Food Hydrocolloids 25 (2011) 1710e1718

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

The influence of gelation rate on the physical properties/structure of salt-induced

gels of soy protein isolateegellan gum
Joice Aline Pires Vilela, Ângelo Luiz Fazani Cavallieri, Rosiane Lopes da Cunha*
Department of Food Engineering, University of Campinas (UNICAMP), P.O. Box 6121, 13083-862 Campinas, SP, Brazil

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

Article history: The cold-set gelation of soy protein isolate (SPI)-gellan gum was induced by the addition of salts (KCl or
Received 3 November 2010 CaCl2) using two different procedures: the direct addition of salts (fast gelation) or the diffusion of salts
Accepted 11 March 2011 through a membrane (slow gelation). The mechanical properties, syneresis and microstructure of the
mixed gels were evaluated, as well as for gellan and SPI gels. The mixed gels induced by calcium diffusion
Keywords: were stronger and more deformable than gels induced by the direct addition of calcium, while the
Soy protein isolate
opposite occurred for potassium-induced gels. All the mixed gels were macroscopically homogeneous,
Cold-set gelation
but at the microscopic level two independent networks could be observed. These two separate networks
Gellan
Microstructure
were more evident for the calcium-induced gels, and the structural characteristics depended strongly on
Mechanical properties the concentration of the protein and the polysaccharide. However an organized microstructure with the
formation of microtubes surrounded by other network was only observed for the mixed gels induced by
calcium diffusion at the higher protein/polysaccharide (10:1) ratio. Thus besides the composition and
concentration of the biopolymers, the results showed that the type of salt and its velocity of incorpo-
ration led to gels with different structures and consequently different mechanical properties.
Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction
Soy proteins have been widely used in the food industry due to
their very mild off-flavor, low cost, high nutritional value and inter-
esting functional properties (Belloque, García, Torre, & Marina, 2002),
such as the ability to form gels by heating or modify the quality of
a solvent. Globulins 7S and 11S represent about 70% of total soybean
storage proteins (Saio & Watanabe, 1978; Wolf, Babcock, & Smith,
1961). Both fractions are extraordinarily complex, consisting of
several subunits which easily associate and dissociate under different
conditions of pH, ionic strength and temperature (Hermansson,1986).
Usually the preparation of globular protein gels consists of three
distinct steps: denaturation, aggregation and gelation. In a typical
heat-set gel, these processes occur simultaneously during the heat
treatment. However, in cold-set gelation denaturation and aggrega-
tion are separate from the gelation step (Bryant & McClements,1998).
A preheating step induces molecular alterations (unfolding of the
protein structure and exposure of the reactive groups), necessary to
obtain the formation of soluble aggregates (Barbut & Foegeding,
1993; Hongsprabhas & Barbut, 1997). The initial pH of the solution
must differ sufficiently from the isoelectric point of the proteins
* Corresponding author. Tel.: þ55 19 3521 4047; fax: þ55 19 3521 4027.
E-mail address: rosiane@fea.unicamp.br (R. Lopes da Cunha).
(Bryant & McClements, 1998), and the ionic strength must be low
enough to avoid an immediate excessive aggregation of the protein
molecules during preheating. The protein concentration during
preheating must also be below a critical value that leads to the
formation of a three-dimensional network. The posterior cold-
induced gelation of the protein can be achieved by a reduction in pH
(Cavallieri & Cunha, 2008) or by adding cations to the protein solution
(Barbut & Foegeding, 1993). In recent years, the ability of soy protein
to form a gel network under cold conditions has aroused renewed
interest in this field because of the possibility of expanding its use in
food applications (Maltais, Remondetto, & Subirade, 2009). A variety
of salts can be added to soy protein isolate-based gels as the coagu-
lating agent, such as calcium chloride (Lee & Rha, 1977) or sodium
chloride to enhance the flavor (Yao, Tanteeratarm, & Wei, 1990). The
investigation of the effect of the partial or complete replacement of
NaCl by KCl on the protein and gel properties is necessary, in order to
maintain or improve the texture, with benefits for health of hyper-
tensive patients (Abernethy, 1979; Braga, Azevedo, Marques,
Menossi, & Cunha, 2006).
The gelling properties of soybean proteins could be improved by
interaction with other gelling agents such as polysaccharides. Gellan
gum is an extracellular polysaccharide produced by the bacterium
Sphingomonas elodea, and composed of repeating tetrasaccharide
(1,3-b-D-glucose, 1,4-b-D-glucuronic acid, 1,4-b-D-glucose, 1,4-a-L-
rhamnose) units containing one carboxyl side group. Gellan gum

0268-005X/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodhyd.2011.03.012
 J.A. Pires Vilela et al. / Food Hydrocolloids 25 (2011) 1710e1718
forms firm, hard and brittle gels, but these properties are dependent
on the ionic strength, type of cation, pH, temperature and polymer
concentration (Moritaka, Nishinari, Taki, & Fukuba, 1995; Sanderson,
1990; Yamamoto & Cunha, 2007). Its gelation mechanism is a two-
step process involving the formation of double helices during cool-
ing from the random coil chains, followed by the cation-mediated
aggregation of pairs of double helices.
In several previous studies, the interactions occurring between
the soy protein isolate and polysaccharides in different systems
have been studied; although most of them were heat-induced gels
(Braga et al., 2006; Ipsen, 1995). Despite the great number of
studies on the mechanism of the cold gelation process for single-
protein systems, the literature on the cold gelation of protein/
polysaccharide mixtures is scarce (Bryant & McClements, 2000;
Cavallieri & Cunha, 2009; Jong, Jan Klok, & Van de Velde, 2009;
Jong & Van de Velde, 2007) as compared to the heat-induced
gels. These studies showed that the mixed gel microstructure and
rheological properties depended on the charge density of the
polysaccharide, and were not only a consequence of the gelation
process, but also the phase separation typical of mixed systems.
Since these two processes were occurring simultaneously, the
gelation rate played an important role in the final gel structures.
The gelation rate in gels induced by acidification can be changed
using glucono-d-lactone, which can cause slow or fast acidification.
Cavallieri and Cunha (2008) verified that under slow acidification
conditions the molecular interactions were occurring simulta-
neously with the lowering of the pH and had sufficient time to
interact and consequently organize the structure, which did not
occur under faster acidification conditions. To the knowledge of the
authors, the change in gelation rate in salt-induced gels has not yet
been studied. In the present work, a method allowing for the slow
incorporation of salts (dialysis membrane) was used, in addition to
direct salt addition, in order to compare different gelation rates in
mixed systems.
Therefore the purpose of this work was to investigate soy protein
gellan gum gelation under cold-set conditions from their mechanical
properties, syneresis and microstructure. Gelation was induced by
direct addition or by diffusion through a membrane of potassium
chloride or calcium chloride. The soy protein isolateesalt and gel-
lanesalt binary systems were also studied in order to clarify the role of
each biopolymer in the mixed network.
2. Material and methods
2.1. Materials
Gellan gum was donated by Kelco Biopolymers (San Diego, CA)
and was used without further purification. The protein (N  6.25)
and moisture contents of the gellan gum powder were, respec-
tively, 0.64  0.11% and 7.23  0.15% (wet basis). The ion contents (%
w/w) of the gellan gum as determined by atomic absorption
spectroscopy were: calcium (0.18), sodium (0.43), magnesium
(0.07) and potassium (4.13). The soy protein isolate was obtained
from the defatted soy flour (The Solae Company, Barueri, Brazil) as
described in Section 2.1.1. The salts used were of analytical grade.
2.1.1. Preparation of soy protein isolate (SPI)
Defatted soy flour was dispersed in distilled water (1:10 w/w)
and the pH adjusted to 8.0 with 2 N NaOH. The dispersion was
gently stirred for 2 h at room temperature and then centrifuged at
10,000 g for 30 min at 4  C in a Sorvall RC5 Plus centrifuge (GSA-
rotor, Dupont, UK). The supernatant was adjusted to pH 4.5 with
2 N HCl and centrifuged at 5000 g (Sorvall GSA-rotor) for 15 min at
4  C. The precipitate was then re-suspended in water, the pH
adjusted to 8.0 with 2 N NaOH and the suspension freeze-dried
1711
(Petruccelli & Añón, 1995). The protein (N  6.25), moisture and ash
contents of the SPI powder were, respectively, 87.77  0.2%,
8.1  0.8% and 3.49  0.01% (w/w). The ion content of the SPI was
determined by atomic absorption spectroscopy, showing the
following composition (%, w/w): calcium (0.02), sodium (1.13),
magnesium (0.01) and potassium (0.06).
2.2. Preparation of biopolymer solutions and gels
2.2.1. Gellan gels
Gellan gels were prepared by the dispersion of gellan gum in
deionized water to form solutions with three final polymer
concentrations (0.3, 0.5 and 0.7% w/w). These solutions were sub-
jected to heat treatment at 90  C for 30 min, and the calcium
chloride or potassium chloride then gently added to the hot gellan
solution in order to obtain the same final ionic strength
(FI ¼ 0.06 M) for all systems. The samples were poured into plastic
cylinders with a diameter of 20 mm and height of 25 mm, which
were rapidly cooled to 10  C and incubated at the same tempera-
ture for 24 h.
2.2.2. Soy protein isolate gels
The SPI suspensions (6, 6.5, 7 and 8% w/w) were subjected to
heat treatment in a water bath for 30 min at 90  C. The solutions
were rapidly cooled to 10  C using an ice bath. The salt-induced gels
were prepared by transferring the solutions to dialysis membranes
(SnakeSkin Dialysis Tubing, 3500 molecular weight cut-off, Pierce,
Rockford, IL, USA), and dialyzing against CaCl2 or KCl solutions for
48 h at 10  C (Barbut & Foegeding, 1993; Kuhn, Cavallieri, & Cunha,
2010). The initial salts solution concentrations were calculated to
achieve equilibrium concentrations of 20 mM of CaCl2 or 60 mM of
KCl, corresponding to an ionic strength of 0.06 M. The slow diffu-
sion of salts using dialysis membranes was necessary to form SPI
gels, because the direct addition of salts led to cluster formation
and prevented the complete homogenization of the systems.
2.2.3. Mixed gels
Stock SPI and gellan gum dispersions were prepared at
concentrations of 7.5% and 1% (w/w), respectively. Each of the
dispersions was dissolved using magnetic stirring and then sub-
jected to heat treatment at 90  C for 30 min. The SPI dispersion was
mixed with the polysaccharide dispersion and diluted to achieve
the desired final concentration. The final concentration of SPI in the
samples was set at 3% while the final concentration of gellan gum
was 0.3, 0.5 or 0.7% (w/w). After mixing the biopolymers, the
dispersions were subjected to magnetic stirring at 90  C until the
complete homogenization of the system.
2.2.3.1. Direct salt addition. Calcium chloride or potassium chloride
solutions at room temperature were gently mixed with the hot
biopolymer mixtures in order to obtain the same final ionic
strength (FI ¼ 0.06 M). Immediately after addition of the salt, the
solutions were transferred to plastic cylinders (20 mm diameter
and 25 mm height), cooled in an ice bath to a temperature of 10  C
and then stored at this temperature for 24 h.
2.2.3.2. Salt diffusion. Mixed gels were also prepared by slow salt
incorporation, where the biopolymer mixtures were cooled after
the heat treatment, put into dialysis membranes (SnakeSkin Dial-
ysis Tubing, 3500 molecular weight cut-off, Pierce, Rockford, IL,
USA), and dialyzed against CaCl2 or KCl solutions with different
concentrations for 48 h at 10  C (Barbut & Foegeding, 1993). The
initial concentrations of the salts solutions were the same as those
used for the soy protein isolate gels (Section 2.2.2).
1712 J.A. Pires Vilela et al. / Food Hydrocolloids 25 (2011) 1710e1718
2.3. Uniaxial compression
The gels were subjected to uniaxial compression measurements
using a TA-XT Plus Texture Analyzer (Stable Micro Systems, UK)
with a 40 mm diameter cylindrical acrylic plate lubricated with
silicon oil to minimize friction between the sample and the plate.
The mechanical properties at rupture were obtained by com-
pressing the gels to 80% of their original height at 25  C, using
a cross-head speed of 1 mm/s. Hencky stress (sH) and strain (3H)
were calculated from the force-deformation data according to
equations (1) and (2), respectively
HðtÞ
sH ¼ FðtÞ$
H0 $A0
 
HðtÞ
3H ¼ ln
H0
where F(t) is the force at time t, A0 and H0 are the initial area and
height of the sample, respectively, and H(t) is the height at time t.
Rupture was associated with the maximum point of the stressestrain
curve. Seven replications were carried out for each gel.
2.4. Scanning electron microscopy (SEM)
Samples (w5 mm  5 mm  3 mm) of gel were fixed overnight
in 2.5% glutaraldehyde in cacodylate buffer (pH 7.2), in order to
minimize structure modification during the posterior drying
treatment. The fixed samples were fractured under liquid nitrogen
and rinsed twice with cacodylate buffer. The samples were then
dehydrated in a graded ethanolic series (20%, 40%, 60%, 70% and
90%). Dehydration was continued in 100% ethanol (three changes
over 1 h) followed by critical point drying (Balzers Critical Point
Dryer CPD03). The dried samples were mounted on aluminum
stubs and coated with gold in a Balzers Sputter Coater SCD 050. At
Fig. 1. Typical macroscopic aspect of the gels. Gellan (A) and SPI (B) calcium-induced gels, mixed gels from direct salt addition with calcium (C) or potassium (D) chloride, and mixed
gels induced by salt diffusion with calcium (E) or potassium (F) chloride.
least three images of the typical structures at a magnification of 500
or 1000 were recorded, using a JEOL JSM 5800 LV (Tokyo. Japan)
operated at 10 kV.
2.5. Syneresis
Syneresis was determined by weighing the water loss from the
gels after being placed on two layers of Whatman n 1 filter paper at
room temperature and left for 1 h (Takeuchi & Cunha, 2008). The
extent of syneresis was estimated according to equation (3):
 
Dw
Syneresisð%Þ ¼ 100$ (3)
(1) w0
where Dw is the weight of liquid separated from the gel, which was
determined by the mass difference before and after 1 h, and w0 is
(2) the initial weight of water in the gel, which was calculated from the
initial sample weight and the known water percentage of the
ingredients. Seven replications were carried out for each gel.
2.6. Statistical analyses
Significant differences (p < 0.05) between gel properties were
determined by a one-way analysis of variance, and the comparisons
between the mean values were evaluated by the Tukey procedure.
The statistical analyses were performed using the software STA-
TISTICA 5.5 (Statisoft Inc., Tulsa, USA).
3. Results and discussion
3.1. Macroscopic visual observations
Fig. 1 shows the differences between the biopolymer gels, which
were dependent on the salt added (potassium or calcium chloride)
and process used (direct salt addition or salt diffusion). Different
concentrations of biopolymers showed similar macroscopic visual
 J.A. Pires Vilela et al. / Food Hydrocolloids 25 (2011) 1710e1718 1713

Fig. 2. Stress at fracture of gels induced by direct salt addition (A) and (C) or salt diffusion (B) and (D). Gellan (A), soy protein (B) and mixed gels (C) and (D). ( ) Calcium chloride
induced gels; ( ) Potassium chloride induced gels. Bars represent standard deviation amongst replications. Different letters mean significant differences (p < 0.05). Capital letters
show the differences amongst the formulations with different biopolymer concentration. Small letters show the differences amongst formulations made with different salts.

Fig. 3. Strain at fracture of gels induced by direct salt addition (A) and (C) or salt diffusion (B) and (D). Gellan (A), soy protein (B) and mixed gels (C) and (D). ( ) Calcium chloride
induced gels; ( ) Potassium chloride induced gels. Bars represent the standard deviation among replications. Different letters mean significant differences (p < 0.05). Capital letters
show the differences amongst the formulations with different biopolymer concentrations. Small letters show the differences amongst formulations with different salts.
1714 J.A. Pires Vilela et al. / Food Hydrocolloids 25 (2011) 1710e1718

appearance. The addition of potassium chloride induced the
formation of transparent gellan gels (result not shown), while the
addition of calcium chloride led to the formation of slightly turbid
gels (Fig. 1A). Gellan gels formed by slow acidification (Yamamoto &
Cunha, 2007) were visually transparent at different final pH values,
similar to the potassium-induced gels. However, gellan gels formed
by direct acidification with HCl were turbid at the same pH values
(Moritaka et al., 1995), similar to calcium-induced gellan gels. SPI
gels could not be formed by direct calcium chloride addition and
thus a dialysis membrane was used to allow for a slow change in he
ionic environment, avoiding cluster formation (Barbut & Foegeding,
1993). A homogeneous, white, opaque gel was formed by CaCl2
diffusion (Fig. 1B), but potassium chloride diffusion using an
ionic strength of 0.06 M was not sufficient for self-supported gel
formation.
Mixed gels formed by the direct addition of calcium chloride
were composed of a continuous less turbid phase (associated with
the gellan gum) with white clusters (associated with the SPI)
dispersed throughout the gel (Fig. 1C), while white, more homoge-
neous gels were formed by diffusion of calcium chloride (membrane
method) (Fig. 1E). However, mixed gels induced by the addition of
potassium chloride showed similar macroscopic appearances,
independent of whether the salt was added directly or by controlled
diffusion (Fig. 1D and F). Previous studies of the acidic gelation of
proteinepolysaccharide systems using GDL showed that the struc-
tures of the gels were a result of the competition between gelation
(aggregation) and phase separation (Jong et al., 2009), both affected
by the gelation rate or charge neutralization. In this work, salt
diffusion through a membrane caused a slow change in ionic
strength of the environment in a way similar to that observed for the
slow acidification produced by the use of glucono-d-lactone (GDL) in
cold-set gels. Thus macroscopic phase separation was not observed
and the slower incorporation of salt allowed for the development of
a more organized structure, but such effect was clearer in the
calcium-induced gels.
3.2. Mechanical properties
Fracture stress reflects the firmness (hardness) of the gel, and the
corresponding strain is an indication of the cohesive properties
(Foegeding, Bowland, & Hardin, 1995) or gel deformability. Stress at
fracture or hardness of the gellan gels increased at higher polymer
concentrations (Fig. 2A), due to the formation of a more densely
linked network structure (Yamamoto & Cunha, 2007). However gellan
gels deformability was independent of the polysaccharide concen-
tration (Fig. 3A). In relation to the type of salt, the gels induced by
adding CaCl2 showed higher stress at fracture and lower strain at
fracture than those induced by KCl, independent of the gellan
concentration. The type of interaction promoted by salts with
different valences can be associated with these results. Divalent
cations can establish direct polyanionecationepolyanion interactions
between the carboxylate groups of adjacent double helices in gellan
gels (Chandrasekaran, Puigjaner, Joyce, & Arnott, 1988). However,
monovalent cations promote indirect crosslinking in the double
helices, forming polyanionecationewaterecationepolyanion link-
ages between adjacent helices (Chandrasekaran et al., 1988) or other
linkages, by shielding the electrostatic repulsion of the carboxylate
groups (Moritaka, Fukuba, Kumeno, Nakahama, & Nishinari, 1991).
From these results one can speculate that calcium bridges are less
deformable and more rigid than the structure formed by polymer-
epolymer interactions due to decreased electrostatic repulsion.
Stress (Fig. 2) and strain (Fig. 3B) at fracture of calcium-induced
SPI gels tended to increase at higher protein concentrations, but
a greater increase was observed between 7 and 8% protein (w/w).
Two types of cold-set SPI gels were observed with direct salt addi-
tion, depending on the Ca2þ concentration (Maltais, Remondetto,

Fig. 4. Syneresis of gellan gels (A), soy protein isolate gels (B), mixed gels induced by direct salt addition (C) and mixed gels induced by salt diffusion (D). ( ) Calcium chloride
induced gels; ( ) potassium chloride induced gels. Bars represent the standard deviation amongst replications. Different letters mean significant differences (p < 0.05). Capital
letters show the differences amongst the formulations made with different biopolymer concentrations. Small letters show the differences amongst formulations induced by
different salts.
 J.A. Pires Vilela et al. / Food Hydrocolloids 25 (2011) 1710e1718
Fig. 5. SEM micrographs of gellan gels induced by direct salt addition and SPI gels induced by salt diffusion. Gellan calcium-induced gel (A); gellan potassium-induced gel (B); 7% SPI
calcium-induced gel (C) and 8% SPI calcium-induced gel (D). Magnification of 2000.
Gonzalez, & Subirade, 2005). Filamentous gels with a translucent
appearance and soft texture were formed at low calcium chloride
concentration (10 mM), since the surface charge was not totally
screened, and the growth of aggregates was promoted preferentially
along linear paths. At higher salt concentration (20 mM), a quasi total
screening of the repulsive forces between structural units occurred,
resulting in particulate gels that were white, granular, brittle and
with clear syneresis (Maltais et al., 2005; Maltais, Remondetto, &
Subirade, 2008). In this work, under similar conditions (6% SPI and
20 mM of calcium chloride) but with salt diffusion through
a membrane, the gels were white, homogeneous, firm and with no
spontaneous syneresis. Thus slow aggregation provided enough time
for the organization of the network structure, allowing for the
production of more homogeneous gels with increased water holding
capacity. As commented before, systems with the addition of KCl at
the same ionic strength did not form self-supported gels, and
because of this, the mechanical properties could not be evaluated.
The mixed gels showed values for stress at fracture similar or
higher (Fig. 2C and D) to those of the systems formed only by SPI
(Fig. 2B), despite the much lower protein content (below the
minimum of 6%, required to form SPI cold-set gels). The minimum
concentration for gelation usually decreases when another
incompatible biopolymer is added, because of the excluded volume
effect in the single-phase mixed solution (Zasypkin, Braudo, &
Tolstoguzov, 1997). The mechanical properties of the mixed gels
induced by direct salt addition showed the same tendency as the
gellan gels, which could indicate a prevailing polysaccharide
network (Fig. 1C) over the protein network. However, the mixed
systems showed lower stress at fracture than gellan gum systems at
the same polysaccharide concentration, showing that the presence
1715
of SPI led to more complex systems, and probably phase separation
exerted an important role. The systems with the addition of calcium
salt showed higher stress at fracture values as compared to the KCl-
induced gels. However, in the same way as gellan gels, the values
for strain at fracture were not dependent on an increase in gellan
concentration, showing that this property was only controlled by
the kind of interaction (or type of salt added) occurring between
the biopolymers.
In general, the mixed gels induced by CaCl2 diffusion showed
higher stress at fracture than the gels induced by direct salt addition.
These results may be related to the greater homogeneity of the mixed
gels induced by the slower increase in ionic strength, but with
potassium chloride, the same process led to weaker gels in compar-
ison to gels induced by direct salt addition. The greatest gel strength
of the gels induced by calcium diffusion was observed at an inter-
mediate gellan concentration (0.5% w/w). An increase in biopolymer
concentration led to a faster gelation rate, but also favored phase
separation or thermodynamic incompatibility between the protein
and the polysaccharide. It seems that a combination of the protein/
polysaccharide ratio (6:1), polysaccharide concentration (0.5% w/w)
and also the slow incorporation of salts, led to gelation prevailing over
phase separation, or even to the hardest gel (maximum stress at
rupture). Above this point it seems that phase separation prevailed
over gelation, leading to weaker gels.
3.3. Syneresis
An increase in gellan concentration decreased the amount of
water released by the gel (Fig. 4A, C and D), regardless of the salt
added, which can be attributed to the greater number of hydrogen
1716 J.A. Pires Vilela et al. / Food Hydrocolloids 25 (2011) 1710e1718
Fig. 6. SEM micrographs of the mixed systems induced by direct addition of salts (A) and (B) or by salt diffusion (C) and (D). Calcium chloride induced gels: 100 (A) and 200 (C).
Potassium chloride induced gels: 200 (B) and 200 (D).
bonds between the carboxylic groups of the polysaccharide and the
water. The gels induced by calcium chloride showed greater
syneresis than those induced by potassium chloride, which can also
be associated with the kind of interaction promoted by salts with
different valences. Divalent cations form bridges between the gel-
lan chains, leaving free water in the pores of the network, while the
monovalent cations only neutralize the negative charges reducing
the electrostatic repulsion, attracting the water molecules and
forming layers of hydration around the polysaccharide (Moritaka
et al., 1991).
Syneresis of the SPI gels decreased with increasing protein
concentration (Fig. 4B), due to the greater number of sites to link
with water, in a similar way as for the polysaccharide gels. The
syneresis of the mixed systems with the direct addition of CaCl2
(Fig. 4C) was greater than for gels induced by slow salt addition
(Fig. 4D), but lower than the gellan gels despite the prevailing
polysaccharide network. This improvement in the water holding
capacity observed for gels containing both biopolymers induced by
the slow addition of CaCl2 could be partly attributed to the more
organized structure that can be achieved with this process.
However, KCl-induced mixed gels formed by direct salt addition
showed visually less syneresis than the slowly formed gels. Quan-
tification of the water released by the latter systems was not
possible because of the weaker network structure, making it diffi-
cult to detach them from the paper. A hypothesis can be raised to
explain the greater gel strength and reduced syneresis of gels
promoted by direct KCl addition as compared to those formed by
the slow incorporation of this salt. This behavior can be associated
with the non-gelation of SPI with the addition of KCl and the
predominant gellan gelation over the decreased thermodynamic
incompatibility between the biopolymers caused by the lower
molecular weight of non aggregated proteins. On the other hand
the slower salt diffusion allowed for the biopolymers to reorganize
and inter-penetrate (especially with decreased incompatibility),
making it difficult for the gellan network to form, consequently
forming a weaker, particulate gel.
3.4. Scanning electron microscopy (SEM)
Fig. 5 shows the microstructure of gellan gels induced by salt
direct addition or soy protein isolate gels induced by salt diffusion.
Two different microstructures were obtained for gellan gels (Fig. 5A
and B), depending on the kind of salt added. A thinner, homoge-
neous network was formed with the addition of calcium salt, while
the addition of the potassium salt led to the formation of two
networks: a thicker network with larger pores interconnected with
a structure with smaller pores.
On the other hand the 7% (w/w) SPI gels showed a particulate
structure (Fig. 5C), similar to a sponge, while the addition of 8%
(w/w) SPI led to a more compact and interconnected structure
(Fig. 5D). These results are consistent with those obtained for the
mechanical properties, because the increase in SPI concentration
led to an increase in stress at fracture, characteristic of a denser
network.
Mixed gels induced by the direct addition of salts (Fig. 6A and B),
showed a structure composed of two independent interpenetrating
networks, one particulate and the other dominant and finer.
Although these networks are not similar to those of the gellan or
SPI gels, the authors believe that the gellan network is the
continuous phase involving the randomly scattered SPI aggregates,
 J.A. Pires Vilela et al. / Food Hydrocolloids 25 (2011) 1710e1718 1717

Fig. 7. SEM micrographs of mixed systems induced by calcium chloride diffusion. (A) Mixed gel of 0.3% (w/w) gellan gum; (B) 0.5% (w/w) gellan gum; and (C) 0.7% (w/w) gellan
gum. Magnification of 200.

because of the visual macroscopic observation of this gel (Fig. 1C).
Larger pores were observed in the mixed systems formed with the
addition of CaCl2 as compared to the mixed gels induced by KCl,
which could explain the results for syneresis (more compact
microstructure leading to less syneresis).
Mixed gels formed by slower salt diffusion showed macro-
scopically homogeneous gels (Fig. 1), but the microscopical results
revealed two types of structure (or microphase separation) in the
CaCl2-induced gels (Fig. 6C). The potassium salt-induced gels did
not show two clearly different networks (Fig. 6D) for all the gellan
concentrations as in the case of CaCl2-induced gels, because of the
longer time taken to form the gels and consequently to form the
network rearrangements.
Fig. 7 shows the effect of increasing the gellan concentration in
mixed gels induced by calcium chloride diffusion. At a 0.3% (w/w)
gellan concentration, the systems showed an organized structure
with microtubes (more details of this structure can be seen in Fig. 8,
with a different view from that shown in Fig. 7A) with approxi-
mately 20 mm of diameter, surrounded by a network similar to the
gellan gel network. The formation of this tubular structure was
probably associated with the radial salt diffusion promoted by the
dialysis method, and was only clearly formed when 0.3% of gellan

Fig. 8. SEM micrographs of mixed gels induced by calcium salt diffusion with 0.3% (w/w) gellan gum: (A) 200 and (B) 2000.
1718 J.A. Pires Vilela et al. / Food Hydrocolloids 25 (2011) 1710e1718
was added to the system. The formation of microtubes was pre-
vented by increasing the gellan concentration to 0.5% w/w, and
a less organized structure with beads was formed in this case,
which presented the highest stress and strain at fracture values of
all the mixed systems. However the gel network formed with 0.7%
w/w gellan showed no organization and a heterogeneous system
with reduced mechanical properties (hardness and deformability)
was formed. It seems that increasing the gellan concentration led to
the formation of a more continuous polysaccharide network, and
did not allow the protein to organize and consequently form
a tubular network.
Despite the unlike microstructures for the calcium diffusion
induced gels, the results for syneresis showed no significant
differences with the increase in gellan concentration. These
different microstructures showed more relevance with respect to
the mechanical properties, since the reduction in stress at fracture
observed in the gels with 0.7% w/w gellan concentration was asso-
ciated with the more heterogeneous structure, and the non-
formation of an organized protein network. Therefore an increase in
the gellan content exerted a great influence on the microstructure/
mechanical properties produced, showing that many types of gel
texture can be formulated by changing the protein/polysaccharide
ratio, the type of salts added and the incorporation rate into the
biopolymer mixture.
4. Conclusions
The results showed that the gelation rate could be changed in
salt-induced mixed gels by using salt diffusion through a membrane
or the direct addition of salt. The membrane process led to slower
gelation and biopolymer organization, which allowed more time for
interactions and the formation of more homogeneous structures.
Moreover, depending on the biopolymer concentration and type of
salt, radial diffusion allowed for the generation of a microstructure
composed of protein microtubes, indicating that new textures could
be created by supplying the adequate process conditions.
Acknowledgments
This research was supported by FAPESP (06/58414-1 and 04/
08517-3) and CNPq (301869/2006-5 and 478691/2007-6).
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