Soluble Casein Research Articles

Structural and compositional characterization of Ca- and β-casein enriched casein micelles

There is an increased interest in understanding the potential of modifying casein micelles composition, or even creating them de novo. In this study, soluble casein fractions were created by dissociation from native casein micelles, and their properties were studied in isolation, and after their addition to native casein micelles. The structure of these enriched casein micelles was investigated by small-angle X-ray scattering (SAXS) to evaluate if the enrichment caused changes in their internal structure. Acidification of native casein micelles from pH 6.8 to 5.6 caused colloidal calcium phosphate dissociation, and SAXS data showed a decrease in intensity at q = 0.08 Å−1, where q is the modulus of the scattering vector. Additionally, the acidification of native casein micelles (to pH 6.0) and incubation at 4 °C caused a decrease in scattering intensity at the intermediate q region (0.01–0.02 Å−1), signifying a loss of heterogeneity among the intermediate structure elements in the casein micelle. This change was caused by micellar dissolution of calcium phosphate and the release of β-casein at 4 °C. After the resuspension of casein micelles with a β-casein enriched serum, the absence of soluble β-casein suggested that most β-casein was re-associated in the colloidal fraction. These samples showed no changes in the structural features probed by SAXS. The work brings new evidence that, while a small extent of dissociation may affect the structure of the casein micelles, the enrichment with β-casein results in similar structures as those of the native micelle.

Structural Properties of Casein Micelles with Adjusted Micellar Calcium Phosphate Content.

Micellar calcium phosphate (MCP) content of skim milk was modified by pH adjustment followed by dialysis. Turbidity, casein micelle size and partitioning of Ca and caseins between the colloidal and soluble phases of milk were determined. Protein structure was characterised by Fourier transform infrared (FTIR) spectroscopy and proton nuclear magnetic resonance (1H NMR), whereas organic and inorganic phosphorus were studied by phosphorus-31 nuclear magnetic resonance (31P NMR). The sample with the lowest MCP content (MCP7) exhibited the smallest particle size and turbidity, measuring 83 ± 8 nm and 0.08 ± 0.01 cm-1, respectively. Concentrations of soluble caseins increased with decreasing MCP levels. At ~60% MCP removal, FTIR analysis indicated a critical stage of structural rearrangement and 31P NMR analysis showed an increase in signal intensity for Ca-free Ser-P, which further increased as MCP concentration was further reduced. In conclusion, this study highlighted the importance of MCP in maintaining micellar structure and its impact on the integrity of casein micelle.

Novel details on the dissociation of casein micelle suspensions as a function of pH and temperature

Membrane filtration is a widespread process for fractionation and recombination of milk components. Although the dissociation of micellar caseins has been studied in detail in skim milk, it is important to better understand the dissociation dynamics occurring between the colloidal and noncolloidal fractions in systems of modified composition. This research aimed at understanding the dissociation of casein proteins in micellar fractions depleted of whey proteins. Casein micelle dispersions were tested at neutral pH and pH 6 (using glucono-δ-lactone as acidulant), after incubation at 4°C or 22°C, and compared with skim milk. The ionic composition of the serum phase was measured using inductively coupled plasma-mass spectrometry, and the protein distribution analyzed using reversed phase-HPLC coupled with mass spectrometry. When incubated at 22°C, there were no differences in casein micelle dissociation between skim milk and whey protein-depleted micelles (∼2.6% dissociated casein). No additional dissociation occurred by lowering the pH from 6.8 to 6 at 22°C, albeit there were more soluble ions at low pH (71% Ca and 65% P). At 4°C, there was an increased amount of β-casein found in the serum phase (23-33% of total β-casein). In addition, there was an uneven dissociation behavior of the various genetic β-casein variants, whereof A2 was more readily released with cooling. In skim milk, approximately 22%, 18%, and 14% of κ-, αS2, and αS1-caseins, respectively, were dissociated from the micellar phase upon cooling and acidification to pH 6.0. This was in contrast to whey protein-depleted casein suspensions, in which only 6%, 5%, and 3% of κ-, αS2, and αS1-caseins, respectively, had dissociated. The results suggested that the whey proteins in the serum phase play a role in the equilibrium between colloidal and soluble caseins in milk. This is of great relevance in processes such as cold membrane fractionation, where more attention should be given to the protein composition in the serum phase, especially when concentration is combined with fractionation of the serum proteins.

Influence of Transglutaminase Crosslinking on Casein Protein Fractionation during Low Temperature Microfiltration.

Low temperature microfiltration (MF) is applied in dairy processing to achieve higher protein and microbiological quality ingredients and to support ingredient innovation; however, low temperature reduces hydrophobic interactions between casein proteins and increases the solubility of colloidal calcium phosphate, promoting reversible dissociation of micellar β-casein into the serum phase, and thus into permeate, during MF. Crosslinking of casein proteins using transglutaminase was studied as an approach to reduce the permeation of casein monomers, which typically results in reduced yield of protein in the retentate fraction. Two treatments (a) 5 °C/24 h (TA) and (b) 40 °C/90 min (TB), were applied to the feed before filtration at 5 °C, with a 0.1 µm membrane. Flux was high for TA treatment possibly due to the stabilising effect of transglutaminase on casein micelles. It is likely that formation of isopeptide bonds within and on the surface of micelles results in the micelles being less readily available for protein-protein and protein–membrane interactions, resulting in less resistance to membrane pores and flow passage, thereby conferring higher permeate flux. The results also showed that permeation of casein monomers into the permeate was significantly reduced after both enzymatic treatments as compared to control feed due to the reduced molecular mobility of soluble casein, mainly β-casein, caused by transglutaminase crosslinking.

Improving rehydration properties of spray-dried milk protein isolates by supplementing soluble caseins

Spray-dried milk protein isolates (MPIs) are important dairy ingredients but may not have desirable rehydration properties for industrial applications. In the present study, rehydration properties of MPIs were improved by spray-drying MPI dispersions containing different amounts of soluble caseins in the form of derivatized MPI (dMPI). dMPI was prepared by alkalizing MPI dispersions to pH 11.0 and subsequently acidifying to pH 6.8 (the pH-cycle). All the spray-dried MPIs had the similar bulk density (around 0.33 g/cm−3), composition, size distribution (1–100 µm), and SEM morphology. However, the decrease of hydrodynamic diameter, dissolution of total solids and proteins, and disruption of particles during the dynamic rehydration were accelerated as the dMPI content increased, indicating the improved rehydration properties. The improvement in rehydration properties was not due to the wettability that decreased as the dMPI:MPI mass ratio changed from 0:8 to 8:0, but resulted from the reduced cross-linking of casein micelles on powder surface and the increased surface porosity during the hydration as observed for partially hydrated samples. The present work may assist industrial applications of spray-dried MPIs.

Sodium caseinate hinders chymosin-induced aggregation of caseins in concentrated milk: The role of soluble caseins and calcium ions

Concentrated casein micelle suspensions show an altered balance between the colloidal and soluble phases compared with native skim milk. The objective of this research was to probe the role of such a change on the chymosin-driven destabilization of casein micelles. Skim milk was gently concentrated by osmotic stressing to increase the volume fraction of the micelles while maintaining a constant ionic composition. In situ turbidity measurements demonstrated that the secondary stage of gelation was hindered in the concentrated suspensions. Addition of ionic calcium overcame this inhibition, whereas restoring the original concentration by redilution did not. This work clearly demonstrated that calcium plays a major role in decreasing electrostatic repulsion in casein micelles, but also showed the importance of noncolloidal proteins in altering the gelling functionality of casein micelles in concentrated milk. Additional calcium induces aggregation of these soluble caseins, restoring gelation in concentrates.

АЛЛЕРГЕННАЯ АКТИВНОСТЬ И ОПАСНОСТЬ ПРОМЫШЛЕННОЙ ПЫЛИ СУХИХ ПРОДУКТОВ ПЕРЕРАБОТКИ КОРОВЬЕГО МОЛОКА

The purpose of the work was to establish in a model experiment the allergenic activity and danger of the extracts obtained from the dust of dry products of cow's milk processing (DPMP), containing complexes of soluble whey (WMP) or casein milk proteins (CMP), as a stage of hygienic regulation of the content of dust DPMP in the air of the working area. Experiments on albino guinea pigs sensitized by the intradermal injection of standard doses of WMP and СМР solutions into the ear revealed the development of severe allergic reactions in the animals of the experimental groups with the prevalence of mixed mechanisms of immediate anaphylactic and delayed cell-mediated types. According to the criteria for the classification of industrial allergens, the WMP and СМР complexes have a strong allergenic activity and are differentiated to the 1-st class of allergenic hazard, which determines the classification of the DPMP dust containing them as extremely dangerous industrial allergens. This is confirmed by the established high levels of indicators of allergic-diagnostic reactions in vivo and in vitro when testing sensitized WMP and СМР animals with a solution of skim milk powder dust, indicating the presence of antigenic determinants of whey and casein milk proteins in it and a real ability to form cross-allergic reactions in the body of workers to dust from all dry milk processing products containing these proteins.

Short communication: Determination of the whey protein index in milk protein concentrates

Milk protein concentrates are common ingredients in the dairy industry, with varying processing histories and composition. The objective of this research was to determine the feasibility of using the whey protein nitrogen (WPN) index, a well-established index for skim milk powder and nonfat dry milk, as a quality parameter for milk protein concentrates. The WPN index is a value based on the moisture-adjusted weight of skim milk powder. We hypothesized that WPN, even when standardized based on protein, may change depending on solubilization conditions of milk protein concentrates because of differences in solubilization conditions or processing history. The WPN was measured for model concentrates with different thermal history or reconstitution conditions. The WPN was not affected by an increased concentration of soluble casein in the dispersions nor after solubilization of the powder at 22 or 60°C. All reconstituted samples were standardized for protein. The WPN was also in full accordance with residual native protein measured by chromatography.

Freshwater Purification by Vacuum Airlift Column Using Methyl Isobutyl Carbinol and Casein

Water purification by foaming in a bubble column is a major challenge for research. It leads to the development of innovative airlift column operated under depression. Sea water clarification by bubble column is well documented, but for freshwater remains insufficiently studied. Extraction of suspended clay particles in freshwater is carried out in an airlift column. The system used is under depression and operates in a closed circuit, thanks to a recirculation tank. Methyl isobutyl carbinol (MIBC) and soluble casein are used as surfactants to allow extraction. Clay used is fine and ultra-ventilated and a turbidimeter is used to measure turbidity. This study showed that the combined use of methyl isobutyl carbinol and casein in the airlift column makes it possible to completely clarify water loaded with clay particles. A turbidity of less than 1 NTU is obtained after 30 minutes of extraction.

Doxorubicin-loaded casein nanoparticles for drug delivery: Preparation, characterization and in vitro evaluation

Casein, a milk protein that self-assembles to form micelles in aqueous solution, can bind to a wide range of drugs (hydrophilic and hydrophobic). Herein, a low cost and facile method was reported to prepare casein nanoparticles loaded with an anticancer drug, doxorubicin (DOX). The particles were fabricated by adding an excess of Ca2+ ions which brings the soluble casein present in the solution into the micellar framework to form dense nanoparticles. The binding between the drug and the macromolecule was confirmed using fluorescence studies. Circular Dichroism (CD) shows that upon addition of excess Ca2+ the protein chains rearrange. The nanoparticles were characterized by transmission electron microscopy (TEM), field emission scanning electron microscopy (FESEM), and dynamic light scattering (DLS). The release at pH 1 was higher than the physiological pH making this formulation potent for delivering the drug to the stomach via the oral route. The DOX attached with casein showed improved efficacy, i.e., better cytotoxicity against human pancreatic carcinoma cell line, PANC 1 cells as compared to the free drug of the same concentration, owing to higher cell uptake of the macromolecule.

The effect of transglutaminase on reconstituted skim milks at alkaline pH

The effect of transglutaminase (TG) treatment on the changes in the physico-chemical properties of reconstituted skim milk (10% w/w) under alkaline conditions (pH ∼6.7 to ∼10) was investigated. The casein micelle hydrodynamic radius, the distribution of proteins between the serum and micelles, and the milk viscosity differed between milk treated with and without TG under alkalinisation. When milks were alkalinised from pH ∼6.7 to ∼8, both TG-untreated and TG-treated casein micelles swelled due to the increase in electrostatic repulsions between the negatively charged casein molecules. In milk without TG, the swelling of the micelles resulted in their progressive dissociation, while in the presence of TG-induced crosslinks, most caseins were maintained in their micellar form. When the pH increased from ∼8 to ∼10, TG-treated casein micelles resisted complete micellar disruption and continued to swell, whereas TG-untreated casein micelles disintegrated into smaller particles, with a marked increase in the soluble casein concentration. Under alkalinisation, only the α-lactalbumin present in the serum of TG treated milk was resistant to denaturation and aggregation. In response to all these micellar and serum changes, the viscosity increased for both TG untreated and treated milks. A simple qualitative model taking into account the extent of casein micelle dissociation and the size of the casein micelles was proposed to describe the behaviour of the viscosity as a function of pH. TG crosslinking of skim milk did not affect the partition of calcium, inorganic phosphorus, magnesium and potassium; and milk alkalinisation did not result in new calcium-phosphate phases.

Effect of pH on dissociation of casein micelles in yak skim milk

The dissociation of yak casein (CN) micelles was evaluated by scanning electron microscopy, particle size, fluorescence properties, and soluble mineral and CN molecule content at pH 4.6 to 8.2. The results showed that the size of CN micelles remained constant with decreasing pH from 8.2 to 5.8 but sharply increased at pH ≤5.4. Casein micelles began to aggregate at pH 5.4, and the serum magnesium, potassium, iron, zinc, copper, and manganese levels had their minimum values at this pH level. During acidification, colloidal calcium phosphate dramatically disassociated from yak CN micelles, but the soluble CN monomer content decreased slightly. During alkalization, the soluble calcium and phosphorus content decreased below pH 6.8 but increased with pH increases from 6.8 to 8.2. However, the soluble CN content increased markedly during alkalization. The emission wavelength of 8-anilino-1-naphthalenesulfonic acid sodium salt fluorescence decreased during both acidification and alkalization from pH 6.6, whereas the opposite was found for intrinsic fluorescence.

Thermal stability of reconstituted milk protein concentrates: Effect of partial calcium depletion during membrane filtration

Milk protein concentrate (MPC) powders are increasingly utilized in manufacturing of protein fortified beverages. Thermal stability of the protein dispersions is of significant importance in such applications. It is known that a decrease in pH can induce partial dissociation of casein micelles and modify the natural equilibrium of calcium and phosphate between the micelles and the serum phase. The presence of soluble casein may improve the rehydration properties of MPC powders, and may impact their thermal stability. The objective of this work was to investigate the effects of partial acidification of milk prior to ultrafiltration on the heat stability of reconstituted MPC dispersions. Milk protein concentrate powders were prepared from skim milk acidified to pH6.0 by addition of glucono-δ-lactone, and then concentrated using ultrafiltration (UF) and diafiltration (DF). The heat stability of the reconstituted MPC dispersions was studied, by determining heat coagulation time, particle size, turbidity, viscosity, soluble and colloidal calcium and phosphate, and non-sedimentable casein both before and after heating at 120°C. Reconstituted MPC powders made with partially acidified skim milk contained lower soluble calcium and phosphate and exhibited very poor thermal stability compared to MPC powders made with skim milk at its natural pH. The thermal stability of the acidified MPC dispersions was not only recovered by restoration of pH and the serum composition through dialysis against skim milk, but it was improved compared to control MPC dispersions. All dialyzed samples had comparable pH, protein content and calcium and phosphate concentration, but the structure of the casein micelles was altered, causing differences in the type of soluble aggregates. It was concluded that the integrity of the casein micelles and the amount of dissociated, non-sedimentable caseins play a major role in determining the thermal stability of the MPC dispersions.

Purification, catalytic, kinetic and thermodynamic characteristics of a novel ficin from Ficus johannis

Abstract Ficin from Ficus johannis was purified to homogeneity by (NH4)2SO4 precipitation, CM-Sepharose and SP-Sepharose chromatography, and gel filtration Sephadex G-25 chromatography. The enzyme, that is characterized by a molecular weight of 25 kDa measured by SDS-PAGE, showed higher affinity towards casein and the highest catalytic efficiency at pH 7.0 and 60 °C. Catalytic hydrolysis rate follows the Michaelis–Menten equation and values of 32.9 s−1 and 13.7 µM for kcat and Km respectively, by Michaelis–Menten, were found. The pKa1 and pKa2 values of the active site ionizable groups controlling Vmax were 4.2 and 8.7, respectively. Thermodynamic parameters for soluble casein hydrolysis and for irreversible inactivation of ficin at different temperatures in the range from 45 to 60 °C were also determined. This is the first report on the thermodynamic parameters of ficin produced by Ficus johannis. These results indicate that Ficin from Ficus johannis may have potential applications in the industrial biotechnology.

Colloidal properties of casein micelles suspensions as a function of pH during concentration by osmotic stressing

The physico-chemical properties of casein micelles in milk adjusted to pH 6 and 5.6 were compared with native pH milk, as a function of concentration. Skim milk was acidified and concentrated using osmotic stressing. The apparent radius of the casein micelles, measured after redilution in their corresponding permeate, decreased with decreasing pH, but showed no differences with concentration. Furthermore, the casein micelles showed free diffusive behaviour at all three pH values; only suspensions at pH 5.6 showed hindered diffusion at 4× concentration. At protein concentrations >6% the extent of caseins dissociation increased in the acidified milk samples. The relative viscosity of the concentrated suspensions (up to 4×) was modeled according to the behaviour of colloidal hard spheres, taking into account hydrodynamic interactions. The voluminosity of the casein micelles was estimated to be 3.5 and 3.1 mg L−1 for pH 6 and 5.6, respectively. The rheological behaviour of casein suspensions at pH 6.0 could still be described as that of colloidal hard spheres. On the other hand, for casein suspensions adjusted at pH 5.6, very low maximum packing volumes were obtained using a hard sphere model. It was suggested that at this pH further rearrangements and aggregation need to occur during concentration >3×, because of dramatic changes in charge, dissociation of calcium and phosphate, a significant increase in soluble casein and the decrease in steric repulsion. Although much is known about the effect of pH on casein micelles in skim milk, very little data is available on the effect of concentration. This research contributed to a better understanding of the colloidal properties of casein suspensions as a function of pH and concentration, allowing better use of modified casein concentrates as functional ingredients.

Changes in particle size, calcium and phosphate solubilization, and microstructure of rehydrated milk protein concentrates, prepared from partially acidified milk

The present work studied the rehydration properties of milk protein concentrates (MPCs), prepared using ultrafiltration (UF) and diafiltration (DF). Milk was acidified to pH 6 with glucono-δ-lactone (GDL) prior to UF to alter the mineral composition of the final concentrates. The particle size distribution and the microstructure of the casein micelles in reconstituted MPCs as well as the partitioning of calcium, phosphate, and proteins between the colloidal and soluble phases were investigated. Reconstituted samples analyzed by electron microscopy showed that, even in partially dissolved particles, the particle surface was porous and similar to its inner portion and had no distinct skin layer. Partial acidification of milk did not have any significant effects on the microstructure; however, it significantly increased the average diameter of the casein micelles for both UF and DF samples and decreased the concentration of total calcium and phosphate. Sodium dodecyl sulfate (SDS)-PAGE analysis of the centrifugal supernatants of reconstituted MPC demonstrated that the amount of soluble caseins present in milk concentrates dramatically increased with acidification, and it further increased after restoring the mineral composition of the serum phase through dialysis against milk. This work contributes to a better understanding of how processing conditions, particularly partial acidification of milk prior to concentration, can alter the composition and physical properties of the caseins and the soluble phase of MPC after rehydration. Such alterations can significantly impact the technological properties of the reconstituted MPC.

Partial calcium depletion during membrane filtration affects gelation of reconstituted milk protein concentrates

Milk protein concentrate powders (MPC) with improved rehydration properties are often manufactured using processing steps, such as acidification and high-pressure processing, and with addition of other ingredients, such as sodium chloride, during their production. These steps are known to increase the amount of serum caseins or modify the mineral equilibrium, hence improving solubility of the retentates. The processing functionality of the micelles may be affected. The aim of this study was to investigate the effects of partial acidification by adding glucono-δ-lactone (GDL) to skim milk during membrane filtration on the structural changes of the casein micelles by observing their chymosin-induced coagulation behavior, as such coagulation is affected by both the supramolecular structure of the caseins and calcium equilibrium. Milk protein concentrates were prepared by preacidification with GDL to pH 6 using ultrafiltration (UF) and diafiltration (DF) followed by spray-drying. Reconstituted UF and DF samples (3.2% protein) treated with GDL showed significantly increased amounts of soluble calcium and nonsedimentable caseins compared with their respective controls, as measured by ion chromatography and sodium dodecyl sulfate-PAGE electrophoresis, respectively. The primary phase of chymosin-induced gelation was not significantly different between treatments as measured by the amount of caseino-macropeptide released. The rheological properties of the reconstituted MPC powders were determined immediately after addition of chymosin, both before and after dialysis against skim milk, to ensure similar serum composition for all samples. Reconstituted samples before dialysis showed no gelation (defined as tan δ=1), and after re-equilibration only control UF and DF samples showed gelation. The gelation properties of reconstituted MPC powders were negatively affected by the presence of soluble casein, and positively affected by the amount of both soluble and insoluble calcium present after reconstitution. This work, testing the chymosin-induced gelation behavior of various reconstituted MPC samples, clearly demonstrated that a decrease in pH to 6.0 during membrane filtration affects the integrity of the casein micelles supramolecular structure with important consequences to their processing functionality.

Disorder in milk proteins: structure, functional disorder, and biocidal potentials of lactoperoxidase.

This article continues a series of reviews on the abundance and roles of intrinsic disorder in milk proteins. Besides caseins, which are the major proteinaceous constituents of any milk that can be isolated by isoelectric precipitation, milk contains a set of soluble whey proteins, such as β-lactoglobulin, α-lactalbumin, serum albumin, immunoglobulins, lactoferrin, lactoperoxidase, glycomacropeptide, and proteose peptone (the last two are soluble casein derivatives). Lactoferrin and lactoperoxidase (LPO) are known to possess prominent biocidal activity, serving as efficient antibiotics and antiviral agents against a wide spectrum of bacteria, fungi, and viruses. LPO is a heme-containing peroxidase expressed as preproprotein. The mature protein has a single catalytic domain, structure of which is known for a protein isolated from several species. Functionally, LPO is a crucial component of the LPO system that includes LPO, hydrogen peroxide (H2O2), and thiocyanate (SCN(-)), being a well-studied, naturally occurring antimicrobial system in milk that is effective against many microorganisms and some viruses. Although various aspects of LPO structure and function are rather well studied and were subjects of several recent reviews, the abundance and potential functional roles of intrinsically disordered regions in this protein have never being addressed as of yet. The major goal of this article is to fill this gap and to show how intrinsic disorder is encoded in the amino acid sequence of LPO, and how intrinsic disorder is related to functions of this important milk protein.

Skim milk protein distribution as a result of very high hydrostatic pressure

This work studies the micellar size and the distribution of caseins, major and minor whey proteins in different fractions of skim milk treated up to 900MPa for 5min. Transmission electron microscopy showed that the smallest casein micelles were formed around 450MPa with no variations at higher pressures. The changes found in micellar size correlated with the concentration of soluble casein, because treatments at 250MPa significantly enhanced the level of non-sedimentable casein while, between 700 and 900MPa, there were no further increases with respect to lower pressures. There was a severe β-lactoglobulin (β-Lg) denaturation at pressures ≥700MPa, which reached 77–87%. α-Lactalbumin (α-La) was stable up to 550MPa, but it denatured at higher pressures. The content of soluble lactoferrin (Lf) decreased with pressure, particularly from 550 to 800MPa, while that of secretory IgA (sIgA) progressively decreased from 250 up to 700MPa. Our results indicated that treatment of milk at very high pressures, from 700 to 900MPa, did not reduce micellar size nor released more soluble casein with respect to treatments at lower pressures (250–550MPa). However, these treatments led to a severe denaturation of the whey proteins, in particular of β-Lg and the minor proteins Lf and sIgA. The possibility of using high hydrostatic pressure to obtain a soluble milk fraction with a casein and whey protein composition similar to that of human milk is discussed.

Influence of calcium depletion on iron-binding properties of milk

We investigated the effects of calcium depletion on the binding of iron in milk. A weakly acidic cation-exchange resin was used to remove 3 different levels (18–22, 50–55, and 68–72%) of calcium from milk. Five levels of iron (5, 10, 15, 20, and 25 mM) were added to each of these calcium-depleted milks (CDM) and the resultant milks were analyzed for particle size, microstructure, and the distribution of protein and minerals between the colloidal and soluble phases. The depletion of calcium affected the distribution of protein and minerals in normal milk. Iron added to normal milk and low-CDM (~20% calcium depletion) bound mainly to the colloidal phase (material sedimented at 100,000×g for 1 h at 20°C), with little effect on the integrity of the casein micelles. Depletion of ~70% of the calcium from milk resulted in almost complete disintegration of the casein micelles, as indicated by all the protein remaining in the soluble phase upon ultracentrifugation. Addition of up to ~20 mM iron to high CDM resulted in the formation of small fibrous structures that remained in the soluble phase of milk. It appeared that the iron bound to soluble (nonsedimentable) caseins in high-CDM. We observed a decrease in the aqueous phosphorus content of all milks upon iron addition, irrespective of their calcium content. We considered the interaction between aqueous phosphorus and added iron to be responsible for the high iron-binding capacity of the proteins in milk. The soluble protein-iron complexes formed in high-CDM (~70% calcium depletion) could be used as an effective iron fortificant for a range of food products because of their good solubility characteristics.