Blackwell Publishing, Ltd.

S YM P O S I U M
C ON TR I BU TI O N Heat stability of milk
HAR JINDER SINGH
Riddet Centre, Massey University, Palmerston North, New Zealand

The heat stability of milk has been the subject of a considerable amount of research for about a century.
This research has been aimed mainly at understanding the effects of compositional and processing
factors on heat stability and elucidating the mechanisms of protein coagulation. This paper provides an
overview of the factors that influence the pH dependence of the heat stability of normal and concentrated
milks. The principal heat-induced changes in the milk system that contribute to coagulation are discussed.
Current knowledge of the mechanisms of heat coagulation in normal and concentrated milks is also
reviewed.
Keywords Casein micelle stability, Concentrated milk, Heat coagulation, Heat stability, Milk proteins,
Milk.

theory, be restored to these milks by appropriately

*Author for correspondence. E-mail: H.Singh@massey.ac.nz

BACKGROUND
The problems of regulating heat stability (that is
the relative resistance of milk to coagulation upon
sterilization) appeared over a century ago in the
manufacture of evaporated (condensed) milk. The
idea of commercially preserving milk by steriliz-
ing dates back to 1856 when Gail Borden was
granted patents in the United States and England
for ‘producing concentrated milk by evaporation
in vacuum without addition of sugars and other
preservatives’.
The commercial production of condensed milk
increased gradually during the First and Second
World Wars, and condensed milk became one of
the major dairy products in the 1920s, because of
easy transport and a long shelf life. The usual prob-
lems faced in those days were that the milk gelled
or coagulated during the heat treatment and exces-
sive thickening of the product occurred during
storage. These problems were controlled by carry-
ing out various heat-stability tests on the raw milk,
and by running pilot sterilization trials on samples
from each batch after the addition of various
amounts of sodium bicarbonate.
Between 1900 and 1960, most of the scientific
research focused on solving heat coagulation prob-
lems in concentrated (condensed) milk. Studies of
factors affecting heat stability were considered to
be important because they could be used to predict
whether a given milk sample would coagulate after
it had been processed into product. Sommer and
*Author for Hart (1919, 1922) showed that mineral balance
correspondence. E-mail:
was important, and that if a milk sample was too
H.Singh@massey.ac.nz
acid (insufficient calcium and magnesium) or too
© 2004 Society of basic (insufficient phosphate and citrate), it would
Dairy Technology be unstable. Heat stability could, according to the
balancing them by adding ‘acids’ (CaCl2 or HCl)
to milks that were too ‘basic’ and vice versa. The
salt balance theory was criticized by Rogers et al.
(1921), who showed that the heat stability of con-
densed milk could not be predicted from the pH
and salt balance of the milk or indeed from the
heat stability of the raw milk. There was certainly
no relationship between the heat stability of raw
milk and that of the condensed milk made from it.
The effect of preheat treatment (i.e. forewarming)
− heat treatment given to the milk prior to
evaporation − was studied in detail in the 1940s
(Webb and Bell 1942). Preheating is now a stand-
ard commercial practice for the manufacture of
condensed or evaporated milk.
From the 1960s, studies on heat stability shifted
from condensed milk to normal (unconcentrated)
milk. This was around the time when the relevance
of pH to heat stability was fully revealed by the
work of Rose (1961). In the 1970s, research con-
centrated on the effects of processing and compo-
sitional factors on the pH dependence of the heat
coagulation time of unconcentrated milk. Some of
the more important findings include understanding
the roles of β-lactoglobulin and κ-casein, milk
salts and urea in heat coagulation. Advances in
analytical methods (e.g. light scattering) and elec-
tron microscopy in the 1980s allowed heat-induced
interactions in milk proteins to be explored in
greater detail and the development of models for
heat coagulation in normal and concentrated milk.
However, some aspects of the mechanism of heat
coagulation have not been completely explained at
a molecular level.
The extensive literature on the heat stability of
milk has been reviewed regularly over the past

Vol 57, No 2/3 May/August 2004 International Journal of Dairy Technology 111
 Vol 57, No 2/3 May/August 2004
40 years (Rose 1963; Fox and Morrissey 1977; Fox
1981a, 1982; Singh 1988, 1995; Van Boekel et al.
1989a,b; Singh and Creamer 1992; O’Connell and
Fox 2003).
ASSESSMENT OF HEAT
STABILITY
The heat stability of milk refers to the ability of
milk to withstand high processing temperatures
without visible coagulation or gelation. The most
widely used method to assess heat stability, at least
for research purposes, involves sealing a milk
sample in a glass tube, which is clipped onto a plat-
form and placed in a temperature-controlled oil bath,
usually at 140°C for normal milk and at 120°C
for concentrated milk. The platform is rocked at a
given rate until a coagulum can be observed visu-
ally. The heat coagulation time (HCT) is defined
as the time that elapses between placing a sample
of milk in an oil bath and the onset of visible
coagulation. Other methods for determining heat
stability include the ethanol test, a whitening test,
a protein sedimentation test and a viscosity determi-
nation. However, the correlations between differ-
ent test methods are generally unsatisfactory and
the HCTs determined by these methods often
correlate very poorly with the stability of milk on
commercial sterilization. From an industry point
of view, the use of a pilot-scale or laboratory-scale
sterilizer provides more reliable results and predic-
tion of behaviour of milk in commercial plants.
H E AT S TA B I L I T Y – pH P R O F I L E
The HCT of milk is affected by a number of factors,
of which pH is the most important. The HCT of
most milks shows a sharp maximum at pH values
around 6.7 followed by a minimum at pH 6.9; the
stability increases again at higher pH value, as
shown in Fig. 1. These milks are classified as
Type A milks. In Type B milks, the HCT increases
as a function of pH, being lower in the region of the
maximum and higher in the region of the minimum
than for Type A milks. The HCT of concentrated
milk (20% non-fat solids) is much lower than that
Table 1 Methods for eliminating the minimum from the HCT–pH profiles of
Type A milks
Conversion of Type A to Type B
Decrease in the assay temperature (150°C to 120°C)
Addition of κ-casein
Removal of whey proteins
Reduction in the levels of soluble salts
Several additives (e.g. aldehydes, oxidizing agents, polyphenols)
Treatment with transglutaminase
112 © 2004 Society of Dairy Technology
Figure 1 Heat coagulation (HCT) vs. pH profile for normal
skim milk heated at 140°C. Type A milk (
), Type B milk (),
serum protein-free casein micelle dispersions () or
concentrated milk (20% total solids) ().
of unconcentrated milk, with the maximum occur-
ring in the pH range 6.4–6.6; the HCT on either
side of the maximum remains very low. The mini-
mum in the HCT vs. pH profile of normal milk can
be eliminated by altering a number of compositional
and processing parameters (Table 1). Artificial
modification of various milk salts influences the
HCT–pH profile; a small reduction in the total
calcium and magnesium ion concentration (from 13
to 11 mm) eliminates the minimum in the HCT–pH
profile whereas addition of these cations decreases
the stability throughout the pH range (Morrissey
1969). The addition of phosphate to milk increases
the HCT, whereas reducing the soluble phosphate
shifts the HCT–pH profile to more alkaline values.
The addition of citrate shifts the maximum to
more acid pH values, and the HCT does not recover
on the alkaline side of the maximum. Removal of
40% of the colloidal calcium phosphate (CCP) in-
creases the HCT in the pH range 6.4–7.4, whereas
removal of 60–100% of the CCP increases the
HCT in the pH range 6.4–7.0 but has a destabiliz-
ing effect at higher pH values (Fox and Hoynes
1975). Increasing the concentration of lactose, to
approximately 150% of its normal level, destabi-
lizes a Type A milk throughout the pH range 6.4–
7.4 and shifts the minimum to more alkaline pH
values (Sweetsur and White 1974).
Urea is the only indigenous constituent of milk
that has been shown to correlate strongly with
natural variations in heat stability. Addition of urea
at low concentrations does not affect the HCT in
the region of the maximum, but at high concentra-
tions increases the HCT of milk. By contrast, addi-
tion of urea to concentrated milk does not affect its
HCT–pH profile (Muir and Sweetsur 1976).
Of the milk proteins, β-lactoglobulin and κ-casein
have the greatest impact on the HCT–pH profile
(Fox and Hoynes 1975; Singh and Fox 1987a).
Vol 57, No 2/3 May/August 2004
β-Lactoglobulin is required for the development
of a Type A HCT–pH profile. The HCT of serum
protein-free casein micelles (SPFCMs), dispersed
in milk ultrafiltrate, increases continuously with
increasing pH. Addition of β-lactoglobulin to an
SPFCM dispersion introduces a maximum and a
minimum into the HCT–pH profile (Fig. 1). Unlike
normal milk, the addition of β-lactoglobulin to
concentrated milk has a destabilizing effect over
the entire pH range. Enrichment of the milk with
κ-casein increases the stability in the region of the
minimum and converts a Type A milk to a Type B
milk (Rose 1961).
In addition to the above factors, the HCT–pH profile
of milk can be modified by numerous additives.
Addition of thiol-blocking agents, such as N-
ethylmaleimide (NEM) or iodoacetamide, markedly
reduces the HCT in the region of the maximum
(Singh and Fox 1987b). Reducing agents such as
β-mercaptoethanol destabilize milk over the
entire pH range whereas oxidizing agents such as
KBrO4 and iodobenzoate eliminate the minimum
in the HCT–pH profile (Singh and Fox 1985b).
Formaldehyde increases the stability throughout
the pH range 6.4–7.4, particularly in the region of
the minimum, which is eliminated (Singh and
Fox 1985a). Addition of anionic detergents, such as
sodium dodecyl sulphate (SDS), to milk increases
the stability in the region of the maximum and
shifts the HCT–pH curve to alkaline pH values
whereas cationic detergents, such as cetylmethyl-
ammonium bromide, shift the HCT–pH profile to
more alkaline values while causing a moderate
increase in the maximum HCT (Fox and Hearn
1978). Polyphenol-rich extracts of tea, coffee, wine,
oak leaves and bark increase the HCT, particularly
in the region of the minimum (O’Connell et al.
1988; O’Connell and Fox 1996). Caffeic acid is
the most effective of the polyphenols examined.
HEAT-INDUCED CHANGES
IN MILK RELATED TO
COAGULATION
The coagulation of milk on extended heating at
high temperatures (120–140°C) is a consequence
Table 2 Changes in milk during heating and their possible impact on heat stability
Changes that promote instability
Decrease in pH
Deposition of calcium phosphate onto micelles
Association of whey proteins with casein micelles
Dephosphorylation of casein
Dissociation and hydrolysis of caseins, in particular κ-casein
Reduction in zeta potential and hydration
Covalent bond formation
© 2004 Society of Dairy Technology
of loss of casein micelle stability, as a result of
numerous physical and chemical changes in its
components. When we consider the stability/insta-
bility of casein micelles, the surface properties
rather than the interiors of the micelles are likely
to be more important. The surface of the micelle
has a number of dissociated carboxyl and some
ester phosphate groups, providing a high negative
charge (the zeta potential at 20°C is −13 mV) and
thus electrostatic stabilization. Then there is a
diffuse surface layer of flexible, hydrophilic poly-
peptide chains consisting mostly of C-terminal
segments of κ-casein, providing steric stabilization
(Holt 1992). This hairy layer of κ-casein provides
a barrier against aggregation unless the hairs are
removed by chymosin treatment or the solvent quality
is reduced (for example by addition of ethanol).
Inside the micelle, the individual casein molecules
are associated by hydrophobic and electrostatic
bonds in which CCP also plays an important role.
Several factors influence the colloidal stability
of milk. Important factors are calcium ions and pH,
both of which diminish electrostatic repulsions
and possibly alter the conformation of κ-casein at
the micelle surface (indirectly reducing steric repul-
sions). Heat treatment markedly changes the serum
phase environment around the casein micelles (e.g.
change in pH and soluble minerals, in particular
calcium ions, breakdown of lactose and urea) as
well as the casein micelles themselves (association of
whey proteins, changes in CCP, dephosphorylation,
casein dissociation). Some of these changes are listed
in Table 2. It is not known exactly which particular
changes are directly responsible for coagulation,
predispose the milk to coagulation or are a conse-
quence of the coagulation process. The initial stages
of the heat coagulation process must involve a change
in colloidal interactions that allows micelles to
approach each other and stay together long enough
for chemical reactions to take place.
CHANGES IN CASEIN MICELLES
Heating milk at the heat stability assay tempera-
tures causes denaturation of whey proteins and
their interactions with casein micelles. κ-Casein on
Changes that enhance stability
Reduction in calcium ion activity
Association of whey proteins with casein micelles
Reduced sensitivity of casein to calcium ions
Thermal degradation products of lactose
113
 Vol 57, No 2/3 May/August 2004
Figure 2 Influence of pH on the formation of
nonsedimentable (100 000 g for 60 min) nitrogen (N) and
nonsedimentable 12% TCA-insoluble N-acetylneuraminic
acid (NANA) (indicative of κ-casein) on heating skim milk at
140°C for 2 min. Unheated milk: N (
) or NANA ().
Heated milk: N () or NANA (). (Data from Singh and
Fox 1985b, 1986).
the surface of casein micelles is involved in the
formation of a specific disulphide-linked complex
with β-lactoglobulin (Singh and Fox 1987a;
Jang and Swaisgood 1990). As the β-lactoglobulin
aggregates or monomers are considered to form
disulphide bonds with κ-casein, the cystine resi-
dues that are located in the para-κ-casein part of
the protein must be relatively accessible to incom-
ing proteins. A question still remains as to how
whey protein aggregates penetrate the hairy layer
to find the disulphide bonds of κ-casein. Perhaps
there are changes in the κ-casein hairy layer during
heating, or heating causes rearrangement of the
micelle surface to allow this interaction to take
place.
The pH of heating has a large effect on the
extent of the association of whey proteins with the
casein micelles (Smits and van Brouwershaven
1980; Singh and Fox 1985a,b). At pH values < 6.8,
a majority of whey protein complexes remain
associated with the casein micelle surface whereas,
at higher pH values, these complexes remain in the
serum. On heating at pH values > 6.8, not only do
the whey protein aggregates remain in the serum
but also micellar κ-casein dissociates in the serum.
It is unclear whether the interaction between κ-
casein and whey proteins occurs in the micelles
and this complex then dissociates into the serum
phase or whether the complex is in fact formed in
the serum. Nevertheless, the presence of whey
protein markedly enhances the dissociation of
micellar κ-casein. Other caseins, αs1-, αs2- and
114 © 2004 Society of Dairy Technology
Figure 3 Effect of heating time on the zeta potential of
particles in skim milk that had been adjusted to pH 6.5 () or
7.1 () and then heated at 140°C. (Data from Anema and
Klostermeyer 1997).
β-caseins, also dissociate from the micelles upon
heating, but to a much lesser extent.
Data obtained by Singh and Fox (1985b, 1986)
on milk and SPFCMs, heated at 140°C for 1 min at
different pH values, are shown in Fig. 2. At pH values
lower than 6.7, the amounts of soluble κ-casein
(nonsedimentable at 100 000 g for 60 min), repre-
sented as 12% TCA-insoluble N-acetylneuraminic
acid (NANA) and total soluble protein from heated
milk, are lower than in a corresponding unheated
milk, whereas at pH values > 6.7, these amounts
are greater than in the unheated milk and increase
with increasing pH. It appears that β-lactoglobulin
prevents the dissociation of micellar κ-casein on
heating at pH values < 6.7 but enhances the release
of micellar κ-casein at higher pH values (> 6.9).
The effect of heat treatment on the zeta potential
is interesting because of similarities between the
effects of β-lactoglobulin on the HCT–pH profile
and the zeta potential–pH profile of SPFCM dis-
persions. Schmidt and Poll (1986) showed that
heating SPFCM dispersions at pH 6.7 for 10 min
at 120°C had only a slight effect on their zeta
potentials at room temperature; additions of β-
lactoglobulin before heating led to an increase in
the zeta potential in the pH range 6.6–6.9 but
reduced it at higher pH values (7.1–7.2). Anema
and Klostermeyer (1996, 1997) showed that
heating milk at 140°C at pH 6.5 caused an initial
increase in the zeta potential, which then decreased
on further heating. However, heating at pH 7.1
markedly reduced the zeta potential initially, which
remained relatively constant thereafter (Fig. 3).
They proposed that initial changes in the zeta
potential are due to association of whey proteins
with casein micelles and precipitation of calcium
phosphate on to the micelles whereas subsequent
changes in the zeta potential are caused by κ-casein
dissociation and dephosphorylation of caseins.
Vol 57, No 2/3 May/August 2004

Clearly, depending on the pH at heating, at least

two different kinds of casein particles with different
structures and stability are produced. This associa-
tion of whey proteins with the micelles at pH values
below 6.8 would certainly modify the surface of the
casein particles, although it is uncertain how this
new, complex surface is stabilized. It is possible
that whey protein aggregates attached to the casein
micelle surface protrude into the serum and thus
act as super-steric stabilizers. It has been shown
recently that the association of denatured whey
protein with the casein micelles increases their size
(Anema and Li 2003) and that the zeta potential of
the whey protein-coated micelles is greater than
that of the native micelles, which would indeed
contribute to the stability of these particles. Conse-
quently, the whey protein-coated micelles are more
stable to heat, calcium ions, ethanol or rennet than
the native casein micelles (Singh and Fox 1986).
The κ-casein-depleted micelles formed by heat-
ing milk at pH > 6.8 have a reduced zeta potential
and show increased sensitivity to calcium ions,
ethanol and heat compared with the native micelles.
κ-Casein in the micelles has been shown to be
present as disulphide-linked polymers of approxi-
mate molecular weight 300– 600 kDa. The deter-
mination of the molecular state of the κ-casein
that dissociates from the micelles is complicated
by the fact that, on heating, whey proteins interact
with it through thiol–disulphide interchange
reactions. It is unknown whether or not κ-casein
polymers are broken into oligomers and monomers Figure 4 (a) Influence of pH on the dissociation of micellar
during the high heat treatment of milk. Reduction κ-casein on heating milks of 10% (), 15% (), 20% () or
of disulphide bonds by treatment with mercaptoeth- 25% (×) total solids at 120°C for 4 min. (b) Influence of
anol tends to promote heat-induced dissociation heating time at 120°C on the dissociation of micellar κ-casein
of micellar κ-casein, indicating that the thermal on heating milks of 10% (), 15% (), 20% () or 25% (×)
breakdown of disulphide bonds (if it occurs) is likely total solids at pH 6.55. (From Singh and Creamer 1991a,
reproduced with permission from Cambridge University
to enhance κ-casein dissociation from the micelles.
Press).
Singh and Latham (1993) analysed the dissociated
protein material by size exclusion chromatography tion on κ-casein may also occur as a result of
and showed that the soluble protein material formed other heat-induced changes, such as Maillard-type
on heating milk at high pH is composed of small- reactions. Alternatively, dissociation may involve
sized whey protein/κ-casein aggregates and some conversion of CCP to an alternative form that is
monomeric proteins. No monomeric κ-casein less capable of binding casein molecules and
could be detected. It is interesting to note that the maintaining the micelle structure (Aoki et al. 1990;
dissociated protein material can generally be sepa- Anema and Klostermeyer 1997). As the binding
rated on SDS gel electrophoresis and gel filtration, of κ-casein to the micelles does not involve CCP,
but that in electrophoresis containing urea buffer it is likely that such a change in the CCP would
systems, these proteins do not separate as discrete influence the dissociation of other caseins but have
bands, indicating modification of charged groups relatively little effect on κ-casein.
and/or more structural changes to the proteins. Concentration of milk prior to heating has a
The reason why κ-casein dissociates from the marked effect on the dissociation of κ-casein; the
micelles at slightly alkaline pH is not clear. It is extent of dissociation of κ-casein at any particular
likely that, when the surface charge reaches certain pH increases, with the dissociation–pH curve shift-
critical values, hydrophobic bonds are insufficient ing towards lower pH values (Nieuwenhuijse et al.
to hold κ-casein on the micelle surface. The disso- 1991; Singh and Creamer 1991a) (Fig. 4). Thus, in
ciation probably occurs as a result of electrostatic concentrated milk, considerable dissociation of
repulsions between κ-casein and other micelle κ-casein occurs on heating at normal pH, i.e. ∼6.5–
components. Modification of the charge distribu- 6.6. In a comprehensive study on casein micelle

© 2004 Society of Dairy Technology 115

 Vol 57, No 2/3 May/August 2004
dissociation, Singh and Creamer (1991b) showed
that the casein composition of the dissociated pro-
tein was dependent on the heating time at 120°C;
only κ-casein dissociated during the initial stages
of heating (up to 6 min) but other caseins also
dissociated with further heating; after heating for
10 min, the dissociated protein was composed of
70% κ-casein, 20% β-casein and 10% αS-caseins.
The dissociated caseins existed as aggregates of
various sizes, including some monomers. Most of
the dissociated κ-casein was covalently linked to
whey proteins. The dissociated κ-casein appeared
to have a charge distribution different from that of
native κ-casein, but α- and β-caseins were essen-
tially the same as in their native state, as indicated by
their separation on urea-containing polyacrylamide
gels. On SDS-containing gels, the dissociated caseins
showed clear, distinct bands, similar to those of native
proteins, indicating that the molecular weights of
the proteins were not affected by the heat treat-
ments, at least during the initial stages of heating.
Another important change in casein micelles is
that the dephosphorylation of caseins would be
expected to influence the casein micelle structure,
as the casein phosphate groups are involved in
interactions of calcium ions and with CCP and
provide negative charges. Loss of phosphate groups
may decrease the binding of caseins with CCP,
resulting in dissociation of caseins. On the other
hand, dephosphorylation of phosphoserine residues
may generate reactive intermediate dehydroalanine,
which may promote casein cross-linking. Although
the real significance of thermal dephosphorylation
is yet to be elucidated, the rate of dephosphoryla-
tion does not appear to correlate directly with the
rate of heat coagulation.
CHANGES IN THE SERUM PHASE
It has long been recognized that a crucial change in
the serum is the decrease in pH, which plays a key
role in creating an environment that favours coag-
ulation of the milk proteins. This is supported by
the observation that, if the pH of milk is readjusted
occasionally to its original value, coagulation of
the milk may be prolonged for at least 3 h (Fox
1981b). The pH of normal milk decreases gradu-
ally with increasing heating time at 140°C; the pH
at coagulation is usually between 5.5 and 6.0 (after
cooling the milk to 20°C). The decrease in pH is
caused by three reactions:
1 thermal oxidation of lactose to organic acids,
which accounts for 50% of the pH decrease;
2 hydrolysis of organic phosphate (from phospho-
serine in casein), which contributes up to 30% of
the decrease in pH;
3 precipitation of tertiary calcium phosphate with
a concomitant release of H+.
116 © 2004 Society of Dairy Technology
Although the pH of milk at the point of coagulation
(estimated to be about 4.9) approaches that of acid
coagulation, heat-induced coagulation is not due
merely to an indirect acid-induced coagulation
mechanism. This is illustrated by the fact that
there is no relationship between the initial pH
and the rate of pH decrease during heating; the
apparent activation energies for acid production
and heat coagulation are very different, and the
coagulum formed on heating cannot be redispersed
by increasing the pH (Van Boekel et al. 1989a,b;
O’Connell and Fox 2003). Nevertheless, the
decrease in pH is likely to reduce electrostatic
repulsions, thus gradually destabilizing the casein
micelle system.
Heat treatment has been shown to reduce the
concentration of soluble phosphate and of both
soluble and ionic calcium. The transfer of calcium and
soluble phosphate from the serum to the micelles
would be expected to shield some of the negative
charges on the micelles, reducing the zeta poten-
tial and decreasing electrostatic repulsions. The
calcium ion activity, which depends on the initial
pH of the milk, also decreases upon heating; this
decrease is reversible and some or all of the cal-
cium ion activity is recovered after heating. How-
ever, this occurs upon cooling, not during heating,
and takes at least 24 h. It is interesting that the
decrease in pH during heating does not result in
an increase in calcium ion activity, but that the
calcium ion activity remains more or less constant
after heating for a few minutes (Van Boekel et al.
1989a,b). Heating has no effect on monovalent ions,
sodium, potassium and chloride, but its effect on
citrate is unclear. Another factor of importance
in the serum phase is the whey proteins. These
proteins are easily denatured by heat treatments
above 70°C and then react with each other or casein
micelles, as discussed earlier.
MECHANISM FOR COAGULATION
OF MILK PROTEIN
Based on many studies, a unified mechanism to
explain the HCT–pH profiles of both normal and
concentrated milks has been developed and largely
accepted, and fits well with most of the experimen-
tal observations (O’Connell and Fox 2003).
The HCT–pH curve is divided into two regions
(Fig. 5); region I (pH values below 6.8) is con-
cerned with the stability of whey protein-coated
micelles, although the amount of whey proteins
that associate with the micelles decreases with pH
in this region. At a pH well below the maximum,
milk coagulates rapidly because of low pH and
high calcium ion activity (decreased electrostatic
repulsions). In addition, relatively high amounts of
whey proteins associated with the micelles at low
pH may promote aggregation of casein particles
Vol 57, No 2/3 May/August 2004
Figure 5 Diagrammatic representation of heat-induced
interactions of proteins in normal milk in relation to pH-
dependence of heat stability. In region I, whey proteins are
associated with the casein micelle. The stability of these whey
protein-coated micelles increases with pH within this region.
In region II, κ-casein/whey protein complexes are largely in
the serum, and the stability of κ-casein-depleted micelles
increases with pH in this region. Coagulation is mainly caused
by aggregation of κ-casein-depleted micelles by calcium.
through cross-linking of whey proteins bound onto
different micelles. The occurrence of a maximum
(at pH 6.7–6.8) in the HCT–pH profile of normal
milk is essentially due to greater stability of whey
protein-coated micelles, as the formation of a com-
plex between κ-casein and β-lactoglobulin on the
surface of the casein micelles alters the steric and
electrostatic interactions and prevents the dissoci-
ation of micellar κ-casein. The details of the coag-
ulation pathway for whey protein-coated micelles
are by no means fully understood, but it is likely
that further heating causes lowering of the pH,
dephosphorylation, covalent bond formation and
other reactions. Consequently, the altered micelles
can make more frequent contact and probably
form covalent cross-links within and between the
micelles, resulting in an irreversibly aggregated
protein material.
At pH values above 6.9 (region II), the stability
decreases due to the dissociation of micellar κ-
casein, thus reducing the stabilizing effect of
this protein. The κ-casein-depleted micelles are
sensitive to calcium ion concentrations. Therefore,
the minimum in the HCT–pH profile is a result of
coagulation of κ-casein-depleted micelles; coagu-
© 2004 Society of Dairy Technology
lation is essentially salt induced, caused by calcium
ions. At higher pH, although dissociation of micel-
lar κ-casein increases, the HCT increases due to
the increase in protein charge and low calcium
ion activity. It is also possible that the dissociated
κ-casein may reassociate during extended heating
because of a heat-induced decrease in pH.
An alternative hypothesis to explain the pH
dependence of the heat stability of milk has been
proposed recently by O’Connell and Fox (2003).
From pH about 6.3 to the pH of maximum stability,
β-lactoglobulin denatures through a calcium-mediated
mechanism and enhances the thermal stability of the
casein micelles by chelating calcium. At pH values
> 6.9, the disulphide bonds of β-lactoglobulin and
κ-casein are reduced on heating, which facilitates
complex formation. Concurrently, the hydrophobic
β-barrel of β-lactoglobulin is exposed, which results
in an increase in the surface hydrophobicity of the
β-lactoglobulin–casein micelle complexes, thereby
sensitizing the casein micelles to the destabilizing
effect of heat-induced calcium phosphate precipi-
tation. At pH values on the alkaline side of the
minimum, stability increases as a function of pH,
possibly because of an increase in protein stability
with increasing pH (micellar zeta potential and
hydration increase as a function of pH) and a decrease
in calcium ion activity with increasing pH. How-
ever, this hypothesis does not explain the marked
dissociation of κ-casein from the micelles at pH >
6.9, as observed by many researchers. In the region
of the minimum, most of the β-lactoglobulin is in
fact found in the serum and is not associated with
the casein micelles. In addition, there is no evidence
to suggest that the disulphide bonds of native
β-lactoglobulin and κ-casein are reduced at high
temperatures, and it is unclear how this reduction
would facilitate complex formation. In such a
situation, no disulphide–sulphydryl interchange
reactions between κ-casein and β-lactoglobulin
would be expected to occur, which clearly is not
the case.
Concentrated milks are considerably less heat
stable than unconcentrated milks. Because of the
lower assay temperatures (120°C), many of the heat-
induced changes in concentrated milk do not pro-
ceed to the same extent as in unconcentrated milk.
For example, the degree of dephosphorylation and
covalent bond formation, and the decrease in pH,
are far smaller than in unconcentrated milk. The
coagulum formed on either side of the maximum
stability is soluble in dissociating buffers, although
some covalent bonds appear to form in the region
of the maximum (Nieuwenhuijse et al. 1991). In
the pH region 6.5–6.7, the dissociation of κ-casein,
the extent of which increases with an increase in
pH, induces coagulation by altering the surface of
the casein micelles, a situation somewhat com-
parable with that existing for the coagulation of
117
 Vol 57, No 2/3 May/August 2004
Table 3 Apparent similarities between the coagulation behaviours of concentrated
milk at pH ∼ 6.6 (region of maximum stability) and normal milk at pH ∼ 6.9 (region
of minimum stability)
Addition of whey proteins causes destabilization
Urea addition up to about 6 mm has no effect
The coagulum is dispersible in 6 m urea buffer
The coagulation is a two-stage process, as revealed by nitrogen-depletion curves
Extensive dissociation of caseins from the micelles, especially κ-casein, occurs
normal milk within the region of minimum stability.
Some of the similarities between the coagulation
behaviour of concentrated milk in the region of the
maximum and that of unconcentrated milk in the
region of the minimum are shown in Table 3. At
pH values below 6.4, the denatured whey proteins,
those in the serum as well as those attached to
casein micelles, are susceptible to heat-induced
aggregation; high calcium ion activity in this pH
range probably promotes the formation of large
whey protein aggregates and network structures.
At higher pH (∼ 7.0), the coagulum appears to
form a gel-like matrix of protein that is probably
derived from whey protein aggregates and the
dissociated protein material (Singh et al. 1995).
PRACTICAL SIGNIF ICANCE OF
HEAT STABILITY
The ability of milk to withstand high-temperature
treatments without loss of its stability is fairly
unique and makes the production possible of many
sterilized milk products with a long shelf life.
These products include UHT milks and creams,
in-can sterilized milks, evaporated milk, sweetened
condensed milk and milk powders, especially those
intended for reconstitution and recombination into
sterilized products (heat-stable powders). Con-
siderable knowledge gained on the heat stability
of milk has allowed most of the practical problems
to be solved relatively easily by manipulating pro-
cessing and compositional variables.
From an industrial viewpoint, the heat stability
of milk of normal concentration has rarely been
a problem. However, in recent years, many new
liquid milks, fortified with high amounts of calcium,
magnesium and zinc, cocoa and tea extracts, have
been introduced into the market. As many of these
additives have a negative impact on heat stability,
these products require very careful manipulation of
the formulation to achieve the desired heat stability
and it is often difficult to achieve the desired shelf
stability. Certain problems regarding the heat
stability of concentrated milk, especially full-fat
homogenized recombined evaporated milk, remain
unsolved. These problems relate largely to seasonal
variations and batch-to-batch variations in heat
118 © 2004 Society of Dairy Technology
stability. Practical solutions to the heat stability
problems include the following:
• manipulation of preheat treatments
• matching the natural pH of milk to that of the
heat stability maximum
• addition of different levels of phosphate
• addition of buttermilk and phospholipids at
appropriate levels
• combination of the above treatments.
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