droplet size dependent solubilization and crystallization of lipids in oil-in-water emulsion

7/29/2019 Droplet Size Dependent Solubilization and Crystallization of Lipids in Oil-In-Water Emulsion

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Chapter 11Droplet-Size DependentSolubilization and

Crystallization ofLipids in Oil-in-Water EmulsionN. Hasuo13, T . Sonoda2, S. Ueno, andK . Sato1*

1Graduate School ofBiosphereScience,Hiroshima University, Higashi,Hiroshima 739-8528, J apan

2Tanabe Pharmaceutical Company L td., Osaka532-8505, J apan3Current address:Meiji Dairies Company, Odawara250-0862, J apan

We measured the rate and extent of solubilization andcrystallizationof lipids (long-chain fatty acids; lauric, myristicand palmtic acids) inol-in-water emulsionwith average oildropletdiameters of120 nm, 170 nm, and 220 nm. We foundthat therate andextent ofthe solubilized materials increasedas theoil concentration in emulsion increased, thediameter ofemulsiondroplets decreased, thetemperature ofsolubilizationincreased, and themelting points of the fatty acids decreased.The crystallizationtemperatures of the fatty acids inemulsionremarkably decreased compared with bulk oil. Thepresentstudy demonstrated that the fatty acids became moresolubilized and less crystallized when they areembedded inthe nm-sizeemulsiondroplets.

2009American Chemcal Society 169

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In Micro/Nanoencapsulation of Active Food Ingredients; Huang, Q., el al.;ACS Symposium Series; American Chemical Society: Washington, DC, 2009.


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170Introduction

Oil-in-water (O/W) emulsion and mcroemulsion have been investigated asan advanced delivery tool of pharmaceutical (/, 2, 3), cosmetic (4) and foodmaterials (5) having water-insouble and oil soluble properties. Ths isbecausethe O/W emulsion droplets are expected to exhbit multiple functions such ascontrolled release (6) and hgh bioavailability (7). In order to make the O/Wemulsion droplets more functional and stable in practical uses, variousphysicochemcal studies are needed; e.g., increased solubilization, controlledrelease, stabilization at elevated temperature for sterilization, and chilledtemperatures for storage. In particular, much attention has been paid to hghysolubilized lipophilic materials in the O/W emulsion droplets in order to increaseloadingefficiency of the lipophilic materials in the emulsion.High solubilizationiscritically important for the oil droplets having diameters around 100 nm, sincesuchdroplets are new toos of delivery systems in nanotechnoogy applicable tofood, cosmetics and pharmaceutical industries.

Solubilization of lipophilic molecules in mcelle soutions has beenthoroughy investigated (8). For solubilizationof oil into mcelle particles fromoil droplets in the O/W emulsion, the solubilization kinetics are thought to bedomnated by interfacial transport processes, whchare affected by temperature,the natureof the oil/water interface, droplet-mcelle interaction, and the chemcalstructure and molecular volume of the oil and surfactant moecules (9). Nizr iand Magdassi studied the solublization kinetics of small lipophilic moleculesintonanoparticles made of surfactants and polymers, and demonstrated that thenanoparticles solubilize the lipophilic moecules at different positions of theparticles depending on the polarity or lipophilicity of the molecules (10). Thesolubilizationof oil in mcroemulsion was also investigated (//). Compared tosolubilization in surfactant-based mcelle soutions and mcroemulsion, fewstudies have been conducted on the effects of droplet sizes on the solubilizationkineticsof lipophilic moecules in O/W emulsions.

Inthe presentwork, we prepared an O/W emulsion containng oil dropletswithdifferent average diameters (120 nm, 170 nm and 220 nm) by changing thehgh-pressure homogenzation conditions. Usually, hgh-melting-temperaturematerial issolubilized into the oil phaseabove its melting point. However, thsmethod does not facilitate precise measurement of the time variation of thesolubilizationand the saturated solubilized quantity in the emulsion. Therefore,we solubilized the solid lipids below their melting point by adding crystalpowders to the emulsion. The time variation of the solubilizedquantity was thusexamned at a constant temperatureover 48 hours. Three long-chain fatty acids(lauric acid ( L A ) , myristicacid ( M A ) and palmticacid (PA)) were employed tomeasure the solubilization properties in the emulsion droplets, whereas lauricacidwas solely examned in the crystallizationexperiments.


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171Materialsandmethods

MaterialsThree fatty acids with purity of 99% were purchased from Sigma A ldrich

(Steinhaim, Germany), and used without further purification. Nanometer-sizedemulsions were made of soybean oil and canola oil mxture as a dispersed oilphase, distilled water as a continuous phase, and polyglycerine fatty acid ester,decaglycerine monostearate (10G1S, Sakamoto Pharmaceutical Co., Osaka,Japan) as an emulsifier. The emulsification was carried out by using a hgh-pressurehomogenzer (DeBEE 2000). The size of the oil droplets was varied bychanging the ratio of oil-water-emulsifier (O-W-E, wt.%) and by changing thepassing time of the emulsification at different emulsification pressures. Theemulsion samples containng the oil droplets with different average diameters(D) were prepared under the following conditions: D-120 30 nm; O - W - E =20-70-10, five passing times at 200 MPa, D-170 30 nm; 20-75-5, five passingtimes at 138M P a , and D-220 30 nm; 20-77-3, five passing times at 100 M P a .A s shown here, the oil concentration was always kept 20 % of the total emulsionsamples. The droplet size was measured withanE L S 8000 (Otsuka Electroncs,Tokyo) using dynamc light-scattering methods.

SolubilizationThe solubilization experiments were conducted using the following

methods. The powder crystal samples of the fatty acid (2 g) were put into theemulsionsample (40 g) placed in a thermostated vessel whose temperature wascontrolled withn0.1C . The emulsion sample containng the crystal powderwas stirredwitha magnetic stirrer (200 rpm). The solubilizedquantity, definedas the fraction (%) of the weight of solubilized materials divided by the totalweight of crystal powder, was obtained by weighng the crystal samples presentin the vessel, whchwere separated from the emulsion by vacuum filtration usinga membrane filter. Samples were weighed up to 48 hours for varying durations(short periods (mnutes) in the initial stage to longer periods (hours) in the laterstageof solubilization). The measurementswere repeated three times to get theaverage values. The solubilizationtemperatures examned were 15 C, 25 Cand 35 C for L A (melting point, 44 C ); 25 C, 35 C and 45 C for PA (meltingpoint, 54 C); and 35 C, 45 C and 55 C for PA (melting pont, 63 C).

For comparison, the solubilities of the fatty acids in the bulk oil weremeasured at the same temperatures as those of the solublization experiments inthe emulsion. The solubilities of the three fatty acids in the water containng


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17210G1S of 2.5 wt.% (aqueous micelle solution) were measured. Samples werevisually observed to determne the saturated concentration of the fatty acids inthe oilphaseand water phasesat constant temperature.

CrystallizationCrystallization behavior of lauric acid solubilized in the bulk oil and

emulsion was observed with DSC by synchrotron radiation X-ray diffraction( SR -XRD) . DSC cooling and heating thermopeaks were measured by coolingfrom50C to 0C, and heating from 0 C to 50 C at a rateof 2 C/min, usingThermoplus 8240(Rigaku, Tokyo). TheS R - X R D measurementswere performedin B L -15A stations of the synchrotron radiation facility, Photon Factory, in theHigh-Energy Accelerator Research Organization ( K E K ) , Tsukuba, Japan. Thedetails of the S A X S / W A X S instrument were reported elsewhere (12). Thewavelength was 0.15 nm, and the X-ray beamshapewas 1.0 mm (horizontal) 0.7 mm (vertical). The emulsion samples were set in sample cellswith internaldimensions of 5 mm ( ) 5 mm (V) 1 mm (W), and the cells were immersedin a temperature control system, L inkam T H M S600 (Linkam ScientificInstruments, Surrey, United Kingdom). The rate of cooling was 100 C/min,with which isothermal crystallizationwas monitored by theS R - X R D technique.

Resultsand DiscussionFigure 1 illustrates the effect of the oil concentration in the emulsion on the

solubilizationquantity of L A in the emulsion samples with threeaverage dropletdiameters measured after stirring up to 5 hours at 35 C. The oil concentrationwas varied bydiluting the initially formed emulsion (oil concentration was 20 %)with water containing the same 10G1S concentrations as those of theemulsification. It was evident that the solubilized quantity increased inproportion to the oil concentration increase in the threetypes of emulsion. Thisdemonstrated that L A was solubilized by the presence of the oil phase of theemulsion, and that the solubilized quantity increased as the droplet diameterdecreased at every oil concentration. From this result, we fixed the oilconcentration for thesolubilizationexperiments at 5 %.

Figures 2 (a), (b) and (c) plot the time variations of the solubilized quantityof L A , M A and PA in the three types of emulsion measured at 35 C. Thefollowing properties were observed in all cases.(a) The solubilized quantity rapidly increased soon after the solubilization

started.


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Oil concentration (%)Figure 1.Solubilized quantityof lauric acidwithvarying oil concentration

in emulsion measured after stirringfor 5hoursat 35 *U .

(b) The solubilized quantity increase began to slow down at around 6 hours,reached a saturated value after 12 hours, and remained constant for up to 48hours.

(c) The solubilizationrate, defined as the time-dependence of solubilizationinthe initial stage, and the extent defined as saturated quantity of solubilizationincreasedwithdecreasing diameter of the oil droplets.

(d) The rate and extent of solubilizationdecreased as the melting point of fattyacid increased; it was the hghest for L A and the lowest for P A .

Althoughnot shown here, the above properties were commony observed in thesolubilizationexperiments of L A at 15 C and25C, inM A at 25 C and 45 C,and inP A at 45 C and 55 C.

Table I summarizes the solubilizationdata of the three fatty acids obtainedin the present study. The solubilized quantities after 48 hours are presented asrepresentative data taken in the emulsion. It must be noted that the solubilizeddata in bulk oil correspond to thermodynamc soubility, whereas the dataobtained in the emulsion do not correspond tosoubility, since the O/W emulsionisnot thermodynamcally stable. Therefore, we refer tosolubilized quantity forthe case of emulsion.

InTable I, the solubilized quantities in emulsion are obviously larger thanthe solubilities in bulk oil for the three fatty acids at all temperatures andemulsion droplet sizes examned. More interestingly, the difference betweenthem increased when the emulsion solubilization temperature and droplet


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Figure 2. Timevariation of solubilized quantityof (a) lauric acid,(b) myristic acid and (c) palmtic acid at 35*C.


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15

Figure 2. Continued.

TableI. Solubilized quantity(g/100gof salad oil) of fatty acids in emulsion,bulk oil and aqueous mcele solution.

Laurie acid Myristic acid Palmtic acid 15 25 35 25 35 45 35 45 55D

120 8 18 70 10 21 56 10 19 62D 170 7 16 62 7 17 48 6 16 53220 6 14 59 4 13 36 5 11 38

3 13 50 2 11 34 2 10 33N O T E : T; temperature (C), D ; emulsion diameter (nm), B : bulk oil.


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176diameter decreased. For example, the solubilized quantity of L A with D-220nm at 25 C was 14 g, and the solubility in bulk oil was 13.37 g, and the samevaluesof P A at 45C were 11 g (emulsion with D-220 nm) and 10.67 g (bulk).However, the solubilizedquantities exceeded the solubilities of the bulk oil by2.8 times (LA, D~120nm at 15 C) and 4 times (MA, D-120 nm at 25 C andP A , D-120 nm at 35 C). From the results summarized in Table I, we mayconclude that the solubility in the O/W emulsion was remarkably increased bydecreasing the emulsion droplet size.

Figure 3 depicts DSC cooling thermopeaks of crystallization of lauric acidsolubilized in bulk oil, and in the emulsion (averagedroplet diameter 120 nm) inwhich lauric acid was solubilized at 35 C. In both cases, the solubilizedquantity of lauric acid was 50 % with respect to the salad oil. An exothermcpeak corresponding to the crystallization of lauric acidappearedat 29.7 C in thebulk oil, whereas a small exothermc peak appeared at 12.1 C in emulsion.Theseresults indicate that crystallization of lauric acid was remarkably retardedin emulsion compared with bulk.

J(a)

04m (b)04m

1 1 1 10 10 20 30 40 50Temperature(C)

Figure 3. DSC cooling thermopeaksof lauric acid, (a) bulksaladoil and (b)emulsionwithaverage dropletdiameterof 120 nm

Figure 4 illustrates the S R - X R D patterns of lauric acid crystals formed inemulsion (D-120 nm), in which lauric acid was solubilized to thequantity of 50wt.% with respect to salad oil. The crystallization occurred at 12 C, and itspolymorphic form was C form, as judged from the W A X S pattern of 0.41 nm


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177and the S A X S pattern of 2.7 nm (13). The C form crystals melted at 34 Cduring heating. These S R - X R D patterns were consistent with the DSCmeasurements. Although not shown here, the reduction in the crystallizationtemperature of lauric acid in emulsion was confirmed for the emulsion dropletscontainingdifferent quantities of lauric acid.

I l l I 1 f

2 3 4 202 (deg.)

Figure 4. Synchrotron radiation-XRD patternsof bulk lauric acid and emulsionwithsolubilized lauric acidtakenduring (a) cooling and (b) heating.

Figure 5. Schematic modelofsolubilization of lipid intoemulsion droplets.


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178The above results indicatethat thesolubility of lipidswas increased and the

crystallization rates in nano-meter size emulsion droplets were retardedcompared with the bulk oil. This may be interpreted by taking into account thesolubilization mechanisms of lipophilic materials in micelles and emultions (8)as illustrated in Figure 5. We assumed that the solubilization of the fatty acidsfrom the crystal powders suspended in theaqueous phasein emulsion might firstoccur through solubilization in the aqueous phaseand then through absorptioninto the emulsion droplets either directly from theaqueous phaseand/or throughsolubilization into the micelle particles made of the emulsifiers, that maytransport the fatty acids into the emulsion droplets. In the oil droplets inemulsion, theremay be two states of solubilization of the fatty acids: in the oi lphaseand at the emulsion membrane surface. The latter statemay be enabledfor the fatty acids because of their amphiphilicity. The excess amount ofsolubilizedquantity of the fatty acids in the emulsion droplets may be ascribed tothe solubilization at the membrane interfaces whose relative importancecompared with the volume size of the oil droplet increases with decreasingemulsion droplet size. Crystallization in the emulsion may be retarded by thesize effects of the droplets as well as by the solubilization at the membraneinterface thatcausedilutioneffects of thecrystallizing materials. Further preciseclarification of theseprocesses is necessary.

AcknowledgementsThis work was supported inpart by a grant from the Food Nanotechnology

Project of theM inistry of Agriculture, Forest and Fisheries.

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Zanetti, M. ;Puel, F. Coll.Surf.., 2007, 299, 93-100.5. Spernath, A . ; A serin, A . Adv. Coll. InterfaceSci., 2006, 128-130, 47-64.6. Malone, M. E .; Appleqvist, I. A. M. J . Control. Release, 2003, 90,227-241.7. Ton, . M.; Chang, C.; Carpentier, Y . A. ; Deckelbaum, R. J. Clin. Nutr.,

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17910. Nizr i , G.; Magdassi, S. J . Coll. InterfaceSc., 2005, 291, 169-174.11. Garti, . ; Yaghmur, . ; Leser, M. E. ; Clement, V. ; Watzke, H. J . J . Agr.

FoodChem, 2001, 49, 2552-2562.12. Higami, M.; Ueno, S.; Segawa, T.; Iwanami, K .; Sato, K .J . Am OilChemSoc., 2003, 80, 732-739.13. Larsson K .; Quinn, P.; Sato, K . ; Tiberg, F. L ipids : Structure physical

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droplet size dependent solubilization and crystallization of lipids in oil-in-water emulsion

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