ARTICLE IN PRESS

International Dairy Journal 18 (2008) 677– 684

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International Dairy Journal

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Review

The casein micelle: Historical aspects, current concepts and significance

P.F. Fox a,, A. Brodkorb b
a
Department of Food and Nutritional Sciences, University College, Cork, Ireland
b
Moorepark Food Research Centre, Teagasc, Moorepark, Fermoy, Cork, Ireland

abstract

The caseins, a group of unique milk-specific acid-insoluble phosphoproteins, represent E80% of the
total protein in the milk of cattle and other commercial dairy species. Owing to their commercial
importance, the caseins have been studied very extensively and are probably the best characterized food
protein system. It has been recognized since the work of Schübler in 1818 that the caseins exist in milk
as large particles suspended in the aqueous phase (milk serum). Initially, the casein particles were
usually referred to as ‘‘calcium caseinate–calcium phosphate particles’’. The term ‘‘casein micelle’’ was
introduced in 1921 and the two terms were used interchangeably for several years but since about 1960,
the latter term has been used exclusively. It has been suggested that the calcium caseinate–phosphate
particles are not true micelles. The term ‘‘micelle’’ was introduced by Nägeli and Schwendener [Nägeli,
C.W., & Schwendener, W. (1877). Das Mikroskop: Theorie und Anwendung Desselben (2nd ed.). Leipzig:
W. Engelmann] for microparticles of cellulose in plant cells visible in the light microscope; later, it was
used for various other types of aggregates. Owing to the importance of the casein micelles for many of the
physico-chemical properties of milk and dairy products, their structure and properties and the effects of
compositional and processing factors thereon have been studied extensively. Since the discovery of the
micelle-stabilizing protein, k-casein, in 1956, several models of the casein micelle have been proposed and
refined. This review will focus on the following aspects: introduction and use of the term ‘‘micelle’’, early
views on the stability of casein in milk, introduction of the term ‘‘casein micelle’’ for the calcium
phosphate–calcium caseinate particles in milk, and the structure and stability of casein micelles.
& 2008 Elsevier Ltd. All rights reserved.

Contents

1. The caseins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 677

2. Micelles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 678
3. Caseinate particles in milk: early studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 678
4. Introduction and early use of the term ‘‘casein micelle’’ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 679
5. k-Casein and the casein micelle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 680
6. Relationship between calcium phosphate and calcium caseinate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 680
7. Structure of the casein micelle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 681
8. Stability of casein micelles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 682
9. Significance of the casein micelle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 682
10. Is the ‘‘casein micelle’’ a ‘‘true micelle’’? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 682
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 683

1. The caseins tance, the caseins have been studied extensively and are probably
the best characterized food protein system (see Fox & McSweeney,
The caseins, a group of unique milk-specific proteins, represent 2003). Research on casein dates from Berzelius (1814), Schübler
E80% of the total protein in the milk of cattle and other (1818) and Braconnet (1830); the early work was reviewed by
commercial dairying species. Owing to their commercial impor- Kastle and Roberts (1909), Beau and Bourgain (1926) and Beau
(1932a).
Casein is usually prepared by isoelectric precipitation at pH
 Corresponding author. Tel.: +353 21 490 2362; fax: +353 21 427 0001. 4.6, a method developed by Hammersten (1883) and improved by
E-mail address: pff@ucc.ie (P.F. Fox). van Slyke and Barker (1918). As early as 1880, Danilewsky and

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678 P.F. Fox, A. Brodkorb / International Dairy Journal 18 (2008) 677–684

Radenhausen reported that isoelectric casein is heterogeneous but 2. Micelles

Hammersten (1883, 1885) concluded that properly prepared
isoelectric casein is homogeneous. Based on differences in Although the word ‘‘micelle’’ is now generally used to describe
solubility in aqueous systems, several authors (e.g., Linderstrøm- aggregates of amphipathic molecules, classically soaps, it has been
Lang, 1925, 1929; Linderstrøm-Lang & Kodama (1925); Osborne & used for at least five, largely unrelated, types of particles. The
Wakeman, 1918), suggested that isoelectric casein is heteroge- word ‘‘micelle’’ or ‘‘micella’’ (diminutive of the Latin, mica,
neous, which was confirmed by the electrophoretic studies of meaning crumb, morsel) was coined by Nägeli and Schwendener
Mellander (1939), who showed that isoelectric casein is a mixture (1877) to describe molecular aggregates or crystalline particles of
of three proteins, which he called a-, b- and g-caseins in order cellulose, which they considered to be the building blocks of plant
of decreasing mobility on free boundary electrophoresis. cells. The quasi-crystalline regions of cellulose are still referred to
These proteins were fractionated by Warner (1944) and by Hipp, as ‘‘micelles’’. Nägeli, who was active in the debate on evolution
Groves, Custer, and McMeekin (1952). Waugh and von Hippel and spontaneous generation, considered micelles to be the first
(1956) used CaCl2 to resolve casein into insoluble and soluble stage in the transition of non-living molecules to living proto-
fractions. The former, which represented 85% of total casein, plasm; he developed a micellar theory of life (Nägeli, 1884; see
contained as- and b-caseins, while the soluble fraction contained a also Strick, 2000).
previously unknown protein, which Waugh and von Hippel (1956) Felix d’Herelle, who discovered bacterial viruses (bacterio-
called k-casein. From the point of view of the stability and phage) in 1915, also developed a micellar theory of life, in which
properties of the casein micelle, k-casein is its most important the term ‘‘micelle’’ was used for colloidal particles, including
component. as-Casein is a mixture of two proteins, as1-casein bacteriophage, which he thought represented the ultimate units
and as2-casein (Annan & Manson, 1969). The four caseins exhibit of living things (d’Herelle, 1924; see also Summers, 1999).
microheterogeneity arising from differences in the degree of d’Herelle’s concept was, in principle, similar to that of Nägeli;
phosphorylation or glycosylation (k-) or genetic polymorphism. both considered micelles to be aggregates of macromolecules
g-Caseins represent C-terminal segments of b-casein while (d’Herelle cited the work and ideas of Nägeli). d’Herelle described
the corresponding N-terminal parts are proteose peptones 5, 8slow a micella as the smallest particle of protein matter and conse-
and 8fast. quently the smallest possible particle of living substance; he
In milk, the caseins exist as large colloidal particles, 50–600 nm proposed that living matter is an ‘‘assemblage of micellae’’ and that
in diameter (mean E150 nm), called ‘‘casein micelles’’. Since ‘‘the protoplasmic micella should be to the cell what the cell is to the
many of the technologically important properties of milk, e.g., its whole organism’’. d’Herelle considered protoplasmic micellae to be
white colour, stability to heat or ethanol and coagulation by polymers of an acid amine (i.e., a protein), a cyclic base (i.e., a
rennet, are due to the properties of the casein micelles, there nucleic acid), a lipid (i.e., a fat globule) or various carbohydrates
has been an economic and technological incentive to characterize (i.e., a polysaccharide). Henderson and Henderson (1920) defined
their properties and elucidate their structure. An extensive a micelle (micella) as an ultra supra-molecular unit of a cell.
literature on these subjects has accumulated and has been In the early years of the 20th century, the term ‘‘micelle’’
reviewed at regular intervals (e.g., de Kruif, 1998, 1999; de was also used to describe aggregates of inorganic molecules,
Kruif & Holt, 2003; Farrell, 1973; Farrell & Thompson, 1974; e.g., hydrated ferric oxide (see Duclaux, 1920; Zsigmondy, 1921).
Fox, 2003; Fox & Kelly, 2004; Garnier, 1973; Garnier & Today, physical chemists use the term ‘‘micelle’’ to describe
Ribadeau-Dumas, 1970; Holt, 1992, 1994; Holt & Horne, 1996; aggregates of amphipathic molecules (e.g., surfactants, detergents,
Horne, 1998, 2002; McMahon & Brown, 1984; McMahon & soaps) that are in dynamic equilibrium with surfactant monomers
McManus, 1998; Morr, 1967; Payens, 1966, 1979, 1982; Rollema, (Dickinson, 1992; Hunter, 1993). These classical micelles were first
1992; Rose, 1969; Ruettimann & Ladisch, 1987; Schmidt, 1980, described by McBain (1913) and since then have been studied
1982; Slattery, 1976; Slattery & Evard, 1973; Tuinier & de Kruif, extensively. In modern textbooks on colloidal or physical
2002; Visser, 1992; Walstra, 1990, 1999; Walstra, Geurts, Noomen, chemistry or biochemistry, the term ‘‘micelle’’ is used exclusively
Jellma, & van Boekel, 1999; Walstra & Jenness, 1984; Waugh, for soap-like micelles without reference to the older and more
1971). general meaning of the word, which is not even acknowledged or
According to Dickinson (1992), the casein particles in milk are referenced (see Dickinson, 1992; Hunter, 1993; Patterson, 1987).
not true micelles because they are irreversibly formed over Williams and Williams (1973) wrote ‘‘The term ‘‘particle’’ is a
normal time-scales; a solution of pure b-casein forms reversible general one and can be applied to any species in or near the size range
aggregates at concentrations above the critical micelle concentra- 1–1000 nm. Some oil droplets, soap particles, and metal oxide
tion in a way that resembles classical surfactant micelles. particles fall within these size dimensions and give rise to systems
Dickinson (1992) urged that, to avoid confusion, the term ‘‘casein traditionally referred to as colloidal dispersions. The particles in these
micelle’’ should never be shortened to ‘‘micelle’’; presumably, he systems (colloidal dispersions) are frequently called micelles as
considered ‘‘soap micelles’’ to be the ‘‘standard/typical micelle’’. opposed to the term macromolecules, which is reserved for individual
However, as discussed below, this is a rather late definition of large molecules or a very low-order polymer of giant molecules’’.
‘‘micelle’’ and in any case, micellar caseins are in equilibrium Dictionaries (e.g., The Oxford Dictionary, 1989 and Tresor de la
with their ‘‘monomeric’’ counterparts in the milk serum (Waugh, Langue Francaise, 1985) credit Nägeli and Schwendener (1877)
1958). Micelle formation and equilibria were studied in depth with introducing the term ‘‘micelle’’, of which they give several
by Waugh and collaborators (see Waugh, 1961, 1971). The term examples, including casein micelles.
‘‘casein micelle’’ was not generally used until about 1960,
mainly through the influence of Waugh, who used it without
explanation. In fact, when and why the term ‘‘casein micelle’’ was 3. Caseinate particles in milk: early studies
introduced does not seem to have been explored. In this review,
the general use and meaning of the term ‘‘micelle’’ will be It has been known since the work of Schubler (1818) that
considered initially, followed by a discussion on when and why casein is ‘‘merely suspended’’ in milk (see Palmer & Richardson,
the term ‘‘casein micelle’’ came into use, and finally a summary 1925). Sheldon (1880) gave the following interesting description
of the current views on the structure and properties of the of casein: ‘‘Casein is a member of the albumin group, about which so
casein micelle. little is known y. Casein is not in solution in the milk, but is swelled
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up by its absorption of water into a kind of very thin jelly. This may be association colloids (micelles) formed by the association of
proved by putting milk into a dialysator, when the casein does not inorganic molecules (Duclaux, 1920; Zsigmondy, 1901, 1909) or
pass the membrane, as it would if it were dissolved’’. Duclaux (1887) amphipathic molecules (McBain, 1913).
referred to casein as a colloid, probably being the first to do so. Although most of the amino acids had been isolated by 1900,
Johnson (1868) makes the following interesting comment: ‘‘When views on the structure of proteins were very unclear until after
casein is separated from milk by rennet, as in making cheese, it 1930 (see Edsall, 1962; Tanford & Reynolds, 2001). It was not
carries with it a considerable portion of the phosphates and other possible to determine the molecular mass of proteins until the
salts of the milk; these salts are not found in the casein precipitated analytical ultracentrifuge was developed in 1925 by Svedberg
by acids, being held in solution by the latter’’. By the end of the 19th and Fahraeus and there was a widely held view that proteins did
century, it was recognized that the casein in milk exists as large not have a clearly defined structure, as did small organic or
colloidal particles that contain calcium phosphate and are inorganic molecules, but were random aggregates of low
retained by Pasteur-Chamberland porcelain filters (see Kastle & molecular mass polypeptides. Beau (1921), who would not have
Roberts, 1909). known the size of casein molecules, may have assumed that the
During the early years of the 20th century, a number of studies casein micelles were actually casein molecules; unfortunately, he
were reported on the ‘‘colloidal chemistry of milk’’ (see Wiegner, did not explain his choice of word and the article is poorly
1914a, 1914b), in which the term ‘‘casein particles’’ or ‘‘large casein referenced.
particles’’ was used to describe the physical state of casein. The term ‘‘micelle’’ was used extensively by Porcher (1923) in
Wiegner (1914b) showed that the particles dissociated if the his review on the chemistry of milk constituents but he did not
calcium therein was replaced by sodium. The stability of these explain why he used this term and the article was not referenced.
particles, especially how they are destabilized during the rennet- In their review of the rennet coagulation of milk, Palmer and
induced coagulation of milk, attracted much attention. It was Richardson (1925) usually used the terms ‘‘colloidal calcium
suggested by Alexander (1910, 1912) and Alexander and Bullowa caseinate’’, ‘‘calcium caseinate’’ or ‘‘calcium caseinate–calcium
(1910) that the casein particles are an irreversible, unstable phosphate’’ to describe the casein system in milk but used the
colloid, stabilized by a reversible, stable, protective colloid term ‘‘micellae’’ once, without explanation, and did not refer to
(Schutzkolloid), which he proposed is ‘‘lactalbumen’’, i.e., whey Beau (1921) or Porcher (1923). It is not clear what Beau (1921),
proteins, and which is destroyed by rennet, leading to the Porcher (1923) or Palmer and Richardson (1925) meant by ‘‘a
coagulation of the casein particles. He suggested that the poor micelle’’ and none of them cited Nägeli and Schwendener (1877),
renneting properties of human and ass’s milk are due to the low Zsigmondy (1901), McBain (1913) or d’Herelle (1924).
level of casein and the high level of lactalbumen in those milks. In a paper on the rennet coagulation of milk, Marui (1926) used
The term Schutzkolloid (protective colloid) was coined by the term ‘‘casein micelle’’ freely but without explanation; the
Zsigmondy (1909) to describe the ability of biopolymers to paper is sparsely referenced and Beau (1921), Porcher (1923) and
prevent the coagulation of a gold sol by electrolytes. The efficacy Palmer and Richardson (1925) were not cited. Marui (1926) used
of biopolymers was expressed as the ‘‘gold number’’, defined as the term ‘‘Schutzkolloid’’ (introduced by Zsigmondy, 1909) in
the amount (mg) of biopolymer that when added to 10 cm3 of a relation to casein micelle stability and rennet-induced coagula-
solution of a gold salt, just prevents its coagulation on addition of tion. Linderstrøm-Lang (1929) also used the term Schutzcolloid in
1 cm3 of a 10% NaCl solution (Zsigmondy, 1901). relation to the casein system although he did not use the term
Destruction of the protective colloid by rennet, essentially the ‘‘casein micelle’’. In their studies on the rennet-induced coagula-
view of Alexander (1910), was also considered by Schryver (1913) tion of milk, Richardson and Palmer (1929) and Hankinson and
and Clayton (1918) to be responsible for the coagulation of milk by Palmer (1943) freely used the terms ‘‘micellae’’, ‘‘caseinate
rennet. Wright (1924) proposed that the coagulation of milk by micellae’’, ‘‘calcium caseinate micellae’’, ‘‘casein/caseinate mi-
rennet is due to an unspecified change in the ‘‘colloidal condition’’ celles’’ and even ‘‘rennin micellae’’. Hankinson and Briggs (1941)
of the calcium salt of caseinogen. Early work on the rennet used the term ‘‘micelle’’ to describe the protein particles in
coagulation of milk was reviewed by Palmer and Richardson sodium caseinate. Clearly, at this time, the term ‘‘micelle’’ was
(1925), who dismissed the protective colloid idea and proposed being used rather indiscriminately to describe proteins/polypep-
that the rennet coagulation of milk is due to the ‘‘precipitation of tides which were sufficiently large to behave as a colloid.
micellae by cations in the presence of a suspensoid which is The casein system, including the colloidal aspects, was
peptidised by the precipitating ion’’. The word ‘‘micellae’’ was used described in considerable detail by Beau and Bourgain (1926)
only once in that paper and no explanation of the term is given; and Beau (1932a, 1932b, 1941). In a book on the colloidal aspects
usually, the term ‘‘calcium caseinate–calcium phosphate com- and rennet-induced coagulation of milk, Porcher (1929) used the
plex’’, which was regarded as an irreversible colloid (suspensoid), term ‘‘micelle’’ widely; this book was serialized in Le Lait, volumes
was used. 9, 10 and 11 (1929, 1930, 1931). The term ‘‘micelle’’ was also used
by Piettre (1931) and Brigando (1933), although they usually used
the term ‘‘colloidal calcium caseinate–calcium phosphate’’ or a
4. Introduction and early use of the term ‘‘casein micelle’’ variant thereof and did not refer to Beau (1921), Porcher (1923) or
Palmer and Richardson (1925).
Duclaux (1920) used the term ‘‘micelle’’ in relation to various Interestingly, most of the early papers in which the term
macromolecules, including casein; he acknowledged that this ‘‘casein micelle’’ was used involved attempts to explain the
term had been introduced by Nägeli. However, as far as we can rennet-induced coagulation of milk, and, with the exceptions of
ascertain, the first author to use the term ‘‘casein micelle’’ was Marui and Palmer, were by French authors, suggesting a common
Beau (1921) who used the term ‘‘lactéine’’ for all milk proteins in origin, perhaps Duclaux or d’Herelle, both of whom worked at the
their native state, which he considered to be aggregates Pasteur Institute in Paris.
comprised of caseins and whey proteins and which he called Sørensen (1930) compiled an extensive review on the forma-
‘‘micelles’’. Beau’s concept of the casein micelle was probably tion of aggregates (bundles, micelles) of macromolecules. He cited
based on the ‘‘protective colloid’’ model (Alexander, 1910, 1912; the work of Meyer (1930) on the formation of cellulose micelles
Alexander & Bullowa, 1910). Beau referred to the work of Duclaux and described his own work on the formation of micelles from
(1920) and was probably familiar with the then current views on various soluble proteins, including casein. In his view, micelles are
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680 P.F. Fox, A. Brodkorb / International Dairy Journal 18 (2008) 677–684

held together by secondary (associating) forces rather than Early investigators (e.g., Hostettler & Imhof, 1951; Knoop &
covalent bonds. Sørensen (1930) proposed that the term ‘‘compo- Wortmann, 1960; Nitschmann, 1949) who applied electron
nent system’’ rather than ‘‘micelle’’ should be used to describe microscopy to milk, used the term Calciumcaseinteilchen exclu-
aggregates of macromolecules. He argued that Nägeli’s ‘‘micelles’’ sively. Probably the first electron microscopy study in which the
corresponded to his ‘‘components’’, i.e., single molecule-like term ‘‘casein micelle’’ was used is that of Barbaro and Calapuj
substances; to quote: Hence to Nägeli the conception micelles is a (1958); Shimmin and Hill (1964) used the term extensively.
unity of the same kind as the molecule conception in the case of Thus, prior to 1955, few authors other than the French,
simple substances, and in the colloid-chemical literature where it is consistently used the term ‘‘casein micelle’’. The term was not
frequently made use of especially by French investigators, the used as a sub-head in the index of Dairy Science Abstracts, first
conception micelle is used in the same sense. We need only refer to published in 1939, until 1955, when von Hippel and Waugh (1955)
J. Duclaux (Les Colloides, Gauthier-Villar, Paris, 1929), who writes ‘‘La was cited.
micelle sera pour le colloı̈de l’analogue de ce le molécule chimique
ordinaire est pour le cristalloı̈de’’. This seems to imply that the term
‘‘micelle’’ was a French concept. Although he cited the work of 5. j-Casein and the casein micelle
McBain briefly, Sørensen (1930) did not acknowledge that McBain
used the term ‘‘micelle’’. Sørensen (1930) considered casein, von Hippel and Waugh (1955) studied the influence of
which had just been shown to be heterogeneous, to be a temperature, pH and protein concentration on the equilibrium
particularly interesting system. However, his discussion on casein between casein monomers and polymers (aggregates, micelles).
related to solutions of the protein in dilute acid or alkali which he They concluded that ‘‘Clearly, there exists in each case a balance
considered to exist as micelles. Surprisingly, Sørensen (1930) did between attractive and repulsive forces dependent on the physical
not discuss the natural colloidal casein particles and did not conditions. These polymerizations bear a marked resemblance to
reference any of the earlier studies in which the term ‘‘micelle’’ those responsible for the formation of soap micelles, as described and
was used or Alexander (1910), Alexander and Bullowa (1910), analysed by Debye’’. None of the previously cited articles in which
Wiegner (1914a, 1914b) or Clayton (1918) in which the colloidal the term casein micelle was used was cited nor was the work of
aspects of casein were discussed. Nägeli or d’Herelle.
The term ‘‘micelle’’ was not used in early textbooks on Dairy Waugh had worked on the association of proteins during the
Science/Chemistry, e.g., Grimmer (1926), Tague (1926), Beau 1940s and published a paper (Langmuir & Waugh, 1940) on the
(1932a), Sutermeister (1927), Associates of Rogers (1928), Rahn surface activity of proteins which contained the following
and Sharp (1928), Davies (1936, 1939), Ling (1946) or Davis and interesting comment ‘‘The spreading of a water-soluble protein to
MacDonald (1953), or in the comprehensive review on milk give an insoluble monolayer depends on the presence of hydrophobic
proteins by McMeekin and Polis (1949). In the second edition of side chains in some of the amino acids of the protein. In aqueous
Fundamentals of Dairy Science (Associates of Rogers, 1935), the solutions of the globular proteins these hydrophobic groups must be
term ‘‘casein micelle’’ was not used in the chapters on Milk inaccessible, probably being enclosed within cage-like protein
Proteins or Physical Equilibria of Milk but was used to a very limited molecules having hydrophilic surfaces; a structure like that of the
extent by L.S. Palmer in his article on the Rennet Coagulation of micelles in solutions of soap and other detergents in which
Milk. Sutermeister and Browne (1939) included several references hydrocarbon chains are packed into the interior of spherical micelles
to the ‘‘casein micelle’’. whose surfaces contain all the polar groups’’. Waugh (1946) also
The analytical ultracentrifuge made it possible to study the used the term ‘‘micelle’’ to describe aggregates of heat-denatured
polydispersity of the casein particles in milk and model systems insulin (alternative terms used were floccules, spherites, fibrils
(see Pedersen, 1936; Svedberg, Carpenter, & Carpenter, 1930a, and micelles). He published a very extensive review (Waugh,
1930b) but the term ‘‘casein micelle’’ was not used by these 1954) on the non-covalent forces involved in protein association
investigators. There was quite an amount of research on the and aggregation, in which the term ‘‘micelle’’ was used widely; he
polydispersity, composition and properties of casein particles described some work on b-lactoglobulin but made no reference to
during the period 1930–1950 (see Lindqvist, 1963; Pyne, 1955) but the caseins. He had also worked on the membrane of erythrocytes
the term ‘‘casein micelle’’ was very rarely used, an exception being and on blood clotting.
Eilers (1947), who used it occasionally. Waugh and von Hippel (1956) isolated k-casein and revolu-
The term ‘‘casein micelle’’ became fairly widely used in the tionized ideas on the structure of the casein micelle. k-Casein, a
1950s. Pyne (1953, 1955) usually used the term ‘‘caseinate–pho- relatively minor component of the casein system (12–15% of
sphate complex’’ but used ‘‘caseinate micelle’’ occasionally; he did whole casein), is soluble in the presence of Ca2+ whereas the
not explain his choice of the term ‘‘micelle’’ and cited none of the remaining 85% of casein are precipitated by Ca2+; k-casein can
earlier authors who had used the term. Berridge (1954) usually stabilize up to 10 times its weight of the Ca-sensitive caseins via
referred to the caseinate particles as ‘‘micelles’’ and stated that: ‘‘it the formation of micelles, thus functioning as the Schutzcolloid
has been known for a considerable time that the casein in milk exists proposed 50 years earlier. This discovery led to a series of
in the form of micelles with calcium phosphate’’. He cited several publications by Waugh and collaborators on the mechanism of
references (Hostettler & Imhof, 1951; Nitschmann, 1949; Ter stabilization and the structure of the casein micelle, which was
Horst, 1947) to support this or similar statements but none of reviewed by Waugh (1971).
these authors referred to the calcium caseinate–calcium phos-
phate particles as micelles.
In a brief description of the milk protein system, Fox and Foster 6. Relationship between calcium phosphate and calcium
(1957) made the following interesting but unreferenced com- caseinate
ment: ‘‘Casein does not occur in milk in true solution, but rather in
the form of large aggregates or micelles of indefinite size, ranging up As discussed previously, it has been known since the end of the
to 2000 Å or more in diameter and averaging perhaps 1000 Å’’. In 19th century that the colloidal caseinate particles contain calcium
their widely acclaimed text-book, Jenness and Patton (1959) phosphate. The relationship between the colloidal casein particles
acknowledged the term ‘‘micelle’’ but decided to use the term and calcium phosphate, now referred to as colloidal calcium
‘‘calcium caseinate–phosphate complex’’. phosphate (CCP), has been the subject of numerous investigations
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P.F. Fox, A. Brodkorb / International Dairy Journal 18 (2008) 677–684 681

Table 1 plans for the structure of the casein micelle? Micelles are expected to
Average characteristics of casein micelles fall into one or more of the categories of (a) single-phase particles, (b)
large chemical compounds or (c) structures having a composition
Characteristic Value
which changes in going from the surface to the center’’. He described
Diameter 120 nm (range: 50– 500 nm) the forces involved in stabilizing colloidal particles and included
Surface area 8  10 10 cm2 some examples, including soap micelles, bacteriophage and
Volume 2.1 10 15 cm3 complex enzyme systems. He continued: In the casein micelle
Density (hydrated) 1.0632 g cm 3
Mass 2.2  10 15 g
system, as will be seen, the micelle state may be the lowest free-
Water content 63% energy state of the system. Of particular interest will be micelle
Hydration 3.7 g H2O g 1 protein structure and the mechanisms which operate in determining micelle
Voluminosity 44 cm3 g 1 size. He proceeded to present a detailed discussion on his views on
Molecular mass (hydrated) 1.3  109 Da
the structure and properties of the casein micelle, based mainly
Molecular mass (dehydrated) 5  108 Da
No. of peptide chains 5  103 on the work of his group on model systems prepared, mainly, from
No. of particles per mL milk 1014– 1016 as- and k-caseins and CaCl2. The review is copiously referenced
Surface of micelles per mL milk 5  104 cm3 but, rather surprisingly, he cited none of the early workers who
Mean free distance 240 nm had used the term ‘‘casein micelle’’.
Since the pioneering work of Waugh (1958), there has been
continuous work on the properties and structure of the casein
during the past 100 years but the matter has not yet been fully micelle, and structural models have been refined progressively.
resolved. The principal issues investigated include: the composi- While views on the detailed structure of the casein micelle are not
tion of CCP; the nature of the association between CCP and casein; unanimous, its general properties are widely accepted. Progress
and the effect of CCP on casein micelle stability and size. These can be followed through the numerous reviews cited earlier and
topics are critical to the structure and properties of the micelles has been summarized by Fox and Kelly (2004). Some key features
but are outside the scope of this review. The reader is referred to and characteristics to be considered are as follows.
Pyne and McGann (1960), McGann and Pyne (1960), Pyne (1962). k-Casein, which is soluble at the calcium concentration in milk
Schmidt (1982), McGann, Buchheim, Kearney, and Richardson and which is about 12% of total casein, can stabilize about 10
(1983) and Holt (1985, 1997, 1998). times its mass of Ca-sensitive caseins (as1-, as2- and b-). The most
obvious organization which would permit this is a core of Ca-
sensitive caseins surrounded by a layer of k-casein, analogous to
7. Structure of the casein micelle the stabilization of lipids by an emulsifier. k-Casein is readily
hydrolysed by chymosin (35 kDa) and reacts via sulphydryl–di-
The general properties of casein micelles are now well sulphide interaction with b-lactoglobulin (dimeric, 36 kDa) on
established and are described in the reviews cited earlier. The heating milk. Both of these reactions suggest either that k-casein
key features are summarized in Table 1. The mechanism by which is exposed on the surface of the micelles or that the micelles are
the casein particles (micelles) in milk are stabilized and very porous so that large molecules can diffuse readily through
destabilized by the action of various agents and conditions have them.
attracted much attention during the 20th century and there is a However, milk is not coagulated by immobilized rennets,
vast volume of information in the literature. One of the important suggesting that the k-casein is not very exposed. Super-polymer-
topics investigated has been elucidation of the structure of the ized aminopeptidase, which cannot diffuse into the micelle,
micelle. releases the N-terminal residue of all four caseins, suggesting
The development of a realistic model of casein micelle that the surface of the micelles is not covered exclusively with
structure became possible only after the isolation and character- k-casein and that some of all four caseins is located on the
ization of k-casein (Waugh & von Hippel, 1956). The first model of surface.
the casein micelle was published by Waugh (1958). This was On removal of colloidal calcium phosphate (by acidification-
followed by a series of papers on the formation and properties of dialysis or a calcium chelator), the micelles disperse into particles
artificial casein micelles, e.g., Waugh (1961), Noble and Waugh of 0.5  106 Da, suggesting that CCP plays an integrating
(1965), Waugh and Noble (1965) and Waugh, Creamer, and role in the micelles. However, the micelles are also dispersed
Slattery (1970). These studies were reviewed by Waugh (1971), by urea, SDS, high pH or ethanol (435%, 470 1C), indicating
who elaborated on a model for the structure of casein sub- that hydrogen bonds, hydrophobic and electrostatic interactions
micelles and explained for his choice of the term ‘‘micelle’’ to are also involved in micelle integrity. Up to 50% of b-casein,
describe the calcium phosphate–calcium caseinate particles in the most hydrophobic of the caseins, dissociates reversibly from
milk. To quote: ‘‘A considerable amount of space could be devoted to the micelles on cooling, indicating the importance of hydro-
the question of the meaning of the word ‘micelle’. In most cases, phobic interactions and suggesting that the micelle is sufficiently
micelle has been used to designate what obviously are colloidal porous to allow the b-casein to diffuse out of the micelle; on
association products. However, the designation has most frequently rewarming, the b-casein appears to form a fuzzy layer on the
been used before even a general understanding of the structure was surface.
available. It is not surprising, at this time, that different micelle types Electron microscopy shows that the micelles have an uneven,
have different structures: the cellulose micelles of von Nägeli (see raspberry-like appearance which was interpreted to mean that
Nägeli & Schwendener, 1877) are different in structure from soap the micelles are built up from sub-micelles, with a core of
micelles (Shinoda, Nakagawa, Tamamushi, & Isemura, 1963) and Ca-sensitive caseins and a k-casein-rich coat. This model under-
from the casein micelle. The micelles of milk are customarily defined went several refinements, e.g., whether the k-casein coat was
as the colloidal association products of the caseins. To write them as uniform or in patches, whether all sub-micelles have a layer
colloidal particles is permissible on the basis that they are large with of k-casein or whether there are k-casein-rich and k-casein-
respect to the constituent monomers but stable with respect to each deficient sub-micelles, with the latter concentrated in the core
other and to the earth’s gravitational field. What might be of the micelle and the former concentrated at the surface, giving
anticipated, from the properties of other systems, as permissible the micelle a k-casein-rich surface. Most, but not all, authors
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682 P.F. Fox, A. Brodkorb / International Dairy Journal 18 (2008) 677–684

proposed that the sub-micelles are held together by micro- 9. Significance of the casein micelle
crystals of CCP.
However, recent electron microscopy studies using improved The casein in the milk of all species studied exists as micelles,
microscopes have failed to confirm the presence of sub-micelles; as indicated by the fact that they are more or less white due to
in one study (Dalgleish, Spagnuolo, & Goff, 2004), the irregula- light scattering, for which the micelles are mainly responsible. The
rities were considered to be microtubules. Three alternatives to widespread or universal distribution of micelles in milk, suggests
the sub-micelle models (Holt, 1994; Horne, 2002; Visser, 1992), that they have some physiological or nutritional significance over
depict the micelle as being made up of casein molecules linked the nutritional value of the proteins per se. Two benefits are
together by CCP microcrystals and hydrophobic bonds but differ apparent:
in detail. Further refinement of these models can be expected,
especially as electron microscopes are improved. 1. Calcium and phosphate are required for the development of
teeth and bone and growth rate is positively correlated with
the concentrations of Ca and Pi. However, calcium phosphate
8. Stability of casein micelles has low solubility at the pH of milk with which it is
supersaturated; therefore, calcium phosphate would be ex-
The colloidal stability of milk is, in many cases, its most pected to precipitate in the mammary gland, with the
important physico-chemical aspect and, consequently, has at- formation of etopic stones that would block the ducts of the
tracted much attention during the past century. In good quality gland, resulting in the death of the organ and perhaps of the
milk, the casein micelles are stable to all processes to which it is animal. By forming micelles, casein maintains the excess
normally subjected: calcium phosphate in a colloidally stable state. Thus, the
casein micelles may be regarded as a device by which to enable
 Concentration by evaporation or ultrafiltration: However, stabi- the secretion of milk with a high concentration of calcium
lity decreases with the degree of concentration, due mainly to phosphate in a ‘‘soluble’’ form.
the closer packing of casein micelles, an increase in [Ca2+] and 2. Presumably, the micelles are designed to be coagulated in the
a decrease in pH due to the precipitation of CaH2PO4 and stomach of the neonate by chymosin, a proteinase designed for
CaHPO4 as Ca3(PO4)2 accompanied by the release of H+. this function. Coagulation delays the entry of milk constituents
 Dehydration: In the absence of heat-induced changes, the into the small intestine, thereby improving digestibility.
micelles in milk powder reconstitute readily and their proper- Furthermore, the coagulum acts as a buffer to facilitate nursing
ties are changed little. at intervals that may be very long (24 h) for some species,
 Freezing: Freezing per se has little, if any, effect on the casein e.g., the hare.
micelles but slow freezing and storage at a temperature in the
range 10 to 20 1C cause destabilization due an increase in
The stability of casein micelles is critical for the technology of
[Ca2+] and a decrease in pH, due to the precipitation of
most dairy products. However, these aspects are incidental and
Ca3(PO4)2; these effects are exacerbated by the crystallization
not the reason why casein micelles evolved.
of lactose.
 Homogenization: Normal homogenization of milk, i.e., up to
20 MPa, has little or no effect on the casein micelles, but high-
pressure homogenization (4200 MPa) or high-pressure treat- 10. Is the ‘‘casein micelle’’ a ‘‘true micelle’’?
ment 4200 MPa cause some dissociation.
 Heat treatment: HTST pasteurization (72 1C  15 s) has little or Today, the term ‘‘micelle’’ is normally used in physical
no effect on the casein micelles but heating at a higher chemistry and biochemistry for aggregates of relatively small
temperature causes denaturation of the whey proteins and amphipathic molecules, such as soaps and bile salts—this is the
their interaction with the casein micelles via sulphydryl– context introduced by McBain (1913). Such micelles can dissociate
disulphide interactions, especially between b-lactoglobulin rapidly and reversibly to monomers on dilution or by changing
and k-casein. This change affects many properties of the other environmental conditions. Casein micelles do not fall into
micelles, e.g., heat stability and rennet coagulation proper- this category, although von Hippel and Waugh (1955) clearly
ties. Severe heating, especially of concentrated milk, causes considered that the casein micelles in milk are in equilibrium with
Maillard browning, a decrease in pH, dissociation of k-casein casein monomers.
from the micelles, and, eventually, coagulation. The term ‘‘micelle’’ was introduced by Nägeli and Schwender-
ener (1877) for aggregates of cellulose or starch which, clearly, are
not amphipathic molecules. Nearly 50 years later, the term was
The micelles are destabilized by a number of factors, some of
used by d’Herelle (1924) for bacteriophage which does not
which are industrially important:
dissociate. The casein monomers are clearly amphipathic mole-
cules (Swaisgood, 2003) and the structure of the casein micelle
 hydrolysis of the k-casein by selected proteinases (rennets), appears to be intermediate between those of the original
which is exploited in the manufacture of most cheese varieties; ‘‘biological micelles’’ and the later ‘‘soap micelles’’. Based on the
 acidification to about pH 4.6, which is exploited in the literal meaning of micelle (small particle, crumb), there seems to
manufacture of some cheeses, fermented milks and functional be no good reason for not using the term ‘‘casein micelle’’ to
caseinate products; describe the calcium phosphate–calcium caseinate particles in
 ethanol (or other alcohol); milk. The term has been used widely for about 50 years and to a
 anionic detergents, e.g., SDS; more limited extent for about 35 years before that. The term
 high pressure. appears to have been introduced by Beau (1921) and to have been
used mainly by French scientists during the period 1921–1932.
The micelles are dispersed by dissolving the colloidal calcium Usage of the term then declined, although it was used occasionally
phosphate (by acidification-dialysis or calcium chelator), at a high as an alternative for calcium caseinate–calcium phosphate
pH, by about 35% ethanol 470 1C, by 44 M urea or by SDS. particles by Eilers (1947), Pyne (1953, 1955) and Berridge
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P.F. Fox, A. Brodkorb / International Dairy Journal 18 (2008) 677–684 683

(1954). The term has been used universally since about 1960 Farrell, H. M., Jr., & Thompson, M. P. (1974). Physical equilibria: Proteins.
owing mainly to the work of Waugh and co-workers. In B. H. Webb, A. H. Johnson, & J. A. Alford (Eds.), Fundamentals of dairy
chemistry, 2nd edn (pp. 442–473). Westport, CT, USA: AVI Publishing Company,
The recommendation of Dickinson (1992) that, in relation to Inc.
the nomenclature of the colloidal calcium phosphate–calcium Fox, P. F. (2003). Milk proteins: General and historical aspects. In Advanced dairy
caseinate particles in milk, the term ‘‘micelle’’ should always be chemistry (Vol. 1), Proteins (3rd ed., pp. 1–48). New York, NY, USA: Kluwer
Academic-Plenum Publishers.
qualified as ‘‘casein micelle’’, in the interest of precision, seems to Fox, S. W., & Foster, J. F. (1957). Protein chemistry. New York, NY, USA: Wiley pp.
be good advice. However, equally, in the interests of precision, 358–359.
aggregates of soap molecules should be referred to as ‘‘soap Fox, P. F., & Kelly, A. L. (2004). The caseins. In R. Yada (Ed.), Proteins in food
processing (pp. 29–71). Cambridge: Woodhead Publishing.
micelles’’. The meaning of the term ‘‘casein micelle’’ is clearly
Fox, P. F., & McSweeney, P. L. H. (2003). Advanced dairy chemistry (Vol. 1), Proteins
understood in Dairy Chemistry and cognate areas and its use (3rd ed.). New York, NY, USA: Kluwer Academic-Plenum Publishers.
is more convenient than alternatives, e.g., ‘‘calcium caseinate– Garnier, J. (1973). Models of casein micelle structure. Netherlands Milk and Dairy
calcium phosphate particles’’. Journal, 27, 240–248.
Garnier, J., & Ribadeau-Dumas, B. (1970). Structure of the casein micelle. Journal of
Dairy Research, 37, 493–504.
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