Journal of Colloid and Interface Science 388 (2012) 95–102

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Journal of Colloid and Interface Science

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Low-energy formation of edible nanoemulsions: Factors influencing dropletsize produced by emulsion phase inversion

Felix Ostertag, Jochen Weiss, David Julian McClements ⇑Biopolymers and Colloids Research Laboratory, Department of Food Science, University of Massachusetts, Amherst, MA 01003, USA

a r t i c l e i n f o

Article history:Received 15 June 2012Accepted 31 July 2012Available online 25 August 2012

Keywords:HomogenizationEmulsionsNanoemulsionsLow energy methodsMicrofluidizationPhase inversionEmulsion phase inversionSpontaneous emulsification

0021-9797/$ - see front matter � 2012 Elsevier Inc. Ahttp://dx.doi.org/10.1016/j.jcis.2012.07.089

⇑ Corresponding author. Fax: +1 413 545 1262.E-mail address: mcclements@foodsci.umass.edu (D

a b s t r a c t

Nanoemulsions can be used for the encapsulation and oral delivery of bioactive lipophilic components,such as nutraceuticals and pharmaceuticals. There is growing interest in the utilization of low-energymethods to produce edible nanoemulsions. In this study, we examined the influence of system composi-tion and preparation conditions on the formation of edible nanoemulsions by the emulsion phase inversion(EPI) method. The EPI method involves titrating an aqueous phase (water) into an organic phase(oil + hydrophilic surfactant). The influence of oil type, surfactant type, surfactant-to-oil ratio (SOR),and initial surfactant location on the particle size distributions of the emulsions was studied. The dropletsize produced by this method depended on: (i) oil type: medium chain triglycerides (MCT) < flavor oils(orange and limonene) < long chain triglycerides (olive, grape, sesame, peanut and canola oils); (ii) sur-factant type: Tween 80 < Tween 20 < Tween 85; (iii) surfactant concentration: smaller droplets were pro-duced at higher SOR; (iv) surfactant location: surfactant initially in oil < surfactant initially in water. Thelow energy method (EPI) was also compared to a high energy method (microfluidization). Small droplets(d < 160 nm) could be produced by both methods, but much less surfactant was needed for the highenergy method (SOR P 0.1) than the low energy method (SOR P 0.7).

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1. Introduction

Colloidal delivery systems are widely used in the food and phar-maceutical industries to encapsulate functional lipophilic compo-nents so that they can be dispersed within aqueous media [1–4].The lipophilic components encapsulated include a variety of differ-ent kinds of molecules with different functional attributes, such astriacylglycerols (clouding agents, carrier oils, nutrients, and bioac-tive lipids), citrus oils (flavoring agents), essential oils (antimicro-bials), phytosterols (nutraceuticals), carotenoids (colorants,antioxidants, and nutraceuticals), oil-soluble vitamins (essentialnutrients) and lipophilic drugs. These lipophilic components varyin their molecular and physicochemical properties, such as polari-ties, surface activities, densities, viscosities, melting points, andboiling points. Consequently, different colloidal delivery systemsare often needed for different kinds of lipophilic components andfor different types of food or pharmaceutical matrices.

Three of the most widely used colloidal delivery systems consistof small lipid particles dispersed within an aqueous phase:microemulsions; nanoemulsions; and, emulsions [5,6]. The maindifferences between these three colloidal systems are their ther-modynamic stability and particle dimensions. Microemulsionsare thermodynamically stable dispersions of oil, water and

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.J. McClements).

surfactant (and possibly cosurfactants) that typically contain lipidparticles with radii less than 100 nm. Nanoemulsions (r < 100 nm)and emulsions (r > 100 nm) are both thermodynamically unstabledispersions that can be distinguished according to their dropletsize. There are certain advantages and disadvantages to the com-mercial utilization of each of these colloidal delivery systems.Microemulsions and nanoemulsions contain small particles thatonly scatter light weakly and so they tend to be optically clear oronly slightly turbid. They also tend to have good stability to grav-itational separation and particle aggregation once they have beensuccessfully formulated due to their small particle size. On theother hand, these systems often require relatively large amountsof surfactant to formulate them. Since microemulsion formationis thermodynamically driven, their existence is governed by intrin-sic and extrinsic thermodynamic variables such as temperature,pressure, and concentration. Microemulsions may thereforebecome unstable if these variables change during processing, pack-aging, transport, storage or at the point of sale. Emulsions containrelatively large droplets that scatter light strongly and so they tendto be optically opaque or highly turbid, which is desirable for someapplications and undesirable for others. They are also usually lessstable to gravitational separation and particle aggregation thanmicroemulsions and nanoemulsions. On the other hand, theamount of surfactant needed to form stable emulsions is usuallyconsiderably less than that required to form microemulsions andnanoemulsions.

96 F. Ostertag et al. / Journal of Colloid and Interface Science 388 (2012) 95–102

In the current study we focus on the formation of edible emul-sions and nanoemulsions suitable for application within the foodand beverage industry. Typically, these colloidal systems are fabri-cated using high energy homogenization methods that require spe-cialized mechanical devices capable of generating intensemechanical disruptive forces, such as high shear mixers, high pres-sure homogenizers, colloid mills, sonicators, or microfluidizers [7–9]. However, emulsions and nanoemulsions can also be producedusing low energy approaches that involve the spontaneous forma-tion of droplets under certain system conditions, such as spontane-ous emulsification and phase inversion methods [5,10,11]. Thepurpose of the present study was to investigate the suitability ofthe emulsion phase inversion (EPI) method for preparing food-grade emulsions and nanoemulsions, and to establish the majorfactors governing the size of the droplets produced using thismethod. A number of previous studies have used the EPI methodfor producing emulsions and nanoemulsions [12–14]. These stud-ies have investigated the role of system composition and process-ing conditions on nanoemulsion and emulsion formation.However, only a limited range of surfactants and oils has previ-ously been investigated, and most of these materials are unsuitablefor food applications. More detailed information on the influence ofoil type, surfactant type, surfactant-to-oil ratio, and preparationconditions on the properties of emulsions and nanoemulsions isessential for establishing a more fundamental scientific frameworkfor rationally designing nanoemulsions using low energy methods.The purpose of this study was therefore to systematically examinethe role of these factors on the formation of nanoemulsions by theEPI method, and to compare the effectiveness of this low energymethod with a high energy method under similar system compo-sitions. In particular, we focused on the utilization of food-gradeoils and surfactants to produce edible nanoemulsions suitable fororal ingestion as there is currently little information in this area.

2. Principle of emulsion inversion point (EPI) method

The formation of nanoemulsions using low-energy approachesrelies on the spontaneous formation of fine oil droplets within

Fig. 1. Schematic representation of the protocol used to carry out the emulsion phase invan organic phase (oil + surfactant) that was continuously stirred in a glass beaker using a moil–water system is also shown illustrating transitional and catastrophic phase inversion

surfactant-oil-water (SOW) mixtures when their compositionand/or environment is altered [10,15,16]. Various physicochemicalmechanisms have been proposed to account for spontaneous drop-let formation, with the precise mechanism depending on systemcomposition and preparation method [17]. A number of low energyapproaches used to prepare oil-in-water nanoemulsions rely oninducing a phase inversion from a W/O to a O/W system, e.g., phaseinversion temperature (PIT), phase inversion composition (PIC),and emulsion inversion point (EPI) methods. These approachescan be further categorized according to their underlying physico-chemical principle as either transitional or catastrophic phase inver-sion methods (Fig. 1). Transitional phase inversion relies onchanges of a formulation parameter (such as the physicochemicalcharacteristics of the surfactant, water, or oil phases), whereas cat-astrophic phase inversion relies on changes in a compositionparameter (i.e., the water-to-oil ratio). In this study, we focusedon the formation of nanoemulsions using the emulsion phaseinversion (EPI) method, which is based on a catastrophic phaseinversion that occurs when water is titrated into a system contain-ing a mixture of oil and a hydrophilic surfactant [11,18]. Previousstudies have shown that a SOW system passes through a numberof different structures when increasing amounts of water are ti-trated into a surfactant–oil–water mixture [12,19]. Initially, a W/O emulsion is formed, then an O/W/O multiple emulsion, and thenan O/W emulsion (Fig. 1). The origin of the structural changesoccurring during catastrophic phase transitions has been relatedto the balance of droplet breakup and coalescence in the system,and the droplet size produced to the formation of the intermediatemultiple emulsion [12,19]. The value of the critical water concen-tration where phase inversion occurs, as well as the size of theoil droplets produced, has been reported to depend on processvariables, such as stirring speed, rate of water addition, and surfac-tant concentration [14,18].

The phase inversion behavior of surfactant–oil–water (SOW)mixtures can be described by formulation–composition maps suchas the one shown schematically in Fig. 1 [13,20]. The x-axis repre-sents changes in the ‘‘composition’’ of the system, which is ex-pressed as the water-to-oil ratio (WOR). The y-axis represents

ersion method. An aqueous phase (buffer solution) was titrated from a burette intoagnetic stirrer (750 rpm). A typical formulation–composition map for a surfactant–

s.

Table 1Physical properties of oils used to prepare emulsions by the EPI method, and meanparticle diameters of emulsions produced using Tween 80 and MCT (SOR = 2.5). Thephysicochemical properties were measured at ambient temperature (�25 �C). Thecorrelation coefficients (r2) were calculated from plots of the mean particle diameterversus the physicochemical property of interest.

Oil type Density(kg m�3)

Viscosity(mPa s)

Interfacial tension(mN m�1)

d32 (nm)

Canola oil 917 ± 3 73.7 ± 0.1 50.36 ± 0.38 1.55 ± 0.01Grape seed

oil921 ± 1 69.7 ± 1.0 28.30 ± 0.30 1.13 ± 0.02

Limonene 843 ± 2 0.81 ± 0.02 39.31 ± 0.35 0.34 ± 0.03MCT 946 ± 5 25.5 ± 0.1 65.83 ± 0.06 0.14 ± 0.04Mineral oil 882 ± 4 152.2 ± 2.0 63.91 ± 0.13 1.00 ± 0.03Olive oil 916 ± 1 78.6 ± 1.0 32.81 ± 0.13 0.64 ± 0.01Orange oil 845 ± 2 0.79 ± 0.01 14.28 ± 0.35 0.26 ± 0.01Peanut oil 914 ± 4 62.9 ± 1.0 35.75 ± 0.67 1.48 ± 0.01Sesame oil 920 ± 3 63.8 ± 0.1 12.78 ± 0.27 1.17 ± 0.04

Correlation(r2)

0.135 0.339 0.004 –

F. Ostertag et al. / Journal of Colloid and Interface Science 388 (2012) 95–102 97

changes in the ‘‘formulation’’ of the system, which can be ex-pressed as the hydrophilic–lipophilic deviation (HLD). The HLD isa measure of the relative affinity of the surfactant for the hydro-philic (water) phase and the lipophilic (oil) phase [21,22]. It is adimensionless parameter that characterizes the behavior of a sur-factant within a specific SOW system, and depends on surfactanttype, oil type, and aqueous phase properties (such as pH, ionicstrength, and cosolvent) [23]. For HLD < 0 a surfactant has: (i) ahigher affinity for water than oil; (ii) forms micelles in water;(iii) stabilizes O/W emulsions. For HLD = 0 a surfactant has: (i) anequal affinity for water and oil; (ii) forms bicontinuous microemul-sions or liquid crystalline phases; (iii) stabilizes neither O/W norW/O emulsions. For HLD > 0 a surfactant has: (i) a higher affinityfor oil than water; (ii) forms reverse micelles in oil; (iii) stabilizesW/O emulsions. Knowledge of the HLD number, water-to-oil ratio(WOR), and formulation–composition map for a particular systemcan be used to rationalize its behavior [23]. This conceptual ap-proach will be used to interpret our experiments on the influenceof various parameters on formation of nanoemulsions using the EPImethod.

Table 2Properties of the surfactants used to prepare emulsions by the EPI method, and meanparticle diameters of emulsions produced using MCT (SOR = 2.5). The values withasterisks were calculated as a weighted average.

Non-ionicsurfactant

Chemical structure Molecularweight (g/mol)

HLBnumber

d32 (lm)

Tween� 20 Polyoxyethylen-20-sorbitan-monolaurate

1228 16.7 0.93 ± 0.15

Tween� 80 Polyoxyethylen-20-sorbitan-monooleate

1310 15 0.14 ± 0.04

Tween� 85 Polyoxyethylen-20-sorbitan-trioleate

1836 11 1.71 ± 0.62

Mixture 1:1:1 T20:T80:T85 1458� 14.2� 0.12 ± 0.00

3. Materials and methods

3.1. Materials

A variety of oil phases were used to prepare the emulsions (Ta-ble 1): medium chain triglycerides, (MCT, Miglyol 812, WarnerGraham, Cockeysville, MD); limonene (L-2129, Sigma–Aldrich, St.Louis, MO); olive oil (O-1514, Sigma–Aldrich); mineral oil (Sig-ma–Aldrich), sesame oil (Baycliff Company, New York, NY); grapeseed oil (AarhusKarlshamm Ltd., Hull, UK); peanut oil (FoodholdUSA, Landover, MD); canola oil (Foodhold, USA); and, orange oil(SC020295, International Fragrances and Flavors, New York, NY).A number of non-ionic surfactants were used to stabilize the emul-sions (Table 2), including Tween� 20, 80 and 85 (Sigma–Aldrich, St.Louis, MO). Tween surfactants consist of a polyoxyethylene headand a fatty acid tail of various lengths with the two moieties beinglinked together via a sorbitol. The aqueous phase used to preparethe emulsions was always sodium phosphate buffer solution(5 mM; pH 7.0). Distilled and deionized water was used to prepareall solutions and emulsions.

3.2. Methods

3.2.1. Emulsion preparation3.2.1.1. Low energy method. Emulsions were prepared usingtheemulsion phase inversion(EPI) method, which involves titratingan aqueous phase into an organic phase with constant stirring.The experiments were performed in a 125 ml beaker at ambienttemperature (�25 �C). The experiments were designed so thatthe final emulsion always had a total mass of 50 g including 5 gof oil (i.e., 10 wt% oil). Preliminary experiments showed that the fi-nal amounts of aqueous phase titrated into the beaker were suffi-cient to induce the water-in-oil to oil-in-water phase inversion inall samples studied. Initially, an organic phase was prepared byadding the surfactant and oil to the beaker and then mixing usinga magnetic stirrer (750 rpm) for 30 min. Aqueous phase was thenadded into the organic phase using a burette with a flow rate of�4 mL/min while continuing to stir the system with the magneticstirrer (750 rpm) for 60 min. Emulsions with different surfactant-to-oil ratios (SOR) were prepared by varying the amount of surfac-tant and water in the system. For example, to prepare a systemwith SOR = 2, 10 g of surfactant and 5 g of oil were mixed together,placed into the beaker, and then 35 g of aqueous phase was titratedin.

For some experiments we examined the influence of the initiallocation of the surfactant (organic versus aqueous phase) on thesize of the droplets produced. In this case, the surfactant was partlyadded to the aqueous phase and partly to the organic phase. Thesurfactant and buffer solution were mixed with a magnetic stirrer(750 rpm) for 30 min prior to adding them to the organic phase.

3.2.1.2. High Energy Method. Oil, water and surfactant were mixedtogether in a beaker, and then blended together using a high-shearmixer (Bamix, Biospec Products, Bartlesville, OK) for 2 min to forma coarse emulsion. The emulsion premix samples were then passedthrough a microfluidizer (M-110L, Microfluidics, Newton, MA)three times at 12,000 psi at ambient temperature using a specificinteraction chamber (F20Y 75 lm, Microfluidics, Newton, MA).

3.2.2. Particle size measurementThe particle size distributions of the emulsions were measured

using a static light scattering instrument (Mastersizer 2000, Mal-vern Instruments Ltd, Malvern, Worcestershire, UK). Mean particlediameters calculated from this data are reported as the surface-weighted mean diameter (d32). A refractive index ratio of 1.08was used in the calculations of the particle size distributions fromthe light scattering patterns.

3.2.3. Interfacial tension and density measurementsThe interfacial tension at the oil–water (buffer solution) inter-

face was measured using droplet shape analysis device (DSA 100,Krüss GmbH, Hamburg, Germany). For oil droplet formation ahook–needle with a diameter of 1.463 mm was used. The density

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Fig. 3. Mean particle diameters (d32) of 10 wt% oil-in-water emulsions withdifferent surfactant-to-oil ratios (SOR) produced by the EPI method.

98 F. Ostertag et al. / Journal of Colloid and Interface Science 388 (2012) 95–102

of the liquids was measured using a digital density meter (Mettler-Toledo Inc., Columbus, OH). All measurements were performed atroom temperature (22 ± 0.5 �C).

3.2.4. Experimental designAll experiments were repeated at least twice with measure-

ments being triplicated. Mean and standard deviations were calcu-lated from this data.

4. Results and discussion

4.1. Influence of SOR on particle size

Initially, we examined the influence of the surfactant-to-oil ra-tio (SOR) on the particle size distribution (Fig. 2) and mean particlediameter (Fig. 3) of emulsions produced using MCT as the oil andTween 80 as the surfactant. At low surfactant concentrations(SOR from 0.1 to 0.65) the droplets formed were relatively large(d32 from 0.5 to 50 lm), but at higher values (SOR from 0.65 to2.5) emulsions were produced with relatively small droplets (d32

from 0.16 to 0.10 lm). There was a slight decrease in mean dropletsize when the SOR was increased from 0.65 to 2.5 (Fig. 3). Visually,the emulsions appeared less turbid as the SOR increased, which isindicative of the formation of smaller droplets that scatter lightless strongly [24]. A decrease in droplet size with increasing SORhas also been reported for the formation of emulsions using thespontaneous emulsification method for a similar system, i.e.,MCT and Tween 80 [25,26].

In our study, O/W emulsions were formed by titrating waterinto a mixture containing a hydrophilic non-ionic surfactant(Tween 80) and oil. The origin of droplet formation can thereforebe attributed primarily to catastrophic phase inversion broughtabout by changing the water-to-oil ratio (WOR), rather than tran-sitional phase inversion brought about by changing the HLD num-ber of the surfactant (Fig. 1). Recently, it has been suggested thatthe creation of multiple emulsions (O/W/O) during the intermedi-ate stages of the titration process is a prerequisite for the formationof fine oil droplets within the final O/W emulsion [12,19]. The for-mation of these multiple emulsions requires that the hydrophilicsurfactant is initially located in the oil phase. At relatively low sur-

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Fig. 2. Particle size distributions of 10 wt% oil-in-water emulsions with differentsurfactant-to-oil ratios (SOR) produced by the EPI method.

factant concentrations the formation of multiple emulsions is sup-pressed and only relatively large oil droplets are produced in thefinal emulsion, which are similar in size to those that would beproduced if the surfactant had been dissolved in the water phaseprior to homogenization [19]. At relatively high surfactantconcentrations, multiple emulsions are formed during the titrationprocess, and the final oil droplet size within the O/W emulsions isdetermined by the size of the inner oil droplets in the intermediateO/W/O emulsions. This phenomenon would therefore account forthe reduction in droplet size with increasing surfactant concentra-tion observed in the current study.

The key to the formation of nanoemulsions using the EPI meth-od therefore appears to be inducing the formation of intermediateO/W/O emulsions that have small inner oil droplets. These smallinner oil droplets are likely to be formed through a spontaneousemulsification process that occurs when an organic phase (oil plushydrophilic surfactant) is brought into contact with an aqueousphase (water) [10,27]. Fernandez and co-workers proposed thatoil droplets were formed spontaneously when water was addedto a surfactant/oil mixture due to a process that involved the con-version of a W/O microemulsion to a bicontinuous system to an O/W emulsion [11]. Mercuri and co-workers proposed that waterpenetrates into the organic phase causing it to swell, which leadsto the formation of W/O microemulsions and then liquid crystal-line phases at the boundary [28]. Fragments of this liquid crystal-line phase then break off from the boundary and move into theaqueous phase, where they dissociate into ultrafine oil droplets.Anton and co-workers proposed that oil droplet formation oc-curred as a result of the movement of surfactant molecules fromthe organic phase into the aqueous phase after the two phaseswere brought into contact [10]. A number of different physico-chemical mechanisms may therefore account for the formation offine oil droplets using the EPI method, and further work is clearlyneeded to provide a more fundamental scientific understandingof the processes involved.

4.2. Influence of oil type on particle size

A variety of different oils may be used to form nanoemulsions inthe food and beverage industries, and therefore we examined theinfluence of oil type on the size of the droplets produced usingthe emulsion phase inversion method. A series of emulsions wasprepared with similar overall compositions (10% oil, 25% surfactant(Tween 80), 65% buffer solution: SOR = 2.5), but using different oils.There were appreciable differences in the size of the droplets that

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Olive

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Canola

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Fig. 4. Particle size distributions of 10 wt% oil-in-water emulsions with constantsurfactant-to-oil ratios (SOR = 2.5) produced by the EPI method using differenttypes of oil. The surfactant used was Tween 80.

F. Ostertag et al. / Journal of Colloid and Interface Science 388 (2012) 95–102 99

could be produced by the EPI method depending on oil type (Figs. 4and 5). We found that the smallest droplets could be producedusing medium chain triglycerides (MCT), and then using flavor oils(orange oil and limonene). Only relatively large droplets(d32 > 0.6 lm) could be formed using mineral oil or long chain tri-glyceride oils (such as olive, grape seed, sesame, peanut and canolaoils).

Initially, we hypothesized that the size of the droplets producedusing the EPI method would depend on the bulk physicochemicalproperties of the oil phase, such as density, viscosity, or interfacialtension. For example, the viscosity of the oil phase may influencethe mass transport rate of surfactant molecules through the oiland into the aqueous phase [29] or droplet disruption within a flowfield [14], whereas the interfacial tension may influence the mobility

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Fig. 5. Mean particle diameters (d32) of 10 wt% oil-in-water emulsions withconstant surfactant-to-oil ratios (SOR = 2.5) produced by the EPI method usingdifferent types of oil. The surfactant used was Tween 80.

of the oil–water interface [29,30] or droplet disruption [14]. Wetherefore characterized the physicochemical properties of the vari-ous oils used in this study (Table 1). Interestingly, we found thatthere was no correlation (r2 < 0.35) between the mean particlediameter produced and the bulk physicochemical properties of theoils. This result suggests that it is not possible to predict the size ofthe droplets formed by the EPI method simply from knowledge ofthe bulk physicochemical properties of the original oil phase. Hence,there must be other factors that are responsible for the influence ofoil type on the size of the droplets produced using this method.

The influence of oil type on droplet size may be partly inter-preted in terms of the formulation–composition map shown sche-matically in Fig. 1. A change in the nature of the oil phase would beexpected to change the HLD number of the surfactant, i.e., its rela-tive affinity for the oil and water phases [13,31]. In turn, a changein the surfactant HLD number would be expected to alter variousphysicochemical properties that impact nanoemulsion formation:the distribution of surfactant molecules between the aqueousand organic phases; the spontaneous formation of fine oil dropletsat the aqueous-organic phase boundary; the coalescence stabilityof the droplets. For example, as the HLD number moves towardszero, there is usually a decrease in oil–water interfacial tensionin a SOW system, which facilitates the spontaneous formation offine oil droplets [29,31,32]. On the other hand, as the HLD numbertends towards zero, the rate of droplet coalescence in SOW systemsusually increases, which would cause any fine droplets formed torapidly grow in size [10,30]. In future studies, it would be usefulto systematically characterize the relationship between the HLDnumbers of different oils and their ability to form fine dropletsusing the EPI method.

4.3. Influence of surfactant type on particle size

A range of different surfactants are available for the formulationof nanoemulsions in the food and beverage industries [33]. Wetherefore examined the influence of surfactant type on the size ofthe droplets produced by the EPI method. In this study, we re-stricted ourselves to a range of food-grade non-ionic surfactants(Tweens) since this type of surfactant is usually considered mostsuitable for the formation of emulsions by low energy methods.These surfactants primarily differ in the nature of the hydrophobictail group (Table 2): Tween 20 has a monolaurate tail (C12:0);Tween 80 has a monooleate tail (C18:1); and Tween 85 has a triool-eate tail (3 � C18:1). A series of emulsions was prepared with sim-ilar overall compositions (10% oil (MCT), 25% surfactant, 65% buffersolution: SOR = 2.5), but using the three individual different surfac-tants (Tween 20, 80 and 85) or a mixed surfactant system (1=3

Tween 20, 1=3 Tween 80, and 1=3 Tween 85).The nature of the surfactant(s) used to form the emulsions

clearly had a major impact on the size of the droplets producedusing the emulsion phase inversion method (Figs. 6 and 7). Thesmallest droplets were produced using the mixed surfactant system(d32 � 0.12 lm), but fine droplets could also be produced usingTween 80 alone (d32 � 0.14 lm). On the other hand, relatively largedroplets were produced when either Tween 20 (d32 � 0.93 lm) orTween 85 (d32 � 1.7 lm) were used as the surfactant. The smallestdroplets appeared to be produced by the surfactants that had inter-mediate HLB numbers (HLB � 15). The fact that the largest dropletswere produced by Tween 85 may have been because this was thelargest and the least hydrophilic of the three surfactants studied.Changes in the molecular characteristics of a surfactant lead tochanges in its HLD number, and therefore the overall phase diagramof the SOW system (Fig. 1). As discussed in Section 4.2, changes insurfactant HLD number would be expected to alter a number ofphysicochemical properties that may influence the formation ofultrafine droplets: the initial distribution of the surfactants

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Fig. 6. Particle size distributions of 10 wt% oil-in-water emulsions with constantsurfactant-to-oil ratios (SOR = 2.5) produced by the EPI method using differenttypes of surfactant. The oil used was MCT.

100 F. Ostertag et al. / Journal of Colloid and Interface Science 388 (2012) 95–102

between the aqueous and organic phases [10]; the phase behaviorof the SOW systems at the aqueous/organic phase boundary [32];the coalescence stability of the droplets in the SOW systems [30].In addition, the kinetics of surfactant movement from the oil tothe aqueous phases would also be expected to decrease as themolecular weight of the surfactant increased, which could alterthe spontaneous formation of oil droplets at the aqueous/organicphase boundary. Overall, differences in the HLD number of the sur-factants may have altered the tendency for O/W/O emulsions to beformed during the titration process, which has been shown to be acritical step in the creation of fine oil droplets using the EPI method[19].

50

4.4. Influence of initial surfactant location on the particle size

It has been suggested that the movement of the surfactant mol-ecules from the oil to the aqueous phase is one of the most impor-tant factors determining the spontaneous formation of oil dropletsusing the EPI method [10,11]. We therefore examined the influence

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Fig. 7. Mean particle diameters (d32) of 10 wt% oil-in-water emulsions withconstant surfactant-to-oil ratios (SOR = 2.5) produced by the EPI method usingdifferent types of surfactant. The oil used was MCT.

of the initial location of the surfactant molecules in the system onthe size of the droplets produced: organic phase versus aqueousphase. A series of emulsions was prepared with similar final compo-sitions (10% oil (MCT), 25% surfactant (Tween 80), 65% buffer solu-tion: SOR = 2.5), but initially having different percentages of thesurfactant in the oil phase and the remainder in the aqueous phase.

Our results clearly show that the initial location of the surfac-tant has a major impact on the particle size distribution and meanparticle diameter (Figs. 8 and 9). The higher the fraction of the sur-factant that was initially present in the organic phase, the smallerthe droplets produced. These results therefore support the hypoth-esis that the movement of the surfactant molecules from the oil tothe aqueous phase plays an important role in the spontaneous for-mation of ultrafine oil droplets at the oil–water boundary. Interest-ingly, the formulation–composition map of a SOW system does nottake into account the initial location of the surfactant in the system(Fig. 1), and therefore cannot be used to predict the relationshipbetween droplet size and surfactant location. Our results supportthe findings of a study using a non-food grade SOW system (poly-oxyethylene nonylphenylether, cyclohexane and water) [19].These authors also found that finer droplets were formed afterwater was titrated into a surfactant–oil mixture when the hydro-philic non-ionic surfactant was initially located in the organicphase, rather than in the aqueous phase. Based on optical micros-copy measurements they suggested that having the surfactant ini-tially in the oil phase promoted the formation of O/W/O emulsionsduring the titration process, which is critical to producing fine oildroplets in the final O/W system.

4.5. Comparison of low-energy and high-energy homogenizationmethods

Our previous results show that emulsions containing very fineoil droplets (d < 0.16 lm) can be produced using the low-energyEPI method. In this section, we compared this low-energy methodwith a high-energy method that is known to be highly effective atproducing ultrafine droplets: microfluidization [34–36]. We com-pared the size of the droplets produced using the two methodsfor systems with different surfactant-to-oil ratios (Figs. 10 and11). At relatively high surfactant levels (SOR > 0.7), both methods

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Particle Diameter (µµm)

100%

75%

50%

25%

0%

Fig. 8. Particle size distributions of 10 wt% oil-in-water emulsions with constantsurfactant-to-oil ratios (SOR = 2.0) produced by the EPI method using MCT as the oiland Tween 80 as the surfactant. The percentage of the Tween 80 initially in theaqueous phase was varied from 0% to 100% (see caption).

0.11 0.110.13

1.74

28.0

0.1

1

10

0 25 50 75 100

Par

ticl

e D

iam

eter

(µ

m)

% Initial Surfactant in Aqueous Phase

Fig. 9. Mean particle diameters (d32) of 10 wt% oil-in-water emulsions withconstant surfactant-to-oil ratios (SOR = 2.0) produced by the EPI method usingMCT as the oil and Tween 80 as the surfactant. The percentage of the Tween 80initially in the aqueous phase was varied from 0% to 100% (see caption).

0

10

20

30

40

50

0.01 0.1 1 10 100 1000

Rel

ativ

e V

olum

e (%

)

Particle Diameter (µµm)

2.5

1.0

0.5

0.2

0.1

SOR

Fig. 10. Particle size distributions of 10 wt% oil-in-water emulsions with differentsurfactant-to-oil ratios (SOR) produced by the microfluidization method.

0.05

0.5

5

50

0.0 0.5 1.0 1.5 2.0 2.5

Par

ticl

e D

iam

eter

(µ

m)

SOR

Low Energy

High Energy

Fig. 11. Mean particle diameters (d32) of 10 wt% oil-in-water emulsions withdifferent surfactant-to-oil ratios (SOR) produced by the low energy (EPI) and highenergy (microfluidization) methods.

F. Ostertag et al. / Journal of Colloid and Interface Science 388 (2012) 95–102 101

could produce ultrafine droplets (d < 0.16 lm). Interestingly, thesize of the droplets produced by the two different methods wasfairly similar in this high surfactant concentration range (Fig. 11).This result suggests that the low energy method is suitable for pro-ducing nanoemulsions without the need of sophisticated or expen-sive manufacturing equipment, provided sufficiently highsurfactant levels are used. On the other hand, the microfluidizerwas capable of producing ultrafine droplets (d < 0.12 lm) even atrelatively low surfactant levels (SOR = 0.1). A major advantage ofthe high energy method is therefore the fact that much lesssurfactant is needed to form a nanoemulsion, which has a numberof economic and practical benefits: lower ingredients costs; re-duced off-flavors associated with surfactants; and less problemswith foaming.

5. Conclusions

Most previous studies using the emulsion phase inversion (EPI)method to create nanoemulsions have focused on materials unsuit-able for food-grade applications [12,13,37,38]. In this study, wehave provided important new information on the influence offood-grade surfactants and oils on the formation and propertiesof edible nanoemulsions using the EPI method. We have shownthat nanoemulsions (d < 200 nm) can be produced by simply mix-ing food grade ingredients (oil, water, and surfactant) together. Thesize of the droplets produced using the EPI method depended on anumber of factors, including oil type, surfactant type, surfactant-to-oil ratio, and initial surfactant location. We did not find a simplerelationship between the bulk physicochemical properties of oils(such as their density, interfacial tension and viscosity) and thesize of the droplets produced using the EPI method. Instead, wefound that a particular surfactant–oil–water (SOW) system hadto be optimized to produce fine droplets using low surfactantconcentrations.

Our results were interpreted in terms of the structural changesreported to occur when water is titrated into an organic phase con-taining a hydrophilic surfactant and oil: W/O to O/W/O to O/W[19]. Previous studies have highlighted that the formation of theintermediate O/W/O emulsion is critical for forming nanoemul-sions using the EPI method [19]. We therefore postulated thatchanges in the nature of the surfactants and oils used in our studyaltered the hydrophilic–lipophilic deviation (HLD) number of thesurfactants, which in turn altered physicochemical phenomenonassociated with the spontaneous formation of fine oil dropletswithin the O/W/O emulsions.

It should be noted that a major disadvantage of the EPI methodwas the need to use relatively high amounts of surfactant to pro-duce small oil droplets, e.g., SOR > 0.7 to get d < 160 nm. This isin contrast to high energy methods that can produce small dropletsat much lower surfactant levels, e.g., d < 120 nm at SOR = 0.1 usingthe microfluidization method employed in this study. This findingis in agreement with another recent study that showed that muchhigher surfactant levels were required to produce nanoemulsionsusing a low energy method (spontaneous emulsification) com-pared to a high energy method (microfluidization) [39]. The largedifference in SOR needed to form fine droplets amongst homogeni-zation approaches can be attributed to differences in the dropletformation mechanisms in low and energy methods. On the otherhand, an advantage of the low energy methods is that expensivespecialized equipment (such as high pressure homogenizers) isnot required to form nanoemulsions. These results may haveimportant implications for the utilization of low energy methodsto form colloidal delivery systems for lipophilic nutraceuticals inthe food and beverage industries.

102 F. Ostertag et al. / Journal of Colloid and Interface Science 388 (2012) 95–102

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