Characterization of a chitosan sample extracted from Brazilian shrimps and its

application to obtain insoluble complexes with a commercial whey protein isolate

Daniele S. Bastos
a, b
, Bianca N. Barreto
b, e
, Hilia K.S. Souza
b
, Margarida Bastos
c
,
Maria Helena M. Rocha-Leo
d
, Cristina T. Andrade
e
, Maria Pilar Gonalves
b,
*
a
Programa Cincia de Alimentos, Instituto de Qumica, Universidade Federal do Rio de Janeiro, Centro de Tecnologia, Bloco A, 21949-900 Rio de Janeiro, RJ, Brazil
b
REQUIMTE, Faculdade de Engenharia, Universidade do Porto, Rua Dr. Roberto Frias, 4200-465 Porto, Portugal
c
Departamento de Qumica, Faculdade de Cincias, Universidade do Porto, Rua do Campo Alegre 687, 4169-007 Porto, Portugal
d
Programa Cincia de Alimentos, Departamento de Engenharia Bioqumica, Escola de Qumica, Universidade Federal do Rio de Janeiro, Centro de Tecnologia,
Bloco E, 21945-970 Rio de Janeiro, RJ, Brazil
e
Programa Cincia de Alimentos, Instituto de Macromolculas Professora Eloisa Mano, Universidade Federal do Rio de Janeiro, Centro de Tecnologia,
Bloco J, 21945-970 Rio de Janeiro, RJ, Brazil
a r t i c l e i n f o
Article history:
Received 26 November 2009
Accepted 18 March 2010
Keywords:
Chitosan
Whey protein isolate
Complex formation
Coacervate
Rheometry
Turbidity
ITC
a b s t r a c t
The rheological behaviour of chitosan solutions in 250 mM acetate buffer was studied at different pHs
(25

C). The intrinsic viscosity decreased from w17 dL/g to w14 dL/g when the pH increased from 4.7 to
6.0. Concentrated solutions (0.5e3.0% w/w) exhibited a shear-thinning behaviour which increased with
increasing chitosan concentration and decreasing pH. A good tting of the experimental data to the Cross
and Carreau ow models was obtained. The elasticity of the solutions decreased with increasing pH and
decreasing chitosan concentration, as a consequence of increased chain exibility.
The interaction of chitosan with whey proteins (WPI) was studied by isothermal titration calorimetry
(ITC) and turbidity measurements, at different pHs (3.0e6.0) and ionic strengths (100 and 250 mM). ITC
results showed that electrostatics is the main driving force for chitosan:WPI interaction, as an increase in
ionic strength lead to a smaller interaction. A pH and chitosan:WPI ratio dependence of aggregate
formation was clearly observed by turbidimetry. At pH 3.0, there was no change in turbidity upon
addition of chitosan, whereas at pH 4.0 and 6.0, the turbidity values varied with chitosan:WPI ratio and
were smaller at 250 mM than those at 100 mM.
The rheology of chitosan:WPI coacervates was studied in acetate buffer (100 and 250 mM), at pH 5.5,
mixing ratios of 0.25:1 and 0.10:1. Time dependent ow behaviour, higher G
/
and G
//
values and higher
elasticity were observed for the coacervates, originating mainly from the electrostatic interactions
between the protein and the polysaccharide chains.
2010 Elsevier Ltd. All rights reserved.
1. Introduction
Complex coacervation, the spontaneous liquideliquid associa-
tive phase separation, which occurs in solutions of oppositely
charged polyelectrolyte, has attracted academic and industrial
interest. It is typically observed in the self-assembly of biological
macromolecules (Ansarian, Derakhshan, Takafugi, & Ihara, 2008;
Dankers & Meijer, 2007). Based on the self-assembly of poly-
cations and polyanions, processes for protein separation and puri-
cation have been developed (Lali, Roshnnie, & Devika, 2000;
McDonald, Victa, Carter-Franklin, & Fahrner, 2008; Mattison,
Brittain, & Dubin, 1995; Porri, Braia, Farrugia, Pic, & Romanini,
2009). However, the most important application of complex coac-
ervation consists of microencapsulation (Ducel, Richard, Saulnier,
Popineau, & Boury, 2004; Junyaprasert, Mitrevej, Sinchaipanid,
Boonme, & Wurster, 2001) of bioactive substances, which other-
wise would be subjected to some kind of degradation, loss of
functionality, or would cause cytotoxic effect to tissues.
In biological systems, proteinepolysaccharide interactions are
usually responsible for the formation of complex coacervates.
Because of their importance, many studies have been devoted to
thermodynamic and structural aspects of such interactions
(Tolstoguzov, 2002; Turgeon, Schmitt, & Sanchez, 2007). The most
studied systems are those in which carboxyl-containing poly-
saccharides function as polyanions, and form complexes with
proteins at pHs below their isoelectric points (Chanasattru, Jones,
* Corresponding author. Tel.: 351 225081684; fax: 351 225081449.
E-mail address: pilarg@fe.up.pt (M.P. Gonalves).
Contents lists available at ScienceDirect
Food Hydrocolloids
j ournal homepage: www. el sevi er. com/ l ocat e/ f oodhyd
0268-005X/$ e see front matter 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodhyd.2010.03.008
Food Hydrocolloids 24 (2010) 709e718
Decker, & McClements, 2009; de Kruif, Weinbreck, & de Vries,
2004; Lutz, Aserin, Portnoy, Gottlieb, & Garti, 2009; Mekhlou,
Sanchez, Renard, Guillemin, & Hardy, 2005; Sanchez, Meklou, &
Renard, 2006; Singh et al., 2007). To the authors knowledge, the
use of a polycationic polysaccharide in complex coacervates with
proteins has been less explored.
Chitosan is a non-toxic, and biocompatible linear poly-
saccharide, obtained from partial deacetylation of chitin, and thus
formed by b-(1,4)-2-amino-2-deoxy-D-glucose and b-(1,4)-2-acet-
amido-2-deoxy-D-glucose repeating units. Contrarily to insoluble
chitin, chitosan is soluble in acid solution, at which conditions the
protonation of amino groups gives rise to its polyelectrolyte char-
acter. Both deacetylation degree (DD) and molar mass have been
shown to inuence the conformation and solution properties of
chitosan (Lamarque, Lucas, Viton, & Domard, 2005). The interaction
of a low molar mass sample of chitosan, with DD = 85%, with
b-lactoglobulin has been studied by isothermal titration calorim-
etry (ITC), electrophoresis and light scattering. ITC revealed an
exothermic interaction between the biopolymers with opposite
charges, in the range of pH 5 and 7 (Guzey & McClements, 2006).
The globular proteins b-lactoglobulin and a-lactalbumin are the
main proteins of whey. In many food products, the functional
properties of whey protein are used to stabilize oil-in-water
emulsions, and to form gels (Gonalves, Torres, Andrade, Azero, &
Lefebvre, 2004; Rocha, Teixeira, Hilliou, Sampaio, & Gonalves,
2009).
The aim of this work was to investigate the formation of
complex coacervates between chitosan and a commercial whey
protein isolate. The chitosan sample was obtained experimentally,
and characterized extensively. As the formation of complex coac-
ervates is known to be dependent on numerous factors, such as pH,
temperature, ionic strength, and molar mass, charge density,
concentration and mixing ratio of components (Burgess, 1994),
isothermal titration calorimetry and UVevis spectrophotometry
were used under different conditions of pH, ionic strength and
components concentrations, so as to ascertain both the energetics
of the interaction and the formation of the aggregates. The rheo-
logical properties of the coacervates were investigated, taking into
consideration its signicance to food product development.
2. Materials and methods
2.1. Materials
Chitosan samples were obtained by partial deacetylation of chitin
from shells of Penaeus schmitti shrimp. WPI (Lacprodan

DI e 9224)
was kindly donated by Arla Foods Ingredients (Denmark). Sodium
acetate trihydrate (CH
3
COONa$3H
2
O) and glacial acetic acid
(CH
3
COOH) were purchased from Merck (99.5%) (Darmstadt,
Germany). Sodium hydroxide (NaOH) from Pronalab (Lisboa,
Portugal). All other chemicals were analytical grade and used
without further purication. Puried water produced by a Milli-Q
ltration system was used for the preparation of all solutions.
2.2. Methods
2.2.1. Chitin extraction from shrimps shells and preparation of
chitosan
Chitin extraction from shrimps shells and the preparation of
chitosan were performed according to the method of Percot, Viton,
and Domard (2003), with some modications. For demineraliza-
tion, shells were treated with 0.25 M HCl in excess at room
temperature (25

C), for 30 min. Chitin deproteinization was
carried out under stirring with 1 M NaOH at room temperature, for
24 h. Pigment elimination was achieved by immersing the sample
in acetone and ethyl alcohol baths. Deacetylation of chitin was
performed in two stages. Heterogeneous alkaline deacetylation of
a-chitin is governed by several factors, such as the chitin source,
alkali concentration, and reaction time and temperature. When
compared to continuous treatments (one stage for long periods of
time), repeated treatments (two or more stages) performed with
b-chitin suggested to lead to lower deacetylation degrees, associ-
ated with less affected molar masses (Tolaimate et al., 2000). In the
rst stage, chitin powder was reacted under stirring with 30% (w/
w) NaOH solution in excess at room temperature, for 30 min. The
reaction product was neutralized by successive washings in baths
of deionized water, ltered, and dried at 50

C for 12 h. The second
stage was carried out under reux and nitrogen bubbling, in the
presence of sodium borohydride (0.1 g/g of deacetylated chitin),
with 50% (w/w) NaOH solution for 5 h. Both deacetylation stages
were performed with a 1/50 (w/v) solid to liquid ratio.
The method of Lavertu et al. (2003) was used to determine the
degree of deacetylation (DD) of the chitosan sample, DD = 93.04%.
2.2.2. Preparation of solutions
Chitosan and whey protein isolate (WPI) stock solutions were
separately prepared in 100 mM or 250 mM CH
3
COOH/CH
3
COONa
(HAc/NaAC) buffer at the desired pH. The relatively high ionic
strengths of the buffers were necessary for them to be effective and
ensure pH stability during the experiments. The dispersions were
gently agitated for at least 2 h at room temperature until complete
polymer dissolutionoccurredandtheir pHs were checked. Finally, the
solutions were stored overnight in the refrigerator, for further use.
2.2.3. Viscosimetry
Viscosity of dilute chitosan solutions, in 250 mM HAc/NaAC
buffer at different pH (4.7 and 6.0), was measured at 25.0 0.1

C
using a glass capillary Ubbelohde viscometer with a capillary
diameter of 0.58 mm. The chitosan solutions were prepared as
described in 2.2.2. The dilute solutions had relative viscosities, h
rel
,
from about 1.2 to 2.0 to assure good accuracy and linearity of
extrapolation to zero concentration. Flow times were measured in
triplicate, for each sample, and their average values were used for
the calculations. The limiting viscosity number (intrinsic
viscosity), [h], was obtained by double extrapolation to zero
concentration of Huggins and Kraemer equations, respectively
h
sp
=C = [h[ k
/
[h[
2
C (1)
(ln h
rel
)=C = [h[ k
//
[h[
2
C (2)
where h
rel
and h
sp
are the (dimensionless) relative and specic
viscosities, k
/
and k
//
are the Huggins and Kraemers coefcients,
respectively, and C is the solution concentration.
Viscosity average molecular masses, M
v
, were calculated using
the MarkeHouwinkeSakurada relationship (3):
[h[ = KM
a
v
(3)
where a and K are constants for the buffer solution and chitosan
system.
These constants were calculated using the two following
equations, proposed by Kasaai (2007) as a model to calculate a and
K for chitosan in any solventetemperature system using visco-
metric constant data previously reported by several research
groups:
a = 0:6202 [0:699x=(0:4806 x)[ (4)
log K$10
5
= 5:7676a 5:9232 (5)
D.S. Bastos et al. / Food Hydrocolloids 24 (2010) 709e718 710
where x =[DA/pH$m], with DA, degree of acetylation of chitosan, pH
of chitosan solution in a solvent with ionic strength of m.
2.2.4. Rheological measurements of chitosan solutions
Chitosan solutions (0.5e3.0% w/w) in 250 mM HAc/NaAC buffer
at 3.0, 4.7 and 6.0 pH were prepared as described in 2.2.2 and used
for rheological measurements.
All rheological measurements were performed at 25

C using
a controlled stress rheometer AR2000 (TA Instruments Inc., New
Castle, DE, USA) tted with a cone-and-plate geometry (2

cone
angle, 40 mm diameter, 54 mm truncation).
Steady-shear data were recorded rst in increasing order and
then in decreasing order of applied torque. The torque was imposed
using a logarithmic ramp, in order to decrease the initial acceler-
ation and the effects due to instrument inertia. Shear rates obtained
were in the 1e300 s
1
range. Frequency sweeps were performed in
the 0.1e100 rad s
1
range, with strain amplitude of 5%, in order to
assure working conditions inside the linear viscoelastic region,
determined by preliminary experiments (strain sweeps). Samples
were covered with a thin layer of parafn oil in order to hinder
evaporation during the experiments.
2.2.5. Turbidity measurements
Turbidity measurements were performed on 0.5% (w/w) WPI
solutions (0.9 mL) contained in a UVeVIS quartz cuvette, titrated
gradually with aliquots of a 0.4% (w/w) chitosan solution. After the
addition of each aliquot the mixture was carefully stirred and the O.
D. (Optical Dispersion) measured using a UVevisible spectropho-
tometer (Agilent 8453 UVeVisible Spectroscopy System, optical
path of 1 cm) at a wavelength of 400 nm (25
B
C). Measurements
were carried out in triplicate for each mixture, and their average is
reported.
2.2.6. Isothermal titration calorimeter (ITC) measurements
The experiments were performed with solutions of chitosan
0.4% (w/w) and WPI 0.5% (w/w) in HAc/NaAC buffer (100 and
250 mM) at different pHs (3.0, 4.0, 5.0 and 6.0), prepared as
described in 2.2.2. These concentrations were chosen according to
previous results in our laboratory (Souza, Bai, Gonalves, & Bastos,
2009).
A twin heat conduction microcalorimeter from ThermoMetric
AB (Jarfalla, Sweden) was used together with a water bath and its
controller, built at Lund University, Sweden, and a 7
1/2
digit HP
nanovoltmeter connected to the calorimetric channel and to the
computer. The calorimetric unit used in this work as well as the
experimental procedure has been described in detail elsewhere
(Bai, Santos, Nichifor, Lopes, & Bastos, 2004; Matos, Lima, Reis,
Lopes, & Bastos, 2004).
Aliquots (6.85 mL) of chitosan solution were sequentially injec-
ted into a 1.0 mL titration cell (ThermoMetric AB (Jarfalla, Sweden))
initially containing either acetate buffer solution or WPI solution
(0.8816 mL), with constant stirring at 90 rpmwith a gold propeller.
Dilution effects were taken care of separately, by titrating the same
chitosan solution into the appropriate acetate buffer solution in the
vessel. The obtained heats in the interaction experiments were
thereafter corrected for the chitosan dilution heat. Each experiment
consisted of 20 consecutive injections. The experiments were per-
formed at (25.000 0.001)

C. Measurements were carried out in
triplicate and the results were reported as their mean.
2.2.7. Preparation and rheological characterization of
chitosaneWPI coacervates
Chitosan and WPI solutions, at 1.0% (w/w) concentration, were
separately prepared in HAc/NaAC buffer (100 and 250 mM), at pH
5.0 or 5.5, as described in 2.2.2. After 24 h of preparation, the
solutions were gently mixed, at different ratios, in test tubes. The
test tubes were maintained under refrigeration overnight to allow
complete phase separation. Then, pictures of the tubes were taken
using a digital camera (Sony

, DSC-W90, China). For some tubes,

the lower (coacervate) phases were separated by centrifugation
(centrifuge Hettich

D e 78532 e Germany), at 3000 rpm, for

30 min, and their moisture content was determined using a venti-
lated oven (Selecta

P, model 210, Spain) at 105

C.
For these coacervates, rheological measurements (dynamic and
steady shear experiments) were performed as described in 2.2.4
but using a steel plate geometry (40 mm diameter, 600 mm gap),
with grooves to avoid slippage.
3. Results and discussion
3.1. Viscosimetry
The intrinsic viscosity, [h], was evaluated in terms of pHat 25

C,
using the Huggins and the Kraemer equations (equations (1) and
(2), respectively). The two extrapolations gave similar results, as
shown in Table 1. [h] was found to be dependent on pH, decreasing
fromw17 dL/g to w14 dL/g when the pH increased from 4.7 to 6.0.
This reduction can be explained taking into account the electro-
static repulsions. At lower pH (more acidic conditions), chitosan
molecules are more charged and take an expanded conformation
while, at pH 6.0, their charge is considerably diminished and the
molecules are more compact due to enhanced chain exibility.
Thus, the macromolecular volume in solution is smaller, at pH 6.0,
resulting in lower [h]. This reversible conformational transition,
from a compact to a more extended conformation and vice versa,
has been observed for other systems, particularly when the
compact conformation is stabilized by intra-molecular forces of
attraction, such as hydrophobic interactions. In the present case,
even though the chitosan sample has a high deacetylation degree
(DD = 99.04%), with a low number of N-acetyl-glucosamine
repeating units, a compact conformation might be expected in the
buffer solution at pH 6 because of the solvent (poor) quality
(Khokhlov & Khalatur, 2005).
An increase in the magnitude of Huggins coefcient, k
/
, with pH
was observed (Table 1). Values of k
/
depend on the state of aggre-
gation of macromolecules and on soluteesolvent interactions.
Values of k
/
w0.3 are obtained for exible macromolecules in
a good solvent but, in case of aggregation, k
/
can be higher than 1.
Our results suggest that the solubility of chitosan molecules
decreased i.e. polymerepolymer interactions or association, were
favored when the pH increased.
Values of [h] obtained for each pH were used in the
MarkeHouwinkeSakurada relationship (equation (3)) to evaluate
the viscosity average molecular mass of the chitosan sample. The
constants K and a were calculated according to Kasaai (2007). The
resulting values are shown in Table 1.
In a good solvent, polymeresolvent interactions are strong and
the conformation of the polymer is extended. Higher values of [h]
and a are then obtained. Most of the reported values for exponent
a lie between 0.7 and 1.0 (Kasaai, Arul, & Charlet, 2000) and were
attributed to conformations varying from extended to linear. A low
Table 1
Physicalechemical parameters of chitosan solutions at 4.7 and 6.0 pH.
pH [h]
a
dL/g [h]
b
dL/g k
/
k
//
K a M
v
/Da
4.7 16.7 17.1 0.58 0.06 8.1 10
4
0.70 1.6 10
6
6.0 13.5 14.1 0.69 0.03 9.8 10
4
0.68 1.2 10
6
a
From Huggins extrapolation.
b
From Kraemer extrapolation.
D.S. Bastos et al. / Food Hydrocolloids 24 (2010) 709e718 711
value of exponent a, related to a random coil and compact
conformation is obtained when a poor solvent is used (Kasaai,
2007). In our case, the pH was varied but the ionic strength was
maintained. As chitosan is a polyelectrolyte, the quality of the
solvent changed with pH. The polymeresolvent interactions and
the polymer conformation depend on the number of positive
charges of the molecule which change with pH. So, the calculated
values of a show that the quality of the solvent was better at pH 4.7
than at pH 6.0.
Values of M
v
calculated using equation (3), were different for
each pH, though of the same order of magnitude, showing that the
equations (4) and (5), proposed by Kasaai (2007) were a reasonable
approach for calculating the parameters K and a. In the literature,
a wide range of chitosan molecular weights can be found (see, for
instance, Berth & Dautzenberg, 2002; Cho, Heuzey, Bgin, &
Carreau, 2006; Kasaai et al., 2000) which makes it difcult to
compare these different results with our own.
3.2. Rheological behaviour of chitosan solutions under dynamic
shear
At the concentrations and oscillatory frequencies studied, the
viscoelastic behaviour of chitosan solutions, independently of pH,
was typical of a system with predominant entanglement networks
between the terminal and plateau zones of frequency response, as
illustrated in Fig. 1 for solutions at pH = 3. At low frequencies
(terminal zone), a liquid-like behaviour is observed, where the loss
modulus, G
//
, is higher than the storage modulus, G
/
, (Fig. 1a) and
the magnitude of the complex viscosity, [h*[, is essentially
frequency independent (not shown in Fig. 1a for the sake of clarity).
At higher frequencies and for higher concentrations, a cross-over is
detected beyond which the elastic contribution predominates
(plateau zone). This cross-over frequency (where G
/
= G
//
or
tan d = G
//
/G
/
= 1) typically moved to lower frequency values when
the concentration increased (Fig. 1a and b) as a consequence of
increasing relaxation times. Values of tan d decreased with
increasing concentration (Fig. 1b) meaning that the elasticity of the
system increased. This kind of behaviour is typical of several
random-coil polysaccharide solutions like galactomannans
(Andrade, Azero, Luciano, & Gonalves, 1999; Sittikijyothin, Torres,
& Gonalves, 2005), and was also observed for chitosan solutions in
0.5 M acetic acid or in 0.5 M acetic acid/0.1 M sodium acetate by
Cho et al., 2006.
In Fig. 2a, the effect of pH on G
/
, G
//
and [h*[ is shown for 1%
chitosan solutions. A decrease of the three parameters with
increasing pH is observed.
In addition, tan d increased (Fig. 2b). As pH increases, the charge
density of chitosan molecules decreases leading to an increase of
chain exibility and a reduction of molecular size. As a consequence
of increased chain exibility, the number of interactions and
entanglements between chitosan molecules may decrease resulting
in a reduction of the elasticity. This kind of reasoning was used by
Cho et al. (2006) to explain the behaviour of chitosan solutions of the
same concentration at different ionic strengths. By increasing
the chitosan concentration, the elasticity increases, as expected, but
the effect of pH is less pronounced (Fig. 2b, for 3% chitosan
concentration) than at lower concentration (Fig. 2b, for 1% chitosan
concentration).
3.3. Steady-shear properties of chitosan solutions
Typical ow curves, at 25

C, for chitosan solutions, at different
concentrations, are shown in Fig. 3, for pH 3.0. Similar curves were
obtained at pH 4.7 and 6.0 (not shown). In all cases, a shear-thin-
ning behaviour was observed.
At low shear rates, the Newtonian ow region with a constant
zero-shear viscosity (h
0
), was attained only for the lowest
concentration studied (0.5%). The shear-thinning behaviour arises
from modications in the macromolecular organization in the
solution as shear rate changes. At low shear rates, the rate of
disruption of inter-molecular entanglements brought about by the
shear force exerted is balanced by that of entanglements newly
formed and a constant zero-shear viscosity can be maintained. For
higher shear rates, disruption predominates over formation of new
entanglements, molecules align in the direction of ow and the
apparent viscosity decreases with increasing shear rate. When the
chitosan concentration increases, the molecules in solution become
more entangled and their mobility decreases. As a consequence, the
time required to form new entanglements to replace those dis-
rupted by the externally imposed deformation increases and the
shear rate corresponding to the transition from Newtonian to
shear-thinning behaviour moves to lower values (Fig. 3).
A comparison between experimental data obtained at different
pH values, showed that the viscosity of chitosan solutions of the
same concentration decreased when the pH increased (Fig. 4). The
increase of chain exibility and reduction of molecular size (see 3.2)
may explain the lower viscosity observed when pH increases
(Muthukumar, 1997). But, the change in viscosity with pH is more
pronounced for lower concentrations (0.5 and 1.0%); for the highest
concentration studied (2%), viscosity values are almost superposed
(compare 0.5% and 2% ow curves in Fig. 4). One possible expla-
nation takes into account the balance between intra- and inter-
Fig. 1. Effect of chitosan concentration, at pH 3.0 and 25.0

C on: (a) storage (G
/
) and loss (G
//
) moduli vs oscillation frequency and (b) tan d vs oscillation frequency. G
/
: full symbols,
G
//
: open symbols. Solutions concentrations: 0.5% (B), 1.0% (,), 2.0% (>), 3.0% (6).
D.S. Bastos et al. / Food Hydrocolloids 24 (2010) 709e718 712
macromolecular interactions. At low concentration, molecules are
less entangled and have more free volume to occupy; if they are
more charged (lower pH), their hydrodynamic volume will increase
because of higher intra-molecular repulsions and the viscosity of
the solution will be higher. When the concentration increases,
molecules become more entangled, with less free space to occupy
and increased inter-molecular interactions. A balance between
intra- and inter-molecular interactions probably implies that the
volume of the macromolecule does not change with pH as much as
it does at lower concentrations.
The owmodels of Carreau (1972, equation (7)) and Cross (1965,
equation (6)) were used to describe the shear-thinning behaviour of
the chitosan solutions:
h
a
= h
N

h
0
h
N
1 (s _ g)
m
(6)
h
a
= h
N

h
0
h
N
h
1 (l _ g)
2
i
N
(7)
where h
a
is the apparent viscosity (Pa s) measured at the shear rate
_ g(s
1
), h
0
is the zero-shear rate viscosity (Pa s), h
N
is the innite
shear rate viscosity (Pa s),s(s) and l(s) are time constants, and m
and N are dimensionless exponents related to the power law
exponent n by m=1 n and N=(1 n)/2 (0 _N<0.5), for the case
h
N
<< h
a
<< h
0
. Since the high shear rate Newtonian viscosity was
never approached in our study, the above equations were simpli-
ed (only three adjustable parameters), assuming h
0
>> h
N
.
A good tting to the experimental data was obtained with both
models (Fig. 3) as illustrated by magnitudes of RE (relative devia-
tion error) (Tables 2 and 3).
As expected, values of h
0
increased with increasing solution
concentration and decreasing pH. In general, h
0
values obtained
using the Cross model were higher than those obtained using the
Carreau model. Values of m and N, for each pH, increased with
increasing concentration meaning that shear-thinning behaviour
increased. Time constants, in general, increased with concentra-
tion, in accordance with the appearance of the shear-thinning
behaviour at lower shear rates, which means, as said before, that
the rate of formation of new entanglements diminishes as
concentration increases.
3.4. Turbidity measurements
The turbidities of mixtures of WPI solutions (0.5% w/w) and
chitosan solutions (0.4%) of varying ratios were measured at
different pH and ionic strength, to evaluate the extent of insoluble
complexes formed.
At pH 4.0, 5.0 and 6.0 (for a buffer concentration of 100 mM)
(Fig. 5), the turbidity of WPIechitosan solutions increased initially
with increasing chitosan concentration until a chitosan:WPI ratio of
0.007:1, 0.005:1 and 0.02:1 for pH 4.0, 5.0 and 6.0, respectively.
After this ratio, for pH 6.0, the turbidity of WPIechitosan solutions
remains relatively constant until the nal mixture tested, where the
ratio was 0.056:1, whereas for pH 4.0 and 5.0 it decreased after the
maximum, albeit more steeply for pH4.0, until a chitosan:WPI ratio
of 0.021:1. After this, it remained constant until the end for pH 4.0,
and for pH 5.0 we can observe another break at a ratio of about
0.055:1. These results indicate that the characteristics (concentra-
tion and dimensions) of the insoluble WPIechitosan complexes
remained constant after chitosan:WPI ratio of 0.02:1 for pH 6.0,
showing that at this pH value an increase of chitosan concentration
does not lead to more or larger aggregates. For the other two
studied pHs, on the other hand, between the rst and second
critical ratios an increase in chitosan concentration decreases either
the size or the concentration of the aggregates initially formed. The
observed concentration and pH dependent behaviour is very
interesting, and has potential practical applications, as it leads to
critical values for the ratios where maximal aggregates are formed.
Fig. 2. Effect of pH on: (a) G
/
, G
//
and [h*[ as a function of oscillation frequency for 1% chitosan solutions at 25

C. (b) tan d as a function of oscillation frequency, for 1% (full symbols)
and 3% (open symbols) chitosan solutions, at 25

C. G
/
: full symbols, G
//
: open symbols. pH: 3.0 (A, >), 4.7 (-, ,), 6.0 (:, 6); [h*[:pH 3.0 (), pH 4.7 (), pH 6.0 (B).
Fig. 3. Flow curves for chitosan solutions of different concentrations, at pH 3.0 and
25

C. Symbols: 0.5% (,), 1.0% (B), 1.5% (>), 2.0% (6); Full lines represent predictions
of the Cross model and dotted lines those of the Carreau model.
D.S. Bastos et al. / Food Hydrocolloids 24 (2010) 709e718 713
At pH 3.0, the interaction between WPI and chitosan was
minimal and the chitosan solution was almost completely trans-
parent, indicating that no signicant insoluble particles were
present.
For a buffer concentration of 250 mM, again the turbidity of
WPIechitosan solutions at all pH values (except pH 3.0) increased
initially with increasing chitosan concentration until a maximumat
0.01:1, 0.009:1 and 0.016:1 for pH 4.0, 5.0 and 6.0, respectively.
After this, the values were only very slightly decreasing until the
end of the titration for pHs 5.0 and 6.0, whereas for pH 4.0 there
was again a sharp decrease after the maximum, reaching
a minimum plateau value at 0.02:1. After this point, a very slight
increasing tendency is observed at this pH until the nal ratio
tested (Fig. 6).
Again at pH 3.0 there was basically no change in turbidity upon
addition of chitosan for all tested chitosan:WPI ratios.
At pH 4.0 and 6.0 the turbidity values obtained at 250 mM were
smaller than at 100 mM. Probably, the higher ionic strength
shielded the charges of both interacting particles, leading to smaller
amount of WPIechitosan aggregates formation. The turbidity
values at pH 5.0 and 250 mM, on the contrary, remain approxi-
mately constant and high, independently of the chitosan:WPI
ratios. Probably, at this pH condition, the WPI being close to its pI
(Isoelectric point) may contribute to a lower solubility and more
turbidity of the WPI solution (see in Fig. 9b, tube 1).
Lee and Hong (2009) reported that the turbidities of 0.6%
a-lactalbuminechitosan (5:1, w/w) complex and 0.6% b-lactoglo-
bulinechitosan (5:1, w/w) complex formation achieved maximum
values at pH values of 6.5, in a pH range varying from 2.0 to 8.0. In
our case, at 100 mM maximum values are obtained also for pH
values of 4 and 6, even at lower chitosan/WPI ratios; at 250 mM, the
maximum observed for pH 5 has no practical application, because,
as explained above, it depends more on WPI itself and not on
complex formation. At this ionic strength, we also nd the best
complex formation at pH6.0, as after the initial increase in turbidity
the values remain constant throughout.
These results show that turbidimetry measurements provide
a very easy and helpful measurement for this kind of systems. It
allows the observation of the pH dependence of aggregate forma-
tion, as well as the critical ratios needed for stable complexes to
form. The obtained O.D. results showed high reproducibility, and
the error of the mean in each measurement is smaller than 2%.
3.5. ITC measurements
The interaction of chitosan with WPI, in HAc/NaAC buffer at
different ionic strength and pH, was characterized by ITC at 25

C.
Interaction proles were obtained for 0.4% (w/w) chitosan solution
titrated into a reaction cell containing either 0.5% (w/w) WPI
solution (100 and 250 mM) or HAc/NaAC buffer (100 and 250 mM).
The observed peaks were integrated to obtain the total enthalpy
change (DH) as a function of chitosan:WPI ratio in the reaction cell.
The interaction enthalpy was found to be non-signicant at
250 mM, at all studied pH values (results not shown). This means
that the interaction is energetically very small, and therefore non-
resolvable within the sensitivity of our ITC instrument. At 100 mM,
although the observed interaction was small, and therefore the
scatter is large, the results were resolvable. The observed enthalpy,
Fig. 4. Flow curves for chitosan solutions at different concentrations (0.5 and 2.0%) and pH 3.0 (,); 4.7 (>) and pH 6.0% (6).
Table 2
Magnitudes of the Cross model parameters for steady simple shearing, obtained for
chitosan in acetate buffer 250 mM at different pH and concentrations.
Samples Conc (w/w) h
0
(Pa s) s (s) m RE
a
Chitosan pH 3.0 2.0 34.8311 0.1378 0.9730 0.0009
1.5 17.4160 0.1868 0.8156 0.0001
1.0 4.7130 0.0665 0.8082 0.0005
0.5 0.5000 0.0178 0.6894 0.0005
Chitosan pH 4.7 2.0 32.0000 0.1101 0.9767 0.0001
1.5 15.0001 0.1108 0.8451 0.0000
1.0 3.2931 0.0500 0.7331 0.0002
0.5 0.2950 0.0072 0.6000 0.0008
Chitosan pH 6.0 2.0 34.4000 0.1457 0.9770 0.0005
1.5 15.0000 0.1486 0.7968 0.0004
1.0 3.2951 0.0620 0.7300 0.0006
0.5 0.1907 0.0061 0.6959 0.0000
a
RE, relative deviation error =
P
n
i =1
[(x
exp;i
x
cal;i
)=x
exp;i
[=n:
Table 3
Magnitudes of the Carreau model parameters for steady simple shearing, obtained
for chitosan in acetate buffer 250 mM at different pH and concentrations.
Samples Conc (w/w)

h
0
(Pa s) l (s) N RE
a
Chitosan pH 3.0 2.0 27.0521 0.1491 0.4423 0.0008
1.5 11.0001 0.1500 0.3732 0.0004
1.0 3.5099 0.0932 0.3260 0.0008
0.5 0.4170 0.0610 0.2075 0.0000
Chitosan pH 4.7 2.0 26.0720 0.1763 0.3885 0.0001
1.5 11.8800 0.1900 0.3261 0.0000
1.0 2.6254 0.1013 0.2699 0.0009
0.5 0.2530 0.0430 0.1532 0.0005
Chitosan pH 6.0 2.0 27.000 0.1800 0.4247 0.0001
1.5 10.1322 0.1500 0.3485 0.0004
1.0 2.6300 0.1501 0.2550 0.0009
0.5 0.1800 0.0686 0.1331 0.0000
a
RE, relative deviation error =
P
n
i =1
[(x
exp;i
x
cal;i
)=x
exp;i
[=n:
D.S. Bastos et al. / Food Hydrocolloids 24 (2010) 709e718 714
DH, showed a different patternwhen chitosan solutionwas injected
into the titration cell containing WPI solution, at pH values 5.0 and
6.0 (Figs. 7 and 8, respectively).
At pH 5.0 and 100 mM, the observed DH was relatively constant
and close to zero, independently of the chitosan:WPI ratio (Fig. 7),
while at pH 6.0 and 100 mM, the enthalpy values were more
negative at low chitosan:WPI ratios, and decreased (in absolute
value) as chitosan:WPI ratios increased (Fig. 8).
At pH 6, bLG (b-lactoglobulin) is negatively charged, as it is
above its isoelectric point, whereas chitosan is positively charged.
Therefore, chitosan and WPI bind to each other due to electrostatic
attraction at this pH value. This justies that the observed values
are more negative at this pH at the start of the titration run. The
decrease in observed enthalpy as the chitosan concentration
increases reects the fact that the amount of free WPI is decreasing.
These results show unequivocally that electrostatics is the main
driving force for chitosan:WPI interaction.
The results obtained at pH 3.0 and 4.0 and 100 mM indicate that
at these pH values, even at the lower ionic strength (i.e. less charge
shielding for both interacting species), the interaction is not
signicant (results not shown).
If we compare the calorimetric results with the ones obtained by
turbidimetry, we can say that in terms of aggregate formation,
turbidimetry showed to be more discriminate, as a result of an
energetically low interaction. On the other hand, the higher
turbidity values observed generally at lower ionic strength
(100 mM) correlate well with the fact that we could only obtain
resolvable measurements at this ionic strength.
3.6. Preparation of chitosan/WPI coacervates
Fig. 9 illustrates the mixtures obtained from chitosan (1.0%) and
WPI solutions (1.0%) in different proportions and conditions. It is
important to emphasize that the pH conditions used to obtain the
Fig. 5. Inuence of chitosan:WPI ratio (mol:mol) on the turbidity of 0.5% (w/w) WPI
solution in 100 mM HAc/NaAC buffer at different pH. Symbols: ,(pH 3.0); B(pH 4.0);
6 (pH 5.0); 7 (pH 6.0).
Fig. 6. Inuence of chitosan:WPI ratio (mol:mol) on the turbidity of 0.5% (w/w) WPI
solution in 250 mM HAc/NaAC buffer at different pH. Symbols: , (pH 3.0); B (pH
4.0); 6 (pH 5.0); 7 (pH 6.0).
Fig. 7. Enthalpy change vs chitosan:WPI ratio (mol:mol) when aliquots of a 0.4% (w/w)
chitosan solution are injected into a titration cell containing buffered 0.5% (w/w) WPI
solution at pH 5.0 (100 mM acetate buffer, 25

C). The results presented are already
corrected for dilution effects, determined in independent titration runs. Open symbols
represent individual experiments and lled symbols represent the average of the three
experiments.
Fig. 8. Enthalpy change vs chitosan:WPI ratio (mol:mol) when aliquots of a 0.4% (w/w)
chitosan solution are injected into a titration cell containing buffered 0.5% (w/w) WPI
solution at pH 6.0 (100 mM acetate buffer, 25

C). The results presented are already
corrected for dilution effects, determined in independent titration runs. Open symbols
represent individual experiments and lled symbols represent the average of the three
experiments.
D.S. Bastos et al. / Food Hydrocolloids 24 (2010) 709e718 715
insoluble complexes were chosen based on the results of turbidity,
previously shown.
It can be seen in Fig. 9a and b that the maximum chitosan:WPI
ratios needed to obtain coacervates after 24 h at pH 5.0, in 100 mM
and 250 mM acetate buffers, are 0.1:1 and 0.06:1, respectively. As
expected, the WPI concentration necessary to ensure the formation
of the coacervate phase, after 24 h, is higher at 250 mM than at
100 mM. The higher ionic strength has a negative contribution to
the establishment of interactions between chitosan and WPI. It was
also observed that, at 250 mM, the coacervates took a longer time to
formwhen compared to the coacervates at 100 mM. Thus, it can be
suggested that, for mixtures with chitosan:WPI ratios >0.06:1, at
250 mM and pH 5.0, the time required for the formation of insol-
uble complexes, if they eventually form, is higher than 24 h.
With respect to Fig. 9c and d, it can be seen that, at pH 5.5, the
maximum chitosan:WPI ratio required to form coacervates was
0.25:1, independently of the ionic strength used. The lower
concentration of WPI required for the formation of the coacervates
at pH5.5 when compared to the one at pH5.0 is probably due to the
fact that, at pH 5.5, the whey proteins are above their isoelectric
points (pI). It is well established that a-lactalbumin and b-lacto-
globulin, the major proteins in WPI, exhibit pI of 4.8 and w5.34,
respectively, according to the relevant literature (Lee & Hong,
2009). So, at pH 5.5, WPI must be negatively charged whereas
chitosan has a positive charge (pH < 6.5) which favors the inter-
actions between the biopolymers (Lee & Hong, 2009). Furthermore,
it was also noted that at pH 5.5 the formation of the coacervates
was faster than at pH 5.0.
In view of these results, we chose pH 5.5 and chitosan:WPI
mixing ratios of 0.25:1 and 0.10:1 for the preparation of the coac-
ervates (see 2.2.7) to be used in the rheological study presented in
the next section. The dry matter content of the coacervates and
respective supernatants were determined (Table 4).
It can be seen that the coacervates prepared with an initial Chit
(chitosan):WPI ratio of 0.1:1, independently of the solvent, were
denser than those prepared with a Chit:WPI ratio of 0.25:1. For the
same Chit:WPI ratio, coacervates formed in 0.10 M HAc/0.10 M
NaAc were denser than those formed in 0.25 M HAc/0.25 M NaAc.
3.7. Rheology of coacervates from chitosan and WPI
3.7.1. Flow behaviour
Fig. 10A and B shows the ow curves, upon increasing and
decreasing shear rate, for the coacervates studied. The viscosity
decreased with increasing shear rate, as observed before with
chitosan solutions (see 3.3). Chitosan was, as expected, the main
molecule responsible for the appearance of this shear-thinning
behaviour. However, contrarily to chitosan alone, the coacervates
exhibited a time dependent behaviour. The initial structure was
practically fully recovered after some time (_2 h, depending on the
coacervate), as illustrated in Fig. 10B for the coacervate 0.10:1,
which means that the behaviour was thixotropic. This complex
behaviour must arise from changes in the structure of the coacer-
vates, induced by shear, which needed time to reform after defor-
mation. Most probably, this structure was due to the electrostatic
interactions between WPI and chitosan at pH 5.5. These electro-
static interactions would lead to an attraction of the protein and
polysaccharide chains which might disturb the deformation of the
polysaccharide (Weinbreck & Wientjes, 2004). For the coacervates
obtained using 0.10 M HAc/0.10 M NaAc as solvent, the viscosity
was higher for the denser coacervate (0.1:1), independently of the
applied shear rate. When 0.25 M HAc/0.25 M NaAc was used as
solvent, the viscosity was similar, at low shear rates, for the 0.25:1
Fig. 9. Images of mixtures from chitosan (1% w/w) and WPI (1.0% w/w) solutions at
different ratios. (a) pH 5.0, 100 mM HAc/NaAC buffer. (b) pH 5.0, 250 mM HAc/NaAC
buffer. (c) pH 5.5, 100 mM HAc/NaAC buffer. (d) pH 5.5, 250 mM HAc/NaAC buffer.
Chitosan:WPI proportions: 1 (0:1); 2 (0.005:1); 3 (0.01:1); 4 (0.02:1); 5 (0.03:1); 6
(0.06:1); 7 (0.1:1); 8 (0.25:1); 9 (0.5:1); 10 (0.7:1) and 11 (1:1).
Table 4
Dry matter content of Chit/WPI coacervates and supernatants.
Solvent Chit:WPI Dry matter (%)
Coacervate
a
Supernatant
0.10 M HAc/0.10 M NaAc pH 5.5 0.25:1 5.3 0.2 0.50 0.03
0.1:1 14.6 0.3 0.57 0.05
0.25 M HAc/0.25 M NaAc pH 5.5 0.25:1 4.0 0.2 0.96 0.03
0.1:1 7.41 0.02 0.67 0.09
a
After correcting for the dry matter content of the solvent.
D.S. Bastos et al. / Food Hydrocolloids 24 (2010) 709e718 716
and the 0.1:1 coacervates but, afterwards, a less shear-thinning
behaviour was observed for the 0.25:1 coacervate, resulting in
higher viscosity at higher shear rates.
3.7.2. Viscoelastic behaviour
Fig. 10C and D shows the frequency sweeps for the coacervates
studied. In Fig. 10C (0.10 M HAc/0.10 M NaAc), a liquid-like behav-
iour is observed at lower frequencies (<1 rad s
1
) for the coacervate
0.25:1 while, for the 0.1:1 coacervate, G
/
>G
//
from the lowest
frequency accessible in the experiment. As G
/
and G
//
are both
frequency dependent and G
/
is not signicantly higher than G
//
for
lower frequencies, the 0.1:1 coacervate forms a weak gel-like
network structure. In Fig. 10D (0.25 M HAc/0.25 M NaAc), the
coacervates are less structured, exhibit lower G
/
values, and are less
elastic (higher tan d values; not shown).
The pure chitosan solutions, as seen in Section 3.2, showed the
typical viscoelastic behaviour of a polymer solution and had much
smaller G
/
and G
//
values as compared to the coacervates with
similar chitosan concentrations; furthermore, they were also less
elastic.
The different behaviour of the coacervates must arise from the
interactions between the protein molecules and the polysaccharide
chains.
The values of G
/
, in Fig. 10C, show that a more compact structure
is favored for the 0.1:1 coacervate (higher G
/
values in the entire
frequency range studied), in accordance with the values of the dry
matter content (Table 4).
Wang, Lee, Wang and Huang (2007) found signicant correla-
tions between the rheological properties and the composition of
b-lactoglobulin (bLg)/pectin (P) coacervates: the increase of the
bLg:P ratio favored the formation of stronger gel-like coacervates.
On the other hand, a salt-enhanced effect (increase in G
/
) was
observed at low salt concentration while the reverse occurred at
high salt concentration. These observations are in accordance with
our results, though our coacervates didnt form strong gel-like
network structures as theirs.
The WPI molecules are polyelectrolytes containing negative and
positive charges while chitosan is a cationic polysaccharide;
therefore, electrostatic attraction and electrostatic repulsion
between the positive and negative charges in these biopolymers
may occur simultaneously. At low ionic strength, the main effect of
the salt may be the screening of the electrostatic repulsion instead
of disturbing the electrostatic attraction (Wang, Lee, Wang, &
Huang, 2007); thus, the WPI and chitosan contents increase in
the coacervates. The higher chitosan content may be the key factor
to explain the higher G
/
and elasticity of the coacervates. On the
contrary, at higher ionic strength, possibly both electrostatic
attraction and repulsion may be screened signicantly, leading to
a looser watery structure, containing less WPI molecules and chi-
tosan chains, with a smaller G
/
and less elasticity.
4. Conclusions
The viscous and the viscoelastic behaviour of chitosan solutions,
in the concentrated domain, were shown to depend on the
concentration and pH: higher solution concentration and lower pH
resulted in higher viscosity and higher elasticity. A decrease of
chain exibility and an increase of molecular size, when the pH
decreases and the charge of chitosan molecules increases, may
explain these observations. For dilute solutions, the intrinsic
viscosity was found to be dependent on pH, decreasing when the
Fig. 10. Flow curves and frequency sweeps of Chit/WPI coacervates, at 25

C. Coacervates were obtained at pH 5.5, using as solvent: 0.10 M HAc/0.10 M NaAc (A, C); 0.25 M HAc/
0.25 M NaAc (B, D). Symbols: (A and B) Viscosity as function of increasing (full symbols) and decreasing (empty symbols) shear rate. (C and D) Storage modulus, G
/
(full symbols),
and loss modulus, G
//
(empty symbols), as function of angular frequency.
D.S. Bastos et al. / Food Hydrocolloids 24 (2010) 709e718 717
pH increased, as the exibility of the chain increased with the
reduction of the charge.
Chitosan and whey proteins interacted, in solution, and the
intensity of this interaction depended on pH, ionic strength and
protein/polysaccharide ratio.
Turbidimetry showed to be a very informative and useful
technique in this type of studies, as it provides an easy and fast way
to determine pH and ratio dependence of aggregate formation, and
further, the critical ratios necessary for an efcient system at each
experimental condition. These results lead to the mixtures to be
studied and characterized by rheology.
In the present case, as opposed to our previous study (Souza
et al., 2009), the interaction was in most cases too low-enthalpy
to be efciently resolved within the sensitivity of our instrument.
This is probably mainly the result of the high molecular weight of
the polymer, and a lower DD, leading to a less favourable interac-
tion between the two species. The observed dependence on ionic
strength justies our conclusion of an interaction mainly based on
electrostatics, as an increase in ionic strength leads to a larger
charge screening, and thus a smaller interaction.
The rheological behaviour of the coacervates was different from
that of the chitosan solutions: time dependent ow behaviour,
higher G
/
and G
//
values and higher elasticity were observed for the
coacervates. The results suggest that the viscous and viscoelastic
behaviours of the coacervates mainly derive from the contribution
of the chitosan chains. However, the observed differences could be
satisfactorily explained on the basis of the electrostatic interactions
between the two species, conrmed by ITC measurements.
Further studies on the structure of the coacervates and on their
possible use for the microencapsulation of bioactive compounds
are envisaged.
Acknowledgements
Coordenao de Aperfeioamento de Pessoal de Nvel Superior
(CAPES) and Fundao para a Cincia e a Tecnologia (FCT) are
gratefully acknowledged for a CAPES/FCT award. FCT is acknowl-
edged for nancial support to REQUIMTE and CIQ(UP), and for
a Post-Doc grant to H.K.S.S. (SFRH/BPD/37514/2007).
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