Food Hydrocolloids 29 (2012) 298e307

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Food Hydrocolloids
journal homepage: www.elsevier.com/locate/foodhyd

Impact of xanthan gum, sucrose and fructose on the viscoelastic properties

of agarose hydrogels
Sania Maurer*, Ann Junghans 1, Thomas A. Vilgis 2
Max-Planck-Institute of Polymer Research, Ackermannweg 10, 55128 Mainz, Germany

a r t i c l e i n f o a b s t r a c t

Article history: Mixed carbohydrate systems are of special interest for the food and non-food industry as they offer
Received 18 August 2011 a versatile range of unique and novel functional properties. However, intense research is required to
Accepted 12 March 2012 understand the complex processes occurring in such systems on a molecular level and to be able to
modify them aim-oriented. In food, characteristic properties are based on the physicochemical functions
Keywords: of the biopolymers added. Thus, small deformation tests and moisture analysis have been applied to
Agarose
study the impact of xanthan gum and two types of sugar on the viscoelastic properties, the solegel
Xanthan gum
transition and the water holding capacity of 1% agarose hydrogels. Agarose gels are very elastic, turbid
Fructose
Sucrose
and prone to synaeresis, which impinges on their mouth feeling. Additions of xanthan gum revealed less
Rheology elastic gels with an unaffected water holding capacity. Progressive addition of two different types of up to
Gelation 40% of sugar yield an increase of the elasticity of agarose gels, whereby sugar concentrations of 60%
partially result in a structural breakdown and thus a signicant lower network structure but better water
holding. In ternary systems, the effect of the sugar concentration and sugar type used is diminished by
xanthan gum. The gelation mechanism of agarose gels with a distinct amount of co-solutes is presumably
mainly affected by the water shortage evolved from the competition for it of all solutes present.
2012 Elsevier Ltd. All rights reserved.

1. Introduction characteristics. In this study, the emphasis is placed on the rheo-

The demand for carbohydrates with unique physicochemical
properties is high and propels the research of such additives to
understand the basic principles of their properties and function as
precise knowledge about the rheological behaviour of thickeners
and gelling agents can be helpful for process design and product
development. Textural characteristics, mouth feeling and the
general appearance of a product, as well as uid ows and ow
conditions during processing are thus more predictable and
controllable (Marcotte, Hoshahili, & Ramaswamy, 2001).
Mixtures of carbohydrates of different properties offer an
immense potential to function as replacer for unfavourable food
ingredients that do not conform well to cultural, ethical, nutritional
and health claims. Currently, alternative gelling agents to gelatine
are of special interest for the food industry. However, a detailed
knowledge about the physicochemical properties of other gelling
additives is essential to full the demands regarding the functional
* Corresponding author. Tel.: 49 6131 379 149.
E-mail addresses: maurer@mpip-mainz.mpg.de (S. Maurer), junghann@mpip-
mainz.mpg.de (A. Junghans), vilgis@mpip-mainz.mpg.de (T.A. Vilgis).
1
Tel.: 49 6131 379 513.
2
Tel.: 49 6131 379 143.
logical behaviour of a ternary system composed of different
carbohydrates unifying three elementary functional properties:
gelation (agarose), thickening (xanthan gum), and sweetening
(fructose and sucrose).
The usage of agarose, the main gelling agent of agareagar
extracted from red algae (Rhodophyceae), in this study is twofold:
First of all, due to its practical relevancy in the food and non-food
industry and secondly due to its relatively simple and neutral
molecular ordered structure that alleviates the modelling of the
gelation mechanism of carbohydrates. The chemical structure of
agarose is composed of alternating repeating agarbiose units,
connected through C-1 and C-3 of b-D-galactopyranose (1e4)
linked to 3,6-anhydro-a-L-galactopyranose. The physical gelation
of such polysaccharides is a complex process of self-assembly
accompanied by biomolecular conformation changes, solution
demixing and molecular cross-linking (Manno et al., 1999; Morris,
2009). Polymer concentration, presence of co-solutes, temperature
history, mixing rate, as well as solvent properties further determine
the mechanism of gelation and the nal gel properties. The precise
gelation mechanism of agarose is not yet fully understood. The
investigation of kinetic and diffusion processes, as well as
concentration occulation involved in the gelation mechanisms
yield different models being based on e.g. spinodal decomposition

0268-005X/$ e see front matter 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodhyd.2012.03.002
 S. Maurer et al. / Food Hydrocolloids 29 (2012) 298e307
and nucleation and growth. In general, the gelation process is
understood in terms of liquideliquid phase separation.
Agarose dissolves in hot water (60e70  C) and gels upon cooling
(35e40  C). Pure agarose gels are highly elastic, hard, clear, ther-
moreversible, relatively prone to synaeresis and provide a poor
mouth feeling (Nordqvist & Vilgis, 2011; Rinaudo, 2008). To modify
these properties, Nordqvist and Vilgis combined agarose with a, in
the physical sense, non-gelling polysaccharide. They reported from
rheological, microscopic and synaeresis measurements that
combinations with xanthan gum, a bacterial hydrocolloid with
unique thickening properties, result in less elastic, softer gels with
a higher water holding capacity and a better mouth feeling. The
plasticising effect of xanthan gum molecules can be referred to the
signicant impact of highly negatively charged stiff rod-like xan-
than molecules on the formation of exible agarose chains to
aggregates. In absence of co-solutes, the gelation process of agarose
is a two-stage process with the formation of agarose helices fol-
lowed by their aggregation into bundles as it has been discussed
recently in detail by Nordqvist and Vilgis (2011). Xanthan mole-
cules, as relatively stiff polyelectrolytes, however undergo a so-
called jamming transition, which is a very general phenomenon in
nature (Liu & Nagel, 1998). The jamming transition of xanthan gum
solutions is induced by random blocking of the dynamics of the
rigid molecules. Indeed such a logjam transition of rigid rods has
been proposed already by Edwards and Vilgis (1986) earlier. The
jamming transition of xanthan solutions can be modied by the
ionic strength. At a critical polyelectrolyte concentration, which is
determined by the chain length and the charges, xanthan solutions
thicken by the jamming process. Combining xanthan and agarose
hinder the aggregation of the agarose helical structure into
bundles. Thus, gelation is reduced to a single-stage process by the
presence of the randomly oriented xanthan molecules (Nordqvist &
Vilgis, 2011).
Besides the texturizing components, jelly gums contain
a considerable amount of sugar. In the case of gelatine, the used
sugars have a synergistic effect on the protein gelation properties
(Kasapis, Al-Marhoobi, Deszczynski, Mitchell, & Abeysekera, 2003).
The purpose of this study was to analyse how different concen-
trations of fructose and sucrose affect the rheological and water
holding properties, as well as the solegel transition of agarose,
respectively agaroseexanthan hydrogels. The experimental results
are underlined with a simple gelation model to emphasis the
special role of water molecules and their involvement in intra- and
intermolecular interactions during the gelation.
Finally, a remark is needed on the choice of the different
constituents. Indeed the choice of agarose, xanthan, and the two
types of sugar has different physical aspects. First, the molecules
show different conformations and physical properties in solution.
While agarose appears to be a relatively exible macromolecule,
xanthan is much stiffer. The persistence length of xanthan is of the
order of the total length of the molecule (Song, 2006). Another
difference comes from the charges of the molecules. Xanthan is
highly charged whereas agarose is practically neutral. These
differences on a molecular level allow studying the interplay with
the low molecular weight sugars, such as the monosaccharide
fructose and the disaccharide sucrose, more systematically.
2. Materials and methods
All materials used in this study are shown in Table 1:
2.1. Gel preparation
A 1% w/v solution of agarose, dispersed in distilled water or in
the appropriate sugar (fructose, sucrose) in water concentration
299
Table 1
Used chemicals (all chemicals were used as received.).
Chemical CAS (chemical Provider Source
abstract service)
Agarose 9012-36-6 Fisher scientic Schwerte, Germany
GmbH
Xanthan gum 11138-66-2 Carl Roth GmbH Karlsruhe, Germany
& Co. KG
D-()-fructose 57-48-7 SigmaeAldrich Steinheim, Germany
Chemie GmbH
Sucrose 57-50-1 Calbiochem Darmstadt, Germany
Merck KGaA
(20, 40, and 60% w/w), was heated up to 68  2  C and kept there
for 1 h while stirring (120 rpm). For 1% w/v agaroseexanthan
solutions (ratio 1:1), agarose and agaroseesugar solutions were
rst stirred for 30 min prior adding to the already prepared xanthan
gum solutions and subsequently further stirred for 30 min at
120 rpm. The solvent evaporation during stirring was minimised by
covering the beaker glass with a glass cover. All hydrogels have
been prepared in 20 ml snap-cap bottles. Mono- and multi-
component hydrogels have been produced by pouring the solutions
into moulds (PVC) of dened dimensions ( 25 mm, h 3 mm) and
allowing the solution to cool down-to room temperature. Samples
were trimmed to the surface of the mould to obtain samples of
a dened height. Due to the relatively unstable texture of
agaroseexanthanesugar hydrogels, such gels were directly poured
onto sand paper being placed under the mould to prevent struc-
tural rupture during the transfer to the rheometer. In general, sand
paper was used for all samples, but usually already stuck onto the
measurement device, to avoid slippage during the rheological
measurements. To achieve a homogeneous hardening of the
agarose gels of different composition, the samples were cooled at
4  C for at least 24 h. All samples were sealed with Paralm to
avoid water evaporation during storage. To adapt to room
temperature, the hydrogels were taken out of the fridge one hour
before measurements.
Xanthan gum solutions of 1% w/v were prepared by dissolving
the xanthan gum in distilled water. Samples were stirred at room
temperature in 20 ml snap-cap bottles at a constant stirring rate of
200 rpm for at least 24 h.
2.2. Dynamic viscoelastic measurements
Dynamic viscoelastic deformation tests of all prepared hydrogel
compositions were performed on a Gemini 200 Advanced
Rheometer (Bohlin Instruments, Malvern, Worcestershire, UK) by
applying parallel plates (PP 25) and the amplitude-sweep mode.
For the delineation of molecular models and pictures of the gelation
process of the agarose hydrogels of different composition, the
storage modulus (G0 ) has been analysed as a function of strain. All
amplitude-sweep tests were performed at constant frequency of
1 Hz under isothermal conditions (T 25  C). To avoid slippage
during measurement, the plates of the rheometer were pasted up
with sandpaper (Flexovit K 500, Wollschlger, Bochum, Germany).
After each run, this paper has been renewed.
2.2.1. Amplitude-sweep tests
For the amplitude-sweep tests, the gap size was set between
2300 and 2800 mm. This range results from the gel nature of the
samples and from the varying water evaporation during storage of
the used components while hardening and systematic errors
during preparation. To avoid slippage or rupture of the sample
during measurement, an appropriate pressure had to be applied on
the sample between the plates. This was monitored by the normal
300 S. Maurer et al. / Food Hydrocolloids 29 (2012) 298e307
force displayed on a scale bar of the rheometer. As the height of all
samples was approximately 3 mm (the height of the mould used for
the sample preparation), the setting of the gap size was started
from 2800 mm and lowered bit by bit until the force rose and thus
indicating an appropriate pressure on the sample in dependence on
the resistance of the sample. The setting has been stopped and the
chosen gap size was proven in a trial measurement as the obtained
G0 values could indicate an inadequate gap. The validated gap has
then been used for the measurements of gels of the same compo-
sition. Samples used for this setting were not included in the
measurement evaluation. This procedure, however, did not work
for soft samples. Here, a dened amount of 2.10 g was weighed in
moulds, already prepared with sandpaper, and analysed by using
a gap of 2800 mm (approx. consistent with the mould height of
3 mm), sufcient to satisfy the criteria mentioned previously. This
had been the case for samples composed of agarose, xanthan and
sugar.
The default strain range was from 0.0005 to 50 for samples
composed of agarose and agarose with sugar. However, this strain
had to be changed for the hydrogels additionally with xanthan to
0.0005 to 0.05, as a higher strain caused structural damages of the
samples.
The descriptive statistical evaluation is based on a fteenfold
determination, which is made up of three replicates of ve samples
of the same hydrogel composition. Thus, the data set of each G0 (G0
at a strain of 0.01 being within the LVE-range) results from the
calculated arithmetic mean value (x) of 15 replicates (n),
P
x 1=n ni 1 xi while the associated error bars indicate the stan-
dard deviation (s), s x2  x2 1=2 .
2.2.2. Temperature-sweep tests
For the temperature-dependent measurements, the solutions
were prepared as usual but the samples were not cooled to form
gels but placed in their sol-state, here the sample amount was
750 ml, on the pre-heated plate (80  C). The transition of the gel-
phase was initiated by cooling down at a rate of 1  C/min to
20  C. The upper plate has been cranked by hand to achieve
a homogenous distribution free of air entrapments of the sol
between the plates. By covering the sample properly with silicon oil
and additionally using a solvent trap, the drying out of the sols/gels
has been slowed down during the cooling run. Also here sand paper
was used to prevent slippage. The gap was set to 1000 mm for each
sample.
2.3. Moisture analysis
A halogen moisture analyser (Mettler Toledo HR73, Giessen,
Germany) has been applied to qualitatively determine the water
holding capacity of pure agarose hydrogels and of agarose hydro-
gels with either xanthan or sugar (fructose, sucrose) or both
components. Sugar concentrations of 20, 40, and 60% w/w were
used. Samples were prepared as already described in paragraph 2.1
but pouring 2  0.2 g of sol into moulds of 50 mm diameter and
1 mm height. Three samples of each hydrogel composition were
produced. In contrast to previous studies (Nordqvist & Vilgis, 2011)
a different analysis for the water holding capacity is used. Never-
theless, for a relative comparison between the samples investigated
here, it is appropriate to use the halogen moisture analyser to
describe the different hydrogel compositions, especially when the
hydrogen bonding of water to the different components of the
hydrocolloids and co-solutes is discussed (see below).
The statistical evaluation of the obtained moisture contents of
the different hydrogel compositions is based on the determination
of the means and the appropriate standard deviation according to
the equations in Section 2.2.1.
The moisture content loss of the hydrogels at constant
temperature of 100  C has been determined in per cent as a func-
tion of time. After 10 min, the measurement was stopped and the
obtained values for the moisture loss during this time have been
used for the qualitative comparison between the hydrogel
compositions. Based on the results, it was assumed that the loss of
water during drying indicates, to some extent, the ability of
different composed gel networks to hold water over a certain
period of time. In this study, the term water holding capacity will
be synonymical used for the ability of the hydrogel network to hold
water when being exposed to a dened temperature. Of course the
values obtained here can be viewed as apparent water holding
capacity, since the absolute values depend on the temperature
which is exposed to the gel samples.
3. Results and discussion
The impact of different sugar types (fructose and sucrose) of
different concentrations (20, 40, and 60% w/w) on the gelation
process and the gel properties of 1% agarose and 1% agaroseexanthan
(1:1) hydrogels have been studied by applying three different
analytical methods. Oscillation amplitude sweeps have been per-
formed to evaluate the modication of the hydrogel viscoelastic
properties when sugar is added. As the viscoelastic behaviour of
agarose gels is affected by the gelation process, temperature-
dependent measurements have been carried out to analyse the
solegel transition. Additional, the loss of water during a dened time
period has been determined to study the water holding capacity of
agarose hydrogels, respectively agaroseexanthan hydrogels con-
taining different concentrations and types of co-solutes.
3.1. Viscoelastic properties
Comparing the elastic modulus of agaroseesugar hydrogels with
the ones of agaroseexanthanesugar gels, distinct differences are
observed (Fig. 1). The mixture of agarose with 20% and 40% of fruc-
tose, respectively sucrose provokes that the resulting gels exhibit
a stronger elasticity. Already 20% of sugar is sufcient to increase the
elasticity of the pure agarose gel (G 5.3  103  7.9  102 Pa) of
about 30% in the case of fructose, and almost 50% for sucrose. A slight,
but not statistical signicant difference is obtained for 20% and 40%
of sucrose (G 9.9  103  5.9  102 Pa), and 40% of fructose with
G 8.9  103  1.5  103 Pa, similar to the value of sucrose. However,
the more sugar is implemented in the agarose gels the more the
particular agaroseesugar compositions differ from each other.
While 60% of fructose results in a gel of an elasticity that is almost
three times higher (G 1.4  104  1.6  103 Pa) than the initial one,
a signicant decrease of G0 of about 33% (G 1.8  103  4.9  102 Pa)
is observed when 60% of sucrose is added. Thus, high amounts of
sucrose reduce the elastic modulus signicant, evolving less elastic
agarose gels.
However, the effect of the sugar concentration on the visco-
elastic properties diminishes rigorously when xanthan gum is also
part of the mixed system. Looking only at the hydrogel samples
devoid of any sugar, the elastic modulus of the binary hydrogel
(G 1.8  103  4.1  102 Pa) is almost three times lower than G0 of
the pure agarose gel. In the case of agaroseexanthan gels with 20%
of fructose, G0 decreases (G 1.3  103  1.4  102 Pa) but exhibits
a reverse effect when 40% (G 2.2  103  3.0  102 Pa) and 60%
(G 2.3  103  2.0  102 Pa) are incorporated, whereby 60% does
not differ signicantly from 40% of fructose. Such a trend cannot be
observed for the different concentrations of sucrose. No change in
G0 is observed for 20% of sucrose and 40% provokes a decrease to
1.4  103  1.2  102 Pa. A pronounced drop of G0 is revealed at
a sucrose concentration of 60%. With 8.1  102  1.0  102 Pa, it is
 S. Maurer et al. / Food Hydrocolloids 29 (2012) 298e307 301

a certain viscosity of the embedded liquid, which acts as a swelling

agent, in this context, a viscosity increase caused by 60% of fructose.
The higher viscosity in the pores presumably acts as a cushion
without affecting the coil-to-helix transition of agarose gels. Similar
effect on the network topology is observed in mixtures of sugar and
agarose gels of up to 40% of sucrose. But in addition, 60% of sucrose
causes a structural breakdown, which will be discussed later in this
section. The discrepancy between both sugar types might refer to
the different impact on the solution viscosity, e.g. based on the
hydration number and the ability of the sugar to change the
structure of water (Galema & Hoeiland, 1991; Uedaira & Uedaira,
1985). These assumptions, concerning the stabilisation of agarose
gels associated with increasing sugar concentration, coincide well
with the results obtained by several scientists (Deszczynski, 2003b;
Normand, 2003; Watase, Nishinari, Williams, & Phillips, 1990). They
suggested that the exclusion of sugar from the surface of the
structure is responsible for this phenomenon. Further congruence
could be found with the results obtained by Kasapis et al. (2003),
who investigated gelling polysaccharides such as kappa-
carrageenan and deacylated gellan gum (in a concentration range
of 0.5e12%). However, the formation of stronger and thermal more
stable gels has only been valid for sucrose, respectively fructose
concentrations up to 40%.
High concentrations of sugar (60% of sucrose) provoke a drastic
drop of the elastic modulus revealing a reverse trend as for the
concentration up to 40% (Fig. 1). The high level of co-solutes in the
agarose solution probably affects the coil-to-helix transition so that
the formation of aggregates is inhibited. This assumption is sup-
ported by results of investigated agarose gels obtained from optical
(Deszczynski, 2003a), calorimetric (Normand, 2003), microscopic
(Nordqvist & Vilgis, 2011; Normand, Lootens, Amici, Plucknett, &
Aymard, 2000), and deformational measurements (Watase et al.,
1990). Accordingly, during the preparation of agarose and
agaroseesugar gels, it was observed that the degree of turbidity
decreases the higher the concentration of sugar is, thus producing
clearer gels (Fig. 2). This change in turbidity is presumably due to
Fig. 1. Average elastic modulus of 1% agarose hydrogel (-) and agaroseexanthan
hydrogel (1:1) (6) with different fructose (A) and sucrose (B) concentrations measured
at of strain of 0.01.

signicant lower than the value obtained for the pure

agaroseexanthan hydrogel. In general, the differences between the
several concentrations, as well as the different sugar types used are
not as distinct as for hydrogels devoid of xanthan gum. It seems that
the magnitude to be able to affect the viscoelastic properties of
agarose gels by sucrose respectively fructose is diminished by the
addition of xanthan gum. Agarose hydrogels with xanthan exhibit
a pronounced turbidity, which does not change macroscopically
with increasing sugar concentration.
As the rheological properties are based on the network char-
acteristics, the impact of the different sugar types on the gelation
process of agarose and thus its structural properties is crucial. Upon
cooling, coils of exible agarose chains transform to helices, stabi-
lised through intra- and intermolecular interactions, with subse-
quent aggregation of such helices to bundles. Due to the high
degree of aggregation, agarose hydrogels appear to be turbid and
distinct elastic (Deszczynski, 2003a). Regarding the mechanical
strengthening, low to relatively high levels of fructose seem to
stabilise the gel structure and the binary mixture evolves into
a stronger, more stress resistant hydrogel. To explain this rheo-
logical behaviour with a schematic but speculative model, helical
brils of agarose form a cohesive, continuous network that extends
over the entire solution and thus embeds the liquid phase. The Fig. 2. Observed changes in turbidity of 1% agarose hydrogels with increasing
network topology reveals a pore, or mesh size, which tolerates concentration of sugar.
302 S. Maurer et al. / Food Hydrocolloids 29 (2012) 298e307
the decreasing part of aggregation and to the smaller correlation
length (average size of the pores). Subsequently, an evolution from
a coarse structure to a ne-stranded gel of homogeneous structure
is suggested (Normand, 2003). Oscillation measurements of the
solegel transition of agarose gels by Nordqvist and Vilgis (2011)
revealed that the gelation mechanism, original being a two step
process, is reduced to a one-step process. As this is based on the
impact of xanthan gum, a similar effect can be attributed to sugar.
It is assumed that xanthan gum has a pronounced impact on the
physical behaviour when immersed in agarose gels. A brief
consideration of the molecular structure of xanthan gum might be
necessary to explain this phenomenon. Due to their chemical
conguration, xanthan molecules are assumed to be highly
charged, stiff rods in absence of salt ions. Such rods tend to undergo
a so-called jamming transition at increasing polymer concentration
due to strong repulsion and the incapability of forming liquid
crystalline phases (Nordqvist & Vilgis, 2011). Therefore, agarose
chains have only restricted space to form bundles of helices as the
diffusion process is prevented by the jammed xanthan rods.
Consequently, a looser gel network with a lower modulus evolves.
Additionally, the high negative charge of xanthan molecules
dominates the effect of sugar molecules to interact with water
molecules. It seems to be reasonable to assume that the reduction
of the modulus is a logical consequence of the water shortage as
there are three components present competing for water.
Extensive studies regarding xanthanesugar solutions of
different concentration (20, 40, and 60% w/w) have also been
conducted.
In general, xanthan gum does not form gels in a physical sense
but can be regarded as a weak gel (Voragen & Challen, 2000). The
addition of moderately concentrations of up to 60% of sugar (fruc-
tose or sucrose) shows a signicant impact on the solution
behaviour of 1% xanthan gum solutions. High amounts of fructose
provoke a signicant reduction of the elasticity modulus, whereby
the increasing addition of up to 60% of sucrose does not have any
further impact (Fig. 3). Fructose and sucrose concentrations of 60%
provoke a slight increase in the viscosity modulus (data not shown)
of 1% xanthan gum solutions. At such high sugar concentrations,
the viscosity of the continuous phase increases signicantly, thus
affecting the distribution of the stiff xanthan molecules in the sugar
in water solution. Concluding, it is assumed that 1% xanthan
Fig. 3. Average elastic modulus of 1% xanthan solutions of different sugar type
(fructose (-) and sucrose (6), zero sugar (,)) and concentration measured at a strain
of 0.01.
solutions with high sugar concentrations exhibit a uid-like rather
than weak-gel-like behaviour.
However, the effects of sugar on the one-component systems
(1% xanthan solution and 1% agarose gel) and on the multi-
component system (1% agaroseexanthan gel (1:1)) have to be
regarded separately as the physical nature of these two systems is
different.
3.2. Solegel transition
Temperature-dependent measurements have been performed to
analyse the solegel transition of agarose and agaroseexanthan gels
with varying fructose or sucrose concentration. Figs. 4 and 5 show
the solegel transition of agarose, respectively agaroseexanthan
hydrogels of 20, 40, and 60% w/w sugar added. Representative
curves have been chosen and slight variations of absolute values of
the elastic modulus might be possible.
For the present, the focus is placed on the curve of the pure
agarose gel as it reects the characteristic gel mechanism of this
gelling polysaccharide. The sigmoidal curve progression
suggests a two-stage process that has already been suggested by
several other scientists (Fernandez et al., 2008; Mohammed,
Hember, Richardson, & Morris, 1998). Two pronounced rises
(steps) in the curve indicate conformational changes of
agarose molecular chains upon cooling. The rst step of increase
of the elastic modulus (G0 ) by lowering the temperature is
referred to the formation of helices, which act as cross-links for
the further formation of such helices to bundles. As shown in
Fig. 4, the rst rise takes place in a temperature range of
76  Ce60  C, where G0 increases from 2 to 38 Pa. Further cooling
to 50  C reveals that the gradient slightly diminishes until
exhibiting a signicant, second rise when a temperature of
30  C is achieved. For a better illustration the two step process
has been indicated and the corresponding measured
temperatures are given (Fig. 4). In Nordqvist and Vilgis (2011)
the physical interpretations of the two steps and the interplay
between the gelling and thickening agents have already been
discussed in detail. The rigorous change of G0 from 1.1  102 to
1.1  104 Pa represents the aggregation of agarose helices to
bundles, thus the second step, driven by entropic restrictions
(Nordqvist & Vilgis, 2011).
Fig. 4. Temperature-dependent measurements of 1% agarose hydrogels with 0 (-), 20
( ,B), 40 ( ,6), and 60 ( ,>) % w/w of fructose (half-lled symbols) or sucrose
(empty symbols) added.  and - - - indicate the two step process of the gelation
mechanism of pure agarose and the corresponding temperatures.
 S. Maurer et al. / Food Hydrocolloids 29 (2012) 298e307 303

Fig. 5. Solegel transition of 1% agaroseexanthan (1:1) hydrogels supplemented with

Fig. 6. Moisture content in per cent of agaroseefructose (-), respectively agar-
0 (-), 20 ( ,B), 40 ( ,6), and 60 ( ,>) % w/w of fructose (half-lled symbols) or
oseesucrose (6) hydrogels being exposed to 100  C over a time period of 600 s ((,)
sucrose (empty symbols).
zero sugar).

As a side remark, it is mentioned that the gelling transition is measurements shown in Fig. 4. Given the two step process the rise
a quite general phenomenon, which has universal features as all of the modulus appears in two steps upon cooling. The rst step,
phase transitions. Universal critical exponents of the percolation already at relatively high temperatures, around 70  C has been
theory (rather than the mean-eld Cayley tree and graph approx- assigned by the formation of helices of the agarose molecules
imations) rule the physics around the gel point. However, to nd (being presumably completed at a temperature of 61  C as indi-
such features here appears quite difcult and is not the scope of the cated in Fig. 4). The molecular connectivity of the sample enhances,
present study. The reasons can be summarized as follows: Critical the modulus rises about two orders of magnitude. The second step
exponents for percolation, e.g. those for correlation length, cluster appears at about 40e45  C (completed around 30  C as shown in
size etc. show up clearly in thermodynamic equilibrium processes, Fig. 4). Here some of the helices aggregate and form microstruc-
i.e. at very low cooling rates, at innite systems. Alone from the tures (as indicated in Fig. 8). Agarose gels are temperature stable up
here present experimental situation (avoiding the drying out of the to 70  C, which is within the same range as the melting of the
samples) it is not possible to reach that state. More down-to -earth helices.
difculties regard the characterization of the gelling agent Looking at the curve progression of the binary hydrogel
(agarose). Its molecular weight (distribution) is not known. Indeed composition in Fig. 4 and of the ternary hydrogel composition in
the dynamics of the gelation process are relatively slow (chain Fig. 5, the addition of further fructose or sucrose has a pronounced
length, helix formation) so that agarose gels are not the best system but different impact on the solegel transition of agarose hydrogels.
for the search of exponents. Regarding agaroseefructose, respectively agaroseesucrose hydro-
Regarding the critical exponents of the modulus as dened by gels, two distinct effects are observed: First, the aggregation step is
Gf(Tg  T)t (Tg being the equilibrium gelation temperature, which shifted to lower temperatures. Secondly, the rst rise in G0 is less
cannot be measured within the framework of the experiments
carried out in this study) similar remarks can be made. The
exponent t depends strongly on the nature of the polymer chains.
For totally exible chains for example, t is identical to the exponent
of the electrical conduction of percolating conducting networks. In
contrast for stiff (more realistic) chains bending forces and torque
forces are involved (as already mentioned by de Gennes, Pincus,
Velasco, & Brochard 1976), which change the universality class.
The latter statements have been investigated by a number of
papers starting by Kantor and Webman (Kantor & Webman, 1984)
and many others; see e.g. (Stauffer & Aharony, 1994). For the
present system that means, depending on the stiffness, which
might change during increasing network connectivity by the
formation of helices in the agarose network, it is expected to see
a crossover behaviour between the two limiting cases. Again, there
is no strict thermal equilibrium during the measurements, which
means that it is unclear how to assign values for critical exponents
in the framework of such connectivity (phase) transition. For the
remarks made in the last paragraph, see e.g. Vilgis, Klppel, and
Heinrich (2009) and references therein, where the situation for
polymer network formation has been summarized.
Indeed in the gelation process of pure agarose, the gelling Fig. 7. Moisture content of agaroseexanthan hydrogels with fructose (-) and sucrose
temperature is not clearly dened according to the rheological (6) of different concentration ((,) zero sugar).
304 S. Maurer et al. / Food Hydrocolloids 29 (2012) 298e307
Fig. 8. Gelation model.
pronounced as for the pure agarose gel. Additionally, the different
sugar types used affect the solegel transition differently. While 20%
of sugar causes slight variations in the formation of agarose chains
to helices, 40% of fructose, respectively sucrose reveals two distinct
different curve progressions. 40% of sucrose in the agarose sol
provokes that G0 moderately increases from 2.1 Pa to 69.3 Pa in
a temperature range of 80  Ce37  C, whereby a pronounced rise in
G0 of agarose with 40% of fructose is already observed at 50  C. The
discrepancy in the gradients of the two previously described curves
is also reected by a nal and second rise of G0 . While 40% of
sucrose still exhibits the distinct sigmoidal curve as obtained for
the lower concentrations, a rather attened slope can be seen in
the curve of 40% of fructose. It seems to be that in the case of the
latter not only the strengthening of the elasticity has been shifted
to a lower temperature but also the formation of helices to
aggregated bundles has been affected. The curve progressions of
the jellied agarose sols combined with 60% of fructose,
respectively sucrose show that high amounts of sugar have
a rigorous impact on the gelation mechanism. First of all, the
elastic modulus is signicantly reduced, being 1.3  102 Pa at
21  C in the case of fructose and 5.5  102 Pa in the case of
sucrose. This is, in comparison to the other concentrations, two
orders of magnitude lower. Nevertheless, the relatively at
increase of the elastic modulus during cooling suggests that the
aggregation of helices has been hindered due to the high level of
sugar present. Regarding these observations, it seems to be that
the gel mechanism is more affected by the addition of fructose
than by the one of sucrose.
Comparing the temperature-dependent measurements with
the oscillation measurements of the cured agarose hydrogels,
some correlations between the evaluations of the obtained results
can be identied. For both applied analytical methods, high
amounts of sucrose reduce the elasticity of 1% agarose gels
signicantly. In contrast, jelled agaroseefructose mixtures have
shown an increase in G0 , while a decrease has been measured in
the oscillation temperature sweep. The discrepancy between
these rheological measurements might be due to the missing
hardening step in the evaluation of the sol-transition. For the
oscillation measurements, the samples were cooled at 4  C for
24 h. In the case of agaroseefructose that might have helped to
strengthen the elasticity of those gels. Concluding, drastic changes
occur when sugar concentrations of 60% are incorporated into
agarose hydrogels. Two factors, already proposed in connection
with the capability of 60% sucrose to reduce the elastic modulus of
the 1% agarose gel (sec. viscoelastic properties), might attribute to
this effect: First, the distinct increase in solution viscosity at high
co-solute levels and secondly, strong competition for water. Due
to the higher solution viscosity, the molecular chains of the dis-
solved agarose are restrained from diffusing freely by the high
amount of present sugar molecules. In terms of a nucleation and
growth mechanism, random coil structures of agarose chains
would rather tend to helix nucleation and the growth process
would be inhibited (Xiong et al., 2005). The transformation from
disordered to ordered structures is affected and presumably
shifted to lower temperatures, equivalent to a slower gelation
process.
 S. Maurer et al. / Food Hydrocolloids 29 (2012) 298e307
The examination of the solegel transition of agaroseesugar gels
has shown that the two step mechanism diminishes further with
increasing sugar concentration. Looking at Fig. 5, the shape of all
curves displayed reveals only one distinct rise in G0 of the
agaroseexanthan hydrogels. This observation coincides well with
the results obtained by Nordqvist and Vilgis (2011). Moreover,
neither the sugar concentration nor the sugar type used show any
signicant effect in the gelation process of the agaroseexanthan
gel. As only representative measurements have been chosen for
the evaluation, precise conclusions according to the impact of the
different sugar types and concentrations used on the gel formation
mechanism are not possible. Apart from that, it is shown that the
elastic modulus of these compositions is one to two orders of
magnitude lower in comparison to agaroseesugar gels free of any
xanthan.
According to the temperature-dependent measurements, it is
assumed that the aggregation of agarose helices to bundles is
inhibited by the highly negative charged xanthan rods. The physical
behaviour of the xanthan molecules seems not to be affected much
by the temperature change during the gelation process, thus still
undergoing jamming transition under the given environmental
conditions. Additionally, the charge of this polyelectrolyte probably
dominates over the effect given by the sugar concentration. Due to
their high afnity to water molecules, interactions between
waterexanthan molecules are presumably favoured so that less
water is available for the formation of hydrogen bonds with agarose
or sugar.
3.3. Moisture holding
Agarose hydrogels are susceptible to synaeresis. The water
holding capacity of biopolymers is, additional to the textural
properties, of great importance to the mouth feeling. Thus, the
percentage loss of moisture content (MC) of simple and mixed
hydrogels exposed to 100  C over a time period of 600 s has been
quantied, as illustrated in Fig. 6 and Fig. 7. 1% agarose hydrogels
devoid of any xanthan or sugar exhibit the lowest moisture content
loss (MC 53  3%) (Fig. 6). The addition of 20% of fructose to
agarose hydrogels seems to have no signicant impact on the water
holding capacity; the moisture content after 600 s is 49  2%. The
same concentration of sucrose tends to lose more water by showing
a MC of 46  4%. After 600 s the residual moisture in the gel of 40%
sugar is 36  2% in the case of fructose and 37  1% for sucrose.
Regarding the different sugar types, no signicant differences can
be observed. The highest capacity to hold water is substantiated for
agarose hydrogels with 60% of fructose or sucrose. Accordingly, the
MC, after the measurement has been stopped, of fructose
is 17  1% and of sucrose is 15  1%, revealing that sucrose holds
water slightly stronger than fructose in this concentration regime.
The results obtained by the evaluation of the water holding
capacity coincide well with the rheological behaviour of the
different agarose gels during the solegel transition. Sugar
concentrations of 20% show little impact on the gelation mecha-
nism, as well as on the water holding capacity. Increasing incor-
poration of sugar into the jelled system reveals that water is
stronger bound verifying the assumption that the helical structure
of agarose has a destabilisation effect due to water shortage
provoked by sugar molecules.
Progressively increasing the concentration of sugar in
agaroseexanthan hydrogels has hardly any impact on the water
holding capacity, except for concentrations of 60%. In general, no
signicant differences exist between 1% agarose gels and those
combined with 50% of xanthan (MC 52  3%) (Fig. 7). This is in
contrast to the previous observations made by Nordqvist and Vilgis
(2011) who reported that already little amounts of xanthan
305
contribute strongly to the improvement of the water holding on the
pure, sugar free gels. The reason for the discrepancy is twofold: On
the one hand, the preparation procedure of the agaroseexanthan
gels has been different (30 min stirring instead of 1 h, different
sample geometries) and on the other hand, the quantication of the
moisture content loss during a certain period of time has been
determined by means of different methods (Nordqvist & Vilgis
(2011) investigated the moisture loss, respectively loss in weight
during 2 h of drying at room temperature). In contrast to the
observed trend of agaroseefructose gels to enhance the water
holding capacity at a sugar level of already 20%, 20% of sucrose
actually shows a slight tendency to impair the water holding
capacity. Thus, after ten minutes of measurement, 20% of fructose
exhibits a moisture loss of 49  2%, while 51  3% is recorded in
the case of 20% of sucrose. The water evaporation is even more
intense when 40% of sucrose is incorporated in the
agaroseexanthan gel. With 55  4% it shows the lowest measured
moisture content in comparison to the other gel samples.
Regarding the relatively large error bars, it is assumed that no
statistical signicant difference can be observed. From this it
follows that the addition of up to 40% of sucrose has probably no
impact on the water holding capacity of agaroseexanthan gels.
A mixture of 1% agaroseexanthan gel with 60% sucrose results in
a gel network, which is able to hold water more strongly
(MC 41  2%). However, 40% and 60% of fructose exhibit a more
pronounced impact on the ability of the gels to hold water,
as reected by a lower moisture loss of 47  3%,
respectively 38  2%. Based on the previously discussed results,
the water holding capacity is more inuenced by sugar concen-
tration and less by xanthan gum. Agarose gels are less susceptible
to synaeresis when immersed in solutions of high co-solute
content. Xanthan used as additional additive in the agarose gel
slightly impinges on the positive impact of sugar. Moreover, it
seems that combinations of agarose, xanthan, and sucrose evolve
into gels with almost unchanged water holding capacity. It is
assumed that the particular components could not be hydrated and
mixed properly with one another. During the preparation of the
ternary solutions, sucrose crystallisation occurred presumably due
to restricted interactions with water molecules. Thus, a rather
inhomogeneous solution composed of three separate components
is generated, producing a gel whose susceptibility is probably
predominately based on the water holding capacity of the gelling
agent agarose.
3.4. Gelation and competition for water
Regarding the competition for water, a schematic model as
illustrated in Fig. 8, has been developed. It is assumed that water
molecules are directly attached to the helical agarose chains or
arrange themselves in form of hydration shells around the poly-
meric agglomerates (Fig. 8 A.1-A.2). Hydrogen bonding between
the solvent and the solute stabilises the helical structure formation
of agarose chains in aqueous solutions thermodynamically (Gekko,
Mugishima, & Koga, 1985). Likewise, the formation of hydration
shells plays an elementary role in the solvation of xanthan gum and
sugar molecules. As outlined in the gelation model, high amounts
of sugar molecules in the agaroseesugar solution react preferably
with water molecules due to their molecular structure (Fig. 8 B.1
and B.2). Thus, the shortage of water probably provokes destabili-
sation of the biopolymeric helices and seems to contribute to
a lower degree of coil-to-helix conformation. The competition for
water molecules used as tools for structural stabilisation would
also explain the alleviated increase of the elastic modulus of the
agaroseefructose, respectively agaroseesucrose hydrogels (sugar
concentration 60%) at the beginning of the cooling process (Fig. 4).
306 S. Maurer et al. / Food Hydrocolloids 29 (2012) 298e307
Additionally, water shortage might force crystallisation of the sugar
molecules involved in the network formation. In the case of
sucrose, it could be observed that small crystals settle out of the
agarose sol during the gel preparation. Presumably, (nano-) crys-
tallisation also occurred in later stages of the gelation process and
might contribute to the clear appearance of the agarose gels.
When additional co-solutes like xanthan gum are implemented
in the agaroseesugar solution, more water molecules seem to be
attracted by the highly negatively charged polyelectrolytes. Thus,
the water molecules are predominately arranged around the stiff
xanthan rods and are not any more available for the formation of
hydrogen bonds between the other co-solutes. In comparison to
agaroseesugar hydrogels of high sugar concentration (60%), the
resulting gel network is looser, less elastic and less turbid (Fig. 8
C.2). It is likely that the impact of sugar type and concentration
used is diminished by the xanthan molecules.
3.5. Effect of water on chain conformation and gelation
The experimental results can be understood, at least qualita-
tively, with some naive physical ideas. The main issues were
already stated: the interplay between the chain conformation,
mainly agarose chains, which appear as relative exible molecules,
the chain rigidity, which addresses xanthan molecules and the
water binding by hydrate shells by polar and charged groups of all
constituents.
Obviously the different sugars have different impact on the
hydrogels. Whereas the modulus of fructose gels rises with
increasing concentration up to 60% the sucrose loaded gels show
a dramatic decrease of the modulus on larger sucrose concentra-
tion. On the other hand, when xanthan is added, both sugars show
no inuence. The increase of the modulus for fructose in the total
range of measured concentrations and sucrose up to 40% may be
addressed to the increase of the viscosity of the solution. On the
other hand, low molecular weight carbohydrates may interact via
hydrogen bonds with the hydrocolloids directly, which may
implicate an apparent stiffening of the exible chains parts, which
are responsible for the entropic part of the elasticity. Stiffer chains
yield larger moduli (de Gennes, 1979), since additional bending
forces are required to deform the exible part of the chains in the
agarose networks. Additionally the helix formation is shifted
slightly to lower temperatures. This is caused by two factors: First
the viscosity of the solvent is enhanced by the presence of the
sugars. The chain segments experience more friction; the dynamics
become slower (Doi & Edwards, 1986). In addition the direct
interaction of the polar sugar molecules slows down the helix
formation between the agarose chains and shifts kinetically the gel
formation to lower temperatures.
The relatively strong inuence of the sugars on the exible
agarose network can be drawn back on the interplay between the
sugar molecules and the monomers of the agarose chains, which
consist of D- galactose connected with 3,6-anhydro-L-galactose. The
monomer unit contains ve OH-groups, which may bind water
molecules in a hydrate shell. Thus, the polymer is basically sur-
rounded by hydrate water, which has some impact on the confor-
mational properties of the polymer, e.g. its exibility, which can be
measured with the local persistence length of the polymer. When
low molecular weight sugars such as fructose and sucrose are
added, a competition for water takes place as the low molecular
carbohydrates need to be hydrated as well. To understand the
nature of the competition better, detailed molecular dynamics on
atomistic levels are required. On an intuitive level, it can be
concluded that sucrose has a higher capability to form hydrate shells
due to a larger number of polar OH-groups (Max & Chapados, 2001).
Thus, the strong decrease for sucrose loaded gels at concentrations
between 30% and 40% can be understood by the competition effect
since the hydration of sucrose is preferred. The strong drop at 60% is
additionally caused by the formation of sucrose crystallites, which
prevent the formation of helix aggregates, which in sugar free
agarose gels yields a further stiffening of the network. Indeed Fig-
s. 1and 4 indicate that the modulus of the sucrose loaded gel is of the
order of magnitude where the helices are formed.
When xanthan is added these effects are more or less ruled out.
First xanthan dominates the physical behaviour by its high charge
degree and secondly by its strong stiffness (Nordqvist & Vilgis, 2011).
Therefore sugars have no strong inuence on the mechanical
behaviour of agaroseexanthan gels, as indicated by the experiments
shown in Fig. 5 and summarized in Fig. 8. This model represents the
two-stage solidication mechanism of agarose hydrogels and the
special role of water involved in this process. The formation of
hydrogen bonds is essential for the formation of agarose chains
(Fig. 8-A.1) to helices (Fig. 8-A.2) and the subsequent aggregation of
them into bundles (Fig. 8-A.3). With the addition of high amounts of
sugar (Fig. 8-B.1), both solutes compete for the solvent water
whereby sugar molecules seem to dominate over agarose mole-
cules. Thus, restrained water molecules by fructose, respectively
sucrose prevent the formation of aggregates and lead to a destabi-
lization of the agarose gel network (Fig. 8-B.2). The resulting gel is
less elastic but clearer than gels devoid of any sugar as shown by the
photographs in Fig. 2. The water shortage by additional co-solutes in
agarose gels becomes even more pronounced when highly negative
charged stiff and rod-like xanthan molecules are present (Fig. 8-C.1).
Presumably, hydration shells are predominately arranged around
the stiff rods and the gel texture is loose and soft.
4. Conclusion
In the course of this work, it could be illustrated that the
rheological properties of 1 % agarose hydrogels are inuenced by
the addition of other carbohydrates, such as fructose, sucrose, and
xanthan gum, veried by strain-dependent and temperature-
dependent oscillation measurements. Thereby, the magnitude of
rheological changes depends partial on the type and on the
concentration of the carbohydrate used. In general, it could be
demonstrated that the addition of high amounts of sugars and/or
highly negatively charged polyelectrolytes evolves less elastic,
clearer gels of looser network structure and better water holding
capacity. The fact, that adding xanthan to hydrocolloids systems
seems to lower the inuence of the amount of added sugar, could
be systematically used for various food applications. In conse-
quence this study shows the detailed interplay between chain
exibility, chain charge fractions and hydrate shells.
Acknowledgement
The authors thank Dr. Harald Pleiner (MPI for Polymer Research)
for valuable discussions and critical reading of the manuscript.
Many regards also to Prof. Dr. Dietrich Knorr (TU-Berlin) for sup-
porting this work. Special thanks to the members of the MPIP Food
Group for fruitful discussions.
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