Enrichment of n-Alkane Assimilation-Deficient Mutants of Candida Yeast by Synergistic Effect of Nystatin and Pyrrolnitrin

In order to clarify further the relationship between the heat stability of casein micelles and the formation of soluble casein upon heating concentrated milk, the effect of formaldehyde was examined. The addition of formaldehyde up to 20 mM markedly increased the heat stability of both concentrated skim milk and concentrated whey protein-free (WPF) milk. The stabilizing effect of formaldehyde was greater for concentrated skim milk than for concentrated WPF milk. The addition of formaldehyde depressed the formation of soluble casein upon heating concentrated milk. No soluble casein was formed on the addition of 20 mM formaldehyde. It was confirmed by Sephadex G-200 gel filtration in the presence of 6.6 M urea that cross-links among the casein components were formed in heated concentrated WPF milk containing formaldehyde. These facts suggest that formaldehyde may introduce cross-links among the casein components and prevent the formation of soluble casein accompanying the release of K-casein from micelles, thus stabilizing the casein micelles.     

Model process for removal of caseins from milk of transgenic animals

We describe a method for selective removal of caseins from milk. The method was developed as a model for transgenic milk processing. Raw cow milk spiked with nonmilk proteins was chosen as the model to resemble transgenic animal milk containing recombinant proteins. The most important elements of the process are (1) "deconstruction" of casein micelles in milk by destroying their Ca(2+) core using a chelating agent (EDTA), thus freeing any protein that might be entrapped in casein aggregates, and (2) "reconstruction" of micelles by providing them with a new Ca(2+) core, thus precipitating them away from the whey proteins, and the protein of interest. Calcium phosphate particles (CAP) were used to reform the disrupted casein micelles. The crystal clear supernatant fraction generated by this method provided >90% recovery and 6- to 13-fold concentration of the desired protein. Product-rich supernatant contained no detectable casein residues, as silver-stained SDS-PAGE and Western blot analyses demonstrated.     

Studies of camel casein micelles: Treatment with soluble and immobilized neuraminidase

Camel casein micelles were obtained from raw uncooled skim milk by ultracentrifugation, washing and then resuspending in UF-skim milk permeate. They were treated with neuraminidase, in both soluble and immobilized forms, to study the location and distribution of the glycosylated portion of camel casein micelle. Kinetic release of sialic acid, soluble in 12% TCA, was studied. Camel casein micelle contains 7·35 mg sialic acid per g casein; 99·3% of it was released with soluble neuraminidase, whereas only 90% was released with immobilized neuraminidase. This implies that about 90% of the glycosylated portion of camel casein micelle (glyco-k-casein-like component) is on the surface of the micelle.     

The disaggregation of calcium-depleted casein micelles

The effect of depletion of Ca2+ on the composition and size distribution of casein micelles in milk has been examined using chemical analysis, size exclusion chromatography, fast protein liquid chromatography, turbidimetry and photon correlation spectroscopy. Partial removal of Ca2+ by EDTA and subsequent dialysis resulted in disaggregation of some of the casein micelles; as the EDTA concentration increased, the proportions of Ca2+ and phosphate relative to protein in the micelles remaining intact decreased. However, the composition of the intact micelles, with respect to the different caseins, and the number-frequency size distribution were essentially unchanged.     

The influence of temperature on the flocculation rate of renneted casein micelles

The flocculation rate constant of completely renneted casein micelles in milk ultrafiltrate was measured by Rayleigh light scattering between 20 and 35 degrees C. In this temperature range an apparent energy of activation of 103 kJ mol (+/-11 kJ mol : n = 50) was measured. At 15 degrees C clotting was not longer perceptible. The activation of the flocculation between 20 and 35 degrees C is explained not so much by the height of the energy barrier separating the clotting micelles, as by the very negative temperature coefficient of that barrier. In line with this conclusion it is suggested that renneted micelles adhere through hydrophobic bonding. The flocculation rate constant of renneted casein micelles is independent of micelle size at the four temperature levels studied.     

Fractionation by size of casein micelles on controlled-pore glass

Casein micelles have been separated from skim milk by chromatography on CPG-10 3000 glass beads. Fractionation of the micelles according to size has been demonstrated. Polyacrylamide gel electrophoresis of urea treated micelles reveals that different relative amounts of the major casein components occur in the various micelle fractions. No discernible dissociation of the micelles into monomeric caseins has been observed.     

Isolation of Tremerogen a-13, a Peptidal Sex Hormone of Tremella mesenterica

Bovine casein micelles were fractionated on controlled pore granule (CPG-10/3000) chromatography by size and the chemical properties of the fractionated micelles were compared. The results indicated the presence of two types of micelles distinguishable as large and small micelles. In skim milk, 72.7% of casein was calculated to be in the form of small micelles, 13.6% in the form of large micelles and 13.8% in non-micellar casein form.

The α_s1-casein content decreased, but β- and κ-casein content increased as the micelle size became smaller. κ-Casein in large micelles had a much higher sialic acid content than in small micelles. It was found that this difference in sialic acid content was due to the presence of non-glycosylated κ-casein in small micelles. In large micelles, non-glycosylated κ-casein was almost undetectable.

The addition of wheat germ lectin to micelles resulted in the formation of aggregates through intermicellar bridges between the carbohydrate chains of κ-casein located on the surface of the micelles. Both large and small micelles formed aggregates after the addition of wheat germ lectin. Large micelles were more sensitive to wheat germ lectin than small ones.     

Biocompatible micro-gel particles from cross-linked casein micelles

The stability of internally cross-linked casein micelles against disruption by urea (which disrupts hydrogen bonds and hydrophobic interactions) and trisodium citrate (which sequesters micellar calcium phosphate) was investigated. Addition of urea (0-6 mol L-1) and/or citrate (0-50 mmol L-1) progressively reduced the turbidity of a suspension of casein micelles cross-linked by transglutaminase and increased particle size (determined by dynamic and static light scattering and small-angle neutron scattering), which was attributed to swelling of the micelles. Furthermore, model calculations, assuming a completely stable casein network, were performed to describe the decreases in turbidity on addition of urea and citrate. Measured and described turbidity values are in agreement, indicating that cross-linking of casein micelles with transglutaminase results in a covalently bound protein network, which is entirely stable to disruption by urea and/or citrate. This may offer potential applications for the use of cross-linked casein micelles as biocompatible protein micro-gel particles.     

Reconstitution of Human Casein Micelle and Its Properties

Human casein micelles were reconstituted from isolated κ- and β-caseins and calcium ions. Micelle formation was recognized in the presence of calcium chloride even at the low concentration of 5mM. At pH levels ranging from 5.5 to 8.0, the re-formed micelles were quite stable so that precipitation of β-casein was not observed. The large micelles were constituted by a higher ratio of β-casein to κ-casein (16:1 by weight) than the small micelles (3: 1). The κ-casein in the small micelles contained carbohydrates to about 43% (w/w) in the molecule, whereas that in the large micelles was only about 25%. When the casein micelles were re-formed from κ-easein and fractionated β-casein components, the extent of phosphorylation of the β-casein component was found to influence the micelle formation; i.e., the β-casein component with no phosphate (the 0-P form) was disadvantageous to form micelles, but the component with 5 phosphates (the 5-P form) formed micelles most easily.     

Selectivity of the insulin-like actions of vanadate on glucose and protein metabolism in skeletal muscle.

The 1H-n.m.r. spectrum of casein micelles consists of a small number of moderately sharp (linewidth approx. 60 Hz) resonances superimposed on the envelope of very broad lines expected for particles of this size. These sharp lines resemble, in chemical shift and relative intensity, the spectrum of the isolated 'macropeptide' released from the micelles by treatment with chymosin. The sharp lines in the casein micelle spectrum are further sharpened by addition of chymosin and broadened markedly by addition of ethanol. These observations are consistent with the proposal that the 'macropeptide' (the C-terminal 64 residues of K-casein) forms flexible 'hairs' on the surface of the micelles.     

Model calculations of casein micelle size distributions.

A previously proposed model for the formation and structure of casein micelles from subunits of variable composition is used to calculate theoretical micelle size distributions. Using the fractional content of k-casein as the only variable but with a value near that observed in a sample of milk serum, the model successfully reproduces experimentally determined distributions. Predicted size distributions are quite sensitive to the value of the variable and shift toward smaller average size as the assumed fractional content k-casein gets larger. Also, there is a discontinuity in the distributions which predicts that there will be essentially no micelles with radii smaller than 25-30 nm. These predictions are all in accord with experimental observations. The good agreement between theory and experimenet supports the micelle structure suggested by the model.     

A lactational study of the composition and integrity of casein micelles from the milk of the tammar wallaby (Macropus eugenii)

The amount of casein found in the milk of the tammar wallaby increases as lactation progresses. The increase is due to increasing amounts of β-casein; the α-casein remains largely constant. The α-casein is the more highly phosphorylated; the most abundant form is the 10-P, throughout lactation. The level of phosphorylation of β-casein shifts to lower average values in late lactation, possibly indicating the enzymatic reaction is overloaded by the increasing amounts of β-casein. Unlike bovine casein micelles, the wallaby micelles are not completely disrupted at pH 7.0 by sequestration of their calcium content with ethylene diamine tetraacetic acid (EDTA). Complete disruption only follows the addition of sodium dodecyl sulphate, indicating considerably greater importance for hydrophobic bonds in maintaining their integrity. This micellar behaviour indicates that, despite the evolutionary divergence of marsupials millennia ago, the caseins of wallaby milk assemble into micelles in much the same fashion as in bovine milk.     

Proteomic characterization of specific minor proteins in the human milk casein fraction

Human milk contains many bioactive proteins that are likely to support the early development of the newborn. The aim of this study was to identify whether there are specific minor proteins associated with the human milk casein micelle prepared by the acid precipitation method. Protein identification was performed by liquid chromatography tandem mass spectrometry analysis. Eighty-two proteins were identified in the casein micelle, 18 of which are not present in their whey compartment. Thirty-two of these proteins specifically associated with the casein micelle have not previously been identified in human milk or colostrum. Proteins involved in immune function comprised the major part (28%) of total proteins, and another significant part is involved in metabolism/energy production (22%). Most of the proteins were of extracellular or cytoplasmic origin (accounting for 50 and 29%, respectively). This study indicates that various soluble proteins should be considered as part of the casein compartment, prepared by the acid precipitation method. The data provide new insight not only into the proteomic profile of the human milk casein micelle and its physiological significance, but also into the proper proportion of casein and casein-associated proteins to use in infant formula.     

A mechanism for the chymosin-induced flocculation of casein micelles

The chymosin-induced flocculation of casein micelles of bovine milk can be explained and calculated in terms of three relationships, which are (i) the action of chymosin upon the κ-casein of the micelles; (ii) the probability that a micelle, with a given proportion of its κ-casein destroyed, will aggregate, and (iii) the aggregation of micelles by a Smoluchowski mechanism. Details of the calculations are given, and the theory is shown to be in good agreement with experimental observations of the dependence of the clotting time with variations in enzyme and substrate concentrations.     

Studies on the properties of cow's-milk tributyrinases and their interaction with milk proteins

1. The tributyrinases in milk are mainly associated with casein micelles. Dilution or addition of sodium chloride increases the enzyme activity, probably by dissociating the micelle–tributyrinase complexes. 2. Tributyrinase activities of milks activated by dilution and sodium chloride addition were in the range 0·2–1·7μequiv. of acid liberated/ml. of milk/min. from tributyrin emulsion at pH8·5 and 25°. The enzymes have a bivalent-cation requirement for full activity and are rather unstable when separated from casein. 3. Ultracentrifugation of skim milks containing sodium chloride (0·75m) gave preparations low in casein but containing about 70% of the milk tributyrinases. The tributyrinases in such preparations appear to be bound in complexes of molecular weight about 350000. Dilution may result in dissociation to give the free enzymes. 4. Pancreatic lipase also formed complexes with casein micelles, but wheat-germ esterase, xanthine oxidase, milk alkaline phosphatase and other enzymes did not.     

Composition and ultrastructure of calcium phosphate-citrate complexes in bovine milk systems

The present studies show that the colloidal calcium phosphate of cow's milk has a (Ca + Mg)/P_i ratio of 1.67 (± 0.10; n = 22) and contains citrate, Mg and Zn at molar ratios to Ca averaging 0.05, 0.03 and 0.003, respectively. The composition of the natural colloidal phosphate of milk is similar to the precipitates formed by neutralization of ultrafiltrates obtained from acidified milks, and to that of the calcium phosphate-enriched fraction produced by extensive enzymic hydrolysis of the casein micelles in milk. Examination by electron microscopy of these artificial preparations of milk calcium phosphate revealed in both a very fine and uniform substructure which consisted of granules having an average, true diameter of approx. 2.5 nm. The size and shape of these tiny granules closely resemble the morphologies reported for the colloidal phosphate particles in native casein micelles, as well as for the subunits of amorphous calcium phosphate observed during calcification in other biological systems such as mitochondria and bone.     

Water interactions with varying molecular states of bovine casein: 2H NMR relaxation studies

The caseins occur in milk as spherical colloidal complexes of protein and salts with an average diameter of 1200 A, the casein micelles. Removal of Ca2+ is thought to result in their dissociation into smaller protein complexes stabilized by hydrophobic interactions and called submicelles. Whether these submicelles actually occur within the micelles as discrete particles interconnected by calcium phosphate salt bridges has been the subject of much controversy. A variety of physical measurements have shown that casein micelles contain an inordinately high amount of trapped water (2 to 7 g H2O/g protein). With this in mind it was of interest to determine if NMR relaxation measurements could detect the presence of this trapped water within the micelles, and to evaluate whether it is a continuum with picosecond correlation times or is associated in part with discrete submicellar structures with nanosecond motions. For this purpose the variations in 2H NMR longitudinal and transverse relaxation rates of water with protein concentration were determined for bovine casein at various temperatures, under both submicellar and micellar conditions. D2O was used instead of H2O to eliminate cross-relaxation effects. From the protein concentration dependence of the relaxation rates, the second virial coefficient of the protein was obtained by nonlinear regression analysis. Using either an isotropic tumbling or an intermediate asymmetry model, degrees of hydration, v, and correlation times, tau c, were calculated for the caseins; from the latter parameter the Stokes radius, r, was obtained. Next, estimates of molecular weights were obtained from r and the partial specific volume. Values were in the range of those published from other methodologies for the submicelles. Temperature dependences of the hydration and Stokes radius of the casein submicelles were consistent with the hypothesis that hydrophobic interactions represent the predominant forces responsible for the aggregation leading to a submicellar structure. The same temperature dependence of r and v was found for casein under micellar conditions; here, the absolute values of both the Stokes radii and hydrations were significantly greater than those obtained under submicellar conditions, even though tau c values corresponding to the great size of the entire micelle would result in relaxation rates too fast to be observed by these NMR measurements. The existence of a substantial amount of trapped water within the casein micelle is, therefore, corroborated, and the concept that this water is in part associated with submicelles of nanosecond motion is supported by the results of this study.     

High pressure-induced changes in bovine milk proteins: a review

High pressure (HP)-induced changes in the proteins of bovine milk have become an area of considerable research interest in recent years; as a result, there is now a detailed understanding of the effects of HP on casein micelles and whey proteins. HP treatment at pressures >400 or >100 MPa denatures the two most abundant whey proteins, alpha-lactalbumin (alpha-la) and beta-lactoglobulin (beta-lg), respectively. The majority of denatured beta-lg in HP-treated milk associates with the casein micelles, although some denatured beta-lg remains in the serum phase or is attached to the milk fat globule membrane; HP-denatured alpha-la is also associated with the milk fat globules. Casein micelles are disrupted on treatment at pressures >200 MPa; the rate and extent of micellar disruption increases with pressure and is probably due to the increased solubility of calcium phosphate with increasing pressure. On prolonged treatment at 250-300 MPa, reassociation of micellar fragments occurs through hydrophobic bonding; this process does not occur at a pressure >300 MPa, leading to considerably smaller micelles in such milk. As a result of HP-induced changes, the size, number, hydration, composition and light-scattering properties of casein micelles in HP-treated milk differ considerably from those in untreated milk.     

Colocalization of α-lactalbumin and a major casein in secretory vesicles of rat mammary epithelial cells

Summary Whether both casein and noncasein (serum or whey) proteins of milk are contained within the same secretory vesicles of milk secreting mammary epithelial cells was explored. Antibodies to a major casein and to -lactalbumin of rat milk were localized in thin sections with colloidal gold-conjugated second antibodies. Antibodies to the casein component bound to an antigen present within lumina of Golgi apparatus cisternae and within secretory vesicles. This antigen was also recognized in structures within secretory vesicles and within alveolar lumina which were ultrastructurally identified as casein micelles. Antigens recognized by antibodies to -lactalbumin also were present in Golgi apparatus cisternae and within secretory vesicles. Both anti-casein and anti--lactalbumin antibodies recognized antigens within the same secretory vesicles. These observations show that one major noncasein protein of rat's milk is present in casein-containing secretory vesicles.