04 colsua shima

Colloids and Surfaces A: Physicochem. Eng. Aspects 238 (2004) 83–90

Effect of the hydrophilic surfactants on the preparation and encapsulationefficiency in course and fine W/O/W type emulsions

Motohiro Shima∗, Yohei Kobayashi, Yukitaka Kimura, Shuji Adachi, Ryuichi Matsuno

Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan

Received 2 July 2003; accepted 26 February 2004

Available online 21 April 2004

Abstract

Although the surfactant plays an important role in the preparation of a water-in-oil-in-water (W/O/W) emulsion, its concentration hasbeen determined empirically. We investigated the location of a hydrophilic surfactant in a coarse W/O/W emulsion and a membrane-filteredfine emulsion. At first, appropriate concentrations of some hydrophilic surfactants to prepare a W/O/W emulsion were investigated from theviewpoints of the median diameter of the oil droplets and the encapsulation efficiency of a hydrophilic substance in the inner-water phase.The location of the surfactants and oil was examined for the W/O/W emulsions, prepared using polyglycerol monolaurate as a hydrophilicsurfactant in the outer-phase solution at 1–10% (w/v) and octanoic acid triacylglycerol, which contained hexaglyceryl condensed ricinoleate asan oil-phase surfactant in the oil phase. The median diameter of the oil droplets in the W/O/W emulsion reached a minimum value at 3% (w/v)decaglycerol monolaurate (ML-750), and was constant over 4% (w/v). The excess decaglycerol monolaurate, that was not adsorbed on theoil-water interface was distributed to the outer-water and oil phases. The oil and hydrophobic surfactant were dissolved in the outer-phasesolution at high concentrations of the hydrophilic surfactant. These results suggest that the hydrophilic-surfactant concentration appropriatefor preparing the W/O/W emulsion would be 3% (w/v) in the outer-phase solution and that the location of the components in the W/O/Wemulsion would also be useful in estimating the adequate concentration of the surfactant.© 2004 Elsevier B.V. All rights reserved.

Keywords:W/O/W emulsion; Surfactant; Drug delivery system; Functional food; Encapsulation efficiency

1. Introduction

Practical application of bioactive substances or phar-maceuticals to foods or medication requires an efficientabsorption system or a controlled-release system[1]. Awater-in-oil-in-water (W/O/W) emulsion, which is an emul-sion of oil droplets in which water droplets are included,is a candidate for a protective carrier or controlled-releasecontainer for these materials[2,3].

A W/O/W emulsion usually contains hydrophobic andhydrophilic surfactants in the oil and outer-water phases,respectively. A nonionic surfactant with a high HLB value isusually recommended to prevent leakage of the inner phasesolution and to stabilize the oil droplets. A commonly usedhydrophilic surfactant, such as Tween 20 (polyoxyethylene

∗ Corresponding author. Tel.:+81-75-753-6288;fax: +81-75-753-6285.

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

(20) sorbitan monolaurate) or a polyglycerol ester, consistsof a polymer as the hydrophilic component and a fatty acidas the hydrophobic one.

Because a commercially available polyglycerol is char-acterized by the hydroxyl value, it is a mixture of glycerolpolymers with various degrees of polymerization. The hy-drophilic surfactant, decaglycerol monolaurate (ML-750),used in this study is also a mixture of polyglycerols boundwith lauric acid. The surfactant is called decaglycerol mono-laurate because the molar ratio of the glycerol residue tolauric acid is about 10. The mixture itself is not always un-suitable for the preparation of emulsions, but it is reportedthat a mixture of some surfactants exhibits a stabilizing ef-fect, for example, as a co-surfactant, in the preparation ofthe microemulsion[4]. Such an effect is not obtained by apurified surfactant.

The hydrophilic and hydrophobic surfactants are thoughtto be adsorbed on the surface of the oil droplet and at theinterface of the inner-water droplet, respectively. However,

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84 M. Shima et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 238 (2004) 83–90

less attention has been paid to the real location of the sur-factants in the W/O/W emulsion. Although many reportshave been published on the preparation of the W/O/Wemulsion, the relationship between the distribution of sur-factants used in the preparation and the stability of theW/O/W emulsion has not been investigated. Therefore, wedetermined the concentrations of the surfactants in eachphase using TLC-FID to discuss the location of the surfac-tants in the W/O/W emulsion. The stability of the W/O/Wemulsion prepared at low surfactant concentrations was alsoexamined.

2. Materials and methods

2.1. Materials

Decaglycerol monolaurate (SY-Glyster® ML-750) wassupplied by Sakamoto Yakuhin Kogyo (Osaka, Japan).Hexaglyceryl condensed ricinoleate (Sunsoft® 818SX),pentaglycerol monolaurate (Sunsoft® A-12E), pentaglyc-erol monomyristate (Sunsoft® A-14E), decaglycerol mono-myristate (Sunsoft® Q-14S), and lysolecithin (Sunlecithin®

A) were gifts from Taiyo Kagaku (Yokkaichi, Japan). Oc-tanoic acid triacylglycerol (abbreviated C8TG) was pur-chased from Sigma (St. Louis, MO, USA). A fluorescentmarker, 1,3,6,8-pyrenetetrasulfonic acid tetrasodium salt(PTSA, Mw 610.42,[5]), was purchased from Molecu-lar Probes (Eugene, OR, USA). Decaglycerol monolau-rate and decaglycerol monomyristate were prepared usingmixtures of linear (mainly) and cyclic polyglycerols withdifferent degrees of polymerization. “Decaglycerol” for thesurfactants was designated based on the hydroxyl valuesof the polyglycerols used in their preparation. Pentaglyc-erol monolaurate and pentaglycerol monomyristate weremanufactured using polyglycerols that contained ca. 60%pentaglycerol as a major component.

Disposable membrane filter cartridges, DISMIC 25CS0-20AN, 25CS045AN and 25CS080AN, which held cellu-lose acetate membranes with pore diameters of 0.2, 0.45and 0.8�m, respectively, were purchased from AdvantecToyo (Tokyo, Japan). A disposable syringe with a capacityof 30 ml (SS-30ES) was purchased from Terumo (Tokyo,Japan).

Table 1HLB and surface excess of the hydrophilic surfactants

Trade name Compound HLBa Surface excessb

(×10−8 g/cm2)

Sunsoft A-12E Pentaglycerol monolaurate 15.6 7.80Sunsoft A-14E Pentaglycerol monomyristate 15.0 4.60SY-Glyster ML-750 Decaglycerol monolaurate 15 17.0Sunsoft Q-14S Decaglycerol monomyristate 14.5 13.4Sunlecithin A Lysolecithin 12 8.85

a The values were provided by the manufacturers.b Surface excess was determined for the interface between Hank’s solution without Ca2+ and Mg2+, and C8TG.

2.2. Preparation of W/O/W emulsion

Selection of a purified water as an outer-phase solutionleads to the increased applicable hydrophilic surfactants, butthe components of the outer-phase solution in the applica-tion fields are some kinds of solution, in many cases, a saltsolution. The preparation profile of W/O/W emulsion usinga physiological saline as an outer-phase solution would notfollow a simple extrapolation from the result of the investiga-tion using purified water as the outer-phase solution, becausesome of the hydrophilic surfactants act differently accordingto the ionic strength in the solution. Furthermore, we alsoplanned for the investigation of the transport enhancementeffect of the W/O/W emulsion using intestinal epithelial cellculture. The cells under investigation were usually main-tained in the physiological saline to prevent the effect of anosmotic pressure. Therefore, we used Hank’s solution as anouter-phase solution in consideration of its application.

Hank’s solution was a physiological saline and wasused as the outer-water phase solution. The solution in-cluded KCl 0.4 g/l, KH2PO4 0.06 g/l, NaCl 8 g/l, NaHCO30.35 g/ l, Na2HPO4 0.0475 g/l, glucose 1 g/l, penicillin G1 × 105 units/l, streptomycin 0.1 g titer/l, and HEPES5.9578 g/l, and its pH was adjusted to 7.3 using a 4N NaOHsolution.

Decaglycerol monolaurate inTable 1 was mainly usedas the hydrophilic surfactant. C8TG containing 10% (w/v)hexaglyceryl condensed ricinoleate was used as the oil-phasesolution. Distilled water containing 10−4 M PTSA was usedas the inner-water solution. A coarse W/O/W emulsionwas prepared through a two-step homogenization usinga rotor/stator homogenizer (Physcotron, Microtec, Tokyo,Japan) at 2.2 × 104 rpm for 2 min in the first step and at1.0 × 104 rpm for 1 min in the second step. A disposablemembrane filter cartridge was connected to a disposablesyringe, and the coarse W/O/W emulsion was loaded in thesyringe [6]. The syringe was then sealed and pressurizedby nitrogen gas to filter the coarse W/O/W emulsion toproduce a fine W/O/W emulsion.

2.3. Median diameter of oil droplets

The particle-size distribution of oil droplets in the W/O/Wemulsion was measured with a laser particle-size analyzer

M. Shima et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 238 (2004) 83–90 85

(SALD-2100, Shimadzu) using 0.1% (w/v) sodium dodecylsulfate solution as a dispersion medium. The median diam-eter was evaluated from the volume-based distribution.

2.4. Encapsulation efficiency of a marker substance

The encapsulation efficiency of PTSA in the inner-waterphase of a W/O/W emulsion was estimated according toour previous method[6]. The efficiency in percentage wasdefined by(m0 − mout)/m0 × 100, wherem0 is the amountof the marker loaded into the inner-water phase andmoutis the amount of the marker leaked into the outer-waterphase. The PTSA concentration was determined with aShimadzu RF-1500 spectrofluorometer (Kyoto, Japan) atexcitation and emission wavelengths of 374 and 404 nm,respectively.

2.5. Measurement of interface tension

Interface tension was measured by an automatic surfacetension meter (CBVP-A3, Kyowa Interface Science, Tokyo),which employed Wilhelmy method[7]. The instrument usesa clean platinum plate hanged from a precision balance. Theplate is touched on the interface or surface of a target solutionand the edge of the meniscus of the target solution at theplate pull it down. The power of the gravity plus interfaceor surface tension is measured by the balance, and the effectof gravity is excluded by initial zero setting.

To measure an interface tension on the oil droplet in theW/O/W emulsion, the outer-water phase solution was care-fully loaded in a clean sample bottle to avoid babble on thesurface, and the oil-phase solution was also carefully loadedon the solution. The platinum plate was immersed in theoil-phase and the buoyant force to the plate in the oil phasewas avoided by the setting indicated by the manufacturer.The plate was contacted at the interface of the two solu-tions. The sample solutions were maintained at 25◦C us-ing water jacket for 1 h and then the interface tension wasmeasured.

2.6. Measurement of osmotic pressure

Osmotic pressures in the inner- and outer-water phasesolutions were measured by an osmometer (OM-802, Vogel,Germany).

2.7. Estimation of outer-phase solution volume

Leakage of the inner-water phase to the outer-water phaseor incorporation of the outer-water phase into the oil phaseresults in a change in the volume of the outer-water phase.Because an exact volume of the outer-water phase was re-quired to estimate the location of the hydrophilic surfactant,it was determined as given further.

A coarse W/O/W emulsion including PTSA in theinner-water phase was prepared. Aliquots of the emulsion

(800�l) were mixed with 400�l of 2 × 10−6 M PTSAdissolved in Hank’s solution. The mixture is called a sam-ple solution. Aliquots of the emulsion (800�l) were alsomixed with 400�l of the Hank’s solution not containingPTSA, and the mixture is designated a reference solutionA. Hank’s solution (800�l) was mixed with 400�l of2 × 10−6 M PTSA in the Hank’s solution. The mixture iscalled reference solution B, which was used for assess-ment of the handling and apparatus derived errors. Thesample solution, reference solutions A and B were gentlyshaken and stored at 4◦C overnight. The oil droplets in thesample solution and reference solution A floated to formcream layers. The outer-water phases of the sample solu-tion and reference solution A were separately withdrawnby a syringe with a needle and were filtered through a dis-posable filter cartridge equipped with a filter of a pore sizeof 0.1�m (Millex-VV (SLVVR25LS), Millipore, Bedford,USA). The filtered solutions (400�l) and 400�l of thereference solution B were separately diluted by an additionof 3600�l of distilled water. The PTSA concentrations inthe sample and reference A and B solutions,CS, CR1 andCR2, were determined. The PTSA amounts in the sampleand reference A and B solutions are given by (V + VA)CS, (V + VA) CR1 and (VH + VA) CR2, respectively.V, VHand VA are the volume of the outer-phase solution in theW/O/W emulsion, the volume of the Hank’s solution in thereference solution B (800�l) and the volume of the PTSAsolution added to the sample solution or the reference so-lution B, respectively.VA also means the volume of thesolution added to the reference solution A, which was thesame volume as the solution added to the sample solutionand reference solution B, but without PTSA. The samplesolution contained PTSA leaked from the inner-water phaseand added after emulsification. The reference solution Acontained PTSA leaked only from the inner-water phase.The reference solution B contained PTSA only added lateras a PTSA solution. Based on the above procedures, thefollowing equation should be considered:

(V + VA)CS = (V + VA)CR1 + (VH + VA)CR2 (1)

The use of the reference solution B enabled us to di-rectly estimate the volume of the outer-phase solution,V, byEq. (2).

V = (VH + VA)CR2

(CS − CR1)− VA (2)

2.8. Analysis of components in the outer-water phase

Two W/O/W emulsions, a coarse W/O/W emulsion anda 0.2�m-filtered one, were used in this study. Aliquots of aW/O/W emulsion were centrifuged at 17,600×g for 10 min.The lower phase was collected using a syringe with a nee-dle and was filtered with a disposable filter of 0.1�m porediameter to eliminate the oil droplets. C8TG and the hy-drophilic and hydrophobic surfactants in the filtrate were


extracted by mixing the filtrate (500�l) in a mixture of chlo-roform and methanol (1:1 by vol., 500�l) with a lab-mixerfor 30 s. The mixture was centrifuged at 17,600× g for10 min, and 1�l of the lower layer solution was applied toa chromarod® (Dia-iatron, Tokyo, Japan), which is a glassrod coated with silica-gel thin-layer for TLC-FID analysis.The chromarod® was first developed with a mixture of ben-zene, chloroform and acetic acid (50:20:0.7 by vol.). Afterthe development, the upper half of the rod was analyzed us-ing Iatroscan MK-5® (Dia-iatron) to determine the C8TGconcentration. The rod was again developed with a mix-ture of benzene, chloroform, diethyl ether and acetic acid(50:10:10:0.2 by vol.), and the upper half of the rod wasanalyzed to determine the concentration of the hydropho-bic surfactant, hexaglyceryl condensed ricinoleate. The rodwas further developed using a mixture of chloroform andmethanol (65:5 by vol.) and the entire rod was analyzed todetermine the concentration of the hydrophilic surfactant,decaglycerol monolaurate.

2.9. Statistical analysis

Error bars in the figures show the standard deviations ofthe measurements. Their numbers are shown in the figurecaptions.

3. Results and discussion

3.1. Effect of the hydrophilic-surfactant concentration onthe median diameter of oil droplets

Fig. 1A and Bshow the median diameters of the coarseand membrane-filtered W/O/W emulsions, respectively. Themedian diameter decreased along with the increase in thesurfactant concentration. The difference in the median diam-eters of the oil droplets in W/O/W emulsions prepared usingdifferent polyglycerol esters shown inFig. 1A would be in-significant, because of the broad distribution of the diameterof the oil droplets in the coarse W/O/W emulsion as shownin the previous report[6]. The diameters of the oil dropletsin the W/O/W emulsions prepared using lysolecithin weremuch smaller than those of the droplets in the emulsionprepared using polyglycerol esters (Fig. 1B). The mediandiameter of the oil droplets in the emulsion prepared withlysolecithin also decreased with the increase in the surfac-tant concentration, but it was difficult to handle at a concen-tration higher than 2% (w/v) because the solution becameviscous.

Fig. 2A and Bshow the distribution of the oil droplet di-ameters in the W/O/W emulsions prepared at different con-centrations of decaglycerol monolaurate and lysolecithin,respectively. The distributions of the oil-droplet diameters inthe W/O/W emulsions prepared at 2–10% (w/v) decaglyc-erol monolaurate were narrow and the median diameterwas smaller at the higher concentration. The distribution

0

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50

0 5 10Hydrophilic Surfactant concentration [% (w/v)]

Med

ian

diam

eter

[µm

]

0

1

2

3

0 5 10

Hydrophilic surfactant concentration [% (w/v)]M

edia

n di

amet

er [

µm]

0

20

40

60

80

Med

ian

diam

eter

for

lyso

leci

thin

[µm

]

1.32

(A)

(B)

Fig. 1. Median diameter of oil droplets in the (A) coarse and (B)membrane-filtered W/O/W emulsions prepared using different hydrophilicsurfactants. Symbols, (�), (�), (�), (�), and (×), indicate that pen-taglycerol monolaurate, decaglycerol monolaurate, pentaglycerol mono-myristate, decaglycerol monomyristate, and lysolecithin were used as thehydrophilic surfactant, respectively. The numbers of measurement aretwo for pentaglycerol monolaurate, two or one for pentaglycerol mon-omyristate and lysolecithin, and one for decaglycerol monolaurate anddecaglycerol monomyristate.

for the emulsion prepared at 1% (w/v) decaglycerol mono-laurate was wide and had two peaks. This suggested thatthe concentration was too low to prepare a W/O/W emul-sion. Fig. 2B shows that the oil-droplet distributions forthe emulsions prepared using lysolecithin. The distribu-tions for the emulsions prepared at 0.1 to 1% (w/v) werealso wide and had two peaks. A membrane filtration atthe low hydrophilic-surfactant concentration thus produceda W/O/W emulsion with both relatively small and largeoil droplets. The large droplets would be formed by thecoalescence of the small droplets. This would be applica-ble to the emulsion prepared using 1% (w/v) decaglycerolmonolaurate shown in Fig. 2A.

3.2. Change in the encapsulation efficiency

The encapsulation efficiency decreased as the surfactantconcentration increased (Fig. 3). Decaglycerol and pen-taglycerol monolaurates showed relatively low encapsula-tion efficiency compared to pentaglycerol and decaglycerolmonomyristates at a high concentration. The difference be-tween the two groups is the hydrophobic acyl-chain length,i.e., the former two surfactants have a 12-carbon-chain


0

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0.1 1 10 100

Diameter [µm]

Freq

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]

(A)

(B)

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5

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15

20

25

30

35

0.1 1 10 100 1000

Diameter [µm]

Freq

uenc

y [%

]

Fig. 2. The distribution of oil-droplet diameters of the W/O/W emulsionsfiltered by a 0.2 �m pore-size membrane. (A) Symbols, (�), (�), (×),(�), and (�), represent 1, 2, 3, 4, and 10% (w/v) decaglycerol mono-laurate, respectively. (B) Symbols, (�), (�), (�), and (�), representlysolecithin concentrations of 0.1, 0.5, 1, and 2% (w/v), respectively.

length and the latter ones have a 14-carbon-chain length.The use of surfactants with longer acyl-chains enabledthe production of an emulsion with higher encapsulationefficiency.

The encapsulation efficiency of the emulsions was slightlydecreased after membrane emulsification (Fig. 3). The en-capsulation efficiencies of the membrane-filtered emulsionsprepared with decaglycerol monolaurate and decaglycerolmonomyristate at lower concentrations (1–4% (w/v)) werelower than those of the coarse emulsions. The encapsula-tion efficiencies of the membrane-filtered emulsions pre-pared with decaglycerol monolaurate and decaglycerol mon-omyristate were also lower than those of the other two.These results suggested that the property of the hydrophilicmoiety of the surfactant would also affect the encapsulationefficiency of the W/O/W emulsions. The encapsulation effi-ciency became constant at the high surfactant concentrationsexcept for pentaglycerol monolaurate. The W/O/W emul-sion prepared using lysolecithin showed a sharp decline inthe encapsulation efficiency with an increase in its concen-tration.

These results suggested that the emulsification methodaffected the relationship between the types of surfactantsand the encapsulation efficiency of the W/O/W emulsion.Suzuki et al. [8] reported that the hydrophilicity of the

90

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0 2 4 6 8 10 12

Hydrophilic surfactant concentration [% (w/v)]

Enc

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]90

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100

0 2 4 6 8 10 12Hydrophilic surfactant concentration [% (w/v)]

Enc

apsu

latio

n ef

fici

ency

[%

]

(A)

(B)

Fig. 3. Encapsulation efficiency for the (A) coarse and (B) membrane-filtered W/O/W emulsions prepared using the different hydrophilic sur-factants. Symbols, (�), (�), (�), (�), and (×), indicate that pentaglyc-erol monolaurate, decaglycerol monolaurate, pentaglycerol monomyris-tate, decaglycerol monomyristate, and lysolecithin were used as the hy-drophilic surfactant, respectively. The numbers of measurement are twofor pentaglycerol monolaurate, two or one for pentaglycerol monomyris-tate and lysolecithin, and one for decaglycerol monolaurate and decaglyc-erol monomyristate.

membrane influenced the properties of the O/W emulsionsproduced using a porous glass membrane. The surfaceexcesses of decaglycerol monolaurate and decaglycerolmonomyristate were larger than those of the other twopolyglycerol esters, as shown in Table 1. The HLB ofthese surfactants are almost the same, i.e., 15 (decaglyc-erol monolaurate), 14.5 (decaglycerol monomyristate), 15.6(pentaglycerol monolaurate), and 15.0 (pentaglycerol mon-omyristate). The decrement in the encapsulation efficiencydue to the membrane-emulsification at the low concentrationof decaglycerol monolaurate and decaglycerol monomyris-tate (lower than 4% (w/v)) would be ascribed mainly to thedifference in the polyglycerol residue of these surfactants,because their hydrophobic residues are the same as those ofpentaglycerol monolaurate and pentaglycerol monomyris-tate, respectively. The relatively large hydrophilic residuesof the decaglycerol monolaurate and decaglycerol mono-myristate would interact with the hydrophilic membraneused in the membrane filtration to prepare a fine W/O/Wemulsion. The encapsulation efficiency of the W/O/Wemulsion prepared using lysolecithin, which was more hy-drophobic than the other four surfactants (Table 1), was


not altered by the membrane emulsification. These resultssuggested that the encapsulation efficiency of the W/O/Wemulsion prepared using the high HLB surfactants at a lowconcentration was more affected by the membrane-filtrationthan that of the emulsion prepared using the relatively lowHLB surfactants.

3.3. Location of surfactant in the W/O/W emulsion

An adequate concentration of a surfactant was determinedempirically, but the actual location of the surfactant duringthe preparation process was scarcely investigated. In order todetermine the behavior of the surfactant during the process,the location of the surfactant during the preparation of theW/O/W emulsion was investigated.

We reported previously [6] that an outer-phase inclusionoccurred in the homogenization process. The outer-phase in-clusion means the incorporation of the outer-phase solutionin the oil droplet during the preparation of a W/O/W emul-sion. Wan and Zhang [9] have recently discussed that thisphenomenon is ascribed to swelling of the W/O/W emulsionprepared by the liquid membrane method, which is called“entrainment swelling” [9]. In our preparation procedures,the observation of the W/O/W emulsions using fluorescentmicroscopy showed that the included water droplets dimin-ished during the filtration of the coarse W/O/W emulsion[6]. The outer-phase volume, which was estimated based onthe PTSA concentration in the outer-phase solution in thisstudy, increased greatly during the preparation of the coarseW/O/W emulsion. The difference in the osmotic pressurebetween the outer-water phase (0.533 ± 0.004 osm/kg, n =3) and inner-water phase (0.005 ± 0.000 osmol/kg, n = 3)would be the main reason for this phenomenon, and the vol-ume of the solution released from the inner-phase to theouter-phase by the osmotic pressure was larger than the vol-ume of the outer-phase solution incorporated in the oil phase(Fig. 4).

Based on the outer-phase volume observed above and thefollowing three assumptions, the locations of the surfactantsand oil in a W/O/W emulsion were estimated. (1) The outersurface of an oil droplet was fully covered by the hydrophilicsurfactant. The residual area per molecule at the interface ofHank’s solution and the oil phase was measured by the Wil-helmy method. (2) Distribution of the hydrophilic surfactantto the oil phase was completed during the homogenizationperiod. (3) The volume of the outer-phase incorporated inthe oil phase was negligibly small compared with the entirevolume of the outer-phase solution after membrane emulsi-fication.

Based on these assumptions, the amounts of the hy-drophilic surfactant in the outer-water, oil and inner-waterphases were estimated from the following equations:

m0 = mA + mB + mC + mD (3)

m0 = m′A + m′

B + m′C (4)

300

400

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0 2 4 6 8 10 12Hydrophilic surfactant concentration

[% (w/v)]

Vol

ume

of t

he o

uter

-pha

se s

olut

ion

[µL

/mL

W/O

/W e

mul

sion

]

Fig. 4. Volume of the outer-phase solution of the coarse W/O/W emul-sions. A dotted line at 500 �l/ml W/O/W emulsion indicates the origi-nal outer-phase solution volume and a broken line at 650 �l/ml W/O/Wemulsion represents the sum of the volume of the outer- and inner-phasesolutions. Measurement was triplicated at each concentration, except at10% (w/v) that was replicated five times.

mA = VpCp (5)

m′A = VaCa (6)

where m0 is the amount of decaglycerol monolaurate used inthe emulsification, mA is the amount of decaglycerol mono-laurate in the outer-phase solution before membrane emul-sification, mB is the amount in the oil-phase solution, mC is

Fig. 5. Schematic diagram of the distribution of the hydrophilic ( ) andhydrophobic surfactants ( ) in the W/O/W emulsion. The upper figurerepresents the location of the surfactants in the coarse W/O/W emulsion,and the lower one represents that of the surfactant in the fine W/O/Wemulsion.


the amount on the surface of the oil droplet, and mD is theamount in the included outer-phase solution (Fig. 5). m′

A,m′

B and m′C are the amounts of decaglycerol monolaurate

in the outer-water and oil phases and on the surface of theoil droplet, respectively, after membrane filtration. Accord-ing to the assumption (1), mC and m′

C can be calculated bydividing the surface areas of the oil droplets in the emul-sion before and after membrane filtration, respectively, bythe residual area per molecule calculated from the surfaceexcess at the interface between outer-water and oil phasesolution from Fig. 6 (8.8 × 10−5 g/m2). From the assump-tion (2), mB is equal to m′

B. Therefore, m′B can be estimated

from m′C using Eqs. (4) and (6). mD was estimated from mB

and mC based on Eqs. (3) and (5). Fig. 7 shows the con-centrations of (A) hexaglyceryl condensed ricinoleate in theouter-phase solution before and after the membrane emulsi-fication, (B) C8TG in the outer-phase solution before and af-ter the membrane emulsification, (C) decaglycerol monolau-rate before membrane emulsification, and (D) decaglycerolmonolaurate after membrane emulsification, at decaglycerolmonolaurate concentrations of 1–10% (w/v).

The concentration of hexaglyceryl condensed ricinoleatein the outer-phase solution slightly increased after the mem-brane emulsification and almost linearly increased with

0

0.01

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0.05

0 2 4 6 8 10 12

0 2 4 6 8 10 12

Decaglycerol monolaurate [% (w/v)]

ML-

750

[g/m

L em

ulsi

on]

0.E+00

2.E-04

4.E-04

6.E-04

8.E-04

1.E-03

ML-

750

[g/m

L em

ulsi

on]

0

0.01

0.02

0.03

0.04

0.05


ML-

750

[g/m

L em

ulsi

on]

0.E+00

2.E-04

4.E-04

6.E-04

8.E-04

1.E-03

ML-

750

[g/m

L em

ulsi

on]

0

0.001

0.002

0.003

0.004

0 2 4 6 8 10 12

0 2 4 6 8 10 12


Sun

soft

818S

X [g

/mL

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sion

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0

0.002

0.004

0.006

0.008

0.01


C8T

G [g

/mL

emul

sion

]

(A)

(B)

(C)

(D)

Fig. 7. Effect of the concentration of a hydrophilic surfactant, decaglycerol monolaurate (ML-750), on the concentration of each component in the W/O/Wemulsions. Symbols, (�) and (�), in A and B represent the concentrations of the hexaglyceryl condensed ricinoleate (Sunsoft® 818SX) or C8TG in theouter-phase solution before and after membrane emulsification using a 0.2 �m pore-size membrane, respectively. Symbols, (�), (�), (�), and (�), in Cand D show the concentrations of decaglycerol monolaurate in the outer-water phase, oil-phase, outer-phase incorporated into the oil-phase, and on theoil-water interface in the W/O/W emulsion, respectively. Figures (C and D) are for the coarse and membrane-filtered emulsions, respectively. Replicationrun numbers were from one to three.

0

1

2

-12 -10 -8 -6 -4 -2 0

Decaglycerol monolaurate [log(M)]

Surf

ace

tens

ion

[mN

/m]

Fig. 6. The Symbol (�) represented the interface tension between aHank’s solution containing indicated decaglycerol monolaurate and C8TGcontaining 10% (w/v) hexaglyceryl condensed ricinoleate. The dotted linerepresents the interface tension (1.5 [mN/m]) when the concentration ofdecaglycerol monolaurate equals zero. The horizontal line represents theinterface tension (0.475 [mN/m]) over CMC. The diagonal line representsthe fitting line, slope is −0.238 [mN/m], intercept is −1.010 [mN/m], witha correlation factor (R2) is 0.999. Replication run numbers were one.

the decaglycerol monolaurate concentration (Fig. 7A). Thehexaglyceryl condensed ricinoleate, which could be passedthrough the pores of the 0.1 �m membrane, would be solu-bilized by the decaglycerol monolaurate in the outer-water


phase. A similar phenomenon was reported by Lezer et al.[10] as a solubilization of the hydrophobic surfactant,which stabilized the oil droplet to prevent swelling break-down. The solubilization of the hydrophobic surfactant bythe hydrophilic one could reduce the concentration of thehydrophobic surfactant in the oil droplet, although the ef-fect seemed to be small in this study because the ratio ofthe amount of the surfactant solubilized in the outer-phasesolution to that presented in the entire system was belowca. 0.9% of the entire hydrophobic surfactant and the os-motic pressure was greater in the outer-phase solution thanin the inner-phase solution. The C8TG concentration alsoincreased after the membrane emulsification. The concen-tration became a minimum value at a decaglycerol mono-laurate concentration of 2–3% (w/v), and the increase inthe C8TG concentration after the membrane emulsificationwas large at the low and high decaglycerol monolaurateconcentrations (Fig. 7B). As shown in Fig. 7C, the amountof decaglycerol monolaurate on the oil-droplet surface wasvery small, and the amount of decaglycerol monolaurate inthe outer-phase inclusion was almost constant at concen-trations over 2% (w/v). The concentration of decaglycerolmonolaurate in the oil phase increased at over 3% (w/v)decaglycerol monolaurate. Fig. 7D shows the location ofdecaglycerol monolaurate after membrane emulsification.The amount of decaglycerol monolaurate located on thesurface of the oil droplet (�) increased after membraneemulsification, and the concentration in the oil phase wasconstant because of assumption (2). The amount of thesurfactant included into the outer-phase was not consid-ered due to assumption (3). Over the concentration of2–3% (w/v) in Fig. 7C and D, the decaglycerol monolau-rate partitioned from the outer-water phase to the oil phaseand the amount of decaglycerol monolaurate located onthe interface between outer-phase solution and oil dropletremained constant. When the surfactant located on the in-terface was supposed to be effective for stabilization ofthe emulsion, a lower concentration of decaglycerol mono-laurate was sufficient for the preparation of the emulsion.The distribution of the oil droplet diameters was, how-ever, broad in the W/O/W emulsion prepared at 1% (w/v)decaglycerol monolaurate, and the distribution became nar-row at decaglycerol monolaurate concentrations higher than2% (w/v) (Fig. 2). These results suggested that the mosteffective concentration of decaglycerol monolaurate was2–3% (w/v). At concentrations higher than 4% (w/v), theencapsulation efficiency and median diameter were almostconstant as shown in Figs. 3 and 2, although the amountof decaglycerol monolaurate distributed in the oil phaseincreased. The distributed decaglycerol monolaurate wouldform reverse micelles, and the micelles would transportthe hydrophilic compound in the inner-water phase to theouter-water phase. Although other factors such as the costand operationality should be considered in selection of ahydrophilic surfactant, the location of the surfactant wouldbe a criterion in the selection.

4. Conclusions

In order to estimate a minimum concentration of hy-drophilic surfactant to successfully prepare a W/O/W emul-sion, the emulsion was prepared at different concentrationsof several polyglycerol fatty acid esters and lysolecithin. Thelocation of the decaglycerol monolaurate was investigatedto obtain its optimum concentration. The concentration es-timated from the location of the surfactant was in accordwith the minimum concentration at which the median diam-eter and encapsulation efficiency of the emulsions remainedconstant. Concentrations lower than 2% (w/v) were too lowto prepare a W/O/W emulsion with a narrow oil-droplet dis-tribution. Distribution of the hydrophilic and hydrophobicsurfactants to the oil-phase and outer-water phase becamesignificant at concentrations higher than 4% (w/v).

Acknowledgements

This work was supported by a grant-in-aid for scientificresearch from the Japan Society for the Promotion of Sci-ence. The surfactants, SY-Glyster® ML-750 and Sunsoft®

818SX, were gifts from Sakamoto Yakuhin Kogyo (Osaka,Japan) and Taiyo Kagaku (Yokkaichi, Japan), respectively.

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