Structure: The Casein Micelle

Most, but not all, of the casein proteins exist in a colloidal particle known as the casein micelle. Its biological function is to carry large amounts of highly insoluble CaP to mammalian young in liquid form and to form a clot in the stomach for more efficient nutrition. Besides casein protein, calcium and phosphate, the micelle also contains citrate, minor ions, lipase and plasmin enzymes, and entrapped milk serum. These micelles are rather porous structures, occupying about 4 mL/g and 6-12% of the total volume fraction of milk. Size ranges from 50-250 nm in diameter.

Micelle image

Casein micelle image from Dalgleish, D. G., P. Spagnuolo and H. D. Goff. 2004. A possible structure of the casein micelle based on high-resolution field-emission scanning electron microscopy. International Dairy Journal. 14: 1025-1031. This micelle is 120 nm in diameter.

There have been many models developed over the years to explain the structure of the casein micelle, based on all of the information available about its composition and reactivity. The casein sub-micelle model was prominent for many years, but there is sufficient evidence now to conclude that there is not a defined sub-micellar structure to the micelle at all. More recent models suggest a more open structure comprised of aggregates of protein around calcium phosphate nanoclusters. Each of the casein proteins has unique abilitites to either bind with CaP or with other caseins, which gie rise to the aggregates. The nanoclusters provide regions of more or less density. The structure is sufficiently porous to hold a considrable amount of water, and for the surface, and even part of the interior, to be reactive to other substances. All models agree that the k-casein is mostly present as a stabilizng layer around the exterior of the micelle. Please see any of the following references for great detail about micelle structures and models.


Selected References

Holt, C. & D. S. Horne. 1996. The hairy casein micelle: evolution of the concept and its implication for dairy technology. Neth. Milk Dairy J. 50: 85-111.

Horne, D. S. 1998. Casein interactions: casting light on the black boxes, the structure in dairy products. Internat. Dairy J. 8: 171-177.

Walstra, P. 1999. Casein sub-micelles: do they exist? Internat. Dairy J. 9: 189-192.

Horne, D. S. 2002. Casein structure, self-assembly and gelation. Current Opinion in Colloid and Interface Sci. 7: 456-461.

Dalgleish, D. G. 2011. On the structural models of bovine casein micelles - review and possible improvements. Soft Matter. 7: 2265-2272. 

Dalgleish, Douglas G. and Milena Corredig. 2012. The Structure of the Casein Micelle of Milk and Its Changes During Processing. Annual Reviews Food Sci. Technol. 3:449–67.

de Kruif , Cornelis G., Thom Huppertz, Volker S. Urban and Andrei V. Petukhov. 2012. Casein micelles and their internal structure. Advances in Colloid and Interface Science 171–172: 36–52.

Casein Micelle Stability

Colloidal calcium phosphate (CCP) acts as a cement between the hundreds or even thousands of submicelles that form the casein micelle. Binding may be covalent or electrostatic. Submicelles rich in kappa-casein occupy a surface position, whereas those with less are buried in the interior. The resulting hairy layer, at least 7 nm thick, acts to prohibit further aggregation of submicelles by steric repulsion. The casein micelles are not static; there are three dynamic equilibria between the micelle and its surroundings:

  • the free casein molecules and submicelles
  • the free submicelles and micelles
  • the dissoved colloidal calcium and phosphate

The following factors must be considered when assessing the stability of the casein micelle:

Role of Ca++:

More than 90% of the calcium content of skim milk is associated in some way or another with the casein micelle. The removal of Ca++ leads to reversible dissociation of ß -casein without micellular disintegration. The addition of Ca++ leads to aggregation.

H Bonding:

Some occurs between the individual caseins in the micelle but not much because there is no secondary structure in casein proteins.

Disulphide Bonds:

alpha(s1) and ß-caseins do not have any cysteine residues. If any S-S bonds occur within the micelle, they are not the driving force for stabilization.

Hydrophobic Interactions:

Caseins are among the most hydrophobic proteins and there is some evidence to suggest they play a role in the stability of the micelle. It must be remembered that hydrophobic interactions are very temperature sensitive.

Electrostatic Interactions:

Some of the subunit interactions may be the result of ionic bonding, but the overall micellar structure is very loose and open.

van der Waals Forces:

No success in relating these forces to micellular stability.

Steric stabilization:

As already noted, the hairy layer interferes with interparticle approach.

There are several factors that will affect the stability of the casein micelle system:

Salt content:

affects the calcium activity in the serum and calcium phosphate content of the micelles.


lowering the pH leads to dissolution of calcium phosphate until, at the isoelectric point (pH 4.6), all phosphate is dissolved and the caseins precipitate.


at 4° C, beta-casein begins to dissociate from the micelle, at 0° C, there is no micellar aggregation; freezing produces a precipitate called cryo-casein.

Heat Treatment:

whey proteins become adsorbed, altering the behaviour of the micelle.


by ethanol, for example, leads to aggregation of the micelles.

When two or more of these factors are applied together, the effect can also be additive.

Casein micelle aggregation

Although the casein micelle is fairly stable, there are four major ways in which aggregation can be induced:

1. Enzymatic - chymosin (rennet) or other proteolytic enzymes as in Cheese manufacturing. 

Chymosin, or rennet, is most often used for enzyme coagulation. During the primary stage, rennet cleaves the Phe(105)-Met(106) linkage of kappa-casein resulting in the formation of the soluble CMP which diffuses away from the micelle and para-kappa-casein, a distinctly hydrophobic peptide that remains on the micelle. The patch or reactive site, as illustrated in the  image below, that is left on the micelles after enzymatic cleavage is necessary before aggregation of the paracasein micelles can begin.

During the secondary stage, the micelles aggregate, as illustrated on the right below. This is due to the loss of steric repulsion of the kappa-casein as well as the loss of electrostatic repulsion due to the decrease in pH. As the pH approaches its isoelectric point (pH 4.6), the caseins aggregate. The casein micelles also have a strong tendency to aggregate because of hydrophobic interactions. Calcium assists coagulation by creating isoelctric conditions and by acting as a bridge between micelles. The temperature at the time of coagulation is very important to both the primary and secondary stages. With an increase in temperature up to 40° C, the rate of the rennet reaction increases. During the secondary stage, increased temperatures increase the hydrophobic reaction. The tertiary stage of coagulation involves the rearrangement of micelles after a gel has formed. There is a loss of paracasein identity as the milk curd firms and syneresis begins.


The Schmidt Model

2. AcidAcidification causes the casein micelles to destabilize or aggregate by decreasing their electric charge to that of the isoelectric point. At the same time, the acidity of the medium increases the solubility of minerals so that organic calcium and phosphorus contained in the micelle gradually become soluble in the aqueous phase. Casein micelles disintegrate and casein precipitates. Aggregation occurs as a result of entropically driven hydrophobic interactions.

3. Heat. Milk is generally very stable to heat up to 90-95oC. At temperatures above the boiling point casein micelles will irreversibly aggregate. On heating, the buffer capacity of milk salts change, carbon dioxide is released, organic acids are produced, and tricalcium phophate and casein phosphate may be precipitated with the release of hydrogen ions.

4. Age gelation. Age gelation is an aggregation phenomenon that affects shelf-stable, sterilized dairy products, such as concentrated milk and UHT milk products. After weeks to months storage of these products, there is a sudden sharp increase in viscosity accompanied by visible gelation and irreversible aggregation of the micelles into long chains forming a three-dimensional network. The actual cause and mechanism is not yet clear, however, some theories exist:

  1. Proteolytic breakdown of the casein: bacterial or native plasmin enzymes that are resistant to heat treatment may lead to the formation of a slow gel forming over a long period of time.
  2. Chemical reactions: polymerization of casein and whey proteins due to Maillard type or other chemical reactions
  3. Formation of kappa-casein-ß -lactoglobulin complexes

An excellent source of information on casein micelle stability can be found in Walstra et al., 2006.