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Food Hydrocolloids 25 (2011) 577e585

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Food Hydrocolloids

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

Designing W1/O/W2 double emulsions stabilized by proteinepolysaccharidecomplexes for producing edible films: Rheological, mechanicaland water vapour properties

M.M. Murillo-Martínez a, R. Pedroza-Islas b, C. Lobato-Calleros c, A. Martínez-Ferez d, E.J. Vernon-Carter a,*aDepartamentos de Biotecnología y Departamento de Ingeniería de Procesos e Hidráulica, Universidad Autónoma Metropolitana-Iztapalapa, San Rafael Atlixco 186,Col. Vicentina, 09340 México, DF, MexicobUniversidad Iberoamericana, Departamento de Ingeniería y Ciencias Químicas, Prolongación Reforma 880, Lomas de Santa Fe, 01210 México, DF, MexicocUniversidad Autónoma Chapingo, Departamento de Preparatoria Agrícola, Km. 38.5 Carretera México-Texcoco, 56230 Texcoco, Estado de México, MexicodDepartamento de Ingeniería Química, Campus Fuentenueva s/n, Universidad de Granada, 18071 Granada, Spain

a r t i c l e i n f o

Article history:Received 25 March 2010Accepted 25 June 2010

Keywords:Double emulsionsProteinepolysaccharide complexesEdible filmsRheological and mechanical propertiesWater vapour permeability

* Corresponding author. Tel.: þ52 55 5804 4648; faE-mail address: jvc@xanum.uam.mx (E.J. Vernon-C

0268-005X/$ e see front matter � 2010 Elsevier Ltd.doi:10.1016/j.foodhyd.2010.06.015

a b s t r a c t

Rheological properties and the droplet mean volume-surface diameter of water-in-oil-in-water doubleemulsions ELMPeWPI and ECMCeWPI, stabilized with low-methoxyl pectin (LMP)ewhey protein isolate(WPI) or sodium carboxymethylcellulose (CMC)eWPI complexes were determined. ELMPeWPI emulsionsshowed smaller droplet diameter (2.47 mm) than ECMCeWPI emulsions (10.68 mm), and higher values ofapparent viscosity, storage modulus and loss modulus. Microstructure and mechanical properties of theFLMPeWPI and FCMCeWPI films were affected by the type of the stabilizing biopolymer complex used in theformulation of the ELMPeWPI and ECMCeWPI double emulsions from which were prepared. The FLMPeWPI

films microstructure consisted of a biopolymer mainframe relatively compact, interrupted by smallemulsion droplets. The FCMCeWPI film mainframe had an open and oriented structure, in which relativelylarge emulsion droplets were packed. FLMPeWPI films presented higher values of transparency, tensilestrength and Young’s modulus, but lower elongation percentage and comparable water vapour perme-ability than FLMPeWPI films.

� 2010 Elsevier Ltd. All rights reserved.

1. Introduction

Edible films can extend shelf life and improve quality of food byproviding barriers to mass transfer, improving the mechanicalintegrity or handling characteristics and/or carrying functionalagents such antimicrobials, antioxidants, probiotics, nutrients,flavours, colours and so on (Hambleton, Debeaufort, Bonnote, &Voilley, 2009; Krochta, 1992; Mei & Zhao, 2003; Salmieri &Lacroix, 2006). The functional properties of edible films dependon the characteristics of the materials used for their preparation.The main film-forming materials used are polysaccharides (i.e.starch, carrageenan, alginate), proteins (i.e. wheat gluten, wheyprotein isolate, caseinate, soy protein), and lipids (i.e. waxes, andfatty acids). Lipid materials, due to their hydrophobic character,provide better moisture barriers than polysaccharides and proteins,but they offer little resistance to gas transfer and have poormechanical strength. On the other hand, hydrocolloids form good

x: þ52 55 5804 4900.arter).

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oxygen and carbon dioxide barriers, but due to their hydrophiliccharacter, provide less effectivemoisture barriers (Kristo, Biliaderis,& Zampraka, 2007; Miller & Krochta, 1997). Thus, incorporation oflipids in hydrocolloids films increase film hydrophobicity andtherefore improve their water vapour permeability (Morillon,Debeaufort, Blond, Capelle, & Voilley, 2002; Rhim, Lee, & Kwak,2005; Yang & Paulson, 2000). Double emulsions as lipid compo-nent incorporated to hydrocolloids films formulation may improvefilm barrier properties, requiring fewer steps and being safer thanlamination of the hydrophilic film with a lipid layer (Pérez-Gago &Krochta, 1999), and may provide further protective and specificdelivery systems of functional food components. Water-in-oil-in-water (W1/O/W2) double emulsions consist of small-sized waterdroplets (W1) contained within larger oil droplets (O) that aredispersed within an aqueous continuous phase (W2). Functionalfood components could be encapsulated within the inner waterphase, the intermediate oil phase, or the outerwater phase, therebymaking it possible to develop a single delivery system containingmultiple functional components, which may be released toa specific site such as themouth, stomach, or small intestine (Weiss,Takhistov, & McClements, 2006).

M.M. Murillo-Martínez et al. / Food Hydrocolloids 25 (2011) 577e585578

Jiménez-Alvarado, Beristain, Medina-Torres, Román-Guerrero,and Vernon-Carter (2009) reported that W1/O/W2 double emul-sions incorporating ferrous bisglycinate in the inner aqueous phaseand stabilized by proteinepolysaccharide complexes (whey proteinconcentrateemesquite gum and whey protein concentrateegumArabic), exhibited good stability, encapsulation efficiencies, protec-tion against oxidation, and slow release rates. All the double emul-sions properties were enhanced when the adsorbed biopolymercomplex was thicker. Proteinepolysaccharide complexes couldimprove the resistance of emulsions to environmental stresses suchas pH, ionic strength, and temperature (McClements, 2005). Never-theless, the characteristics of adsorbed complexes, and the struc-tures of mixed biopolymer interfaces, are still poorly understood(Dickinson, 2008). A proper understanding and control of the pro-teinepolysaccharide interactions should help in designing emulsi-fied films with properties comparable to those of bilayered films,and, in this sense, rheological analysis may provide a basic under-standing about the film microstructure and barrier properties inrelation to the double emulsions composition tested (Xu, Liu, &Zhang, 2006).

The aim of this work was to: (a) Obtain double W1/O/W2

emulsions stabilized by two different protein:polysaccharidecomplexes, and evaluate their rheological properties (stationaryand dynamic), and mean volume-surface droplet size; and (b)Obtain edible films by casting of the double emulsions, and eval-uate their mechanical properties, water vapour permeability,contact angle, transparency, colour and microstructure.

2. Materials and methods

Mineral oil (MO; NF-85 food grade) was obtained from Mate-riales y Abastos Especializados, S.A. de C.V. (Mexico City, Mexico).The water-soluble surfactant (WS; Panodan SDK, esters of mono-glycerides and diglycerides of diacetyl tartaric acid) and the oil-soluble surfactant (OS; Grindsted PGPR 90, esters of polyglyceroland polyricinoleate fatty acids) were from Danisco Mexico, S.A. deC.V (Mexico City, Mexico). Whey protein isolate (WPI; Bipro�)containing 90% protein in dry basis was from Davisco Foods Inter-national, Inc. (Le Sueur, MN, USA). Low-methoxyl pectin (LMP;Grindsted LC-950) was from Dannova Quimica, S.A. de C.V. (MexicoCity, Mexico). Sodium carboxymethylcellulose (CMC; Blanose�

Cellulose Gum 7LF-pH) was from Aqualon, a Division of HerculesIncorporated (Widnes, Cheshire, UK). Glycerol, hydrochloric acid(HCl) and sodium hydroxide (NaOH) were from J.T. Baker (Xalostoc,State of Mexico, Mexico). Deionized water was used in all theexperiments, and sodium azide (Hycel de Mexico, S.A. de C.V.,Mexico City, Mexico) was used as preservative.

2.1. Preparation of emulsions

2.1.1. Conditions leading to the formulation of W1/O/W2

double emulsions2.1.1.1. Zeta potential. Aqueous solutions 5.0% w/w WPI, 0.3% w/wCMC and 0.7% w/w LMP were prepared and stored at 4 �C during24 h in order to allow their complete hydration. The zeta potentialof the biopolymers aqueous solutions was determined at differentpH values using a Malvern Zetasizer Nano ZS (Malvern InstrumentsLtd., Malvern, Worcestershire, UK). The pH of the aqueous solutionsof the biopolymers was adjusted by the addition of 0.1 N HCl and/or0.1 N NaOH. The pH where the maximum stoichiometric differenceof the electrostatic charges between protein (Pr) and poly-saccharide (Ps) occurred (pHS) was determined (Jiménez-Alvaradoet al., 2009).

2.1.1.2. Equivalence point of biopolymers solutions. The equivalencepoint of the biopolymers stock solutions (5.0% w/w WPI, 0.3% w/wCMC and 0.7% w/w LMP) was determined from the potentiometrictitration curves, using 0.5 mL of NaOH 0.1 N aliquots with contin-uous stirring. The precise concentration of base was determined bytitratingwith a standard 0.01 NHCl solution. A 60 s time lag elapsedbetween two doses to allow the reaction to reach equilibrium.Biopolymers solution pH was determined with a vernier pH-BTA(Beaverton, OR, USA) at 25 �C (Espinosa-Andrews, Sandoval-Castilla, Vázquez, Vernon-Carter, & Lobato-Calleros, 2010).

2.1.1.3. Formulation and preparation of the double emulsions. Thedouble emulsions were prepared at room temperature using a two-stage emulsification procedure (Rodríguez-Huezo, Pedroza-Islas,Prado-Barragán, Beristain, & Vernon-Carter, 2004). In the first stage,0.5 dispersed phase mass fraction (4W1=O) water-in-oil (W1/O)primary emulsions were prepared employing a total surfactantconcentration of 10% w/w and OS:WS ratio of 6:4. The total surfac-tants concentration and ratio were established based on the resultsreported by Jiménez-Alvarado et al. (2009) using the same surfac-tants and 4W1=O as in this work. The aqueous inner phase (W1)(deionized water þ WS) was added drop-wise to the oil phase (O)(MO þ OS) using a high shear Ultra Turrax homogenizer (model T50basic, IKA Labortechnik, Staufen, Germany) at 7600 rpm for 15min. Inthe second stage, the W1/O primary emulsion was re-emulsified at4000 rpm for 5min using theUltra Turrax homogenizer into aqueoussolutions of CMCþWPI and LMPþWPI, with the biopolymers ratiosestablished based on the equivalence point. Immediately after theemulsion formation, the pH of double emulsions was adjusted to thepHS, where the formation of Pr:Ps complex was maximized asdetermined by the zeta potential measurements, using 0.1 N HCl or0.1 N NaOH in order to allow the formation of biopolymers complexat the outer oilewater interface. The dispersed phase mass fraction(4W1=O=W2

) in the (W1/O/W2) emulsions was 0.2. The W1/O/W2emulsions ECMCeWPI and ELMPeWPI, and were done in triplicate.

2.2. Rheological properties of the double emulsions

A Physica MCR 301 rheometer (Anton Paar, Messtechnik, Stutt-gart, Germany), with a cone-plate geometry, in which the rotatingcone was 50 mm in diameter, and cone angle of 2� was used forperforming all the rheological measurements. Temperature mainte-nance was achieved with Physica TEK 150P temperature control andmeasuring system.Double emulsion sampleswere carefully placed inthe measuring system, and left to rest for 1 h for structure recoveryand temperature equilibration. A solvent trap was used to preventdehydration. All the measurements were performed at 25 �C. Rheo-logical characterization was carried out using stationary shear flowand oscillatory tests. In the steady-shear measurements, shear ratesfrom 0.001 to 100 s�1 were applied and apparent viscosity wasrecorded as a function of shear rate. Determination of the viscositycurve over awide range of shear rate values is essential for solving theflowequations. This is usually donebyfitting the datapoints to oneofthe viscosity models. Our experimental data showed that the visco-sityeshear rate curves exhibited a zero shear viscosity region, fol-lowed by high shear rate behaviour, but never reaching a limitingviscosity value. The modified Carreau model in which the infiniteshear viscositywas considered negligiblewas used formodelling ourviscosityeshear rate curves (Chen, Kuo, & Lai, 2009):

h ¼ h0

h1þ �

l _g�2iðn�1Þ

2(1)

where h is the apparent viscosity (Pa s), _g is the shear rate (s�1), h0(Pa s) is the low shear rate limiting viscosity, l (s) is a characteristic

M.M. Murillo-Martínez et al. / Food Hydrocolloids 25 (2011) 577e585 579

time constant related to the relaxation times of the flocculateddouble emulsions, and n (dimensionless) is the power-law behav-iour index.

Dynamic amplitude sweeps (0.01e100% strain) were performedunder a constant frequency (1 rad s�1), in order to determine thelinear viscoelastic range, where rheological properties are not strainor stress dependent. From the linear viscoelastic range a strain levelof 0.5%was chosen to perform frequencies sweeps at 0.1e100 rad s�1.The storage modulus (G0), loss modulus (G00), and complex viscosity(h*) were recorded as a function of frequency (Steffe, 1996). In allcases, the data was analyzed using the equipment’s Rheoplus/32V2.62 software. All measurements were done in triplicate.

2.3. Determination of the average droplet size

A Malvern particle size and distribution analyzer series 2600(Malvern Instruments, Ltd., Worcestershire, UK) was used todetermine the mean volume-surface diameter (d32) of the doubleemulsions. All double emulsions were analyzed in triplicate.

2.4. Films preparation

Within 6 h of preparation, each double emulsion was spread onglass plates using an eight path square wet film applicator (Paul N.Gardner Company, Inc., Pompano Beach, FL, USA), which has oneach side a different under gap that allows for the casting of films.The spread double emulsions were dried at 25 � 1 �C in a convec-tive oven (Felisa, model FE, Mexico City, Mexico) for 5 h, resulting inedible films FLMPeWPI and FCMCeWPI. The films were removed fromthe glass surface with a thin spatula. Circular samples from eachfilm were cut for the water vapour permeability testing, whereasrectangular samples were cut for the mechanical properties testing(Bósquez-Molina, Guerrero-Legarreta, & Vernon-Carter, 2003).

2.5. Film thickness measurements

Film thickness was measured using a Digimatic Indicator(Mitutoyo, Tokyo, Japan) at five random positions around the film,by slowly reducing the micrometer gap until the first indication ofcontact. The mechanical properties and water vapour permeabilitywere calculated using the average thickness for each film replicate(Villagómez-Zavala et al., 2008).

2.6. Mechanical properties

A texture analyzer (TA, XT2, Texture Technologies Corp., Scars-dale, NY, USA) was used to measure the tensile properties of theedible films according to ASTM standard method D882 (ASTM,2000). Prior to the test, films were cut in rectangular strips80mm long and 25mmwide and stored inside desiccators over thesodium bromide saturated solution for 10 days at 25 �C (Phan The,Debeaufort, Voilley, & Luu, 2009). Initial grip separation andcrosshead speedwere set at 50mm and 0.8 mmmin�1, respectively(Osés et al., 2009). Force and distance were recorded during theextension of the strips and elongation % (E), tensile strength (TS)and Young’s modulus (ME) were calculated as outlined in ASTMD882 (2000). At least 10 samples of each film were analyzed.

2.7. Water vapour permeability

The gravimetric Modified Cup Method based on ASTM E96-92(McHugh, Avena-Bustillos, & Krochta, 1993) was used to determinewater vapour permeability (WVP). A cabinet containing a fan to testfilm WVP was employed. Fan speeds were set to achieve an airvelocity of 150 m/min to ensure uniform relative humidity (RH)

throughout the cabinet. Prior to each experiment, the cabinet wasequilibrated to 0% RH using silica gel (Merck, Darmstadt, Germany).

Circular test cups were made of polymethylmethacrylate with5.08 cm internal diameter and 1.1 cm height. Cup walls were thickenough tomake the cups impermeable towater vapour. Filmswithoutpinholes or any defects were sealed to the cup mouth with silicon(Bluestar Silicones, Lyon, France) and a polymethylmethacrylate ringsymmetrically located around the cup circumference and held withfour screws. Previously, 6 mL of deionized water was placed at thebottom of the test cups to expose the film to a high RH inside them.Average stagnantairgapheightbetweenthewater surfaceandthefilmwere determined. After assembly, the cups were put in the 0% RHcabinet. Within 2 h, steady state had been achieved; 5 weights werethen taken for each cup at 3 h intervals. At sampling time, cups weretaken out of the cabinet forweighing and the fanwas stopped in ordernot to affect the cabinet RH (Osés et al., 2009). Three replicates of eachfilmwere tested. WVP of filmwas calculated as described byMcHughet al. (1993).

2.8. Wetting properties

The wettability of the edible films was determined from contactangle measurement using a Contact Angle Meter (TANTEC,Schaumburg, IL, USA). A droplet of distilled water (w0.033 mL) wasplaced on the film surface with a precision syringe. Contact anglebetween the baseline of the drop and the tangent at the dropboundary was manually measured by projecting the drop profile ona large display screen that allowed for a bright clear image of the dropto be viewed. The contact angle was then measured with the help ofa protractor. Measurements at the centre and edge of the films,immediately and after 3 min of placing the water droplet on the filmsurface, were taken. Measurements were done in duplicate.

2.9. Film colour

The films colour index (CI*) was determined with the followingequation:

CI* ¼ ða*1000Þ=ðL*b*Þ (2)

Films specimenswere placed on awhite standard plate (L*¼ 100)and the CIELab scale parameters of lightness (L*) and chromaticityparameters a* (redegreen) and b* (yelloweblue) were measured atfive different locations with three repetitions for each film, usinga Minolta colorimeter CM 2500d (Minolta Camera Co., Ltd, Osaka,Japan).

Sample colour varies in accordance to CI* values as follows(Vignoni, Césari, Forte, & Mirábile, 2006):

(a) If CI* is negative (�40 to�20), sample colour ranges from blue-violet to dark green.

(b) If CI* is negative (�20 to �2), sample colour ranges from darkgreen to greenish-yellow.

(c) If CI* is in between (�2 to þ2), sample colour is yellowish-green.

(d) If CI* is positive (þ2 to þ20), sample colour ranges from paleyellow to intense orange.

(e) If CI* is positive (þ20 to þ40), sample colour ranges fromintense orange to deep red.

2.10. Transparency

Film transparency was determined according to ASTM D1746(ASTM, 1997). The films were cut into 2 mm � 40 mm rectangular

Fig. 1. Changes in the apparent viscosity a function of shear rate for the doubleemulsions.

M.M. Murillo-Martínez et al. / Food Hydrocolloids 25 (2011) 577e585580

shapes and placed on the internal side of a spectrophotometer cell.Transparency of films wasmeasured using a spectrophotometer DU650 (Beckman Instruments, Inc., Fullerton, CA) at 560 nm. Threereplicates of each filmwere tested. The environment in which testswere conducted was held at 20 � 2 �C and a 50 � 5% RH duringtesting. The percent transparency (TR) was calculated as follows:

TR ¼ Ir=I0 � 100 (3)

where Ir is the light intensity with the specimen in the beam, and I0is the light intensity with no specimen in the beam.

2.11. Scanning electron microscopy

Edible films microstructure was observed using a JEOL-JSM-6390LV scanning electron microscope (Jeol, Ltd., Akishima, Japan),operated at 20 kV. Films were fixed on the support using doubleside adhesive tape, horizontally and with and angle of 90� to thesurface, which allowed observing the films surface and cross-section (Phan The et al., 2009). The samples were covered witha thin layer of gold with a Denton Vacuum Deposition System LLC1259 (Moorestown, NJ, USA).

2.12. Statistical analyses

Analyses of variance (ANOVA) and Duncan’s means comparisontest with a significance level of 0.05 was applied in all cases usingthe SPSS version 12.0 software.

3. Results and discussion

3.1. Conditions leading to double emulsion formulation

Electrophoretic mobility measurements of the individualbiopolymers aqueous solutions indicated that the negative chargedensity of the 0.7%w/w LMP solutionwas always greater than that ofthe 0.3% w/w CMC solution, irrespective of pH. The absolutemaximum stoichiometric difference of the electrostatic chargesbetween protein and polysaccharide (jDjmV) was 53.7 mV at pH 3.5for LMPeWPI and 38.3 mV at pH 4.0 for CMCeWPI. The equivalencepoint of biopolymers stock solutions was determined from theinflexion point of the titrations curves (data not shown). Thebiopolymers stock solutions, i.e.WPI (5wt%), CMC (0.3wt%) and LMP(0.7 wt%), had 0.90, 1.05 and 1.00 milliequivalents of NaOH, respec-tively, so that the relative amounts between the biopolymers used forstabilizing the double emulsions were 14.3:1 for CMC:WPI and 6.4:1for LMP:WPI. The most suitable total concentration of the biopoly-mers blends used in the outer aqueous phase (W2) for doubleemulsion formation was 7.5 wt% because it allowed for an easiercasting of the double emulsions and readier formation of the ediblefilms than at either lower (double emulsion was too thin) or higher(double emulsions were too thick) biopolymers concentrations.

3.2. Droplet size of emulsions

Both double emulsions were made up by spherical oil dropletscontaining within them a large number of water droplets, so thatthe double emulsions had type C morphologies (Lobato-Calleroset al., 2008). The biopolymers blends used as stabilizers in theexternal aqueous phase affected d32 that was 2.47� 0.04 mm for theELMPeWPI and 10.68 � 0.28 mm for the ECMCeWPI double emulsions.In both biopolymer blends the amount of polysaccharide pre-dominated over the amount of protein, so that upon vigorousmixing, both the polysaccharide and the protein, diffused to andarrived to the newly formed droplet oilewater interfaces in

a similar proportion as that occurring in the biopolymers blends(Bergenståhl, 1995). Whey protein concentrates and isolates aregood at forming emulsions and giving short-term stability withinthe homogenizer, as they have the ability to adsorb and unfoldrapidly at the nascent oilewater interface (Euston & Hirst, 2000).The action of CMC is that of a stabilizing agent rather than emul-sifying agent, acting as a macromolecular thickening agent,increasing the viscosity of the aqueous phase and modifying itsrheology (Coffey, Bell, & Henderson, 1995). It seems that a combi-nation stabilizing mechanisms took place in the ECMCeWPI doubleemulsion. On one hand, adsorption of WPI molecules at the dropletsurface took place, but not in enough numbers to cover entirely thedroplets surfaces, so that when mixing was terminated incipientdroplet coalescence occurred, with oil droplet size increasing.Jayasundera, Adhikari, Aldred, and Ghandi (2009) calculated themono-molecular layer concentration of b-lactoglobulin ona 0.75 mm fat droplet to be 2.27 mg per m2 assuming 100% surfacecoverage and 1.95 mg per m2 assuming 86% coverage. Likewise, theformation of a barrier of hydrated macromolecules of CMC aroundeach emulsion droplet took place, but the apparent viscosity of themacromolecular barrier around the oil droplets was at its minimumdue to the shear-thinning behaviour of CMC. At the end of theagitation, viscosity increased retarding collision and aggregation ofthe droplets.

Leroux, Langendorff, Schick, Vaishnav, and Mazoyer (2003)reported that pectin possesses surface-active properties attrib-uted to protein residues present within the pectin molecules. Thusin the case of the ELMPeWPI double emulsion, also some LMPmolecules adsorb together with WPI molecules at the oil dropletssurfaces, preventing to a larger extent incipient coalescence,resulting in a smaller mean d32 than in the case of the ECMCeWPIdouble emulsion.

3.3. Rheological properties of double emulsions

3.3.1. Steady-shear rheological propertiesFig. 1 shows the apparent viscosity versus shear rate plot for the

ELMPeWPI and ECMCeWPI double emulsions. The steady-shearbehaviour of both double emulsions indicated that they behaved astypical structural materials, showing a Newtonian region at lowshear rate range and a shear-thinning region at higher shear raterange. This typical viscosity characteristic of many non-Newtonianfluids (e.g. polymeric fluids, flocculated dispersions, colloids) is

1 10 1000.01

0.1

1

10

100

G´, G

" (P

a)

ω (rad/s)

G´ E CMC-WPI

G" E CMC-WPI

G´ E LMP-WPI

G" E LMP-WPI

Fig. 2. Changes in storage (G0) and loss (G00) moduli during frequency sweep at 0.5%strain for the double emulsions.

Shear rate (1/s), ωω (rad/s)

0.001 0.01 0.1 1 10 100

η, η

∗ (P

a s

)

η∗ (P

a s

)

0.01

0.1

1

η∗η

10

100

1000

η∗η

a

b

M.M. Murillo-Martínez et al. / Food Hydrocolloids 25 (2011) 577e585 581

probably due to a reversible “structure” or network that forms inthe “rest” or equilibrium state. Shearing of the material causes thestructure to break down, resulting in a shear-dependent behaviour(Darby, 1996). The experimental data of Fig. 1 fitted well by themodified Carreau model (R2 > 0.979) and the values for the rheo-logical parameters h0,l and n of the double emulsions aresummarized in Table 1. ELMPeWPI exhibited significantly higher h0,but significantly lower l and n values than the ECMCeWPI emulsion.Higher values of h0 suggest that stronger interactions occurredbetween the adsorbed biopolymer complexes between neigh-bouring oil droplets for the ELMPeWPI emulsion than for theECMCeWPI emulsion. In the latter emulsion, the number of dropletsper unit volume is substantially lower than for the ELMPeWPIemulsion, and the interactions between neighbouring oil dropletsare less. This fact seems to be corroborated by the lower n valuedisplayed by the ELMPeWPI emulsion, which required a higher shearstress for structure break down to occur (Chen et al., 2009), anda lower time (lower l) for structure recovery.

3.3.2. Dynamic shear rheological propertiesThe changes in G0 and G00 as a function of frequency of the double

emulsions are shown in Fig. 2. The ELMPeWPI double emulsionexhibited a higher G00 than G0 values at low (w<1 rad s�1) and high(w6 rad s�1) frequencies, but similar values at frequencies inbetween 1 and 5 rad s�1. The ECMCeWPI double emulsion exhibitedhigher G00 than G0 values over the whole frequency range. Bothmoduli showed significant frequency dependence, increasing asfrequency rose. The variations of G0 with increasing amplitudeoscillation are more consistent with the behaviour of weak, topo-logical interactions of polymer chains, than of normal gels. G0 didnot overcome G00 at higher frequencies, as is expected of entan-glement network system (Peressini, Bravin, Lapasin, Rizzotti, &Sensidoni, 2003). While the ECMCeWPI double emulsion showeda predominantly liquid like behaviour in the frequency rangeassayed (Fissore, Matkovic, Wider, Rojas, & Gerschenson, 2009), theELMPeWPI double emulsion tended to exhibit a more complexbehaviour than the ECMCeWPI double emulsion. This behaviour couldbe in part due to the difference in droplet sizes shown by the doubleemulsions. While its overall behaviour was more liquid than solid,it displayed a frequency range where G0 and G00 overlappingoccurred, and this behaviour is more in line with that of a networkembedded in a softer matrix, and rigidity in those regions can beproduced by chemical or physical cross-linking (Rodriguez-Gonzalez, Ramsay, & Favis, 2004).

3.3.3. Test of the CoxeMerz ruleA well-known empiricism in the rheology of polymer melts is

the Cox and Merz (1958) rule, which simply indicates that themagnitude of the complex viscosity (h*) is equal to that of shearviscosity (h) at equal values of radial frequency (u) and shear rate( _g). The application of the rule has great value in polymer rheologyas it allows to predict h( _g) from oscillatory measurements or jh*(u)jfrom steady state viscosity data (Goodwin & Hughes, 2000). A good

Table 1Modified Carreau model rheological parameters for the double emulsions.

Double emulsion code R2 n (dimensionless) l (s) h0 (Pa s)

ELMPeWPI 0.99 0.44a 17.5a 9.9a

ECMCeWPI 0.97 0.65b 67.6b 0.8b

ELMPeWPI ¼ water-in-oil-in-water double emulsion stabilized by low-methoxylpectin (LMP)ewhey protein isolate (WPI) complex. ECMCeWPI ¼ water-in-oil-in-water double emulsion stabilized by sodium carboxymethylcellulose (CMC)ewheyprotein isolate (WPI) complex. h0 ¼ low shear rate limiting viscosity,l ¼ characteristic time constant, n ¼ power-law behaviour index. Superscripts withdifferent letters in same column indicate significant differences (p � 0.05).

deal can be learned about the microstructure of materials from thedegree to which they adhere to the rule.

Fig. 3 shows the CoxeMerz plots for ELMPeWPI and ECMCeWPIdouble emulsions. Superposition of the dynamic and steady-shear viscosities occurred over a wide u and _g range for theELMPeWPI double emulsion (Fig. 3a), but not for the ECMCeWPI

η,

Shear rate (1/s), ω (rad/s)

0.001 0.01 0.1 1 10 1000.1

1

Fig. 3. Test of the CoxeMerz rule for the double emulsions: (a) Stabilized with low-methoxyl pectinewhey protein isolate complex (ELMPeWPI), and (b) Stabilized withsodium carboxymethylcelluloseewhey protein isolate complex (ECMCeWPI).

Table 2Edible films thickness, transparency %, colour index and contact angle.

Film code Thickness (mm) Transparency (%) Colour index Contact angle

Centre (t ¼ 0 min) Centre (t ¼ 3 min) Edge (t ¼ 0 min) Edge (t ¼ 3 min)

FCMCeWPI 0.185 � 0.006a 6.81b � 0.92 �4.62b � 0.04 50.0 � 0.0Ba 27.5 � 0.7Bb 42.0 � 0.0Ba 30.5 � 0.7Bb

FLMPeWPI 0.200 � 0.010a 16.39a � 0.35 �2.98a � 0.34 52.5 � 0.7Aa 50.0 � 0.0Ab 48.0 � 0.0Aa 38.5 � 0.7Ab

FCMCeWPI¼ edible film casted from double emulsion stabilized with sodium carboxymethylcellulose (CMC)ewhey protein isolate (WPI) complex. FLMPeWPI¼ edible film castedfrom double emulsion stabilized with low-methoxyl pectin (LMP)ewhey protein isolate (WPI) complex. Superscripts with different lower case letters in same row indicatesignificant differences (p � 0.05). Superscripts with different upper case letters in same column indicate significant differences (p � 0.05).

M.M. Murillo-Martínez et al. / Food Hydrocolloids 25 (2011) 577e585582

double emulsion (Fig. 3b). CoxeMerz superposition of steady-shear viscosity, from large deformation measurements, anddynamic viscosity, from small deformation analyses, is consistentwith topological entanglement interactions of individual species(Haque & Morris, 1993).

Although rheological and structural properties correlations arein general complex, and are beyond the scope of this work, theassociation of small deformation and steady-shear rheologicalmeasurements combined with other methods, such as differentmicroscopy techniques, can provide important insights for estab-lishing suitable conditions for manufacturing double emulsions forobtaining edible films for specific applications.

3.4. Film appearance

Both films were flexible and homogeneous. Their surfaces weresmooth without pores and cracks visible to the naked eye. Thick-ness differences were insignificant varying between 185 and200 mm (Table 2). The films were easily detached from the casting

Fig. 4. SEM micrographs of edible film (FCMCeWPI) casted from double emulsion stabilized wi(b) Cross-section of film with magnification 500� and 2000�, respectively, and (c) surface

glass. Transparency of both films was very low (Table 2), but wassignificantly higher for the FLMPeWPI film than for the FCMCeWPI film.This low transparency was probably due to the morphology of thedouble emulsions, whose oil droplets had relatively large sizes,which on turn contained multiple dispersed water droplets in theirinterior, causing a large degree of deflection of the spectropho-tometer light beam. The colour index values for both films (Table 2)indicate that their value was related to colours that range from darkgreen (�20) to yellow-green (�2) (Vignoni et al., 2006), and thatthe FLMPeWPI film had a significantly higher yellow component thanthe FCMCeWPI film.

3.5. Films microstructure

Cross-section of the FCMCeWPI (Fig. 4a) and FLMPeWPI (Fig. 5a)films observed under Scanning ElectronMicroscopy (SEM) revealeda microstructure consisting of closely packed elongated doubleemulsion droplets into a biopolymers mainframe. Progressivedistortion of oil droplets in emulsions has been associated with

th sodium carboxymethylcellulose (CMC)ewhey protein isolate (WPI) complex: (a) andview of film.

Fig. 5. SEM micrographs of edible film (FLMPeWPI) casted from double emulsion stabilized with low-methoxyl pectin (LMP)ewhey protein isolate (WPI) complex: (a) and (b) Cross-section of film with magnification 500� and 2000�, respectively, and (c) surface view of film.

M.M. Murillo-Martínez et al. / Food Hydrocolloids 25 (2011) 577e585 583

evaporation of the aqueous continuous phase. Evaporation of thewater continuous phase caused compression of the emulsion withprogressive distortion of the oil drops and thinning of the waterfilms separating them (Aranberri, Binks, Clint, & Fletcher, 2004).Although the FCMCeWPI and FLMPeWPI films presented the samemainstructural features, they showed differences in the structuralcharacteristics of their biopolymer mainframes and the size andspatial distribution of their emulsion droplets. FCMCeWPI filmmainframe (Fig. 4a) showed an oriented microstructure, consistingmainly of fibrous-like structures attributed to aggregates of thebiopolymers forming it, surrounding emulsion droplets (evidencedby the empty spaces originally occupied by the relatively large sizeddroplets corresponding to the ECMCeWPI emulsion). In contrast, theFLMPeWPI mainframe (Fig. 5a) was more compact and less orderedthan that of the FCMCeWPI film (Fig. 4a), and it was interrupted bysmaller emulsion droplets corresponding to the ELMPeWPI emulsionthan those observed in the FCMCeWPI film. Smooth appearance ofthe films mainframes and differences in the size and the spatialdistribution of the emulsion droplets were more evident whenlooking SEMmicrographs at higher magnification (Figs. 4b and 5b).It seems that a greater degree of cross-linking occurred betweenthe adsorbed biopolymers at the interface when the emulsiondroplets were smaller, resulting in a more closed film mainframe(FLMPeWPI film). Films surfaces, in general terms, had a homogenousand smooth appearance (Figs. 4c and 5c). However, some particleswith granular-like formwere observed on the films surfaces. Waterloss by evaporation from the emulsion surface during the filmformation could have led to a gradient in the water concentration,causing insolubility of the bulk solution biopolymers, resulting intheir deposition on the surface.

3.6. Wetting properties

Table 2 shows the contact angles obtained for both filmsimmediately and after 3 min of depositing the water droplet ontheir surface. Initial contact angle (qt¼0) and after 3 min (qt¼3) weresignificantly higher for the FLMPeWPI film than for the FCMCeWPI film,and were significantly higher at the centre than at the edge. Thedifference in the values of q at t ¼ 0 and t ¼ 3 minwas lower for theFLMPeWPI film than for the FCMCeWPI film. Both films possessedhydrophilic surfaces as they exhibited contact angles q < 65 at alltimes (Hambleton, Debeaufort, et al., 2009). The gaps in qt¼0 andqt¼3 shown by the films can be probably attributed to modificationof their surfaces, due to solvation, hydration or swelling, which onturn could have caused spatial rearrangement and coalescence ofthe double emulsion droplets (internal and external). Thesephenomena usually reach metastable equilibrium during the first20e30 s (Hambleton, Fabra, Debeaufort, Dury-Brun, & Voilley,2009).

3.7. Edible films mechanical properties

Films based on biopolymer complexes are viscoelastic materialsthat possess characteristics of both solids and liquids. A tridimen-sional matrix, constructed by interaction between the biopolymersforming the complex, is presumably the supporting structure thatdictates the mechanical properties of the films. Interactionsbetween the biopolymers, the solvent, plasticizers, lipids, and otheradditives dispersed in the space of thematrix, also contribute to themechanical properties of the film (Chen, 1995).

M.M. Murillo-Martínez et al. / Food Hydrocolloids 25 (2011) 577e585584

Mechanical properties are important to edible films, becauseadequate mechanical strength ensures the integrity of the film andits freedom from minor defects such as pinholes. Quantitativeinformation of mechanical parameters of edible films is essentialfor their adequate design. Tensile strength (or more accurately,ultimate tensile strength) is the ultimate tensile strength that thefilm can sustain. Elongation is usually taken at the point of breakand is expressed as the percentage of change of the original gaugelength of the specimen. Modulus of elasticity, or Young’s modulus,is the ratio of stress to strain in the linear region and measures theintrinsic stiffness of the film.

Table 3 shows mechanical properties values of both edible films.Differences in the mechanical properties of the FLMPeWPI andFCMCeWPI films were observed, which can be attributed to the effectof the biopolymers on the size of the emulsion droplets and theinteractions taking place among biopolymers molecules formingthe mainframe structure of the films. Thus, the FLMPeWPI filmformed by relatively small emulsion droplets and a great cross-linking biopolymer mainframe (Fig. 5b) exhibited higher tensilestrength and Young’s modulus, but lower elongation % than thoseobserved for the FCMCeWPI film. The latter had a structure formed byrelatively large emulsion droplets immersed in a relaxed andordered biopolymer mainframe (Fig. 5a).

The tensile strength results are in close accordance with therheological (stationary and dynamic) results given above. Thevalues of TS and E exhibited by our films were of the same order ofthose shown by edible films based whey protein isolate, wheyprotein concentrate or calcium caseinate added with differentplasticizers, lipid phases or nutraceuticals (Chen, 1995; Mei & Zhao,2003; Villagómez-Zavala et al., 2008). However, TS and E weremuch lower (one or two orders of magnitude lower) than thevalues corresponding to synthetic polymers (high and low-densitypolyethylene) based films (Chen, 1995). Materials show differenttensile patterns. For example, methylcellulose films are of hightensile strength and low elongation, and protein films are ofmoderate tensile strength and greater elongation. Methylcellulosefilm is hard and brittle, and protein film is soft and tough. Thesedifferences can be attributed to different molecular structures.Proteins are generally of sophisticated configuration because ofcomplex inter- and intra-molecular interactions and a variety ofside-chain groups. The structure of polysaccharides backbone isusually linear (Chen, 1995). Young’s modulus for the FLMPeWPI filmwas about half, and that of the FCMCeWPI film about a fourth of thevalues for this parameter for films obtained from binary blends ofhydrocolloids (mesquite gumewhey protein concentrate; sodiumalginateekappa carrageenan; whey protein concentrateesodiumalginate; whey protein concentrateekappa carrageenan) addedwith glycerol. It seems that both, the FLMPeWPI and FCMCeWPI films,tended to display mechanical properties that resembled more theproperties of protein based films than those of polysaccharidebased films, despite that in both cases, the proportion of poly-saccharide to protein was overwhelmingly on the polysaccharideside. These findings tend to confirm that WPI was the biopolymer

Table 3Mechanical properties and water vapour permeability (WVP) of the edible films.

Film code Tensile strength(MPa)

% Elongation Young’sModulus (MPa)

WVP(gmm/m2 h kPa)

FCMCeWPI 0.93 � 0.11b 6.11 � 0.87a 48.76 � 4.90b 1.5 � 0.2a

FLMPeWPI 1.49 � 0.17a 2.15 � 0.43b 119.74 � 48.61a 1.6 � 0.1a

FCMCeWPI ¼ edible film casted from double emulsion stabilized with sodiumcarboxymethylcellulose (CMC)ewhey protein isolate (WPI) complex.FLMPeWPI ¼ edible film casted from double emulsion stabilized with low-methoxylpectin (LMP)ewhey protein isolate (WPI) complex. Superscripts with differentletters in same column indicate significant differences (p � 0.05).

that adsorbed more strongly at the oilewater interface and isresponsible to a greater degree of the properties imparted thedouble emulsions.

3.8. Water vapour permeability

The WVP values of the FCMCeWPI and FLMPeWPI were not signif-icantly different (Table 3). These results mean that the structure ofthe adsorbed biopolymer complex layer around the double emul-sions did not eventually affect WVP of the films. Both filmsexhibited WVP values (1.5e1.6 g mm m�2 h�1 kPa�1) lower thanthose of other WPI based films added with different compounds(calcium lactateegluconate blend, a-tocopheryl acetate, sorbitol,beeswax or potassium sorbate) (Mei & Zhao, 2003; Ozdemir &Floros, 2008), or made with heated and unheated WPI (Guckian,Dwyer, O’Sullivan, O’Riordan, & Monahan, 2006).

4. Conclusions

Double emulsions stabilized by proteinepolysaccharidecomplexes can yield edible films with mechanical propertiescomparable with those exhibited by hydrophilic films, but withwater vapour permeability comparable with those displayed byhydrophobic films. Additionally, the manufacturing of edible filmsfrom double emulsions offers the possibility for incorporatingantimicrobials, nutraceuticals, probiotics, and many othercompounds, providing the basis for designing a new generation ofedible films with enhanced functional properties.

Acknowledgements

The authors wish to thank the partial financing this project theConsejo Nacional de Ciencia y Tecnologia (CONACyT) of Mexico forthrough grant U-81157-Z, and to the Agencia Española de Cooper-ación Internacional para el Desarrollo (AECID) through project“Optimización de técnicas para el desarrollo de emulsiones doblesestabilizadas con polímeros naturales y sintéticos: Aplicaciónpotencial y funcionalidad”.

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