The least number of phosphate groups for crosslinking of casein by colloidal calcium phosphate.

Artificial casein micelles were formed with whole human casein at 20 mM calcium, 17 mM phosphate, and 10 mM citrate. The casein micelles disaggregated by 6 M urea were separated by high performance gel chromatography on a TSK-GEL G4000SW column into crosslinked and monomeric fractions. When the crosslinked casein fraction was analyzed by high performance ionexchange chromatography on a TSK-GEL DEAE-5PW column, a small peak, representing the 3-P component of human beta-casein, and distinct peaks of the 4-P and 5-P components were found. Artificial casein micelles were formed from mixtures of each purified component of human beta-casein and bovine kappa-casein, disaggregated by urea, and separated on a TSK-GEL G4000SW column. The casein aggregates crosslinked by colloidal calcium phosphate were formed in artificial casein micelles of the 3-P and 4-P components. In contrast, no crosslinkage was formed in artificial micelles of the 1-P and 2-P components. The results indicate that at least three phosphate groups are needed for crosslinking of casein by colloidal calcium phosphate.     

High pressure effects on the colloidal calcium phosphate and the structural integrity of micellar casein in milk. Part 1. High pressure dissolution of colloidal calcium phosphate in heated milk systems

Results of this study confirm that high temperature (118°C/15 min) and high pressure (400 MPa/5 min) processing of skim milk, skim milk ultrafiltration and ultracentrifugation fractions, and model milk salt solutions cause dramatic shifts in their colloidal and soluble Ca phospate equilibrium that affect their pH, dissolved Ca content, turbidity, and casein micelle microstructure. The relations between high temperature and high pressure processing-induced changes in the colloidal and soluble Ca phosphate equilibrium were evaluated in raw, pasteurized, and high temperature treated skim milk, ultrafiltration retentate and permeate of pasteurized skim milk, clear ultracentrifugation infranatant of pasteurized skim milk, and synthetic milk ultrafiltrates with and without lactose or Ca. The magnitude of the pH and dissolved Ca shifts caused by high temperature and high pressure processing was a function of casein micelle concentration. Ultrafiltration permeate exhibited the most drastic shifts in pH and dissolved Ca contents due to high temperature and high pressure processing. Although high temperature processing reduced the pH of ultrafiltration permeate from 6.59 to 6.03 and the dissolved Ca from 100% to 58%, high pressure processing reversed both of these changes. These changes in high temperature and high pressure processed milk, milk fractions, and model milk salt solutions were related to microstructural changes in the casein micelles as revealed by electron microscopy.     

In vitro studies of adsorption of milk proteins onto tooth enamel

The aim of this investigation was to study in vitro adsorption of milk proteins onto tooth enamel. In vitro “milk protein pellicles” were formed on enamel specimens incubated in fluid milk products: skimmed milk (pH 6.7), acidified skimmed milk (pH 4.2), yoghurt and neutralized yoghurt (pH 6.7). The enamel specimens were used as such or pre-incubated in saliva. The “milk pellicles” were desorbed from enamel surfaces, and proteins separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were identified by their pattern of migration in SDS-PAGE or by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) fingerprinting. Neutral “milk protein pellicles” (skimmed milk and neutralized yoghurt) mainly consisted of caseins. At pH values close to their isoelectric point, caseins remained unabsorbed, and lactoferrin and bovine serum albumin were the most abundant protein species in the acidified “skimmed milk pellicle”. Virtually no proteins in yoghurt were able to adsorb onto enamel. Pre-incubation of enamel specimens in saliva did not inhibit or affect “milk pellicle” formation indicating that in vivo adsorption of milk proteins may occur on tooth surfaces in the oral cavity and have implications in oral hygiene.     

含乳(钙奶)饮料中钙含量的测定

目前对含乳饮料中钙含量的测定一般采用GB12398-90<食物中钙的测定方法>,有原子吸收分光光度法和EDTA滴定法.     

Determination of molecular weight of a purified fraction of colloidal calcium phosphate derived from the casein micelles of bovine milk

Colloidal calcium phosphate (CCP) plays a key role in the formation and integrity of casein (CN) micelles. However, limited information is available on the molecular weight (M_w) of CCP. Recently, we theoretically derived the M_w of CCP and the objectives of this study were to experimentally determine the M_w of CCP. We used 2 methods to prepare CCP fractions: skim milk was enzymatically digested with either trypsin or a combination of papain and proteinase enzymes to remove most CN. The CN phosphopeptides are resistant to trypsin hydrolysis. Digestion was carried out in a membrane tube that was dialyzed against the same bulk milk used in sample preparation to remove small peptides and to minimize perturbation of CCP. After digestion, the protein contents of the enzyme-treated milks were 0.92 and 0.36% for the trypsin and papain-proteinase treatments, respectively. Size-exclusion chromatography, coupled with multi-angle laser light scattering, was used to separate the CCP-phosphopeptide fraction from the digested mixture. Simulated milk ultrafiltrate was used as a mobile phase during size-exclusion chromatography separation to try to preserve the integrity of CCP. Size-exclusion chromatography peaks, which had higher Ca and P contents than the baseline, were identified as the likely fractions containing the phosphopeptide-stabilized CCP; this peak eluted with retention times of 100 to approximately 110 min for trypsinated samples. The papain-proteinase treatment caused excessive loss of CN that were needed to stabilize CCP, which resulted in no obvious peak that had elevated Ca and P contents. Debye plots at these retention times indicated that the weight-average M_w for the fraction prepared by trypsin was 17,450 g/mol. Attempts to estimate the M_w of the phosphopeptides associated with CCP using sodium dodecyl sulfate-PAGE were not successful, as we did not observe any peptide bands in these gels, presumably because of their low concentration in the isolated, unconcentrated fraction. Assuming that 4 CN phosphopeptides stabilized each CCP and if the M_w of each of these phosphopeptides was about 2,500 g/mol, then the M_w of CCP would be around 7,450 g/mol. This experimental value was close to the theoretically-derived M_w of 4,897 and 9,757 g/mol for tetrahedron and bi-pyramid shaped objects, respectively, when using the brushite form of calcium phosphate.     

Mineral and casein equilibria in milk: effects of added salts and calcium-chelating agents

We have investigated the effects of adding a range of mineral salts and calcium-chelating agents on the distribution of casein and minerals between the non-pelleted and pelleted phases of milk obtained upon centrifugation at 78000 g for 90 min. Adding CaCl2 or mixtures of NaH2PO4 and Na2HPO4 to reconstituted skim milk (90 g milk solids/kg) at pH 6.65 increased both pelleted casein and pelleted calcium phosphate. Opposite effects were obtained by adding citrate or EDTA. The change in pelleted calcium phosphate was not simply related to casein release from the micelle. Upon adding 5 mmol EDTA/kg milk, 20% of the pelleted Ca, 22% of the pelleted phosphate and 5% of the micellar casein were removed. Increasing the concentration of EDTA to 10 mmol/kg milk decreased the pelleted Ca by 44% and the pelleted phosphate by 46%, and caused 30% of the micellar casein to be released. The effects of adding phosphate, citrate or EDTA at pH 6.65, followed by the addition of CaCl2, demonstrated the reversibility of the dissolution and formation of the micellar calcium phosphate. There were limits to this reversibility that were related to the amount of colloidal calcium phosphate removed from the casein micelles. Adding CaCl2 to milk containing > or = 20 mmol EDTA or > or = 30 mmol citrate/kg milk did not result in complete reformation of casein micelles. Light-scattering experiments confirmed that the dissolution of moderate amounts of colloidal calcium phosphate had little effect on micellar size and were reversible, while the dissolution of larger amounts of colloidal calcium phosphate resulted in large reductions in micellar size and was irreversible.     

The equilibria of calcium and phosphate ions with the micellar calcium phosphate in cow's milk.

Calorimetric measurements of the heat of solution of micellar Ca phosphate suggest that this material more closely resembles OH-apatite than amorphous Ca phosphate or any other crystalline form. Preliminary computer calculations of the ionic equilibria of milk salt solutions indicate that, over a wide range of pH and temperature, such solutions are supersaturated with respect to OH-apatite and to various other Ca salts under certain conditions.     

Acid-induced gelation of milk protein concentrates with added pectin: Effect of casein micelle dissociation

The dynamics of the formation of the acid gel network for mixtures of milk protein concentrate (MPC) and low methoxyl amidated (LMA) pectin were studied using rheological measurements. The results as a function of pectin content and casein micelle integrity, from neutral pH to approximately pH 4.2, together with the microstructural changes observed in some of these systems, are presented.The gelation profiles of a mixture of 4% w/v MPC and LMA pectin (0–0.075% w/v) after the addition of 1.2% w/v glucono-δ-lactone showed a gradual decrease in the shear modulus with the incorporation of pectin. The effects of casein micelle integrity on casein–pectin interactions were studied, by preparing MPC dispersions containing various levels of micellar casein. A gradual change in the shear modulus, from a disrupting effect of pectin added to MPC, in which the casein micelles are intact, to a clear synergistic effect of pectin added to dissociated casein systems, was found in the acid-induced milk gels.     

Experimental evidence for previously unclassified calcium phosphate structures in the casein micelle

¹H-³¹P Cross-polarization magic angle spinning (CP-MAS) measurements of 40-d-old Mozzarella cheese and 20 mM EDTA-treated casein micelles revealed that each sample had immobile phosphorus with the same spectral pattern, which did not match that of native casein micelles. To identify the immobile phosphorus bodies, ¹H-³¹P CP-MAS spectra and cross-polarization kinetics measurements were undertaken on native casein micelles, EDTA-chelated casein micelles, and reference samples of β-casein and hydroxyapatite. The results showed that the immobile phosphorus bodies in the mature Mozzarella cheese had the following characteristics: they are immobile phosphoserine residues (not colloidal calcium phosphate); they are not the product of phosphoserine to colloidal calcium phosphate bridging; the phosphate is complexed to calcium; their rigidity is localized to a phosphorus site; their rigidity and bond coupling is unaffected by protein hydration; and the immobile bodies share a narrow range of bond orientations. Combining these observations, the best explanation of the immobile phosphorus bodies is that bonding structures of phosphorus-containing groups and calcium exist within the casein micelle that are not yet clearly classified in the literature. The best candidate is a calcium-bridged phosphoserine-to-phosphoserine linkage, either intra- or inter-protein.     

Performance and digestion by calves from limestone added to milk replacers containing soy protein concentrate

Sixteen male Holstein calves were fed milk replacers containing milk protein or 50% of the protein from soy protein concentrate for study of limestone as a buffer in the small intestine. Experimental diets were: A) 19% of crude protein as milk protein; B) same as A but containing .8% limestone; C) 19% crude protein, 9.5% milk protein, and 9.5% soy protein; and D) same as C but containing .8% limestone. Milk replacers were reconstituted with water to 14% solids and fed at 8, 9, 10, 11, 12, and 12% body weight from 1 to 6 wk of age. Calves were fed twice daily, and clean fresh water was available at all times. Soy protein concentrate in the replacer resulted in 20% lower weight gains, lower dry matter and protein digestibilities. Apparent crude protein retention also was reduced, but intake of nutrients, feed efficiencies, fecal scores, and rectal temperatures were not different between protein sources. Limestone did not change any of these measurements. Xylose absorption tests at 3 and 6 wk of age showed differences from age but not treatment. Analysis of digesta from different sections of the gut of 6-wk-old calves sacrificed 6 h after feeding revealed that pH throughout the small intestine was above 6 for both proteins, which may explain the ineffectiveness of added limestone. Compared to milk protein, soy protein concentrate produced a lower pH in the large intestine and feces.     

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.     

Fat digestion in veal calves fed milk replacers low or high in calcium and containing either casein or soy protein isolate

The hypothesis tested was that the inhibitory effect of dietary soy protein versus casein on fat digestion in veal calves would be smaller when diets were fed with high instead of low calcium content. Male calves, 1 wk of age, were fed 1 of 4 experimental milk replacers in a 2 x 2 factorial design. There were 19 animals per dietary group. The milk replacers contained either casein or soy protein isolate as variable protein source and were either low or high in calcium. Body weight gain was not significantly affected by the experimental diets. Soy protein isolate versus casein significantly reduced apparent fat digestibility. High versus low calcium intake also depressed fat digestion. The protein effect was smaller (2.9% units) for the high than the low calcium diets (3.6% units), but the interaction did not reach statistical significance. Soy protein isolate versus casein raised fecal bile acid excretion and so did high versus low calcium intake. The difference in bile acid excretion between the soy and casein containing diets was significantly greater for the high than low calcium diets. The absorption of phosphorus, calcium, and magnesium was higher for the casein diets than for the soy-containing diets. This study shows for the first time that soy protein isolate versus casein depressed fat digestion and raised fecal bile acid excretion in veal calves.     

牛乳液酪蛋白的CO2沉淀分离

以CO2为沉淀剂,从牛乳液中分离酪蛋白,考察了牛乳液浓度、CO2压力和温度对酪蛋白产率的影响.结果表明,在相对低温的条件下,用低压CO2处理低浓度牛乳液,可能得到较高的酪蛋白产率.据此提出了可供参考的牛乳酪蛋白分离工艺条件:牛乳液浓度为1/40纯乳;CO2压力为0.5MPa;温度为10～25℃.     

双缩脲法测定乳中酪蛋白含量

双缩脲法测定乳中酪蛋白是作为一种判定牛乳质量的指标.酪蛋白含有肽键,肽键在碱性溶液中可与双缩脲试剂中的铜离子形成紫红色化合物,用光谱测定法,可求得酪蛋白含量.     

Dephosphorylation of bovine casein by milk alkaline phosphatase.

The pH of optimum activity of alkaline phosphatase from cow's milk depended on the substrate, being 10-1 for rho-nitrophenylphosphate, 8-6 for phosphoserine, 8-0 for phosvitin and 6-8 for casein. Individual casein components were dephosphorylated more rapidly than mixtures of alphas- and beta-caseins or of alphas-, beta-and kappa-caseins and micellar casein. Mixtures of 2 components involving kappa-casein were more readily dephosphorylated than alphas- and beta-casein mixtures. At pH 6-8, lactose, whey proteins and phosphate ions had an inhibitory effect. beta-Lactoglobulin had an inhibitory effect only when the pH of the reaction was lower than the optimum pH value of the enzyme. Mg2+ and Zn2+ were not inhibitory. The optimum conditions for dephosphorylation of casein are described.     

凯氏定氮法测定牛乳中酪蛋白含量

酪蛋白是牛乳中的特征性成分,其含量可作为牛乳掺假检验的定量指标。实验结果表明等电点法结合凯氏定氮法能够有效地检测牛乳中酪蛋白含量,测定结果准确,重复性好。实验验证了纯牛乳中酪蛋白含量为2.5%。     

Serum protein and casein concentration: effect on pH and freezing point of milk with added CO2

The objective of this study was to determine the effect of protein concentration and protein type [i.e., casein (CN) and serum protein (SP)] on pH (0 degree C) and freezing point (FP) of skim milk upon CO2 injection at 0 degree C. CN-free skim milks with increasing SP content (0, 3, and 6%) and skim milks with the same SP content (0.6%) but increasing CN content (2.4, 4.8, and 7.2%) were prepared using a combination of microfiltration and ultrafiltration processes. CO2 was injected into milks at 0 degree C using a continuous flow carbonation unit (230 ml/min). Increasing SP or CN increased milk buffering capacity and protein-bound mineral content. At the same CO2 concentration at 0 degree C, a milk with a higher SP or a higher CN concentration had more resistance to pH change and a greater extent of FP decrease. The buffering capacity provided by an increase of CN was contributed by both the CN itself and the colloidal salts solublized into the serum phase from CN upon carbonation. Skim milks with the same true protein content (3%), one with 2.4% CN plus 0.6% SP and one with 3% SP, were compared. At the same true protein content (3%), increasing the proportion of CN increased milk buffering capacity and protein-bound mineral content. Milk with a higher proportion of CN had more resistance to pH change and a greater extent of FP decrease at the same carbonation level at 0 degree C. Once CO2 was dissolved in the skim portion of a milk, the extent of pH reduction and FP depression depended on protein concentration and protein type (i.e., CN and SP).     

The role of calcium, phosphate and citrate ions in the stabilization of casein micelles.

To obtain greater insight into the interaction of Ca, citrate and phosphate ions with casein, 31PNMR measurements were performed on combinations of these ions with alphas- and kappa-caseins. It was found that addition of alphas-casein to a Ca phosphate solution in D2O at 27 degrees C and pD 6.4 resulted in a downfield shift of the 31P singlet. An almost identical shift was observed with kappa-casein, but no shift was found when only phosphate ions were present or when Ca2+ were added to phosphate ions in the absence of casein. Separate experiments with poly-L-lysine, mol. wt approx. 35,000, resulted in similar downfield 31P chemical shifts of Ca phosphate as with both caseins, whereas no shift was observed when poly-glycine was added. From these results it can be concluded that Ca and phosphate ions associate with casein in a co-operative manner, probably in the way described by ter Horst (1963) as a complex with the NH3+-groups of lysine or arginine in a structure such as: (casein--NH3+)--PO43---Ca2+. The formation of this complex may be enhanced by citrate ions, since preliminary results have shown that addition of Na citrate to a solution of alphas-casein with added Ca phosphate produces a broadening of the 31P signal as well as a chemical shift.     

Influence of added calcium chloride on the heat stability of unconcentrated and concentrated bovine milk

The influence of added calcium chloride (1–10 mmol/L) on the heat-induced coagulation of skim bovine milk was examined. Unconcentrated milk displayed a pH-heat coagulation time (HCT) profile with a maximum at pH 6.6 and minimum at pH 7.0. Adding calcium chloride to unconcentrated milk progressively reduced the HCT at the maximum, increased the pH at which the maximum occurred and reduced the HCT at pH > 7.0. For concentrated milk, the shape of the pH-HCT profile, that is, a maximum at pH 6.6, was not altered by added calcium chloride, but HCT was reduced progressively with increasing concentration of calcium chloride. Preheating (90°C for 10 min) shifted the maximum in the pH-HCT profile of unconcentrated milk to a more acidic pH, and addition of 5 mmol/L calcium chloride to preheated milk induced changes in heat stability similar to those noted for unheated milk. Addition of calcium chloride to milk prior to preheating strongly reduced the stability of milk against heat-induced coagulation. These data suggest that calcium has a strong destabilizing effect on the stability of milk systems against heat-induced coagulation.