Food Chemistry 138 (2013) 2327–2337

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Food Chemistry

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Interaction of curcumin with phosphocasein micelles processed or notby dynamic high-pressure

Amal Benzaria a, Marc Maresca b, Nadira Taieb c, Eliane Dumay a,⇑a Université Montpellier 2, UMR 1208, Ingénierie des Agropolymères et Technologies Emergentes, Equipe de Biochimie et Technologie Alimentaires cc023,2, Place Eugène Bataillon, 34095 Montpellier Cedex 5, Franceb Aix-Marseille Université, CNRS, iSm2, UMR 7313, Avenue Normandie-Niemen, 13397 Marseille Cedex 20, Francec Aix-Marseille Université, Laboratoire PPSN, EA 4674, Equipe IMSM, Avenue Normandie-Niemen, 13397 Marseille cedex 20, France

a r t i c l e i n f o a b s t r a c t

Article history:Received 24 April 2012Received in revised form 29 November 2012Accepted 3 December 2012Available online 27 December 2012

Keywords:PhosphocaseinsCurcuminDynamic high-pressureFluorescenceIn vitro digestionCell metabolic activityCell membrane integrity

0308-8146/$ - see front matter � 2012 Elsevier Ltd. Ahttp://dx.doi.org/10.1016/j.foodchem.2012.12.017

⇑ Corresponding author. Tel.: +33 467 143 351; faxE-mail address: Eliane.Dumay@univ-montp2.fr (E.

The binding of curcumin to native-like phosphocaseins (PC) dispersed in simulated milk ultrafiltrate atpH 6.6 was assessed by fluorescence spectrophotometry. Curcumin binds to native-like PC micelles with�1 binding site per casein molecule, and a binding constant of 0.6–5.6 � 104 M�1. Dynamic high pressure(or ultra-high pressure homogenisation, UHPH) at 200 MPa did not affect the binding parameters of cur-cumin to processed PC. UHPH-processing of PC dispersions at 300 MPa was followed by a slight but sig-nificant (p = 0.05) increase in the binding constant of curcumin to processed PC, which may result fromthe significant UHPH-induced dissociation of initial PC micelles into neo-micelles of smaller sizes, andfrom the corresponding 1.5–2-fold increase in micelle surface area. PC–curcumin complexes were resis-tant to pepsin but were degraded by pancreatin, providing the possibility of a spatiotemporally controlledrelease and protection of bound biomolecules. UHPH-processed PC did not induce TC7-cell damage ormajor inflammation as assessed by LDH release or IL-8 secretion, respectively, compared with native-likePC. PC micelles could provide a valuable submicron system to vectorise drugs and nutrients.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

In general, oral delivery is one of the most convenient ways toadministrate drugs or other bio-compounds to patients due to itssimplicity and safety compared to injection delivery. However, oralbioavailability is poor for water-insoluble hydrophobic compoundsand for labile biomolecules susceptible to alteration by the gastro-intestinal environment (Acosta, 2009). In addition, interactions be-tween drugs and food matrix components may also decrease drugbioavailability.

Strategies to improve oral delivery of drugs, vitamins or nutra-ceuticals with low oral bioavailability include their protection byencapsulation and/or incorporation into liposomes, oil droplets,matrix particles or molecular assemblies (Chen, Remondetto, &Subirade, 2006; Gonnet, Lethuaut, & Boury, 2010; Patel & Velikov,2011). Thus, encapsulation of bio-compounds into nano/submicronparticles increased their oral delivery through higher solubility andresistance to the gastrointestinal environment. Loading curcumininto polylactic-co-glycolic acid (PLGA) particles (<200 nm) im-proved its dispersibility in aqueous buffer and rate of release inrat intestinal juice, enhancing oral bioavailability by 5.6-fold com-

ll rights reserved.

: +33 467 143 352.Dumay).

pared to non-encapsulated curcumin (Xie et al., 2011). Encapsula-tion/inclusion into nano/submicron particles could also protectbiocompounds against oxidation. Indeed, Zimet, Rosenberg, andLivney (2011) showed that sodium caseinate binds omega-3 fattyacid (DHA) allowing some protective effect against oxidationthroughout shelf-life studies. Chitosan–b-lactoglobulin particlesresistant to pepsin degradation have been proposed as oral carriersfor nutraceutical compounds (Chen et al., 2006).

Although nanoparticles seem promising as drug delivery sys-tems, debate exists regarding their environmental hazard andaccumulation, particularly for metal-containing systems (Elsaesser& Howard, 2012; Sharifi et al., 2012). To overcome this problem,safer and biodegradable nano/submicron particles are needed.Such particles can be formulated from synthetic compounds, suchas polyethylene glycol (PEG), polycyanoacrylates or PLGA, or fromnatural macromolecules, such as polysaccharides (chitosan, gumarabic, starch, pectin) or proteins (albumin, gelatin, whey proteins,zein) (Ko & Gunasekaran, 2006; MaHam, Tang, Wu, Wang, & Lin,2009; Ofokansi, Winter, Fricker, & Coester, 2010; Patel, Hu, Tiwari,& Velikov, 2010; Sneharani, Karakkat, Singh, & Rao, 2010).

Caseins could also be used as submicron carriers (Elzoghby, AboEl-Fotoh, & Elgindy, 2011; Livney, 2010; Sahu, Kasoju, & Bora,2008; Semo, Kesselman, Danino, & Livney, 2007). Casein micellesare large self-assemblies with high hydrophobic character and dis-play an open fully hydrated structure containing 3.7–4 g water/

2328 A. Benzaria et al. / Food Chemistry 138 (2013) 2327–2337

g protein (Horne, 2002; McMahon & Oommen, 2008; Qi, Brown, &Farrell, 2001). Sodium caseinate, aS1-casein or milk casein micellesbind hydrophobic compounds, such as curcumin (Sahu et al., 2008;Sneharani, Singh, & Rao, 2009), vitamin D (Semo et al., 2007) orDHA (Zimet et al., 2011). Similarly, casein microspheres (>1 lm)cross-linked using glutaraldehyde improved oral delivery of anti-cancer drugs (Knepp et al., 1993; Willmott, Magee, Cummings, Hal-bert, & Smyth, 1992). Native-like phosphocasein micelles preparedon an industrial scale by microfiltration plus diafiltration at mildtemperatures, while maintaining the milk mineral environment,have not been previously evaluated and appear to be suitable fororal delivery applications. In addition, phosphocasein micelleshave been ingested by humans for centuries without any knownside effects.

In a previous study Regnault, Thiebaud, Dumay, and Cheftel(2004) observed dissociation/reassociation phenomena of phos-phocasein micelles induced by isostatic high-pressure (HP) atambient or cold temperature. Micelles were dissociated into neo-micelles with diameters half those of the original micelles. Similardissociation phenomena may also be induced by ultra-high pres-sure homogenisation (UHPH, also called dynamic high-pressure),a physical process studied in recent years as a means to break par-ticles down to the submicron range as well as to inactivate micro-organisms (Dumay et al., in press). Trying to include hydrophobiccompounds in native-like or pressure-processed phosphocaseinmicelles appeared to be beneficial.

In the present study, we investigated the binding of curcumin tophosphocasein micelles as a potential colloidal delivery system inhumans. Curcumin binding to phosphocasein micelles, native-likeor UHPH-processed, was followed by fluorescence spectroscopy.In addition, experiments involving biological tools were per-formed: in vitro simulated gastric or intestinal digestion, uptakeof bound curcumin by TC7-cell monolayers, safety evaluation ofphosphocaseins on intestinal epithelial cells. The behaviour of na-tive-like or UHPH-processed phosphocaseins was compared.

2. Materials and methods

2.1. Materials

Native-like phosphocasein (PC) was industrially prepared byIngredia Dairy Ingredients (Arras, France) at mild temperature bymicrofiltration and diafiltration using milk ultrafiltrate to maintaina quasi-native character to casein micelles. The PC powder (Pro-milk 852B) obtained by spray-drying contained 95% (w/w) dry sol-ids and, on a dry weight basis, 87% total proteins (�80%phosphocaseins), 0.8% fat, 5% lactose and 7.3% minerals including�2% calcium.

Dulbecco’s modified Eagle medium (DMEM) with Glutamax,Dulbecco’s phosphate buffer saline (DPBS) ± Ca2+ and Mg2+, Hank’sBalanced Salt Solution (HBSS), penicillin–streptomycin mixture,non-essential amino acid and foetal bovine serum (FBS) were ob-tained from Invitrogen (Villebon-sur-Yvette, France). Human inter-leukin-8 ELISA kit came from BD-BioSciences (Le Pont-de-Claix,France). Curcumin, pepsin, pancreatin, b-nicotinamide adeninedinucleotide hydrate (NAD), Trizma-base (Tris), L-lactic acid,bicinchoninic acid (BCA) solution, copper sulphate (III) pentahy-drate solution and iodoacetamide came from Sigma–Aldrich(Saint-Quentin Fallavier, France). Triton X-100 came from Merck(Darmstadt, Germany). All other chemicals were of analytical gradeand came from Carlo Erba (Milan, Italy).

2.2. Preparation of phosphocasein micelles

PC dispersion was prepared at 2.5% proteins (�2.3% phospho-caseins, w/w) and pH 6.6, in lactose-free simulated milk ultrafil-

trate (SMUF) (Jenness & Koops, 1962) by gentle stirring for30 min at 22 ± 2 �C. After storage overnight at 4 �C, PC dispersionwas heated at 40 �C for 1 h in a water bath to complete powderhydration and allow native micelles to be restored with regard tob-casein and calcium-phosphate migration. PC dispersion was keptat 22 ± 2 �C for further analyses or rapidly cooled to 14 �C beforeUHPH-processing. The density of PC dispersion in SMUF equalled1016 ± 3 kg m�3 at 20 �C.

2.3. Ultra-high pressure homogenisation (UHPH)

PC dispersions were processed at an initial temperature (Tin) of14 �C, using a UHP-homogeniser (model FPG7400H, Stansted FluidPower Ltd., Harlow, UK) specially equipped with thermocouples,manometers and pressure gauges to follow temperature and pres-sure changes at different locations of the homogeniser during thewhole process. Pressure and temperature were recorded before(T1, P1) and after (T2, P2) the high-pressure valve (HP-valve orfirst-stage), and after the rapid cooling of the processed fluiddownstream of the HP-valve (T3) (Picart et al., 2006). The secondstage of the homogeniser (or low-pressure valve) was not used inthe present study. PC dispersions were UHPH-treated at 200 or300 MPa (single-pass) and collected at the homogeniser outletafter the first 600 mL corresponding to three-times the mean resi-dence time of a particle in the whole homogeniser. Initial milktemperature in the feeding tank (Tin) and milk outlet temperature(T4) were measured with thermistors. After UHPH-processing,samples were stored at 4 �C until further analyses.

2.4. Physicochemical characterisation of phosphocasein micelles

Particle size distribution was determined by photon correlationspectroscopy (PCS) using a Nano-series Zetasizer (Nano-ZS, Mal-vern Instruments, Malvern, UK) equipped with a 4 mW He–Ne la-ser light (wavelength 632.8 nm) and a detecting angle of 173�.After a 10-fold dilution with SMUF to avoid multiple diffusion phe-nomena, particle size distributions were determined at 25 �C usingpolystyrene 4-sided polished cuvettes (Sarstedt, Nümbrecht, Ger-many), for both filtered (through 0.8 lm cellulose-acetate mem-brane, Sartorius, Göttingen, Germany) and non-filtered dilutedsamples to ascertain the absence of disturbing particles and no lossof protein particles due to the filtration step. Indeed, filtrationthrough 0.8-lm cellulose acetate membrane eliminated possibledust, and also air bubbles in samples by air expansion at the filteroutlet while allowing passage of micelles up to �800 nm. No sig-nificant difference could be seen between the size distributioncurves of filtered and non-filtered samples. Data were assessedby NNLS algorithm (Zetasizer software 6.01, Malvern Instruments).The dispersant viscosity and refractive index were 0.89 mPa sand 1.33 at 25 �C, respectively. The imaginary and real refractiveindex of PC equalled 0.004 and 1.36, respectively (Regnault et al.,2004).

PCS measurements were performed for non-processed PC dis-persion (control A), PC-dispersion processed in the homogeniserwithout applying high-pressure (control B), and UHPH-processeddispersions at 200 or 300 MPa (Tin = 14 �C). Size distribution curveswere given in light intensity or particle number fraction (%), calledsize distribution in light intensity or in particle number frequency.For each independent experiment carried out on different days,mean distribution curves in intensity and in number were calcu-lated from six PCS measurements per PC sample. Mean particlediameters (arithmetical means, nm) were calculated from the dis-tribution curves in intensity or in number. Mean particle surfaceareas (nm2) were calculated from the size distributions in volume,using voluminosity values of 4.7 nL ng�1 for control samples and3.9 nL ng�1 for UHPH-processed samples as previously determined

A. Benzaria et al. / Food Chemistry 138 (2013) 2327–2337 2329

(Chevalier-Lucia, Blayo, Gràcia-Julià, Picart-Palmade, & Dumay,2011). Molecular weight (MW) distributions and MW arithmeticalmeans were calculated from the size distributions in volume, usingEq. (1) (Dewan & Bloomfield, 1973):

MW ¼ ð4=3Þpðd=2Þ3NA=V ð1Þ

where d is the hydrated diameter as determined by PCS in number;V is the hydrated volume of particle per g of phosphocasein (orvoluminosity); NA is the Avogadro’s number.

Zeta-potential (f) was measured at 25 �C using the Nano-seriesZetasizer and zeta-cells (DTS 1060C, Malvern Instruments). PCsamples were diluted in SMUF to maintain micelle organisation.Dispersant dielectric constant was taken as 79 at 25 �C. Each f-va-lue was an average from six determinations using the monomodaltreatment of the Nano-series Zetasizer software.

2.5. Evaluation of phosphocasein–curcumin interaction byfluorescence spectroscopy

The binding of curcumin to PC micelles was evaluated by fluo-rescence spectroscopy at 22 ± 2 �C using a Tecan Infinite M200(Männedorf, Switzerland) microplate-reader (bandwidth of 5 nmfor excitation and 20 nm for emission) and 96-well-microplates(Nunc flat black, VWR, Fontenay-sous-Bois, France) according toRiihimäki et al. (2006). Curcumin was solubilised at 5 mg mL�1 inabsolute ethanol. It was checked that the final ethanol concentra-tion in protein dispersions (<2%, v/v) did not affect PC intrinsicfluorescence.

Protein intrinsic fluorescence was measured at a PC concentra-tion of 13.5 lM, and increasing curcumin concentration (0–30 lM)using a wavelength of 280 nm for excitation (kexc) and 330 nm foremission (kem). The quenching data were analysed according to La-kowicz (1999):

log10½ðF0 � FÞ=F� ¼ log10 Kb þ n log10½curcumin� ð2Þ

where F and F0 are the respective fluorescence intensity in the pres-ence or absence of curcumin; [curcumin] is the quencher concen-tration; Kb is the binding constant; n is the number of bindingsites per phosphocasein molecule. A mean casein MW of21,500 Da was taken for calculations.

Curcumin fluorescence (kexc 430 nm; kem 510 nm) was mea-sured at a curcumin concentration of 5 lM, and increasing PC con-centration (0–20 lM). The binding parameters were evaluatedaccording to Wang and Edelman (1971):

1=DFI ¼ ð1=DFImaxÞ þ 1=ðKb � DFImax � ½PC�Þ ð3Þ

where DFI is the change in curcumin fluorescence intensity in thepresence or absence of PC; DFImax is the maximal change in curcu-min fluorescence intensity corresponding to the plateau value of FIvs. [PC]; Kb is the binding constant; [PC] is the phosphocaseinconcentration.

A mean fluorescence intensity (FI) was calculated from thereading of three plate-wells. No correction was applied to fluores-cence intensity measured using microplate-reading. The sameprotocols were used to evaluate PC–curcumin binding afterUHPH-processing at 200 or 300 MPa (Tin = 14 �C) of PC micelles.Results were the means obtained from two to three independentexperiments carried out on different days, with triplicate measure-ments per independent experiment.

Fluorescence spectra of PC–curcumin mixtures were recordedusing a Cary Eclipse spectrofluorimeter (Varian, Melbourne, Aus-tralia) and polystyrene 4-sided polished cuvettes of high quality(Sarstedt; 1 cm path length). Intrinsic fluorescence spectra of PC(kexc 280 nm) were recorded over 290–400 nm in the presence ofcurcumin at increasing concentration (bandwidth of 5 nm for both

excitation and emission). Curcumin fluorescence spectra were re-corded over 460–600 nm (kexc 430 nm) in the presence of PC atvarious concentrations (bandwidth of 5 nm for excitation and10 nm for emission). For each PC–curcumin combination, the mea-sured fluorescence intensity was corrected for inner filter effects(i.e., light absorption) using the correction factor 10ðAexcþAemÞ�0:5

(Muresan, van der Bent, & de Wolf, 2001) where Aexc and Aem arethe mixture (PC + curcumin) absorbances at the corresponding kexc

and kem, respectively; 0.5 cm is half of the cuvette path length ap-plied to correct light absorption (Varian spectrofluorimeter).Absorbance of PC–curcumin mixtures was recorded over 220–600 nm (UV–Vis Unicam UV2 spectrometer) and corrected forthe turbidity of PC dispersions, to get absorbance values at 280,330–340, 430 and 510 nm. The fluorescence intensity of SMUF(blank) was subtracted from that of samples after light absorptioncorrection. Fluorescence spectra from two independent experi-ments carried out on different days were recorded in duplicate.Binding parameters were calculated using Eqs. (2) and (3).

Fluorescence spectra were quantified by specifying the centre ofemission spectral mass (CSM) as defined by Silva, Moles, and We-ber (1986):

CSM ¼Xðmi � FiÞ=

XFi ð4Þ

where mi(1/ki) is the wave number (cm�1); Fi is the fluorescenceintensity at mi and

PFi is the spectral integral in the spectrum

range. CSM corresponds to the spectrum barycentre and reflectsthe global changes in exposure of aromatic residues to their micro-environment. Changes in the spectrum barycentre can be expressedin wave number (cm�1, CSM) or nanometres (nm, 1/CSM).

2.6. Preparation of phosphocasein–curcumin complexes

PC samples (control A or UHPH-processed) were incubated at aprotein concentration of 100 lM with 50 lM curcumin (final con-centrations) with gentle shaking for 1 h. After centrifugation(6000g for 10 min) in 1.5-mL Eppendorf tubes to separate thePC–curcumin complexes (pellets) from free curcumin (superna-tants), pellets were washed with 1 mL SMUF, then centrifugedagain (same conditions). Pellets containing PC–curcumin com-plexes were (i) re-suspended in the appropriate buffer for furtheranalyses (Sections 2.7–2.9), or (ii) re-suspended in 1.5 mL metha-nol to extract then quantitate curcumin bound to phosphocaseins.After 30 min shaking then centrifugation (16,000g for 5 min) toprecipitate PC micelles, bound curcumin was quantified in metha-nolic supernatants at 450 nm (EL-311SX UV–Vis spectrometer, Bio-Tek, Colmar, France) using the calibration curve of curcumin (0–200 lM) in methanol. Incubation and centrifugation steps wereperformed at 22 ± 2 �C.

2.7. In vitro digestion

Enzymatic digestion of PC–curcumin complexes was performedaccording to Puyfoulhoux et al. (2001) with some modifications.Briefly, PC–curcumin complexes re-suspended in pH 7.4 DPBS con-taining 0.9 mM calcium and 0.5 mM magnesium (called PBS2+)were incubated with porcine pepsin (25 mg mL�1 final) in PBS2+

brought to pH 2.0 with HCl, or with porcine pancreatin (2 mg mL�1

final) in PBS2+ at pH 7.4, to mimic gastric or intestinal digestion,respectively. As negative or positive control for curcumin release,PC–curcumin complexes were respectively incubated in PBS2+, orin DPBS free of calcium and magnesium but containing 100 mMEDTA. PC–curcumin complexes were incubated at 37 �C under gen-tle shaking for 1, 5, 15, 30, 60 or 180 min.

After appropriate incubation-time, the released curcumin wasseparated from the curcumin bound to non-digested PC micelles

0

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300 MPa

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Fig. 1. Size distribution curves of phosphocasein (PC) micelles as calculated in lightintensity (A) or in particle number frequency (B) for non-processed PC dispersion(native-like PC or control A), PC dispersion processed through the homogeniserwithout applying high-pressure (control B), or PC dispersion processed by ultra-high pressure homogenisation (UHPH) at 200 or 300 MPa (Tin = 14 �C). PC disper-sions containing 2.5% (w/w) protein at pH 6.6 were diluted with SMUF then filteredbefore laser diffractometry (Malvern Nano-ZS) measurements (for experimentaldetails see Sections 2.3 and 2.4). For each sample, mean curves from sixmeasurements are shown.

2330 A. Benzaria et al. / Food Chemistry 138 (2013) 2327–2337

(pellets) by centrifugation (16,000g, 22 ± 2 �C, 15 min). Curcuminwas extracted from pellets with 1.5 mL methanol, as explained inSection 2.6. Curcumin release was expressed as:

100� ð½curcumin�t0 � ½curcumin�tiÞ=½curcumin�t0Þ ð5Þ

where [curcumin]t0 and [curcumin]ti are curcumin concentrationmeasured in the pellets at t0 and after the various incubation times(ti), respectively. Results were the means of three independentexperiments carried out on different days, with duplicate measure-ments per independent experiment.

2.8. Cellular uptake of curcumin

Cellular uptake of curcumin bound to non-processed or UHPH-processed PC micelles was determined using human intestinal epi-thelial TC7-cells, kindly provided by Dr. Rousset, Centre de Recher-che des Cordeliers (Paris). TC7-cells (passage 39) were cultured inDMEM supplemented with 20% FBS, 1% penicillin–streptomycinand 1% non-essential amino acids (%, v/v). TC7-cells were seededat 250,000 cells/well, in 24-well plates (Nunc, Fontenay-sous-Bois,France), then incubated for 19 days in a humidified incubator(8000DH, Thermo Electron, Herblain, France) at 37 �C, 8% CO2,92% air, 100% RH (relative humidity), to reach confluence.

For uptake experiments, TC7-cells were washed three timeswith PBS2+ then incubated at 37 �C for 15 min with PC–curcumincomplexes deposited on cells at curcumin concentrations of 0,2.5, 5, 10 and 20 lM, after adequate dilution with HBSS. The cul-ture medium was then removed and plate-wells were washedthree times with ice-cold PBS2+. Cell-internalised curcumin was ex-tracted using methanol (1 mL/plate-well) then quantitated at450 nm (Multiscan Spectrum, Thermo Electron, Vintaa, Finland).Curcumin concentration loaded into TC7-cells was normalised toprotein content in plate-wells measured using the BCA procedure(Smith et al., 1985).

2.9. Evaluation of cell membrane integrity and cell inflammation

TC7-cells were exposed to PC samples free of curcumin, atincreasing PC concentration (0.001, 0.01, 0.1, 1 and 10 mg mL�1)in DMEM. After 24 h incubation (37 �C, 8% CO2, 92% air, 100%RH), the culture supernatants were collected. Cell membraneintegrity was assessed using the lactate dehydrogenase (LDH) re-lease assay according to Mahfoud, Maresca, Garmy, and Fantini(2002), and cell inflammation, using interleukin-8 (IL-8) secretionaccording to Maresca et al. (2008).

For LDH activity determination, 25 lL of culture supernatantwas added to 250 lL of reaction solution (112 mM Tris–HCl, pH9.3; 172 mM KCl; 56 mM L-lactic acid; 1.72 mM NAD final) in96-well microplates (Maxisorp™ microplates, VWR International,Fontenay-sous-Bois, France). Absorbance was recorded at 340 nm(EL-311SX UV–Vis spectrometer, Bio-Tek, Colmar, France) immedi-ately and after 10 min incubation at 37 �C. A positive control wasincluded using 1% (v/v) Triton X-100 solution in DMEM, leadingto �100% LDH-release by lysing the cells completely. LDH releasewas expressed as follows:

LDH release ð%Þ ¼ ðDAbs of the sampleÞ=ðDAbs of the positive controlÞ� 100

ð6Þ

where DAbs is the difference between absorbance measured imme-diately and 10 min after depositing the reaction solution.

Interleukin-8 (IL-8) secretion was assessed using a commercialELISA kit. Briefly, 96-well Maxisorp™ microplates were coatedwith anti-human IL-8, overnight at 4 �C. Plate-wells were thenwashed and blocked with 10% (v/v) FBS in PBS for 1 h at

20 ± 1 �C. After three successive washings, well-plates were incu-bated with IL-8 kit standards or samples for 2 h at 20 ± 1 �C. Afterfive additional washings, the detector reagent (biotinylated anti-human IL-8 + streptavidin–horseradish peroxidase (HRP)) wasadded to plate-wells, and left for 1 h at 20 ± 1 �C. The colourHRP-reaction was developed by addition of Sigma-Fast™ OPD solu-tion (30 min in the dark), then stopped by addition of H2SO4 (4 N)in plate-wells. Absorbance was read at 450 nm (EL-311SX micro-plate reader). IL-8 concentration was calculated using IL-8 stan-dard curve in the 3.1–200 pg mL�1 range. A positive control wasincluded in the series, with 20 ng mL�1 of human recombinantIL-1b (Peprotech, Neuilly-sur-Seine, France) incubated for 3 h.

2.10. Statistical analyses

Statistical analyses of experimental data were carried out usingFisher’s and Student’s tests for p = 0.05. Values were the means ±standard deviation for independent experiments carried out ondifferent days.

3. Results and discussion

3.1. Physicochemical characteristics of phosphocasein micelles

The size distribution curves of PC micelles as determined byPCS, in light intensity or particle number frequency (%) are shownin Fig. 1A and B, for the non-processed PC dispersion (control A),after running the PC dispersion in the homogeniser without ap-plied pressure (control B), or after UHPH-processing at 200 or

A. Benzaria et al. / Food Chemistry 138 (2013) 2327–2337 2331

300 MPa. Control A displayed a wide monomodal distribution over60–615 nm, with a peak maximum at 200 nm (in light intensity,Fig. 1A), or 110 nm (in particle number frequency, Fig. 1B), inaccordance with previous results obtained with bovine milk orphosphocasein micelles (Regnault et al., 2004). Control B displayedsimilar distribution curves to control A. PCS revealed a weak de-crease in micelle size after UHPH-processing at 200 MPa, but amarked size decrease after UHPH-processing at 300 MPa, accom-panied by a shift of the peak-maximum from 200 to 140 nm (inlight intensity, Fig. 1A) or from 110 to 73 nm (in particle numberfrequency, Fig. 1B). Mean micelle sizes calculated from distribu-tions in light intensity significantly (p = 0.05) decreased at bothUHPH-pressure levels compared to the non-processed PC (Table 1).Mean micelle sizes calculated from distributions in number fre-quency indicated a significant (p = 0.05) difference between controlA and PC processed at 300 (but not 200) MPa (Table 1). Such reduc-tions in micelle size after UHPH at 300 MPa result from dissocia-tion of phosphocasein micelles pressure-induced during thepressure-build up in the homogeniser intensifier, upstream of theHP-valve, as already suggested (Chevalier-Lucia et al., 2011).

Fluid temperatures T1 and T2 measured before or immediatelyafter the HP-valve during UHPH-processing, equalled respectively19.1 ± 0.1 or 50.3 ± 0.3 �C for UHPH at 200 MPa, and 20.7 ± 0.1 or67.4 ± 0.2 �C for UHPH at 300 MPa. The residence time of the pro-cessed fluid at temperature T2 was <0.25 s in the present homoge-niser (Picart et al., 2006), and the fluid was rapidly cooled down to32.4 ± 0.4 (200 MPa) or 39.4 ± 0.2 �C (300 MPa) using a cooling de-vice located just at the outlet of the HP-valve, thus avoiding over-heating of PC dispersions.

Mean surface area of PC micelles calculated from size distribu-tions assuming spherical particles, was close to 1.4 � 1014 nm2/ng phosphocasein, and did not significantly (p = 0.05) differ for na-tive-like PC (controls A and B) and UHPH-processed sample at200 MPa (Table 1), while the mean particle surface area increased1.5–2-fold up to 2.1 � 1014 nm2 ng�1 for neo-micelles of reducedsizes after UHPH at 300 MPa (Table 1), which led us to expect anincrease in the probability of contact between curcumin moleculesand neo-micelles.

Distribution curves of micelle MW were calculated from thesize distributions in volume using Eq. (1). MW values widely variedbetween 107 and 109 Da with means at 3.1 � 108 (native-like PC),and 2.5 � 108 or 7.1 � 107 Da for UHPH-processed sample at 200or 300 MPa, respectively (Table 1). The MW value calculated for

Table 1Physicochemical characteristics of phosphocasein (PC) micelles and parameters of curcumiPC, and PC processed by UHPH at 200 or 300 MPa (Tin = 14 �C).

Samples Native-like PC

Mean diameter in intensity (nm)A 218 ± 20(a)

Mean diameter in number (nm)A 141 ± 29(a)

Mean volume (nm3)B 2.42 ± 0.50 � 106(

Mean surface area (nm2 per ng of PC)B 1.43 ± 0.29 � 1014

Mean molecular weight (Da)B 3.1 ± 0.69 � 108(a

Number of individual PC per micelleC 14.4 ± 3.2 � 103

Kb (M�1)⁄ 0.56 ± 0.12 � 104(

Kb (M�1)⁄⁄ 2.40 ± 0.15 � 104(

Number of binding sites of curcumin (n)⁄ per phosphocasein 0.73 ± 0.16(a)

Estimated number of binding sites of curcumin per micelle 10.5 � 103

Zeta-potential value (mV) �15.8 ± 0.42(a)

(⁄) (⁄⁄)Apparent binding constant (Kb) and number of binding sites (n) calculated uMeans ± standard deviation calculated from three independent experiments for native300 MPa. For each independent experiment, triplicates were performed.Different letters (a, b, c) in the same line indicate significant difference for p = 0.05.

A Arithmetical means ± standard deviation calculated from six PCS distribution curvesB Arithmetical mean of the size distribution in volume, the surface area or the M.W. dis

For details, see Section 2.4.C A mean molecular weight of 21,500 Da was taken for individual phosphocaseins.

native-like PC agrees with data published for defatted milk (Dewan& Bloomfield, 1973; Mathieu, 1998). Micelle MW significantly(p = 0.05) decreased after UHPH at 300 MPa, as expected, accompa-nied by a decrease in the number of individual phosphocaseinmolecules per mole of micelle (Table 1). However, despite the�1.5-fold decrease in micelle size induced by UHPH at 300 MPa,no significant (p = 0.05) change in particle f-potential values wasnoticed for micelles dispersed in SMUF (Table 1).

3.2. Binding affinity of curcumin to native-like phosphocaseins

Fluorescence emission spectra of native-like PC micelles dis-played a maximum close to 337–340 nm (kmax) (Fig. 2A), indicatingthat excitation at 280 nm caused the emission of mainly Trp resi-dues, and that the contribution of tyrosine residues over 305–310 nm, i.e., wavelength-range corresponding to free tyrosineemission, or emission for a protein dominated by tyrosine (Esmailiet al., 2011; Ruan & Balny, 2002; Ruan, Tian, Lange, & Balny, 2000)was low. A kmax value at 337–340 nm suggested that Trp residuesin native-like PC were located in a relatively apolar environment.Indeed, the emission maximum wavelength of free tryptophan orNATA excited at 280 nm in aqueous solution at 0.1 MPa, is closeto 352 nm (Ruan et al., 2000), but shorter (i.e., blue shifted) in apo-lar solvent or hydrophobic microenvironment such as inside a pro-tein core (Ruan & Balny, 2002). Individual phosphocaseins contain1–2 Trp residues per molecule: two at positions 164/199 and 109/193 for aS1 and aS2-casein, respectively; one at position 143 and76, for b- and j-caseins, respectively (Farrell et al., 2004).

Adding curcumin at increasing concentration caused a progres-sive quenching of PC fluorescence intensity (Fig. 2A) suggesting theformation of PC–curcumin complexes which changed the fluores-cence intensity of any or all Trp residues (Lakowicz, 1999). Con-comitantly, CSM value increased from 29,000 (absence ofcurcumin) to 29,795 cm�1 (30 lM curcumin), corresponding to ablue shift of spectrum barycentre from 344.8 to 335.6 nm, and sug-gesting that the microenvironment of aromatic residues andmainly Trp, became more apolar through curcumin binding. Abinding constant (Kb) of (5.24 ± 0.08) � 104 M�1 and a number ofbinding sites (n) of 0.89 ± 0.003 were obtained using Eq. (2), as cal-culated from fluorescence emission intensity measured at 330 or340 nm and corrected for inner effects (Fig. 2B, insert). It was con-cluded that there was �1 binding site of curcumin per individualphosphocasein molecule in native-like PC micelles.

n binding to phosphocaseins as evaluated by fluorescence spectroscopy for native-like

PC UHPH-processed at 200 MPa PC UHPH-processed at 300 MPa

188 ± 15(b) 148 ± 8.5(c)

130 ± 20(a) 85 ± 10(b)

a) 1.65 ± 0.25 � 106(a) 0.46 ± 0.05 � 106(b)

(a) 1.40 ± 0.22 � 1014(a) 2.14 ± 0.25 � 1014(b)

) 2.5 ± 0.51 � 108(a) 0.71 ± 0.12 � 108(b)

11.6 ± 2.3 � 103 3.3 ± 0.5 � 103

a) 0.59 ± 0.05 � 104(a) 1.12 ± 0.25 � 104(b)

a) 2.39 ± 0.14 � 104(a) 3.78 ± 0.14 � 104(b)

0.74 ± 0.07(a) 0.80 ± 0.18(a)

8.5 � 103 2.6 � 103

�15.6 ± 0.74(a) �15.2 ± 0.81(a)

sing Eq. (2) (⁄) or (3) (⁄⁄) and microplate-reading. For details, see Section 2.5.-like PC or from two independent experiments for UHPH-processed PC at 200 or

in intensity or in number per PC dispersion sample. For details, see Section 2.4.tribution, calculated from the mean size distribution in number for each PC sample.

a

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Fig. 2. Fluorescence emission induced by the binding of curcumin to phosphocasein (PC): (A) Intrinsic fluorescence emission spectra of native-like PC (13.5 lM) in thepresence of increasing concentration of curcumin (0–30 lM, a–k). Excitation wavelength (kexc) was set at 280 nm. (B) Intrinsic fluorescence emission of native-like PC(13.5 lM) at 330 nm (kexc = 280 nm) as a function of increasing concentration of curcumin up to 30 lM. The double logarithmic plot of PC–curcumin binding is shown in theinsert. (C) Fluorescence emission spectra of curcumin (5 lM) in the presence of increasing concentration of native-like PC (0–40 lM, a–k). Excitation wavelength was set at430 nm. (D) Fluorescence emission of curcumin at 510 nm (kexc = 430 nm) as a function of increasing concentration of native-like PC up to 40 lM. The double reciprocal curve1/DFI vs. 1/[PC] is shown in the insert.

2332 A. Benzaria et al. / Food Chemistry 138 (2013) 2327–2337

In parallel, curcumin fluorescence emission was strongly in-creased in the presence of added PC (Fig. 2C and D), whichstrengthened the indication of PC–curcumin complex formation.CSM value increased from 18,696 to 19,275 cm�1, which corre-sponded to a blue shift of spectrum barycentre from 534.9 nm(curcumin alone) to 518.8 nm (40 lM PC), and suggested that cur-cumin molecules were transferred to more apolar domains in PCmicelles. Kb value obtained using Eq. (3) (Fig. 2D, insert) equalled(5.56 ± 0.34) � 104 M�1 in accordance with Kb value obtained fromquenching experiments (Eq. (2)). However, Kb values remainedweak for both estimations, suggesting that curcumin binding toPC was not very specific. PC micelles are highly hydrated proteinassemblies opened to the solvent, with a rheomorphic character.It looks like that phosphocasein assemblies were able to capturecurcumin and retain it in the vicinity of hydrophobic areas and/or buried inside, without a strong specificity.

In the present study, the number of binding sites was calculatedper mole of phosphocasein using a mean MW of 21,500 Da, whichwas precisely done from the phosphocasein content in PC disper-sions. This allows the comparison between UHPH-processed ornon-processed PC samples, added at the same concentration inPC–curcumin mixtures, independently of PC micelle size andtherefore micelle number in PC–curcumin mixtures. Furthermore,binding calculations using Eq. (2) or (3) are designed for proteinmonomers or small oligomers, but not high MW protein assem-blies for which diffusion phenomena and internal viscosity effectsare likely to exist and complicate the binding process. Apparentnumbers of curcumin binding sites per PC micelle are proposedin Table 1.

Sahu et al. (2008) reported Kb values of 1.48–11.3 � 104 M�1,depending on whether they measured curcumin or protein fluores-cence emission, and assuming that curcumin binds to casein in a1:1 M ratio. In that study, casein micelles from skimmed milk weredispersed in pH 7.4 Tris-buffer containing 10 mM CaCl2, instead ofSMUF at pH 6.6 in the present study. Esmaili et al. (2011) observedthat purified camel b-casein (pH 7.0 phosphate buffer, 80 mMNaCl) exhibited 0.9–1.3 binding sites for curcumin, with Kb valuesof 1.8–8.3 � 104 M�1, depending on the temperature (25–37 �C), asassessed by intrinsic fluorescence of tyrosine residues (Trp resi-dues were not present in camel b-casein). Sneharani et al. (2009)found higher Kb value (2.0 � 106 M�1) for curcumin binding topurified aS1-casein in pH 7.4 HEPES-buffer at 27 �C, as assessedby curcumin fluorescence emission.

3.3. Binding affinity of curcumin to UHPH-processed phosphocaseins

Fluorescence emission intensity of PC samples at 330 nm (kexc

280 nm) upon curcumin binding is shown in Fig. 3A. In parallel,Fig. 3B displays curcumin fluorescence emission at 510 nm (kexc

430 nm) as a function of increasing PC concentration. Both kindsof results indicated similar behaviour for native-like PC or PCUHPH-processed at 200 MPa. Nevertheless, it looks like curcuminbinding was slightly more efficient for UHPH-processed PC at300 MPa, with slightly greater fluorescence intensity values(Fig. 3B). Binding parameters determined using Eq. (2) or (3) andthe microplate-reading method are indicated in Table 1. Generallyspeaking, Kb and n values obtained using microplate readingwere lower compared to cuvette fluorescence measurement.

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Fig. 3. Binding of curcumin to PC UHPH-processed at 200 MPa (4) or at 300 MPa( ) (Tin = 14 �C), compared to the curcumin binding to native-like PC (s). (A)Intrinsic fluorescence emission of PC (13.5 lM) at 330 nm (kexc = 280 nm) as afunction of increasing curcumin concentration (0–100 lM). (B) Fluorescenceemission of curcumin (5 lM) at 510 nm (kexc = 430 nm) as a function of increasingPC concentration (0–20 lM). Results were the means from two to three indepen-dent experiments carried out on different days, with triplicate measurement foreach independent experiment.

A. Benzaria et al. / Food Chemistry 138 (2013) 2327–2337 2333

Nevertheless, Kb calculated from curcumin fluorescence emission(Eq. (3)) for the non-processed PC remained of the same order ofmagnitude with both methods.

Non-significant difference in n or Kb was observed between PCUHPH-processed at 200 MPa and native-like PC, while PC UHPH-processed at 300 MPa displayed a slight but significant (p = 0.05)increase by 1.5–2-fold in Kb (Table 1), which may result fromUHPH-induced decrease in micelle sizes at 300 MPa, and subse-quent increase in particle surface area (Section 3.1), favouring thusprotein–ligand encounter then ligand ad/absorption to PC neo-mi-celles at atmospheric pressure; n did not significantly change (Ta-ble 1). Such increases remained weak and should be compared tothose obtained when curcumin is added to PC dispersions beforeUHPH-processing.

Few studies have dealt with effects of high-pressure on phos-phocasein–ligand binding. Semo et al. (2007) studied vitamin D2

encapsulation into micelles reconstituted from sodium caseinateby salt addition at pH 6.7–7.0, and its protection against UV-light.Casein dispersions were processed by homogenisation at 185 MPa,but effects of processing per se in vitamin encapsulation yield werenot considered. The following studies focused more on high-pres-sure effects on bioactive-compound entrapment: Chevalier-Lucia

et al. (2011) observed an increase in a-tocopherol entrapment effi-ciency by 1.5–3-fold after UHPH of PC–a-tocopherol mixtures at200–300 MPa (Tin 14 or 34 �C), compared with binding propertiesof untreated sample at atmospheric pressure. By comparison, theligand curcumin was added to PC micelles after UHPH-processingof PC dispersions in the present study; then PC–ligand bindingwas evaluated at atmospheric pressure. Menéndez-Aguirre et al.(2011) studied vitamin D2 encapsulation into micellar casein,through isostatic high-pressure treatment at 600 MPa and 37 �Cfor 60 min, followed by fast or slow pressure release (20 or600 MPa min�1) in the presence (or not) of added calcium phos-phate. At atmospheric pressure, vitamin D2 entrapment was en-hanced by 4-fold in the presence of added calcium phosphatethrough micelle aggregation, as expected. Interestingly, HP-treat-ment in the absence of calcium phosphate followed by slow-pres-sure release induced a 6.7-fold increase in vitamin D2 entrapmentthrough slow casein reassembly. Indeed, in both studies, pressureappeared as a physical tool able to modulate ligand entrapmentwithout including any chemical processing-step.

3.4. In vitro digestibilty of phosphocasein–curcumin complexes

PC–curcumin complexes were obtained by PC incubation withcurcumin (Section 2.6). Amounts of 2.30 ± 0.27, 2.36 ± 0.45 or2.59 ± 0.25 nmol curcumin were found per mg of PC for native-likePC, PC UHPH-processed at 200 or 300 MPa, respectively, which wasnot significantly different (p = 0.05) despite the slight increase ob-served at 300 MPa.

PC–curcumin complexes were subjected to in vitro pepsin orpancreatin digestion at 37 �C (Fig. 4A–D). In the absence of diges-tive enzymes, all PC–curcumin complexes were quite stable whencalcium and magnesium was included in the buffer (PBS2+)(Fig. 4A). Indeed, only 11–28% of total curcumin was released with-in 5–60 min incubation. Conversely, in calcium/magnesium-freePBS containing 100 mM EDTA, 83–92% of total curcumin was re-leased within similar incubation times, due to phosphocaseinassembly dissociation through calcium sequestration by EDTA(Fig. 4D). PC–curcumin complexes were quite resistant to pepsintreatment simulating stomach digestion (Fig. 4B). By comparison,pancreatin treatment (Fig. 4C) caused greater and faster curcuminrelease, showing 38–77% of total curcumin released within 5–60 min incubation, and �81% after 3 h incubation, which was notso far from the 95% released in the presence of EDTA (Fig. 4D).

Native-like PC and PC processed at 200 MPa gave similar results(Fig. 4A–D) in accordance with fluorescence emission results(Fig. 3). Nevertheless, PC UHPH-processed at 300 MPa displayedlower curcumin release (even non-significant for p = 0.05) throughpancreatin-treatment within 60 min incubation (Fig. 4C) and sug-gested a trend of slower ligand release compared to native-likePC. In the case of phosphocasein assembly deconstruction throughchelation by EDTA (Fig. 4D), most curcumin was released withinthe first 5 min incubation. The present study showed that PC–cur-cumin complexes were resistant to pepsin but not to pancreatindigestion. This interesting finding demonstrates that PC could beused as a gastro-resistant encapsulation system for biomoleculesand drugs, allowing its further delivery mainly in the smallintestine.

Few studies concern the behaviour of caseins used as biomole-cule carriers, under digestion conditions. Encapsulation of hydro-phobic drugs such as mitoxantrone or paclitaxel with b-caseinwas proposed for gastric carcinoma treatment to stabilise the drugagainst further aggregation/precipitation and retain its cytotoxicactivity under in vitro gastric digestion (Shapira, Assaraf, Epstein,& Livney, 2010; Shapira, Davidson, Avni, Assaraf, & Livney, 2011).

In the present study PC casein micelles, natural protein assem-blies with GRAS characteristics and able to spontaneously bind

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Fig. 4. Kinetics of in vitro curcumin release from native-like PC native (d), PC UHPH-processed at 200 MPa ( ) or at 300 MPa ( ). Incubation was carried out as follows: (A) inpH 7.4 phosphate buffer saline, containing 0.9 mM Ca2+ and 0.5 mM Mg2+ (PBS2+); (B) with porcine pepsin in phosphate saline solution (PBS2+) at adjusted pH 2; (C) withporcine pancreatin in PBS2+ at pH 7.4; (D) in calcium/magnesium-free PBS containing 100 mM EDTA, at pH 7.4. PC–curcumin complexes were incubated under gentle shakingat 37 �C for 1–180 min. At each incubation time, extraction of curcumin was performed as explained in Section 2.7. Results were the means ± s.d. of three independentexperiments carried out on different days, with duplicate measurement for each independent experiment.

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2334 A. Benzaria et al. / Food Chemistry 138 (2013) 2327–2337

hydrophobic compounds, may be used to carry biomolecules with-out including any chemical modification in the ligand binding pro-cess. The slight difference observed between non-processed PCmicelles and micelles UHPH-processed at 300 MPa could resultfrom the lower voluminosity and/or higher interfacial area of pro-cessed particles that could influence bioaccessibility for digestiveenzyme, and further curcumin release.

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3.5. Cellular uptake of PC-bound curcumin

Curcumin cellular uptake was evaluated by incubation of TC7-cells with PC–curcumin complexes at increasing curcuminconcentration (0–20 lM) in the culture medium (Fig. 5). The15 min exposure time was chosen on the basis of preliminarystudies with Caco-2 cells (not shown). Fig. 5 showed a significant(p = 0.05) concentration-dependent increase in curcumin uptakeabove 2.5 lM curcumin. Surprisingly, at the highest curcuminconcentrations, curcumin uptake was significantly (p = 0.05) lowerfor UHPH-processed than native-like PC, indicating that micellesize reduction through UHPH-processing was not followed byuptake improvement.

Some studies have showed that nanoparticles (100 nm) dis-played greater uptake than submicron or micro-particles of largersizes (Desai, Labhasetwar, Walter, Levy, & Amidon, 1997; McCleanet al., 1998). Cellular uptake of nanoparticles may be influenced byexperimental conditions (incubation time, particle concentration)and particle characteristics (size and surface properties) (He, Hu,Yin, Tang, & Yin, 2010; Win & Feng, 2005; Yue et al., 2011). Couma-

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Fig. 6. LDH release (A) and IL-8 secretion (B) evaluated from differentiated TC7-cells incubated at 37 �C for 24 h with increasing phosphocasein concentration:native-like PC (j); PC UHPH-processed at 200 MPa PC ( ) or 300 MPa PC ( ). Apositive control (h) was included in LDH release and for IL-8 secretion assays (seeSection 2.9 for details). Mean values ± s.d. of three independent experiments carriedout on different days with duplicate measurements for each independent exper-iment. In the same figure (A or B), values affected with different letters weresignificantly different for p = 0.05.

A. Benzaria et al. / Food Chemistry 138 (2013) 2327–2337 2335

rin-6-loaded PLGA nanoparticles (150–500 nm) coated with poly-vinyl alcohol (PVA) or TPGS (D-a-tocopherol-polyethylene-glycolsuccinate) were compared with uncoated fluorescent polystyrenebeads (50–1000 nm) (Win & Feng, 2005). Results obtained withpolystyrene fluorescent beads indicated maximal uptake efficiencyfor the 100–200 nm particles, but particle coating increased uptakeefficiency in the following order: TPGS-coated PLGA > PVA-coatedPLGA > uncoated polystyrene beads of corresponding size. In thepresent study, the decrease in PC particle size was not accompa-nied by an increase in cellular uptake of bound curcumin. Kineticstudies are needed to know if curcumin delivery is delayed dueto a tightened binding to the processed-PC micelles, or not.

Yue et al. (2011) prepared chitosan-based particles with mono-modal size distribution (215 nm in diameters) and f-potential val-ues over �46/+39 mV. Particle surface charge affected the rate andamount of cellular uptake. Positive charge promoted internalisa-tion rate and further uptake amount, probably due to facilitatedadhesion of positive-charged nanoparticles with the negativelycharged cell surface, via electrostatic interactions. He et al.

(2010), using chitosan-based nanoparticles and various non-phag-ocytic cell lines concluded that particles of 150 nm in size andbearing a slight negative charge, displayed the highest probabilityof cell internalisation without being rapidly taken up and de-stroyed by macrophages.

In the present study, PC micelles diluted in SMUF displayedmoderate f-potential values close to�16 mV whatever was PC par-ticle size. Charge effects cannot therefore explain the PC micellebehaviour. Furthermore, PC micelles (a protein biodegradablematerial) may display different behaviour to polysaccharide orpolystyrene beads. The detailed mechanisms involved in curcumincellular uptake from PC–curcumin complexes require furtherinvestigations.

3.6. Effect of PC micelles on TC7 cell integrity or cell inflammation

The behaviour of PC micelles on TC7-cell monolayers was as-sessed by: (i) LDH release. Indeed, LDH release from cytosol indi-cates a loss of cellular membrane integrity leading to further celldeath. (ii) IL-8 secretion, a reliable marker of gut inflammation(Maresca et al., 2008).

Fig. 6A demonstrates that native-like PC micelles did not causeany cell damage even after 24 h incubation at the highest concen-tration (10 mg mL�1) in the culture medium. The present resultsagree with those obtained by Shapira et al. (2011) who found nocytotoxic activity (as measured by the XTT-based colorimetric as-say for cell metabolic activity) of b-casein deposited at 1 mg mL�1

on N87 human gastric epithelial-cells for 72 h. UHPH-treatment ofPC micelles did not affect cellular integrity, since no significant dif-ferences were noticed in LDH release between UHPH-processedand non-processed samples (Fig. 6A).

Fig. 6B shows that PC micelles caused a significant (p = 0.05) butmoderate IL-8 secretion at the highest concentration (10 mg mL�1)after 24 h exposure. Accordingly, phosphopeptides generated bycasein digestion have been reported to induce IL-6 expression (an-other pro-inflammatory cytokine) by human intestinal cells(Kawahara & Otani, 2004; Kitts & Nakamura, 2006). However, PCinflammatory effect remained low compared to the one causedby the pro-inflammatory cytokine IL-1 (Fig. 6B) included in thepresent study as positive control, and is mainly observed after24 h of TC7-cell exposure to the highest PC dose, which is a longtime compared to the 3–6 h of a digestion process. NeverthelessUHPH-processing did not induce an inflammatory response com-pared to the non-processed sample of higher particle sizes(Fig. 6B), which is positive in view of its application to the NovelFood Regulation for food ingredient development.

4. Conclusions

In the present study, PC micelles were assessed as a potentialbioactive compound delivery system. Curcumin binds to PC mi-celles with �1 binding site per phosphocasein molecule and abinding constant of 0.6–5.6 � 104 M�1. Such apparent binding con-stants remain weak compared to Kb values found for protein mono/dimers such as b-lactoglobulin that display a specific binding siteand Kb values of 106–108 M�1 (Kontopidis, Holt, & Sawyer, 2002).

UHPH-processing of PC dispersion at 200 MPa did not affect thefurther binding parameters of PC with curcumin. UHPH-processingat 300 MPa induced only a slight increase in the binding constant,probably in relation with the 1.5–2-fold increase in the surfacearea of processed micelles. It will be interesting to compare thesefirst results with the binding of curcumin added to PC dispersionsbefore UHPH-processing to check if the process could increase ornot the yield of bound curcumin as already observed for tocopherol(Chevalier-Lucia et al., 2011).

2336 A. Benzaria et al. / Food Chemistry 138 (2013) 2327–2337

PC–curcumin complexes (i) were resistant to pepsin providingsome protection against gastric acidic conditions, and (ii) were de-graded by pancreatin providing the possibility of spatiotemporallycontrolled release. Our results confirmed that PC micelles repre-sent a valuable submicron particular system to vectorise hydro-phobic drugs and nutrients, although the binding process doesnot seem very specific. Some kinetics of curcumin uptake byTC7-cells or other cell lines vs. incubation times are needed tobring additional insight to the adhesion and internalisation mech-anisms of PC micelles.

Acknowledgements

We wish to thank the French National Research Agency (ANR,Paris) for funding the present study including the salary of one ofus, Dr. Benzaria, within the framework of the NANOLIA project(ALIA Program). Dr. Benzaria thanks the Biochimie & TechnologieAlimentaire team (Université Montpellier 2) for UHPH experi-ments, and Dr. Chahinian (Université Paul Cézanne) for advices.

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