Food Hydrocolloids 24 (2010) 502–511

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

Interactions between milk proteins and gellan gum in acidified gels

Carolina Siqueira Franco Picone, Rosiane Lopes da Cunha*
Department of Food Engineering, Faculty 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 mechanical properties, microstructure and water holding capacity of systems formed from whey
Received 18 May 2009 protein concentrate (0–3% WPC w/w), sodium caseinate (0–2% w/w), and gellan gum (0.1–0.3% w/w) in
Accepted 23 December 2009 the coil or helix conformational state (Coil/Helix), were investigated. This polymer combination resulted
in bi-polymeric or tri-polymeric systems, which were slowly acidified to pH 4.0 by the addition of GDL in
Keywords: order to favor electrostatic protein–polysaccharide interactions. The properties of the tri-polymeric
Gellan gum
systems differed considerably from the bi-polymeric ones. At high polymer concentrations the WPC-
Milk proteins
gellan samples showed incompatibility and microphase separation, which resulted in weaker and less
Mechanical properties
Polymer interactions deformable gels. However, in systems with coil gellan the incompatibility was less intense, which was
attributed to the formation of electrostatic complexes between the protein and the polysaccharide
during the mixing process. In caseinate–gellan systems, complex formation was observed and an
increase in the gel mechanical properties as the caseinate concentration rose, although the water holding
capacity decreased at higher gellan concentrations. The caseinate–gellan coacervate was not visualized in
the tri-polymeric systems and the incompatibility between the biopolymers was intensified, although
the mechanical properties were considerably higher than in the bi-polymeric gels.
Ó 2009 Elsevier Ltd. All rights reserved.

1. Introduction
Food products are complex multi-component mixtures which
hinders comprehension of the role of each ingredient in their
interactions and thus their influence on the properties of the final
product. The use of model systems is a helpful way of correlating
the physical and sensory properties of materials without the
constraints of a particular food product (Tolstoguzov, 1996). Rele-
vant food structure models can be obtained by the combination of
native or denatured proteins with polysaccharides (Brownsey &
Morris, 1988; De Jong & Van De Velde, 2007; Tolstoguzov, 2000).
The interactions between these polymers depend on their charac-
teristics and environmental conditions such as temperature, ionic
strength, pH and previous treatments (Panouillé & Larreta-Garde,
2009; Valim, Cavallieri, & Cunha, 2009). Associative interactions
typically occur between negatively charged polysaccharides and
positively charged proteins by electrostatic attraction, often
resulting in complexation or coacervation (Dickinson, 2003).
Repulsive interactions lead to incompatibility between proteins
and polysaccharides as a result of differences in their molecular
properties, such as shape, size or charge (e.g. when both polymers
* Corresponding author. Tel.: þ55 19 3521 4047; fax: þ55 19 3521 4027.
E-mail address: rosiane@fea.unicamp.br (R.L. da Cunha).
have the same charge) and may cause phase separation (De Jong,
Klok, & Van De Velde, 2009). In gelling systems the balance
between phase separation and the gelation process determines
their structure and mechanical properties. In general, the relative
concentration of a biopolymer mixture is crucial for the gelling
process. Increasing the polymer concentration can enhance the
gelling process, since the macromolecules become closer to each
other, facilitating aggregate formation and contributing to the
strengthening of the structure (Yamamoto & Cunha, 2007).
However, above a critical value, thermodynamic incompatibility
takes place and phase separation is observed (De Jong & Van De
Velde, 2007).
Protein gelling can be achieved by chemical or enzymatic cross-
linking, salt addition, lowering the pH of the solution, pressure
application or heat treatment (Totosaus, Montejano, Salazar, &
Guerrero, 2002). Heat treatment and acidification have particular
importance in dairy products, since they are common steps in the
processing of milk products. The heat treatment exposes reactive
groups on the protein which were previously inaccessible.
Lowering of the pH reduces electrostatic repulsion, enhancing
interactions between the molecules.
Gelling polysaccharides such as carrageenans, gellan gum and
agar show helix (ordered)–coil (disordered) transition with
increasing temperature, which corresponds to the gel–sol transi-
tion. Gellan gum is a linear and anionic heteropolysaccharide

0268-005X/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodhyd.2009.12.007
 C.S.F. Picone, R.L. da Cunha / Food Hydrocolloids 24 (2010) 502–511 503

produced by Sphingomonas elodea (Nishinari, 1999). Gellan has
a pKa value near 3.5, which is given by its monomer, glucuronic
acid. In solutions where the pH > pKa, gellan gum is a polyanion
and there is an increase in electrostatic repulsion between the
molecules. It is a relatively low charge density polysaccharide with
an average charge density of 0.25 negative charges/mol of mono-
saccharide in the form of the carboxylic acid group (De Jong & Van
De Velde, 2007). At temperatures above 50  C, gellan polymers in
aqueous solutions are in the disordered single coiled state, but
when cooled, threefold left-handed double helixes are formed
(Chandrasekaran & Radha, 1995). After this coil–helix transition,
the gellan double helix can aggregate to form junction zones.
Hydrogen bonds between the junction zones are induced by
lowering the pH or adding salts, resulting in macroscopic gel
formation (Yamamoto & Cunha, 2007).
The conformational state of the polysaccharide may also
determine interactions with other biopolymers, such as proteins.
Proteins usually interact with the polysaccharide junction zones in
the helix state if the pH value is low. However, if the protein is
added to a coil polysaccharide, it can interact with a single
polysaccharide molecule, decreasing the polysaccharide helicity
(Burova et al., 2007). Burova et al. (2007) studied complex
formation between carrageenans and b-casein, and noticed that it
was related to the protein concentration and pH. Studies on gellan
gum – milk proteins are scarce (De Jong et al., 2009; Sosa-Herrera,
Berlib, & Martı́nez-Padilla, 2008) and the influence of the gellan
conformational state in the interactions with such proteins is
unknown. Moreover, there is no information about the behavior of
complex structures such as tri-polymeric systems containing gel-
lan gum.
Thus, the aim of the work was to investigate the mechanical
properties, microstructure and water holding capacity of gels
formulated with gellan gum, whey proteins and sodium caseinate,
as well as the influence of the conformational state of the poly-
saccharide on these properties. Therefore, bi and tri-polymeric
systems formulated using different polymer concentrations and
gellan gum conformations and acidified to pH 4.0 by adding GDL,
were studied.
2. Material and methods
2.1. Materials
Deacylated gellan gum powder (KelcogelÒ F) (3.42% moisture
content) was kindly donated by Kelco Biopolymers (San Diego,
CA). Acid digestion (nitric and hydrochloric acid) followed by
Inductively Coupled Plasma (ICP) Emission Spectroscopy (Perkin
Elmer – Optima 3000 DV, Waltham, MA, USA) was carried out to
characterize the gellan gum. The following ion composition was
obtained (% w/w): Kþ ¼ 4.13, Naþ ¼ 0.43 and Ca2þ ¼ 0.18. Whey
protein concentrate (WPC) (2.30% moisture content, 77.62% total
protein content) was provided by Fonterra (Clandeboye, New
Zealand) and showed the following ion composition as deter-
mined by IPC (% w/w): Kþ ¼ 0.46, Naþ ¼ 0.38 and Ca2þ ¼ 0.41.
prepared by dissolution of the casein in deionized water, with
constant magnetic stirring for 5 h at a maximum temperature of
50  C. The pH was constantly adjusted to 6.7 with 10 M NaOH. The
7% (w/w) WPC stock solution was prepared by dispersing the
powder in deionized water and leaving for 1 h at room tempera-
ture. The solution was then heated at 80  C for 30 min in a jacketed
vessel with mild agitation, to induce protein denaturation, and then
cooled to 10  C in an ice bath. The 0.7% (w/w) gellan gum stock
solution was also prepared by stirring the gellan powder in
deionized water at 80  C for 30 min in a jacketed vessel, followed by
cooling to 10  C in an ice bath.
2.2.2. Gel preparation
The coil samples (CS) were prepared by mixing the stock
solutions of WPC and gellan gum (just before cooling) with
caseinate stock solution and water (for dilution to desired
concentration) at 80  C for 5 min, in order to obtain the final
concentrations shown in Table 1. The samples were then cooled to
10  C in an ice water bath and the necessary amount of GDL added
with magnetic stirring for 2 min, to reduce the pH to 4. The
homogeneous mixtures were poured into cylindrical plastic tubes
(21 mm diameter  21 mm height) and maintained at 10  C for
72 h for gel formation.
The helix samples (HS) were prepared by mixing the stock
solutions and water at 10  C for 5 min. After mixing, GDL was
added and the same procedure used as applied in the formation of
CS gels. The polymer concentration range evaluated is also shown
in Table 1.
2.3. Mechanical properties
The uniaxial compression of the gels was carried out using
a TA-XT Plus Texture Analyser (Stable Microsystems Ltd., Surrey,
England) equipped with a 40 mm diameter cylindrical acrylic
plate lubricated with silicon oil to minimize friction between
the sample and the probe. The gels were compressed to 80%
of their original height at 10  C, using a crosshead speed of
1 mm/s.
Fracture stress (sH) and strain (3H) were calculated from the
force-deformation data according to Eqs. (1) and (2), respectively
(Steffe, 1996):
 
HðtÞ
sH ¼ FðtÞ (1)
H0 A 0
 
HðtÞ
3H ¼ ln (2)
H0
Table 1
Gel compositions: gellan concentration of each system according to the whey
protein (WPC) and sodium caseinate (CN) concentrations, for helix samples (HS) and
coil (CS) samples.

Casein (6.25% moisture content, 86.29% total protein content) and % WPC Gellan (% w/w)
glucono-d-lactone (GDL) were purchased from Sigma–Aldrich (w/w)
HS/CS
Corporation (St. Louis, USA). The ion content of the casein, as
0% CN 1% CN 2% CN
determined by ICP was (% w/w): Kþ ¼ 0.08, Naþ ¼ 0.16 and
(w/w) (w/w) (w/w)
Ca2þ ¼ 0.14.
0 0.1 0.1 0.1
0.3 0.3 0.3
2.2. Sample preparation
1 0.1 0.1 0.1
0.3 0.3 0.3
2.2.1. Stock solutions
In order to prepare the samples, a stock solution of each polymer 3 0.1 0.1 0.1
0.3 0.3 0.3
was prepared. The sodium caseinate (CN) solution (17% w/w) was
504 C.S.F. Picone, R.L. da Cunha / Food Hydrocolloids 24 (2010) 502–511

A B
30 30
Bb Bb
25 Bb
Cb 25
20 20
σR (kPa)

σ R (kPa)
15 A 15 A
10 10
Ba Ca Aa Aa
5 5
NG GN 0.3 % NG GN 0.3 %
0 0
GN 0.1 % GN 0.1 %
WPC 0% WPC 0%
WPC 1% WPC 1%
WPC 3% WPC 3%

C D
50 50

40 40

30 30

Ε (kPa)
Ε (kPa)

Ba Ba Ba Ba
20 20
A Ba A
Ba
10 10
Aa Aa
NG GN 0.3 % NG GN 0.3 %
0 0
GN 0.1 % GN 0.1 %
WPC 0% WPC 0%
WPC 1% WPC 1%
WPC 3% WPC 3%

E F
0.7 0.7 Aa
A Ab A Aa
0.6 0.6
0.5 0.5
Aa Ba
0.4 0.4
εR

Aa Bb
εR

Ba
0.3 0.3
0.2 0.2
0.1 0.1
NG GN 0.3 % NG GN 0.3 %
0.0 0.0
GN 0.1 % GN 0.1 %
WPC 0% WPC 0%
WPC 1% WPC 1%
WPC 3% WPC 3%

Fig. 1. Mechanical properties of gels composed of whey proteins (WPC) (0, 1 and 3% w/w) and gellan (GN) (0.1 and 0.3% w/w). A), c), and e) helix samples (HS) and b), d) and f) coil
samples (CS). A) and b) fracture stress; c) and d) young’s modulus and e) and f) fracture strain. Means with different letters show significant differences at p < 0.05: capital letters
(ABC) in the axis x and small letters (ab) in the axis z. Ng:non self-supported sample.

Where F(t) is the force at time t, A0 and H0 are the initial sample area
and height, respectively, and H(t) is the height at time t.
The stress (sR) and strain (3R) at fracture were obtained from the
maximum point of the stress–strain curve, while the Young’s
modulus was the slope of the initial linear region of this curve. All
the measurements were made with five replicates.
2.4. Water holding capacity (WHC)
The water holding capacity of the gels was determined using an
centrifuge (Allegra 25 – R Beckman Coulter, Germany) in 5  10 min
steps (400 g; 1500 g; 3500 g; 6200 g; 10 000 g), in an adaptation of
the Schkoda, Hechler, and Kessler (1999) method. Approximately
20 g of each sample was evaluated in triplicate experiments. The
water holding capacity was calculated according to Eq. (3).
" !#
waterreleased ðgÞ
WHC ¼ 100$ 1 
watergel ðgÞ
where waterreleased is the amount of water released after centrifu-
gation and watergel is the initial amount of water in the sample.
2.5. Scanning electron microscopy (SEM)
Pieces of gel (approximately 10 mm  2 mm  2 mm) were fixed
overnight in 2.5% glutaraldehyde in cacodylate buffer (0.1 M) at pH
7.2. After rinsing in cacodylate buffer (0.1 M), the samples were
fractured in liquid nitrogen, followed by another rinse with
cacodylate buffer. The fractured samples were post fixed overnight
in 1% buffered osmium tetroxide and then dehydrated in a graded
ethanolic series (30, 50, 70, 90 and 100% v/v). In order to avoid
structural damage, the samples were dried at the CO2 critical point
(Critical Point Dryer CPD03 Balzers) and then mounted on
aluminum stubs and coated with gold in an SCD 050-Balzer Sputter
Coater. At least three images of typical structures were recorded at
(3) a magnification of 1.000, using a JEOL JSM 5800 LV (Tokyo, Japan)
microscope operating at 10 kV.
 C.S.F. Picone, R.L. da Cunha / Food Hydrocolloids 24 (2010) 502–511 505

A B
30 30
25 25
Cb Cb
20 20
σR (kPa)

σR (kPa)
Bb Bb
15 A 15 A
10 10
5 5
NG Aa GN 0.3 % NG Aa Ba GN 0.3 %
0 Ba 0
GN 0.1 % GN 0.1 %
CN 0% CN 0%
CN 1% CN 1%
CN 2% CN 2%

C D
50 50 Cb
Cb
40 40
Bb Bb

Ε (kPa)
30
Ε (kPa)

30

20 20
A A
10 10
NG Aa Aa GN 0.3 % NG Aa Ba GN 0.3 %
0 0
GN 0.1 % GN 0.1 %
CN 0% CN 0%
CN 1% CN 1%
CN 2% CN 2%

E F
0.7 0.7
A A
0.6 0.6
Aa
0.5 0.5 Ba Ba
Ba Ba
0.4 0.4
εR

εR

Aa Aa 0.3 Aa
0.3
0.2 0.2
0.1 0.1
NG GN 0.3 % NG GN 0.3 %
0.0 0.0
GN 0.1 % GN 0.1 %
CN 0% CN 0%
CN 1% CN 1%
CN 2% CN 2%

Fig. 2. Mechanical properties of gels composed of sodium caseinate (CN) (0, 1 and 3% w/w) and gellan (GN) (0.1 and 0.3% w/w). A), c), and e) helix samples (HS) and b), d) and f) coil
samples (CS). A) and b) fracture stress; c) and d) young’s modulus and e) and f) fracture strain. Means with different letters show significant differences at p < 0.05: capital letters
(ABC) in the axis x and small letters (ab) in the axis z. Ng:non self-supported sample.

Table 2 Table 3
Fracture stress of tri-polymeric systems composed of gellan gum with different Fracture strain for tri-polymeric systems composed of gellan gum with different
conformations. conformations.

%WPC %GN Hencky stress (kPa) %WPC %GN Hencky strain

Helix Coil Helix Coil

1% CN 2% CN 1% CN 2% CN 1% CN 2% CN 1% CN 2% CN
1 0.1 14.81Aa 15.27Aa 11.85Ab 12.89Ab 1 0.1 0.47Aa 0.39Ab 0.44Aa 0.43Aa
0.3 56.75Ba 63.37Bb 49.62Bc 62.35Bb 0.3 0.54Ba 0.51Bab 0.55Bac 0.53Ba

3 0.1 15.26Aa 11.70Cb 11.02Ab 8.95Cc 3 0.1 0.31Ca 0.28Ca 0.27Ca 0.29Ca
0.3 42.86Ca 47.65Db 38.15Cc 36.45Dc 0.3 0.29Ca 0.30Da 0.31CDa 0.28Da

Different capital letters in the same column and different small letters in the same Different capital letters in the same column and different small letters in the same
row mean significant differences (p < 0.05). Concentrations in % (w/w). row mean significant differences (p < 0.05). Concentrations in % (w/w).
506 C.S.F. Picone, R.L. da Cunha / Food Hydrocolloids 24 (2010) 502–511

Table 4 showing the essential role of protein in gel network formation.

Young’s modulus for tri-polymeric systems composed of gellan gum with different However, higher rupture values were achieved with higher gellan
conformations.
concentrations. At higher concentrations, the gellan chains are
%WPC %GN Young’s modulus (kPa) closer to each other, enhancing the probability of aggregation and
Helix Coil the formation of junction zones. Similar behavior was reported by
De Jong and Van De Velde (2007) for whey protein isolate – gellan
1% CN 2% CN 1% CN 2% CN
gels at pH 4.8 and by Yamamoto and Cunha (2007) for pure gellan
1 0.1 20.03Aa 21.38Aa 17.81Aa 17.75Aa
0.3 40.99Ba 58.13Bb 33.68Bc 48.45Bb
gels, who related this to a densely linked structure. Moreover, the
bi-polymeric systems were considerably more rigid and elastic
3 0.1 30.21Ca 30.09Ca 26.90Cab 26.22Cb
than the pure gels composed of 0.3% gellan (w/w), but less
0.3 20.06Aa 22.05ACabc 26.24Cb 17.22Ac
deformable (Figs. 1 and 2).
Different capital letters in the same column and different small letters in the same
Distinct tendencies were observed with increasing amounts of
row mean significant differences (p < 0.05). Concentrations in % (w/w).
WPC, according to the gellan concentration in the HS bi-poly-
meric systems. With 0.1% (w/w) gellan gum, the gel strength
2.6. Statistical analyses (Fig. 1A) increased considerably with increases in WPC concen-
tration, but gel deformability (Fig. 1E) was not statistically
The results were evaluated using an analysis of variance affected. On the other hand, the samples formed with the higher
(ANOVA) and the significant differences (p < 0.05) between gellan concentration (0.3% w/w) showed a decrease in gel
different samples determined by the Tukey procedure using the hardness and deformability (Fig. 1A and E) at the highest WPC
software STATISTICA 5.5 (Statsoft Inc., Tulsa, USA). concentration (3% w/w), but the increased gel elasticity
observed in samples with the lower gellan concentration was not
3. Results significant (Fig. 1C). Differently from WPC–gellan gels, caseinate–
gellan systems presented the same tendencies independent of
3.1. Mechanical properties the polysaccharide concentration, showing an increase in frac-
ture stress and strain with increased protein concentration
All the systems formed self-supported gels except the pure gel (Fig. 2).
with the lowest polysaccharide content (0.1% w/w) (Figs. 1 and 2),

Fig. 3. SEM micrographs of whey proteins (WPC) and gellan (GN) gels. (a) WPC 0% and GN 0.3% HS, (b) WPC 1% and GN 0.3% HS, (c) WPC 3% and GN 0.1% HS, (d) WPC 3% and GN
0.3% HS, (e) WPC 3% and GN 0.3% CS. Scale bar ¼ 10 mm.
 C.S.F. Picone, R.L. da Cunha / Food Hydrocolloids 24 (2010) 502–511 507

In general, the HS and CS samples showed the same tendencies
with respect to fracture stress and the elasticity modulus with
increasing caseinate concentration (Fig. 2A and D), however, this
was not the case with fracture strain (Fig. 2E and F). The systems
prepared with helix gellan showed no significant differences in
deformability with changing protein concentration (Fig. 2E),
whereas with CS, an increase in protein concentration led to more
deformable gels (Fig. 2F). Similar tendencies for the fracture strain
values and Young modulus were observed in the CS and HS systems
with increases in WPC concentration, although the reduction in gel
deformability of the HS systems with the lowest gellan concen-
tration was not statistically significant (Fig. 1B). The fracture stress
of coil WPC–gellan systems was less influenced by the variation in
WPC concentration than HS samples (Fig. 1).
The rigidity of the tri-polymer systems was about two-three
times greater than the corresponding bi-polymer system (Figs. 1
and 2; Table 2). In general, similar to the bi-polymer systems, more
rigid samples were obtained in the tri-polymer systems at the
highest gellan concentration (Table 2) (Figs. 1 and 2). However,
increases in the WPC concentration of the tri-polymeric systems
led to lower values for the mechanical properties (Tables 2–4),
except for the Young modulus at the lowest gellan concentration.
Increasing caseinate concentrations resulted in different tendencies
for the mechanical properties (Tables 2–4). With 0.3% gellan (w/w),
more rigid and elastic gels (Tables 2 and 4) were obtained with
increasing caseinate concentration, although the deformability of
the gels remained the same (Table 3), confirming the results
obtained with the caseinate–gellan systems. The deformability of
the CS and HS systems was not different (Table 3), but gels prepared
with helix gellan were more rigid and strong (Tables 2 and 4) than
the coil samples.
3.2. Water holding capacity and microstructure
Pure gellan samples formed a homogeneous structure with
a great number of pores, which conferred a spongy aspect on the
gel network (Figs. 3 and 4A). The pores were homogeneously
distributed throughout the structure, and presented a wide range of
sizes forming a structure similar to that observed by Yamamoto and
Cunha (2007), who studied gellan gels formed by GDL acidification
to pH 2.5. The different pore sizes of the pure gellan network could
be responsible for the high water holding capacity of such gels
when compared to the bi-polymeric (Fig. 5) and tri-polymeric
(Table 5) samples. The addition of caseinate modified the pored,

Fig. 4. SEM micrographs of gellan (GN) and sodium caseinate (CN) gels. (a) CN 0% and GN 0.3% HS, (b) CN 1% and GN 0.3% HS, (c) CN 2% and GN 0.1% HS, (d) CN 2% and GN 0.3% HS,
(e) CN 2% and GN 0.3% CS. Scale bar ¼ 10 mm.
508 C.S.F. Picone, R.L. da Cunha / Food Hydrocolloids 24 (2010) 502–511

A B
100 A 100 A
Bb Ca Bb Cb
75 75
Ba Ba

WHC (%)
W HC ( % )

Aa
50 50 Aa

25 25

NG GN 0.3 % NG GN 0.3 %
0 0
GN 0.1 % GN 0.1 %
WPC 0% WPC 0%
WPC 1% WPC 1%
WPC 3% WPC 3%

C D
100 A
100 A

Bb 75 Bb
75
WHC (%)

WHC (%)
Cb Cb
50 50

25 25
Aa Ba Aa
GN 0.3 % Bb
NG NG GN 0.3 %
0 0
GN 0.1 % GN 0.1 %
CN 0% CN 0%
CN 1% CN 1%
CN 2% CN 2%

Fig. 5. Water holding capacity (WHC) of gels. Helix samples (HS) composed by gellan gum (GN) (0.1 and 0.3% w/w) and a) whey proteins (WPC) (0, 1 and 3% w/w) or c) sodium
caseinate (CN) (0, 1 and 3% w/w), respectively. Coil samples (CS) composed by gellan gum (GN) (0.1 and 0.3% w/w) and b) whey proteins (WPC) (0, 1 and 3% w/w) or
d) sodium caseinate (CN) (0, 1 and 3% w/w), respectively. Means with different letters show significant differences at p < 0.05: capital letters (ABC) in the axis x and small letters (ab)
in the axis z. Ng:non self-supported gel.

spongy gellan network to a more compact structure, with small
spheres surrounded by an entangled network (Fig. 4B–E). Such
spheres corresponded to complexes formed by caseinate, positively
charged at pH 4.0, and the anionic gellan gum. Increasing the
caseinate concentration from 1 to 2% (w/w) in systems containing
the highest gellan concentration (Fig. 4B–E), resulted in an increase
in ramification and compacting of the background network, while
the number of spheres decreased, resulting in a decrease in WHC
(Fig. 5C and D). Reducing the amount of gellan, the number of
spheres decreased greatly and their shape became less uniform,
since there was less polysaccharide available to interact with the
protein (Fig. 4C). This led to a reduction in WHC of the gels when
compared to high gellan concentration gels (Fig. 5C and D).
The addition of whey protein concentrate to the gellan
samples led to the formation of flatter and more continuous
structures (Fig. 3B–E) with a higher WHC than the caseinate–
gellan systems. However, the SEM micrographs showed
Table 5
Water holding capacity of tri-polymeric systems composed of gellan gum with
different conformations.
%WPC %GN Water holding capacity (%)
Helix Coil
microphase separation at higher polymer concentrations, espe-
cially for the HS systems. Due to such phenomena, the protein
and polysaccharides were forced into localized areas leading to
the formation of a heterogeneous structure formed by two
different networks (Fig. 3B and D). The first showed a ramified
aspect, similar to the gellan network (Fig. 3A) and remained
above the other one, which was flatter and continuous and
formed by prevailing whey proteins. Since such a microstructure
had smaller pores and larger numbers of free hydrophilic sites
due to the increase in protein concentration, it showed a slightly
higher water holding capacity (Fig. 5A and B) than the samples
composed of 1% WPC (w/w) and 0.3% gellan (w/w).
The tri-polymer system structures are shown in Fig. 6. The
structures visualized differed widely from those of the bi-polymer
samples and changed considerably according to the polymer
concentration. In general the networks were more compact and the
pores more elongated than in the bi-polymer samples (Figs. 3 and
4) which resulted in higher WHC values (Table 4). Although all
tri-polymeric samples were composed of caseinate and gellan gum,
the presence of spheres, previously identified as complexes, was
not observed. Overall, the water holding capacity of bi-polymeric
CS systems was lower than that of the HS samples, but the opposite
was observed for the tri-polymeric systems (Table 5).

1% CN 2% CN 1% CN 2% CN 4. Discussion
1 0.1 66.67Aa 67.96Aab 68.88Ab 74.90Ac
0.3 69.88Ba 71.15Bb 67.21Bc 71.91Bb Mixed gels were formed by segregative or attractive interactions
3 0.1 74.28Ca 74.94Cb 71.20Cc 79.77Cd between the proteins and polysaccharides, depending on the
0.3 72.07Da 77.80Db 77.07Dc 79.08Cd concentration of each biopolymer and type of milk protein. At pH
Different capital letters in the same column and different small letters in the same values below their isoeletric point, the whey protein and caseinate
row mean significant differences (p < 0.05). Concentrations in % (w/w). molecules became positively charged which enhanced their
 C.S.F. Picone, R.L. da Cunha / Food Hydrocolloids 24 (2010) 502–511 509

Fig. 6. SEM micrographs of whey proteins (WPC), sodium caseinate (CN) and gellan (GN) gels. (a) CN 2%, WPC 1% and GN 0.3% HS, (b) CN 2%, WPC 1% and GN 0.3% CS, (c) CN 2%, WPC
3% and GN 0.3% HS, (d) CN 2%, WPC 3% and GN 0.3% CS, (e) CN 1% WPC 3% and GN 0.3% HS, (f) CN 2%, WPC 3% and GN 0.1% HS. Scale bar ¼ 10 mm.

interactions with gellan gum (an anionic polysaccharide) leading to
more rigid, elastic and less deformable gels (Figs. 1 and 2). SEM
micrographs (Fig. 3) showed that the samples containing protein
were more continuous than the pure gellan samples. WPC–gellan
structures showed a flatter gel network in comparison with the
caseinate–gellan structures (Figs. 3 and 4) which presented small
spheres (Fig. 4B–E) corresponding to complexes formed by protein,
positively charged at pH 4.0, and the anionic gellan gum.
Overall, as the biopolymer concentrations increased, so the
number of interactions between the macromolecules increased,
resulting in denser, more rigid and elastic networks (Figs. 1–4).
However, when the biopolymer concentrations increase such that
they exceed a certain critical value, the large size and rigidity of the
macromolecules may lead to thermodynamic incompatibility and
further phase separation (Tolstoguzov, 2003). The free energy
produced by mixing proteins and polysaccharides contains both an
interaction term (incompatibility between the two polymers) and
a translation entropy term. Phase separation is observed when the
entropy term is reduced as a consequence of an increase in
molecular mass of a component, such as protein aggregation (De
Jong et al., 2009) induced by heating. This phenomenon was
observed in the bi-polymeric systems at the highest WPC and
gellan concentrations (3 and 0.3% w/w, respectively) due to the
high polymer concentration and great WPC molar mass. When
denaturated, the whey proteins expose reactive groups allowing for
protein intermolecular exchanges (Mulvihill & Donovan, 1987),
aggregation and further gel formation, leading to aggregates of up
to 200 kDa (Cavallieri, Costa-Neto, Menossi, & Cunha, 2007). In the
present case, macroscopic phase separation was not observed, but
since both the whey proteins and gellan gum are gelling agents, the
gelling process was faster than phase separation and only micro-
phase separation occurred. The formation of two interpenetrating
networks led to a heterogeneous structure, with greater fragility
and lower deformability as compared to samples with lower
510 C.S.F. Picone, R.L. da Cunha / Food Hydrocolloids 24 (2010) 502–511
protein contents. Since such samples contained higher whey
protein concentrations, they showed higher WHC values (Fig. 5).
Differently from the WPC–gellan gels, the number of interac-
tions between the caseinate and the gellan gum tended to increase
as the biopolymer concentration rose, and no incompatibility
seemed to occur between these biopolymers. As commented
before, thermodynamic compatibility between polymers is closely
related to their molecular weight. Caseinate molecules have
molecular weights of about 23 kDa (Allen, Dickinson, & Murray,
2006), ten times lower than the WPC, which explains the opposite
tendency observed between gel rigidity and deformability when
the protein concentration was increased (Figs. 1 and 2). The water
holding capacity of these systems showed opposite behaviors at
higher polymer concentrations when compared to the WPC–gellan
systems (Fig. 5). Whey proteins are extremely hydrophilic mole-
cules (Cavallieri et al., 2007), since they are mainly formed of polar
amino acids (Cheftel, Cuq, & Lorient, 1996; Sgarbieri, 2005). Thus
the WHC of WPC systems increased with protein concentration
because of thermodynamic incompatibility, which reduced the
interactions between both biopolymers such that more hydrophilic
sites remained available to bind water. In caseinate–gellan systems,
the increase in protein resulted in an increase in biopolymer
interactions and a decrease in water binding sites, but with 0.1%
gellan (w/w) the observed increase in WHC was probably related to
the excess of hydrophilic protein sites available to bind water and
not linked to the gellan.
In tri-polymer systems, the presence of both proteins led to
much more rigid structures than with the corresponding bi-poly-
mer system (Figs. 1 and 2; Table 2). In this case, besides the increase
in protein–polysaccharide interactions, the proteins also interacted
between themselves through sulphydryl–disulphide reactions,
together with hydrophobic interactions which contributed to an
increase in the gel mechanical properties (Vasbinder, Alting, & De
Kruif, 2003), even though the incompatibility at high polymer
concentration was more intense, since the polymer concentration
was greater (Table 2). In the same way as with fracture stress, the
difference in WHC between the tri-polymer systems and caseinate–
gellan ones was statistically higher than between the tri-polymer
and WPC–gellan samples. This confirms that the presence of WPC
was more relevant than that of caseinate with respect to the gel
properties (Figs. 1, 2 and 5 and Tables 2 and 5).
The conformational state of the gellan also affected the gel
properties (Figs. 1–6 and Tables 2–5). The protein interacted more
easily with the unordered (coil) gellan, as observed by De Jong et al.
(2009), reducing the gellan helicity after cooling. Such bindings
resulted in an increase in the interactions between positively
charged protein groups and negatively charged gellan sites after
reaching a pH value lower than the pI, forming electrostatic
complexes which decreased phase separation during cooling. This
phenomenon had already been observed by Burova et al. (2007) for
carrageenans and b-caseins, and depended on the system pH and
polymer concentration. At higher polymer concentrations, electro-
static complex formation was enhanced, increasing gel hardness
and decreasing water holding capacity (Figs. 1 and 5). For polymer
mixtures prepared at low temperatures (HS), protein–gellan elec-
trostatic interactions were promoted, especially at gellan junction
zones, which seemed to increase the thermodynamic incompati-
bility between WPC and gellan. These hypotheses were corrobo-
rated by the SEM micrographs (Fig. 3). While samples with higher
WPC and gellan concentrations presented two overlapping
networks in the HS systems, the CS systems only showed one
network, with no strands and with reduced numbers of pores. In CS
caseinate–gellan systems (Fig. 4E), the background network was
more compact and closed up due to the increase in protein–poly-
saccharide interaction, which resulted in increased deformability
and elasticity of such samples (Fig. 2D and F). In tri-polymeric
systems, electrostatic complexes were probably formed preferen-
tially between the gellan and caseinate, due to the lower molecular
weight of the latter as compared to the whey protein aggregates,
resulting in a closer network (Fig. 6A–D). Therefore the excluded
WPC increased thermodynamic incompatibility and also the
number of hydrophilic sites available to bind water. As a result, the
CS tri-polymeric systems showed more compact structures (Fig. 6),
but were significantly less rigid and elastic, with higher water
holding capacity than the HS samples (Tables 2, 4 and 5), contrasting
with the results observed for the bi-polymeric systems (Figs. 1–6).
5. Conclusions
The bi-polymeric systems showed different mechanical prop-
erties, structures and water holding capacities than the tri-poly-
meric systems. The gel properties were greatly influenced by the
polymer concentrations, more than by the gellan conformational
state. An increase in whey protein concentration led to thermo-
dynamic incompatibility between the biopolymers, especially in
the tri-polymeric samples, reducing the gel strength and deform-
ability. Microphase separation was observed in the WPC-helix
gellan systems at high polymer concentrations, whilst the
caseinate–gellan samples formed complexes. In bi-polymeric
systems the use of coil gellan led to the formation of electrostatic
complexes between the polysaccharide and proteins, with
increased gel rigidity and deformability but reduced water holding
capacity. However, in the tri-polymeric systems, the interactions
led to weaker and less elastic gels, which were more capable of
holding water. Thus, the characteristics of the multi-compound
systems were shown to be dependent not just on the concentra-
tions of each polymer, but also on the preferential interactions
between them, showing the complexity of food colloid science,
especially with an increasing number of ingredients.
Acknowledgements
This work was supported by The Fundação de Amparo à Pes-
quisa e Desenvolvimento de São Paulo – Brazil (FAPESP, Grant No.
2004/08517-3 and 2006/02390-7) and by The Conselho Nacional de
Desenvolvimento Cientı́fico e Tecnológico - Brazil (CNPq, Grant No.
301869/2006-5).
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