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Food Hydrocolloids 23 (2009) 536–547

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Characterization of chitosan–oleic acid composite films

Maria Vargas�, Ana Albors, Amparo Chiralt, Chelo Gonzalez-Martınez

Department of Food Technology, Institute of Food Engineering for Development, Universidad Politecnica de Valencia,

Camino de Vera s/n, 46022 Valencia, Spain

Received 30 November 2007; accepted 26 February 2008

Abstract

Edible films based on high molecular weight chitosan (CH) and different concentrations of oleic acid (OA) were prepared.

Film-forming dispersions (FFD) were characterized in terms of rheological properties, surface tension, particle size distribution and

z-potential. In order to study the impact of the incorporation of OA into the CH matrix, the water sorption isotherms, water vapour

permeability (WVP), mechanical properties, and optical properties of the dry films were evaluated. Results showed that the increase in

OA promoted changes in the size and surface charge of the FFD particles, which had an impact on the rheological properties of the FFD.

As regards the film properties, the higher the OA content, the lower the WVP and the moisture sorption capacity. In general, the addition

of OA into the CH matrix leads to a significant increase in gloss and translucency and a decrease in the tensile strength, elongation

at break and elastic modulus of the composite films. The mechanical and optical properties of the films were related with their

microstructure, which was observed by SEM.

r 2008 Elsevier Ltd. All rights reserved.

Keywords: Chitosan; Film; Oleic acid; Water vapour permeability; Transparency; Mechanical properties; Microstructure

1. Introduction

Chitosan is a biodegradable cationic polysaccharidewith antimicrobial activity (Cuero, 1999; Jung & Kim,1999; No, Park, Lee, & Meyers, 2001; Tharanathan &Kittur, 2003; Zheng & Zhu, 2003) and excellent film-forming ability (Domard & Domard, 2001; Li, Dunn,Grandmaison, & Goosen, 1992). This makes it particularlysuitable for the formulation of edible coatings, which haveproved to be effective at extending the shelf-life of fruitsand vegetables (Durango, Soares, & Andrade, 2006; Han,Zhao, Leonard, & Traber, 2004; Vargas, Albors, Chiralt, &Gonzalez-Martınez, 2006).

Chitosan films have a selective permeability to gases(CO2 and O2) and good mechanical properties. However,the fact that they are highly permeable to water vapourlimits their uses (Butler, Vergano, Testin, Bunn, & Wiles1996; Caner, Vergano, & Wiles, 1998), which is an

ee front matter r 2008 Elsevier Ltd. All rights reserved.

odhyd.2008.02.009

ing author. Tel.: +3496 387 7000x73642;

73 69.

ess: mavarco@tal.upv.es (M. Vargas).

important drawback since an effective control of moisturetransfer is a desirable property for most foods.The functional properties of chitosan-based films can be

improved by combining them with other hydrocolloids(Park, Marsh, & Rhim, 2002; Xu, Kim, Hanna, & Nag,2004). In this sense, Hoagland and Parris (1996) developedchitosan/pectin laminated films by interacting cationicgroups of chitosan with the anionic groups of pectin. Xuet al. (2004) observed a decrease in water vapourtransmission rates (WVTRs) by combining chitosan withtwo thermally gelatinized corn starches.In order to improve water barrier properties, lipid

compounds such as fatty acids, natural waxes, surfactantsand resins are frequently incorporated into hydrocolloid-based films (Baldwin, Nisperos, Hagenmaier, & Baker,1997; Fabra, Talens, & Chiralt, 2008; Kester & Fennema,1986). For example, Wong, Gastineau, Gregorski, Tillin,and Pavlath, (1992) added saturated fatty acids and fattyacid esters to chitosan-based films, obtaining a higherresistance to water vapour transmission when lauric andbutyric acids were used; Park and Zhao (2004) incorpo-rated Vitamin E into the chitosan matrix obtaining a

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significant decrease in the water vapour permeability(WVP), an increase of opacity and a significant reductionin the tensile strength of the composite films. The effect ofadding some saturated fatty acids, such as palmitic andstearic, has also been investigated (Srinivasa, Ramesh, &Tharanathan, 2007). Nevertheless, to the best of ourknowledge, there are no reported data on the effect ofunsaturated fatty acids, such as oleic acid, on the propertiesof chitosan-based films and film-forming dispersions(FFD).

In formulations of films and coatings based on mixturesof hydrocolloids and lipids, surfactants are usuallyincorporated to promote emulsion formation and inter-facial stabilization. In these dispersions polysaccharidesand proteins act as stabilizers through the increase of theviscosity of the continuous phase (polysaccharides), orthrough the formation of an adsorbed layer on theoil–water interface (proteins) that promotes steric stabili-zation. In this sense, chitosan can enhance the stability ofFFD by an electro-steric effect, since it is positivelycharged in acid media, as well as by a viscous effect, thuspromoting emulsion stability and decreasing the rate ofcreaming (Rodrıguez, Albertengo, & Agullo, 2002).

The stability of the FFD plays an important role in theproperties of lipid–hydrocolloid composite films. The lipidparticle size greatly affects the development of the systemduring the film drying process (Morillon, Debeaufort,Blond, Capelle, & Voilley, 2002). Water evaporation leadsto flocculation, coalescence and the creaming of lipiddroplets, depending on droplet size and distribution,among other factors, such as viscosity of the continuousphase, interactions between droplets (electrostatic, van derWaals and steric interactions) and rheological properties ofthe O/W interface.

The stability of the FFD greatly affects the finalmicrostructure of the film matrix, which defines to agreater extent its functional properties. In this sense,the characterization of some stability factors of thedispersions could contribute to understand the differencesin functional properties of the final film. In particular,the analysis of the surface charge of the particles andtheir size distribution allows us to obtain criteria of thestability for FFD (McClements, 2007). The analysis ofrheological behaviour is useful not only to establishstability criteria, but also to define the applicationtechnique when the FFD are applied to a particularproduct (Fellows, 1990). Surface properties of the FFD(contact angle) give information about the phenomenon ofwetting or non-wetting of a product surface by a FFDand thus, about the uniformity of coating when appliedto a particular solid surface (Karbowiak, Debeaufort, &Voilley, 2006).

Among functional properties of edible films, opticalproperties are essential to define their ability to be appliedover a food surface, since these affect the appearance of thecoated product, which is an important quality factor. Inthis sense, it is important to ensure that gloss and

transparency of the films are in an adequate range for aspecific target application.The aim of this work is to analyse the effect of oleic acid

incorporation on the functional characteristics of chitosan-based films such as water sorption, mechanical, optical andbarrier properties together with structural characteristics.Some relevant properties (contact angle and particle sizedistribution) of the FFDs were also characterized andrelated with the film properties.

2. Materials and methods

2.1. Materials

High molecular weight chitosan (CH), with a deacety-lation degree of 82.7%, and a viscosity of 919 cPs at 1%w/w in 1% w/w acetic acid solution (Batch 14624DB,Sigma-Aldrich Quımica, Madrid, Spain), 98% glacialacetic acid, 1N NaOH, oleic acid (OA) and Tween80 (Panreac, Barcelona, Spain), were used to preparethe FFD.

2.2. Preparation of the film-forming dispersions

Chitosan (1% w/w) was dispersed in an aqueous solutionof glacial acetic acid (1% v/w), at 40 1C. Tween 80 at 0.1%(v/w) was added to improve wettability (Choi, Park, Ahn,Lee, & Lee, 2002). After 8 h, oleic acid was added to thechitosan solution to reach a final concentration of 0%, 1%,2% and 4% (v/w). These FFDs were named CH,CH–1OA, CH–2OA and CH–4OA, respectively. Thesemixtures were emulsified at room temperature using arotor–stator homogenizer (Ultraturrax DI 25 basic-Yellowline, Janke & Kunkel, Staufen, Germany) at13,500 rpm for 4min. In order to guarantee the stabilityof the FFD, the pH was adjusted to 5.2 with NaOH 1N(Jumaa, Furkert, & Muller, 2002).

2.3. Characterization of the film-forming dispersions

2.3.1. Density, pH, surface tension and contact angle

Density of the FFD was measured by means of a digitaldensimeter DA-110M (Mettler Toledo, Barcelona, Spain).A pH-meter C831 (Consort, Tumhout, Belgium) was usedto determine the pH of the FFD at 20 1C.Surface tension (g) was measured by means of the

‘‘pendant drop method’’ (McClements, 2005a). Thismethod is based on the analysis of the shape of a liquidpendant droplet, which is in both interfacial and hydro-dynamic equilibrium governed by

g ¼gDr ðdeÞ

2

H(1)

where g is the acceleration of the gravity, Dr thedensity difference between the drop and the air, de theequatorial diameter of the drop, and H the correctionfactor (Wu, 1974).

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Measurements of surface tension and contact angle wereperformed in a Video-Based Contact Angle Meter modelOCA 20 (DataPhysics Instruments GmbH, Filderstadt,Germany). Image analyses were carried out by SCA20software. The Laplace–Young method was selected toobtain surface tension values. Contact angle measurementswere obtained by analysing the shape of a sessile drop afterit had been placed over a candelilla wax plate for 60 s(Hagenmaier & Baker, 1993).

2.3.2. Rheological behaviour

The rheological behaviour of FFD was analysed intriplicate at 25 1C by means of a rotational rheometer(HAAKE Rheostress 1, Thermo Electric Corporation,Karlsruhe, Germany) with a type Z34DIN Ti sensorsystem of coaxial cylinders. Rheological curves wereobtained after a stabilization time of 5min at 25 1C.The shear stress (s) was measured as a function ofshear rate (_g) from 0 to 512 s�1 in the following way:5min to reach the maximum shear rate and 5min toattain zero shear rate. The power law model (Eq. (2)) wasapplied to determine the consistency index (K) and theflow behaviour index (n). Apparent viscosities werecalculated at 100 s�1.

s ¼ K _gn (2)

2.3.3. Particle size and z-potential measurements

Particle size analysis of the FFD was carried out by usinga laser diffractometer (Mastersizer 2000, Malvern Instru-ments, Worcestershire, UK). The samples were diluted inacetic acid buffer (175mM, pH ¼ 5.2) at 2000 rpm until anobscuration rate of 10% was obtained. Mie theorywas applied by considering the following optical propertiesfor OA droplets: a refractive index of 1.46 and absorptionof 0. Three samples of each FFD were measured inquintuplicate.

In order to perform z-potential measurements, FFDwere diluted to a droplet concentration of 0.02% OAusing acetic acid buffer (175mM, pH ¼ 5.2). z-Potentialwas determined by using a Zetasizer nano-Z(Malvern Instruments, Worcestershire, UK). The Smolu-chowsky mathematical model was used to convert theelectrophoretic mobility measurements into z-potentialvalues.

2.4. Preparation of films

FFD were poured into a framed and leveled polytetra-fluorethylene (PTFE) plate (f ¼ 15 cm) and were dried inatmospheric conditions for 48 h. Film thickness wascontrolled by pouring in the PTFE plate the amount ofFFD that will provide a surface density of solids in thedry films of 56 g/m2 in all formulations. Dry films werepeeled off from the casting surface and preconditioned indesiccators at 5 1C and 58.5% relative humidity (RH) priorto testing.

A hand-held digital micrometer (Palmer-Comecta,Spain, 70.001mm) was used to measure film thickness inat least three different points of the same sample, asdescribed by Park and Zhao (2004).

2.5. Water sorption isotherms

For the adsorption experiments, film pieces of about3 cm in diameter were transferred to vacuum chamberscontaining P2O5 to complete drying. Afterwards, filmspecimens, in triplicate, were placed at 5 1C in hermeticchambers containing saturated salt solutions with differentwater activity (aw): LiCL (0.11), MgCl2 (0.34), K2CO3

(0.43), Mg(NO3)2 (0.59), NaBr (0.64), KI: 0.73, NaCl:(0.76), KCl (0.88). Samples were weighed periodically till aconstant value (DmE0.0005 g) was reached, where theequilibrium was assumed to be achieved (Spiess & Wolf,1983). Finally, the equilibrium moisture content wasdetermined by drying them to a constant weight in avacuum oven at 70 1C.

2.6. Water vapour permeability

WVP was measured in dry film discs (f ¼ 7 cm,equilibrated at 58.5% RH) according to the ‘‘watermethod’’ of the ASTM E-96-95 (ASTM, 1995), usingpolymethylmethacrylate cups, following the design pro-posed by Gennadios, Weller, and Gooding (1994).Deionized water was used inside the testing cup to achieve100% RH on one side of the film, while an oversaturatedmagnesium nitrate (58.5% RH) or oversaturated potas-sium iodide (73.3% RH) solutions were used to control theRH on the other side of the film. During WVP testing, theside of the film in contact with the PTFE plate was placedin contact with that part of the test cup having the highestRH. This situation tries to simulate the case of a filmapplied on the wet surface of a fresh cut vegetable or fruit.A fan placed on the top of the cup was used to reduceresistance to water vapour transport. WVTR measure-ments were performed at 5 1C to reproduce the storageconditions of fresh cut coated products. To calculateWVTR, the slopes of the steady-state period of the curvesof weight loss as a function of time were determined bylinear regression. For each type of film, WVP measure-ments were replicated three times and WVP was calculatedaccording to Villalobos, Hernandez-Munoz, and Chiralt(2006).

2.7. Mechanical properties

Mechanical properties were measured by using a TextureAnalyser TA-XT-plus (Stable Micro Systems, Surrey, UK),with a 50N load cell equipped with tensile grips (A/TGmodel). Sample films were cut into 25.4mm wide and100mm long strips, according to the ASTM D-882standard (ASTM, 2001). Grip separation was set at50mm, with a cross-head speed of 50mm/min. Tensile

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Table 1

Density (r), surface tension (g) and contact angle (y) of the FFD at 25 1C

FFD r (g/cm3) g (mN/m) y60s

Solvent 0.996 (0.001) 77.6 (1.6)a 58 (8)a

CH 1.008 (0.076) 51 (3)b 36 (5)c

CH–1OA 1.006 (0.002) 52 (1.4)b 41 (2)b,c

CH–2OA 1.004 (0.0001) 41 (2)c 43 (4)b

CH–4OA 1.002 (0.001) 37.1 (0.9)d 43 (4)b

Mean values and standard deviation.

Solvent: Acetic acid buffer (175mM).a,b,c,dDifferent superscripts within a column indicate significant differ-

ences among formulations (po0.05).

M. Vargas et al. / Food Hydrocolloids 23 (2009) 536–547 539

strength (TS) and percentage of elongation (%E) at break,and elastic modulus (EM) were evaluated in eight samplesfrom each type of film.

2.8. Optical properties

Gloss was measured using a flat surface gloss meter(Multi-Gloss 268, Minolta, Langenhagen, Germany) at201, 601, and 851 angles from the normal to the film surfaceaccording to the ASTM standard D523 (ASTM, 1999).Prior to gloss measurements, films were conditioned indesiccators at 5 1C and 58.5% RH. Gloss measurementswere performed over a matte standard plate and were takenin quintuplicate. Results were expressed as gloss units,relative to a highly polished surface of black glass standardwith a value close to 100.

Colour measurements were carried out on film speci-mens equilibrated at different RH. The transparency of thefilms was determined through the surface reflectancespectra in a spectrocolorimeter CM-3600d (Minolta Co,Tokyo, Japan) with a 10mm illuminated sample area.Measurements were taken from three samples in eachformulation by using both a white and a black back-ground. The transparency was determined by applying theKubelka–Munk theory for multiple scattering to thereflection spectra. As each light flux passes throughthe layer, it is affected by the absorption coefficient (K)and the scattering coefficient (S). Transparency (K/S) wascalculated, as indicated by Hutchings (1999), from thereflectance of the sample layer backed by a knownreflectance background and the reflectance of the layerwith an ideal black background. Moreover, CIE-L* a* b*coordinates (CIE, 1986) were obtained by the infinitereflection spectra of the samples, using D65 illuminant/101observer in order to calculate the whiteness index (WI) ofthe samples

WI ¼ 100� ðð100� L�Þ2 þ a�2 þ b�2Þ0:5 (3)

2.9. Scanning electron microscopy

Microstructural analysis of the surface and crosssections of the dry films (previously conditioned indesiccators with P2O5 for at least 15 days) wascarried out using SEM technique in a JEOL JSM-5410(Japan) electron microscope. Pieces of 5� 1mm2 werecut from films and mounted in copper stubs. Sampleswere gold coated and observed using an acceleratingvoltage of 10 kV.

2.10. Statistical analysis

Results were analysed by multifactor analysis of variancewith 95% significance level using StatgraphicssPlus 5.1.Multiple comparisons were performed through 95% LSDintervals.

3. Results and discussion

3.1. Characterization of the film-forming dispersions

3.1.1. Density, surface properties and particle

size distribution

Density, surface tension and contact angle of the FFDare reported in Table 1. CH solution showed a surfacetension of 61.8mN/m before the addition of Tween 80,which is in the same range of values observed by otherauthors (Choi et al., 2002; Garcia, Pinotti, Martino, &Zaritzky, 2004). The addition of Tween 80 at 0.1% causedan additional decrease in the surface tension in accordancewith the results reported by Choi et al. (2002) when addingthe same surfactant. Every FFD showed surface tensionvalues which were significantly lower than the solvent(po0.05) after CH, Tween 80 and OA were added. Theincorporation of small concentrations of OA did not causea significant decrease in the surface tension of CHdispersions, which can be associated with the formationof complexes with the polymer or with a micellar structure.In fact, the OA concentration in the CH–1OA system is3.1mM, which is in the Critical Micelle Concentrationrange reported by Murakami, Chang, & Routtenberg(1986) for OA. However, at higher concentrations of OA, asignificant surface effect was observed.Contact angle was measured over a candelilla wax plate,

which was used as a model surface that simulates thenaturally waxy fruit surface. The reported values werelower than 901, which indicates that all FFD wouldpartially wet the model surface spontaneously (Hagenmaier& Baker, 1993). Moreover, these results showed that inorder to define the extensibility and wetting properties ofthe FFD, it is not enough to obtain their surface tension,since the lowest surface tension value did not match thelowest contact angle value. The latter also indicates thatboth the development of molecular interactions among thecomponents of the FFD and the waxy surface affect thewetting properties of the FFD. In the present study, it isremarkable that the presence of OA reduces the wettingproperties of the FFD over the wax plate, regardless of itsconcentration.

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The typical average particle size distributions of theCH–OA FFD are plotted in Fig. 1a, together with theaverage z-potential distributions (Fig. 1b). All CH–OAFFD showed a monomodal and polydisperse distribution,with particle size values between 0.4 and 10 mm. As shownin Table 2, the mean particle size was expressed asvolume–length diameter (d43) and the area–volume meandiameter (d32). The average surface area of dropletsexposed to the continuous phase per unit volume ofemulsion (AN), and the number of particles perm3 of FFD(N) were estimated through d32 (Table 2). The increase inOA content significantly increased the mean particle size(po0.05), at the same time as increasing the interfacial areaof the dispersed phase. The incorporation of OA also led toa decrease in z-potential of the particles when compared tothe values of the CH dispersion at the same pH. Thus, theincrease in OA content led to bigger emulsified dropletswith a lower electrical net charge (Table 2). The reductionof the electrical net charge of CH–OA particles (decrease inz-potential) together with the increase in OA content couldbe explained by the electrostatic interactions between CHand OA at the pH of the FFD (5.2). At this pH, the amino

Fig. 1. (a) Particle size distribution of CH–OA film-forming dispersions,

(b) z-potential distribution of CH–OA film-forming dispersions

(n CH–1OA; � CH–2OA, J CH–4OA).

Table 2

z-potential, particle size (d43 and d32), average surface area (AN) and number

FFD z-Potential d43 (mm) d32

CH 62 (2)a – –

CH–1OA 58.3 (1.3)b 1.9 (0.3)a 1.3

CH–2OA 54.1 (0.9)c 2.3 (0.3)b 1.5

CH–4OA 52 (3)c 3.25 (0.11)c 2.1

Mean values and (standard deviation).a,b,c,dDifferent superscripts within a column indicate significant differences a

group of chitosan (pKa NH+3 /NH2E6.5) is positively

charged and could be partially neutralized through theinteractions with the carboxylate function of oleic acid(pKa COOH/COO�E4.8), which is negatively charged(Ham-Pichavant, Sebe, Pardon, & Coma, 2005).In order to clarify this hypothesis, the particle size and

z-potential of a dispersion prepared with Tween 80 (0.1%v/w) and OA (1% v/w) at pH 5.2 were measured. Inthe absence of CH, particles were negatively charged(z-potential ¼ �3.12mV) and showed a bimodal distribu-tion, with two peaks at 0.17 and 1.49 mm, respectively, andmean particle size values (d43 ¼ 0.61 mm, d32 ¼ 0.18)significantly lower than those reported for CH–1OAFFD. The particle size distribution points out to thepresence of two types of particles in the system: oleic acidmicelles and oleic acid–Tween 80 micelles, which arenegatively charged and which can interact with CH toform larger particles. Therefore, the hypothesis for thestructure of CH–OA–Tween 80 system is that CH chainsare adsorbed on the OA or Tween 80–OA micelles, leadingto bigger particles positively charged, showing a mono-modal distribution. The steric stabilization promoted bythe CH interfacial adsorption and the high value of theparticle z-potential (significantly higher than +30mV)ensures the stability of the emulsified system (Roland, Piel,Delattre, & Evrard, 2003) and, as it has been proved inadditional experiments (data not shown), particles remainwell dispersed for a long time, without evidences offlocculation or creaming.

3.1.2. Rheological behaviour

Every FFD showed a pseudoplastic behaviour (no1)and no thixotropic effects were observed from thecomparison of the up and down curves. Rheological datawere fitted to the Ostwald de Waale model and the modelparameters are shown in Table 3, together with apparentviscosity (Zap) values at a shear rate of 100 s�1. Theaddition of OA did not promote changes in the rheologicalpattern of the FFD.The rheological behaviour of dispersions depends on

several factors that are related to the disperse phasecharacteristics (volume concentration, particle size, dis-tribution and shape of the particles and surface electricalcharge) together with the presence of surfactants andthickening agents (Rao, 1977). As shown in Table 2,

of particles/m3 (N) of the FFD at 25 1C

(mm) AN (m2) N� 10�20 (particles/m3)

– –

1 (0.17)a 4.29 (0.05)a 2.31 (0.09)a

(0.3)b 7.13 (0.09)b 2.82 (0.05)a

(0.2)c 11.06 (0.04)c 2.22 (0.02)a

mong formulations (po0.05).

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Fig. 2. Water sorption isotherms (experimental points and GAB fitted

model) of CH and CH–OA composite films at 5 1C (&pCH; n CH–1OA;

� CH–2OA, J CH–4OA).

Table 3

Ostwald de Waale model parameters and apparent viscosity of the FFD

FFD 0p_gp512 s�1

N k (Pa s)n Zap (100 s�1) (Pa s)

CH 0.811 (0.009)a 0.292 (0.003)a 0.126 (0.001)a

CH–1OA 0.75 (0.02)b 0.50 (0.06)b 0.162 (0.005)b

CH–2OA 0.785 (0.016)c 0.37 (0.04)c 0.141 (0.003)c

CH–4OA 0.77 (0.02)c 0.49 (0.09)c 0.176 (0.018)d

Mean values and (standard deviation).a,b,c,dDifferent superscripts within a column indicate 99.99% significant

difference among formulations.

M. Vargas et al. / Food Hydrocolloids 23 (2009) 536–547 541

important changes occurred in the dispersed phase of thesamples under study as a function of the OA content:changes in the volume fraction together with modificationsof the size, number and electrical net charge of theparticles.

As shown in Table 3, the increase in OA concentration inthe CH dispersions caused a significant increase (po0.05)in Zap, consistency index (k) and more marked pseudoplas-tic behaviour (lower n). It could be expected that the Zapvalues increase in line with the volume fraction of OA, ashas been previously observed in concentrated emulsions(Campanella, Dorwards, & Singh, 1995; Walstra, Geurts,Noomen, Jellema, & Van Boekel, 1999). However, anirregular increase in Zap and K values was observed as theratio OA:CH increased, and unexpected apparent viscosityvalues were observed for the FFD containing a ratio of 2:1.This indicates that the rheological properties of these FFDwere also notably influenced by other factors, apart fromthe disperse phase volume fraction, such as the electricalnet charge of the particles. The lower electrical charge(lower z-potential) measured in CH–2OA FFD couldexplain the reported decrease in its apparent viscosity incomparison with CH–1OA, as has been explained by someauthors (McClements, 2005b). Moreover, this change inz-potential could also imply changes in the shape of theparticles, which directly affect the flow behaviour of theFFD. On the other hand, CH–2OA and CH–4OA showedno differences in terms of electrical net charge, and thus,the increase in apparent viscosity observed in CH–4OAwhen compared with a CH–2OA sample could be explai-ned by the increase in disperse phase concentration(McClements, 2005b).

3.2. Characterization of the films

3.2.1. Water sorption isotherms

The water sorption isotherms (WSI) (equilibrium moist-ure content in dry basis vs. water activity) of the CH andCH–OA composite films at 5 1C are shown in Fig. 2.

The WSI of a CH film is sigmoid in shape, showing aslower increase in water content till aw 0.6, after which maybe observed a steep rise in moisture content, due tophenomenon of solubilization. This sigmoid shape was also

observed by other authors in CH films (Del Nobile,Buonocore, & Conte, 2004; Gocho, Shimuzu, Tanioka,Chou, & Nakajima, 2000; Wiles, Vergano, Bunn, & Testin,2000). According to Cervera et al. (2004) CH has threepredominant adsorption sites: hydroxylpropil group,amine group and polymer chain end (made up of ahydroxyl group or an aldehyde group).As regards the incorporation of OA into the films, there

were detected no differences in water sorption capacitybetween CH and CH–1OA at aw lower than 0.6. When theaw was higher, the equilibrium moisture content (We) ofCH–1OA was higher than the We of CH, which could beexplained by an enhancement of the interactions betweenCH and OA at these aw levels, leading to more hygroscopicparticles with more polar groups that interact with watermolecules.The BET molecular model of adsorption (fitted for

awo0.6) and the Guggenheim–Anderson–de Boer (GAB)model (fitted in the entire aw range) were used to fit thewater adsorption data of the films (Table 4). To fit theGAB model, the second-degree polynomial equation wasused. BET equation constants, which have a thermody-namic base, were used to analyse the differences in samplebehaviour and the interaction between the components andwater molecules. The monolayer moisture content (Wo) ofthe CH film at 25 1C was higher than the values found inthe literature (Cervera et al., 2004), which is attributed to atemperature effect or to the different nature of the chitosanused, since the degree of deacetylation and the molecularweight of CH is directly related to the number ofadsorption sites.As concerns the CH–OA composite films, a significant

decrease in the monolayer moisture content was observedwhen the amount of OA in the film increased. This isattributable to the fewer sites available for water sorption

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Fig. 3. Comparison between experimental values of the equilibrium

moisture content (We) and those predicted by the model (Eq. (4)) for

CH–OA composite films (& CH; n CH–1OA; � CH–2OA; J CH–4OA).

Fig. 4. Water vapour permeability of films (T ¼ 5 1C) at 100/59 RH

gradient (continuous line) and 100/73 RH gradient (dashed line). Mean

values and 95% LSD intervals. Different letters (a, b, c) indicate

significant differences (po0.05).

Table 4

BET and GAB parameters obtained from isotherms of CH and CH–OA

composite films

Film BET GAB

W0 C R2 W0 C K R2

CH 0.146 105.2 0.96 0.168 7.49 1.008 0.81

CH–1OA 0.142 2218 0.96 0.147 160 1.001 0.71

CH–2OA 0.106 240 0.86 0.085 1136 1.076 0.93

CH–4OA 0.065 441.9 0.75 0.055 71.41 1.05 0.77

W0 (g H2O/g d.b.).

M. Vargas et al. / Food Hydrocolloids 23 (2009) 536–547542

in the polymeric matrix due to the increase in thehydrophobic chains from the fatty acid. Likewise, the CHmolecule could be less hydrophilic due to the electrostaticneutralization of CH amino groups with the carboxylategroup of OA, as mentioned above. Constant C, related tothe water–substrate interaction energy, did not show aclear trend with the change in OA concentration. Never-theless, a significant drop was detected when OA was notpresent in the film. This seems to indicate that, as the filmbecomes more hydrophilic, the water molecules areadsorbed with less energy in the active sites. This coincideswith how HPMC-based films behaved when the hydro-colloid/surfactant ratio increased, as observed by Villalo-bos et al. (2006).

In order to analyse the role of OA in the adsorptionbehaviour of CH in the composite films, the equilibriummoisture content of these films at each aw was comparedwith the value obtained by applying Eq. (4). In thisequation, We was obtained from the equilibrium value ofpure CH and its proportion in the dried film, by assumingthat the lipophilic character of OA meant that it adsorbedno water molecules

W estjaw ¼ XCH W eCH (4)

where W estjaw is the equilibrium moisture content of thecomposite film (d.b.) at a constant aw; XCH the massfraction of chitosan in the composite dry film; and WeCH

the equilibrium moisture content of pure chitosan film (d.b)at a constant aw.

Fig. 3 shows experimental versus estimated equilibriummoisture content of CH–OA composite films. At a loweraw range (Weo0.4 gw/g d.b), predicted and experimentalWe values for CH–2OA and CH–4OA films are located onthe diagonal, which indicates that the interactions betweenfilm components are insignificant and that they are mostlyin separate phases. For CH–1OA films, the experimentalvalues are greater than the predicted ones, which indicatethat OA enhanced the hydrophilic character of the filmthrough interactions with CH. At high levels of wateractivity, experimental We values were higher than thosepredicted, especially for CH–1OA formulation. The lattersuggests that, above a certain hydration level, watermolecules promote the mobility of the system by partiallydissolving CH to form a gel in which the interactions

among the polar groups of CH and OA are promoted, thusmodifying the supramolecular structure of the films andleading to significant changes in its water sorption capacity.This behaviour has also been observed in hydroxypropilcellulose-based films containing surfactants (Seo &Kumacheva, 2002; Villalobos et al., 2006).

3.2.2. Water vapour permeability

The RH conditions used for measurement of WVP(100/73 and 100/59) of the films were established tosimulate the environmental conditions when the films areapplied as a coating for vegetables stored at 5 1C. Theaverage thickness of the CH and CH–OA composite filmswas 54 mm (s ¼ 14). As seen in Fig. 4, at the RH gradient

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Fig. 5. (a) K/S values at 450 nm and (b) Whiteness index (WI) of CH and

CH–OA composite films as a function of aw. Mean values and 95% LSD

intervals (& CH; n CH–1OA; � CH–2OA; J CH–4OA).

M. Vargas et al. / Food Hydrocolloids 23 (2009) 536–547 543

of 100/73, adding OA to the films had no significant effecton their WVP. At this high level of RH, water acts as astrong plasticizer in hydrophilic polymers such as chitosan,decreasing their barrier properties, regardless of thepresence of OA. In fact, at this RH gradient, the averageaw in the films would be around 0.88. At this highhydration level, water molecules partially dissolve CH toform a gel in which the interactions among the polargroups of CH and OA are promoted thus leading to asignificant increase in its water sorption capacity, ashas been described above for the water vapour isothermsof the films.

On the other hand, the WVP values for the 100/59 RHgradient were in the range of those reported by Parkand Zhao (2004). Moreover, WVP showed a significantdecrease in line with the increase in OA concentration,following a linear trend described by Eq. (5) (r2 ¼ 0.971),although the differences were only significant betweenCH–1OA and CH–4OA composite films (po0.05)

WVP ¼ �24:058� Rþ 158:51 (5)

where WVP is the water vapour permeability,gm�1 s�1 Pa�1; R is the OA:CH ratio, gOA/g CH.

3.2.3. Optical properties

The gloss and transparency of the films are relevantproperties since they have a direct impact on theappearance of the coated product. Fig. 5a shows K/Svalues at l ¼ 450 nm as a function of aw level. Filmtransparency was evaluated through the Kubelka–MunkK/S coefficient, defined as the ratio between light absorp-tion and scattering. An increase in K/S can be assumed asan increase in transparency, although the effect of theselective absorption of components may also be considered(Hutchings, 1999). K/S vales at l ¼ 450 nm were signifi-cantly affected by the amount of OA, and the water activityas well as the interaction between both factors (po0.05).CH–OA composite films were in general less opaque thanCH films, although the differences depended on thehydration level of the films.

The different transparency level is linked to the internalstructure developed throughout drying process, which isgreatly affected by the initial structure of the FFD, i.e.volume fraction of the dispersed phase and size of CH–OAaggregates. During drying, changes in the solvent proper-ties of the continuous phase do occur, leading tocoalescence and creaming phenomena as water is elimi-nated. At the same time, the viscosity of the continuousphase increases, thus limiting aggregation and creaming,which contributes to ensuring a specific dispersion level oflipid components in the polymer matrix and a certaindegree of anisotropy in the physical properties throughoutthe matrix (Villalobos, Hernandez, Chanona, Gutierrez, &Chiralt, 2005).

The observed results can be explained by consideringthat the structure of CH film is dense, closed and opaque.In the composite films, however, the OA droplets are

inserted between chitosan chains, interrupting the matrixand developing a more open structure, especially at highwater activities, where hydration also promotes the filmtransparency at the same time as increasing the molecularmobility. The interaction with OA and water moleculesmodifies the refractive index of CH and the differencesbetween the film phases’ refractive indices, thus affectingthe film transparency.Fig. 5b shows the whiteness index (WI) values, obtained

from the infinite reflection spectra, of CH and CH–OAcomposite films at different water activity levels. Asoccurred with the K/S parameter, WI was significantlyaffected by OA content, and water activity, as well as bythe interaction between factors (po0.05). The addition ofOA promoted a significant decrease in the WI of thecomposite films (compared with CH film), which isexplained by the light selective absorption of OA at lowwave lengths that imparts yellowness to the films. Ingeneral, the increase in aw did not cause significantchanges in the WI of samples for aw levels up to 0.59.However, at aw 0.73, the WI values of every film signi-cantly dropped (po0.05) and there were no differences

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Fig. 6. Gloss at 60 and 851 of CH and CH–OA composite films. Mean values and 95% LSD intervals.

Table 5

Percentage of elongation at break (E), tensile strength (TS) and elastic

modulus (EM) of CH and CH–OA composite films

Film E (%) TS (MPa) EM (MPa)

CH 17 (8)a 12 (8)a 310 (256)a

CH–1OA 15 (8)a,b 18 (5)b 495 (282)b

CH–2OA 11 (7)a,b 7 (2)a 202 (126)a

CH–4OA 11 (5)b 7.4 (1.9)a 223 (51)a

Mean values and (standard deviation).a,b,c,dDifferent letters in the same column indicate significant differences

among formulations (po0.05).

M. Vargas et al. / Food Hydrocolloids 23 (2009) 536–547544

among formulations. This can be attributed to the highdegree of molecular hydration, which modifies theirabsorption spectra.

The gloss values of the films measured at incidence anglevalues of 601 and 851 are shown in Fig. 6. These angles areenough to distinguish among low gloss surfaces, due to theenhancement of specular reflection when the incidenceangle increases (Hutchings, 1999). This effect can be clearlyobserved in Fig. 6, where the higher the incidence angle, thegreater the gloss. The behaviour of gloss as a function ofOA concentration is similar for low incidence angle values,although the effect was more significant at an incidenceangle of 851. The higher the OA content in the films, themore gloss, this effect being particularly intense forCH–4OA films.

The gloss of the films is related with the surfacemorphology reached during film drying. In general, thesmoother the surface, the higher the gloss (Ward &Nussinovitch, 1996). In this sense, the increase in gloss inline with the increase in OA content could be explained bythe coalescence and creaming of the OA droplets duringdrying. This leads to a decrease in surface roughness andan increase in specular reflectance in the air–film interface,since OA is in a liquid state and can fill the small surfacevoids generated during the film formation.

3.2.4. Mechanical properties

Table 5 shows the percentage of elongation at break(E%) and tensile strength (TS) and elastic modulus (EM)of films equilibrated at 58.9% RH at 5 1C. These valuesare, in general, lower than those found in the literature forCH films which were around 20MPa and 42%, for TS andE, respectively (Caner et al., 1998). The addition of OAcaused a decrease in tensile strength, elongation at breakand elastic modulus when the ratio OA:CH was higherthan 1:1, which coincides with the results reported bySrinivasa et al. (2007) when adding saturated fatty acids toa CH matrix. However, for CH–1OA films, an oppositeeffect was detected, since EM and TS significantlyincreased (po0.05). At the 1:1 OA–CH ratio molecularinteractions could enhance the structural bonds in thepolymer network leading to an increase in the TS of thefilms, but decreasing its ability to stretch. This cross-linking

effect between CH and OA can be also deduced from thewater sorption isotherms pattern, where particular inter-actions between the fatty acid and the polymer wereobserved for CH–1OA films. In this sense, cross-linkingeffects between CH and plasticizers (polyethylene glycoland propylene glycol) have been also described at lowlevels of plasticizers (95:5 CH–plasticizer ratio), causing areduction in the free volume and the molecular mobility ofthe polymer (Suyatma, Tighzert, Copinet, & Coma, 2005).

3.2.5. Microstructure

The final structure that was developed by the differentFFD after drying is influenced by the structural arrange-ment of the different components (CH, Tween 80 and OA)in the initial dispersion, and their development during thedrying process: flocculation, coalescence and creaming.Fig. 7 shows SEM micrographs of the surface of the films.If compared with CH film (Fig. 7a), composite CH–OAfilms (Fig. 7b–d) showed remarkable differences in terms ofsurface microstructure. While a smooth, continuousstructure was observed for the CH film surface, thepresence of OA caused discontinuities associated with theformation of two phases (lipid and polymer) in the matrix:lipid droplets embedded in a continuous polymer phase.The increase in the OA content of the film leads to anappreciable change in the surface topography, revealing adifferent amount of lipid particles on the surface and,particularly for the film CH–4OA, a bigger particle size,which coincides with the greater lipid concentration andthe bigger particle size in the FFD. Fig. 8 shows the SEM

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Fig. 7. SEM micrographs of the surface of films: (a) CH; (b) CH–1OA; (c) CH–2OA; (d) CH–4OA.

Fig. 8. SEM micrographs of the cross-sections (top and bottom parts) of the films: (a) CH; (b) CH–1OA; (c) CH–4OA.

M. Vargas et al. / Food Hydrocolloids 23 (2009) 536–547 545

micrographs of the cross sections of the films where thepresence of lipid particles (or their voids originated duringthe film fracture) can also be appreciated in the formula-tions containing OA. A greater ratio of droplets can beseen in the top part of the films than in the bottom part,which confirms the occurrence of creaming during the filmdrying. A greater accumulation of lipids near the filmsurface was observed in the CH–4OA film (Fig. 8c), whichcoincides with that observed in the surface micrographs. Inthis case, lipid droplets show a high degree of flocculationand partial coalescence. In fact, an almost continuous layerof partially coalesced droplets can be observed on the

surface which may explain the exceptional gloss of thesefilms and the lower WVP.

4. Conclusion

Interactions between CH and OA in the FFD and theirdevelopment during film drying affected the final structureof the composite films and their physicochemical char-acteristics. The increase in OA concentration in the FFDleads to lower surface tension values and an increase inviscosity, the latter being greatly affected by the electricalnet charge of the disperse particles. The addition of OA to

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CH supposed a decrease in the moisture adsorptioncapacity and the WVP, as well as in the stiffness of thecomposite films. Nevertheless, when the ratio CH:OA was1:1, special interactions among components were detectedwhich made the film’s resistance to break greater, and alsoincreased the film’s water sorption capacity. In general, if acomparison is made with pure CH films, an increase in OAcontent leads to glossier and more translucent films, whichcan be explained in terms of the film microstructure.

Acknowledgements

The authors acknowledge the financial support providedby Conselleria de Empresa, Universidad y Ciencia (Spain)by the project GV040-380 and Ministerio de Educacion yCiencia (Project AGL2004-01009). Author M. Vargasthanks Ministerio de Educacion y Ciencia (Spain) for aFPU grant. Authors also thank M. Planes and J. L. Moyafrom UPV Electronic Microscopy Service for theirassistance in the use of SEM.

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