Harjinder Singh

Heat stability of milk: pH-dependent dissociation of micellar κ-casein on heating milk at ultra high temperatures

Preheating milk at 140 °C for 1 min at pH 6·6, 6·8, 7·0 or 7·2 shifted the heat coagulation time (HCT)/pH profile to acidic values without significantly affecting the maximum stability. Whey proteins (both β-lactoglobulin and α-lactalbumin) co-sedimented with the casein micelles after heating milk at pH 6·9) resulted in the dissociation of whey proteins and κ-casein-rich protein from the micelles and the residual micelles were unstable, without a maximum–minimum in the HCT/pH profile. Preformed whey protein–casein micelle complexes formed by preheating (140 °C for 1 min) milk at pH 6·7 dissociated from the micelles on reheating (140 °C for 1 min) at pH > 6·9. The dissociation of micellar-κ-casein, perhaps complexed with whey proteins, may reduce the micellar zeta potential at pH ≃ 6·9 sufficiently to cause a minimum in the HCT/pH profile of milk.

Heat stability of milk: further studies on the pH-dependent dissociation of micellar κ-casein

Whey protein complexed and became co-sedimentable with casein micelles after heating milk at ≥ 90°C for 10 min at pH ≤ 6·9 while at higher pH values (7·3) whey proteins and κ-casein-rich protein dissociated from the micelles on heating. κ-Casein-deficient micelles were more sensitive to heat, Ca 2+ or ethanol than whey protein-coated or native micelles and were readily coagulable by rennet. Isolated κ-casein added to skim milk before preheating (90°C for 10 min) did not associate with the micelles at pH ≥ 6·9. Sodium dodecyl sulphate increased the level of both non-sedimentable N (NSN) and N -acetylneuraminic acid (NANA) and shifted the NSN-pH and NANA-pH curves to more acidic values while cetyltrimethylammonium bromide had the opposite effect. It is suggested that the pH-dependent dissociation in micellar κ-casein, which appears to be reversible, depends on the surface charge on the micelles; at a certain negative charge, disruption of hydrophobic and electrostatic forces could result in the dissociation of κ-casein from the casein micelles.

Heat stability of milk: influence of colloidal and soluble salts and protein modification on the pH-dependent dissociation of micellar κ-casein

Reducing the colloidal calcium phosphate (CCP) content of milk by 40% or increasing it by 20% did not significantly affect the heat-induced pH-dependent dissociation of micellar κ-casein. However, changes in soluble Ca and phosphate affected the dissociation of κ-casein markedly; decreasing the phosphate concentration or increasing the Ca concentration reduced the formation of non-sedimentable N (NSN) and non-sedimentable 12% TCA-insoluble N -acetyl-neuraminic acid (NANA). Dialysis of milk against water for short periods (∼ 5 h) reduced the formation of both NSN and non-sedimentable 12% TCA-insoluble NANA, as did NaCl at concentrations above 0·05 M. Modification of protein amino groups by succinylation promoted the release of κ-casein while amidation of carboxyl groups had the opposite effect. It appears that the pH-dependent dissociation of κ-casein produced on heating milk above 90°C is controlled by electrostatic interactions. The effects of soluble ions such as Ca 2+ or Na + appear to be due to shielding of such negatively-charged groups as seryl phosphate and carboxyl on the protein, thus reducing the release of κ-casein.

Binding of zinc to bovine and human milk proteins

Zn binding by whole bovine and human casein and by purified bovine caseins and whey proteins was investigated by equilibrium dialysis. Bovine alpha s1-casein had the greatest Zn-binding capacity (approximately 11 atoms Zn/mol). Protein aggregation was observed as Zn concentration was increased and the protein precipitated at a free Zn concentration of 1.7 mM. Zn binding increased with increasing pH in the range 5.4-7.0 and decreased with increasing ionic strength. Competition between Zn and Ca was observed for binding to alpha s1-casein indicating common binding sites for these two metals. Bovine beta-casein bound up to 8 atoms Zn/mol and precipitated at a free Zn concentration of approximately 2.5 mM, while kappa-casein bound 1-2 atoms Zn/mol. Whole bovine and human casein bound 5-8 atoms Zn/mol and precipitated at a free Zn concentration of approximately 2.0 mM. Scatchard plots for Zn binding to caseins showed upward convexity, possibly due to Zn-induced association of caseins. Apparent average association constants (Kapp) for all caseins were similar (log Kapp 3.0-3.2). Enzymic dephosphorylation of alpha s1- or whole bovine casein markedly reduced, but did not eliminate, Zn binding. Thus, phosphoserine residues appeared to be the primary Zn-binding sites in caseins. With the exception of bovine serum albumin, which bound over 8 atoms Zn/mol, the bovine whey proteins, beta-lactoglobulin, alpha-lactalbumin and lactotransferrin, had little capacity for Zn binding.

Zinc binding in bovine milk

About 90% of the Zn in bovine skim milk was sedimented by ultracentrifugation at 100,000 g for 1 h. About half of the non-sedimentable Zn was non-dialysable, indicating that it was associated with protein, probably non-sedimented casein micelles. Casein micelles incorporated considerable amounts of Zn added to skim milk as ZnCl2, and at Zn concentrations greater than or equal to 16 mM coagulation of casein micelles occurred. Ca was displaced from casein micelles by increasing ZnCl2 concentration and approximately 40% of micellar Ca was displaced by 16 mM-ZnCl2. Micellar Zn, Ca and Pi were gradually rendered soluble as the pH of milk was lowered and at pH 4.6 greater than 95% of the Zn, Ca and Pi were non-sedimentable. These changes were largely reversible by readjustment of the pH to 6.7. About 40% of the total Zn in skim milk was non-sedimentable at 0.2 mM-EDTA and most of the remainder was gradually rendered soluble by EDTA over the concentration range 1-50 mM. This indicates that there are two distinct micellar Zn fractions. No micellar Ca or Pi was solubilized at EDTA concentrations up to 1.0 mM, indicating that both colloidal calcium phosphate (CCP) and casein micelles remained intact under conditions where the more loosely bound micellar Zn fraction dissolved. Depletion of casein micelles of colloidal Ca and Pi by acidification and equilibrium dialysis resulted in removal of Zn, and in colloidal Pi-free milk non-dialysable Zn was reduced to 1.2 mg/l (approximately 32% of the original Zn). Thus, approximately 32% of the Zn in skim milk is directly bound to caseins, while approximately 63% is associated with CCP. Over 80% of the Zn in colloidal Pi-free milk was rendered soluble by 0.2 mM-EDTA, indicating that the casein-bound Zn is the loosely bound Zn fraction in casein micelles. A considerable fraction of the Zn in acid whey (pH 4.6) co-precipitated with Ca and Pi on raising the pH to 6.7 and heating for 2 h at 40 degrees C, indicating that insoluble Zn phosphate complexes form readily under these conditions. Studies on dialysis of milk against water, or dilution of milk or casein micelles with water, showed that CCP and its associated Zn is very stable and dissolves only very slowly at pH 6.6. The nature of Zn binding in casein micelles may help to explain the lower nutritional bioavailability of Zn in bovine milk and infant formulae compared with human milk.

Heat stability of milk: the mechanism of stabilization by formaldehyde

The increase produced by formaldehyde (HCHO) in the heat stability of milk did not occur when milk was treated with HCHO at temperatures up to 60°C followed by dialysis at 5°C. However, the minimum in the heat coagulation time (HCT)–pH curve was irreversibly removed if the milk was preheated at 80–C for 10 min in the presence of 5 mM-HCHO. Although this treatment blocked the e-amino groups of lysyl residues, the stabilizing mechanism is considered to be due to the cross linking action of HCHO which reduced the level of non-sedimentable, κ-casein-rich protein dissociated from the micelles on heating. The specific crosslinking agent, dimethyl suberimidate, modified the HCT-pH profile of milk in a manner similar to preheating at 80°C for 10 min with 5 mM-HCHO, supporting the crosslinking hypothesis. The results of this study appear to lend some support to the proposal of Kudo (1980) that the minimum in the HCT-pH curve of milk is due to the dissociation of κ-casein from the micelles on heating at high temperatures at pH values greater than 6η7.

Heat stability of milk: influence of modifying sulphydryl-disulphide interactions on the heat coagulation time–pH profile

Addition of reducing agents such as 2-mercaptoethanol (2-ME), dithio-threitol and Na sulphite to milk markedly reduced its heat stability at pH values below 7·1. 2-ME reversibly destabilized milk or serum protein-free casein micelle dispersions and promoted the release of κ-casein-rich protein from the micelles. Reduction of either casein micelles or β-lactoglobulin (β-lg) with 2-ME and subsequent blocking of the newly formed –SH groups with N-ethylmaleimide irreversibly reduced the maximum to minimum ratio in the heat stability profile. 2-ME disrupted κ-casein/β-lg complexes and stripped κ-casein from the micelles on heating. The milk or caseinate systems were thus destabilized. Addition of KBrO 4 or iodosobenzoate to milk at 5 HIM eliminated the minimum but destabilized milk in the region of the maximum. However, KIO 3 at 5 mm had a strong stabilizing effect throughout the pH range 6·5–7·3.

Improved Zinc Bioavailability from Colloidal Calcium Phosphate-Free Cow’s Milk

The lower bioavailability of zinc in cow’s milk compared with human milk is well documented, but the basis of this difference has yet to be adequately explained. Over 90% of the Zn in cow’s milk is associated with casein micelles and it has been shown (Lonnerdal et al., 1985) that the bioavailability to rats of this fraction of Zn is similar to that in cow’s milk, but lower than that of human skim milk. We have recently obtained evidence that 60–70% of the Zn in bovine casein micelles is associated with colloidal calcium phosphate (CCP), while the remainder is directly bound to casein (Singh et al., 1987). The object of this study was to investigate whether the CCP fraction influences the bioavailability of Zn in cow’s milk.

Binding of Zinc to Colloidal Calcium Phosphate in Human and Cow’s Milks

The lower bioavailability of Zn in cow’s milk compared with human milk is well documented. It is generally agreed that this is due to differences in the nature and/or concentrations of Zn-binding ligands in the two milks and in the interactions of Zn with these ligands or their digestion products in the gastrointestinal tract. Lonnerdal et al. (1985) have shown that the bioavailability to rats of the Zn associated with bovine casein micelles is lower than that in human milk. However, the nature of Zn binding in casein micelles is poorly understood. The object of this study was to investigate the distribution of Zn in human and bovine milks and to examine the nature of Zn binding in casein micelles.