Carl Holt

Effects of colloidal calcium phosphate content and free calcium ion concentration in the milk serum on the dissociation of bovine casein micelles

The strength of binding of the individual caseins and the nature of the bonding within bovine casein micelles were examined through dissociation of the micelles by dialysis of skim milk either against phosphate-free buffers containing 3 or 6 mm-CaCl 2 , or against buffers that were nearly saturated with respect to micellar calcium phosphate, but which had a free Ca 2+ concentration in the range 0·4–5·9 mm. Dissociation was followed by ultracentrifuging the dialysed milks and determining the partition of the total and the individual caseins between the pellet and serum. During dialysis against the phosphate-free buffers both colloidal Ca and P i in the milks decreased and about 30 % of the P i could be removed without significant casein dissociation. With further loss of P i , however, increasing dissociation occurred and the proportions of the individual caseins retained in the casein pellet were in the order α s2 - > α s1 - > β- ≈ κ-casein. Dialysis against the calcium phosphate buffers resulted in no loss of colloidal P i but colloidal Ca increased with the free Ca 2+ concentration of the buffer. Little change in the casein partition occurred in the presence of more than 1 mm free Ca 2+ , but serum casein increased markedly at lower levels, and the strength of binding of the individual caseins in the pelleted casein was in the order α s2 -> α s1 - > β- > κ-casein. In both types of buffer, dissociation is considered to occur through the breaking of linkages between the caseins and inorganic constituents. Analysis of the amino acids in a calcium phosphate-rich material obtained after exhaustive proteolytic digestion of casein micelles suggests that these linkages involve the phosphate centres of the caseins.

Inorganic constituents of milk. III. The colloidal calcium phosphate of cow's milk.

Experiments involving the depletion of milk calcium phosphate (MCP) with EDTA have shown that it has a Ca to phosphate ratio of 1.61 +/- 0.04, close to that of dental enamel. The calcium phosphate also contains small amounts of citrate (citrate/Pi = 0.097 +/- 0.011 and Mg (Mg/Pi = 0.044 +/- 0.011). These experiments and model calculations indicate that the Ca binding capacity of casein is not reduced by mineralization with calcium phosphate and hence that ion binding sites and nucleation sites are distinct, or nearly so. Calculations of the ionic equilibria in the aqueous phase of milk show that there is an invariant ion activity product for a molecular formula close to that of dicalcium phosphate. It is suggested that MCP is a mixture of salts, with dicalcium phosphate a precursor of more basic salts such as octacalcium phosphate and hydroxyapatite.

Inorganic constituents of cheese: analysis of juice from a one-month-old Cheddar cheese and the use of light and electron microscopy to characterize the crystalline phases

SUMMARY. A method of extracting cheese juice is described. Juice from a single onemonth-old Cheddar cheese was used to investigate the relationship between the composition of the cheese juice and the occurrence of crystalline aggregates in the cheese. Two classes of crystalline aggregates were identified by light microscopy and also by electron microscopy in combination with X-ray microanalysis: the larger class contained both Ca and P whereas the smaller class contained Ca but not P. Concentrations of ions in the cheese and cheese juice were determined and corrected for the excluded volume of co-solutes and other phases present. When this was done, comparison of the concentrations of Na, K, Cl and lactate in the cheese and the cheese juice indicated that they were virtually entirely in solution, whereas appreciable amounts of the total Ca, P and Pj in the cheese were not extracted in the cheese juice. The amounts of the non-extractable salts were consistent with the volume fractions of the crystalline aggregates found by microscopy; calculations of the ion equilibria in the cheese juice showed that it was supersaturated with respect to various Ca phosphate salts and to tricalcium citrate. It is concluded that the physicochemical origin of the crystalline aggregates was the nucleation and growth of crystals from a supersaturated solution, though the sites of nucleation may have been formed by the microbial activity in the cheese. Of the free amino acids found in the juice, none was at a sufficiently high concentration to form a saturated solution, indicating that in this relatively young cheese the crystalline aggregates were not formed from amino acids. Cheese may be thought of as a multi-phase system normally comprising a paracasein gel in which fat, a salty acidic whey, enzymes and bacteria are held. Also, during maturation, various crystalline phases of salts may form in and on this gel system. The bacterial colonies and enzymes may be considered to inhabit the aqueous phase or at least to be adsorbed at an interface with it. The chemical composition of this water phase largely determines the types and numbers of bacteria that may grow in cheese, since bacteria take up nutrients from and pass metabolic products to the aqueous phase. Similarly, enzymes act on substrates in the aqueous phase and at water-protein and water-lipid interfaces, so its chemical composition

Inorganic constituents of milk: I. Correlation of soluble calcium with citrate in bovine milk

A high correlation was found between soluble calcium and citrate concentrations during a study of seasonal changes in the composition of the commercial milk supply to South-west Scotland. A simple physicochemical model of the ion equilibria in the aqueous phase of milk suggests that the observed correlation is causal. This hypothesis was corroborated by an analysis of data on individual cow milks and by experiments involving the addition of citrate to milk at constant pH.

Measurements of the size of bovine casein micelles by means of electron microscopy and light scattering

Abstract Casein micelles from the milk of two cows were examined by electron microscopy and light-scattering methods. Samples prepared by freeze etching and negative staining were examined and size distributions were determined by counting about 3000 images. Average micellar sizes were also calculated from sedimentation, diffusion, and elastic light-scattering results. In the literature there is a general discrepancy between the average size of micelles determined by electron microscopy and light scattering. In this work we have found that there is a very long tail at the large particle end of the size distribution curve of casein micelles. Failure to detect and characterize this tail region could be responsible for the usual observation that average sizes measured by electron microscopy are lower than those found by light scattering. We obtained reasonably consistent average sizes with all the techniques, making it unnecessary to postulate a large, water-rich, coat region for the micelles as has previously been done. The great differences in the shapes of the size distributions found by electron microscopy and reported in the literature can be understood by supposing that the smaller particles are detected and classified with varying effectiveness, depending on the method used to prepare the samples.

Natural variations in the average size of bovine casein micelles: II. Milk samples from creamery bulk silos in south west Scotland

Samples of bulk silo milk from 5 creameries in the south west of Scotland were taken over a 16-month period. The average radii of casein micelles were determined by measuring the wavelength dependence of the turbidity of skim-milk diluted with its own ultrafiltrate. In addition, the concentrations of casein, soluble and colloidal Ca and P 1 were measured. The average size of casein micelles followed a pronounced seasonal trend with smaller average sizes in the summer compared to the winter period, confirming earlier observations made on milks from individual cows. The average size of casein micelles correlated positively ( r = +0·777, P 1 per unit weight of casein and negatively ( r = −0·77, P r = −0·61, P

Natural variations in the average size of bovine casein micelles: III. Studies on colostrum by electron by microscopy and light scattering

Bovine casein micelles in pre- and post-partum colostrum have been characterized by both electron microscopy and light scattering. Attempts have been made to relate the changes in average micelle size to the partitioning of Ca and inorganic orthophosphate betwen the colloidal and aqueous phases. Pre-partum colostrum, and to a decreasing extent post-partum colostrum, contain casein micelles several microns in diameter. These giant micelles often contain closed cavities suggesting that they are formed by aggregation of particles comparable in size to normal casein micelles. Electron microscopy of mammary gland alveoli from nearterm pregnant heifers shows clear differences in average size between micelles immediately before and after secretion from Golgi vesicles. It appears that micelles aggregate in the alveolar lumen, possibly as a result of continuing co-precipitation of casein with Ca phosphate.

Comparison of the structure of micellar calcium phosphate in milk from six species by extended X-ray absorption fine structure spectroscopy

Extended X-ray absorption fine structure spectra, above the Ca K absorption edge, were recorded from lyophilized casein micelles obtained from the milk of cow, pig, goat, rabbit and rat. A spectrum was also obtained from the calcium phosphate prepared from the casein micelles of human milk. Comparison of these spectra showed that the short-range environment of calcium in the micellar calcium phosphate was the same in all six species. Furthermore, these spectra closely resembled that of brushite (CaHP0 4 . 2H 2 0). However, X-ray diffraction showed no evidence for an extensive crystal lattice in micellar calcium phosphate.

The inorganic constituents of milk IV. Diffusible calcium and magnesium concentrations in goat's milk and the effect of starvation

The concentrations of diffusible Ca and Mg in milk diffusates from 3 goats were determined before, during and after a 2-d period of starvation. Free Ca-concentrations were determined by a murexide method and compared with values calculated from a detailed model of the ion equilibria. It was shown that diffusible Ca increased as citrate increased during fasting and both decreased after refeeding, but there was a difference of about half a day in the response curves, with citrate following Ca. The difference was accounted for mainly by a change in free Ca2+ concentration which in turn was related mainly to changes in milk pH. Diffusible Mg underwent changes similar to those of diffusible Ca.

Post-secretory aggregation of caseins.

Particles as large as several micrometer diam. have been observed occasionally in normal milk and commonly in prepartum and postpartum colostrum. These particles can be dissociated by EDTA and their appearance closely resembles that of normal casein micelles. However, they are often too large to have been completely formed within the Golgi vesicles of mammary epithelium and hence some degree of post-secretory aggregation of caseins is thought to occur. Two possible mechanisms of post-secretory aggregation of caseins are: (1) a continuation of the normal processes of micelle assembly in the alveolus and (2) aggregation as a result of limited proteolysis of the caseins during the time the milk is in the mammary gland. Incubation of milk with fibrinolysin, however, failed to produce aggregation of normal micelles.