DAIRY CHEMISTRY AND BIOCHEMISTRY known about them. rowland observed that when milk was heated to 95.c for 10 min, 80% of the nitrogenous compounds in whey were denatured and co-precipitated with the casein when the pH of the heated milk was adjusted ubsequently to 4.6. He considered that the heat-denaturable whey proteins represented the lactoglobulin and lactalbumin fractions and designated the remaining 20%proteose-peptone. The proteose-peptone fraction was precipitated by 12% trichloracetic acid(TCA) but some nitrogenous com- pounds remained soluble in 12% TCA and were designated as nonprotei based on that of Rowland, is shown in Figure 4y groups of milk proteins, A scheme for the fractionation of the principal 4.3 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 4.3.1 Acid ( isoelectric) precipitation Acidification of milk to about pH 4.6 induces coagulation of the casein Aggregation occurs at all temperatures, but below about 6.C the aggregates are very fine and remain in suspension, although they can be sedimented by low-speed centrifugation. At higher temperatures(30-40oC), the aggregates are quite coarse and precipitate readily from solution at above about 50 C, the precipitate tends to be stringy and difficult to handle. For laboratory-scale production of casein, HCI is usually used for cidification; acetic or lactic acids are used less frequently. Industrially, HCI is also usually used; H, SO4 is used occasionally but the resulting whey is not suitable for animal feeding(MgSO4 is a laxative). Lactic acid produced in situ by a culture of lactic acid bacteria is also widely used, especially in New Zealand, the principal producer of casein time is allowed for solution, isoelectric casein is essentially free of calcium phosphate. In the laboratory, best results are obtained by acidifying skim milk to pH 4.6 at 2C, holding for about 30 min and then warming to 30-35.C. The fine precipitate formed at 2 C allows time for the colloidal calcium phosphate to dissolve( Chapter 5). A moderately dilute acid(1 M) is preferred, since concentrated acid may cause localized coagulation. Acid production by a bacterial culture occurs slowly and allows time for colloidal calcium phosphate to dissolve. The casein is recovered by filtration or centrifugation and washed repeatedly with water to free the casein of lactose and salts. Thorough removal of lactose is essential since even traces of152 DAIRY CHEMISTRY AND BIOCHEMISTRY known about them. Rowland observed that when milk was heated to 95°C for 10 min, 80% of the nitrogenous compounds in whey were denatured and co-precipitated with the casein when the pH of the heated milk was adjusted subsequently to 4.6. He considered that the heat-denaturable whey proteins represented the lactoglobulin and lactalbumin fractions and designated the remaining 20% 'proteose-peptone'. The proteose-peptone fraction was precipitated by 12% trichloracetic acid (TCA) but some nitrogenous com￾pounds remained soluble in 12% TCA and were designated as nonprotein nitrogen. A scheme for the fractionation of the principal groups of milk proteins, based on that of Rowland, is shown in Figure 4.4. 4.3 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. 4.3.1 Acid (isoelectric) precipitation Acidification of milk to about pH 4.6 induces coagulation of the casein. Aggregation occurs at all temperatures, but below about 6°C the aggregates are very fine and remain in suspension, although they can be sedimented by low-speed centrifugation. At higher temperatures (3O-4OcC), the aggregates are quite coarse and precipitate readily from solution. At temperatures above about 5OCC, the precipitate tends to be stringy and difficult to handle. For laboratory-scale production of casein, HCl is usually used for acidification; acetic or lactic acids are used less frequently. Industrially, HC1 is also usually used; H,SO, is used occasionally but the resulting whey is not suitable for animal feeding (MgSO, is a laxative). Lactic acid produced in situ by a culture of lactic acid bacteria is also widely used, especially in New Zealand, the principal producer of casein. The inorganic colloidal calcium phosphate associated with casein in normal milk dissolves on acidification of milk to pH 4.6 so that if sufficient time is allowed for solution, isoelectric casein is essentially free of calcium phosphate. In the laboratory, best results are obtained by acidifying skim milk to pH 4.6 at 2"C, holding for about 30min and then warming to 30-35°C. The fine precipitate formed at 2°C allows time for the colloidal calcium phosphate to dissolve (Chapter 5). A moderately dilute acid (1 M) is preferred, since concentrated acid may cause localized coagulation. Acid production by a bacterial culture occurs slowly and allows time for colloidal calcium phosphate to dissolve. The casein is recovered by filtration or centrifugation and washed repeatedly with water to free the casein of lactose and salts. Thorough removal of lactose is essential since even traces of