Colloidal Calcium Phosphate Research Articles

Relationship between thermal treatment of milk and hysteresis of the milk protein system. II. Studies on casein micelles (author's transl)

The association of colloidal calcium phosphate with the casein micelles is the principal reason for hysteresis of the milk protein system. Because of this association the secondary calcium phosphate cannot be titrated at its pK = 7.2, but only at lower pH values depending on the previous thermal treatment of the milk. However after overcoming certain energy barriers it can be titrated (between pH 5.8 and 5.2), but then calcium phosphate is separated irreversibly from the micelles. Therefore in the subsequent base titration other molecular conditions are passed through than in acid titration. The consequence is a hysteresislike titration curve. By heating, the surface of the micelles are covered successively with serum proteins. Due to this coating additional diffusion barriers are constructed. The diffusion of the protons into the interior of the micelles is measurably retarded. The expansion of the hysteresis loop can be determined quantitatively.

Heat stability characteristics of ovine, caprine and equine milks

SummaryOvine and caprine milks showed a marked stability maximum at ~ pH7 in their heat-stability/pH curves, but became very unstable at higher pH values. A low level of κ-casein appears to be responsible for this low stability of ovine milk; β-lactoglobulin offsets the stabilizing influence of κ-casein at lower pH values. Removal of colloidal calcium phosphate from ovine or caprine milks had very little influence on their heat stabilities and it was necessary to reduce the concentration of soluble salts to a very low level before an effect was observed. At the pH of maximum stability ovine and caprine milks, although quite variable, had stabilities in the same range as bovine milks. Equine milk was very unstable to heat and the shape of the heat-stability/pH curve of most individual samples was similar to ovine and caprine milks.

Colloid and Crystal Formation in Parotid Saliva of Cystic Fibrosis Patients and Non-Cystic Fibrosis Subjects. I. Physicochemistry

Two types of turbidity were found in parotid saliva from both cystic fibrosis (CF) patients and non-CF subjects. On cooling saliva, a rapidly forming, reversible, cold-dependent turbidity appeared in increasing amounts with decreasing temperature and increasing protein concentration. At 37 degrees, a slowly forming, stable turbidity appeared in increased amounts in parotid saliva samples containing increased amounts of calcium. The 2 degree centrifuged pellet consisted predominantly of protein, whereas the 37 degree pellet contained calcium, inorganic phosphate, and protein. The cold-dependent turbidity at 2 degrees was not inhibited by EDTA, but 37 degrees turbidity was dramatically inhibited. Urea and guanidine hydrochloride reduced 2 degree turbidity, and, to a lesser extent, inhibited 37 degree turbidity. The tendency towards higher levels of protein, amylase, and calcium in CF compared with child control parotid saliva (4, 6) causes a greater incidence and degree of turbidity formation in saliva of CF patients. In this paper only the nature of the turbidity has been investigated, not its relative occurrence in each group of subjects.

Heat stability of milk: influence of colloidal calcium phosphate and β-lactoglobulin

SummaryReduction of the level of colloidal calcium phosphate (CCP) progressively increased the heat stability of milk at pH values <~7·0 and increased the pH of maximum stability. Removal of 40% CCP also stabilized the system at the pH of minimum stability, but removal of ≥60% CCP rendered milk very unstable at pH values >7·2, an effect not offset by a 4-fold increase in κ-casein concentration. Doubling CCP had a slight destabilizing effect in the pH range 6·5–7·5.Addition of β-lactoglobulin to serum protein-free casein micelles had a marked destabilizing effect at pH values > ~6·8, but increased stability in the pH range 6·4–6·8. β-Lactoglobulin had a similar and more apparent effect on the heat stability of Na caseinate dissolved in milk diffusate.It is suggested that rather than being a stabilizing factor responsible for the maximum in the heat stability-pH curve, the true effect of β-lactoglobulin is to shift the curve to more acid pH values (reason unknown) and to sensitize the caseinate system to heat-induced Ca phosphate precipitation at pH values > ~7·0. Low stability at ~pH 7·0 introduces an apparent maximum in the heat stability-pH curve at ~pH 6·8, but this has no independent existence. At pH values >7·2, increased protein charge more than off-sets the influence of heat-precipitated CCP and stability again increases in micellar but not in soluble casein systems.

Some physico-chemical properties of porcine milk

SummaryThe principal results of an investigation of the major physico-chemical properties of porcine milk were:Heat stability: all individual porcine milks examined had poor heat stability and some coagulated in 2 min at 95°C; the Q10°Cwas high and variable and was 40 for some samples. Dialysis of porcine milk against bulk bovine milk did not influence its heat stability; removal of colloidal calcium phosphate (CCP) from porcine milk increased its heat stability 4-fold, but it was still very unstable. Unlike bovine milk, porcine milk did not exhibit a maximum-minimum in the heat stability-pH curve, but rather resembled bovine casein micelles suspended in milk dialysate. Addition of isolated bovine β-lactoglobulin to porcine milk did not influence its heat stability-pH curve.Rennet coagulation: under similar conditions, porcine milk coagulated about 4 times faster than bovine milk and the resultant curd was about twice as soft. Dialysis of porcine milk against a large excess of bulk bovine milk did not influence its rennet coagulability nor did removal of 50% of its CCP, but complete removal of the CCP rendered porcine milk non-coagulable by rennin. Porcine milk was readily coagulated by rennin down to 5°C; its second-stage coagulation was independent of temperature – a characteristic due mainly to its high level of CCP.Alcohol stability: porcine milk was coagulated by 66–68% (v/v) ethanol, as compared with 74% (v/v) for bovine milk.Calcium stability: porcine milk was stable to at least 300 mM-CaCl2at both 2 and 37°C.Solvation: porcine milk contained significantly less water of hydration than bovine milk, 1·7g H2O/g casein compared with 2·0g H2O/g casein.

Reaction of melting salts upon casein micelles and their effects on calcium, phosphorus and bound water.

Casein micelles with and without colloidal calcium phosphate were isolated from skim milk. Sodium salts of ortho, pyro, tripoly and polyphosphoric acid and also sodium citrate were added and their effects upon calcium and phosphorus distribution were investigated along with the changes of bound water.Melting salts of phosphate origin increased both non-diffusible ester and colloidal phosphorus, while decreased both diffusible ester and inorganic phosphorus.Bound water of casein micelles was increased with the addition of polyphosphate or citrate, while decreased with the addition of pyro- or tripolyphosphate.Taking into consideration of the viscometric studies, melting salts might be divided into three categories, namely orthophosphate, pyro- or tripolyphosphate and polyphosphate or citrate group, respectively.

Influence of aggregation on the susceptibility of casein to proteolysis

SummaryThe susceptibility of the casein in milk to proteolysis was shown to be greatly influenced by its state of aggregation. In normal milk, where the casein is largely in micellar form, the αs1- and β-caseins are almost inaccessible to proteolysis. On removal of the colloidal phosphate, the casein micelles disintegrate, rendering the components, especially the αs1-casein, accessible to proteolysis. The role of colloidal calcium phosphate in the casein micelle is believed to be that of a non-specific aggregating agent which can be effectively replaced by calcium. Dissolved colloidal phosphate can be effectively reformed by elevation of the pH of colloidal phosphate-free (CPF) milk before equilibrium dialysis. Addition of κ-casein to CPF milk also causes aggregation of the component caseins but the micelles formed are smaller than those of normal milk.The behaviour of micellar β-casein differs considerably from that of micellar αs1-casein. The evidence suggests that part of the β-casein freely dissociates either outside or within the micelle when the temperature is reduced. The temperature dependence of the susceptibility of β-casein to proteolysis was similar in skim-milk and in solutions of sodium caseinate, and increased as the temperature was reduced. αs1-Casein was quite resistant to proteolysis in normal milk but became susceptible when the micelle structure was disrupted on removal of colloidal phosphate.It is concluded that limited proteolysis may prove a valuable technique in the study of casein micelle structure.

Association of lipases with micellar and soluble casein complexes

SummaryThe association of lipase with casein micelles and soluble casein complexes was investigated by gel-filtration on Sephadex G-200 and Sepharose 2B columns which were equilibrated with synthetic milk serum. Gel-filtration indicated that the molecular weight of casein micelles in milk is > 108whereas the casein in colloidal phosphate-free milk is present as soluble complexes of molecular weightca.2×106containing αs-, β- and κ-casein. The soluble complexes appear to be stabilized in the micelle by colloidal calcium phosphate linkages. On addition of pancreatic lipase to milk, activity was impaired due to binding of the enzyme both to micellar and to soluble casein complexes. The enzyme dissociated from the latter during gel-filtration on Sepharose 2B columns. The binding of lipase to casein was not dependent on the presence of colloidal phosphate and consequently complete micellar structure is not essential for association of lipase with casein. Binding of the lipase to phosvitin did not result in a loss of enzyme activity. Lipases in milk appear to be involved in the equilibrium between micellar and soluble casein. The activity of lipases in milk is apparently influenced by this equilibrium. Some problems encountered in the use of gel-filtration to study the interactions of lipases with caseins are described.

Zonal Ultracentrifugation of β-Lactoglobulin and κ-Casein Complexes Induced by Heat

A zonal ultracentrifuge was used to isolate the heat-induced complexes of β-lactoglobulin and κ-casein. The complexes formed at 80C in sodium cacodylate buffer, pH 6.65, ionic strength 0.08, varied in size and composition, but moved as a single band in starch-gel electrophoresis. The maximum ratio in the complex was estimated to be 3 β-lactoglobulin:1 κ-casein. When a 1:1 mixture was heated at 110C the major component banded in 6.9% sucrose, had a sedimentation coefficient of 2.35, and moved as a single band in starch gel. At 140C, κ-casein was degraded extensively, and no complex could be detected. The complex formed at 90C from a 1:1 mixture in a synthetic serum containing calcium had a sedimentation coefficient of 39. For other mixtures in the presence of calcium the unreacted proteins could not be quantitatively separated from the complex. Zonal ultracentrifugation of heated skimmilk and colloidal calcium phosphate free milk failed to show a discrete complex peak.

Relation Between Micellar and Serum Casein in Bovine Milk

Little or no additional casein dissolved when milk was diluted with large volumes of milk dialysate. Some dissolution of micellar to serum casein occurred when sedimented micelles were redispersed in ultra-filtrate mechanically, but the concentration of serum casein remained far below that of the original milk. It is concluded that micellar and serum casein do not form an equilibrium system controlled by the solubilities of the various caseins.αs-, β-, and κ-Casein dissolved in the serum when milk was cooled, with β-casein accounting for about 55% of the total increase in serum casein. Serum casein content decreased by addition of calcium, but was increased by addition of phosphate, by addition of acid in the range from pH 6.7 to 5.3, and by removal of colloidal calcium phosphate. The results suggest that, at a fixed level of calcium caseinate, the calcium phosphate content of the micelles and the degree of polymerization of temperature-sensitive casein components (mostly β-casein) are the major factors controlling the proportion of casein present in micellar form. Loss of either calcium phosphate or of temperature-sensitive casein from the micelle releases additional casein to the serum; loss of both has a more than additive effect.

Internal Structure of Casein Micelles from Bovine Milk

The internal structure of casein micelles from bovine milk has been reinvestigated. Observations from electron microscopy, X-ray diffraction, and enzymatic digestion of the micelles followed by centrifugation suggested that electron-dense particles in the micelles are probably granules of colloidal calcium phosphate and not protein aggregates. These granules are embedded in a surrounding matrix of caseins to form the micelle. The foci of formation of the calcium phosphate granules may be either the ester phosphate groups or the ɛ-amino groups of lysine.

Precipitation of Casein by the Addition of Sodium Carboxymethylcellulose to Skimmilk

The influence of sodium carboxymethylcellulose (CMC) on the casein micelle in milk at the neutral pH was studied. CMC precipitated part of casein in milk. From the relative areas of electrophoretic patterns and sialic acid contents of casein fractions, it was calculated that the CMC-precipitated casein contained 51% αs-, 17% κ-, and 32% β-casein, while the distribution of the whole casein was 44% αs-, 19% κ-, and 34% β-casein.Measurements by ultracentrifugal method indicated that added CMC dissolved a portion of calcium and inorganic phosphorous. But a slight decrease in the dialyzable and ionized calcium was determined by equilibrium dialysis of skimmilk containing CMC. Interaction between CMC and the precipitated casein was not observed. The precipitation of casein did not occur in the skimmilk of which all of the calcium was removed by the addition of potassium oxalate. It appears that CMC combines with the calcium ion and dissociates the colloidal calcium phosphate in skimmilk, and causes the considerable decrease in the stability of the casein micelle to calcium.

Micellar Changes in Skimmilk Treated with Alkali or Acid

Samples of skimmilk were adjusted to various pH levels with N-KOH or N-HCl, and after 3 hr at 20C were restored to the original pH. The rennet coagulation times of milks transiently above pH 10 increased logarithmically with increasing pH. Milks transiently at acid pH's coagulated more rapidly. Since soluble calcium is an important variable influencing coagulation time, it was measured by a dialysis procedure to determine its role. Colloidal calcium content was nearly constant in the milks of the higher pH range, but gradually decreased in milks of the lower pH range.Turbidity decreased when the samples were adjusted to above pH 8.7 and below pH 5.4. In the alkali range the micelles were swollen and altered by pH change, whereas colloidal calcium phosphate was not disturbed.Accordingly, the rapid decrease of rennet coagulation time in milks transiently at lower pH was attributed to the increase of soluble calcium. The logarithmic increase in coagulation time in milks treated above pH 10 was attributed to protein changes that altered micellar interaction and H2S liberation.

Factors Affecting the Heat Stability of Milk

Summary Removal of colloidal calcium phosphate from milk by acidification and dialysis increased heat stability of the milk and eliminated the minimum from the heat stability-pH relation. If dissolution of the colloidal phosphate had approached completion, the normal heat stability-pH relation was not recovered by reprecipitation of the phosphate by neutralization before dialysis. Increasing the degree of forewarming progressively decreased coagulation time at 140C at all equivalent points on the heat stability-pH curve, but the cumulative heating time decreased markedly in only one of five milks tested. The behavior of an individual milk after forewarming depended upon its original pH relative to the pH of maximum heat stability. With milks from 20 individual cows, both maximum and minimum heat stability (min) were significantly correlated with β -lactoglobulin content (mg n/ml) ( r = +0.50 and −0.52, respectively). Inorganic components, and ratios of these, were not correlated with maximum heat stability.

Seasonal Changes in Interrelationships among Milk Constituents

Summary Since total soluble phosphorus, soluble organic and inorganic phosphorus, citric acid, and magnesium show important seasonal changes, differences in the relationships of these constituents to other milk constituents have been studied. Important changes in correlation between constituents are shown to occur with changes in season. The correlation coefficients between total soluble phosphorus and many other milk constituents change from highly significant to not significant with changes in season. The data show marked changes in relationships with casein, calcium, and phosphorus. The relation between soluble inorganic phosphorus and the soluble calcium also shows major shifts with changes in season. The magnitude of these shifts is sufficiently great that changes in stability of the milk would be expected. The soluble organic phosphorus is not closely associated with any of the major milk constituents. There is little evidence to suggest that changes in this constituent could account for seasonal changes in the properties of milk. Citric acid is normally quite independent of the other milk constituents. However, in the spring it is highly significantly correlated with casein, colloidal phosphorus, and the insoluble calcium. Association of citrate with the casein micelles and the colloidal calcium phosphate appear to be seasonal phenomena. Magnesium also shows its most significant relationships in the spring. In other seasons, its changes are independent of the other major constituents. No relationship between magnesium and soluble calcium is indicated at any season.

Relation of Milk Serum Proteins and Milk Salts to the Effects of Heat Treatment on Rennet Clotting

A study was made of the effects of heat at 85 or 90°C. for 30min. on the rennet coagulability of skimmilk and of artificial systems composed of caseinate centrifuged from skimmilk and dispersed in milk dialysate with and without added milk serum proteins. Such heat treatments of skimmilk cause an immediate prolongation of the rennet clotting time at 35°C. and a further prolongation (hysteresis) when the heated sample is held following heating. These effects occurred with the artificial systems only if they contained β-lactoglobulin. Furthermore, 0.2% β-lactoglobulin added to skimmilk enhanced the magnitude of both the immediate prolongation and the hysteresis. Thus, heat treatment appears to alter β-lactoglobulin or to cause it to complex with casein in such a way that it inhibits rennet clotting. The rate of action of rennet at 16°C. is slower on heated than on raw milk, hence the heat-induced reactions involving β-lactoglobulin appear to interfere with the primary (enzymatic) action of rennet.Heat treatment of eight samples of skimmilk at 85°C. for 30min. caused an average transfer of 6mg. of calcium and 4mg. of phosphorus per 100ml. from the dissolved to the colloidal state. The dissolved calcium and phosphate tended to return to their original concentrations when heated milk was held cold. Dialysis of heated milk against a large volume of raw milk increased the colloidal phosphate and partially restored the rennet coagulability. These changes in state of calcium and phosphate occurred in the absence of β-lactoglobulin and were not enhanced in magnitude by added (0.2%) β-lactoglobulin. Thus, they seem to be independent of that protein. The hysteresis phenomenon may result from loss of colloidal calcium phosphate from the caseinate particles on holding after heating.

The colloidal phosphate of milk: II. Influence of citrate

SummaryA new method for determining the composition of the colloidal phosphate of milk has been developed, based on analysis of milk free from colloidal phosphate. Preparation of this material is described.The results suggest that the so-called colloidal calcium phosphate of milk should be more properly described as a colloidal phosphate—citrate.This colloidal phosphate—citrate shows considerable analytical resemblance to the precipitate formed on neutralizing acidified milk serum. Both can be represented approximately by the empirical formula of a hypothetical citrate apatite.

Studies on the Colloidal Proteins of Skimmilk. II. The Effect of Heat and Disodium Phosphate on the Composition of the Casein Complex

Summary The molar ratio of calcium to phosphorus in the so-called colloidal calcium phosphate of the casein complex was calculated to be approximately 1.5 in raw skimmilk. This ratio was reduced to 1.25 when 0.15% Na 2 HPO 4 was added before heating to 88° C. for 15 minutes, indicating that some diphosphate was formed. The composition of the sedimented complex with respect to calcium, phosphorus, and nitrogen was practically constant regardless of particle size. The calcium in the serum phase was reduced to about one-third its original value by adding 0.15% Na 2 HPO 4 before preheating skimmilk.

265. On the use of various sera for the determination of the soluble calcium and phosphorus of milk

A comparison of the value of various milk sera for the determination of the soluble phosphate and calcium of milk, using dialysis at 0–5° C. as a standard method, gave the following results:(i) Rennet whey was the most satisfactory; with fresh milk it yields fairly accurate values for calcium, slightly low (average 5 %) for phosphate. With increasing acidity of the milk both sets of values tend to rise somewhat relatively to those of the dialysate, phosphate approaching the true value and calcium rising slightly above it.(ii) Mercuric chloride borax serum gives high results for the soluble phosphate of fresh milk With increasing acidity of the milk, the results fall steadily.(iii) Papain whey gives somewhat low results for both the phosphate and calcium of raw and pasteurized milk, with the same tendency as rennet whey to yield higher and improved results as the acidity of the milk: increases. With boiled milk it gives abnormally high results for both constituents. The additional phosphate and calcium appear to arise from the solution of colloidal calcium phosphate.

The Relation of Calcium Proteinate and Colloidal Calcium Phosphate to the Partition of Calcium in the Blood Stream.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTThe Relation of Calcium Proteinate and Colloidal Calcium Phosphate to the Partition of Calcium in the Blood Stream.David M. Greenberg and Clarence E. LarsonCite this: J. Phys. Chem. 1939, 43, 9, 1139–1150Publication Date (Print):September 1, 1939Publication History Published online1 May 2002Published inissue 1 September 1939https://doi.org/10.1021/j150396a004RIGHTS & PERMISSIONSArticle Views37Altmetric-Citations6LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (550 KB) Get e-Alerts