseins precipitate (143). This phenomenon should be kept in mind during some of the following discussions. 

White, amorphous powder or granules without odor or taste. Very sparingly sol in water and in nonpolar organic solvents sol in aqueous so]ns of alkalies, levorotatory. The isoelectric zone is around pH 4.7 sol in coned HCI with light violet color. Amphoteric forms salts with both acids and bases. Present in bovine milk as neutral calcium caseinate and in human milk as potassium caseinate. Precipitated from solns satd with metallic salts. Forms a hard, inso] plastic with formaldehyde. 

Casein, precipitated from cow s milk by acetic acid, and purified by washing with HjO, solution in very dilute NaHO, precipitation with acetic acid, washing with alcohol and ether and drying, is a snow-white powder, almost insoluble in pure HaO, soluble in alkalies and alkaline phospbates and carbonates. It is distinctly acid in reaction and decomposes the carbonates of Ca, Mg and Ba, suspended in HaO, with evolution of COa. It also dissolves in lime or baryta water, and the solutions so formed do not coagulate when boiled. 

The emulsions formed using a - and ( caseins appear to be understandable in terms of similarity to the behaviour of the proteins themselves. However, it appears that more attention must be paid to the behaviour of caseins in mixed systems, especially in terms of the factors which govern the exchange of caseins on the interfaces. Such interchange can influence the behaviour of the particles by altering the nature of the surface layer (e.g. ( casein precipitates less readily than -casein v en subjected to Ca " ). 

In order to reach an analogous separation, Hammarsten proposes the use of casein to purify the lab extract of calf stomach lining Casein precipitates pepsin, while reimet remains in solution. The maceration of stomach lining is made in o 24 per cent HCl. The precipitation is caused by the addition of a 4 per cent solution of sodium caseate. To 130 c.c. of acid maceration are added, slowly and with shaking, 200 c.c. of a neutral solution of sodium caseate. Then N/10 NaOH is added until no further precipitation occurs. This precipitate contains the pepsin, while the rennet remains in solution. 

Milk, whey proteins prepared by casein precipitation diluted (1 4) with 8.25 mM borate buffer, 0.1% Tween 20, pH 8.0 Various cereals, extraction with 50% 1-propanol, glutenins with 50% 1-propanol, 1% DTT... 

The separation of dairy proteins by CE has been generally carried out by CZE and has been exhaustively covered in several review papers, - - thus Table 30.8 only presents the key methodologies that offer the reader an overview of their most distinctive features. Basically, dairy protein analysis has been performed in whole milk for the simultaneous determination of caseins and whey proteins, or in fractions isolated from milk after casein precipitation. The first approach being used when the quantitative determination of the major proteins is required for the calculation of casein/whey protein ratios or for authentication purposes where an analysis of the whole protein profile is required. In both cases, accurate quantitative data must be derived. However, few studies have addressed the analysis of both groups of proteins in a single run by presenting quantitative data based on calibration curves constructed with analytical standards and good recovery of all proteins from milk samples. 

Finally, a third method was based on MEKC, where proteins were separated after complete denaturation with SDS and oL-dithiothreitol in uncoated capillaries at pH 9.5. Although the method had the advantage of being very rapid (separation completed in less than 90 s), it was not quantitative (Table 30.8). The second approach reported for the analysis of dairy proteins was the analysis of the whey fraction after casein precipitation. Unlike the methods described earher, separations were carried out in uncoated capillaries using polymeric additives, a high ionic strength, and high pH... 

Other whey protein fractions were extracted after casein precipitation with 1 M HCl until pH 4.6, centrifugation at 700 X g (10 min), and filtration of the supernatant. 

Rennet casein n. A type of casein precipitated from milk to means of rennet, the dried extract of stomach secretions firom calves or other ruminants containing the enzyme reimin. 

The pH of fresh milk is 6.5 6.75. When the pH decreases to 4.6, due to lactic acid formation, caseins precipitate, leaving a yellowish solution of whey (serum). The acidification (fermentation of lactose to lactic acid) is not only the spontaneous activity of contaminating microorganisms during storage of milk, but also a result of the activity of arange of cultural microorganisms used in dairy industry. 

The isoionic point of a protein depends upon its amino acid composition. At its isoionic point, a protein has no net charge, and its solubility is at a minimum. As a consequence, a protein tends to precipitate form solution at its isoionic point. For example, casein, a protein in milk, has a negative charge at pH 6.3. Casein has many glutamic acid and aspartic acid residues. If acid is added to milk, these side chains are protonated, and casein precipitates. Casein is used in making cheese, and it is obtained by adding acid to milk or by adding bacteria that make lactic acid, which has the same effect. 

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