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Food Hydrocolloids 27 (2012) 316e323

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Food Hydrocolloids

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Stability and release properties of double emulsions for food applications

Lanny Sapei, Muhammad Ali Naqvi, Dérick Rousseau*

Department of Chemistry and Biology, Ryerson University, 350 Victoria St., Toronto, ON M5B 2K3, Canada

a r t i c l e i n f o

Article history:Received 31 January 2011Accepted 11 October 2011

Keywords:Double emulsionSaltGelatinStabilityControlled releaseModelling

* Corresponding author. Tel.: þ1 416 979 5000x215E-mail address: rousseau@ryerson.ca (D. Rousseau

0268-005X/$ e see front matter � 2011 Elsevier Ltd.doi:10.1016/j.foodhyd.2011.10.008

a b s t r a c t

Water-in-oil-in-water (W1/O/W2) double emulsions (DEs) containing gelatin and sodium chloride (NaCl)in the inner aqueous phase were developed for controlled release applications. Emulsions were preparedwith water and canola oil, as well as with polyglycerol polyricinoleate and polysorbate 80 as emulsifiersfor the primary water-in-oil (W1/O) emulsion and secondary W1/O/W2 emulsions, respectively. All DEscontaining both NaCl and gelatin were stable against sedimentation for the month-long study whereascontrol emulsions (with either no NaCl or gelatin) showed visual phase separation. The average oilglobule size in freshly-prepared DEs grew from w45 to 70 mm with an increase in salt load from 2 to 8%(w/w), and changed little after 1 month. Besides its role in stabilization, NaCl was also used as a markerto evaluate DE release behaviour. The salt diffusion coefficient obtained using Fujita’s model rose from4.7 to 6.0 � 10�11 cm2/s with increasing NaCl concentration in the DEs from 2 to 8% (w/w). All stable DEsshowed a high salt retention in the inner aqueous phase (>94%) after 1 month of storage at 4 �C. Theseresults demonstrated the synergistic action of a gelling agent and electrolyte in stabilizing and modu-lating the release behaviour of NaCl from W1/O/W2 DEs.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction

Water-in-oil-in-water (W1/O/W2) double emulsions (DEs)consist of a water-in-oil (W1/O) emulsion dispersed as dropletswithin an aqueous phase (Garti, 1997; Sagalowicz & Leser, 2010).Due to the presence of two aqueous domains separated by an oillayer, the inner aqueous compartment offers great potential for theencapsulation and controlled release of hydrophilic bioactiveingredients. However, such emulsions are more difficult to prepareand control than simple emulsions as they typically consist ofrelatively large droplets that coalesce either quiescently or due tocommonly-encountered processing regimes (e.g., shear, steriliza-tion), and have a strong tendency to release entrapped compoundsin an uncontrolled manner (Garti, 1997). Furthermore,commercially-sterile DEs may be difficult to manufacture, partic-ularly when numerous ingredients are present (Dalgleish, 2001).Usage of W1/O/W2 DEs for food applications is further limited bythe lack of suitable food-grade emulsifiers and stabilizers for theinner and outer emulsions.

W1/O/W2 emulsions have been used in cosmetics and pharma-ceuticals for applications such as drug controlled release and tar-geted delivery (Gallarate, Carlotti, Trotta, & Bovo, 1999; Laugel,Chaminade, Baillet, Seiller, & Ferrier, 1996; Vaziri & Warburton,

5; fax: þ1 416 979 5044.).

All rights reserved.

1994; Vlaia, Vlaia, Miclea, Olariu, & Coneac, 2009). Other applica-tions have included the removal of toxic materials via entrapmentand solubility enhancement of poorly-soluble materials (Yan & Pal,2001). W1/O/W2 DEs have also been investigated for various foodapplications, including the encapsulation of vitamin/minerals(Benichou, Aserin, & Garti, 2007; Bonnet et al., 2009; O’Regan &Mulvihill, 2010), aroma and flavour release (Malone, Appelqvist, &Norton, 2003) and the production of low-calorie foods (DeCindio& Cacace, 1995), e.g., low-fat dressing (Taki, 2008).

Given their lack of kinetic stability, the widespread applicationof W1/O/W2 DEs in the food industry remains elusive. Garti’sgroup attempted to improve the stability and release character-istics of W1/O/W2 DEs using steric stabilizers (Garti, Aserin, &Cohen, 1994; Lutz, Aserin, Wicker, & Garti, 2009), Pickeringstabilization with fat crystals (Garti, Binyamin, & Aserin, 1998) andprotein-polysaccharide hybrids (Benichou et al., 2007). Muschio-lik’s group examined the incorporation of gelatin, NaCl and poly-glycerol polyricinoleate (Pgpr) in the primary emulsion, the use ofsodium caseinate-dextran conjugates as an external emulsifier,and membrane emulsification, all to better harness DE stabilityand controlled release patterns (Fechner, Knoth, Scherze, &Muschiolik, 2007; Muschiolik, 2007; Muschiolik et al., 2006).Others have focused on reducing Pgpr concentration by incorpo-rating sodium caseinate in the inner aqueous phase (Su, Flanagan,Hemar, & Singh, 2006) or by adding a highly-concentrated modi-fied gum Arabic in the external aqueous phase (Su, Flanagan, &Singh, 2008).

L. Sapei et al. / Food Hydrocolloids 27 (2012) 316e323 317

The objective of this researchwas to formulate a food-gradeW1/O/W2 DE with good stability and controllable release properties.The DEs herein developed were kinetically-stable for the durationof the study (1 month) with the release of NaCl incorporated asa marker triggered through changes in osmotic environment.

2. Materials and methods

2.1. Materials

Deionized water with a resistivity of >15 megohm-cm (Barn-stead E-Pure, Ottawa, ON, Canada) was used for the aqueous phase.Gelatin (porcine skin, type A, Bloom w300) and the water-tendingemulsifier polysorbate 80 were purchased from SigmaeAldrich(Oakville, ON, Canada). Sodium chloride (NaCl) was purchased fromFisher Scientific (Ottawa, ON, Canada). Canola oil (acid value < 0.2)was purchased from a local supermarket (Toronto, ON, Canada).The oil-tending emulsifier polyglycerol polyricinoleate (Pgpr) wasobtained from Nealanders (Mississauga, ON, Canada). All chemicalswere used without further purification.

2.2. Double emulsion preparation

The inner aqueous phase (W1) containing gelatin (0%, 3% or 10%w/w) and NaCl (0e8% w/w) was hydrated for 30 min followed bymixing at 65 �C for 25 min. The oil phase (O) containing 6% (w/w)Pgpr was also mixed at 65 �C for 25 min. Finally, the outer aqueousphase (W2) containing 1% (w/w) polysorbate 80 was mixed 25 �Cfor 30 min.

Food-grade W1/O/W2 DEs were prepared via a simple, repro-ducible two-stage process. The primary water-in-oil emulsion (W1/O) [40% w/w inner aqueous phase (W1) and 60% w/w oil phase (O)]was homogenized at 65 �C using a rotor-stator with a homogeni-zation generator (d ¼ 1.2 cm) (Polytron� PT 10/35, Kinematic, CH-6010, Switzerland) at 27,000 rpm for 3 min. The resulting emul-sion was immediately quench-cooled to 4 �C in a waterbath totrigger the solegel transition of the gelatin present in the dispersedaqueous phase. Prior to second-stage homogenization, thetemperature was slowly raised to 26 �C to improve emulsificationefficacy, as preliminary results showed inefficient emulsification atlower temperatures. The secondary emulsion (W1/O/W2) wasprepared by gradually adding the W1/O emulsion (20% w/w) to theouter aqueous phase (W2) followed by mixing with the rotor-statorat 10,000 rpm for 2 min and subsequent quench-cooling in a 4 �Cwaterbath.

2.3. Stability evaluation

2.3.1. SedimentationFreshly-made DEs were poured into 40 ml glass vials

(ID ¼ 25 mm; length ¼ 95 mm; Fisher Scientific, Nepean, ON,Canada) to a height of w6 cm and stored at 4 �C. The height of theopaque emulsion phase was measured weekly over 1 month (ht)and compared to the initial emulsion height (h0) to determinesedimentation stability (S):

S ¼�h0 � ht

h0

�� 100% (1)

2.3.2. MicroscopyBrightfield light microscopy was used to examine DE emulsion

morphology. Samples were placed onto microscope slides (FisherScientific, Nepean, ON, Canada) and gently coveredwith a cover slip(Fisher Scientific, Nepean, ON, Canada). A Zeiss Axiovert 200M

inverted light microscope (Zeiss Inc., Toronto, ON, Canada) witha 20� objective (combined with a 1.6� magnifier lens) was used.Images captured with a Q-Imaging CCD camera were analyzedusing Northern Eclipse software (version 7.0, Empix Imaging,Mississauga, ON, Canada). Emulsions were characterized at roomtemperature (25 �C).

2.3.3. Oil globule size determinationThe mean oil globule size distribution in the DEs (W1/O) was

characterized for 1 month with a Malvern particle size analyzer(Mastersizer 2000S, Malvern Instruments Ltd., Malvern, Worces-tershire, UK), equipped with a HeeNe laser (l ¼ 633 nm). Theoptical parameters selected were: dispersed phase refractive indexof 1.47; globule absorbance of 0.01 and a dispersant liquid(deionized water) refractive index of 1.33. Measurements werecarried out in triplicate on each emulsion and the results are re-ported as the typical globule size distribution (in mm), and thevolume-weighted mean globule size D (4,3):

Dð4;3Þ ¼P

nid4iPnid3i

(2)

where ni is number of particle i and di is diameter of particle i (mm).The oil globule size distribution was determined from the best

fit between the experimental measurements and prediction usingthe Mie light scattering theory. As the inner aqueous droplets wereconsidered encapsulated within the oil globules, their contributionto scattering was not significant.

2.4. Release properties

2.4.1. NaCl release and kineticsNaCl release from the inner aqueous phase towards the external

aqueous phasewas measured using a conductivity meter (model HI98188, Hanna Instrument, Romania) equipped with an integrateddata logger and associated transfer software (HI 92000 version5.0.7). To carry out measurements, the conductivity meter wasdipped into a 15 ml aliquot of W1/O/W2 DE placed in a 50 ml Falcontube (Fisher Scientific, Nepean, ON, Canada). The time-dependentconductivity values were converted into NaCl concentration usinga calibration curve (not shown). The fraction of NaCl released (FR) inthe external aqueous phase was defined as the ratio of NaClreleased into W2 at a specific time (Mt) relative to the total amountpresent in the external aqueous phase if all NaCl were releasedðMNÞ.

FRð%Þ ¼ Mt

MN� 100% (3)

Release experiments for all samples containing salt and gelatinwere conducted in triplicates, whereas for the control samples (nogelatin), the experiments were conducted in duplicates due to lackof emulsion stability. Experiments were conducted at 4 �C.

2.4.2. Encapsulation efficiencyEncapsulation efficiency (EE) was defined as the percentage of

NaCl still entrapped within the inner aqueous phase (W1):

EEð%Þ ¼ 100� FRð%Þ (4)

2.4.3. Modelling NaCl releaseFick’s second law in a spherical coordinate system was used to

describe NaCl transport in the DEs (Eq. (5)). The system wassimplified mathematically into a two-phase system consisting ofthe NaCl ‘reservoir’ (in the inner W1/O emulsion) and the

Fig. 1. Role of salt concentration in the inner aqueous phase on the sedimentation ofW1/O/W2 double emulsions stabilized with 3% gelatin. (a) 0% NaCl; (b) 2% NaCl; (c) 4%NaCl; (d) 6% NaCl; (e) 8% NaCl. All double emulsions are 1 month old.

Fig. 2. Role of gelatin concentration in the inner aqueous phase on the sedimentationof W1/O/W2 double emulsions containing 2% NaCl. (a) 0% gelatin; (b) 3% gelatin; (c)10% gelatin. All double emulsions are 1 month old.

L. Sapei et al. / Food Hydrocolloids 27 (2012) 316e323318

continuous phase (W2). The mass transfer was explained usingconcentration-dependent diffusivities where Ct is the NaClconcentration in the continuous phase at time t, r is the radialcoordinate discretized along reservoir radius (specified using oilglobule size data) into 50 intervals and D is the concentration-dependent diffusion coefficient of NaCl from the inner emulsion.

vCtvt

¼ v

vr

�DvCtvr

�(5)

Fujita’s model of free volume was used to model salt releasekinetics (Eq. (6)) (Fujita, 1952).

D ¼ Deqexp�� b

�1� Ct

Ceq

��(6)

where Deq is the diffusion coefficient of NaCl in an equilibrated DE,b is a dimensionless scaling parameter that relates the initial andequilibrium diffusivities and Ceq is the equilibrium NaCl concen-tration in the continuous phase. The equations were integratedusing a finite-difference method in Athena Visual Studio modelingsoftware v.14 (Athena Visual Software, Inc, 2009, Evanston, IL, USA).Shown below are the initial and boundary conditions.

Initial conditions:

t ¼ 0;Ct ¼ 0

Boundary conditions:

r ¼ 0;vCtvr

¼ 0r ¼ Req;Ct ¼ 1

where Req is the equilibrium oil globule size based on light scat-tering experiments. The sum of NaCl released from the emulsionat each discretized radial interval was equal to the NaCl increase inthe continuous phase. The model was only concerned with theexcipient NaCl content and hence the release concentration aswell as rate of release was zero at t ¼ 0 and r ¼ 0. The sphericalcoordinate system in Athena Visual Studio considers only a singleplane of the sphere to integrate the diffusion model equationaccording to a finite-difference method. Sink conditions wereassumed to exist at the surface of the reservoirs. Emulsiondecomposition, swelling and contraction were ignored in themodel.

2.5. Statistics

All experiments were carried out in triplicate and the results areexpressed as mean � standard deviation. Statistical analysis wasconducted using SigmaStat 4 integrated in SigmaPlot 11 (SystatSoftware, Chicago, IL, USA). The average oil globule size andencapsulation efficiency were analyzed using t-tests or one-wayanalyses of variance (ANOVA) and the Holm Sidak post hoc test.The goodness of fit of the NaCl release kinetics modelled withFujita’s model was based on correlation coefficients (r2) and their95% confidence interval. Statistical analyses were deemed signifi-cant at p < 0.05.

3. Results

3.1. Sedimentation

The DEs prepared with both NaCl (Fig. 1bee) and gelatin (Fig. 2band c) were stable against sedimentation for the month-long study.In contrast, control DEs with either no NaCl (Fig. 1a) or gelatin(Fig. 2a) showed signs of phase separation immediately after

preparation. The sedimentation stability (S) of emulsions contain-ing gelatin but no NaCl decreased fromw45% on day 1 tow30% onday 29 whereas those with only NaCl showed a higher sedimen-tation stability (w90% on day 1 and w80% on day 29) (Table 1). AllDEs containing salt and gelatin were visually more viscous than thecontrol DEs, but still pourable.

3.2. Double emulsion morphology

The morphology of all formulations was similar after 1 day(Fig. 3I) and 1 month (Fig. 3II), with numerous internal aqueousdroplets present within oil globules themselves dispersed ina continuous aqueous phase. The internal aqueous droplets in theDEs with 8% (w/w) NaCl were somewhat larger after 1 month

Table 1Double emulsion sedimentation stability and average oil globule sizes after days 1 and 29.

Double emulsion Sedimentation stability (S), % Average oil globule size (D (4,3)), mm

Day 1 Day 29 Day 1 Day 29

0% NaCl, 3% gelatin 44.8 � 0.4 28.5 � 0.4 12.35 � 0.34a,1 13.08 � 0.91a,1

2% NaCl, 3% gelatin 100 100 46.37 � 1.27a,2 46.02 � 0.53a,2

4% NaCl, 3% gelatin 100 100 56.64 � 0.82a,3 57.28 � 1.40a,3

6% NaCl, 3% gelatin 100 100 65.79 � 0.87a,4 65.66 � 1.53a,4

8% NaCl, 3% gelatin 100 100 70.50 � 0.77a,5 70.73 � 0.90a,5

2% NaCl, 10% gelatin 100 100 47.70 � 0.35a,6 48.56 � 0.44b,6

2% NaCl, 0% gelatin 90.2 � 0.4 78.5 � 0.5 41.81 � 0.74a,7 41.98 � 1.55a,7

a,b Means within the same row without a common letter are significantly different (p < 0.05).1,2 Means within the same column without a common number are significantly different (p < 0.05).

L. Sapei et al. / Food Hydrocolloids 27 (2012) 316e323 319

(Fig. 3c I vs. II) compared to the other formulations [i.e, 2e6% (w/w)NaCl], where no obvious size differences were apparent. Theinternal aqueous droplets in the DEs sans gelatin were slightlylarger than in gelatin-containing emulsions (Fig. 3d vs. aec). Oilglobule flocculation was pronounced in the DEs without NaCl(Fig. 3a), with a high dispersed oil globule packing density in allsalt-containing systems (Fig. 3bed).

3.3. Droplet size characteristics

There were no changes in the oil globule size distribution of anyDE after 1 month of storage (not shown). The initial oil globule sizedistributions (Fig. 4) of all DEs containing NaCl were bimodal witha dominantw15e200 mmdistribution and a small distribution in thew2e15 mm range. In the DEs prepared without NaCl, the dominantmode was at 2e20 mm, with several smaller modes. Thus, these DEdroplet size distributions were not bimodal. The initial average oilglobule size [D (4,3)] increased from w45 mm to w70 mm with anincrease in salt concentration from 2% (w/w) to 8% (w/w) (Table 1),which was corroborated by the oil globule size distributions alsoshifting to larger values with more added NaCl (Fig. 4a) (p < 0.05).DEs containing 2% (w/w) NaCl stabilized with varying amounts ofgelatin demonstrated similar oil globule size distributions, witha dominant 10e100 mm distribution and a small distribution in the2e10 mm range (Fig. 4b). In all DEs, the average oil globule sizes afterdays 1 and 29 were not statistically significantly different (p > 0.05),except for the sample with 2% NaCl and 10% gelatin (p < 0.05)(Table 1). Thus, all DEs were considered kinetically-stable.

3.4. NaCl release and kinetics

NaCl release for 48 h after DE preparation showed an initiallyrapid flux followed by a gradual rise towards a plateau (Fig. 5).Release after 30 min was 2.6e2.9% with plateau values lower at 8%than 2% (w/w) salt (4.5 vs. 5.1%) (Fig. 5a). Fig. 5b shows thatincreasing the gelatin concentration within the internal aqueousphase slowed salt release. However, release from these DEs wasonly recorded for 20 h as the gelatin-free DEs phase-separated.

The release profiles of all salt-containing DEs stabilized withgelatin were normalized and fitted using Fujita’s model (curvefitting examples shown in Fig. 6) to determine the diffusionparameters Deq and b (Table 2). Application of this model yieldedcorrelation coefficients (r2) > 0.99 for all release curves. The NaCldiffusion coefficient increased from 4.7 � 10�11 cm2/s for the DEwith 2% (w/w) NaCl to 6.0 � 10�11 cm2/s in the 8% (w/w) NaCl DE.The impact of gelatin concentration on NaCl release rate was slightwith Deq diminishing from 4.7� 10�11 cm2/s for the DE with 3% (w/w) gelatin to 4.1 � 10�11 cm2/s in the 10% (w/w) gelatin DE.b parameter estimates were very similar and could not be used todistinguish differences in release profiles between the various DEs.

3.5. NaCl encapsulation efficiency

The NaCl encapsulation efficiency of all DEs remained at 94e95%during the month-long study irrespective of NaCl load and gelatinconcentration in the inner aqueous phase (Table 3). However, thesmall variations observed (DEE < 1%) still proved to be statisticallysignificant (p < 0.05).

4. Discussion

The presence of gelatin and salt played a key role in DE stabilityand release (Fig. 7). Salt (Fig. 7a) likely enhanced emulsifier efficacyand affected Laplace pressure. Aronson and Petko (Aronson &Petko, 1993) reported that electrolytes could increase emulsifieradsorption density at the oil/water interface and reduce interfacialtension in W/O emulsions. Scherze et al. showed that addition ofNaCl to the dispersed phase of Pgpr-stabilized W/O emulsions wasessential in preventing dispersed water droplet coalescence(Scherze, Knoth, & Muschiolik, 2006). Parallel experiments in ourlab (results not shown) also established a synergistic effectbetween NaCl and Pgpr on canola oil-based W/O emulsions, withthese demonstrating remarkable sedimentation and droplet sizestability. Such stable primary (W/O) emulsions were crucial toobtaining stable double W1/O/W2 emulsions.

Rosano et al. found that electrolytes could stabilize W/O emul-sions by counter-balancing the Laplace pressure differencesbetween water droplets, thus preventing Ostwald ripening(Rosano, Gandolfo, & Hidrot, 1998). Similarly, Kanouni et al.(Kanouni, Rosano, & Naouli, 2002) found that DE stability requireda balance between the Laplace and osmotic pressures (betweenW1droplets in O and between W1 droplets and the external aqueousphase W2). Thus, the presence and concentration of salt in W1played a critical role in balancing these effects, with excess saltresulting in water migration from W2 to W1 and subsequentswelling of the W1/O droplets (Lutz & Garti, 2006; Rosano et al.,1998).

In DEs with gelatin and no salt (Fig. 7b), gelation of the inneraqueous phase enhanced DE stability (Fechner et al., 2007;Muschiolik et al., 2006) and increased NaCl encapsulation effi-ciency. Instability of ungelled DEs was also evident based on NaClrelease behaviour, which was much more rapid in comparison togelatin-stabilized DEs (Fig. 5b).

The amount of gelatin present also altered DE release behaviourand stability. The overall fraction release of NaCl for the first 20 hwas much slower in DEs stabilized with 10% gelatin compared towith 3% gelatin (Fig. 5b), likely due to a firmer inner aqueous gelledphase that retarded possible salt release during secondaryhomogenization. The encapsulation efficiency of the DEs with 10%(w/w) gelatin was slightly higher than with 3% (w/w) gelatin(p < 0.05).

Fig. 3. Double emulsion microstructure prepared using various NaCl and gelatin concentrations incorporated in the inner aqueous phase. (a) 0% NaCl and 3% gelatin; insets arehigher magnification regions of the oil globules; (b) 2% NaCl and 3% gelatin; (c) 8% NaCl and 3% gelatin; (d) 2% NaCl and 0% gelatin. (I) day 1; (II) day 29. The W1/O/W2 doubleemulsions prepared using 3% gelatin combined with 4% and 6% NaCl or with 10% gelatin and 2% NaCl are not shown due to their similar appearance to the samples shown. Bar is40 mm.

L. Sapei et al. / Food Hydrocolloids 27 (2012) 316e323320

Salt-gelatin interactions also affected DE stability and releasebehaviour (Fig. 7c). For example, inner water droplets containingboth salt and gelatin were densely-packed within the oil dropletsdue to enhanced gelatin swelling in the presence of salt.

Kawashima et al. noted that W1/O/W2 emulsions with a hyper-tonic inner aqueous phase were stable due to a swelling-inducedincrease in viscosity compared to emulsions with an isotonic orhypotonic inner aqueous phase (Kawashima, Hino, Takeuchi, &

Fig. 4. Initial oil globule size distributions [D (4,3)] of W1/O/W2 double emulsions. (a)W1/O/W2 double emulsions stabilized with 3% gelatin combined with various NaClconcentrations entrapped in the inner aqueous phase. (C-C) 0% NaCl; (V-V) 2% NaCl;(---) 4% NaCl; (>->) 6% NaCl; (:-:) 8% NaCl. (b) W1/O/W2 double emulsionscontaining 2% NaCl stabilized with various gelatin concentrations. (C-C) 0% gelatin;(V-V) 3% gelatin; (---) 10% gelatin.

Fig. 5. Release patterns of W1/O/W2 double emulsions examined via conductivity. (a)W1/O/W2 double emulsions stabilized with 3% gelatin combined with various NaClconcentrations entrapped in the inner aqueous phase. (C-C) 2% NaCl; (V-V) 4% NaCl;(---) 6% NaCl; (>->) 8% NaCl. (b) W1/O/W2 double emulsions containing 2% NaCl inthe inner aqueous phase stabilized with various gelatin concentrations. (C-C) 0%gelatin; (V-V) 3% gelatin; (---) 10% gelatin.

Fig. 6. Normalized release profile of W1/O/W2 double emulsions fitted with Fujita’smodel. (C-C) 2% NaCl and 10% gelatin; (B-B) 8% NaCl and 3% gelatin. (d) Fit fromFujita’s model.

L. Sapei et al. / Food Hydrocolloids 27 (2012) 316e323 321

Niwa, 1992). The increase in oil globule size with more NaCladded resulted from the higher osmotic pressure gradientexperienced between the two aqueous phases, implying greaterwater diffusion from the external to the internal aqueous phase(Mezzenga, Folmer, & Hughes, 2004). This water flux resulted inthe swelling and expansion of the oil globules and a consequentincrease in their packing density (Chanamai & McClements,2000) because the polydispersed aqueous droplets effectivelyfilled the available space within the oil globules (Das & Ghosh,1990). In this regard, NaCl screening of the charged sitespresent on the gelatin polymer chains allowed them to morefreely re-organize and swell further (Chatterjee & Bohidar, 2006).Yet, this mechanism applied only to DEs containing 3% gelatinand 2e8% (w/w) NaCl [which all showed unchanging oil globuledistributions over time (p > 0.05) (Table 1)]. For DEs with 10% (w/w) gelatin, there was a significant difference in D (4,3) betweendays 1 and 29 (p < 0.05), suggesting that an increase in gelatinbeyond a critical concentration resulted in an osmotic pressureimbalance between the two aqueous phases and oil globulecoalescence during storage.

Table 2Diffusion parameter estimates based on the Fujita model. Error reported as 95%confidence intervals.

Double emulsion Deq � 1011 (cm2/s) eb r2

2% NaCl, 3% gelatin 4.68 � 0.13 2.28 0.9974% NaCl, 3% gelatin 5.25 � 0.11 2.28 0.9986% NaCl, 3% gelatin 5.29 � 0.18 2.45 0.9948% NaCl, 3% gelatin 5.97 � 0.22 2.38 0.9922% NaCl, 10% gelatin 4.12 � 0.05 2.50 1.000

Table 3NaCl encapsulation efficiency after days 2 and 29.

Samples Encapsulation Efficiency, %

Day 2 Day 29

2% NaCl, 3% gelatin 94.86 � 0.02a,1 94.70 � 0.06b,1

4% NaCl, 3% gelatin 94.95 � 0.02a,2 94.10 � 0.04b,2

6% NaCl, 3% gelatin 95.07 � 0.03a,3 94.35 � 0.11b,3

8% NaCl, 3% gelatin 95.30 � 0.00a,4 94.22 � 0.02b,2,3

2% NaCl, 10% gelatin 95.75 � 0.06a,5 95.07 � 0.14b,4

a,b Means within the same row without a common letter are significantly different(p < 0.05).1,2 Means within the same column without a common number are significantlydifferent (p < 0.05).

L. Sapei et al. / Food Hydrocolloids 27 (2012) 316e323322

The incorporation of NaCl as a marker in the internal aqueousphase induced an osmotic gradient that led to the migration ofexternal aqueous phase into the internal droplets. The osmoticgradient increased from w685 to 2735 mOsmol by the

Fig. 7. Proposed (de-)stabilization mechanisms of W1/O/W2 double emulsions. (a) no ge

incorporation of 2e8% (w/w) NaCl. This was 4e15� higher than therecommended osmotic gradient range (180e200 mOsmol) forobtaining stable DEs (Muschiolik et al., 2006). However, theseemulsions still showed long-term stability and no oil globulebreakdown or phase separation was visible upon swelling. Thisindicated that the Pgpr likely provided an expandable andcompressible interfacial layer that covered the internal aqueousdroplets after swelling.

Initial NaCl release from the DEs increased from 2.6% to 2.9%with an increase in NaCl concentration from 2% to 8% (w/w) (Fig. 5),which was presumably due to loss of NaCl from the internalaqueous phase during second-stage homogenization. Additionally,it has been reported that low molecular weight compounds mayundergo rapid release as a result of a high osmotic and/or increasedconcentration gradient (Lakkis, 2007), so that an initial release ofsolute was foreseeable in this system. However, the DEs stillretained >94% salt after 1 month (Table 3).

Salt diffusion from the inner aqueous droplets to the externalaqueous phase was successfully modelled with the Fujita model(r2 > 0.99 at all salt loads), which is based on Fick’s second law. Asthe internal aqueous droplet and oil globule size and numberhardly changed over a month, this suggested that NaCl release wasgoverned by diffusion and not by droplet rupture or erosion(Bonnet et al., 2009; Magdassi & Garti, 1984).

The DEs with the higher salt loads reached lower plateauconcentrations, despite their higher initial release (Fig. 5). As thegelatin concentration was constant [3% (w/w)], this may have beendue to an increased osmotic gradient that induced water dropletswelling, and consequently lengthened the diffusion path of theentrapped salt. The DEs with no gelatin showed a higher release

latin and with NaCl; (b) no NaCl and with gelatin; (c) with gelatin and with NaCl.

L. Sapei et al. / Food Hydrocolloids 27 (2012) 316e323 323

rate that was highly variable due to phase separation. Conversely,DEs stabilized with 10% gelatin showed a highly-reproduciblelower initial release and a more delayed release profile than DEsstabilized with 3% gelatin (Fig. 5b; Table 2).

5. Conclusions

We designed food-grade W1/O/W2 DEs with long-term stabilityand desirable controlled release behaviour using a simple, repro-ducible approach. DE stability was dependent on the presence ofNaCl and gelatin in the inner aqueous phase, with the diffusion-controlled release behaviour of these DEs governed by Fickiandiffusion. These results clearly demonstrated that the presence ofa gelling agent and electrolyte could be used to modulate therelease behaviour of incorporated compounds from W1/O/W2 DEs.With their high encapsulation efficacy, the potential use of theseDEs for food applications appears promising. However, shelf-lifestability at higher temperatures, exposure to typical unit opera-tions (e.g. heat treatment) and organoleptic properties must beascertained prior to their successful usage in foods.

Acknowledgements

Financial support from the Natural Science and EngineeringResearch Council (NSERC) of Canada, the Advanced Foods andMaterials Network (AFMNet), and Ryerson University isacknowledged.

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