Food Chemistry 132 (2012) 799–807

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Food Chemistry

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Nanoemulsion- and emulsion-based delivery systems for curcumin:Encapsulation and release properties

Kashif Ahmed, Yan Li, David Julian McClements, Hang Xiao ⇑Biopolymers and Colloids Research Laboratory, Department of Food Science, University of Massachusetts, Amherst, MA 01003, United States

a r t i c l e i n f o

Article history:Received 20 June 2011Received in revised form 28 September 2011Accepted 8 November 2011Available online 15 November 2011

Keywords:CurcuminBioavailabilityBioaccessibilityDigestionpH-statEmulsionsNanoemulsionsDelivery systems

0308-8146/$ - see front matter � 2011 Elsevier Ltd. Adoi:10.1016/j.foodchem.2011.11.039

⇑ Corresponding author. Tel.: +1 413 545 2281; faxE-mail address: hangxiao@foodsci.umass.edu (H. X

a b s t r a c t

Curcumin has been reported to have many biological activities, but its application as a functional ingredientis currently limited because of its poor water-solubility and bioaccessibility. This study investigated theimpact of different lipid-based formulations on curcumin encapsulation and bioaccessibility. Oil-in-waternanoemulsions (r < 100 nm), or conventional emulsions (r > 100 nm), were prepared with different lipids:long, medium, and short chain triacylglycerols (LCT, MCT and SCT, respectively). An in vitro model simulat-ing small intestine digestion conditions characterised rate and extent of lipid phase digestion. A centrifu-gation method determined fraction of curcumin released into mixed micelles after digestion(bioaccessibility). Initial digestion rate decreased in the order SCT > MCT > LCT, while final digestion extentdecreased in the order MCT > SCT > LCT. The bioaccessibility of curcumin decreased in the orderMCT > LCT� SCT and appeared to be slightly higher in conventional emulsions than in nanoemulsions.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction

Emulsion-based delivery systems are being used increasingly forencapsulating lipophilic bioactive components in the pharmaceuti-cal industry (Porter, Trevaskis, & Charman, 2007). Oil-in-water(O/W) emulsions, or nanoemulsions, can be prepared by solubilisingthe lipophilic bioactive components within the oil phase, and thenhomogenising this phase with an aqueous phase containing awater-soluble emulsifier. The size of the droplets produced dependson the composition of the system and the homogenisation methodused. O/W conventional emulsions and nanoemulsions are boththermodynamically unstable systems that consist of emulsifier-coated lipid droplets dispersed within an aqueous medium (Solans,Izquierdo, Nolla, Azemar, & Garcia-Celma, 2005). However, the twosystems can be distinguished from each other based on their particlesizes: the mean droplet radius is <100 nm for nanoemulsions, but>100 nm for conventional emulsions. The relatively small size ofthe droplets in nanoemulsions means that they often have differentphysicochemical and biological properties than conventional emul-sions. Nanoemulsions have been reported to have better stability toparticle aggregation and gravitation separation due to their smalldroplet sizes (Solans et al., 2005; Sonneville-Aubrun, Simonnet, &L’Alloret, 2004). Nanoemulsions that are optically transparent canbe prepared by making the droplet size much smaller than the

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wavelength of light so that they only scatter light weakly. The bio-availability of encapsulated lipophilic components within the gas-trointestinal tract may be increased by using nanoemulsionsbecause of their relatively small droplet size (Patel & Sawant,2007). One of the objectives of the present study was therefore todetermine the influence of droplet size on the digestion and releasecharacteristics of emulsion-based delivery systems.

The digestion of lipids within the gastrointestinal tract is a com-plex process and its impact on the release and uptake of any encap-sulated lipophilic components is not fully understood. In vitrodigestion models have recently gained much attention as a tool forunderstanding the basic physicochemical processes that occur dur-ing lipid digestion and the release of encapsulated components(McClements & Li, 2010). In particular, in vitro lipolysis models(‘‘pH-stat methods’’) have been developed as a powerful means ofquantifying the lipid digestion process (Pouton, 2006, Zangenberg,Mullertz, Kristensen, & Hovgaard, 2001a, 2001b) and the release oflipophilic substances into the various colloid phases formed duringlipid digestion, e.g., mixed micelles (Porter et al., 2007). The fractionof a lipophilic component released into the mixed micelle phaseafter lipid digestion can be taken as a marker of its bioaccessibility.The bioactivity of a component, which is a measure of its specific bio-logical affect, is determined by the fraction available for absorption.Ultimately, a successful in vitro digestion model should be able topredict in vitro–in vivo correlations because this is a prerequisitefor rational development of effective delivery systems (Frickeret al., 2010).

800 K. Ahmed et al. / Food Chemistry 132 (2012) 799–807

In this study, we used curcumin as a model lipophilic compo-nent that could be incorporated into functional food products.Curcumin is a natural polyphenolic phytochemical extractedfrom the powdered rhizomes of turmeric (Curcuma longa) spice.Curcumin has been reported to have a number of potentiallybeneficial biological activities, e.g., anti-tumour, anti-oxidant,anti-microbial and anti-inflammatory properties (Duvoix et al.,2005). However, curcumin has an extremely low water solubility(11 ng/ml), which makes it difficult to incorporate into manyfood products (Tonnesen, Masson, & Loftsson, 2002), as well asa low bioavailability, which means that its beneficial attributesmay not be realised even if it is ingested (Maiti, Mukherjee, Gan-tait, Saha, & Mukherjee, 2007). Many attempts have thereforebeen made to improve the water solubility and bioavailabilityof curcumin. Studies have shown curcumin can be encapsulatedwithin complexes formed using either polysaccharides or phos-pholipids (Liu, Lou, Zhao, & Fan, 2006; Tonnesen et al., 2002).Recent studies have shown that emulsion-based delivery systemscan also be used to encapsulate curcumin (Bisht et al., 2007;Mukerjee & Vishwanatha, 2009; Wang et al., 2008; Yu & Huang,2010). These studies indicate that emulsion-based systems cangreatly increase the bioavailability of curcumin compared tocrystalline curcumin dispersed within water.

In the present study we use an in vitro digestion model to sys-tematically examine some of the major factors that impact the bio-accessibility of curcumin encapsulated within emulsion-baseddelivery systems. In particular, we examined the influence of oildroplet size (emulsions versus nanoemulsions) and lipid phasecomposition (triacylglycerol molecular weight) on lipid digestionand curcumin bioaccessibility. The knowledge gained from thisstudy should facilitate the rational design of delivery systems forenhancing the bioavailability of curcumin and other health-promoting lipophilic components.

2. Materials and methods

2.1. Materials

Curcumin (1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadi-ene-3,5-dione) was used as a model lipophilic nutraceutical compo-nent. Curcumin (�95.2% pure, from Curcuma longa) was purchasedfrom Bepharm Ltd. (Shanghai, China) and used without furtherpurification. Powdered b-lactoglobulin (b-Lg) was provided byDavisco Foods International Inc. (BioPURE, Le Sueur, MN). Lipidphases with predominantly long, medium, and short chain triacyl-glycerols (LCT, MCT, and SCT, respectively) were used. Corn oil (LCT)was purchased from a local supermarket, Miglyol� 812 (MCT) waspurchased from SASOL (Houston, TX), and Tributyrin (98%) (SCT)was purchased from Sigma–Aldrich (St. Louis, MO). All were usedwithout further purification. Lipase (from porcine pancreas pancre-atin) and bile extract (porcine) were also obtained from Sigma–Aldrich. Double deionized water was used in all experiments. A5 mM pH 7 phosphate buffer solution (PBS) was produced by dis-persing 0.011% of anhydrous NaH2PO4 (Sigma–Aldrich, St. Louis,MO) and 0.058% of anhydrous Na2HPO4 (Fisher) in water.

2.2. Curcumin solubility

The maximum solubility of curcumin in each lipid phase wasdetermined using a spectrophometry method. Excess powderedcurcumin was added to an oil phase heated to 60 �C, magneticallystirred for 10 min, and then sonicated for 20 min. A temperature of60 �C was used based on previous studies of curcumin dissolutionin oils (Wang et al., 2008). The resulting crystalline slurry was cen-trifuged at 1750 rpm (1170g) using a bench top centrifuge (Model

225A, Fisher Scientific), at ambient temperature, for 10 min to re-move non-dissolved (crystalline) curcumin, and then the superna-tant was collected. Preliminary experiments indicated that thecurcumin remained dissolved in all the oil phases when stored at25–60 �C for 24 h or more. The supernatant was then diluted toan appropriate concentration to be analysed by a UV–VIS spectro-photometer (Ultraspec 3000 Pro, GE Healthcare). A cuvette con-taining pure oil was used as a reference cell to zero thespectrophotometer. Wavelengths used, to obtain kmax, were depen-dent upon oil type and ranged from 415 to 420 nm. A calibrationcurve was prepared for each oil phase by measuring the absor-bance as a function of curcumin concentrations. Results are re-ported as the average of three measurements on freshly preparedsamples.

2.3. Nanoemulsion and conventional emulsion preparation andcharacterisation

Oil-in-water conventional emulsions, or nanoemulsions,containing curcumin were prepared. Concentrations are expressedas weight percentage (wt.%), which is equal to 100 multiplied bythe mass of the component divided by the total mass of the sample.An aqueous emulsifier solution containing 1 wt.% protein was pre-pared by dispersing b-lactoglobulin into PBS and stirring for at leasttwo hours to ensure complete hydration. The oil phase was preparedby adding 0.15 wt.% curcumin into the heated oil (60 �C), magneti-cally stirring for 10 min, and then sonicating for 20 min. Coarseemulsions were prepared by homogenising 10 wt.% oil phase, with90 wt.% aqueous emulsifier solution, using a M133/1281 high-speedblender (Biospec Products Inc., ESGC, Switzerland) for 2 min at roomtemperature. For some experiments these coarse emulsions wereused as conventional emulsions, for other experiments they werefurther homogenised to produce nanoemulsions. Nanoemulsionswere prepared by passing the coarse emulsions through a high pres-sure homogeniser five times at 9000 psi (�620 bars) (MicrofluidicsM-110Y Microfluidizer™, MFIC Corporation, Newton, MA, USA)with a F20 Y 75 lm interaction chamber. After preparation, allemulsions were stored at 4 �C.

The particle size distribution (PSD) of the nanoemulsions andconventional emulsions was measured using dynamic light scat-tering (Zetasizer Nano-ZS, Malvern Instruments, Worcestershire,U.K.), with each individual measurement being the average of 12runs. Results were reported as the average diameter (Z-average)and width (polydispersity index-PDI). Emulsions were dilutedprior to analysis using buffer solution (5 mM phosphate, pH 7.0)to avoid the effects of multiple scattering.

2.4. In vitro digestion

The dynamic in vitro digestion model used was a modification ofthose described previously (Zangenberg et al., 2001a, 2001b; Mun,Decker, Park, Weiss, & McClements, 2006). Emulsion and buffersolution (30.0 ml in total) were placed in a clean beaker, in a waterbath, at 37.0 �C for 10 min and adjusted to pH 7 with NaOH solu-tion (range from 0.05 to 1 M). Then, 4.0 ml of bile extract solution,containing 187.5 mg of bile extract (pH 7.0, PBS) and 1.0 ml ofCaCl2 solution, containing 110 mg of CaCl2 (pH 7.0, PBS), was addedinto the 30 ml emulsion under stirring. The resultant mixture wasthen adjusted to pH 7. Finally, 2.5 ml of freshly prepared pancrea-tin suspension, containing 60 mg of lipase (pH 7.0, PBS), was addedto the mixture. The bile solution and lipase solution concentrationswere 5.0 and 1.6 mg/ml, respectively. At this point, an automatictitration experiment was started. The pH-stat (Metrohm, USAInc.) was used to monitor and control the pH (at pH 7) of the diges-tion solution. The volume of added NaOH solution (500 mM) wasassumed to be equal to the amount of free fatty acids generated

K. Ahmed et al. / Food Chemistry 132 (2012) 799–807 801

by lipolysis of the initial triacylglycerols. Digestion experimentswere performed for 2 h.

The amount of free fatty acids released was calculated using thefollowing equations:

VMax ¼ 2ðmoilÞðMWoilÞ

ð1000ÞðCNaOHÞ

� �ð1Þ

%FFA Released ¼ VExp

VMax� 100% ð2Þ

Here, moil is the total mass of oil present in the reaction vessel (g),MWoil is the molecular weight of the oil (g per mol), CNaOH is theconcentration of sodium hydroxide in the titration burette (molper 1000 cm3), and VMax is the volume of NaOH titrated into thereaction vessel to neutralise the FFA released assuming that all thetriacylglycerols are converted to two free fatty acids. Finally, VExp

is the actual volume of NaOH titrated into the reaction vessel to neu-tralise the FFA released during the experiment. A relatively highcalcium level (20 mM) was used in the simulated small intestineconditions to ensure that all of the lipid phases were completelydigested within the experimental timeframe (Li & McClements,2010).

2.5. Bioaccessibility determination

After in vitro digestion, emulsions were centrifuged (12,500 rpm,29,700g), Sorvall RC 6C Plus, DuPont), at 25 �C for 30 min. Theemulsions separated into an opaque sediment phase at the bottom,a clear micelle phase in the middle, and sometimes an oily orcreamed phase at the top. Aliquots (5 ml) of the micelle phase werecollected using a syringe, vortexed with 5 ml of chloroform, and thencentrifuged at 1750 rpm (1170g), at room temperature, for 10 min(Fisher Scientific 225A, Fisher). The bottom chloroform layer wascollected, while the top layer was vortexed with another 5 ml ofchloroform, and centrifuged (1750 rpm, 1170g), at room tempera-ture, for 10 min. The second bottom chloroform layer was addedto the previously set aside chloroform layer, mixed, and analysedby UV–VIS spectrophotometer (Ultrospec 3000 Pro, GE). A cuvettecontaining pure chloroform was used as a reference cell to zerothe spectrophotometer. The concentration of curcumin extractedfrom a sample was then determined from a previously preparedcalibration curve of absorbance versus curcumin concentration inchloroform.

2.6. Structural characterisation of micelle phase

The size of particles present in the micelle phase after digestionwas characterised by dynamic light scattering. After in vitro diges-tion, emulsions were centrifuged (12,500 rpm, 29,700g Sorvall RC6C Plus, DuPont) at 25 �C, for 30 min. Aliquots were taken fromthe micelle phase and were either filtered (0.45 lm), or left unfil-tered. The particle size distribution of the micelle phase was thenmeasured using dynamic light scattering (Zetasizer Nano-ZS,Malvern Instruments, Worcestershire, UK), with each individualmeasurement being the average of 12 runs. The samples were di-luted prior to analysis using buffer solution (5 mM phosphate, pH7.0) to avoid the effects of multiple scattering.

Table 1Particle size characteristics of 10% oil-in-water nanoemulsions and emulsionscontaining 0.15% curcumin prepared using different lipid phases.

Oil type Z-average (nm) PDI (nm)

LCT 181 ± 9 0.19 ± 0.02MCT 174 ± 2 0.16 ± 0SCT 1981 ± 1061 0.75 ± 0.31LCT:SCT 182 ± 0 0.13 ± 0.01

3. Results and discussion

3.1. Curcumin solubility in oil phase

Curcumin has a relatively low water-solubility (<0.005 wt.%)and a high oil–water partition coefficient (logP = 3.1), and so themaximum amount of curcumin that can be incorporated into an

emulsion-based delivery system is mainly determined by the max-imum amount that can be incorporated into the lipid phase. Curcu-min has a relatively high melting temperature (�183 �C) and istherefore crystalline at ambient temperatures. Consequently, itmust be dissolved within the lipid phase prior to making an oil-in-water emulsion, since it is very difficult to homogenise lipidphases containing crystalline material, due to their tendency toblock homogeniser channels and promote droplet coalescence.The amount of a highly lipophilic material that can be dissolvedin an oil phase depends on the molecular characteristics of theoil (e.g., molecular weight, polarity, and interactions). For this rea-son, we initially measured the maximum amount of curcumin thatcould be solubilised within different lipid phases at 60 �C:LCT = 0.30 ± 0.10 wt.%; MCT = 0.79 ± 0.2 wt.%; SCT = 2.98 ± 0.18wt.%; LCT:SCT = 1.94 ± 0.44 wt.%.

The maximum amount of curcumin that could be solubilised in-creased as the average molecular weight of the carrier oil moleculesdecreased. This phenomenon can be attributed to differential inter-action and excluded volume effects (Huyskens & Haulaitpirson,1985). Shorter chain triglycerides have more polar groups (oxygen)per unit mass than longer chain triglycerides, and hence thereshould be more dipole–dipole interactions between polar groupson the carrier lipid and curcumin molecules for SCT, which may fa-vour solubilisation. There is also an excluded volume effect associ-ated with incorporation of curcumin molecules into a lipid phase.When a curcumin molecule is introduced into a lipid phase therewill be a region around it from which the centre of the lipid mole-cules are excluded. The concentration of lipid molecules in this ex-cluded zone is therefore effectively zero, whereas theirconcentration in the bulk lipid phase is equal to the number of lipidmolecules per unit volume. The presence of an exclusion zone isthermodynamically unfavourable due to an osmotic effect associ-ated with this concentration gradient. The thickness of the depletionzone increases with increasing molecular weight of the lipid mole-cules, hence this effect may be greater for LCT than SCT. The solubil-ity of the curcumin in the LCT:SCT mix was close to the average of itssolubility in pure LCT and SCT. In other words, there was no signifi-cant synergistic or antagonistic effect on curcumin solubility bymixing the two oils. Based on these results, we used 0.15 wt.% curcu-min in the lipid phase to prepare all of the conventional emulsionsand nanoemulsions, so that they could be compared based onsimilar, fully dissolved curcumin concentrations.

3.2. Influence of oil type on nanoemulsion characteristics

A series of oil-in-water nanoemulsions was produced by micro-fluidization that contained 10 wt.% lipid phase and 0.15 wt.% curcu-min. The mean particle size (Z-average diameter) and polydispersityindex (PDI) of these nanoemulsions are summarised in Table 1, andselected particle size distributions are shown in Fig. 1. The particlesize distributions of the MCT and LCT:SCT nanoemulsions were sim-ilar to that of the LCT nanoemulsions.

Nanoemulsions containing relatively small particles (d <200 nm) with narrow particle size distributions (PDI < 0.2) couldbe formed when LCT or MCT were used as the lipid phase. On theother hand, emulsions containing relatively large particles

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802 K. Ahmed et al. / Food Chemistry 132 (2012) 799–807

(d > 1900 nm) with broad particle size distributions (PDI > 0.6) wereformed when SCT was used as the lipid phase. After 24 h storage, theLCT and MCT nanoemulsions maintained a homogeneous opticallyopaque yellowish appearance, whereas the SCT emulsions rapidlyseparated into a yellow creamy or oily layer, on top of a transparentserum layer. These results showed that the SCT emulsion was highlyunstable to droplet growth and creaming. Recent research hasattributed this effect to Ostwald ripening because of the relativelyhigh water solubility of the low molecular weight triacylglycerol(Li, Le Maux, Xiao, & McClements, 2009). Ostwald ripening is theprocess where larger droplets grow at the expense of smaller onesdue to molecular diffusion of oil through the aqueous phase. Thedriving force for droplet growth is the increase in oil solubility inthe aqueous phase for droplets with high curvatures (small sizes).

The sample containing 50:50 LCT:SCT contained relatively smalllipid droplets (d < 200 nm) with a narrow particle size distribution(PDI < 0.2), which were stable to particle growth and gravitationalseparation. This effect can be attributed to the fact that Ostwaldripening can be inhibited in emulsions prepared from oils with rel-atively high water solubility (such as SCT), by mixing them withoils with relatively low water solubility (such as LCT) (Kabalnov& Shchukin, 1992; Li et al., 2009). The physicochemical origin ofthis effect is due to a compositional ripening process (which arisesdue to an imbalance in molecular compositions of different drop-lets) that counteracts the Ostwald ripening process (which arisesdue to an imbalance in droplet curvatures).

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3.3. Influence of oil type & concentration on in vitro lipid digestion

In this section, we quantified the influence of carrier oil typeand concentration on the rate and extent of lipid digestion usingan in vitro lipid digestion model. A series of oil-in-water nanoemul-sions containing 0.15 wt.% curcumin in the lipid phase was pro-duced with different types and amounts of lipid. The kinetics oflipid digestion was then monitored by measuring the amount offree fatty acids released over time after the nanoemulsions wereintroduced into the simulated small intestinal fluid.

Lipid type: Initially, we compared the rate and extent of lipiddigestion for emulsions prepared using different carrier oil phases,at a constant carrier oil concentration (1 wt.% in the reactionvessel). The nature of the carrier oil type clearly had a major impact

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Fig. 1. Particle size distributions for 10 wt.% oil-in-water emulsions, containing10 wt.% lipid phase (LCT or SCT) and 0.15% curcumin. The PSD of MCT and LCT:SCTwere similar to those for LCT.

on the kinetics of lipid digestion (Fig. 2). In general, the FFA releaseprofiles followed a similar trend: an initial rapid increase in FFAduring the first 2000 s, followed by a more gradual increase atlonger digestion times (Fig. 2a). The initial rate of FFA release ap-peared to be faster for the emulsions containing either SCT orLCT:SCT than for the emulsions containing either MCT or LCT(Fig. 2b). There are a number of possible explanations for this phe-nomenon. First, SCTs have some water solubility and thereforesome digestion may occur in the aqueous phase. Second, SCT mol-ecules may protrude more into the aqueous phase than MCT or LCTmolecules because of their short hydrocarbon chains, thereby facil-itating the ability of lipase molecules to hydrolyse the ester bonds.Third, the relatively small digestion products of SCTs may be ableto rapidly leave the droplet surfaces.

The final amount of FFAs produced also depended strongly onlipid type: �68% for LCT; �90% for SCT and LCT:SCT; �113% for

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Fig. 2. Effect of carrier lipid type on rate and extent of lipid digestion, measuredusing an in vitro digestion model that simulates small intestine conditions. A finallipid concentration of 1 wt.% in the simulated small intestinal fluid was used.Results with ‘‘�’’ were statistically different from the LCT samples. The reactionvessel contained 5 mg/ml bile, 1.6 mg/ml lipase, and 20 mmoles calcium. Theoverall profile is shown in Fig. 2a and the initial states in Fig. 2b.

K. Ahmed et al. / Food Chemistry 132 (2012) 799–807 803

MCT. The relatively low amount of LCT digested may be due to thefact that long chain fatty acids tend to accumulate at dropletsurfaces unless there is a physicochemical mechanism to removethem, such as bile salt solubilisation or calcium precipitation(Sek, Porter, Kaukonen, & Charman, 2002). The accumulation ofFFA at the droplet surface has previously been shown to inhibit li-pase digestion of emulsified triglycerides. It is possible that therewas insufficient bile salts or calcium present in the reaction vesselto remove all of the long chain FFA produced by digestion. Short ormedium chain FFAs are known to be more readily dispersible inaqueous phases than long chain FFAs, and therefore do not tendto accumulate at the droplet surfaces and inhibit lipid digestion(Sek et al., 2002). This would account for the relatively highamount of FFA digestion that occurred when the lipid phase waseither SCT or MCT. Nevertheless, it does not account for the factthat the amount of FFA released by digestion was appreciably high-er than 100% for the MCT sample. The calculation of the percentageof FFA released from the lipid droplets during digestion assumes:(i) that only two FFA molecules are released per TAG molecule;(ii) no other components release protons during the digestion pro-cess. It is possible that some of the medium chain monoacylgylce-rols (MAGs) produced during digestion were broken down to FFAsand glycerol, thereby meaning that more than two FFAs were pro-duced per TAG. Alternatively, other components in the reactionvessel may have produced protons during the digestion process,such as phospholipids or proteins, although this effect should havebeen similar for all the lipid phases studied. Other researchers havealso reported that lipid digestion rates were substantially higher onMCT, as compared to LCT emulsions (Deckelbaum et al., 1990). Thesamples containing SCT and LCT:SCT were almost fully digestedwithin the digestion period studied. Interestingly, the amount ofLCT:SCT digestion (�90%) was not the average of the LCT digestion(�68%) and SCT digestion (�90%), suggesting that the short chaintriglycerides might facilitate the digestion of the LCT.

Lipid concentration: The amount of lipid present in the smallintestine may vary depending on the type and amount of food in-gested. We therefore investigated the influence of lipid concentra-tion on the rate and extent of lipid digestion. The full digestionprofiles for the emulsions containing LCT, MCT and SCT are shownin Fig. 3. The amount of FFA released after 120 min of digestion issummarised for all of the samples in Table 2.

For the emulsions containing LCT, MCT and LCT:SCT there was asignificant decrease in the amount of FFA produced as the total li-pid concentration was increased from 1 to 2 wt.% (Fig. 3a and b andTable 2). This effect may be attributed to a number of physico-chemical mechanisms: (i) as the lipid concentration increased,the amount of lipase per unit surface area of oil droplets decreased;(ii) the amount of bile salts present may have been insufficient tosolubilise all of the digestion products (FFA and MAG) producedat higher lipid concentrations; (iii) at the higher lipid concentra-tions, the amount of calcium present may have been insufficientto precipitate all the FFAs produced by digestion and remove themfrom the droplet surfaces. For the emulsions containing SCT, therewas no significant effect of total lipid concentration on the rate orextent of lipid digestion (Fig. 3c). This may have been because ofthe much greater water dispersibility of the shorter chain fattyacids produced by digestion. Short chain FFAs can readily moveinto the aqueous phase even in the absence of bile salts andcalcium.

3.4. Influence of oil type & concentration on in vitro bioaccessibility

In this section, we quantified the influence of carrier oil typeand concentration on the bioaccessibility of curcumin measuredusing an in vitro digestion model. It was assumed that the fractionof the original curcumin that ended up in the micelle phase after

digestion was a measure of curcumin bioaccessibility. The trans-parent micelle phase was collected after ultracentrifugation ofthe digested emulsion and extraction via chloroform.

The bioaccessibility of curcumin clearly depended on the typeand amount of carrier lipid present in the nanoemulsion-baseddelivery systems, ranging from as low as 1% to as high as 58% (Ta-ble 2). The curcumin had a very low bioaccessibility (�1%) when itwas encapsulated in an emulsion containing only SCT as the lipidphase. This effect can be attributed to the fact that short chain fattyacids, such as those produced by digestion of SCT, do not formmixed micelles that are capable of solubilising highly lipophiliccomponents (Fatouros & Mullertz, 2008). Presumably, the curcu-min precipitated from solution when there were no mixed micellesavailable to solubilise and transport them, thereby decreasing theamount incorporated into the micelle phase. These results suggestthat utilisation of SCT in lipid-based delivery systems should beavoided because any encapsulated components may not bebioavailable.

On the other hand, emulsions containing MCT or LCT within thecarrier lipid were able to substantially increase the bioaccessibilityof curcumin, which can be attributed to their ability to form mixedmicelles capable of solubilising highly lipophilic components. Forthese systems, the bioaccessibility of curcumin tended to increaseas the total lipid concentration increased, due to the increase inmixed micelles available to incorporate the curcumin. The onlyexception to this trend was the emulsion containing LCT, wherethe bioaccessibility of curcumin was similar at 1.5 and 2.0 wt.% li-pid (Table 2). This may have been because an appreciable amountof the LCT was not digested at the highest lipid concentration, andhence some of the curcumin may not have been released from thedroplets. In summary, we postulate that there are two competingphysiochemical mechanisms that determine the dependence ofcurcumin bioaccessibility on lipid concentration. First, bioaccessi-bility may increase with increasing lipid content, because moremixed micelles are formed to solubilise the released curcumin.Second, the bioaccessibility may decrease with increasing lipidcontent (for LCT systems), because a greater fraction of lipid phaseremains non-digested, which means some of the curcumin is notreleased from the droplets into the surrounding micelle phase.

Overall, our results suggest that MCT is the most effective lipidphase for increasing the bioaccessibility of curcumin, whereas SCTis the most ineffective lipid phase.

3.5. Influence of oil droplet size on in vitro digestion andbioaccessibility

It has been suggested that the bioavailability of lipophilic com-ponents is greater in nanoemulsions than conventional emulsions(Acosta, 2009). We therefore compared the in vitro digestion andcurcumin bioaccessibility of two delivery systems with the sameoverall composition, but very different droplet sizes: nanoemul-sions (d < 200 nm) and conventional emulsions (d > 10 lm). Itshould be noted that the nanoemulsions prepared using SCT werehighly unstable to droplet growth, so that the droplet size rapidlyincreased above this 1 lm limit.

The rate and extent of FFAs released from emulsions containing1%, 1.5% or 2% of LCT, MCT, SCT or LCT:SCT was measured for thenanoemulsions and conventional emulsions. Representative datafor the systems containing 1% MCT as the lipid phase is shown inFig. 4. The total amount of FFAs produced after 120 min of lipiddigestion for all the nanoemulsions and conventional emulsionsstudied are reported in Tables 2 and 3, respectively. We found nomajor differences between the digestion profiles of thenanoemulsions and conventional emulsions (Fig. 4). Previous stud-ies have found that the rate of lipid digestion increased when thedroplet size decreased, which was attributed to the increase in the

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Fig. 3. Effect of lipid concentration (1, 1.5 or 2 wt.%) on the rate and extent of lipid digestion, measured using an in vitro digestion method’’ (a) LCT; (b) MCT; (c) SCT. Resultswith ‘‘�’’ were statistically different from the 1% lipid samples. The reaction vessel contained 5 mg/ml bile, 1.6 mg/ml lipase, and 20 mmoles calcium.

Table 2Influence of lipid type and concentration on the bioaccessibility of curcumin and the total amount of FFA released after 120 min of digestion of oil-in-water nanoemulsions. Theasterix (�) show samples that are significantly different from the samples containing 1% lipid. The initial mean particle diameters (Z-average) are also shown for each sample.

Lipid concentration (%) LCT (Z-av. = 181 ± 9 nm) MCT (Z-av. = 174 ± 2 nm) SCT (Z-av. = 1981 ± 1061 nm) LCT:SCT (Z-av. = 182 ± 0 nm)

Bioavailability (%) FFA (%) Bioavailability (%) FFA (%) Bioavailability (%) FFA (%) Bioavailability (%) FFA (%)

1 20 ± 10 92 ± 9 8 ± 1 135 ± 9 1 ± 0 93 ± 3 3 ± 1 99 ± 81.5 40 ± 6⁄ 80 ± 9 20 ± 7⁄ 120 ± 6 1 ± 1 97 ± 15 9 ± 3⁄ 91 ± 22 41 ± 4⁄ 68 ± 6 58 ± 6⁄ 113 ± 6 1 ± 1 92 ± 8 20 ± 3⁄ 89 ± 3

804 K. Ahmed et al. / Food Chemistry 132 (2012) 799–807

surface area of the lipid phase exposed to the aqueous phase (Li &McClements, 2010; Lundin & Golding, 2009). The reason that wedid not see a similar effect maybe because different experimentalconditions were used: lipid concentrations; emulsifier type; andsimulated small intestinal fluid composition. For example, if thelipid concentration is already relatively high compared to the

amount of lipase present (i.e., so not all of the surfaces can be satu-rated with lipase), then increasing the surface area further may nothave a major impact on the digestion rate.

In general, the trends in the bioaccessibility of the curcumin weresimilar for the conventional emulsions (Table 3) as for thenanoemulsions (Table 2): at the highest lipid concentration,

0

20

40

60

80

100

120

140

0 1000 2000 3000 4000 5000 6000 7000 8000

% F

FA R

elea

sed

Digestion Time (s)

Nanoemulsion

Emulsion

Fig. 4. Comparison of in vitro digestion profiles of a nanoemulsion and aconventional emulsion containing 1 wt.% MCT.

Fig. 5. Creaming stability of conventional emulsions (left) and nanoemulsions(right) after 4 h storage at ambient temperature (before digestion).

K. Ahmed et al. / Food Chemistry 132 (2012) 799–807 805

curcumin bioaccessibility decreased in the following order MCT >LCT� SCT; curcumin bioaccessibility increased with increasing li-pid concentration, for emulsions containing MCT or LCT. Neverthe-less, there were some appreciable differences between thenanoemulsions and conventional emulsions. In particular, the cur-cumin bioaccessibility appeared to be appreciably higher whenLCT was used as the lipid phase in the conventional emulsions (Table3) than in the nanoemulsions (Table 2). This effect may have beenbecause a greater fraction of the LCT appeared to have been digestedin the conventional emulsions (Table 3) than in the nanoemulsions(Table 2), leading to a greater release of curcumin into the aqueousphase. The reason that a greater amount of lipid phase was digestedin a conventional emulsion than in an equivalent nanoemulsion iscurrently unknown. It is possible that the interfacial layer of globularproteins (b-lactoglobulin) that initially coated the lipid droplets hadsome impact on lipid digestion or release (Lee & McClements, 2010).The protein layer may have inhibited the ability of lipase to comeinto close contact with the lipids, or it may have inhibited the releaseof the digested lipids. Globular proteins tend to form a two-dimen-sional network of cross-linked molecules at oil–water interfaces. Asdigestion proceeds, this protein coating may have collapsed andformed a thick crinkled layer that trapped any remaining lipid, or li-pid digestion products, inside (Lee & McClements, 2010). This effectwould be more important for LCT than the other lipids because theyare least dispersible in water.

Previous studies have also found that the bioaccessibility of lipo-philic substances in lipid-based delivery systems is more closelycorrelated to the solubilisation capacity of the lipid digestion prod-ucts than to the initial droplet size (Porter, Kaukonen, Boyd, Ed-wards, & Charman, 2004; Sek, Boyd, Charman, & Porter, 2006).Our results suggest that there is no advantage, from a bioaccessibil-ity point of view, in using a nanoemulsion, rather than a

Table 3Influence of lipid type and concentration on the bioaccessibility of curcumin and the total aasterix (�) show samples that are significantly different from the samples containing 1% l

Lipid concentration (%) LCT (d43 = 20 ± 11 lm) MCT (d43 = 22 ± 6 lm)

Bioavailability (%) FFA (%) Bioavailability (%)

1 38 ± 22 98 ± 16 3 ± 01.5 56 ± 9 88 ± 8 17 ± 62 50 ± 10 73 ± 10 59 ± 5⁄

conventional emulsion, to encapsulate a highly lipophilic compo-nent such as curcumin. Indeed, there may even be an advantagein using larger droplet sizes in lipid-based delivery systems con-taining LCT as the lipid phase in terms of maximum achievable bio-accessibility. Nevertheless, there may be other advantages to usingnanoemulsions rather than conventional emulsions to encapsulateand deliver lipophilic functional components. During storage atambient temperatures, conventional emulsions were observed tocream within a few hours, while nanoemulsions were much morestable (Fig. 5). After 4 h storage, the conventional emulsion con-sisted of a yellowish cream layer on top of a slightly turbid serumlayer, whereas the nanoemulsion consisted of a homogeneous opti-cally opaque system.

Nanoemulsions containing LCT or MCT as the lipid phase werestored in a refrigerator (4 �C), for up to 10 days, without any notice-able phase separation, i.e., droplet creaming or curcumin sedimen-tation. On the other hand, nanoemulsions containing SCT showedphase separation within a few hours, which was due to the Ost-wald ripening instability mechanism discussed earlier. The stabil-ity of the SCT nanoemulsions could be greatly improved byincorporating 50% LCT into the lipid phase as described in an ear-lier section.

3.6. Influence of lipid type & concentration on micelle phase structure

Finally, we examined the influence of lipid type and concentra-tion on the structure of the micelle phase formed at the end of thelipid digestion process. The particle size distribution of the micellephase was measured using dynamic light scattering, and the meanparticle size (Z-average diameter) and polydispersity index (PDI)are reported in Table 4. The mean particle diameters were rela-tively large (d > 100 nm) and the distributions were relativelybroad (PDI > 0.2). Particles ranging in diameter from 10 to over1000 nm were observed in the particle size distributions, whichsuggested that the micelle phase contained a mixture of micellesand vesicles. The relatively large mean diameters can be attributed

mount of FFA released after 120 min of digestion of oil-in-water coarse emulsions. Theipid. The initial mean particle diameters (d43) are also shown for each sample.

SCT (d43 = 14 ± 2 lm) LCT:SCT (d43 = 19 ± 5 lm)

FFA (%) Bioavailability (%) FFA (%) Bioavailability (%) FFA (%)

139 ± 9 0 ± 0 110 ± 6 5 ± 3 104 ± 4132 ± 13 0 ± 0 110 ± 3 20 ± 6⁄ 103 ± 5123 ± 8 0 ± 0 107 ± 4 45 ± 7⁄ 103 ± 4

Table 4Particle size characteristics of the structures in the micelle phase collected from digested nanoemulsions prepared with different lipid types and concentrations.

Oil type 1% Lipid 1.5% Lipid 2% Lipid

Mean diameter (nm) PDI Mean diameter (nm) PDI Mean diameter (nm) PDI

LCT 99 ± 14 0.26 ± 0.03 113 ± 5 0.37 ± 0.18 161 ± 10 0.42 ± 0.09MCT 161 ± 39 0.51 ± 0.08 121 ± 13 0.20 ± 0.01 68 ± 1 0.30 ± 0.04LCT:SCT – – 131 ± 19 0.26 ± 0.04 120 ± 13 0.29 ± 0SCT – – – – – –

806 K. Ahmed et al. / Food Chemistry 132 (2012) 799–807

to the fact that larger particles scatter light much more effectivelythan small ones, and therefore tend to dominate the signal in dy-namic light scattering experiments.

As well as forming mixed micelles, bile salts, phospholipids, andlipid digestion products may assemble into various other types ofassociation colloids during the lipid digestion process, includingvesicles and lamellar structures. The concentration and structureof these association colloids is likely to change throughout thedigestion process and to depend on the molecular characteristicsof the carrier oil, which may affect their ability to incorporate lipo-philic bioactive ingredients such as curcumin. In future studies, ittherefore would be useful to characterise the concentration andstructure of the various association colloids formed throughoutthe lipid digestion process in more detail, e.g., using electronmicroscopy or X-ray diffraction methods.

4. Conclusions

This study has shown that physically stable curcumin-loadednanoemulsions can be prepared using a variety of different lipidphases: LCT; MCT; LCT:SCT. On the other hand, stable nanoemul-sions could not be prepared using pure SCT because of Ostwald rip-ening effects associated with the relatively high solubility of thislipid in water. The maximum amount of curcumin that could beloaded into the lipid phase was shown to decrease as the molecularweight of the lipid phase increased: SCT > MCT > LCT. The rate andextent of lipid digestion also depended on lipid type. The initialrate decreased in the following order SCT > MCT > LCT, while thetotal amount of FFA produced at the end of digestion decreasedin the following order MCT > SCT > LCT. The bioaccessibility of cur-cumin, assumed to be equal to the fraction of curcumin incorpo-rated into the micelle phase after digestion, decreased in thefollowing order MCT > LCT� SCT. The bioaccessibility of curcuminappeared to be slightly higher in conventional emulsions than innanoemulsions, but nanoemulsions had much better physical sta-bility. The bioaccessibility results are interpreted in terms of theinfluence of lipid type and concentration on: (i) the formation ofmixed micelles available to solubilise the digestion productsformed – an increase in the number of mixed micelles leads toan increase in bioaccessibility; (ii) the amount of undigested lipidremaining after the digestion period – an increase in the amountof undigested lipid leads to a decrease in bioaccessibility. These re-sults have important implications for the design and fabrication ofdelivery systems to encapsulate and release highly lipophilic func-tional ingredients.

Acknowledgement

This work was supported in part by an EPA-NSF-NIFA (AFRI)joint Grant (2010-05266) program, NIH Grant CA139174, a specialcall Grant from Massachusetts Center for Agriculture, and a CVIPGrant from the University of Massachusetts Amherst. The authorshave declared no conflict of interest.

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