Food Structure 13 (2017) 45–55

Contents lists available at ScienceDirect

Food Structure
journal homepage: www.elsevier.com/locate/foostr

Effect of polysaccharide concentration and charge density on

acid-induced soy protein isolate-polysaccharide gels using HCl
Wee M.S.M.* , Yusoff R., Lin L., Xu Y.Y
Food Innovation & Resource Centre, Singapore Polytechnic, 500 Dover Road, 139651, Singapore

A R T I C L E I N F O A B S T R A C T

Article history:
Received 30 March 2016 Mixed biopolymer gels of soy protein isolate with k-carrageenan, l-carrageenan and alginate were
Received in revised form 4 July 2016 produced via acidification with simulated gastric fluid (HCl) at pH 1.2. The gels could be formed
Accepted 22 August 2016 instantaneously via dripping into the acid, or over 24 h via a dialysis membrane. The main factors studied
Available online 23 August 2016 were charge density of the polysaccharide and polysaccharide concentration (0.25
1.0% w/w). Their
effects on the physical properties of the gel i.e. gel mass formed, protein concentration in supernatant,
Keywords: hardness, storage modulus (G0 ), water holding capacity (WHC) and microstructure were evaluated.
Soy protein isolate Overall, with higher charge density of the polysaccharide (at pH 1.2) and/or increasing polysaccharide
Carrageenan
concentration, the gel formed more rapidly and had greater mechanical strength due to a denser network
Alginate
between the biopolymers. As a result, WHC was also increased with improved freeze-thaw stability as
Mixed biopolymer gel
Simulated gastric fluid observed under confocal microscopy.
Acid gelation ã 2016 Elsevier Ltd. All rights reserved.

1. Introduction several reasons. Soy (plant) protein is a viable alternative to meat

Single biopolymer gels such as whey protein isolate (WPI) gels
are formed by heating, whereby the proteins are denatured,
unfolded and rearranged to form a gel network (Alting, 2003).
With the introduction of another biopolymer i.e. a polysaccharide,
interpenetrating, coupled and phase-separated networks could be
formed (Turgeon & Beaulieu, 2001). These mixed biopolymer gels
could enhance the mechanical and other functional properties of
the gel such as taste profile and digestive breakdown (Bradbeer,
Hancocks, Spyropoulos, & Norton, 2015; Turgeon & Beaulieu,
2001). Electrostatically-interacted protein-polysaccharide gels can
be made by reducing the pH to below the isoelectric point of the
protein such that a positively-charged protein interacts with a
negatively-charged polysaccharide and traps water within the gel
network. This mechanism has been explored via the concept of
‘intragastric gelation’ (Zhang & Vardhanabhuti, 2014; Zhang,
Zhang, & Vardhanabhuti, 2014), since the acidic environment of
gastric fluid (pH 2) would make the proteins positively charged
when the solution is ingested and thereby forming gels.
In this study, soy protein isolate (SPI)-polysaccharide (PS)
(k-carrageenan (KC), l-carrageenan (LC) and alginate (Alg)) gels
were studied. Soy protein was selected as the protein source due to
* Corresponding author.
E-mail addresses: may_wee_sui_mei@sp.edu.sg, maywee@gmail.com
(M.S.M. Wee).
or dairy proteins, it is representative in the Asian context, and it is
also relatively unexplored as compared to whey protein, especially
in mixed biopolymer gels. Soy protein isolate is generally
compatible with polysaccharides and is able to form mixed
biopolymer gels with polysaccharides under certain conditions.
For example, cold-set SPI-gellan gums were formed using
potassium and calcium salts (Pires Vilela, Fazani Cavallieri, & da
Cunha, 2011), heat-induced SPI-KC gels at high (>8% w/w) protein
concentrations (Fazani Cavallieri, Garcez, Takeuchi, & da Cunha,
2010), and acid-induced gels of SPI with xanthan or guar gum using
GDL (Chang, Li, Wang, Bi, & Adhikari, 2014). k-carrageenan, LC and
Alg are all polyelectrolytes but differ in physical properties such as
charge density, molecular weight and conformation. On its own, KC
is a polysaccharide which gels in the presence of potassium (K+)
ions after heating and re-cooling. Alginate gels in the presence of
calcium (Ca2+) ions. l-carrageenan, however, is non-gelling.
The resultant gel structure and properties are usually governed
by competition between phase separation and gelation (de Jong,
Klok, & van de Velde, 2009). At the high concentrations used in this
study i.e. 5% w/w SPI and 0.25-1.0% w/w polysaccharide, phase
separation should occur as a result of thermodynamic incompati-
bility. However, due to the high viscosity of polysaccharides, they
also provide a stabilising effect which delays phase separation by
slowing down molecular mobility (Huang, Kakuda, & Cui, 2001).
Many mixed biopolymer gels found in the literature are often in the
low concentration range e.g. 1–5% w/w for the protein and 0.01–

http://dx.doi.org/10.1016/j.foostr.2016.08.001
2213-3291/ã 2016 Elsevier Ltd. All rights reserved.
46 M.S.M. Wee et al. / Food Structure 13 (2017) 45–55
0.3% w/w for the polysaccharide, where phase separation occurs
more readily especially with neutral polysaccharides (de Jong &
van de Velde, 2007). Furthermore, acid-induced gels in other
studies are usually formed using glucono-delta-lactone (GDL),
whereby a longer time period is required for the pH to decrease and
allowing phase separation to take place at the same time (de Jong
et al., 2009). This current study differs in terms of acidification rate

the microstructure is arrested immediately upon contact with
the acid, leaving minimal time for phase separation. The pH at
which gelation occurs is also considerably lower in this study i.e. 1.2
as compared to 3.5 to 4.8 for GDL-induced gels.
The objectives of this study were to characterise acidified soy
protein-polysaccharide gels formed using simulated gastric fluid
(SGF), using three different polysaccharides of different charge
densities (KC, LC and Alg) at concentrations from 0.25 to 1.0% w/w.
The macro- and microstructures and physical properties of the gels
were evaluated. The use of SGF allowed the observation of the acid
gel formed under simple simulated gastric conditions. However,
gastric enzymes i.e. pepsin were not included in the SGF in order to
isolate the effects of acid-induced gelation without protein
breakdown in the process. Understanding the basic structure of
the gel would help to provide further insight to its breakdown
during digestion. In-vitro and in-vivo digestion studies of these gels
will be reported in a separate study.
2. Materials and methods
2.1. Materials
The protein used in this study was native soy protein isolate
(SPI) powder (PRO-FAM 974 ADM, Illinois, USA) with a protein, fat,
ash and moisture content of approximately 90, 4, 5 and 6% w/w
respectively according to the material specifications. Polysacchar-
ides used in this research were k-carrageenan (Grindsted
Carrageenan CL 220, Danisco), l-carrageenan (Satiagum ADC 25,
Cargill) and alginate (Grindsted Alginate FD 155, Danisco).
Simulated gastric fluid (SGF) was prepared according to the US
Pharmacopeia 33-28NF by diluting 7 ml of concentrated hydro-
chloric acid in 1000 ml of milli-Q water to approximately pH 1.2,
with an ionic strength of 0.034 M (2 g/l) NaCl (sodium chloride).
2.2. Zeta-potential
Zeta-potential of the individual protein and polysaccharide
solutions (0.1% w/w) was measured using a Zetasizer Nano ZS
(Malvern Instruments Ltd, Worcestershire, UK) based on electro-
phoresis and laser Doppler velocimetry techniques. The samples
were measured using universal folded capillary cells (DTS1060C;
Malvern Instruments Ltd, Worcestershire, UK) at 25  0.02  C in
triplicates.
2.3. Acidification by SGF
2.3.1. Dripping (Extrusion) Method
Protein (10% w/w) and polysaccharide (2% w/w) stock solutions
were prepared by hydrating the protein or polysaccharide in milli-
Q overnight under continuous stirring at room temperature
(20  C). The protein-polysaccharide mixture was made by adding
appropriate amounts of protein and polysaccharide stock solutions
to achieve the required final concentrations. Mixtures for
constructing the phase diagram had varied protein concentrations
from 0 to 7.5% w/w and polysaccharide concentrations from 0 to 1%
w/w. The protein concentration for all other experiments were
fixed at 5% w/w. The mixture was heated at 85  C in a waterbath for
30 min with continuous stirring, and cooled to room temperature
before acidification. To simulate simple intragastric gelation of the
PR-PS mixture, the mixture (9 g) was extruded into SGF (15 g)
under continuous stirring with a 10 ml syringe and syringe pump.
This extrusion process allowed the PR-PS gel beads to form on
contact with SGF, and therefore produce a more uniform size and
surface area of the beads.
2.3.2. Membrane Dialysis
In order to measure physical properties such as hardness and
storage modulus, the gels should have a consistent dimension
suitable for measurements. Gels for textural measurements were
therefore prepared by filling the protein-polysaccharide mixtures
in dialysis tubes (MWCO 12,000 25 mm, SpectraPor, USA), and
dialysed against SGF for 24 h. The resultant cylindrical gel was cut
into circular discs measuring approximately 10 mm in diameter
and 10 mm in height for measurement.
2.4. Protein concentration in supernatant & gel weight
Based on a fixed amount of PR-PS mixture (9 g total; 5% w/w SPI
concentration), gel beads were formed in a fixed amount of SGF
(15 g) with the dripping method and the protein content in the
supernatant was quantified after centrifuging. The protein content
in the supernatant would indicate the amount of protein which
was involved in formation of the gel matrix. After gel formation,
the mixture was centrifuged at 4000g (Sorvall Centrifuge, Thermo
Scientific, Waltham, MA) for 10 min at 25  C to separate the gel
beads from the SGF (supernatant) and filtered through a 0.2 mm
syringe filter (Sartorius Minisart). The protein concentration of the
supernatant was measured in UV cuvettes (FischerScientific, USA)
at an absorbance wavelength of 280 nm (Shimadzu Spectropho-
tometer UV-1800, Japan) after a 20-fold dilution. Protein concen-
tration standards were measured using bovine serum albumin
(Sigma Aldrich, USA) from a concentration of 0–1600 mg/ml. Wet
gel weight was obtained by weighing gels after decanting the
supernatant. Dry gel weight was obtained by drying the wet gels in
an oven (Memmert,USA) at 70  C for 24 h and then weighing it.
2.5. Texture analysis
Textural attributes i.e. hardness was measured using a texture
analyser (TA-XT2I/25 Texture Analyzer, Stable Micro System) with
a cylindrical aluminium probe (f = 36 mm; 36R). The cylindrical
gels (f  10 mm  10 mm) prepared using the dialysis method
were compressed at a strain of 20% and test speed of 1 mm/s. The
peak force at compression was recorded as the hardness of the
sample. Five cylindrical gels were measured from each sample, and
each sample was prepared and measured twice.
2.6. Rheological measurements
Viscosity (rotational) measurements of the protein-polysac-
charide solutions (all at 5% w/w SPI and 0.25–1.0% w/w
polysaccharide) were made using a Paar Physica rheometer MCR
301 (Anton-Paar, Graz, Austria) in controlled shear rate (CSR) mode
at 25  0.1  C (unless otherwise stated) with a cup and bob
geometry. The viscosity was determined in the shear rate range of
0.01–1000 s
1 and measurement points were recorded using the
no time setting option (equilibrium mode). The cylindrical gels
prepared using the dialysis membrane method (section 2.3) were
used to measure for storage modulus. For oscillatory rheological
measurements of the gels, amplitude sweeps were carried out
between 0.01 and 1000% strain at a constant frequency of 1 Hz at
25  0.1  C (unless otherwise stated) with a serrated parallel plate
geometry (PP25/S, Anton-Paar, Graz, Austria) at a gap height of
10 mm. Storage modulus (G0 ) was obtained at 10% strain within the
linear viscoelastic region for all samples. Measurements were
 M.S.M. Wee et al. / Food Structure 13 (2017) 45–55 47

carried out in duplicates. The gels measured were all fixed at 5% w/

w SPI concentration with polysaccharide concentrations from 0.25
to 1.0%.

2.7. Water holding capacity (WHC)

The cylindrical gels prepared using the dialysis membrane

method (section 2.3) were used to measure water holding capacity
(WHC). The gels were placed on a filter paper (Whatman #1,
55 mm; Maidstone, UK) and inserted into a centrifuge tube. The
gels were then centrifuged at 180g (Sorvall Centrifuge, Thermo
Scientific, Waltham, MA) for 10 min at 25  C, and then removed
from the filter paper and centrifuge tube. The water loss from the
gel was calculated based on the difference in weight of the
centrifuge tube with the filter paper before and after centrifuging.
Water holding capacity was calculated based on the water retained
per weight of the gel:

Water holding capacity ¼ 1
Water loss f rom gel
Gel weight
 100%

2.8. Confocal scanning laser microscopy

Soy protein was labelled with rhodamine B (Sigma Aldrich,

USA) by adding a few drops of 0.1% w/v rhodamine B to the gel
surface. Gel samples were observed using a confocal scanning laser
microscope (CSLM; FluoView 1000, Olympus, USA) at an excitation
wavelength of 543 nm. The gels were placed on a microscope slide
with a raised well to prevent structural damage to the gel when
fixing the coverslip. The absorption and emission wavelength
maxima of rhodamine B are 540 and 625 nm respectively. At least
six images were taken for each gel sample at various magnifica-
tions (x10, x20, x40 and x60) to confirm that a representative
image was taken. The experiment was also repeated twice and
similar images were obtained to check for consistency.

3. Results and discussion

3.1. Phase diagrams

A simple phase diagram was constructed for SPI-KC, SPI-LC and

SPI-Alg mixtures to determine the concentration boundaries
where acidic gelation occurs at room temperature (25  C) i.e. gel
point, with a pre-heating step at 85  C for 30 min. Pre-heating
partially unfolds the protein which exposes more functional
groups for association and therefore gelation (Perrechil, Braga, &
Cunha, 2013). The minimum SPI concentration for heat-induced
gelation is 6% w/w (Li, 2005). However, this value would be
considerably altered in the presence of another biopolymer i.e.
polysaccharide (Fig. 1).
Three outcomes (by visual observation) were possible with the
SPI-PS mixtures after subjecting to SGF via the dripping method.
The solutions either did not form gels upon contact with SGF (),
formed gels upon contact with SGF (*), or gelled upon heating and
cooling (). For non-gelling mixtures, there would be no distinct
separation between the supernatant and mixture, hence turning
the entire system turbid on gentle perturbation. In general, at low
SPI (2.5% w/w) and PS (< 0.25% w/w) concentrations, gels could not
be formed when added to SGF. As protein concentration increased
(> 5% w/w), a lower PS concentration (< 0.25% w/w) was required
to gelation. When protein and polysaccharide concentrations were
both sufficiently high (e.g. 0.75 and 1.0% w/w respectively),
gelation occurred after preheating and cooling.
For SPI-LC and SPI-Alg mixtures, weak gels were formed after
heating and cooling at SPI and LC or Alg concentrations above 5 and
0.5% w/w respectively. However, they were easily broken by
shearing (stirring at 200 rpm for approximately 5 min) and
Fig. 1. Phase diagrams of SPI with a) KC, b) LC and c) Alg; the mixtures were heated
at 85  C for 30 min and then subjected to acidification with SGF via the dripping
method; outcomes were determined by visual observation (non-gelling: ; gelling:
*; heat-gelling: ) and the dotted lines serve as visual aid for the phase boundaries.
reversed back to solution form. Although LC and Alg (in the
absence of Ca2+ ions) are non-gelling polysaccharides, they have a
higher viscosity (Fig. 2) as compared to SPI-KC mixtures. This may
have contributed to reduced mobility within the system, therefore
facilitating gelation after heating and cooling (Ako, Durand, &
Nicolai, 2011) more easily as compared to SPI-KC mixtures.
3.2. Macroscopic observations
Based on visual observations (Fig. 3), the gel beads were
generally off-white in colour and slightly translucent at low (0.25%
w/w) polysaccharide concentrations. For SPI-KC gels, the gel beads
were soft, fragile and not easily separated from SGF at the lowest
KC concentration (0.25% w/w) with a swollen appearance. As
concentration of KC increased to 1.0% w/w, the gel beads appeared
more distinctive and discrete. The SPI-LC gels were visually similar
to SPI-KC gels on increasing polysaccharide concentration. On the
48 M.S.M. Wee et al. / Food Structure 13 (2017) 45–55
Fig. 2. Viscosity (at 10s
1) of SPI-PS mixtures with varying polysaccharide
concentrations at 25  C (all SPI concentrations at 5% w/w).
other hand, the physical appearance of SPI-Alg gels did not differ
appreciably with increasing Alg concentration. As Alg concentra-
tion increased, the gels were less swollen but were soft and fragile
Fig. 3. Visual appearance of SPI-KC (row 1), SPI-LC (row 2) and SPI-Alg (row 3) gel beads at increasing polysaccharide concentrations from 0.25 to 1.0% w/w (left to right) (all
SPI concentrations at 5% w/w).
even at 1% w/w Alg. Nevertheless, Alg was able to form gels with
SPI in the absence of calcium ions which further supports the
presence of electrostatic interactions involved in gelation.
3.3. Zeta-potential
The main factor attributing to the difference in physical
properties between gels was likely to be the polysaccharide
charge density as demonstrated in other literature (de Jong & van
de Velde, 2007; Zhang, Zhang et al., 2014). Zeta-potential (ZP) of
SPI and the individual polysaccharides were measured at different
pH values. Interaction between SPI and the negatively charged
polysaccharide is greatest when pH is below isoelectric point of the
protein. Below the isoelectric point of SPI (pI  4.6; ZP = 0), the
protein becomes positively charged (ZP > 0) and therefore electro-
static attraction between biopolymers can take place. No
electrostatic interaction takes place between protein and neutral
polysaccharides (e.g. guar gum) therefore the system does not form
gels via opposite charges (Zhang, Zhang et al., 2014). The degree of
electrostatic interaction between the protein and polysaccharide
can be estimated from their ZP. Fig. 4a shows ZP of SPI, KC, LC and
Alg individually at different pH values.
Overall, LC is most negatively charged at pH < 4.6 followed by
KC and then Alg. Both KC and LC carry charged sulphate (OSO3
)
groups, whereby KC carries one sulphate group per repeating
 M.S.M. Wee et al. / Food Structure 13 (2017) 45–55
Fig. 4. a) Zeta-potential of 0.1% w/w SPI, KC, LC and Alg solutions at various pH; shaded grey area represents the pH range at which the protein is positively charged and
therefore electrostatic attraction can take place b) Absolute zeta-potential (obtained by multiplying zeta-potential of protein and the polysaccharide) at various pH.
disaccharide unit, and LC carries three sulphate groups per
repeating diasaccharide unit. The charge densities for KC and LC
are therefore 0.5 and 1.5 mol/mol (mol negative charge per mol
monosaccharide) respectively (de Jong & van de Velde, 2007).
Although the charge density of Alg is also relatively high at 1 mol/
mol, the pKa of Alg at 3.5 (Harnsilawat, Pongsawatmanit, &
McClements, 2006) results in partial or complete protonation at
pH < 3.5. As a result, electrostatic charges between SPI and Alg
were not as strong as KC and LC at pH of the SGF. Fig. 4b shows the
absolute ZP between SPI and the respective polysaccharide. The
greatest interaction would be between SPI and LC at pH 2.5 since
the absolute ZP is highest at this pH. The trend is similar for KC,
although the absolute values were lower than LC at all pH values
below 3.5. Alginate has the smallest degree of interaction with SPI
at pH < 3.5.
3.4. Protein concentration in supernatant
Fig. 5 shows the protein concentration remaining in the
supernatant with increasing polysaccharide concentration for
SPI-KC, SPI-LC and SPI-Alg gels. All three polysaccharides showed
the same trend of decreasing protein concentration in the
Fig. 5. Protein concentration in supernatant (mg/ml) after formation of SPI-KC, SPI-
LC and SPI-Alg gels (all SPI concentrations at 5% w/w).
49
supernatant as polysaccharide concentration increased. Overall,
SPI-Alg gels had the highest protein concentration in the
supernatant while SPI-LC gels had the lowest. Since SPI-Alg gels
were the weakest and had the least charged interactions with the
polysaccharide therefore more protein remained in the superna-
tant. Conversely, SPI-LC had the strongest interaction and therefore
the lowest supernatant protein concentration. There were no
obvious differences between SPI-KC and SPI-LC gels in terms of
supernatant protein concentration, although it was slightly lower
for SPI-LC gels. As seen from Fig. 5, there was a slight increase in
supernatant protein concentration at 1.0% w/w LC. By visual
observation, the supernatant turned slightly turbid after forming
the gels. This was likely due to saturation of charges between the
protein and polysaccharide, which resulted in excess protein and/
or polysaccharide in the supernatant and hence the turbid
appearance.
3.5. Gel weight
Apart from protein concentration in supernatant, the gel weight
was also used as an indicator for the interaction between protein
and polysaccharide. Fig. 6a shows the wet gel weight of the beads
with increasing polysaccharide concentration. The wet gel weight
overall decreased with increasing polysaccharide concentration as
a result of less water being adsorbed by the gel beads (Fig. 3). The
dry gel weight (Fig. 6b) however, increased with polysaccharide
concentration. This was in agreement with the supernatant protein
concentration (Fig. 5), as more protein (and also polysaccharide)
would be entrapped in the gel matrix, resulting in a higher gel
mass.
For SPI-LC gels, there was a slight decrease in dry gel weight at
1.0% w/w LC, along with an increase in supernatant protein
concentration (Fig. 5). Further increasing the polysaccharide
concentration to 1.0% w/w could lead to repulsion of negative
charges amongst polysaccharides in the solution and thus a looser
network between protein and polysaccharide when forming the
gel. For LC which has a higher charge density, it is possible that
saturation of the positive charges on the protein was reached at a
lower polysaccharide concentration. As mentioned, the superna-
tant turned turbid at this LC concentration.
The higher dry gel weight observed at 0.25% w/w is not well
understood at this moment. It could be due to the lower mixture
viscosity (Fig. 2) which allowed better interaction between protein
and polysaccharide as the biopolymer molecules have greater
mobility. Although with increasing polysaccharide concentration
there would be more interaction with the protein, this may come
with other opposing effects to gel formation, such as increasing
50 M.S.M. Wee et al. / Food Structure 13 (2017) 45–55

Fig. 6. a) Wet weight and b) dry weight of SPI-KC, SPI-LC and SPI-Alg gels with increasing polysaccharide concentration (all SPI concentrations at 5% w/w).

electrostatic repulsion between polysaccharides or higher viscosi-
ty of the continuous phase.
3.6. Hardness and storage modulus (G0 )
In order to measure physical properties such as hardness and
storage modulus, the gels were prepared via dialysis membrane to
obtain a cylindrical gel shape. The resultant appearance of the gels
are as shown on Fig. 7. Only SPI-KC and SPI-LC gels were analysed
as the SPI-Alg gels were non-self-supporting and could not be
taken out of the dialysis tubing intact. It was expected that the gels
produced using the dialysis method may have certain microstruc-
tural differences from those by gel bead extrusion (Fig. 3), since
gelation did not occur instantaneously and other effects such as
phase separation may have been predominant. This will be
examined using confocal microscopy in a later section.
For the visual appearance of SPI-KC gels, increasing polysac-
charide concentration led to a firmer gel although the cross-
sectional surface became coarser and uneven. Less syneresis was
also observed. The same was seen for SPI-LC gels, although a
hollow centre was found at 0.75 and 1.0% LC concentrations. This
hollow centre was likely a result of rapid gelation on the exterior,
thus preventing the diffusion of acid into the interior of the gel
(within 24 h). It could also be due to the higher viscosity (Fig. 2) of
SPI-LC gels which slowed down the rate of acid diffusion into the
gel centre.
The mechanical strength of the gels were tested at both large
(nonlinear) and small (linear) deformations measuring hardness
and storage modulus respectively. The hardness of the SPI-KC gels
increased with polysaccharide concentration. However, the
hardness of SPI-LC gels did not increase with polysaccharide
concentration but rather decreased dramatically above 0.75% w/w
LC. For both SPI-KC and SPI-LC gels, G0 increased exponentially with
increasing polysaccharide concentration, and SPI-LC gels had an
overall higher G0 than SPI-KC gels except at 1% w/w polysaccharide.
An increasing hardness or G0 with polysaccharide concentration
indicated a denser gel network with more interactions (Yamamoto
& Cunha, 2007) since there would be more negatively charged
groups on the polysaccharide available to bind to the positively
charged groups on the protein. This was also observed in many
protein-polysaccharide gel systems, including heat-induced SPI-
KC gels which had increasing rupture stress with increasing
polysaccharide concentration (Fazani Cavallieri et al., 2010), and
acid-induced WPI-xanthan gum and WPI-carrageenan gels which

Fig. 7. Visual appearance of SPI-KC (top row) and SPI-LC (bottom row) gels at 0.25, 0.5, 0.75 and 1.0% w/w polysaccharide concentrations formed via dialysis against SGF for
24 h (f  10 mm x 10 mm) (all SPI concentrations at 5% w/w).
 M.S.M. Wee et al. / Food Structure 13 (2017) 45–55
had higher G0 as polysaccharide to protein ratio increases (Zhang,
Zhang et al., 2014). The type of interactions formed during
complexation e.g. electrostatic, covalent (sulphide) or hydrophobic
also determines the strength of the gel (Alting, Hamer, de Kruif, &
Visschers, 2003).
At 0.75–1.0% w/w KC however, there was an unexpected
increase in gel hardness as well as G0 . The gel strength was
expected to plateau with increasing polysaccharide concentration
since the electrostatic interaction between charges would saturate
and no further interactions could be formed. Therefore it is
possible that there is a synergistic effect between KC and SPI and
not between LC and SPI. It has been found in other studies that KC
may exhibit synergy with proteins such as soy and pea protein
(Ipsen, 1995), and dairy proteins (Ould Eleya & Turgeon, 2000).
Furthermore, KC is a gelling polysaccharide while LC is non-gelling.
Since KC gels upon heating and cooling, the preheating step prior to
acidification may have resulted in partial gelation of a KC network,
which contributed to additional elasticity of the gel at sufficiently
high KC concentrations (Ako et al., 2011).
The discrepancy between small and large deformation behav-
iour for SPI-LC gels may be due to the difference in length scales
measured under small and large deformation (Vliet, 1995), where
stress is applied in the form of compression (uniaxial to strain) for
hardness vs. shear (parallel to strain) for storage modulus. Gel
hardness is determined by both the number of effective strands in
the gel and the modulus of the protein strands (van Vliet, 1999),
while the elastic modulus (G0 ) is governed by the number of elastic
effective junctions between strands (Alting et al., 2004). Under
compression (large deformation), it is likely that the hollow
centres of the SPI-LC gels at 0.75 and 1.0% w/w contributed
significantly to their structural weakness as observed in their
visual appearances (Fig. 7). For G0 , since the deformation is within
the linear region at mm length scales, the gel maintains its
structure and is not affected at length scales of the hollow centre
for the SPI-LC gels (i.e. >1 mm).
Charge density, and therefore consequently the gelation rate
were likely to have played an important factor in determining the
mechanical strength of the gels. l-carrageenan, which has a higher
charge density than KC, has an overall higher G0 for SPI-LC gels than
SPI-KC gels due to more interactions between the protein and
polysaccharide. At the same time, however, gelation then occurred
more rapidly for SPI-LC as compared to SPI-KC gels as manifested
by the hollow centre in the cylindrical gels (Fig. 7). This in turn led
to a weaker gel strength under compression. Although Alg has a
high charge density at neutral pH, the total absolute charges
between SPI and Alg were low at acidic pH (Fig. 4b), due to the high
pKa of Alg (pKa  3.5) (Draget, Moe, Skjak-Braek, & Smidsrod,
Fig. 8. a) Hardness at g = 20% based on compression test (large deformation) and b) G0 at g = 1% based on oscillatory rheological test of SPI-KC and SPI-LC gels (f  10 mm x
10 mm) (all SPI concentrations at 5% w/w).
51
2006). As a result, the SPI-Alg gels formed were weak and non-self-
supporting. On the other hand, the OSO3
groups in KC and LC have
a lower pKa (<2) than alginate (Gu, Decker, & McClements, 2004).
Furthermore, the OSO3
groups have a strong attraction to the local
protein amino groups (NH3+) which contributed to the compati-
bility of the biopolymers and therefore the strength of the gels
(Grinberg & Tolstoguzov, 1997).
This is somewhat in agreement with Zhang, Hsieh et al. (2014);
Zhang, Zhang et al. (2014); de Jong and van de Velde’s (2007) work
on the effects of charge density on gel strength, whereby the
polysaccharide with a higher charge density formed stronger gels.
In de Jong et. al.’s (2007) work, it was found that for
polysaccharides with a high charge density (>0.7 mol/mol) i.e.
LC, increasing polysaccharide concentration in the mixed gel also
increased the gel fracture stress. On the other hand, polysacchar-
ides with intermediate charge density (0.3–0.7 mol/mol) i.e. KC,
have decreasing fracture stress with increasing polysaccharide
concentration. Their results do not reflect the findings in this paper,
possibly due to the different acidification mechanism, as well as
the concentration range investigated. In their paper, the authors
used GDL to form the gels which would have led to phase
separation in the duration in which the pH was decreased.
Additionally, the polysaccharide concentrations used was up to
0.3% w/w, where viscosity effects were not as pronounced. The
high viscosity (Fig. 2) would have helped to slow down phase
separation and formed a different microstructure. Therefore this
method of rapid acidification using HCl may be a viable and
effective method for making gels at high polysaccharide concen-
trations.
3.7. Water holding capacity (WHC)
The water holding capacity (WHC) of SPI-KC and SPI-LC gels
(prepared via dialysis method) are as shown on Fig. 9. SPI-Alg gels
were not determined for WHC as the gels were not self-supporting
and prone to disintegration. The WHC increased with polysaccha-
ride concentration for both KC and LC, although the SPI-KC gels still
had an overall lower WHC than SPI-LC gels. The gels at 0.25% w/w
polysaccharide were also observed to exude more serum under its
own weight when left to stand (Fig. 7).
If one were to take the weight difference between the wet
(Fig. 6a) and dry (Fig. 6b) gels, there would be a decreasing amount
of water loss to the contrary of an increasing WHC with increasing
polysaccharide concentration. Therefore the WHC would refer to
the water tightly bound to the protein-polysaccharide gel which is
not removed by centrifugation whereas parameters such as
swelling ratio or water-binding capacity would take into account
52 M.S.M. Wee et al. / Food Structure 13 (2017) 45–55
Fig. 9. Water holding capacity (%) of SPI-KC and SPI-LC gels (all SPI concentrations
at 5% w/w).
the water adsorbed by the gel. It has been reported that with
smaller crosslink densities there would be increased swelling due
to solubilisation of the whey protein (Peters, Luyten, Alting, Boom,
& van der Goot, 2015). At lower polysaccharide concentrations,
there would also be fewer protein-polysaccharide interactions and
therefore more sites on the protein and/or polysaccharide are
available to bind free water (Zhang, Hsieh, & Vardhanabhuti, 2014),
giving the swollen gel appearance with a high wet gel weight.
The hardness and G0 of the gels correlated with its WHC i.e.
increasing hardness or G0 and WHC with polysaccharide concen-
tration. Furthermore, LC with a higher charge density (and firmer
gel) (Fig. 8) formed gels with higher WHC as compared to the SPI-
KC gels. Higher polysaccharide concentrations and charge density
resulted in a denser (firmer) gel network which is able to trap more
Fig. 10. Microstructure of SPI-KC (top row), SPI-LC (middle row) and SPI-Alg (bottom row) gel beads; scale bar represents 100 mm (image size: 210  210 mm).
bound liquid within the gel network that cannot be removed under
the current centrifugation conditions. The hydrophilic nature of
polysaccharides would also contribute to increased water-binding.
However, an increase in polysaccharide concentration does not
always necessarily translate to a higher WHC in mixed protein-
polysaccharide gels. The gelation mechanism governs the micro-
structure of the gel which in turn affects the WHC. In whey protein-
pectin gels, increasing pectin concentration led to a decrease in
WHC due to thermodynamic incompatibility resulting in a coarse
micro-phase separated network. Therefore the WHC of these gels
will be further discussed in the next section in relation to its
microstructure.
3.8. Microstructure
The microstructure of SPI-KC, SPI-LC and SPI-Alg gel beads are
as shown on Fig. 10. The red (bright) areas represent the gel
(protein-polysaccharide network) stained by Rhodamine B while
the dark areas represent the pores.
The SPI-KC and SPI-LC gels were fairly similar in their
microstructure with increasing polysaccharide concentrations.
There were distinct pores in the gel network at polysaccharide
concentrations from 0.25 to about 0.625% w/w, which initially
increased in size (from 5 to 20 mm) but decreased in pore
numbers within this concentration range. These pores were no
longer visible at 0.75 or 1.0% w/w within the red areas. Instead,
there were large regions of dark areas which were likely to be due
to the coarseness and uneven surface of the gel viewed on a single
focal plane. It may appear at first glance that phase inversion has
occurred at 1.0% w/w KC and LC. However, based on visual
observations, the gel beads took shape more rapidly at 1.0% w/w (as
compared to 0.25% w/w) KC and LC, which contradictorily reduces
the time for phase separation before gelation. This rapid gelation
combined with the high solution viscosity may have prevented
slow rearrangement of the gel structure after gelation, hence the
coarse and uneven texture appearing as dark areas.
The SPI-Alg gels have a distinctively different microstructure
from SPI-KC and SPI-LC gels. The pores were much larger (20 mm
compared to 5 mm for SPI-KC and SPI-LC gels) especially at 0.375-
 M.S.M. Wee et al. / Food Structure 13 (2017) 45–55
Fig. 11. Microstructure of 5% SPI-0.375% Alg gel; scale bar represents 100 mm
(image size: 317  317 mm).
Fig. 12. Microstructure of SPI-KC (top row), SPI-LC (middle row) and SPI-Alg (bottom row) gels (image size: 210  210 mm).
53
0.5% w/w concentration, where the pores were enclosed by a web-
like thin-stranded network distributed among gel clusters. The
overall microstructure was better viewed at x10 magnification
(Fig. 11). Numerous pores (10 mm) formed a web-like structure
around clusters of the gel. It is unclear whether the clusters were
solely protein-rich clusters or the strands were made of interacted
protein and polysaccharide complexes. The web-like strands
running through the gel network may be the most important
factor contributing to its weak mechanical strength and hence not
self-supporting gel when made via the dialysis method. At 0.75 and
1.0% w/w Alg, the network became particulate-like (bright red
dots, possibly concentrated dye spots) which could be due to
protein aggregation as a result of phase separation in excess
polysaccharide concentration (van den Berg, Rosenberg, van
Boekel, Rosenberg, & van de Velde, 2009).
The microstructures of SPI-KC and SPI-LC were not distinctively
different, and their other properties also had similar trends and
values. Increasing polysaccharide concentration reduced the
porosity of the gel, which correlated with an improvement in
WHC, hardness and G0 . Coarsening of the gel texture and reduced
pore sizes suggests an increase in localised protein concentration
of the gel complex, which could improve the mechanical strength
of the gel (de Jong & van de Velde, 2007) since the hardness of the
gel is related to the number of effective protein strands within the
matrix (van Vliet, 1999). The polysaccharide may be bound and
buried within large clusters of protein aggregates upon interaction,
which results in larger pores when interaction is stronger and
54 M.S.M. Wee et al. / Food Structure 13 (2017) 45–55
smaller pores evenly distributed in gel network when interaction is
not as strong (Zhang, Zhang et al., 2014). However, it should be
noted pore sizes do not necessarily always correlate to mechanical
strength
gels with similar cluster and pore sizes may have
contrasting rheological behaviour depending on network connec-
tivity, therefore pore sizes may not be a good indicator on gel
strength (Eugenia Hidalgo et al., 2015; Olsson, Langton, &
Hermansson, 2002).
Microstructures of the gel beads were also taken before freezing
and after thawing (Fig. 12). On freeze-thaw, the microstructure of
the gels at 0.25% w/w polysaccharide appeared to have undergone
extensive rearrangement with the appearance of large dark
regions. Pores were also no longer visible within the red (bright)
areas and became smoother as well. These dark areas were likely to
be porous regions created by the freezing and melting of ice
crystals, which is commonly observed in freeze-thawed matrices
(Charoenrein, Tatirat, Rengsutthi, & Thongngam, 2011). Since ice
Fig. 13. Microstructures of SPI-KC (rows 1 & 2) and SPI-LC (rows 3 & 4) gels at 0.25
and 1.0% w/w polysaccharide concentrations made via fast (left) and slow (right)
acidification.
has a larger volume than water, this was likely to result in an
excluded volume effect on the gel, thus concentrating the gel
regions and therefore tightening the pores. At low polysaccharide
concentrations, the gels had a weaker network i.e. poorer hardness,
G0 and WHC, which exacerbated the freeze-thaw effect as the gel
structure could not maintain its integrity on formation of ice
crystals. In contrast, the microstructure of the gels at 1.0% w/w
polysaccharide appear unchanged by freeze-thaw. Therefore the
polysaccharide concentration plays a critical role in the gel
network structure and integrity, whereby a denser network would
result in better mechanical strength and water-binding.
The microstructures of the gels made by two different methods
i.e. dripping (fast acidification) and membrane dialysis (slow
acidification) were also compared (Fig. 13). The difference between
slow and fast acidification were most prominent at low polysac-
charide concentrations i.e. 0.25% w/w. For SPI-KC gels at 0.25% w/w
KC, the microstructure with slow acidification had more dark
regions with less connectivity seen in the microstructure without
the pores which could be due to phase separation during the
process of gelation. Similar observations were made for the SPI-LC
gels at 0.25% LC, whereby the microstructure was coarser and more
phase separated with slow acidification. At 1% w/w polysaccharide
concentration, however, the microstructure of the gel did not differ
much in terms of the non-protein rich areas for both SPI-KC and
SPI-LC gels. It should also be noted that the cylindrical gel samples
made from slow acidification were taken from within the cross-
sectional area whereas the gel beads made from fast acidification
were viewed from its surface. The difference in sampling methods
may have contributed to differences in microstructure.
4. Conclusion
Polysaccharide concentration and charge density were the
main factors determining the mechanical strength, physical
properties and microstructure of the gels. Polysaccharide concen-
tration correlated well with the extent of interaction and therefore
mechanical strength of the gels. With increasing polysaccharide
concentrations, there were less protein in the supernatant and
greater gel mass formed. The gels were also harder and more
elastic, which improved its WHC and freeze-thaw stability. The
presence of numerous pores distributed among the gel network
may be the contributor of poor gel strength. Nevertheless, factors
such as rate of acidification and viscosity of the protein-
polysaccharide mixture should also be taken into account since
they affect the overall thermodynamic stability of the system and
hence final microstructure. The polysaccharide with the highest
negative charge density at pH 1.2 i.e. LC also formed the strongest
gels under small deformation, followed by KC, and Alg which has a
low charge density at pH 1.2 did not form self-supporting gels
using the dialysis method. Overall, this method of forming acid-
induced gels using HCl would be suitable for systems with high
polysaccharide concentrations (therefore viscosity). It can also be
used for simple simulation of intragastric gelation. Future work
include in-vivo and in-vitro digestion studies of these gels, as well
as using a different plant protein i.e. pea protein to form these acid
gels.
Industrial relevance
The formation of soy protein isolate-polysaccharide gels in the
presence of simulated gastric fluid shows a possibility of conferring
satiety in consumers. It is also an alternative method of forming
acid-induced gels as opposed to using glucono-delta-lactone
(GDL). Soy-based products are very popular in Asia and therefore
this research would be even more relevant in the Asian context.
 M.S.M. Wee et al. / Food Structure 13 (2017) 45–55
Acknowledgements
The authors would like to acknowledge the Ministry of
Education, Singapore for the financial support under the transla-
tional innovation fund (TIF) (Grant No.: J030) as well as Singapore
Polytechnic, Singapore for the internal TIEFA grant. The authors
would also like to thank colleagues Chiang Jiehong and Grace
Foong En Yi for their kind assistance in this project, as well as
Kelvin Goh and Lara Matia-Merino of Massey University for their
insightful advice.
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