Food Hydrocolloids 14 (2000) 391–394

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Interaction of pectin and casein micelles

A. Maroziene a,*, C.G. de Kruif b,c
a
Lithuanian Food Institute, Taikos pr. 92, Lt-3031 Kaunas, Lithuania
b
NIZO food research, P.O. Box 20, 6710 BA Ede, The Netherlands
c
Van ‘t Hoff Laboratory for Physical and Colloid Chemistry, Debye Research Institute, University of Utrecht, Padualaan 8, 3584 CH Utrecht, The Netherlands

Abstract
The influence of pectin (low methoxyl—LM, low methoxyl amidated—LMA, high methoxyl—HM) on the stability of milk was
investigated using the dynamic light scattering and viscosimetric methods. At pH 6.7, a depletion flocculation of the casein micelles was
observed. This mechanism involves the exclusion of the polymer pectin chains from the space between colloidal particles (casein micelles),
which induces an effective attractive interaction between the colloidal particles. If the attraction is strong enough a phase separation occurs in
agreement with the theoretical predictions.
At pH 5.3 the pectin molecule adsorbs onto the casein micelles; at low concentrations of pectin, a bridging flocculation is observed. On
increasing the pectin concentration further the casein micelles become fully coated and attraction between the particles is lowered. On
increasing the pH from 5.3 to 6.7, pectin desorbs. These experiments thus show that depending on the interaction between protein and
polysaccharide, different ‘instabilities’ are observed. 䉷 2000 Elsevier Science Ltd. All rights reserved.
Keywords: Depletion interactions between casein micelles and pectin; Flocculation of casein micelles; Adsorption and bridging of pectin onto/between casein
micelles

1. Introduction charged particles and that the stability is better explained

Both proteins and polysaccharides contribute to the
structural and textural properties of food products. It is
therefore of interest to study their interactions. Pectin can
prevent aggregation of casein micelles or be the cause of it.
These phenomena are related to the interaction of pectin
with the proteins; the outcome depends on whether the
pectin adsorbs onto the casein particles or not. The aim of
this study was to investigate the interaction of pectin (high
methoxyl—HM, low methoxyl—LM, and low methoxyl
amidated—LMA) with the casein micelles in skim milk.
Firstly, we studied the system of pectin–casein micelles
at a neutral pH where the pectin does not adsorb onto the
micelles. Non-adsorbing pectin leads to a segregative phase
separation while adsorption may lead to a bridging floccu-
lation. By lowering the pH of the samples, complexes of
pectin and casein micelles are formed. Ambjerg Pedersen
and Jorgensen (1991) proposed that the binding of pectin to
the casein micelle is of electrostatic nature, as the binding is
pH dependent, which is not the case for a hydrophobic
binding. Parker, Boulenguer and Kravtchenko (1994)
found that the zeta potential is too small to account for a
stability caused by electrostatic repulsion between the
* Corresponding author.
0268-005X/00/$ - see front matter 䉷 2000 Elsevier Science Ltd. All rights reserved.
PII: S0268-005 X( 00)00 019-9
by assuming a steric stabilisation, i.e. by adsorbing the
pectin molecules onto the colloidal particles (Napper,
1983). At a low pH, pectin starts to adsorb onto the casein
micelle.
2. Materials and methods
Skim milk was reconstituted from dried skim milk
powder (NILAC, NIZO food research, Ede, The Nether-
lands). The weight percentage of casein micelles in skim
milk is about 2.5%, which leads to an effective volume
fraction of casein micelles of about 0.11.
Solutions of pectin were prepared at 70⬚C. We used three
different pectin samples: (a) 73% methylated pectin (HM);
(b) 35% methylated pectin (LM); (c) 35% methylated-20%
amidated pectin (LMA). Hercules, Copenhagen, Denmark,
kindly supplied these pectin samples. Mixtures of pectin and
skim milk were prepared at 60⬚C. Solutions of 5 N HCl and
5 N NaOH were used to adjust the pH.
Measurements of the apparent particle size were
performed with a dynamic light scattering (DLS) at right
angles and using a red 5 mW He–Ne laser (Malvern 2C
equipment, Malvern, UK). Prior to the measurements the
samples were diluted 100-fold using permeate from an
392 A. Maroziene, C.G. de Kruif / Food Hydrocolloids 14 (2000) 391–394
Fig. 1. Depletion interaction between two colloidal particles induced by a
dissolved polymer. The polymer molecules are excluded from the (overlap)
volume in between the two particles. For LM pectin about half of the
amount is required to induce phase separation.
ultra-filtered skim milk. Viscosity measurements were made
using a capillary Ubbelohde viscometer (Schott gerate).
3. Results and discussion
Pectin does not adsorb onto casein micelles in skim milk
at its natural pH of 6.7. This conclusion follows from the
DLS measurements. On adding pectin (HM, LM, LMA) to
skim milk the apparent particle size did not change up to
concentrations of 0.1% w/w. Note that the weight per-
centage is on the basis of skim milk but that the samples
were diluted 100-fold for the DLS measurement.
However, the undiluted samples showed a phase sepa-
ration. The system consisted of two layers: a white concen-
trated but liquid layer in the lower part of the sample tube
and a transparent white-yellow upper layer. With increasing
concentration of pectin in the mixtures, the upper layer
became more transparent, and the height of the lower
layer decreased. As a result of the segregative interaction
between the casein micelles and pectin, the solution of
macromolecules separates into two phases above a certain
concentration of pectin. At a lower pectin concentration, the
Fig. 2. Phase diagram of casein micelles/pectin mixtures. The shape of the
two-phase line is typical for depletion flocculation. At high pectin concen-
trations the pectins (LMA and LM) form a gelled network.
Fig. 3. The apparent particle size of the casein micelles at pH ˆ 5:3 as a
function of the added pectin. The peaks are taken as the concentration of
half-coverage of the casein micelles. (o HM, D LMA, ⫹ LM).
phase volume occupied by the pectin molecules is low. With
an increasing pectin concentration, the occupied volume
and the osmotic pressure of the pectin increases, which
induces a stronger flocculation of the casein micelles. In
case of the HM pectin the upper part of the mixtures was
liquid. At very high pectin concentrations of 0.6% both LM
and LMA pectin formed a gel in the upper layer; the upper
phase was not homogeneous.
Since the pectin is non-adsorbing at pH ˆ 6:7 we
attribute the phase separation to the so-called depletion
interactions. This phenomenon is schematically presented
in Fig. 1. The pectin (polymer) molecules are depleted
from the surface of the casein micelles. Overlap of such
depletion layers makes available an extra volume to the
pectin molecules. Stated differently, in the overlap volumes
the effective osmotic (polymer) pressure is lowered and this
results in an effective attraction of the casein micelles. If
the depletion attraction is strong enough the system phase
separates.
In Fig. 2 we present the phase diagram for the system
pectin–skim milk at pH ˆ 6:7: The experimental points
were obtained by making a large number of samples and
observing the boundary between the stable and phase sepa-
rated samples. The drawn lines serve to guide the eye. As
can be seen from Fig. 2 the behaviour of the HM and LMA
pectin is very similar. In order to induce phase separation in
skim milk at pH ˆ 6:7 about 0.2% pectin (HM, LMA) is
needed. For LM only 0.1% is needed for phase separation.
This is consistent with the theoretical predictions (Tuiner &
De Kruif, 1999; Vrij, 1976); as the radius of gyration of LM
is larger, the LM pectin is a more expanded molecule.
Theoretical calculations by Tuiner and De Kruif (1999)
on a system with exopolysaccharides show a very similar
phase behaviour. So the non-adsorbing pectin leads to segre-
gative phase behaviour caused by the so-called depletion
interaction.
In practice, pectin is used in low-pH systems. We there-
fore carefully lowered the pH. At pH ˆ 5:3 the milk is still
stable and both DLS and viscosimetric measurements can be
 A. Maroziene, C.G. de Kruif / Food Hydrocolloids 14 (2000) 391–394
Fig. 4. Viscosity of skim milk at pH ˆ 5:3 as function of the added HM
pectin. The peak corresponds to half-coverage of the micelles.
made. In Fig. 3 we present the DLS measurements of par-
ticle size as a function of the pectin concentration. The trace
is very typical and was observed for carrageenans as well
(Langerdorff, Cuvelier, Launay & Parker, 1997). The
apparent particle size shows a typical maximum and then
levels off to a (higher) constant value. This behaviour is
attributed to adsorption and in particular the bridging
flocculation of the pectin. The interconnection or bridging
of pectin between the micelles causes a lower diffusivity of
the casein micelles, which is translated into an effective larger
diameter. The maximum occurs at a polymer concentration
corresponding to 50%, i.e. 50% coverage of the particles.
At full coverage the particles are sterically stabilised
again and this explains the function of pectin in acidified
milk drinks. The amount of pectin needed for full coverage
increases from HM ⬍ LMA ⬍ LM. A similar behaviour
was observed for carrageenans l ⬎ i ⬎ k-carrageenan
(Langerdorff et al., 1997). Clearly the charge density of
the pectins (as with the carrageenans) seems to determine
the strength of the adsorption (Pereyra, Schmidt & Wicker,
1997). However, the amount of adsorbed pectin will depend
on the conformational properties of the pectin. The adsorp-
tion and bridging of the pectin onto/between the micelles
also influences the viscosity of the dispersion.
In Fig. 4 we have given the viscosity of skim milk with
added HM pectin. There is a clear increase in viscosity,
which shows a characteristic maximum. It should be noted
Fig. 5. Viscosity of skim milk–pectin as a function of time after raising the
pH from 5.3 to 6.7. The particle size decreases as a result of the gradual
desorption of the pectin molecules.
393
that the addition of pectin to skim milk UF-permeate hardly
increases the viscosity. The capillary viscosimeters operate
at relatively high shear rates (O) 100–1000 s ⫺1. Therefore it
is expected that considerable shear thinning will be present
if a bridging flocculation occurs. Once the pectin has fully
covered the casein micelles, they are stable again with a low
viscosity. The apparent particle size is increased by 60 nm
for the HM pectin. Parker et al. (1994) found a similar value
from sedimentation experiments in a centrifuge.
When the amount of pectin is further increased beyond
full coverage the system becomes phase separated again due
to the depletion flocculation. Still further increasing the
pectin concentration dramatically lowers the depletion
layer thickness (Fleer, Scheutjens & Vincent, 1984). As a
result the casein–pectin system re-stabilises again, although
the viscosity is extremely high with the high pectin concen-
trations. So with increasing pectin concentration in skim
milk at pH ˆ 5:3 one can observe the following sequence:
stable—bridging flocculation—stable—depletion floccu-
lation—stable/gel.
Once the casein micelles are fully covered the system is
stable; a further increase of the pectin concentration may
lead to depletion flocculation. A similar view is held by
Syrbe, Bauer and Klostermeyer (1998). However, the
strength of the depletion force also depends on the thickness
of the depletion layer, which decreases with increasing
pectin (polymer) concentration. If the depletion interaction
is strong enough the system may gel since the interactions
become short ranged at these high concentrations of pectin.
In other words the depletion forces induce a ‘continuous’
network of casein micelle particles, which, however, will
disperse on dilution. Also at these high pectin concentra-
tions, in particular the LM pectin and LMA pectin may form
gelled polymer networks.
On increasing the pH from 5.3 to 6.7 it is to be expected
that the pectin desorbs again. The pH was increased from
pH ˆ 5:3 to pH ˆ 6:7; the normal pH of a skim milk–HM
pectin mixture (0.09%). We measured the viscosity in an
Ubbelohde viscosimeter. Initially the viscosity remains at a
high level associated with the bridging flocculation and then
it drops (with some noise) to a new and constant level (see
Fig. 5).
In Fig. 6 we plot the apparent particle size as a function of
time after the pH increase. Clearly the pectin desorbs,
although the time scale is rather long with a half decay
time of about 1 h. It is known that adsorption is very fast
while desorption requires a ‘co-operativity of chain
elements’ and thus is inherently slow.
From these experiments we conclude that the adsorption
of pectin at a low pH on casein micelles is a reversible
process.
4. Conclusions
When pectin is added to skim milk at a neutral pH, a
394 A. Maroziene, C.G. de Kruif / Food Hydrocolloids 14 (2000) 391–394
Fig. 6. The apparent particle size of casein micelles as a function of time,
after raising the pH from 5.3 to 6.7. Particle size decreases as a result of the
gradual desorption of pectin molecules.
depletion type of phase separation may occur if the amount
of pectin and the concentration of casein micelles is
sufficient. At pH 6.7, 0.2% of the LMA and HM pectin in
skim milk caused depletion flocculation. In the case of LM
pectin the concentration was lower, 0.1%. This is explained
by the differences in effective volume of the pectin
molecule. Pectin is a non-adsorbing polymer when it is in
solution with skim milk at pH 6.7.
On lowering the pH to 5.3, adsorption of pectin onto the
casein micelles took place. Bridging flocculation was
observed at a pectin concentration that was not enough for
full coverage of the casein micelles. On increasing the
pectin concentration further, the system re-stabilised. The
pectin molecules fully coated the casein micelles. Full
coverage by the HM pectin occurred at a lower concen-
tration than full coverage by the LM and LMA pectin.
The adsorbed amount depends on the conformational
properties of the pectin.
When the pH of the system was increased from 5.3 to 6.7,
the process of desorption of pectin molecules from the
casein micelles and the decrease in the viscosity of the
samples with increasing pectin concentration provided
the evidence of the reversibility of the pectin adsorption.
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