Characterization of a Chitosan Sample Extracted From Brazilian Shrimps

8/12/2019 Characterization of a Chitosan Sample Extracted From Brazilian Shrimps

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Characterization of a chitosan sample extracted from Brazilian shrimps and itsapplication 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, Brazilb REQUIMTE, Faculdade de Engenharia, Universidade do Porto, Rua Dr. Roberto Frias, 4200-465 Porto, Portugalc Departamento de Qumica, Faculdade de Cincias, Universidade do Porto, Rua do Campo Alegre 687, 4169-007 Porto, Portugald 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

TurbidityITC

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 fromw17 dL/g tow14 dL/g when the pH increased from 4.7 to6.0. 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 anddecreasing 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). ITCresults 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 aggregateformation 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 andwere 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 0 and G 00 values and higherelasticity 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 oppositelycharged polyelectrolyte, has attracted academic and industrialinterest. It is typically observed in the self-assembly of biologicalmacromolecules (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 offunctionality, 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 tothermodynamic 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 withproteins at pHs below their isoelectric points (Chanasattru, Jones,

* Corresponding author. Tel.: 351 225081684; fax: 351 225081449.

E-mail address: [email protected] (M.P. Gonalves).

Contents lists available atScienceDirect

Food Hydrocolloids

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . co m / l o c a t e / f o o d h y d

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
mailto:[email protected]://www.sciencedirect.com/science/journal/0268005Xhttp://www.elsevier.com/locate/foodhydhttp://www.elsevier.com/locate/foodhydhttp://www.sciencedirect.com/science/journal/0268005Xmailto:[email protected]


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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, theuse 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 thusformed 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 theprotonation of amino groups gives rise to its polyelectrolyte char-acter. Both deacetylation degree (DD) and molar mass have beenshown to inuence the conformation and solution properties of

chitosan (Lamarque, Lucas, Viton, & Domard, 2005). The interactionof 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 oppositecharges, in the range of pH 5 and 7 (Guzey & McClements, 2006).

The globular proteinsb-lactoglobulin anda-lactalbumin are the

main proteins of whey. In many food products, the functional

properties of whey protein are used to stabilize oil-in-wateremulsions, 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 wheyprotein 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 spectrophotometrywere used under different conditions of pH, ionic strength andcomponents 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 intoconsideration its signicance to food product development.

2. Materials and methods

2.1. Materials

Chitosan samples were obtained by partial deacetylation of chitinfrom shells ofPenaeus schmittishrimp. WPI (Lacprodan DIe9224)was kindly donated by Arla Foods Ingredients (Denmark). Sodiumacetate trihydrate (CH3COONa$3H2O) and glacial acetic acid(CH3COOH) were purchased from Merck (99.5%) (Darmstadt,

Germany). Sodium hydroxide (NaOH) from Pronalab (Lisboa,

Portugal). All other chemicals were analytical grade and usedwithout 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

chitosanChitin extraction from shrimps shells and the preparation of

chitosan were performed according to the method ofPercot, Viton,

and Domard (2003), with some modications. For demineraliza-tion, shells were treated with 0.25 M HCl in excess at roomtemperature (25 C), for 30 min. Chitin deproteinization wascarried 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 wasperformed 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. Whencompared to continuous treatments (one stage for long periods oftime), 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 bathsof deionized water, ltered, and dried at 50 C for 12 h. The secondstage was carried out under reux and nitrogen bubbling, in thepresence of sodium borohydride (0.1 g/g of deacetylated chitin),

with 50% (w/w) NaOH solution for 5 h. Both deacetylation stageswere performed with a 1/50 (w/v) solid to liquid ratio.

The method ofLavertu et al. (2003) was used to determine thedegree of deacetylation (DD) of the chitosan sample, DD 93.04%.

2.2.2. Preparation of solutionsChitosan and whey protein isolate (WPI) stock solutions were

separately prepared in 100 mM or 250 mM CH3COOH/CH3COONa

(HAc/NaAC) buffer at the desired pH. The relatively high ionicstrengths of the buffers were necessary for them to be effective andensure pH stability during the experiments. The dispersions were

gently agitated for at least 2 h at room temperature until completepolymerdissolutionoccurred and their pHs were checked.Finally,thesolutions were stored overnight in the refrigerator, for further use.

2.2.3. ViscosimetryViscosity 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 Cusing a glass capillary Ubbelohde viscometer with a capillary

diameter of 0.58 mm. The chitosan solutions were prepared asdescribed in2.2.2. The dilute solutions had relative viscosities,hrel,from about 1.2 to 2.0 to assure good accuracy and linearity of

extrapolation to zero concentration. Flow times were measured intriplicate, for each sample, and their average values were used forthe calculations. The limiting viscosity number (intrinsicviscosity), [h], was obtained by double extrapolation to zeroconcentration of Hugginsand Kraemer equations, respectively

hsp=C h k0h2C (1)

lnhrel=C h k00h2C (2)

where hrel and hsp are the (dimensionless) relative and specicviscosities, k 0 and k 00 are the Huggins and Kraemers coefcients,respectively, andC is the solution concentration.

Viscosity average molecular masses, Mv, were calculated using

the Marke

Houwinke

Sakurada relationship(3):

h KMav (3)

wherea and Kare constants for the buffer solution and chitosansystem.

These constants were calculated using the two followingequations, proposed byKasaai (2007)as a model to calculateaand

K for chitosan in any solventetemperature system using visco-metric constant data previously reported by several researchgroups:

a 0:6202 0:699x=0:4806 x (4)

logK$105 5:7676a 5:9232 (5)

D.S. Bastos et al. / Food Hydrocolloids 24 (2010) 709e718710


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wherex [DA/pH$m], with DA, degree of acetylationof chitosan, pHof chitosan solution in a solvent with ionic strength ofm.

2.2.4. Rheological measurements of chitosan solutionsChitosan 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 in2.2.2and usedfor rheological measurements.

All rheological measurements were performed at 25 C usinga controlled stress rheometer AR2000 (TA Instruments Inc., NewCastle, 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 torquewas imposedusing a logarithmic ramp, in order to decrease the initial acceler-

ation and the effects due to instrument inertia. Shear rates obtainedwere in the 1e300 s1 range. Frequency sweeps were performed inthe 0.1e100 rad s1 range, with strain amplitude of 5%, in order toassure working conditions inside the linear viscoelastic region,

determined by preliminary experiments (strain sweeps). Sampleswere covered with a thin layer of parafn oil in order to hinderevaporation during the experiments.

2.2.5. Turbidity measurementsTurbidity 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 theaddition 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 (25BC). Measurementswere carried out in triplicate for each mixture, and their average isreported.

2.2.6. Isothermal titration calorimeter (ITC) measurementsThe 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 asdescribed in 2.2.2. These concentrations were chosen according toprevious 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 itscontroller, built at Lund University, Sweden, and a 71/2 digit HPnanovoltmeter connected to the calorimetric channel and to thecomputer. 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 rpm with a gold propeller.Dilution effects were taken care of separately, by titrating the same

chitosan solution into the appropriate acetate buffer solution in thevessel. The obtained heats in the interaction experiments werethereafter 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 intriplicate and the results were reported as their mean.

2.2.7. Preparation and rheological characterization of

chitosaneWPI coacervatesChitosan 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 allowcomplete phase separation. Then, pictures of the tubes were takenusing 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 andsteady 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 pH at25 C,

using the Huggins and the Kraemer equations (equations (1) and(2), respectively). The two extrapolations gave similar results, asshown in Table 1. [h] was found to be dependent on pH, decreasingfromw17 dL/g tow14 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), chitosanmolecules are more charged and take an expanded conformationwhile, 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 ofattraction, 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-glucosaminerepeating 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,k0, with pHwas observed (Table 1). Values ofk0 depend on the state of aggre-

gation of macromolecules and on soluteesolvent interactions.Values of k0 w0.3 are obtained for exible macromolecules ina good solvent but, in case of aggregation,k0 can be higher than 1.Our results suggest that the solubility of chitosan molecules

decreasedi.e. polymerepolymer interactions or association, werefavored when the pH increased.

Values of [h] obtained for each pH were used in theMarkeHouwinkeSakurada relationship (equation(3)) to evaluate

the viscosity average molecular mass of the chitosan sample. TheconstantsKand a were calculated according toKasaai (2007). The

resulting values are shown inTable 1.In a good solvent, polymeresolvent interactions are strong and

the conformation of the polymer is extended. Higher values of [h]anda are then obtained. Most of the reported values for exponent

alie 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 k0 k00 K a Mv/Da

4.7 16.7 17.1 0.58 0.06 8.1 104 0. 70 1.6 106

6.0 13.5 14.1 0.69 0.03 9.8 104 0. 68 1.2 106

a From Huggins extrapolation.b

From Kraemer extrapolation.

D.S. Bastos et al. / Food Hydrocolloids 24 (2010) 709e718 711


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value of exponent a, related to a random coil and compactconformation is obtained when a poor solvent is used (Kasaai,

2007). In our case, the pH was varied but the ionic strength wasmaintained. As chitosan is a polyelectrolyte, the quality of thesolvent changed with pH. The polymeresolvent interactions and

the polymer conformation depend on the number of positivecharges of the molecule which change with pH. So, the calculatedvalues ofashow that the quality of the solvent was better at pH 4.7than at pH 6.0.

Values of Mv calculated using equation (3), were different for

each pH, though of the same order of magnitude, showing that theequations(4) and (5), proposed by Kasaai (2007) were a reasonableapproach for calculating the parameters Kand 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 tocompare these different results with our own.

3.2. Rheological behaviour of chitosan solutions under dynamic

shear

At the concentrations and oscillatory frequencies studied, theviscoelastic behaviour of chitosan solutions, independently of pH,

was typical of a system with predominant entanglement networksbetween the terminal and plateau zones of frequency response, asillustrated in Fig. 1 for solutions at pH 3. At low frequencies(terminal zone), a liquid-like behaviour is observed, where the loss

modulus, G00, is higher than the storage modulus, G0, (Fig. 1a) andthe magnitude of the complex viscosity, jh*j, is essentiallyfrequency independent (not shown in Fig.1afor 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 G0 G00 ortandG00/G0 1) typically moved to lower frequency values whenthe 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 thesystem increased. This kind of behaviour is typical of severalrandom-coil polysaccharide solutions like galactomannans

(Andrade, Azero, Luciano, & Gonalves, 1999; Sittikijyothin, Torres,& Gonalves, 2005), and was also observed for chitosan solutions in0.5 M acetic acid or in 0.5 M acetic acid/0.1 M sodium acetate byCho et al., 2006.

InFig. 2a, the effect of pH on G0, G00 and jh*j is shown for 1%chitosan solutions. A decrease of the three parameters withincreasing pH is observed.

In addition, tandincreased (Fig. 2b). As pH increases, the chargedensity of chitosan molecules decreases leading to an increase of

chain exibility and a reduction of molecular size. As a consequenceof increased chain exibility, the number of interactions andentanglements between chitosan molecules may decrease resulting

in a reduction of the elasticity. This kind of reasoning was used byCho etal. (2006) to explain thebehaviour of chitosan solutionsof thesame concentration at different ionic strengths. By increasingthe chitosan concentration, the elasticity increases, as expected, but

the effect of pH is less pronounced (Fig. 2b, for 3% chitosanconcentration) than at lower concentration (Fig. 2b, for 1% chitosanconcentration).

3.3. Steady-shear properties of chitosan solutions

Typical ow curves, at 25 C, for chitosan solutions, at differentconcentrations, are shown inFig. 3, for pH 3.0. Similar curves wereobtained 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 constantzero-shear viscosity (h0), was attained only for the lowestconcentration studied (0.5%). The shear-thinning behaviour arisesfrom modications in the macromolecular organization in the

solution as shear rate changes. At low shear rates, the rate ofdisruption of inter-molecular entanglements brought about by theshear force exerted is balanced by that of entanglements newlyformed and a constant zero-shear viscosity can be maintained. For

higher shear rates, disruption predominates over formation of newentanglements, molecules align in the direction of ow and theapparent viscosity decreases with increasing shear rate. When thechitosan concentration increases, the molecules in solution become

more entangled and their mobility decreases. As a consequence, thetime required to form new entanglements to replace those dis-rupted by the externally imposed deformation increases and theshear rate corresponding to the transition from Newtonian to

shear-thinning behaviour moves to lower values (Fig. 3).

A comparison between experimental data obtained at differentpH values, showed that the viscosity of chitosan solutions of thesame 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 morepronounced 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 inFig. 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 (G0) and loss (G00) modulivsoscillation frequency and (b) tandvsoscillation frequency. G0: full symbols,

G

00

: open symbols. Solutions concentrations: 0.5% (B

), 1.0% (,

), 2.0% (>

), 3.0% (6

).



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macromolecular interactions. At low concentration, molecules are

less entangled and have more free volume to occupy; if they aremore charged (lower pH), their hydrodynamic volume will increase

because of higher intra-molecular repulsions and the viscosity ofthe solution will be higher. When the concentration increases,molecules become more entangled, with less free space to occupyand increased inter-molecular interactions. A balance betweenintra- and inter-molecular interactions probably implies that the

volume of the macromolecule does not change with pH as much asit does at lower concentrations.

The ow models ofCarreau (1972, equation (7)) and Cross (1965,equation (6)) were used to describe the shear-thinning behaviour of

the chitosan solutions:

ha hN h0hN1 s _gm

(6)

ha hN h0hNh1 l _g

2iN (7)

wherehais the apparent viscosity (Pa s) measured at the shear rate_gs1, h0 is the zero-shear rate viscosity (Pa s), hN is the inniteshear rate viscosity (Pa s),ss and ls 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

hN ), 2.0% (6); Full lines represent predictions

of the Cross model and dotted lines those of the Carreau model.



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At pH 3.0, the interaction between WPI and chitosan wasminimal and the chitosan solution was almost completely trans-

parent, indicating that no signicant insoluble particles werepresent.

For a buffer concentration of 250 mM, again the turbidity ofWPIechitosan solutions at all pH values (except pH 3.0) increased

initially with increasing chitosan concentration until a maximum at0.01:1, 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 theend of the titration for pHs 5.0 and 6.0, whereas for pH 4.0 there

was again a sharp decrease after the maximum, reachinga minimum plateau value at 0.02:1. After this point, a very slightincreasing tendency is observed at this pH until the nal ratiotested (Fig. 6).

Again at pH 3.0 there was basically no change in turbidity uponaddition of chitosan for all tested chitosan:WPI ratios.

At pH 4.0 and 6.0 the turbidity values obtained at 250 mM weresmaller than at 100 mM. Probably, the higher ionic strength

shielded the charges of both interacting particles, leadingto smalleramount of WPIechitosan aggregates formation. The turbidityvalues 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 moreturbidity of the WPI solution (see inFig. 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 maximumvalues 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 pHvalues 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 bestcomplex formation at pH 6.0, 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. Itallows the observation of the pH dependence of aggregate forma-

tion, as well as the critical ratios needed for stable complexes toform. The obtained O.D. results showed high reproducibility, andthe 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 atdifferent ionic strength and pH, was characterized by ITC at 25 C.

Interaction proles were obtained for 0.4% (w/w) chitosan solutiontitrated 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 enthalpychange (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 meansthat 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) h0(Pa s) s(s) m REa

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 0.00001.0 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 Pni 1jxexp;ixcal;i=xexp;ij=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) h0(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 0.00080.5 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 Pni

1

jxexp;ixcal;i=xexp;ij=n:



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DH, showed a different pattern when chitosan solution was injectedinto 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 DHwas 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 absolutevalue) as chitosan:WPI ratios increased (Fig. 8).

At pH 6, bLG (b-lactoglobulin) is negatively charged, as it isabove its isoelectric point, whereas chitosan is positively charged.

Therefore, chitosan and WPI bind to each other due to electrostaticattraction at this pH value. This justies that the observed valuesare more negative at this pH at the start of the titration run. Thedecrease in observed enthalpy as the chitosan concentration

increases reects the fact that the amount of free WPIis decreasing.These results show unequivocally that electrostatics is the maindriving 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 higherturbidity 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. 9illustrates the mixtures obtained from chitosan (1.0%) andWPI solutions (1.0%) in different proportions and conditions. It isimportant 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 changevschitosan: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 andlled symbols represent the average of the threeexperiments.

Fig. 8. Enthalpy changevschitosan: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 andlled symbols represent the average of the three

experiments.



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insoluble complexes were chosen based on the results of turbidity,

previously shown.It can be seen inFig. 9a and bthat the maximum chitosan:WPI

ratios needed to obtain coacervates after 24 h at pH 5.0, in 100 mMand 250 mM acetate buffers, are 0.1:1 and 0.06:1, respectively. As

expected, the WPI concentration necessary to ensure the formationof the coacervate phase, after 24 h, is higher at 250 mM than at100 mM. The higher ionic strength has a negative contribution tothe establishment of interactions between chitosan and WPI. It was

also observed that, at 250 mM, the coacervates took a longer timeto

form when compared to the coacervates at 100 mM. Thus, it can be

suggested that, for mixtures with chitosan:WPI ratios >0.06:1, at250 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 toFig. 9c and d,it can be seen that, at pH 5.5, themaximum chitosan:WPI ratio required to form coacervates was0.25:1, independently of the ionic strength used. The lowerconcentration of WPI required for the formation of the coacervates

atpH 5.5 whencompared totheoneat pH 5.0 is probablydueto thefact that, at pH 5.5, the whey proteins are above their isoelectricpoints (pI). It is well established that a-lactalbumin and b-lacto-globulin, the major proteins in WPI, exhibit pIof 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 coacervateswas faster than at pH 5.0.

In view of these results, we chose pH 5.5 and chitosan:WPImixing ratios of 0.25:1 and 0.10:1 for the preparation of the coac-ervates (see2.2.7) 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 thesame Chit:WPI ratio, coacervates formed in 0.10 M HAc/0.10 MNaAc 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 anddecreasing shear rate, for the coacervates studied. The viscositydecreased with increasing shear rate, as observed before with

chitosan solutions (see 3.3). Chitosan was, as expected, the mainmolecule responsible for the appearance of this shear-thinningbehaviour. However, contrarily to chitosan alone, the coacervatesexhibited a time dependent behaviour. The initial structure was

practically fully recovered after some time (2 h, depending on thecoacervate), as illustrated in Fig. 10B for the coacervate 0.10:1,which means that the behaviour was thixotropic. This complexbehaviour 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 electrostaticinteractions 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 thepolysaccharide (Weinbreck & Wientjes, 2004). For the coacervatesobtained using 0.10 M HAc/0.10 M NaAc as solvent, the viscositywas 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 assolvent, 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/NaACbuffer. (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 (%)

Coacervatea 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.



9/10

and the 0.1:1 coacervates but, afterwards, a less shear-thinningbehaviour was observed for the 0.25:1 coacervate, resulting in

higher viscosity at higher shear rates.

3.7.2. Viscoelastic behaviourFig. 10C and D shows the frequency sweeps for the coacervates

studied. InFig. 10C (0.10 M HAc/0.10 M NaAc), a liquid-like behav-

iour is observed at lower frequencies (G00 from the lowestfrequency accessible in the experiment. As G0 and G00 are bothfrequency dependent andG0 is not signicantly higher thanG00 forlower frequencies, the 0.1:1 coacervate forms a weak gel-like

network structure. In Fig. 10D (0.25 M HAc/0.25 M NaAc), thecoacervates are less structured, exhibit lowerG0 values, and are lesselastic (higher tan d values; not shown).

The pure chitosan solutions, as seen in Section3.2,showed the

typical viscoelastic behaviour of a polymer solution and had muchsmaller G0 and G00 values as compared to the coacervates withsimilar chitosan concentrations; furthermore, they were also lesselastic.

The different behaviour of the coacervates must arise from theinteractions between the protein molecules and the polysaccharidechains.

The values ofG0, inFig.10C, show that a more compact structureis favored for the 0.1:1 coacervate (higher G 0 values in the entire

frequency range studied), in accordance with the values of the drymatter 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 G0) wasobserved at low salt concentration while the reverse occurred at

high salt concentration. These observations are in accordance withour 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 repulsionbetween the positive and negative charges in these biopolymersmay occur simultaneously. At low ionic strength, the main effect ofthe 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 inthe coacervates. The higher chitosan content may be the key factorto explain the higher G 0 and elasticity of the coacervates. On thecontrary, at higher ionic strength, possibly both electrostatic

attraction and repulsion may be screened signicantly, leading toa looser watery structure, containing less WPI molecules and chi-tosan chains, with a smallerG0 and less elasticity.

4. Conclusions

The viscous and the viscoelastic behaviour of chitosan solutions,in the concentrated domain, were shown to depend on theconcentration and pH: higher solution concentration and lower pH

resulted in higher viscosity and higher elasticity. A decrease ofchain exibility and an increase of molecular size, when the pHdecreases and the charge of chitosan molecules increases, mayexplain 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 0 (full symbols),

and loss modulus, G00 (empty symbols), as function of angular frequency.



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pH increased, as the exibility of the chain increased with thereduction of the charge.

Chitosan and whey proteins interacted, in solution, and theintensity of this interaction depended on pH, ionic strength andprotein/polysaccharide ratio.

Turbidimetry showed to be a very informative and usefultechnique in this type of studies, as it provides an easy and fast wayto determine pH and ratio dependence of aggregate formation, andfurther, the critical ratios necessary for an efcient system at each

experimental condition. These results lead to the mixtures to bestudied 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-enthalpyto be efciently resolved within the sensitivity of our instrument.This is probably mainly the result of the high molecular weight ofthe polymer, and a lower DD, leading to a less favourable interac-

tion between the two species. The observed dependence on ionicstrength justies our conclusion of an interaction mainly based onelectrostatics, as an increase in ionic strength leads to a largercharge screening, and thus a smaller interaction.

The rheological behaviour of the coacervates was different fromthat of the chitosan solutions: time dependent ow behaviour,

higherG0 andG00 values and higher elasticity were observed for thecoacervates. The results suggest that the viscous and viscoelasticbehaviours of the coacervates mainly derive from the contribution

of the chitosan chains. However, the observed differences could besatisfactorily explained on the basis of the electrostatic interactionsbetween 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 compoundsare 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 fora Post-Doc grant to H.K.S.S. (SFRH/BPD/37514/2007).

References

Andrade, C. T., Azero, E. G., Luciano, L., & Gonalves, M. P. (1999). Solution propertiesof the galactomannans extracted from the seeds ofCaesalpinia pulcherrimaandCassia javanica: comparison with locust bean gum. International Journal ofBiological Macromolecules, 26, 181e185.

Ansarian, H. R., Derakhshan, M., Takafugi, M., & Ihara, H. (2008). The impact ofsupramolecular chemistry in medicine: removing the border between infec-tious and non-infectious diseases. Medical Hypotheses, 71, 881e885.

Bai, G., Santos, L. M. N. B. F., Nichifor, M., Lopes, A., & Bastos, M. (2004). Thermo-dynamics of the interaction between a hydrophobically modied poly-electrolyte and sodium dodecyl sulfate in aqueous solution. Journal of Physical

Chemistry B, 108, 405e

413.Berth,G., & Dautzenberg,H. (2002). Thedegreeof acetylationof chitosanand itseffect

onthe chain conformationin aqueoussolution. Carbohydrate Polymers,47, 39e51.Burgess, D. J. (1994). Complex coacervation: microcapsule formation. In P. Dubin,

J. Bock, R. Davis, D. N. Schulz, & C. Thies (Eds.), Macromolecular complexes inchemistry and biology (pp. 285e300). Berlin: Springer Verlag.

Carreau, P. J. (1972). Rheological equations from molecular network theories.Transactions of the Society of Rheology, 16, 99e127.

Chanasattru, W., Jones, O. G., Decker, E. A., & McClements, D. J. (2009). Impact ofcosolvents onformationandproperties ofbiopolymernanoparticlesformedby heattreatmentof b-lactoglobulinepectincomplexes.FoodHydrocolloids,23, 2450e2457.

Cho, J., Heuzey, M.-C., Bgin, A., & Carreau, P. J. (2006). Viscoelastic properties ofchitosan solutions: effect of concentration and ionic strength. Journal of FoodEngineering, 74, 500e515.

Cross, M. M. (1965). Rheology of nonNewtonian uids: a new ow equation forpseudoplastic systems. Journal of Colloid Science, 20 , 417e437.

Dankers, P. Y. W., & Meijer, E. W. (2007). Supramolecular biomaterials. A modularapproach towards tissue engineering. Bulletin of the Chemical Society of Japan,

80, 2047e

2073.

Ducel, V., Richard, J., Saulnier, P., Popineau, Y., & Boury, F. (2004). Evidence andcharacterization of complex coacervates containing plant proteins: applicationto the microencapsulation of oil droplets. Colloids and Surfaces A, Physico-chemical and Engineering Aspects, 232, 239e247.

Gonalves, M. P., Torres, D., Andrade, C. T., Azero, E. G., & Lefebvre, J. (2004).Rheological study of the effect ofCassia javanica galactomannans on the heat-set gelation of a whey protein isolate at pH 7. Food Hydrocolloids, 18, 181e189.

Guzey, D., & McClements, D. J. (2006). Characterization ofb-lactoglobulinechitosaninteractions in aqueous solutions: a calorimetry, light scattering, electropho-retic mobility and solubility study. Food Hydrocolloids, 20, 124e131.

Junyaprasert, V. B., Mitrevej, A., Sinchaipanid, N., Boonme, P., & Wurster, D. E.(2001). Effect of process variables on the microencapsulation of vitamin Apalmitate by gelatineacacia coacervation. Drug Development and IndustrialPharmacy, 2, 561e566.

Kasaai, M. R. (2007). Calculation of MarkeHouwinkeSakurada (MHS) equation visco-metric constants for chitosan in any solventetemperature system using experi-mental reported viscometric constants data. Carbohydrate Polymers, 68, 477e488.

Kasaai, M. R., Arul, J., & Charlet, G. (2000). Intrinsic viscosity emolecular weightrelationship for chitosan.Journal of Polymer Science: Part B: Polymer Physics, 38,2591e2598.

Khokhlov, A. R., & Khalatur, P. G. (2005). Solution properties of charged hydrophobic/hydrophilic copolymers. CurrentOpinionin Colloidand Interface Science,10, 22e29.

de Kruif,C. G.,Weinbreck,F., & de Vries, R. (2004).Complex coacervationof proteinsandanionicpolysaccharides.CurrentOpinionin Colloid andInterfaceScience, 9,340e349.

Lali, A., Roshnnie, N., & Devika, T. (2000). Reversible precipitation of proteins oncarboxymethyl cellulose. Process Biochemistry, 35, 777e785.

Lamarque, G., Lucas, J.-M., Viton, C., & Domard, A. (2005). Physicochemical behaviorof homogeneous series of acetylated chitosans in aqueous solution: role ofvarious structural parameters. Biomacromolecules, 6, 131e142.

Lavertu, M., Xia, Z., Serreqi, A. N., Berrada, M., Rodrigues, A., Wang, D., et al. (2003). Avalidated 1H NMRmethodfor thedeterminationof thedegreeof deacetylation ofchitosan. Journal of Pharmaceutical and Biomedical Analysis, 32 , 1149e1158.

Lee,A.-C., & Hong, Y.-H. (2009).Coacervateformationofa-lactalbuminechitosanand b-lactoglobulinechitosan complexes. Food Research International, 42(5e6),733e738.

Lutz, R., Aserin, A., Portnoy, Y., Gottlieb, M., & Garti, N. (2009). On the confocalimages and the rheology of whey protein isolated and modied pectins asso-ciated complex. Colloids and Surfaces B: Biointerfaces, 69, 43e50.

McDonald, P., Victa, C., Carter-Franklin, J. N., & Fahrner, R. (2008). Selectiveantibody precipitation using polyelectrolytes: a novel approach to the puri-cation of monoclonal antibodies. Biotechnology and Bioengineering, 102,1141e1151.

Matos, C., Lima, J. L. C., Reis, S., Lopes, A., & Bastos, M. (2004). Interaction of anti-inammatory drugs with EPC liposomes. Calorimetric study in a broadconcentration range.Biophysical Journal, 86, 946e954.

Mattison, K. W., Brittain, I. J., & Dubin, P. L. (1995). Proteinepolyelectrolyte phaseboundaries.Biotechnology Progress, 11, 632e637.

Mekhlou, G., Sanchez, C., Renard, D., Guillemin, S., & Hardy, J. (2005). pH-induced

structural transitions during complexation and coacervation ofb-lactoglobulinand acacia gum. Langmuir, 21, 386e394.Muthukumar, M. (1997). Dynamics of polyelectrolyte solutions.Journal of Chemical

Physics, 107, 2619e2635.Percot, A., Viton, C., & Domard, A. (2003). Optimization of chitin extraction from

shrimp shell. Biomacromolecules, 4, 12e18.Porri, M. C., Braia, M., Farrugia, B., Pic, G., & Romanini, D. (2009). Precipitation

with polyacrylic acid as a trypsin bioseparation strategy. Process Biochemistry,44, 1046e1049.

Rocha, C., Teixeira, J. A., Hilliou, L., Sampaio, P., & Gonalves, M. P. (2009). Rheo-logical and structural characterization of gels from whey protein hydrolysates/locust bean gum mixed systems. Food Hydrocolloids, 23, 1734e1745.

Sanchez, C., Meklou, G., & Renard, D. (2006). Complex coacervation between b-lactoglobulin and acacia gum: a nucleation and growth mechanism. Journal ofColloid and Interface Science, 299, 867e873.

Singh, S. S., Siddhanta, A. K., Meena, R., Prasad, K., Bandyopadhyay, S., &Bohidar, H. B. (2007). Intermolecular complexation and phase separation inaqueous solutions of oppositely charged biopolymers. International Journal ofBiological Macromolecules, 41, 185e192.

Sittikijyothin, W., Torres, D., & Gonalves, M. P. (2005). Modelling the rheologicalbehaviour of galactomannan aqueous solutions. Carbohydrate Polymers, 59,339e350.

Souza, H. K. S., Bai, G., Gonalves, M. P., & Bastos, M. (2009). Whey protein iso-lateechitosan interactions: a calorimetric and spectroscopy study. Thermochi-mica Acta, 495, 108e114.

Tolaimate, A., Desbrires, J., Rhazi, M., Alagui, A., Vincendon, M., & Votter, P. (2000).On the inuence of deacetylation process on the physicochemical characteris-tics of chitosan from squid chitin. Polymer, 41, 2463e2469.

Tolstoguzov, V. (2002). Thermodynamic aspects of biopolymer functionality in bio-logicalsystems,foods, and beverages.CriticalReviews in Biotechnology, 22, 89e174.

Turgeon, S. L., Schmitt, C., & Sanchez, C. (2007). Proteinepolysaccharidecomplexes and coacervates. Current Opinion in Colloid and Interface Science,12, 166e178.

Wang, X., Lee, J., Wang, Y.-W., & Huang, Q. (2007). Composition and rheologicalproperties of b-lactoglobulin/pectin coacervates: effects of salt concentrationand initial protein/polysaccharide ratio. Biomacromolecules, 8, 992e997.

Weinbreck, & Wientjes, R. H. W. (2004). Rheological properties of whey protein/gum arabic coacervates. Journal of Rheology, 48, 1215e1228.


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