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Rheological and structural study of electrostatic

cross-linked xanthan gum hydrogels induced by
Cite this: Soft Matter, 2013, 9, 3063
b-lactoglobulin†
Xuan T. Le and Sylvie L. Turgeon*

Received 2nd November 2012
Accepted 9th January 2013
DOI: 10.1039/c3sm27528k
www.rsc.org/softmatter
This study examines for the first time the role of xanthan gum (XG) and b-lactoglobulin (blg) in network
formation induced by electrostatic attractive interaction. The gelation processes of blg–XG mixtures
were monitored by viscoelastic measurements as a function of the blg–XG ratio and blg and XG
concentrations. The structural characterization of the gels was addressed by means of confocal laser
scanning microscopy. It was found that the initial tenuous network of XG provided a frame for gel
organization, the blg aggregated along the XG chains and could be regarded as a crosslinking agent,
and more elastic gels were obtained at high XG concentrations. The lowest XG concentration at which
gelation is possible is estimated to be 4.8
103 wt%. The blg–XG ratio strongly affects gelation
kinetics and is a main factor controlling the gelation process and the gel structure. The optimal ratio for
electrostatic interaction at final pH of gels was estimated to be approximately 3.5. The decreasing
repulsive interaction between XG chains and the increasing attractive interaction between blg and XG
with decreasing pH resulted in the formation of soluble complexes followed by the formation of
interpolymer complexes. The electrostatic cross-linking of XG chains by blg results in a sol–gel transition
at the point of gelation. This mechanism may be applicable in a wide variety of protein–polysaccharide
systems in which the structure of these systems is mainly stabilized by electrostatic attractive interaction.

Introduction several protein–polysaccharide mixtures has been associated

The gelation of naturally occurring macromolecules is of great
interest with respect to the production of popular biomaterials
due to their high biocompatibility and large variety of applica-
tions in the cosmetics, pharmaceutical, biomedical, food, and
coating industries.1,2 The simultaneous use of proteins and
polysaccharides is commonly encountered in food products to
control the structure, texture, and stability of the products.3–8
Several types of gel structures can be formed depending on the
characteristics of the proteins and polysaccharides used and on
the environmental conditions. Mixed gels containing more than
one gelling agent can be classied into four types: swollen
networks, interpenetrating networks, phase-separated
networks, and coupled gels.9,10 The gelation mechanism of
STELA Dairy Research Center and Institute of Nutraceuticals and Functional Foods,
Faculty of Agriculture and Food Science, Université Laval, Pavillon Paul-Comtois,
2425 rue de l0 agriculture, Québec, Canada G1V-0A6. E-mail: Sylvie.Turgeon@fsaa.
ulaval.ca; Fax: +1-418-656-3353; Tel: +1-418-656-2131 ext. 4970
† Electronic supplementary information (ESI) available: Details of a hydrogel
formation process for the mixture at a ratio of 5, and 0.30 wt% total solid
concentrations by the confocal laser scanning microscope and for the mixture
at a ratio of 2, and 0.10 wt% total solid concentrations by a phase contrast
optical microscope (Olympus, Tokyo, Japan) and the time dependence of
storage modulus and loss modulus during gelation for some blg–XG mixtures
are provided. See DOI: 10.1039/c3sm27528k
with interpenetrating5 and phase-separated networks.4,11 An
interpenetrating network is formed when both biopolymers
associate independently to form separate networks. This type of
network is observed in BSA–LM pectin mixtures in the presence
of calcium ions. Proteins aggregate by heating to form a very
weak gel interpenetrated by an LM pectin network, which is
formed by linkages between LM pectin chains via calcium ions.5
If some degree of demixing occurs prior to gelation, then both
biopolymer networks will be separated spatially, resulting in a
phase-separated network. Generally, the gelation of mixtures of
proteins and polysaccharides in aqueous dispersion occurs
aer phase separation and arrests phase separation.12–14 Phase
separation is either segregative (thermodynamic incompati-
bility) or associative (thermodynamic compatibility), depending
mainly on the electrical charges of the associated biopolymers
and thus on the factors affecting them, such as the ionic
strength and pH.7,15 The microstructure of gels is the result of a
balance between demixing and gelation processes.11,16 A
coupled network is formed under associative conditions when
both biopolymers are linked together to form junction zones.
Some studies have explored coupled networks of two poly-
saccharides. A typical example of this network has been repor-
ted for galactomannan–xanthan binary gels.17,18 Fewer studies
have reported the gelation of protein–polysaccharide mixtures
under associative conditions and complex, coacervate

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Soft Matter
structures are frequently obtained for such mixtures. There is
therefore a need to understand the gelation mechanisms of
protein–polysaccharide mixtures under associative conditions
to produce new materials for the improvement of conventional
foods and for the development of novel so biomaterials.
Recently, it has been found that electrostatic gels can be
obtained from the associative interaction between native
proteins and polysaccharides at very low concentrations.12 The
novelty of this study resides in the fact that no thermal, enzy-
matic or any other denaturing treatment was applied to the
protein or the mixture at any time. Gelation was induced by
in situ acidication at room temperature to a pH at which the
molecules carry net opposite charges. The authors also noted
potential uses of these gels in the food industry to enhance the
stability of foods and to protect micronutrients, and in the
pharmaceutical industry to deliver and to protect drugs or active
molecules. However, microstructural details of this gel remain
unknown until now and the molecular mechanisms by which
proteins interact with XG chains are not fully understood.
Therefore, understanding the sol–gel transition of these
protein–polysaccharide systems is of great interest so that the
desired structure and texture can be established in the design of
new products. Specically, reversible hydrogels are sought for
diverse applications, including cell delivery, tissue engineering,
coating, and in the elds of biomedicine, cosmetics and phar-
maceuticals.19–21 Here, we report on the gelation of mixed
aqueous solutions of the globular protein b-lactoglobulin (blg)
and polysaccharide xanthan gum (XG). Both biopolymers are
extensively used because they are naturally occurring. blg is the
main protein component of the whey fraction and has a molar
mass of 18.6 kg mol1. It has an isoelectric point (Ip) of 5.1.22
Above a critical protein concentration, irreversible aggregation
leads to the formation of a gel when a blg dispersion is heated
above approximately 60  C. The gelation of a blg dispersion has
not been observed at ambient temperatures, even when the pH
has been brought to the isoelectric point of the protein.12 blg is
able to form electrostatic complexes with anionic poly-
electrolytes at pH levels above its Ip through its positively
charged patches.23
XG is an anionic polysaccharide produced commercially by
bacterial fermentation. It is widely used in the food industry as
a stabilizer and thickener of food products due to its specic
physical (viscosity, pseudo-plasticity) and chemical (water
solubility, pH stability) properties. This polysaccharide
consists of a linear (1–4)-b-D-glucose backbone with a charged
trisaccharide side chain on every second glucose residue. XG
exhibits pronounced pseudoplastic behavior due to its strong
self-associative behavior.24 The chain–chain association is
modulated by changes in the conformation of xanthan gum
with salt content and temperature. In distilled water, XG
appears elongated due to the electrostatic repulsion from the
charged groups in the lateral chains; the coiling or bending of
XG molecules appeared to occur when some salt was present.25
Heating a XG solution above the transition temperature, Tm,
results in the melting of the ordered structure. This structure
returns to its original state upon cooling, depending on the
salt environment.26
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A previous study on b-lactoglobulin–xanthan gum (blg–XG)
as a model system proposed that the gelation process is initi-
ated by the formation of soluble complexes followed by inter-
polymeric electrostatic complexes that are able to form junction
zones, which result in a sol–gel transition at the point of gela-
tion.12 The system studied had a total concentration of 0.1 wt%.
Light scattering is largely used to monitor the kinetics of gela-
tion but is limited to low concentrations because of the very
strong multiple scattering events that are observed at high
concentrations and the large uctuations in intensity and
relaxation time due to the onset of the gel network.27 Some
complementary techniques are needed to verify the gelation
mechanism of mixed blg–XG systems. Techniques that are
frequently used for structural characterization, e.g., gas/vapor
adsorption, mercury porosimetry, and conventional scanning
electron microscopy (SEM), can only be applied to a dry mate-
rial. However, drying hydrogels of such low concentration can
result in the shrinkage, deformation and collapse of the entire
pore network.28
Confocal laser scanning microscopy (CLSM) and small-
deformation rheology have oen been used to monitor the
structural development of protein–polysaccharide mixed
systems without altering the gel structures.4,5,11,29 The objective
of this study was to investigate the role of blg and XG in gel
formation and to describe in greater detail the gelation mech-
anism of blg–XG mixtures under associative conditions using
both techniques to monitor the structural development of
coupled gels as they are affected by the biopolymer concentra-
tion and ratios. We show that the role of XG is to provide a
framework for gel organization, whereas the role of blg is to
participate in network development as a crosslinking agent.
Experimental section
Materials
Whey protein isolate (High-Beta, lot # JE 002-8-922, 98.2 wt%
protein, 85 wt% of which is blg, 1.8 wt% minerals, and 4 wt%
moisture) was obtained from Davisco Foods International Inc.
Due to the high content of blg in this powder, it was assumed
that its behavior was governed by that of blg;12,30 therefore, this
powder will be referred to as blg hereaer. Xanthan gum (XG)
(Keltrol RD, lot # 4G3367K, 96.36 wt% total sugar, 3.02 wt%
protein) was provided by CP. Kelco UK Ltd. Glucono-d-lactone
(GDL, lot # 70H0163, USA) and rhodamine B isothiocyanate (lot
# G39595, USA) were purchased from Sigma and J.T. Baker,
respectively.
Sample preparation
XG and blg powders were dissolved in ltered deionized water
(Modulab Analytical, Fisher Scientic) with continuous stirring
at 25  C for at least 1 h for protein and 2 h for polysaccharide
and le overnight at 4  C. Before preparing the mixtures for
analysis, the initial pH of the protein and polysaccharide
dispersions was adjusted to 6.60  0.08 using 0.1 N HCl and
0.1 N NaOH. Four blg–XG ratios (r) were studied: 2 : 1, 5 : 1,
10 : 1 and 20 : 1 (r ¼ 2, 5, 10, and 20, respectively). The mixed
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dispersions were prepared at (1) a xed total solid concentration
of 0.3 wt% and different blg–XG ratios, (2) a xed XG concen-
tration of 0.03 wt% with different concentrations of blg (0.06,
0.15, 0.30 and 0.60 wt%), (3) a xed protein concentration of 0.3
wt% and different XG concentrations, and (4) different total
solid concentrations to study the effect of protein–poly-
saccharide ratio and protein, polysaccharide and total solid
concentration on gel formation. Protein and polysaccharide
dispersions were mixed at the desired r and stirred gently for 30
min. The gelation of the mixed system was induced by the
addition of GDL. The amount of GDL used in each formulation
was related to the total biopolymer concentrations in the
mixture to obtain a nal pH of 4.4  0.08 at room temperature.
The pH of the dispersions was continuously measured using a
DL53 titrator (Mettle Toledo, Switzerland).
Dynamic oscillatory measurements
The small-deformation properties of the mixed gels during
acidication were determined in dynamic oscillation mode with
a controlled-strain rheometer (ARES-100, TA-Instrument, Pis-
cataway, NJ, USA) equipped with a circulating water bath
temperature controller. A Couette device with a cup (33.93 mm
diameter) and bob system (32.05 mm diameter, 33.29 mm
length) was used. Thirty seconds aer GDL addition, the
samples were transferred to the rheometer. The development of
the storage modulus (G0 ) and the loss modulus (G00 ) during
gelation was recorded for 15 h at 25  C at a frequency of 0.1 Hz
and a strain of 0.5%. Following oscillation, a strain sweep test
was recorded at increasing strains from 0.1 to 100% to verify the
linear region. Samples were covered with a thin lm of castor oil
to prevent evaporation during measurement. The interconnec-
tion point, pH4, at which interpolymeric complexes formed,
was determined as the last point before the exponential
increase in G0 , and the gelation point, pHg, was arbitrarily
chosen as the pH at which G0 was equal to 1 Pa (ref. 31) (see
more details in the Gelation process section).
Turbidity measurements
The development of turbidity in the mixed dispersions aer
GDL addition was monitored by measuring absorbance varia-
tions as a function of time at 800 nm using an HP 8453 UV-
visible spectrophotometer (Agilent Technologies, Inc. Santa
Clara, CA, USA) at 25  C. A blank correction was made under the
same conditions in the absence of both biopolymers. The
inection point in the turbidity curves was taken as the inter-
connection point, pH4.30
Confocal laser scanning microscopy
The structural features of the gels were investigated using a
Nikon Eclipse TE2000-E Confocal Laser Scanning Microscope
(Tokyo, Japan) equipped with an inverted microscope. The
objective lens used was 60X WI, NA 1.20. The excitation wave-
length was 543 nm, with an emission maximum at 605/675 nm.
Because proteins do not exhibit intrinsic uorescence at this
wavelength, proteins were stained by adding rhodamine B iso-
thiocyanate (RITC) to protein dispersions under magnetic
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stirring for 1 h before mixing with XG dispersions. The FA-XG
prepared according to the method of Donato et al.5 was
provided by Laneuville and was used in the mixture with RITC-
blg in order to determine the location of blg and XG in the gel.
Preliminary trials revealed that labeling did not change the
rheological behavior of the systems (results not shown).
Mixtures of RITC-blg–XG and RITC-blg–FA-XG were prepared as
described above. Aer GDL addition, the sample was poured
between a 0.5 mm deep well concavity slide and a cover slip,
which was hermetically sealed with nail enamel. Micrographs
were taken during gelation and approximately 24 h aer GDL
addition. The previous study reported that the structure of the
gels was fully developed aer z18 h of acidication.12 Digital
images were acquired at a pixel resolution of 512
512 or
1024
1024.
z-Potential measurement
To estimate the optimal ratio of blg–XG for electrostatic inter-
action, the net charge of complexes formed under shear
conditions was measured. The electrical charges (z-potential) of
individual biopolymers and biopolymer complexes as a func-
tion of pH and at the nal pH of the systems in an aqueous
solution were determined using a Zetasizer 2000 (Malvern
Instruments Ltd, Worcs, UK). Before analysis, samples were
diluted in ltered deionized water to obtain solutions of 0.05
wt% solid total concentration. Measurements were carried out
in duplicate, and the results are reported as means and stan-
dard deviations.
Results and discussions
Gelation process
A typical evolution of G0 , G00 versus pH is shown in Fig. 1a. Three
stages (denoted as I, II, and III in Fig. 1a) can be clearly iden-
tied. In stage I, when the pH is higher than 5.5 and the protein
net charge is negative, the mixture remains in the sol state.
Although both G0 and G00 showed large deviations due to low
torque signals, G0 appeared to be higher than G00 and neither
changed during stage I. However, the absorbance was observed
to increase slightly (Fig. 1b) in stage I.
As pH is further reduced (stage II), G0 , G00 and the absorbance
increase more rapidly and then approach a plateau (stage III).
No crossover between G0 and G00 was observed during the gela-
tion process; in fact, G0 was already higher than G00 at the
beginning of the measurement. It may be possible that the
mixture was already highly structured well before the gelation
point. This behavior may be derived from the pseudo-plastic
properties of XG over a wide range of pH, through which XG
solutions exhibit dominant elastic responses to frequency.24
Previous studies12,32 reported that regardless of the experi-
mental conditions (under shear or quiescent conditions), elec-
trostatic attraction between blg and XG can occur at pHc [ Ip
of blg due to positively charged patches on the protein molecule
surface, which form soluble complexes followed by the forma-
tion of interbiopolymer complexes with a further decrease
in pH.23,33 The pHc for this system under shear was equal to
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Fig. 1 pH dependence of (a) storage modulus and loss modulus, and (b)
absorbance for the blg–XG mixture at r ¼ 5 and a total solid concentration of 0.30
wt%. The white squares (Fig. 1a) indicate the point at which the gel is stable and
the deviation of maximum G0 does not exceed 5%.
5.76  0.03. Similar effects have also been observed in other
32
protein–polyelectrolyte systems.30,34–36 Thus, the slight increase
in turbidity observed in stage I may correspond to the formation
of soluble complexes and the rapid increase in G0 , G00 , and the
turbidity observed early in stage II may reect the formation of
interbiopolymer complexes. In addition, Fig. 1a and b show that
turbidity measurements are more sensitive to structural
changes than rheological measurements in stage I, stage II was
initiated at pH4 ¼ 5.66 (Fig. 1b) instead of pH4 ¼ 5.50 (Fig. 1a).
To determine the gelation point, Winter and Chambon's
criteria37 seem to be the most rigorous among those prescribed
by several methods. However, as sol–gel transition takes place
from the solution state, this method is oen very difficult to
apply due to the low viscoelastic moduli and low torque signals
near the gelation point.38 Therefore, another method, i.e., one
that tracks the time or the pH at which the G0 rose above a
threshold value of 1 Pa, was used. This method is now widely
used in the literature.31,39,40 The gelation pH, pHg, represents a
gelation point determined as a function of pH (point 2 in
Fig. 1a) and tg is the corresponding time of gelation (point 2 in
Fig. S1†).
The corresponding microstructures obtained for a blg–XG
mixture at r ¼ 5 and a total solid concentration of 0.30 wt%
during acidication are shown in Fig. 2.
Fig. 2 clearly shows that macroscopic phase separation
occurs at pH 5.50 and that there is a coarsening of the network
structure. This coarsening of the network with decreasing pH is
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paralleled with the increase in gel strength observed by
rheology, indicating interpolymeric formation by the intercon-
nection of soluble complexes. In the rheological prole (Fig. 1a
and S1 – see ESI†), this point is characterized by an initial
increase in G0 (point 1); the associated pH and time values were
denoted as pH4 and t4, respectively. Beyond this pH value, open
and stringy structures are observed at pH 5.46 (Fig. 2) and G0
exceeds the value of 1 Pa at which gelation occurs. Comparing
inset (a) and inset (d) in Fig. 2, we can nd that a highly inter-
linked, permanent network instead of the sol state is formed at
pH 4.4, which strongly agrees with the high G0 observed in stage
III (Fig. 1a and S1†). The data of frequency sweep tests showed
that storage modulus and loss modulus were slightly dependent
on frequency which indicated an almost uniform solid response
over the frequency window studied.41 This is consistent with
network bond relaxation moving to much longer timescales and
G00 /G0 ratio varies from 0.01 to 0.1 suggesting that the gel
properties are intermediate between entanglement network and
true gel, and so are characteristic of a weak gel.42
The microstructure during the unraveling of the gelation
processes (see ESI†) revealed that the nal microstructure was
obtained at the end of stage II. In addition, no change in G0 was
observed in stage III (Fig. 1a and S1†); thus, stage III is called the
quasi-equilibrium stage. The pH at which G0 did not change is
called the electrical charge equivalence pH (EEP). De Jong et al.
also reported no further change in the microstructure of mixed
whey protein–polysaccharide cold-set gels aer gelation.11
Effects of blg–XG ratio
The effects of the biopolymer ratio on gelation processes are
presented in Fig. 3. The evolution of storage modulus as a
function of time with different ratios is also presented in Fig. S2
(see ESI†)
As the ratio increases, interpolymer complexes are formed
and gelation occurs at higher pH and sooner (see also Table 1
and Fig. S2†). This behavior can be explained by the faster
diffusion of proteins to polysaccharide chains in systems
featuring smaller densities of polysaccharides with lower
viscosities. In addition, more proteins were available for inter-
action, and the systems were neutralized sooner.12,30,32 More-
over, stronger gels were obtained as the XG fraction
incorporated into the gel formulation increased. This result
suggests that the gel network may be based on XG chains. In
addition, Fig. 3 shows that a maximum and constant G0 is
obtained for r ¼ 5 and r ¼ 10 at the electrical charge equivalence
pHs, indicating the existence of an electrostatic equilibrium in
the gel, where different biopolymers carry similar but opposite
charges and their interaction is maximal, and the gel structure
may be stabilized at this pH and does not change upon the
decrease of pH; The electrostatic equilibrium is attained more
quickly (higher EEP is obtained at higher ratios – Table 1) as the
ratio increases due to the availability of a greater number of
proteins for interaction. However, the gradual evolution of G0
continued to take place for r ¼ 2 (Fig. 3), and G0 did not expe-
rience a plateau aer reaching the Ip of the proteins. This is
possibly due to the interaction between protein-unsaturated XG
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Fig. 2 Microstructure formation for RITC-blg–XG mixture at r ¼ 5 and total solid concentration 0.30 wt% observed by confocal laser scanning microscopy during
acidification. The scale bar is 20 mm in all images. The light areas are protein rich.

concentration. The concentration of blg strongly affects the

gelation kinetics (see Fig. S3 in the ESI† for time evolution of
storage modulus).
An increase in protein concentration from 0.06 to 0.6 wt%
results in an increase in pHg from pH 5.0 to pH 5.5 and a
decrease in tg from 210 minutes to 35 minutes (Fig. S3†). In
addition, in the blg concentration range between 0.06 and 0.30
wt%, which corresponds to a range of blg–XG ratios from 2 to
10, the gel obtained at r ¼ 5 presented the highest solid-like
character, indicating that this ratio is close to the optimal ratio
that fosters the blg–XG interaction. The weakness of the gel at a
ratio of 10 may be due to the effect of excess protein,12 which
Fig. 3 Evolution of the storage modulus during gelation for blg–XG mixtures at
promoted the association of proteins with individual XG
a total solid concentration of 0.30 wt% and different ratios. molecules to form individual complexes, with a consequent
reduction in the junction zones of the gel network. However, at
very high protein concentrations (0.60 wt%), the gel obtained
Table 1 Gelation kinetic parameters for blg–XG mixtures at a total solid was stronger than at r ¼ 5, which may be explained by the
concentration of 0.30 wt% and different ratios cooperative binding of proteins at a pH close to the Ip of the
protein. At this pH and as the local concentration of protein
blg–XG
increases, the repulsive forces between protein molecules are
ratios t4 (min) tg (min) pH4 pHg EEPa
negligible, which may favor the hydrophobic/hydrogen inter-
2:1 100 110 5.37  0.03 5.22  0.05 N/Ab actions between protein particles resulting in the formation of a
5:1 98  4 108  4 5.42  0.05 5.37  0.05 4.55  0.03 second protein network in a network of coupled gels. The
10 : 1 60  7 72  17 5.60  0.14 5.55  0.17 4.62
a b
EEP (electrical charge equivalence pH). Not attained under the
current condition.

chains by hydrogen bonds or to the higher viscosity of this

system,12 which slowed down the interactions between inter-
polymeric complexes. To verify the role of viscosity and the role
of protein availability on the kinetics of gelation, some blg–XG
mixtures at a xed polysaccharide concentration and different
blg concentrations were tested by dynamic oscillatory
measurements.

Effects of protein concentrations

Fig. 4 shows the protein concentration dependence of gelation Fig. 4 Evolution of storage modulus as a function of pH for blg–XG mixtures at a
processes for blg–XG mixtures at a xed polysaccharide fixed polysaccharide (0.03 wt%) and different protein concentrations.

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attractive interaction between blg molecules, especially as they
complex with pectin at pH levels ranging from 4 to 6, was
reported by Girard, Turgeon et al. 2003.23 Faster gelation at high
protein concentrations (see Fig. S3†) indicates that as more
protein is available, more of the gel structure is rapidly devel-
oped. This also suggests that the crosslinking function of
proteins in network development and the availability of
proteins are determining factors for gelation kinetics.
The z-potentials of individual components and three mixed
blg–XG solutions under shear conditions as a function of pH
and at pH 4.40 are shown in Fig. 5.
As XG is an acidic polysaccharide having the carboxyl groups
on the side chains, the negative zeta potential decreased with
decrease in pH. At pH 4.8, the net charge on blg is close to zero
because this pH is near to the electrostatic point of the
protein.22 When the pH of solution was decreased from 6.0 to
4.4 the z-potentials of mixed solutions were more positive than
that of the pure XG solution, indicating that the electrostatic
complexes had been formed. Moreover, at higher ratios blg–XG
(>2), the systems were neutralized sooner at the electrical charge
equivalence pHs (EEPs), and aer these pHs the net charges of
the mixed solutions were similar to that of the protein, indi-
cating that the electrostatic complexes had been neutralized
and the net charge of systems came from that of the free
proteins in the mixed solutions. Fig. 5b suggests that at pH 4.4
the stoichiometric equivalence ratio blg–XG at which an initially
Fig. 5 z-Potentials for individual biopolymers, blg–XG complexes at different
ratios and a constant polysaccharide concentration of 0.03 wt% (a) as a function
of pH and (b) at pH 4.4.
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negative net charge on the XG molecules should drop to zero
and the electrostatic interaction is maximal under shear
conditions may occur between 2 and 5. At a ratio of 2, the
insoluble complexes exhibited a net negative charge, indicating
that the negative charge of the XG dominated the charge of the
blg. At higher ratios (r $ 5), the net charge of all systems is
positive and similar to that of the protein, indicating an excess
of blg. As the gel is mainly stabilized by the electrostatic inter-
action, the maximum of electrostatic interaction should give a
gel with the highest solid-like character. As expected, the
highest solid-like character was obtained at r ¼ 3.5 for nal gels
(Fig. 6), so the optimal ratio for electrostatic interactions was
estimated to be approximately 3.5. The highest G0 obtained at
r ¼ 2 in Fig. 3 is explained by the highest proportion of XG in the
gel formulation where the total solid concentration was xed at
0.30 wt%.
Effects of polysaccharide concentrations at a xed protein
concentration
The gelation processes as a function of XG concentrations are
shown in Fig. 7. At 0.03 wt% XG and a ratio of 10, the gelation
was reached at pHg 5.5, which is higher than that (pHg 5.3)
reached by 0.06 wt% XG at a ratio of 5. This behavior is
Fig. 6 Storage modulus at pH 4.4 for b-lg–xanthan gels at a fixed concentration
of xanthan (0.03 wt%) and different blg–XG ratios. Different letters indicate
significant difference between ratios evaluated by the Tukey procedure (p < 0.05).
Fig. 7 Evolution of storage modulus as a function of pH for blg–XG mixtures at
fixed protein (0.3 wt%) and different XG concentrations.
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explained by the availability of blg at a ratio of 10 as described
above.
The doubling of the XG concentration from 0.03 wt% to 0.06
wt% increased the G0 6.8-fold, from 53 to 362 Pa. The stronger
gel obtained for an XG concentration of 0.06 wt% again suggests
that XG plays a key role in the formation of gel networks.
Effects of total solid concentrations at ratio 5
The effects of the solid total concentration on the gelation
processes and the effects of the XG concentration on the storage
modulus at the nal pH are presented in Fig. 8a and b,
respectively.
The gelation point, pHg, seemed to be independent of the
solid total concentration; gelation occurred at pH z 5.3. The
ratio or the availability of proteins was the same for all formu-
lations. The effect of solid total concentration is a combination
of the effects of the viscosity of a mixture and the volume
occupied by XG. At higher concentrations, the gelation may slow
down due to high viscosity, but it may also be enhanced by the
volume occupied by XG. However, stronger gels were obtained
0
at higher biopolymer concentrations. The dependence of GpH4.4
on the XG concentration was marked, particularly at low
0
concentrations (Fig. 8b). The value of GpH4.4 was extrapolated to
1 Pascal for an XG concentration of 4.8
103 wt%. This value
(Co) can be taken as an estimate of the lowest XG concentration
at a ratio of 5 at which gelation is possible; the corresponding
Fig. 8 (a) Evolution of storage modulus as a function of pH for blg–XG mixtures
at ratio 5 and different solid total concentrations. (b) G0 as a function of XG
concentrations for gels at pH 4.4 and a ratio of 5.
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concentration of blg is 0.024 wt%. This critical XG concentra-
tion is in the vicinity of the overlap concentration for an XG
solution at which the intermolecular interactions between XG
polymer chains through hydrogen bonding and polymer
entanglement result in the formation of a complex network of
entangled rod-like molecules.43–45 The overlap concentration
has been reported to be as low as 103, 5.0
103 and 12.6

103 wt%.44,46,47 This indicates that the XG chain overlap is a pre-
requisite for mixtures to gel.
Role of the protein and polysaccharide in network formation
Fig. 9 shows different networks formed from different quanti-
ties of XG and blg in the gel formulations. The bright zones
correspond to zones enriched in proteins and dark zones
correspond to zones devoid of blg.
Fig. 9a and b show a gel obtained with the same quantity of
blg but a different quantity of XG. The network became denser
with an increase in the XG concentration, which explains the
higher G0 observed at pH 4.4 for the gel at an XG concentration
of 0.06 wt% and a ratio of 5 (Fig. 7). A network with larger dark
zones is obtained in the gel formulation with high protein
content at a constant polysaccharide concentration (compare
Fig. 9b with Fig. 9c). The network density is proportional to the
XG concentration, which suggests that the blg aggregated on XG
chains, so dark zones may correspond to zones devoid of
biopolymers, and pores of the gel network. An open network
shown in Fig. 9b may have resulted from the effect of excess
protein in the system. At a ratio of 10, the repulsive forces
caused by the excess proteins in the porous network are more
predominant, which may result in a larger pore size. This result
is in agreement with those presented in Fig. 4, where the gel at a
ratio of 5 is observed to be stronger than the gel at a ratio of 10.
At a very low XG concentration (CXG ¼ 4.50
103 wt% < Co)
and a high blg concentration (4.50
102 wt%), a complex was
formed instead of a gel (Fig. 10a). However, at a higher XG
concentration (CXG ¼ 8.30
103 wt% > Co), the gelation of the
blg–XG mixture occurred (Fig. 10b).
To complete these observations and to ensure that the blg
aggregated on XG chains, observations on mixtures containing
both RITC-blg and FA-XG were performed. Fig. 11 shows
the nal microstructures obtained for the mixture of 0.06 wt%
FA-XG and 0.30 wt% RITC-blg.
The uorescence of each polymer was coded: green for XG
uorescence, red for blg uorescence. The red and green
networks obtained in Fig. 11a and b respectively correspond to
protein and polysaccharide networks. Fig. 11c shows the nal
microstructure obtained for 0.30 wt% blg–0.06 wt% XG mixture
when the two networks were superimposed. The three networks
are identical. So, dark zones correspond to zones devoid of
biopolymers and proteins and XG being located in the same
location. These observations conrmed that XG provided a
frame for gel organization, and blg aggregated along the XG
chains and could be regarded as crosslinking agents. Iijima
et al.48 reported that aer annealing of XG solution at 1.0 wt%
for 24 h at 40  C and subsequently cooling it, a homogeneous
network structure was obtained that is comparable to the
Soft Matter, 2013, 9, 3063–3073 | 3069

Soft Matter
Fig. 9 Confocal micrograph of RITC-blg–XG gels obtained under different conditions. The images represent an area of 91.87
91.87 mm. The light areas are protein
rich.
Fig. 10 Confocal micrograph of RITC-blg–XG: (a) complex formation at a total
solid concentration of 0.05 wt% and a ratio of 10; (b) gel formation at 0.05 wt%
and a ratio of 5. The images represent an area of 212.17
212.17 mm. The light
areas are protein rich.
network obtained in our study by CLSM (Fig. 9). In distilled
water and at a very dilute solution (0.02
103 wt%) XG chains
exist as single strands and isolated molecules are observed by
AFM.49 The molecules observed are approximately 0.8 mm in
length, 2 nm in width and 8 nm in thickness. In their quiescent
state and at the critical overlap concentration (c*), the inter-
molecular interactions between XG polymer chains through
hydrogen bonding and polymer entanglement result in the
formation of a complex network of entangled rod-like mole-
cules.43–45 This concentration has been reported to be as low as
Fig. 11 Localization of blg and xanthan in the mixture of 0.30 wt% blg and 0.06 wt% XG observed by confocal laser scanning microscopy. Scale bar corresponds to
30 mm and is appropriate to all images.
3070 | Soft Matter, 2013, 9, 3063–3073
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103, 5.0
103 and 12.6
103 wt%.44,46,47 In addition, the
highly ordered network of entangled stiff molecules results in a
XG solution with the viscoelastic characteristics of a weak gel
and a dominant elastic response to frequency.24 Moreover,
Iijima et al.48 reported that large-scale molecular assemblies are
formed and act as colloidal dispersions when XG is dissolved in
water. Kobori et al.50 also reported the aggregation of sodium
caseinate along XG chains at a pH level below the Ip of this
protein.
These results conrm the hypothesis that the gel networks
are based on XG chains. Therefore, the density of the gels
increased with increasing XG content (Fig. 9a and b). This is not
in agreement with the previous result that indicated that the
density of the gels, based on fractal dimensions of systems,
increased with increasing protein content.12 In recent report, we
found that, by image analysis, the fractal dimension increases
with increasing XG concentration.41 These ndings suggest also
that there exists a limiting molecular weight of XG required for
the formation of a gel below which particulate electrostatic
complexes instead of a stable network will form, as reported by
Laneuville et al.51
Identication of gelation mechanism
It is commonly accepted that XG does not gel on its own
through the typical gelation mechanism, although there is
considerable evidence of strong intermolecular interactions in
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solution.24,25,46,48,50,52–57 In the presence of divalent or trivalent
metal ions, the sol–gel transition occurred.58–60 These authors
consider the metal ion crosslinking agents that link the nega-
tively charged xanthan chains to the gel network. However, the
limiting concentration of XG58 needed for gelation with 5 mM
Cr(III) at 20  C is higher than that in this study (0.35 wt% (ref. 58)
versus 4.8
103 wt%) which may indicate that the size of the
cross-linking agent may be important for XG hydrogel forma-
tion. Recently, some authors have reported the formation of XG
gels by annealing48 and freezing and thawing XG solutions.61
These authors interpreted the gelation of XG as the result of
intermolecular interactions during processing.
Based on the previously discussed CLSM, the rheological
proles and earlier reports in the literature, the gel formation
mechanism is proposed as follows (see Fig. 12). In aqueous
solution, xanthan gum exhibits pseudoplastic behavior due to
the presence of large-scale molecular assemblies.48 The assem-
blies of xanthan gum molecules occur by an end-to-end asso-
ciation52 or by a side–side association.62
As the pH decreases, the repulsive interaction between XG
chains decreases, while the attractive interaction between blg
and XG increases. The aggregation of proteins through the
polysaccharide chains and the decrease in pH may enhance the
intermolecular interaction between XG chains through blg.
When the pH approaches the Ip of the protein and is still higher
than pH4, the interaction between negatively charged groups on
XG chains with positively charged patches on the blg results in
the formation of soluble complexes, followed by the formation
of interpolymer complexes as the pH decreases further. The
interpolymer complexes may also be soluble up to the point
where large-scale interactions are forged. The association that
occurs as electrostatic repulsion decreases results in a sol–gel
transition at the point of gelation. At high blg–XG ratios, larger
strands were observed in the gel structures (Fig. 9c and d); thus,
the gels may have multiple layers of proteins that aggregate
along XG chains. It should be noted that the gels obtained in
this study are pH-reversible. The gels melt rapidly when sus-
pended in phosphate buffer at pH 7 above the Ip of the protein
(results not shown); however, they can gel again when the pH
decreases below the Ip of the protein, which affirms that the
junction zones are electrostatic in nature. This proposition is
slightly different from that reported previously.12 In both
propositions, the gelation process is stated to initiate with the
Fig. 12 Schematic of the interaction and gel formation process between blg (circles) and XG (rod sharp); rectangle presents aggregation zones of blg on XG chains,
arrow presents electrostatic cross-linking zones of XG chains by blg.
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Soft Matter
formation of soluble and interpolymeric electrostatic
complexes. However, the interconnection between the inter-
polymeric complexes seems to spontaneously occur during the
second stage due to the entanglement of XG chains and the
decrease in the repulsive interaction between them as the pH is
reduced. Moreover, in this study, the role of both biopolymers
in network formation induced by the electrostatic attractive
interaction was reported for the rst time, blg may be regarded
as a crosslinking agent at pH4, i.e., there may exist at least two
XG molecules that share the same blg molecules to support
electrostatic interactions, as reported by Girard, Turgeon et al.;23
Fogolari, Ragona et al.63 that blg may likely interact with nega-
tively charged macromolecules through more than one amino
acid zone. Therefore, the linkage of XG chains though blg seems
to be identical to a polymerization process, which is supported
by the lamentous nature of the gel structures (Fig. 9 and 11).
This is contrary to the aggregate nature reported in the previous
study.12 Due to the reduced mobility of XG chains in the system,
each XG chain may be considered a nucleus. Furthermore, as
pH is reduced, the proteins will diffuse to the XG chain and
aggregate, due to attractive interactions that support the
formation of soluble complexes, followed by the formation of
interpolymeric complexes. The growth of soluble complexes by
the aggregation of blg through XG chains indicates that there is
an increase in the size of structural domains, which is consis-
tent with the increase in the scattered light intensity observed in
the previous study during gelation.12 Understanding the
mechanism of this type of structuration is important because it
could be representative of other protein–polysaccharide asso-
ciative behaviors found in various biological systems (protein–
hyaluronic acid,64,65 chitosan–protein,66 etc.) or because it could
be used to develop materials for tissue-engineering applica-
tions67 and in the control of the rheological properties of some
food products. For example, the production of very low
concentrations of exopolysaccharide by lactic acid bacteria
signicantly affects the properties of fermented product gel and
water-retention capacity.68,69 Protein-anionic polysaccharides
may become as important as cationic–anionic biopolymers in
materials development.70 Moreover, the gels formed in this
study are pH-reversible hydrogels due to the electrostatic nature
of the junction zones. This reversibility is attractive for the
design of physical gels geared toward biomedical applications19
or controlled release of bioactive molecules.71
Soft Matter, 2013, 9, 3063–3073 | 3071

Soft Matter
Conclusions
The gelation kinetics and structural evolution of electrostatic
blg–XG gels are followed by rheological measurement and
confocal scanning microscopy. The blg aggregated along the XG
chains that initially provided a tenuous network for gel struc-
turation and could be regarded as a crosslinking agent. The
gelation process followed three stages: induction stage, gelation
stage, and quasiequilibrium stage, corresponding to the
formation of soluble complexes, the formation of junction
zones followed by the formation of interpolymer complexes,
and the stabilization of the gel at the nal pH, respectively. The
gel strength mainly depended on the XG content. The gel
structure stabilized at an electrical charge equivalence pH
during the acidication and did not change aer this pH (EEP),
and this EEP value depends on the biopolymer ratio. The blg–
XG ratio was observed to be an important factor in controlling
the gelation process, the gel strength, and the gel structure, and
an optimal ratio for electrostatic interaction was estimated.
This novel hydrogel could be used for encapsulation of active
molecules.
Acknowledgements
ACDI/PCBF is acknowledged for its fellowship for Xuan Thang
Le along with Diane Gagnon and Sandra I. Laneuville for their
technical assistance and fruitful discussion. This work has been
funded by NSERC discovery grant.
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