ARTICLE IN PRESS

LWT 40 (2007) 99–106

www.elsevier.com/locate/lwt

Effect of pH at heat treatment on the hydrolysis of k-casein

and the gelation of skim milk by chymosin
Skelte G. Anemaa,b,, Siew Kim Leea,1, Henning Klostermeyera
a
Lehrstuhl für Chemie der Biopolymere, Technische Universität München, 85350 Freising, Germany
b
Riddet Centre, Massey University, Private Bag 11222, Palmerston North, New Zealand
Received 10 March 2005; accepted 17 August 2005

Abstract

Skim milk was adjusted to pH values between 6.5 and 7.1 and heated at 90 1C for times from 0 to 30 min. After heat treatment, the
samples were re-adjusted to the natural pH (pH 6.67) and allowed to re-equilibrate. High levels of denatured whey proteins associated
with the casein micelles during heating at pH 6.5 (about 70–80% of the total after 30 min of heating). This level decreased as the pH at
heating was increased, so that about 30%, 20% and 10% of the denatured whey protein was associated with the casein micelles after
30 min of heating at pH 6.7, 6.9 and 7.1, respectively. Increasing levels of k-casein were transferred to the serum as the pH at heating was
increased. The loss of k-casein and the formation of para-k-casein with time as a consequence of the chymosin treatment of the milk
samples were monitored by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The loss of k-casein and the
formation of para-k-casein were similar for the unheated and heated samples, regardless of the pH at heating or the heat treatment
applied. Monitoring the gelation properties with time for the chymosin-treated milk samples indicated that the heat treatment of the milk
markedly increased the gelation time and decreased the firmness (G0 ) of the gels formed, regardless of whether the denatured whey
proteins were associated with the casein micelles or in the milk serum. There was no effect of pH at heat treatment. These results suggest
that the heat treatment of milk has only a small effect on the primary stage of the chymosin reaction (enzymatic phase). However, heat
treatment has a marked effect on the secondary stage of this reaction (aggregation phase), and the effect is similar regardless of whether
the denatured whey proteins are associated with the casein micelles or in the milk serum as nonsedimentable aggregates.
r 2005 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved.

Keywords: Milk; Heat treatment; pH; Chymosin; Casein micelles; Gelation

1. Introduction This arrangement accounts for the high stability of the

In bovine casein micelles, the k-casein is located
predominantly at the micelle surface as disulphide-linked
polymeric species. The hydrophobic N-terminal region is
associated with the micelle core whereas the hydrophilic,
negatively charged C-terminal region protrudes from the
micelle surface as a highly charged flexible hair (Holt &
Horne, 1996; Horne, 1998; Schmidt, 1982; Walstra, 1990).
Corresponding author. Fonterra Research Centre, Private Bag 11029,
Palmerston North, New Zealand. Tel.: +64 6 350 4649;
fax: +64 6 356 1476.
E-mail address: skelte.anema@fonterra.com (S.G. Anema).
1
Fonterra Research Centre, Private Bag 11029, Palmerston North, New
Zealand.
casein micelles because the flexible hair provides steric and
electrostatic stabilization against aggregation. Although
the casein micelles are extremely stable, they can be
destabilized by processes such as acidification to the
isoelectric point, the addition of solvents such as ethanol
or by certain enzymes (Holt & Horne, 1996; Horne, 1998;
Walstra, 1990).
Enzymatic destabilization of the casein micelles is the
basis of the cheese-making process. Traditionally, the
enzyme extract used is rennet, obtained from the fourth
stomach of the young calf; it contains a number of
enzymes, of which chymosin (E.C. 3.4.23.4) is the major
enzyme responsible for the coagulation of the milk
(Dalgleish, 1992; Hyslop, 2003; Walstra & Jenness, 1984).

0023-6438/$30.00 r 2005 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.lwt.2005.08.002
 ARTICLE IN PRESS
100 S.G. Anema et al. / LWT 40 (2007) 99–106
The reaction occurring after the chymosin has been added
to the milk can be divided into distinct steps or phases
(Dalgleish, 1992; Hyslop, 2003; Walstra & Jenness, 1984).
The first phase is the enzymatic process in which the
enzyme hydrolyses k-casein, forming two peptides as
reaction products. One peptide, the N-terminal para-k-
casein, remains associated with the casein micelle, whereas
the C-terminal peptide, the glycomacropeptide (GMP), is
liberated into the serum phase. In effect, the action of
chymosin cleaves the flexible hair from the surface of the
casein micelles, reducing the surface charge (Anema &
Klostermeyer, 1996; Pearce, 1976) and removing the steric
‘‘hairy’’ layer (Anema, 1997; Holt & Horne, 1996; Walstra
& Jenness, 1984; Walstra, 1990). This results in the
destabilization of the casein micelles. The second phase
involves micelle aggregation, which occurs when sufficient
k-casein has been hydrolysed and if the temperature and
the calcium ion activity are sufficiently high (Dalgleish,
1992; Hyslop, 2003; Walstra & Jenness, 1984). This
secondary phase results in the formation of a gel. Some
reports suggest a further tertiary stage in the reaction,
which involves processes such as syneresis, nonspecific
proteolysis and structural rearrangements of the gel
network (Dalgleish, 1992).
The heat treatment of milk results in the denaturation of
the whey proteins and interaction between the denatured
whey proteins and the k-casein at the micelle surface. The
interaction involves thiol–disulphide bond interchange
reactions between the free thiols of the denatured whey
proteins (particularly b-lactoglobulin) and the disulphide
bonds of k-casein. As the disulphide bonds of k-casein are
found in the para-k-casein region, the denatured whey
proteins will remain with the para-k-casein (micelles) after
hydrolysis of k-casein by chymosin. Therefore, the heat
treatment of milk prior to cheese-making is of considerable
interest, as it appears to have the potential to substantially
increase yield by incorporating the whey proteins in the
cheese curd. As a consequence, there has been considerable
research into the effects of heating on the cheese-making
properties of milk (Dalgleish, 1992; Lucey, 1995; Hyslop,
2003; Walstra & Jenness, 1984).
It is generally recognized that the heat treatment of milk
at temperatures sufficiently high to denature the whey
proteins results in an increased time for coagulation of the
milk by chymosin (Dalgleish, 1992; Lucey, 1995; Hyslop,
2003; Walstra & Jenness, 1984). However, there are
conflicting views on the mechanism for this retardation in
coagulation time. Most reports suggest that the interaction
of denatured whey proteins with the k-casein inhibits the
enzyme action of chymosin on k-casein (Dalgleish, 1992;
Hyslop, 2003; Leaver, Law, Horne, & Banks, 1995; Lucey,
1995; Reddy & Kinsella, 1990; Walstra & Jenness, 1984;
Wheelock & Kirk, 1974; Wilson & Wheelock, 1972).
However, other reports suggest a negligible effect of
heating on the primary phase of the chymosin reaction,
and it has been suggested that the secondary phase of the
coagulation process is inhibited by the presence of
denatured whey proteins (Marshall, 1986; van Hooydonk,
de Koster, & Boerrigter, 1987; Vasbinder, Rollema, & de
Kruif, 2003) or by the reduced calcium ion activity induced
by heating (Schreiber, 2001; van Hooydonk, Hagedoorn, &
Boerrigter, 1986). A recent report by Vasbinder et al.
(2003) compared several methods for the analysis of the
enzymatic reaction products. This study found that the
level of GMP isolated from heat-treated milk was
dependent on the method used to isolate the GMP. It
was concluded that whey protein denaturation had only a
small effect on enzyme activity, and that the release of
GMP (or the formation of para-k-casein) was similar in
heated and unheated milks.
Most studies on the effect of heat treatment on the
chymosin reaction have been on milk at the natural pH.
Recent studies have shown that the adjustment of the pH
of milk prior to heat treatment alters the level of
interaction of denatured whey proteins with the casein
micelles. At low pH (about pH 6.5), about 70–80% of the
whey proteins associate with the casein micelles on the heat
treatment of milk. As the pH of the milk is increased prior
to heating, progressively higher levels of the whey proteins
are in the serum as nonsedimentable aggregates, so that
only about 30% of the denatured whey proteins are
associated with the casein micelles at pH 6.7 (Anema & Li,
2003a, b). pH-dependent dissociation of the casein micelles
occurs as the pH of milk is increased. About 10–15% of the
k-casein is nonsedimentable when milk at pH 6.5 is heated,
and this level progressively increases as the pH is increased,
so that about 30%, 45% and 60% is nonsedimentable at
pH 6.7, 6.9 and 7.1, respectively (Anema & Klostermeyer,
1997). As the pH is increased above pH 6.7, most of the
denatured whey protein remains in the serum as non-
sedimentable aggregates (Anema & Klostermeyer, 1997;
Anema, Lee, Lowe, & Klostermeyer, 2004; Singh &
Creamer, 1991; Singh & Fox, 1985). By manipulating the
milk pH prior to heating, it is possible to produce casein
micelles with altered composition, in particular the amount
of whey protein associated with k-casein on the micelle
surface and the distribution of k-casein between the
colloidal and serum phases. These changes may alter
the reactivity of chymosin with the casein micelles and the
gelation characteristics of the milk. This study therefore
examined whether the pH-dependent, heat-induced
changes to casein micelles affect the primary phase of the
renneting reaction in heated milk by monitoring the action
of chymosin on k-casein, and the secondary phase of this
reaction by monitoring the rheological properties of the
chymosin-treated milks.
2. Materials and methods
2.1. Milk supply
Experimental skim milk samples were prepared by
reconstituting low heat skim milk powder (Fonterra Co-
operative Group, New Zealand) to 10 g/100 g in water that
 ARTICLE IN PRESS
S.G. Anema et al. / LWT 40 (2007) 99–106
had been purified by reverse osmosis followed by filtration
through a Milli-Q apparatus (Millipore Corp., Bedford,
MA, USA). The reconstituted skim milk samples were
allowed to equilibrate at ambient temperature (about
20 1C) with gentle stirring for 24 h before further treatment.
Sodium azide (0.01 g/100 g) was added to the milk as a
preservative.
2.2. Adjustment of pH, heat treatment and re-adjustment of
pH
Sub-samples of skim milk were adjusted to pH values in
the range 6.5–7.1 by the slow addition of 1 mol/l HCl or
1 mol/l NaOH to well-stirred solutions. The milk samples
were allowed to equilibrate for approximately 3 h at
ambient temperature. Samples of milk at each pH were
transferred to glass vials and heated, with continuous
rocking, for the required time in a thermostatically
controlled water-bath preset to 90 1C. After heat treatment,
the milk samples were cooled to room temperature by
immersion of the glass vials in cold running water. The
heated milk samples were held at ambient temperature for
24 h and then re-adjusted to the natural pH (pH 6.67) with
1 mol/l HCl or 1 mol/l NaOH. The samples were held
for 6 h, with periodic pH checks and re-adjustments,
before use.
2.3. Monitoring of the action of chymosin on milk samples
Milk samples (2 ml) in stoppered tubes were placed in a
water-bath preset to 30 1C and allowed to equilibrate to
temperature for 60 min. Chymosin (99% purity, P99,
Hansen, Lübeck, Germany) was initially diluted (1:300)
with water. For each milk sample, diluted chymosin (50 ml)
was added to each tube, and the tubes were gently shaken
by upturning. The reaction was allowed to proceed for
40 min. Sub-samples of the chymosin-treated milk were
taken at 5-min intervals and immediately diluted into
sample buffer used for sodium dodecyl sulphate poly-
acrylamide gel electrophoresis (SDS-PAGE), which
stopped the reaction. Samples were analysed for k-casein
and para-k-casein by SDS-PAGE. Addition of chymosin to
skim milk in sample buffer and analysis by SDS-PAGE
confirmed that the action of chymosin was prevented when
added to this buffer system.
2.4. Rheology
The rheological properties of the chymosin-treated milks
were monitored with time using low amplitude dynamic
oscillation. A Carrimed CSL100 rheometer (TA Instru-
ments UK, Cirencester, Gloucestershire, England) and a
cone (4 cm, 41) and plate arrangement were used for all
experiments, as has been described previously (Anema et
al., 2004). All measurements were performed at 30 1C, with
a strain of 0.01 and at a frequency of 0.1 Hz. Chymosin was
initially diluted (1:3) with water. The diluted chymosin
101
(40 ml) was added to a sample of the milk (1300 ml), which
was gently shaken by upturning, and the milk was
immediately placed on the rheometer and the experiment
was started. Each experiment was performed for a total of
60 min and experimental points were collected at intervals
of 2.5 min.
2.5. Centrifugation
Milk samples (1 ml) were placed in small plastic tubes of
1.5 ml total volume. These samples were centrifuged at
14,000 rev/min (25,000g average) for 1 h at 20 1C in an
Eppendorf Centrifuge Type 5417C, as has been described
previously (Anema & Li, 2003a, b). The protein content
and composition of the supernatants were determined by
native-PAGE and SDS-PAGE.
2.6. Gel electrophoresis and densitometry
Native-PAGE and SDS-PAGE were performed as
described previously (Anema & Klostermeyer, 1997).
Native-PAGE and SDS-PAGE gels were scanned using a
Molecular Dynamics Model P.D. computing densitometer
(Molecular Dynamics Inc., Sunnyvale, CA, USA). The
integrated intensities of the protein bands of interest were
determined using the Imagequant software associated with
the densitometer. The quantity of each protein in the
centrifugal supernatants was determined as a percentage of
that in the original milk sample.
3. Results and discussion
Chymosin hydrolyses a specific bond of k-casein,
converting the protein into two peptides, para-k-casein
and GMP. The reaction can be followed by monitoring the
loss of k-casein or the formation of (either or both) the
peptide products. By using SDS-PAGE analysis, it is
possible to simultaneously monitor the loss of the k-casein
substrate and the formation of the para-k-casein product.
Fig. 1A compares the separation patterns for untreated
skim milk, partially hydrolysed skim milk and skim milk at
the point at which chymosin-induced coagulation was first
observed. The bands corresponding to k-casein (peak 3)
and para-k-casein (peak 5) show the separation between
these species and the other milk protein components. The
loss of k-casein and the formation of para-k-casein were
clear and easily monitored. The relationship between the
levels of k-casein and para-k-casein was, as expected,
linear and inversely correlated (Fig. 1B). These results
indicate that the SDS-PAGE method should be suitable
for monitoring the action of chymosin on k-casein in
skim milk.
Milk samples were adjusted to pH values from 6.5 to 7.1
and were heated at 90 1C for times up to 30 min, and then
the pH was re-adjusted back to the natural pH (pH 6.67).
Sub-samples were centrifuged and the supernatants
were analysed by native-PAGE, to determine the level of
 ARTICLE IN PRESS
102 S.G. Anema et al. / LWT 40 (2007) 99–106

100
2500
2

1 80

Whey protein (% of total)

2000

60
Intensity (arbitrary units)

4
1500
5
40
3 6
40 min
1000 20
25 min

0 (A)
500 10 min
100

0 min
0 80
0 10 20 30
(A) Migration distance (mm)
κ-casein (% of total)

60
100

40
para-κ-casein (% of maximum level)

80

20
60

40
20
0 nonsedimentable k-casein (filled symbols) in skim milk heated at 90 1C for
0 20 40
(B) κ-casein (% of initial level)
Fig. 1. (A) Electrophoretic traces of skim milk treated with chymosin for
times from 0 to 40 min. Peak 1: aS-casein; peak 2: b-casein; peak 3: k-
casein; peak 4: b-lactoglobulin; peak 5: para-k-casein; peak 6: g-casein. (B)
Relationship between k-casein and para-k-casein levels in chymosin-
treated skim milk. Milk samples adjusted to pH 6.5 (K), pH 6.7 (J), pH
6.9 (.) or pH 7.1(,), and then re-adjusted back to the natural pH (pH
6.67) before chymosin treatment.
denatured whey protein, and by SDS-PAGE, to determine
the level of nonsedimentable whey protein and k-casein.
Unless otherwise stated, the whey protein reported
corresponds to a-lactalbumin and b-lactoglobulin com-
bined. In all samples, about 80% of the whey protein was
0 (B)
0 5 10 15 20 25 30
Heating time (min)
Fig. 2. (A) Level of native (filled symbols) and nonsedimentable (open
symbols) whey protein (a-lactalbumin and b-lactoglobulin combined) in
skim milk heated at 90 1C for times from 0 to 30 min. (B) Level of
times from 0 to 30 min. The skim milk was adjusted to pH 6.5 (K, J), pH
60 80 100 6.7 (., ,), pH 6.9 (’, &) or pH 7.1(~, }), either unheated or heated,
and then re-adjusted back to the natural pH (pH 6.67) before
centrifugation and analysis.
denatured after 5 min of heating, and this increased to close
to 100% after 30 min of heating (Fig. 2A). There was only
a small effect of pH, as samples at any given heating time
had similar levels of whey protein denaturation regardless
of the initial pH of the samples. The levels of denaturation,
and the effect of pH, are in agreement with previous
reports (Anema et al., 2004; Anema & McKenna, 1996;
Dannenberg & Kessler, 1988).
In contrast to denaturation, there was a marked effect of
pH on the level of whey protein associating with the
casein micelles (Fig. 2A). At pH 6.5, about 70–80% of the
 ARTICLE IN PRESS
S.G. Anema et al. / LWT 40 (2007) 99–106
denatured whey protein was found to be associated with
the casein micelles after 30 min of heating. Lower levels of
denatured whey protein were associated with the micelles
as the pH was increased, so that about 30%, 20% and 10%
was associated with the micelles at pH 6.7, 6.9 and 7.1,
respectively. Low levels of k-casein (approximately 20%)
were nonsedimentable in the unheated milk. On heating at
pH 6.5, the level of nonsedimentable k-casein decreased
slightly compared with the unheated samples. Heating at
pH 6.7 increased the level of nonsedimentable k-casein
from about 20% in the unheated sample to 30% in the
heated samples, whereas about 40% and 60% of the
k-casein was nonsedimentable at pH 6.9 and 7.1 after heat
treatment. There was little effect of heating time on the
level of serum k-casein at all pH values (Fig. 2B). This
effect of pH on whey protein association with the casein
micelles and dissociation of k-casein is in agreement with
previous reports (Anema & Klostermeyer, 1996, 1998;
Anema & Li, 2003a, b).
Samples of the unheated and heated milks were treated
with chymosin at a level that induced gelation after a
period of about 40 min at 30 1C in the unheated milk. Sub-
samples were taken at set time periods and the loss of
k-casein and the formation of para-k-casein were mon-
itored using SDS-PAGE. Note that all milk samples were
re-adjusted to pH 6.67 before chymosin treatment. The
level of k-casein decreased and the level of para-k-casein
increased with time after chymosin addition (Fig. 3). The
rate of loss of k-casein and the rate of formation of para-k-
casein in all heated samples were similar to those in the
unheated samples, regardless of the pH of the samples at
heat treatment or the duration of the heat treatment
applied (Fig. 3). The small variation amongst the samples
was probably a consequence of small variations in pH
between the samples at the time of chymosin addition, or
experimental error in adding chymosin or in the determi-
nation of the concentration of k-casein and para-k-casein
in the milk samples by the SDS-PAGE method.
The average levels of k-casein and para-k-casein for all
unheated milk samples in comparison with those for all
heated milk samples are shown in Fig. 3B. These results
clearly demonstrate that there was little difference in the
loss of k-casein or the formation of para-k-casein between
unheated and heated milk samples. Examination of the
results in Figs. 2 and 3 clearly indicates that the heat
treatment of milk, including the denaturation of the whey
proteins and their subsequent interactions with serum or
colloidal components, has only a small effect on the
primary phase of the reaction of chymosin on k-casein in
skim milk.
The gelation of the unheated and heated milk samples
was monitored by dynamic oscillatory rheology. In all milk
samples, the pH was re-adjusted to the natural pH (pH
6.67) before the reaction was commenced, so that the
rheological behaviours were directly comparable. For the
rheology experiments, the chymosin concentration was
increased to a level that induced gelation in the unheated
103
(A)
100
80
60
40
κ-casein or para-κ-casein level
(% of original or maximum)
20
0
100 (B)
80
60
40
20
0
0 10 20 30 40
Time after chymosin addition (min)
Fig. 3. (A) Loss of k-casein (open symbols) and formation of para-k-
casein (filled symbols) on chymosin treatment of skim milk samples. The
skim milk samples were either unheated at pH 6.7 (K, J) or heated at
901C for 15 min at pH 6.5 (., ,), pH 6.7 (’, &), pH 6.9 (~, }) or pH
7.1 (m, n). (B) Average loss of k-casein (open symbols) and formation of
para-k-casein (filled symbols) for unheated samples at all pH values (J,
K) and for heated samples at all pH values and heating times (., ,). The
error bars show the standard deviation. In all cases, the skim milk samples
were adjusted to the desired pH, either heated or unheated, and then re-
adjusted back to the natural pH (pH 6.67) before chymosin treatment.
milk after a period of about 5 min, and the experiment was
allowed to proceed for a total period of 60 min. The
gelation curves for the unheated and heated milk samples
are shown in Fig. 4 and key gelation parameters are
summarized in Table 1. In all cases, the G0 versus time
curves had similar shapes, with an initial period in which
the G0 was very low, followed by a period in which the G0
increased. Unless otherwise stated, the final G0 refers to the
G0 value measured after 1 h of gelation time, whereas the
gelation time refers to the time at which the samples had a
G0 X1 Pa.
For the unheated milk samples (Fig. 4A, Table 1), the
gelation times and the final G0 values were very similar
regardless of the pH adjustment steps prior to chymosin
treatment. This indicates that the pH adjustment from the
natural pH (pH 6.67) to pH 6.5–7.1 and subsequent re-
adjustment back to the natural pH had little effect on the
primary and secondary phases of the chymosin treatment
of the milk. On heat treatment of the milk, the gelation
 ARTICLE IN PRESS
104 S.G. Anema et al. / LWT 40 (2007) 99–106

(A)
5 min maintained slightly higher final G0 values and shorter
100 gelation times than those heated for longer periods. There
was little effect of the pH at heat treatment on the gelation
80 times or the final G0 , although the samples at pH 6.7 had a
slightly higher final G0 than those at the other pH values
60 (Fig. 4, Table 1). These results indicate that the heat
treatment of milk markedly affects the gelation of milk by
40 chymosin. However, the results also indicate that the pH at
heating, and therefore the distribution of denatured whey
proteins and k-casein between the micelle and serum
20
phases, does not influence this final gelation as the
behaviour of all heated samples was similar.
0 In the literature, there are conflicting reports as to
100 (B) whether the primary (enzymatic) phase of the renneting
reaction is affected by heat treatment. Many studies
suggested a delay in the primary phase when milk was
Storage modulus (G', Pa)

80
heated, and it was thought that the interaction between
denatured whey protein and k-casein at the casein micelle
60 surface inhibited access of the chymosin enzyme to the
susceptible bond on k-casein (Dalgleish, 1992; Hyslop,
40 2003; Leaver et al., 1995; Lucey, 1995; Reddy & Kinsella,
1990; Walstra & Jenness, 1984; Wheelock & Kirk, 1974;
20 Wilson & Wheelock, 1972). However, other studies found
little or no effect of heating on the primary phase of the
0
renneting reaction (Marshall, 1986; van Hooydonk et al.,
1987; Vasbinder et al., 2003). These studies suggested that
(C) the heat treatment of milk reduced the ability of the casein
100
micelles to aggregate and that this effect may be due to
80 steric hindrance or the increased charge as a consequence
of the whey proteins associating with the casein micelles.
60
A recent study by Vasbinder et al. (2003) showed that the
experimental methods for determining the extent of the
enzymatic reaction by measuring the level of GMP gave
40
variable results. This variability was largely due to the type
and the concentration of the acid used to precipitate the
20 other milk proteins away from the GMP. This variability
may account for the apparent retardation of the primary
0 phase. When the most reliable technique was used, only
very small effects of heating on the primary phase of the
0 10 20 30 40 50 60
Time (min)
renneting reaction were observed. In addition, the study by
Vasbinder et al. (2003) showed that the heat-precipitated
Fig. 4. Changes in storage modulus (G0 ) with time after chymosin calcium phosphate had little effect on the primary phase of
addition for unheated and heated skim milk samples. (A) unheated skim the renneting reaction as the effects of chymosin on
milk; (B) skim milk heated at 90 1C for 15 min; (C) skim milk heated at
90 1C for 30 min. Skim milk samples were adjusted to pH 6.5 (K), pH 6.7
unheated and heated whey-protein-free milk were virtually
(J), pH 6.9 (.) or pH 7.1 (,), either heated or unheated, and then re- identical.
adjusted back to the natural pH (pH 6.67) before chymosin treatment. In the current study, milk samples were heated at
different pH values. This produced samples in which the
denatured whey proteins were distributed between the
colloidal and serum phases (Fig. 2). At low pH (pH 6.5),
time increased markedly and the final G0 decreased the denatured whey proteins were almost entirely asso-
markedly (Figs. 4B and C, Table 1). The gelation time ciated with the casein micelles, whereas, at higher pH, the
increased from about 6 min for the unheated milk to over denatured whey proteins were largely in the serum phase,
20 min for the milk samples that were heated for 30 min, either as nonsedimentable whey protein aggregates or with
and the final G0 was only about 5–15 Pa in these samples considerable levels of dissociated k-casein. Rennet treat-
compared with about 90 Pa for the unheated milk (Fig. 4, ment of the heated milks at the natural pH (pH 6.67)
Table 1). Similar effects were observed for all heating times showed almost identical rates of formation of para-k-
from 5 to 30 min (Table 1), although the samples heated for casein and loss of k-casein (Fig. 3). This clearly shows that
 ARTICLE IN PRESS
S.G. Anema et al. / LWT 40 (2007) 99–106
Table 1
Gelation times and final storage modulus for unheated and heated skim milk samples treated with chymosin
Heating time pH of milk sample at heating
pH 6.5 pH 6.7
Gelation time Final G0 Gelation time
(min) (Pa) (min)
Unheated 6 90.0 7 88.2
5 min 14 12.6 11
10 min 21 10.9 19
15 min 21 11.7 17
30 min 21 8.3 21
Skim milk samples were adjusted to the desired pH, heated and then re-adjusted back to the natural pH (pH 6.67) before chymosin treatment.
the heat treatment of milk, and in particular the denatura-
tion of the whey proteins, has little effect on the primary
phase of the renneting reaction, regardless of whether the
whey proteins are associated with the casein micelles or in
the serum as nonsedimentable aggregates.
It is generally accepted that the secondary or gelation
phase of the renneting reaction is retarded by heat
treatment (Dalgleish, 1992; Lucey, 1995; Marshall, 1986;
Singh, Shalabi, Fox, Flynn, & Barry, 1988; van Hooydonk
et al., 1987; Vasbinder et al., 2003). One explanation for
this retardation of gelation is that, as b-lactoglobulin forms
disulphide bonds with the para-k-casein region, this
complex will remain attached to the casein micelles after
the GMP has been removed, and this b-lactoglobulin/para-
k-casein complex can sterically hinder the aggregation of
the renneted casein micelles (Lucey, 1995; Singh et al.,
1988; van Hooydonk et al., 1987;Vasbinder et al., 2003).
In the current study, adjustment of the pH of the milk
prior to heat treatment allowed manipulation of the
distribution of denatured whey proteins and k-casein
between the serum and colloidal phases (Fig. 2). In all
cases, the gelation of the milk by chymosin was retarded by
the heat treatment, and little difference in gelation
behaviour was observed regardless of whether the dena-
tured whey proteins were predominantly in the serum
phase or associated with the casein micelles (Fig. 4). This is
in broad agreement with van Hooydonk et al. (1987) and
Singh et al. (1988), as both these studies found that the
rennet coagulation time of milk was increased on heating,
regardless of the pH at heating. These results suggest that
the retardation of gelation is unlikely to be due to a steric
stabilization of the casein micelles through attachment of
denatured whey proteins to the micelle surface.
Centrifugation of selected rennet-treated milks and
analysis of their supernatants for protein components
showed that, after chymosin treatment, the level of
nonsedimentable denatured whey proteins and (para-)k-
casein was markedly reduced when compared with the
untreated milks, regardless of the pH at heating (results not
shown). This suggests that the nonsedimentable denatured
whey protein and (para-)k-casein were associated with the
aggregating casein micelles during rennet treatment. It
105
pH 6.9 pH 7.1
Final G0 Gelation time Final G0 Gelation time Final G0
(Pa) (min) (Pa) (min) (Pa)
6 90.4 6 91.1
26.0 14 16.5 14 18.7
21.6 19 13.0 19 15.3
15.0 22 9.0 24 6.3
15.1 24 6.1 25 3.5
therefore appears that the denatured whey proteins,
whether attached to k-casein at the micelle surface or in
the serum phase as nonsedimentable aggregates (with k-
casein), can inhibit or interfere with the aggregation
process, slowing the rate of aggregation and therefore
retarding the gelation process and forming weaker gels.
This is supported by the study of Vasbinder et al. (2003),
who examined the particle size changes of skim milk during
renneting using diffusing wave spectroscopy (DWS). DWS
can give information on the early stages of the aggregation
reactions leading to gelation. It was shown that the start of
aggregation was similar in heated milk and unheated milk,
indicating that the primary phase of the reaction was
relatively unaffected by the heat treatment. However, it
was observed that the rate of subsequent aggregation was
markedly slower in the heated milk, particularly at heating
temperatures above about 75 1C. These results confirm that
the casein micelles in unheated milk and heated milk were
similarly destabilized by rennet treatment; however, the
heating process retarded the subsequent aggregation of
the casein micelles. Vasbinder et al. (2003) concluded that
the retardation of the gelation of heated milk systems by
chymosin is entirely attributable to the association of
denatured whey proteins with the casein micelles, which
supports one of the hypotheses proposed in the literature
(Singh et al., 1988; Lucey, 1995; van Hooydonk et al.,
1987).
However, in our study, a retardation in gelation was
observed regardless of whether the denatured whey
proteins were associated with the casein micelles or in the
serum phase (Fig. 4). This suggests that the retardation of
the gelation process is not specifically due to steric effects
of the whey proteins that are associated with k-casein at the
casein micelle surface. It appears that the denatured whey
proteins, whether on the casein micelles or in the serum as
complexes with k-casein, interfere with the aggregation
process and therefore increase the gelation time. The
conversion of k-casein to para-k-casein and GMP was
similar in heated milk and unheated milk regardless of the
pH at heating (Figs. 3 and 4), and this hydrophobic para-k-
casein, along with the associated denatured whey proteins,
was incorporated into the aggregates formed by the
 ARTICLE IN PRESS
106 S.G. Anema et al. / LWT 40 (2007) 99–106
chymosin-treated casein micelles. The incorporation of
these para-k-casein/denatured whey protein complexes
may inhibit further aggregation and therefore retard
gelation.
Acknowledgements
The authors would like to thank Mike Boland for helpful
discussions and Claire Woodhall for proofreading the
manuscript. The Foundation for Research, Science and
Technology, contract number DRIX0201, provided finan-
cial support. Author Anema was supported by a research
fellowship from the Alexander von Humboldt Stiftung.
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