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24 Chemistry and Biochemistry of Milk Constituents 455coagulation of milk and hence in the structure and properties of
the Ca-caseinate particles (for review, see Fox and Kelly 2003,
Fox and McSweeney 2003). The term casein micelle appears to
have been first used by Beau (1921). The idea that the rennet-
induced coagulation of milk is due to the destruction of a pro-
tective colloid (Schutz colloid) dates from the 1920s; initially, it ̈
was suggested that the whey proteins were the ‘protective col-
loid’. The true nature of the protective colloid, the structure of
the casein micelle and the mechanism of rennet coagulation did
not become apparent until the pioneering work on the identifi-
cation, isolation and characterisation ofκ-casein by Waugh and
von Hippel (1956). Since then, the structure and properties of
the casein micelle have been studied intensively. The evolution
of views on the structure of the casein micelle was described by
Fox and Kelly (2003), Horne (2003, 2011) and Fox and Brod-
korb (2008). Current knowledge on the composition, structure
and properties of the casein micelle and the key features thereof
are summarised later.
The micelles are spherical colloidal particles, with a mean
diameter of approximately 120 nm (range, 50–600 nm). They
have a mean particle mass of approximately 10^8 Da, that is there
are about 5000 casein molecules (20,000–25,000 Da each) in
an average micelle. On a dry weight basis, the micelles contain
approximately 94% protein and approximately 6% non-protein
species, mainly calcium and phosphate, with smaller amounts
of Mg and citrate and traces of other metals; these are collec-
tively called colloidal (or micellar) calcium phosphate (CCP
or MCP). Under the conditions that exist in milk, the micelles
are hydrated to the extent of approximately 2 g H 2 O/g protein.
There are approximately 10^15 micelles/mL milk, with a total sur-
face area of approximately 5× 104 cm^2 ; the micelles are about
240 nm apart. Owing to their very large surface area, the surface
properties of the micelles are of major significance and because
they are quite closely packed, even in unconcentrated milk, they
collide frequently due to Brownian, thermal and mechanical
motion.
The casein micelles scatter light; the white appearance of
milk is due mainly to light scattering by the micelles, with
a contribution from the fat globules. The micelles are gener-
ally stable to most processes and conditions to which milk is
normally subjected and may be reconstituted from spray-dried
or freeze-dried milk without major changes in their properties.
Freezing milk destabilises the micelles, due to a decrease in pH
and an increase in [Ca^2 +]. On very severe heating, for example at
140 ◦C, the micelles shatter initially, then aggregate and eventu-
ally, after approximately 20 minutes, the system coagulates (see
Chapter 25)
The micelles can be sedimented by centrifugation; approxi-
mately 50% are sedimented by centrifugation at 20,000×gfor
30 minute and approximately 95% by 100,000×gfor 60 minute.
The pelleted micelles can be redispersed by agitation, for exam-
ple by ultrasonication, in natural or synthetic milk ultrafiltrate;
the properties of the redispersed micelles are not significantly
different from those of native micelles.
The casein micelles are not affected by regular (∼20 MPa)
or high-pressure (up to 250 MPa) homogenisation (Hayes and
Kelly 2003). The average size is increased by high-pressuretreatment at 200 MPa, but they are disrupted (i.e., average size
reduced by∼50%) by treatment at a somewhat higher pressure,
that is≥400 MPa (Huppertz et al. 2002, 2004).
The micro-structure of the casein micelle has been the subject
of considerable research during the past 40 years, that is since
the discovery and isolation of the micelle-stabilising protein,
κ-casein; however, there is still a lack of general consensus.
Numerous models have been proposed, the most widely sup-
ported initially was the sub-micelle model, first proposed by
Morr (1967) and refined several times since (Fox and Kelly
2003, Horne 2003, 2011, Fox and Brodkorb 2008). Essentially,
this model proposes that the micelle is built up from sub-micelles
(MW∼ 5 × 106 Da) held together by CCP and surrounded and
stabilised by a surface layer, approximately 7 nm thick, rich inκ-
casein but also containing some of the other caseins (Fig. 24.3A).
It is proposed that the hydrophilicC-terminal region ofκ-casein
protrudes from the surface, creating a hairy layer around the
micelle and stabilising it through a zeta potential ofca−20 mV
and by steric stabilisation. The principal direct experimental ev-
idence for this model is provided by electron microscopy, which
indicates a non-uniform electron density; this has been inter-
preted as indicating sub-micelles.
However, several authors have expressed reservations about
the sub-unit model and three alternative models have been pro-
posed. Visser (1992) suggested that the micelles are spherical
aggregates of casein molecules randomly aggregated and held
together partly by salt bridges in the form of amorphous calcium
phosphate and partly by other forces, for example hydrophobic
interactions, with a surface layer ofκ-casein. Holt (1992) pro-
posed that the Ca-sensitive caseins are linked by micro-crystals
of CCP and surrounded by a layer ofκ-casein, with itsC-
terminal region protruding from the surface (Fig. 24.3B). In the
dual-binding model of Horne (2003, 2011), it is proposed that
individual casein molecules interactviahydrophobic regions in
their primary structures, leaving the hydrophilic regions free and
with the hydrophilicC-terminal region ofκ-casein protruding
into the aqueous phase (Fig. 24.3C). Thus, the key structural
features of the sub-micelle model are retained in the three alter-
natives, that is the integrating role of CCP and aκ-casein-rich
surface layer.
The micelles disintegrate:
when the CCP is removed, for example by acidification
to pH 4.6 and dialysis in the cold against bulk milk, or by
addition of trisodium citrate, also followed by dialysis;
removal of>60% of the CCP results in disintegration of the
micelles;
on raising the pH to approximately 9.0, which does not
solubilise the CCP and presumably causes disintegration by
increasing the net negative charge;
on adding urea to>5 M, which suggests that hydro-
gen and/or hydrophobic bonds are important for micelle
integrity.The micelles are precipitated by ethanol or other low MW al-
cohols at levels of≥approximately 35% at 20◦C. However, if the
temperature is increased to≥ 70 ◦C, surprisingly, the precipitated
casein dissolves and the solution becomes quite clear, indicating