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.