Food Chemistry 139 (2013) Contents lists available at SciVerse ScienceDirect. Food Chemistry

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1 Food Chemistry 139 (213) Contents lists vilble t SciVerse ScienceDirect Food Chemistry journl homepge: Modulting b-crotene bioccessibility by controlling oil composition nd concentrtion in edible nnoemulsions L. Slvi-Trujillo, C. Qin b, O. Mrtín-Belloso, D.J. McClements b, Deprtment of Food Technology, University of Lleid, Lleid 25198, Spin b Deprtment of Food Science, University of Msschusetts, Amherst, MA 3, USA rticle info bstrct Article history: Received 5 November 212 Received in revised form 21 December 212 cepted 12 Februry 213 Avilble online 19 Februry 213 Keywords: b-crotene Nnoemulsions Oil type LCT MCT Ft content Digestibility Bioccessibility Diets rich in crotenoids hve been correlted with reduced risk of developing certin types of chronic diseses, but these bioctive components hve low intestinl bsorption due to their hydrophobic nture. The im of this work ws to study the effect of crrier oil composition (medium-chin triglyceride (MCT) to long-chin triglyceride (LCT) rtio) nd totl crrier oil concentrtion (1% or 4% w/w) on the physicl stbility, lipid digestibility nd bioccessibility of b-crotene-loded nnoemulsions, using simulted digestion process. Lipolysis led to n pprecible increse in the size nd negtive chrge on the prticles in the system. The totl frction of tricylglycerols converted to free ftty cids decresed s the percentge of LCT within the lipid phse incresed, prticulrly for the nnoemulsions with higher ft contents. There ws n increse in b-crotene bioccessibility s the LCT within the lipid phse incresed for low ft nnoemulsions, which ws ttributed to the incresed solubilistion cpcity of mixed micelles formed by LCT. b-crotene bioccessibility showed complex reltionship on LCT content for high ft nnoemulsions, due to the opposing effects of lipid digestion nd micelle solubilistion. These results my fcilitte the optimistion of delivery systems for lipophilic bioctive compounds for food or phrmceuticl pplictions. Ó 213 Elsevier Ltd. All rights reserved. 1. Introduction Corresponding uthor. Tel.: ; fx: E-mil ddress: mcclements@foodsci.umss.edu (D.J. McClements). b-crotene is one of the mjor crotenoids present in fruit nd vegetbles tht exhibits pro-vitmin A ctivity once ingested by humns (Johnson, 22). It is recognised s plying n importnt role in the prevention of certin humn diseses due to its ntioxidnt nd non-ntioxidnt ctivities (Bendich & Olson, 1989; Ro & Ro, 27). The ingestion of sufficient levels of b-crotene hs been relted to reduced risk of developing certin types of cncer, crdiovsculr diseses, photosensitivity diseses, nd ctrcts (Myne, 1996). Therefore, there is growing interest mong consumers nd the food industry to incorporte this compound into functionl foods nd beverges. The orl biovilbility of highly lipophilic compounds, such s crotenoids, plys mjor role in determining their overll biologicl ctivity. Studies hve shown tht the bsorption of ingested dietry crotenoids my be gretly improved when they re consumed in the presence of lipids (Mini et l., 29; Yeum & Russell, 22). Indeed, improvements in the orl biovilbilities of poorly wter-soluble drugs, when they re co-dministered with lipid excipients, re well-estblished in the phrmceuticl industry (Dhn & Hoffmn, 28; Huss, 27; Porter, Pouton, Cuine, & Chrmn, 28; Pouton & Porter, 28). Knowledge of the physicochemicl nd physiologicl processes tht occur within the gstrointestinl trct fter ingestion of dietry lipids cn be used to design effective delivery systems to improve the orl biovilbility of bioctive compounds in foods (McClements, Decker, Prk, & Weiss 29; Singh, Ye, & Horne 29). In mny emulsified foods, crotenoids re initilly present within triglyceride droplets dispersed within n queous medium (Rigotti, 27). As these foods pss through the digestive trct, the triglyceride molecules re digested, due to the ctions of gstric nd pncretic lipses, leding to the formtion of free ftty cids nd monocylglycerols. These lipid digestion products my remin t the ft droplet surfces, or they my move into the surrounding queous phse nd form colloidl structures with bile slts nd phospholipids ( mixed micelles ), cpble of solubilising lipophilic molecules. As result of these processes, crotenoid molecules my move from the originl oil droplets into the mixed micelle phse formed by digestion. After solubilistion, crotenoids re trnsported to the intestinl cells, where they re bsorbed nd then pcked into chylomicrons tht re secreted into the lymphtic system (Furr & Clrk, 1997; Rigotti 27; Yonekur & Ngo, 27). The bioccessibility nd biovilbility of crotenoids depend on severl fctors, including the type nd mount of crotenoids consumed, the composition nd structure of the food mtrix, nd /$ - see front mtter Ó 213 Elsevier Ltd. All rights reserved.

2 L. Slvi-Trujillo et l. / Food Chemistry 139 (213) the nture of the processing conditions used (Cstenmiller & West, 1998; Reboul et l. 26). Mny studies hve shown tht the totl mount of dietry ft consumed plys n importnt role in crotenoid bsorption, with the biovilbility normlly incresing with incresing ft content (Dimitrov et l., 1988; Jyrjn, Reddy, & Mohnrm, 198; Prince, Frisoli, Goetschkes, Stringhm, & LMurgli, 1991). There is lso strong evidence tht the nture of the tricylglycerol molecules present in food influences crotenoid biovilbility (Borel et l., 1998; Huo, Ferruzzi, Schwrtz, & Fill, 27). Recent studies hve shown tht the shorter the ftty cid chins in triglyceride molecules, the lower the bioccessibility of crotenoids (Huo et l., 27). Medium-chin triglycerides (MCT) re being used s ft source in severl food nd beverge products rther thn long-chin triglycerides (LCT) becuse they my lower cholesterol secretion, crdiovsculr disese risk, nd weight gin (Bch, Ingenbleek, & Frey, 1996; Mrten, Pfeuffer, & Schrezenmeir, 26). However, the potentil influence of the ftty cid chin length of co-ingested fts on the orl biovilbility of lipophilic bioctive molecules (such s crotenoids) my hve some undesirble consequences. A greter understnding of the interction between lipophilic bioctive compounds nd co-dministered crrier oils during pre-bsorptive intrluminl processing my led to more rtionl selection of the optimum crrier lipids for incorportion into lipid-bsed delivery systems (Sek, Porter, Kukonen, & Chrmn, 22). Recently, the interest in developing lipid-bsed delivery systems to incorporte crotenoids into functionl foods hs incresed (Boon, McClements, Weiss, & Decker, 21; Nik, Corredig, & Wright, 211; Nik, Lngmid, & Wright, 212; Nik, Wright, & Corredig, 211). Nnoemulsions re emerging s strong lterntive to conventionl emulsions to protect nd deliver lipophilic functionl components since they present enhnced physicl stbility, improved opticl clrity, nd incresed bioccessibility (McClements, 211; Nik et l., 212). Indeed, recent studies indicted tht nnoemulsions my improve the bioccessibility of lipophilic bioctive compounds such s b-crotene (Nik, Corredig, et l., 211; Nik, Wright, et l., 211; Nik et l., 212; Wng, Liu, Mei, Nkjim, & Yin, 212). The purpose of the present work ws to study the potentil biologicl fte of b-crotene-loded nnoemulsions using simulted gstrointestinl conditions (Li, Hu, & McClements, 211; Li & McClements, 21). In prticulr, our object ws to determine the influence of crrier lipid concentrtion nd type (MCT nd LCT) on triglyceride digestibility nd b-crotene bioccessibility. 2. Mteril nd methods 2.1. Mterils b-crotene, lipse (porcine pncres, type II, L3126, Btch # 96K747), bile extrct nd clcium chloride were purchsed from Sigm Aldrich (St. Louis, MO). Miglyol 812 (MCT) nd corn oil (LCT) were purchsed from SASOL (Houston, TX) nd from locl supermrket, respectively. All queous solutions were prepred using purified wter from Mili-Q filtrtion system Methods Nnoemulsion formtion An oil phse ws prepred by dispersing.5% (w/w) b-crotene into crrier lipid (MCT or LCT) nd then sonicting for 1 min nd pplying mild heting (<5 C for 5 min) so tht complete dissolution ws chieved. An queous phse contining surfctnt ws prepred by dispersing 1.5% (w/w) Tween 2 in queous buffer solution (1. mm phosphte buffer,.1% (w/w) sodium zide, ph 7.). A corse emulsion ws prepred by blending the oil phse (4% w/w) nd queous phse (96% w/w) together using highsher mixer t 1, rpm for 2 min. Nnoemulsions were then obtined by pssing the corse emulsion three-times through microfluidizer (Model 11, Microfluidics, Newton, MA) working t 9 psi. For some experiments, nnoemulsions were prepred from crrier lipids tht consisted of different mss rtios of MCTto-LCT (w/w): :, 75:25, 5:5, 25:75 or :. The finl systems contined 4% (w/w) totl oil nd were referred to s highft nnoemulsions. We lso crried out experiments on lowft nnoemulsions contining 1% (w/w) oil by diluting the highft nnoemulsions with buffer solution Nnoemulsion chrcteristion The men prticle chrge (f-potentil) nd size (men volume dimeter) of initil nnoemulsions, rw digest, nd mixed micelles were mesured. The prticle size distribution ws mesured by dynmic light scttering (DLS) (Zetsizer NnoZS, Mlvern Instruments Ltd., Worcestershire, UK) t wvelength of 633 nm nd temperture of 25 C using bcksctter detector (173 ). Smples were diluted prior to nlysis with buffer solution (1:1) to void multiple scttering effects nd to chieve n ttenution of the lser bem between 5 nd 1. The f-potentil of the prticles ws mesured by phse-nlysis light scttering (Zetsizer NnoZS, Mlvern Instruments Ltd, Worcestershire, UK). Smples were diluted with buffer solution (1:1) nd plced in cpillry cell equipped with two electrodes to ssess the electrophoretic mobility of the prticles In vitro digestion The digestion of the lipid droplets, under simulted smll intestinl conditions, ws monitored using ph-stt device (Metrohm USA, Riverview, FL) (McClements & Li, 21). An liquot of 3 ml of the nnoemulsions ws plced in temperture-controlled (37 C) chmber nd the ph ws set t 7. using NOH solution. Then, 4 ml of bile extrct (46.87 mg/ml) nd 1 ml of clcium chloride (11 mg/ml) solutions, dissolved in phosphte buffer, were dded to the smple nd the ph ws djusted to 7., if necessry. 2.5 ml of freshly prepred lipse suspension (24 mg/ml), dissolved in phosphte buffer, ws then dded to the mixture. The ph of the mixture ws monitored nd the volume of NOH (ml) necessry to neutrlise the free ftty cids (FFA) relesed from lipid digestion, so s to mintin the ph t 7., ws recorded during 2 h. The concentrtion of NOH used ws.25 M nd 1 M for the emulsions contining 1% (w/w) nd 4% (w/w) of oil phse, respectively. The following eqution ws used to clculte the percentge of FFAs relesed during the digestion process, ssuming tht ech molecule of triglyceride (TAG) genertes two molecules of FFA when completely digested (Mu & Høy, 24): FFAðtÞ ¼ V NOHðtÞC NOH M Oil 2 m Oil where V NOH (t) is the volume of NOH solution required to neutrlise the FFAs produced t digestion time t (L), C NOH is the molrity of the NOH solution used to titrte the smple (M), M oil is the moleculr weight of the oil (g mol 1 ), nd m oil is the totl mss of oil initilly present in the incubtion cell (g). The moleculr weights of the corn oil nd the MCT oil were tken to be 8 g mol 1 nd 4 g mol 1, respectively. The moleculr weight of the oil phse in the mixed emulsions ws clculted proportionlly to the mss rtio of the two oils in the blend. Blnks were crried out in the bsence of oil in the smples nd subtrcted from the reported vlues. ð1þ

3 88 L. Slvi-Trujillo et l. / Food Chemistry 139 (213) Bioccessibility determintion The bioccessibility of b-crotene in the nnoemulsions ws determined fter the in vitro digestion process using method described previously (Qin, Decker, Xio, & McClements, 212). An liquot of the rw digest ws centrifuged (CL1 centrifuge, Thermo Scientific) t 4 rpm for 4 min t 25 C. The superntnt ws collected nd ws tken to be the micelle frction in which the bioctive compound ws solubilised. In some smples, top lyer of tiny droplets of non-digested oil ws observed nd ws completely removed from the micelle frction, prior to nlysis, with the id of glss pipette. The micelle phse ws then collected using nother glss pipette. Aliquots of 5 ml of rw digest or micelle frction were mixed with 5 ml of chloroform, vortexed nd centrifuged t 175 rpm for 1 min t 25 C. The bottom lyer, with the solubilised b-crotene, ws collected while the top lyer ws mixed with 5 ml of chloroform nd the sme procedure ws followed. The bottom chloroform lyer ws dded to the previous one nd the bsorbnce ws mesured t 45 nm using spectrophotometer (Ultrospec 3 pro, GE Helth Sciences, USA). A cuvette contining pure chloroform ws used s reference cell. The concentrtion of b-crotene extrcted from smple ws determined from clibrtion curve of bsorbnce versus b-crotene concentrtion in chloroform. The bioccessibility ws clculted using the following eqution: C Micelle Bioccessibility ¼ ð2þ C RwDigest Where, C micelle nd C RwDigest re the concentrtions of b-crotene in the micelle frction nd in the overll smple (rw digest) fter the ph-stt experiment, respectively Sttisticl nlysis All experiments were ssyed in duplicte, nd results were expressed s the men nd the stndrd devition. Sttisticl nlysis softwre (MP 8 Mcintosh Pckge, SAS Institute Inc.) ws used to perform the nlysis of vrince. The Student s t-test ws run to determine significnt differences t 5% significnce level (p <.5). 3. Results nd discussion 3.1. Influence of oil composition on prticle chrcteristics Initilly, we exmined the influence of oil phse composition on the physicl properties of the lipid nnoprticles in the low-ft (1% w/w) nnoemulsions. The men volume dimeter of the prticles ws mesured in the initil nnoemulsions, in the overll digest fter lipid digestion, nd in the micelle frction collected from the digest (Fig. 1). The men dimeter of the initil nnoemulsions incresed, from round 146 to 415 nm, s the LCT content in the mixed oil phse (MCT:LCT) incresed from % to % (w/w). The reson for this increse in droplet size cn be ttributed to chnges in the dispersed phse viscosity of the nnoemulsions (McClements, 25). The efficiency of droplet disruption within high pressure homogenizer usully increses s the viscosity of the disperse phse decreses. LCT hs n pprecibly higher viscosity thn MCT, nd therefore droplet brekup during homogenistion becomes less efficient s the LCT frction increses, leding to lrger droplets. The men prticle size mesured fter the nnoemulsions hd been subjected to in vitro digestion incresed pprecibly regrdless of the initil oil phse composition (Fig. 1). The droplet size distribution of the digest smples ws monomodl for ll oil phse compositions with the exception of the system formulted with % LCT, which ws bimodl (Fig. 2). The increse in Men Volume Dimeter (nm) 1 1 Bb Bb Bb Initil Rw Digest Micelle : 75:25 5:5 25:75 : Fig. 1. Volume men dimeter (nm) of nnoemulsions formulted with different MCT:LCT rtios before nd fter simulted gstrointestinl model: (i) initilly; (ii) totl digest; (iii) micelle phse. The finl lipid content in the digest ws 1% ( lowft ). Different cpitl letters men significnt differences (p <.5) in droplet dimeters for different MCT:LCT rtios, wheres different lowercse letters men significnt differences for similr MCT:LCT rtios. (A) Reltive Volume (%) (B) Reltive Volume (%) : Ab Prticle Dimeter (nm) Ab : 25:75 5:5 75:25 : 1 1 Prticle Dimeter (nm) : 25:75 5:5 75:25 Fig. 2. Effect of MCT:LCT rtio on the prticle size distribution of smples collected fter nnoemulsions hd been subjected to n in vitro digestion model: (A) totl digest; (B) micelle phse. The finl lipid content in the digest ws 1% ( low-ft ).

4 L. Slvi-Trujillo et l. / Food Chemistry 139 (213) droplet size fter simulted digestion conditions my be due to floccultion nd/or colescence phenomen depending on the nture of the system (Mun, Decker, & McClements, 27). These destbilistion mechnisms my be promoted by chnges in the composition nd properties of the lipid droplet surfces within the gstrointestinl trct, e.g. due to displcement or digestion of the originl emulsifiers (Grgouri, Julien, Bois, Verger, & Srd, 1983; Singh et l., 29). Moreover, the lipse ctivity t the oil droplet surfce my lso contribute to chnges in interfcil properties leding to decrese in colescence stbility (Reis, Holmberg, Wtzke, Leser, & Miller, 29). It is known tht complex ssocition colloids, such s mixed micelles, vesicles nd lmellr structures, re formed in the digestion medium s result of the interctions of lipid digestion products, bile slts, phospholipids, nd clcium (Kossen, Boyd, Porter, & Chrmn, 23). In this study, we found tht the prticles in the micelle phse hd monomodl distribution (Fig. 2b) with volume men dimeter < 1 nm (Fig. 1), which suggests tht they were predominntly mixed micelles. The prticle size of the mixed micelles did not depend strongly on the initil oil phse composition (Figs. 1 nd 2b). Visully, the micelle frction of ll the smples ws lmost trnsprent, which cn be ttributed to wek light scttering by the smll prticles present. Recent studies, using curcumin-loded nnoemulsions, lso found tht the size of the prticles in the micelle frction (1 nm) did not depend strongly on the initil oil phse composition (MCT or LCT) (Ahmed, Li, McClements, & Xio, 212). On the other hnd, other reserchers hve reported slight increse in micelle size (from 3 to 7 nm) s the chin length of the ftty cids in the lipid phse incresed (Christensen, Schultz, Mollgrd, Kristensen, & Mullertz, 24). The f-potentils of the prticles in the initil nnoemulsions, the overll digest, nd the micelle frction were lso mesured (Fig. 3). The negtive chrge of the initil nnoemulsions incresed slightly with incresing LCT content in the oil phse, with the vlues going from 4.7 to 5.8 mv. Even though the initil nnoemulsions were stbilized by non-ionic surfctnt, (Tween 2) it hs been reported tht they cn give negtive chrge to oil droplets, e.g. due to preferentil dsorption of hydroxyl ions from the queous phse or due to the presence of nionic impurities : 75:25 5:5 25:75 : such s free ftty cids in the surfctnt or oil phses (McClements, 25). It is possible tht the LCT contined more nionic impurities thn the MCT, which ment tht the droplets were slightly more negtively chrged. The prticles in the digest ( 15 nd 2 mv) hd n pprecibly higher negtive chrge thn those in the initil nnoemulsions ( 4 nd 5 mv). The mgnitude of the negtive chrge on the prticles in the digest nd in the micelle phse incresed pprecibly s the frction of LCT in the initil oil phse incresed (Fig. 3). The higher negtive chrge on the prticles in the smples contining higher mount of LCT might be due to severl resons. When lipid droplets re exposed to simulted intestinl conditions, nionic bile slts my displce the originl surfctnt molecules from their surfces, thereby, ltering their surfce chrge (Wickhm, Grrood, Leney, Wilson, & Fillery-Trvis, 1998). In ddition, the hydrolysis of triglycerides by pncretic lipse t the surfce of the lipid droplets, leds to the relese of nionic free ftty cids, which lso lters the surfce chrge (McClements et l., 29; Singh et l., 29). It is known tht medium chin ftty cids produced during digestion of MCT re ble to rpidly migrte into the surrounding queous phse; wheres, long chin ftty cids produced by LCT tend to ccumulte t the oil wter interfce (Ahmed et l., 212; Li et l., 211; Pouton & Porter, 28). Consequently, more nionic ftty cids my hve remined t the prticle surfces for the systems contining LCT thn those contining MCT Influence of oil composition on triglyceride digestibility The influence of initil oil composition (MCT:LCT) on the rte nd extent of triglyceride digestion in the nnoemulsions with low ft content (1% w/w) using the ph-stt method (Fig. 4) ws exmined. For ll nnoemulsions studied, the mount of free ftty cids (FFA) liberted incresed steeply during the first 4 min fter dding pncretic lipse to the digestion medium. The mount of FFAs relesed incresed slower fter 4 min of digestion. The totl mount of FFAs produced fter 2 h of digestion decresed s the frction of LCT in the initil oil phse incresed. For exmple, the totl mount of FFA relesed decresed from 123% to 88% s the LCT concentrtion incresed from % to % (Fig. 4). There re number of resons why the finl mount of FFA relesed ws greter thn % for the MCT systems. First, there might hve been some conversion of monocylglycerols into glycerol nd FFAs ζ-potentil (mv) Ab AB Bb CD BCc ABb BC CDc BCb Initil Rw Digest Micelle D Db Cb Fig. 3. f-potentil (mv) of initil nnoemulsions formulted with different MCT:LCT rtios before nd fter simulted gstrointestinl model: (i) initilly; (ii) totl digest; (iii) micelle phse. The finl lipid content in the digest ws 1% ( low-ft ). Different cpitl letters men significnt differences (p <.5) in droplet dimeters for different MCT:LCT rtios, wheres different lowercse letters men significnt differences for similr MCT:LCT rtios. Free Ftty ids Relesed (%) : 75:25 5:5 25:75 : Digestion Time (min) Fig. 4. Influence of MCT:LCT rtio on the percentge of free ftty cids (FFA%) relesed from nnoemulsions formulted with different MCT:LCT rtios. The finl lipid content in the digestion medium ws 1% ( low-ft ).

5 882 L. Slvi-Trujillo et l. / Food Chemistry 139 (213) (Crey, Smll, & Bliss, 1983), which ws not tken into ccount in the clcultions. Second, there my hve been other components in the systems tht were hydrolysed nd contributed to the ph stt mesurements, such s phospholipids, proteins, or surfctnts in the digestion medium (Li et l., 211). It is known tht medium chin ftty cids re ble to migrte rpidly to the surrounding queous phse; wheres, long chin ftty cids tend to ccumulte t the oil wter interfce nd therefore inhibit lipse ctivity, which would ccount for the observed decrese in digestibility t high LCT levels (Dhn & Hoffmn, 28; Sek et l., 22). Our results re in greement with other recent studies, tht hve reported higher digestibility of MCT compred to LCT when they re incorported in emulsions or nnoemulsions (Ahmed et l., 212; Li & Mcclements, 21; Li et l., 211) Influence of oil composition on b-crotene bioccessibility Concentrtion (%) c A bc AB b AB Bioccessibility FFA Relesed : 75:25 5:5 25:75 : Fig. 5. Effect of MCT:LCT rtio on the percentge of free ftty cid relesed (FFA%) fter 12 min of digestion nd on the bioccessibility (%) of b-crotene incorported in nnoemulsions. The finl lipid content in the digestion medium ws 1% ( lowft ). Different cpitl letters men significnt differences (p <.5) in n the percentge of free ftty cids relesed fter 12 min. Different lowercse letters men significnt differences (p <.5) on the bioccessibility of b-crotene. b B C The influence of oil phse composition on b-crotene bioccessibility in the low ft (1% w/w) nnoemulsions ws determined by mesuring the b-crotene concentrtions in the micelle phse nd in the overll digest (Fig. 5). As comprison, the percentge of FFAs relesed by the end of the lipid digestion step is plotted on the sme figure. A significnt increse in b-crotene bioccessibility ws observed s the frction of LCT in the initil oil phse incresed. For exmple, the bioccessibility of b-crotene incresed from round 14% to 86% s the LCT percentge in the oil phse incresed from % to %. The bsorption of dietry crotenoids in the humn body hs been reported to be similr to tht of triglycerides (Rigotti, 27). Crotenoids incorported within oil droplets re trnsferred into mixed micelles formed by bile slts nd lipolysis products fter the triglycerides re digested (Yonekur & Ngo, 27). Our study, using nnoemulsion-bsed delivery systems, therefore supports previous work tht hs shown tht the presence of ft is essentil for crotenoid solubilistion nd bsorption (Furr & Clrk, 1997), nd tht the mount of lipophilic compounds solubilised in mixed micelles tends to increse with incresing ftty cid chin length (Christensen et l., 24). This ltter effect my be relted to the fct tht the solubilistion cpcity of mixed micelle depends on how esily lipophilic compound cn be incorported into its structure (Lher & Brrowmn, 1983). One would expect tht reltively long chin, non-polr molecules like b-crotene would be more esily ccommodted into mixed micelles formed by long chin ftty cids thn those formed by medium chin ftty cids. Hving sid this, our dynmic light scttering mesurements suggested tht the mixed micelles formed by digestion of different kinds of triglycerides were firly similr in dimensions (Fig. 1). This my hve been due to limittions of the dynmic light scttering method when used to nlyse system tht contins vrious kinds of colloidl structures, such s mixed micelles, vesicles, nd lmellr structures. Alterntively, the colloidl structures tht solubilised the b-crotene my hve been different in systems contining different types of oil phses. Studies with phrmceuticl preprtions hve shown tht hydrophobic drugs dministered with LCT re solubilised within mixed micelles fter digestion, wheres those dministered with MCT re trpped within vesicles (Kossen et l., 23). It is therefore possible tht some or ll of the reltively lrge vesicles in the digest were removed during the centrifugtion process, thereby reducing the mount of b-crotene mesured in the micelle phse. Other recent studies in our lbortory hve reported lower bioccessibility of lipophilic bioctive compounds encpsulted within MCT oils thn in LCT oils (Ahmed et l., 212; Qin et l., 212) Effect of high ft content on digestion nd bioccessibility Finlly, the influence of reltively high initil ft content on triglyceride digestibility nd b-crotene bioccessibility ws exmined. This ws chieved by using nnoemulsions contining 4% (w/ w) totl oil phse in the digestion medium, rther thn 1% (w/w). This study ws crried out becuse the totl mount of ft present in the humn gstrointestinl trct my chnge depending on the type nd mount of food consumed. In ddition, this study lso highlights the importnce of using n pproprite ft level in in vitro digestion studies. The overll chnges in prticle size nd chrge for the high-ft nnoemulsions were similr to those for the low-ft nnoemulsions (dt not shown). The rte nd extent of free ftty cid relese ws much more dependent on the initil oil phse composition for the systems contining high ft contents (Fig. 6) thn those contining low ft contents (Fig. 4). For exmple, the finl percentge of FFAs relesed fter two hours of digestion decresed from 89% to 34% for the high ft system nd from 123% to 88% for the low ft system s the LCT content in the oil phse Free Ftty ids Relesed (%) : 75:25 5:5 25:75 : Digestion Time (min) Fig. 6. Influence of MCT:LCT rtio on the percentge of free ftty cids (FFA%) relesed from nnoemulsions formulted with different MCT:LCT rtios. The finl lipid content in the digestion medium ws 4% ( high-ft ).

6 L. Slvi-Trujillo et l. / Food Chemistry 139 (213) incresed from % to %. Overll, the frction of FFAs relesed from the triglycerides ws considerbly lower for the high-ft nnoemulsions thn the low-ft ones, which hs lso been reported by other uthors (Li et l., 211). This phenomenon might occur due to severl resons ssocited with the fct tht the composition of the simulted smll intestinl fluids ws kept constnt s the ft content ws incresed. First, the rtio of lipse-to-triglyceride in the digestion medium decreses s the totl ft content incresed, nd so the enzyme ctivity of the lipse my hve decresed (Li et l., 211). Second, the rtio of bile slts-to-triglyceride digestion products decresed, nd so there my not hve been sufficient mixed micelles formed to solubilise ll of the FFAs relesed from the high ft systems (Li et l., 211). Third, the rtio of clcium-to-triglyceride digestion products lso decresed nd so there my not hve been sufficient clcium ions present to precipitte long chin ftty cids generted t the droplet surfces (Li et l., 211). The removl of FFAs from lipid droplet surfces during digestion by solubilistion in bile slts or precipittion by clcium ions is known to ply n importnt role in promoting full hydrolysis of triglycerides (Dhn & Hoffmn, 28; Pouton & Porter, 28). It should be noted tht even though the frction of FFAs relesed ws lower in the high ft systems, the bsolute mount of FFAs relesed would be higher becuse of the higher initil concentrtion of triglycerides present. The influence of oil phse composition on b-crotene bioccessibility nd the finl mount of FFAs relesed fter digestion is compred in Fig. 7 for the high ft systems. The behviour of the high ft system ws quite different from tht of the low ft system (Fig. 5). The totl mount of FFAs relesed decresed pprecibly with incresing LCT in the lipid phse. As mentioned erlier, this effect my hve been due to the lck of sufficient mounts of bile slts nd clcium ions in the simulted smll intestinl fluids to solubilise nd precipitte the long chin FFAs generted t the droplet surfces during lipid digestion. For the pure MCT system, the bioccessibility of the crotenoid ws reltively high (84%) in the high ft system (Fig. 7) wheres it ws reltively low (14%) in the low ft system (Fig. 5). This effect my be ttributed to the fct tht greter bsolute mount of mixed micelles nd other ssocition colloids cpble of solubilising b-crotene were formed fter the high ft systems were digested. However this cnnot be the only reson since the bioccessibility incresed sixfold (from 14% to 84%) when the totl initil ft content incresed fourfold (from 1% to 4%). There my, therefore, hve lso been chnge in the structure of the ssocition colloids formed in the micelle phse fter digestion of the high ft systems, which ltered their solubilistion cpcities. In the high ft systems, b-crotene bioccessibility decresed s the LCT content in the oil phse incresed from % to 5%, but then it incresed when the LCT content ws incresed from 5% to % (Fig. 7). This behviour differs from the progressive increse in b- crotene bioccessibility observed in the low ft systems with incresing LCT content (Fig. 5). We ttribute the reltively complex behviour observed in the high ft systems to number of competing physicochemicl phenomen. First, the totl mount of nondigested triglycerides in the system increses s the LCT content increses, which my inhibit the incorportion of b-crotene into mixed micelles becuse crotenoids were trpped within the non-digested oil droplets (Nik, Corredig, et l., 211). This effect would led to decrese in bioccessibility with incresing LCT content, nd my ccount for the decrese observed between nd 5% LCT (Fig. 7). Second, the solubilistion cpcity of the mixed micelles increses s the LCT content within them increses (Section 3.3). This effect would led to n increse in bioccessibility with incresing LCT content, nd my ccount for the increse observed between 5% nd % LCT (Fig. 7). The fct tht the b-crotene bioccessibility ws reltively high (79%) in the pure LCT system contining high ft content, even though n pprecible mount of the triglycerides hd not been digested (67%) suggests tht crotenoid molecules were ble to move from the non-digested oil phse nd into the micelle phse. Presumbly, this occurred becuse of the reltively high bsolute concentrtion nd solubilistion cpcity of the mixed micelles formed t high LCT levels. These experiments highlight the importnce of using pproprite levels of ft in simulted digestion studies, nd lso highlight the potentil importnce of non-digested ft on the biovilbility of lipophilic substnces. However, it should be noted tht in prctise, the humn body cn respond to higher ft contents by slowing down the emptying of the stomch, or by secreting more bile slts nd lipse into the smll intestine, nd therefore these results should be treted with some reservtions. Concentrtion (%) A Bioccessibility FFA Relesed b B c : 75:25 5:5 25:75 : C Fig. 7. Effect of MCT:LCT rtio on the percentge of free ftty cid relesed (FFA%) fter 12 min of digestion nd on the bioccessibility (%) of b-crotene incorported in nnoemulsions. The finl lipid content in the digestion medium ws 4% ( highft ). Different cpitl letters men significnt differences (p <.5) in the percentge of free ftty cids relesed fter 12 min. Different lowercse letters men significnt differences (p <.5) on the bioccessibility of b-crotene. D E 4. Conclusions We hve shown tht bioctive crotenoid (b-crotene) cn successfully be incorported into food-grde nnoemulsions with different oil phse compositions. Nnoemulsions contining different rtios of MCT nd LCT in the oil phse behved firly similrly in simulted digestion model: there ws n pprecible increse in men size nd negtive chrge of the prticles fter digestion. Nevertheless, there were some differences in the physicl chrcteristics of the prticles produced by digestion depending on initil oil composition. The negtive chrge on the prticles in the overll digest nd in the mixed micelle phse incresed s the LCT content incresed, which ws ttributed to the ccumultion of long-chin ftty cids t the prticle surfces. The overll extent of triglyceride hydrolysis fter digestion ws lso influenced by oil composition, decresing with incresing LCT in the lipid phse for both low-ft (1%) nd high-ft (4%) nnoemulsions. The influence of oil composition on b-crotene bioccessibility depended on the initil totl ft content. For low-ft nnoemulsions, the bioccessibility progressively incresed with incresing LCT content, which ws ttributed to the greter solubilistion cpcity of mixed micelles contining long chin ftty cids. For high-ft nnoemulsions, the bioccessibility

7 884 L. Slvi-Trujillo et l. / Food Chemistry 139 (213) decresed nd then incresed with incresing LCT content, which ws ttributed to chnges in the mount of non-digested oil present, s well s in the solubilistion cpcity of the micelle phse. Overll, this study provides vluble informtion tht cn be used to design nutrceuticl delivery systems tht optimise the bioccessibility of lipophilic compounds. For exmple, high b-crotene bioccessibility cn be chieved either by using low-ft LCT nnoemulsions or high-ft MCT nnoemulsions. The bioccessibility in mixed oil nnoemulsions ppers to be highly dependent on oil composition. 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