Phosphate in milk

Horst, M. G. ter. 1947. The condition and mutual relationship of calcium caseinate and calcium phosphate in milk. Neth. Milk Dairy J. 1, 137-151. 

In recent years, a considerable body of research has been conducted on the cariostatic effects of cheese. Early work (Shaw et al., 1959 Dreizen et al., 1961) demonstrated that the incorporation of dairy products into the diet greatly reduced the development of dental caries in rats. Reynolds and Johnson (1981) confirmed these findings. Later work (Weiss and Bibby, 1966 Jenkins and Ferguson, 1966) indicated that if enamel is treated with milk in vitro and subsequently washed, the solubility of the enamel is greatly reduced. This effect was attributed to the high levels of calcium and phosphate in milk (Jenkins and Ferguson, 1966) or to the casein (Weiss and Bibby, 1966). Later work supports both viewpoints. 

P NMR spectroscopy is a useful tool to discriminate between phosphorylated molecules in liquid or amorphous/solid-like sample with respect to their nature and dynamics. The major advantage of the NMR technique is that the sample can be analysed without pretreatment or extraction, and can be recovered since NMR is non-destructive. Phosphates in milk and in isolated casein micelles have been widely investigated using liquid-state P NMR spectroscopy As the restricted motion induced by the large colloidal structure of casein micelles does not permit the obtaining of hi ly resolved spectra, only the mobile phosphates (a part of easein phosphoprotein residues, the dissociated inorganic phosphate and the milk fat phospholipids) found in the soluble phase were detected by liquid-state NMR. 

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). 

Van Emon et al. ° developed an immunoassay for paraquat and applied this assay to beef tissue and milk samples. Milk was diluted with a Tween 20-sodium phosphate buffer (pH 7.4), fortified with paraquat, and analyzed directly. Fortified paraquat was detected in milk at less than 1 pgkg , a concentration which is considerably below the tolerance level of 10 pg kg Ground beef was extracted with 6 N HCl and sonication. Radiolabeled paraquat was extracted from ground beef with recoveries of 60-70% under these conditions. The correlation coefficient of ELISA and LSC results for the ground beef sample was excellent, with = 0.99, although the slope was 0.86, indicating a significant but reproducible difference between the assays. 

CCP in milk is mentioned in connection with casein above (Section VI.C). Fluorapatite is a major constituent of phosphate rocks, and a constituent, probably important, of human tooth enamel for those whose drinking water contains significant amounts of naturally occurring or added fluoride. Fluorapatite is significantly less soluble than hydroxyapatite - the relationship between the solubilities of fluorapatite and hydroxyapatite parallels (but is much less extreme than) that between calcium fluoride (Ksp — 3.9 x 10 11 mol3 dm-9) and calcium hydroxide (Ksp = 7.9 x 10 6 mol3 dm 9). Calcium diphosphate, Ca2P207, is believed to be the least soluble of the calcium phosphates. 

We now look more closely at the structure of casein. It is a long molecule with different ends one end is polar and the other is nonpolar. In milk, the polar group (ending with a phosphate group) is positioned to face the polar water, and the non-polar end faces the oil. In effect, each particle of oil has a double coating the inner layer is the non-polar end of the casein emulsifier, and the outer layer is a sheath of polar phosphate groups. 

G.l.c.—May be used for the analysis of pesticide residues,212 phosphates in water,212 phosphamide in milk and meat,213-214 and phosphatidylcholines.216... 

Drugs that are not absorbed from the gastrointestinal tract of the baby, such as warfarin, are safe for the mother to take. The administration of antibiotics to a breast-feeding mother usually poses no concern for the newborn infant. However, metronidazole, which has mutagenic properties, reaches concentrations in milk that equal or exceed maternal plasma levels. Caution is also advised with sulphonamides, nitrofurantoin, or naladixic acid since these can cause haemolysis in infants with glucose-6-phosphate dehydrogenase deficiency. 

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 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 of... 

Because they occur as large aggregates, micelles, most (90-95%) of the casein in milk is sedimented by centrifugation at 100000 g for 1 h. Sedimentation is more complete at higher (30-37°C) than at low (2°C) temperature, at which some of the casein components dissociate from the micelles and are non-sedimentable. Casein prepared by centrifugation contains its original level of colloidal calcium phosphate and can be redispersed as micelles with properties essentially similar to the original micelles. 

Casein may be coagulated and recovered as rennet casein by treatment of milk with selected proteinases (rennets). However, one of the caseins, K-casein, is hydrolysed during renneting and therefore the properties of rennet casein differ fundamentally from those of acid casein. Rennet casein, which contains the colloidal calcium phosphate of milk, is insoluble in water at pH 7 but can be dissolved by adding calcium sequestering agents, usually citrates or polyphosphates. It has desirable functional properties for certain food applications, e.g. in the production of cheese analogues. 

Calcium and magnesium. Some calcium and magnesium in milk exist as complex undissociated ions with citrate, phosphate and bicarboante, e.g. Ca Citr-, CaP04, Ca HCOj. Calculations by Smeets (1955) suggest the following distribution for the various ionic forms in the soluble phase ... 

The murexide method measures Ca2+ only Mg2+, at the concentration in milk, does not affect the indicator appreciably. Calculation of Mg2 + concentration is possible when the total calcium and magnesium (obtained by EDTA titration) is known. This is based on the assumption that the same proportion of each cation is present in the ionic form, which is justifiable since the dissociation constants of their citrate and phosphate salts are virtually identical. 

Alkaline phosphomonoesterase (EC 3.1.3.1). The existence of a phosphatase in milk was first recognized in 1925. Subsequently characterized as an alkaline phosphatase, it became significant when it was shown that the time-temperature combinations required for the thermal inactivation of alkaline phosphatase were slightly more severe than those required to destroy Mycobacterium tuberculosis, then the target micro-organism for pasteurization. The enzyme is readily assayed, and a test procedure based on alkaline phosphatase inactivation was developed for routine quality control of milk pasteurization. Several major modifications of the test have been developed. The usual substrates are phenyl phosphate, p-nitrophenyl-phosphate or phenolphthalein phosphate which are hydrolysed to inorganic phosphate and phenol, p-nitrophenol or phenolphthalein, respectively ... 

Milk contains a range of groups which are effective in buffering over a wide pH range. The principal buffering compounds in milk are its salts (particularly soluble calcium phosphate, citrate and bicarbonate) and acidic and basic amino acid side-chains on proteins (particularly the caseins). The contribution of these components to the buffering of milk was discussed in detail by Singh, McCarthy and Lucey (1997). 

A. Baudrimont and J. Pelouze (1833) fused the sodium sulphate with galena or zinc blende and formed the alkali plumbate or zincate, and J. B. M. P. Closson boiled a soln. of sodium sulphate with milk of lime and lead oxide. The plumbate can be decomposed by sulphide, carbon dioxide, or by electrolysis. The St. Gobain Co. patented a process in which sand, coal, and sodium sulphate are heated together water-glass is formed and a soln. or suspension of that salt in water is decomposed by carbon dioxide or by milk of lime. J. Simpson (1890), J. C. Ody (1892), N. Basset and W. von Baranofi (1894) decomposed a soln. of sodium sulphate by calcium phosphate in dil. acid. The soluble sodium phosphate which is formed... 

In this assay, milk samples could be analyzed without dilution, but tissue and egg white samples should be homogenized in 5% trichloroacetic acid, centrifuged, and brought to pH 7 prior to analysis. Egg yolks required a separate treatment involving mixing with a pH 7.4 phosphate-EDTA buffer, incubation for 3 h at 25 C with cephalexin antiserum and enzyme-label cephalexin, and centrifugation. The assay could detect cephalexin down to 30 ppb in milk, 60 ppb in egg yolk, and 400 ppb in hen tissue. 

Capillary electrophoresis has been applied by Chen and Gu (281) for simultaneous determination of oxytetracycline, tetracycline, chlortetracycline, and doxycycline residues in bovine milk. Separation was performed on a noncoated capillary column, 57 cm total length with 50 cm effective length, 75 m internal diameter (i.d.) and 375 m outside diameter (o.d.), using a mobile phase containing 10 mM sodium dodecyl sulfate, 50 mM borate, and 50 mM phosphate, pH 8.5. Under these conditions, concentrations below 10 ppb could be determined in milk using an ultraviolet spectrophotometer set at 370 nm. 

---