CHEMISTRY OF MILK

Module 3. Milk proteins

Lesson 7
CASEINS: FRACTIONATION, STRUCTURE AND CONFORMATION, ANS PHYSICO CHEMICAL PROPERTIES

7.1 Definition

The behaviour of individual milk proteins and their concentration will be known only when they are fractionated from milk. This can be done by chemical or by several other means. A classical scheme for fractionation of milk proteins by chemical method was developed by Rowland (1938) which was further modified by Aschaffenburg and Drewry (1959).

7.2 Rowland Method of Fractionation

In this method of fractionation the total protein in milk will be fractioned initially into protein nitrogen and non protein nitrogen using the insoluble property of NPN compounds in trichloro acetic acid (TCA). The casein components are precipitated by the utilizing solubility of non-casein proteins in 10 % acetic acid. The NPN components are further fractioned by utilizing the insolubility of the components with variation.

Fig. 7.1 Rowland fractionation

(Source: Rowland,J.Dairy Res,1938)

7.3 Aschaffenburg and Drewry Method

In the modified method suggested by Aschaffenburg and Drewry (1959) the solubility of some fractions of the proteins in sodium sulphate solutions is utilized where the total albumin along with NPN compounds will be soluble. While in saturated sodium sulphate the albumins get precipitated (Figure 7.2).

Fig.7.2 Protein fractionation by aschaffenburg and drewry (1959)

(Source: Aschaffenburg and Drewry,Int.Dairy congr,1959)

7.4 Differential Solubility Methods

Several 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. Hipp et al (1952)^* developed two procedures which have been used extensively or partly incorporated into other methods. The first one is based on the differential solubility of casein in 50% alcohol in the presence of ammonium acetate by carrying the pH temperature and ionic strength. The second procedure involves the dispersion of whole casein in 6.6M urea and separation of casein fractions by dilution, pH adjustment and finally addition of (NH₄)₂ SO₄. The order of precipitation of the casein in both methods is α-casein , β-casein and then γ- casein. Warner’s casein fractions are now identified as follows, α- casein is a mixture of α_s and κ - caseins.

The α_s caseins and κ - caseins have been prepared largely from either whole casein or the α-casein fraction of Hipp and co workers. Crude α_s -casein is prepared from casein obtained by CaCl₂ precipitation at 37°C by removing calcium with oxalate to solubilize the casein and reprecipitation with 0.25M CaCl₂ at 37°C and pH 7.0. The κ-caseins are removed from the supernatant by precipitation with Na₂SO_{4 ,} followed by re-precipitation from 50% ethanol with ammonium acetate or by using calcium oxalate as a precipitate to enhance the removal of the other caseins. Adjustment of the pH of whole casein dispersions in urea also has been used to precipitate the α_s -casein either by adjusting 6.6 M urea dispersion to pH 1.3 to 1.5 with H₂SO₄ or by adjusting a 3.3M urea to pH 4.5. The crude k caseins can be obtained from supernatant of the H₂SO₄ method, by precipitating with (NH₄)₂ SO₄ and purified by reprecipitation from aqueous ethanol. More than 90% of α_s1 -caseins have been recovered from acid casein by precipitation with 75mM CaCl₂ at 5°C.

Fig. 7.3 Fractionation of casein by Hipp et. al (1952) base on urea solubility
(Source: Hipp et. al., J. of Dairy Sci., Solubility)

Starting with α -casein fraction of Hipp and co workers the α_s -caseins can be precipitated by Ca Cl₂ treatment and the κ-caseins can be removed from the supernatant by pH adjustment to 4.7. Some research workers have added 12% tricholoro acetic acid (TCA) to a 6.6 M urea dispersion of the same fraction at 3°C and precipitated α_s-fraction . After removal of the urea and TCA from the supernatant they adjusted the pH to 7.0 added CaCl₂ to 0.25 M and removed the precipitate. κ-casein was finally obtained from the supernatant at pH 4.4. Wake (1959 ) prepared κ-casein from the supernatant remaining from the β- casein precipitation by the first procedure of Hipp and his co-workers by adjusting the pH to 5.7. A κ-casein concentrate has been prepared from commercial casein based on the differential solubility’s of the caseins in CaCl₂ solutions.

The major β-casein component can be prepared by a simplification of the urea method of Hipp and his co workers. Whole casein is dispersed in 3.3M urea at pH 7.5 and adjusted to pH 4.6 which precipitates the bulk of α_s1 and κ-caseins . The supernatant is adjusted to pH 4.9 diluted to 1.0M urea and warmed to 30°C precipitating the major β- casein.

7.5 Physico Chemical Characteristics of Caseins

The physico-chemical parameters for the casein fractions are shown in Table 7.1. Number of these parameters is derived from the composition based on determination of the primary structure.

Table 7.1 Physico-chemical characteristics of casein

(Source: Development in Dairy Chemistry, Fox, 1982)