Food Research International 64 (2014) 831–838

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Food Research International

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Lycopene and β-carotene transfer to oil and micellar phases duringin vitro digestion of tomato and red carrot based-fractions

Paola Palmero, Agnese Panozzo, Dorothy Simatupang, Marc Hendrickx, Ann Van Loey ⁎Laboratory of Food Technology, Leuven Food Science and Nutrition Research Centre (LFoRCe), Department of Microbial and Molecular Systems (M2S), KU Leuven, Kasteelpark Arenberg 22,3001 Leuven, Belgium

⁎ Corresponding author at: KU Leuven, DepartmenSystems, Laboratory of Food Technology, Kasteelpark AreLeuven, Belgium. Tel.: +32 16 32 15 67; fax: +32 16 32

E-mail address: ann.vanloey@biw.kuleuven.be (A. Van

http://dx.doi.org/10.1016/j.foodres.2014.08.0220963-9969/© 2014 Elsevier Ltd. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 17 June 2014Accepted 24 August 2014Available online 30 August 2014

Keywords:Lycopeneβ-CaroteneStructural barriersDigestionTransfer efficiency

The natural structural barriers (i.e. chromoplast substructure and cell wall) in conjunction with the carotenoidhydrophobicity were investigated as factors that may play a role governing the carotenoid transfer efficiencyfrom the tomato and red carrot matrices into the oil and micellar phases during digestion. In addition, the effectof thermal processing on the carotenoid transfer during digestion was studied. While the carotenoid transfer ef-ficiency from the matrix into the oil phase was found to be highly determined by the level of bio-encapsulation,the carotenoid hydrophobicity, being lower for lycopene than for β-carotene,was themain factor influencing thetransfer from the oil into the micelles. Overall, the incorporation into the micellar fraction represents the criticalstep for carotenoid bioaccessibility. Moreover, the effect of thermal treatments on these systems depended oncarotenoid species, being negative for lycopene and positive for β-carotene.

© 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Several studies have focused on understanding the relationship be-tween potentially active micronutrients and their health benefits.Among these micronutrients, carotenoids have gained attention dueto their antioxidant activities that account for the reduction of somediseases that have lately increased worldwide. The protection againstthe development of cardiovascular diseases andcancer is themost statedhealth benefits related to the consumption of carotenoids (Gerster,1992; Kohlmeier & Hastings, 1995; Krinsky & Johnson, 2005). Thesecomponents cannot be synthesized by humans, hence emphasizing theimportance of consuming carotenoid containing foods such as fruitsand vegetables. However, the actual health-related benefits of caroten-oids strongly depend on their bioaccessibility (i.e. the fraction of aningested nutrient that is released from the food matrix and made avail-able for intestinal absorption).

In order to become bioaccessible, carotenoids must be firstly re-leased from the food matrix in where they are embedded. Afterwards,they need to solubilize into the oil phase either during processing and/or during the gastric digestion. In this context, the co-ingestion of oiland carotenoids is very important (Huo, Ferruzzi, Schwartz, & Failla,2007; Unlu, Bohn, Clinton, & Schwartz, 2005). Once the carotenoidsare solubilized in the oil phase, they might be transferred into themixedmicelles formed in the small intestine. In fact, themixedmicelles

t of Molecular and Microbialnberg 23 postbox 2457, B-300119 60.Loey).

constitute the principal transfer vehicle for the carotenoids to beabsorbed through the intestinal mucosa (Rich, Faulks, Wickham andFillery-Travis, 2003).

From the above, it is clear that the release from thematrix, the disso-lution into the oil phase and the incorporation into the micelles consti-tute critical steps in the carotenoid absorption process (Yeum & Russell,2002). However, each of these steps is conditioned by different factorsthat can be controlled/modified in order to improve carotenoid bioac-cessibility (Fernández-García et al., 2012). Carotenoids in fruit andvegetable matrices are located inside the chromoplast organelles ina specific sub-structure of crystalline, membranous or globular natureembedded in a cellular structure (Schweiggert, Mezger, Schimpf,Steingass, & Carle, 2012). Consequently, the chromoplast sub-structureand the cell wall are the twomain natural structural barriers that governcarotenoid release from the matrix (Jeffery, Holzenburg, & King, 2012;Palmero et al., 2013). Throughout the subsequent steps of the digestionprocess, the carotenoid hydrophobicity (Tyssandier et al., 1998), carot-enoid interactions (Van den Berg, 1999) and the presence of dietaryfat and fiber (Castenmiller & West, 1998) are among the main factorsaffecting the transfer of carotenoids into the oil phase and finally intothe micellar phase. All of these factors are considered to be responsiblefor determining the amount of carotenoids that can potentially reachthe organs to exert physiological functions. It seems reasonable tohypothesize that the interplay of these factors eventually results in a dif-ferent carotenoid transfer efficiency from the matrix into the oil phaseand into the mixed micelles. However, the specific role of the naturalstructural barriers, carotenoid species, and potential interaction on therelease and incorporation into the oil and the micellar phases of thecarotenoids has not been fully elucidated yet.

832 P. Palmero et al. / Food Research International 64 (2014) 831–838

Processing can be exploited to favor/improve carotenoid bioaccessi-bility (Van Buggenhout et al., 2010). Much circumstantial evidenceindicates that carotenoid bioaccessibility can be strongly affected bythermal treatments. Though the effects of thermal treatments on carot-enoid bioaccessibility have been largely investigated, contradictory re-sults are available in the literature. In particular, thermal treatmentswere found to promote the disruption of the food matrix, leading toan increase in β-carotene bioaccessibility in carrot pieces (Hedren,Diaz, & Svanberg, 2002; Lemmens, Van Buggenhout, Oey, Van Loey,& Hendrickx, 2009) as well as in lycopene bioaccessibility in tomatoquarters and pulp (Ryan, O'Connell, O'Sullivan, Aherne, & O'Brien,2008; Colle, Lemmens, Van Buggenhout, Van Loey, & Hendrickx,2010). In contrast, thermal treatments were also found to negatively af-fect β-carotene and lycopene transfer into themicelles during digestionof carrot and tomato based products due to the possible encapsulationof the carotenoids within the matrix (Tydeman et al., 2010; Karakaya& Yılmaz, 2007; Palmero, Lemmens, Hendrickx, & Van Loey, 2014).

Therefore, the aim of the present work was to better understand themain factors governing the transfer of carotenoid from the food matrixinto the oil and the micellar phases during digestion. For this purpose,carotenoid transfer efficiency from thematrix into the oil and themicel-lar phases was determined. The effect of the natural structural barriers(i.e. cell wall and chromoplast substructure) in determining carotenoidrelease from the matrix, oil solubility and subsequent transfer into themicellar phase, was investigated by decomposing tomato and red carrotmatrices into a chromoplast and a cell cluster fraction. Within the twomatrices both lycopene and β-carotene were considered to study theeffect of carotenoid hydrophobicity on their transfer into the oil phaseand further transfer into themicelles. In addition, the impact of thermalprocessing on subsequent transfer efficiencieswas investigated in orderto understandwhether a specific factor and/or step during the digestionprocess constitutes an important limitation for increasing carotenoidbioaccessibility upon thermal processing.

2. Materials and methods

2.1. Materials

Tomatoes (Lycopersicon esculentum cv. Prunus) and red carrots(Daucus carota unknown Indian variety) were obtained fresh fromlocal shops in Belgium. They were washed, cut and blanched (8 minfor tomatoes and 5 min for carrots at 95 °C). The pieces of tomatoesand carrots were subsequently frozen with liquid nitrogen, and storedat−40 °C until use for the experiments.

2.2. Preparation of tomato and carrot based-fractions

2.2.1. Chromoplast fractionChromoplast enriched fractions from tomatoes and carrots were

obtained as previously described by Palmero et al. (2013). Tomato andcarrot pieces were mixed (Waring Commercial, Torrington, CT, USA)at low speed for 5 s with 50% of 0.05M EDTA solution. The obtained pu-rees were gently filtrated using a cheesecloth and further centrifuged(Beckman, J2-HS Centrifuge, Palo Alto, CA, USA) at 27,200 g, 4 °C for30 min. The collected pellet was re-suspended in 100 ml of deionizedwater (further indicated as chromoplast fraction).

2.2.2. Cell cluster fractionCell cluster fractions were prepared following the procedure de-

scribed by Palmero et al. (2013). Firstly, tomato and carrot pureeswere obtained. In the case of tomatoes, the pieces were mixed (BüchiB-400 mixer, Flawil, Switzerland) three times for 5 s. Carrot puree wasobtained by mixing (Waring Commercial, Torrington, CT, USA) the car-rot pieces with 50% of deionized water for 1 min. The obtained pureeswere sieved using a wet sieving equipment (Retsch AS200, Haan,Germany). The fractions between 40–250 μm and 160–500 μm were

collected for the tomato and carrot cell cluster fractions, respectively.These particle sizes correspond to previously measured sizes of thetomato and carrot cells.

2.3. Thermal treatments

Thermal treatments of the isolated chromoplast and cell cluster frac-tions from carrots and tomatoes were performed immediately aftertheir isolation. The chromoplast fraction was placed in sealed plasticbags and thermally treated in water bath at a temperature of 95 °C for10 min. Stainless steel tubes (5 mm internal diameter, 12 mm externaldiameter and 100 mm length) were used to perform the thermal treat-ments of the cell cluster fractions. The treatment was performed at125 °C for 25 min in an oil bath.

2.4. Carotenoid extraction

The isolated fractions and the thermally treated fractions wereimmediately subjected to the carotenoid extraction procedure inorder to determine the initial amount of carotenoids. Carotenoidswere extracted following the procedure described by Sadler, Davis,and Dezman (1990). The procedure was performed by mixing 1 ml(chromoplast fraction) or 1 g (cell cluster fraction) with 25 ml of[hexane:acetone:ethanol (50:25:25 v/v/v) with 0.1% of BHT] and 1 gof NaCl. Afterwards, themixturewas stirred for 20min at 4 °C, followedby the addition of 7.5 ml of reagent grade water (18.2 MΩ·cm). Themixture was thenmixed for 10more minutes at the same temperature.

The mixtures were then placed in separation funnels to collect theorganic phase. The isolated organic phase was filtered (Chromafil PETfilters, 0.2 μm pore size–25 mm diameter) and transferred into a darkvial for HPLC analysis.

The identification and quantification of carotenoidswere performedusing a HPLC system equipped with a C30-column (3 μm × 150 mm ×4.6 mm, YMC Europe, Dinslaken, Germany) and a diode array detector(Agilent Technologies 1200 Series, Dinslaken, Belgium). The tempera-ture of the column was kept constant at 25 °C during the analyses.A linear gradient, using methanol (A), methyl-t-butyl-ether (B) andreagent grade water (18.2 MΩ·cm) (C), was applied. The starting con-ditions were 81% A, 15% B and 4% C and the ending conditionscorresponded to 16% A, 80% B and 4% C. The flow rate was set at1 ml/min and the gradient was built up in 44 min. Identification andquantification were performed at 472 nm for all-E-lycopene and at450 nm for all-E-β-carotene. Standards of all-E-lycopene and all-E-β-carotene (CaroteNature, Lupsingen, Switzerland) were used to buildup calibration curves for the quantification.

2.5. In vitro digestion procedure

The in vitro digestion was performed according to the proceduredescribed by Lemmens et al. (2009).

In order to investigate the effect of different initial carotenoidamounts on subsequent bioaccessibility, the amounts of sample sub-jected to the in vitro digestion procedure corresponded to 1 ml of thechromoplast fractions with different amounts of re-suspended pellets(0.5, 1, 1.5, 2 g). In the case of the cell cluster fraction, 0.5, 1, 2 and 3 gwere evaluated. To investigate the impact of thermal processing on ca-rotenoid transfer efficiencies to oil andmicellar phases duringdigestion,1 ml of the chromoplast fraction and 1 g of the cell cluster fraction wereconsidered as starting material.

To all the samples, 10ml of an oil in water emulsion was added. Theoil in water emulsion was prepared by dissolving 1% L-α-phosphatidyl-choline as the emulsifier and 5% of olive oil in deionizedwater. After ho-mogenizing for 10 min at 9500 rpm (Ultraturrax® T25, IKA®-WerkeGMBH & CO.KG, Staufen, Germany), the emulsion was stabilized ap-plying high pressure homogenization (Gea Niro Soavi, Parma, Italy)at 1000 bar, 4 °C for one cycle.

833P. Palmero et al. / Food Research International 64 (2014) 831–838

Thereafter, 5 ml of electrolyte solution (0.3% NaCl, 0.11% KCl, 0.15%CaCl2 · 2H2O, 0.05% KH2PO4 and 0.07% MgCl2 · 6H2O) was added tothe samples. The pH was then adjusted to 4 ± 0.05 and 5 ml of gastricpepsin solution (0.52% porcine pepsin in electrolyte solution) wasadded. The headspace of the tubes was flushedwith N2 and the sampleswere incubated at 37 °Cwhile shaking end-over-end for 30min. The pHwas then modified to 2 ± 0.05 in order to simulate gastric conditions.The headspace of the samples was again flushed with N2. The sampleswere incubated again at 37 °C while shaking end-over-end for 30more minutes.

Once the gastric digestionwas concluded, the pH of the sampleswasadjusted to 6.9 ± 0.05 in order to simulate the small intestine condi-tions. Sixmilliliters of pancreatin/bile solution (0.2% lipase fromporcinepancreas, 0.4% porcine pancreatin, 2.5% bile extract, 0.5% pyrogallol and1% tocopherol) was added to each sample. The headspace of the tubeswas flushed with N2 and the samples were again incubated for 2 h atthe same conditions as previously.

In order to separate the oil, the micellar and insoluble phases, thesamples were ultracentrifuged (Beckman, L7 Ultracentrifuge, PaloAlto, CA, USA) at 165,000 g, 4 °C for 1 h and 7 min. The supernatant(which corresponds to the micellar phase) and the pellet (which corre-sponds to the insoluble phase) were collected and the carotenoids wereextracted and quantified according to the method described above.The micellar phase was filtrated (Chromafil PET filters, 0.2 μm poresize-25 mm diameter) before the extraction.

The non-digested oil phase could not be quantitatively collected dueto the small amount present; therefore, the amount of the carotenoidsremaining in the oil phase was calculated according to Eq. (1).

Coil ¼ Ci–Cmicellar–Cinsoluble ð1Þ

where Coil corresponds to the amount of carotenoids in the oil phase, Cito the initial amount of carotenoids in the sample (before digestion),Cmicellar to the amount of carotenoids in the supernatant and Cinsoluble

to the amount in the pellet.As the carotenoid concentration in the micellar fraction from toma-

toes was low, the extracts were up-concentrated. The up-concentrationprocedure was performed by adding 300 μl of β-apo-8′-carotenal as in-ternal standard and evaporating the hexane phase at 30 °C for 35 minusing a rotary evaporator. The concentrated extract was then re-dissolved in 400 μl of hexane:dichloromethane (4:1) for the injectionto the HPLC system.

2.6. Light microscopy

Unstained tomato and red carrot chromoplast and cell clusterfractions were examined under an Olympus BX-41 light microscope(Olympus, Tokyo, Japan) equipped with an Olympus XC50 CCD camera(Olympus, Tokyo, Japan). Micrographs were taken using differential in-terface contrast (DIC) and image analysis software Cell* (Olympus,Tokyo, Japan). The cell cluster fractions were analyzed with 10× objec-tive lens and the chromoplast fraction with 40× objective lens.

2.7. Data analysis

A linear regression procedure was applied in order to evaluate sig-nificant differences among the amount of carotenoids present in eachphase after the in vitro digestionwith different initial amounts of the ca-rotenoids. The parameters were estimated using the software packageSAS Version 9.3 (Cary, NC, USA). A parameterwas considered significantat P b 0.05. Samples were analyzed in triplicate.

In order to evaluate significant differences among the amount of ca-rotenoids present in each phase after the application of the thermaltreatments, a Tukey's Studentized Range Test was applied. The samesoftware as for the linear regression procedure was used. The level of

significance was also set at P b 0.05. All analyses were carried out intriplicate.

3. Results and discussion

3.1. Lycopene and β-carotene transfer efficiency from thematrix into the oiland micellar phases

In order to gain an insight in the effect of the natural structuralbarriers on the transfer efficiency of the carotenoids into the oil andeventually into the micelles, different amounts of the chromoplast orcell cluster fractions from tomatoes and red carrots were subjected tothe in vitro digestion procedure. Afterwards, the soluble and insolublephases were collected and the lycopene and β-carotene content wasquantified. The amount of the carotenoids in the non-digested oilphase was calculated (Eq. (1)).

It is important to remark that a fixed amount of oil corresponding to5%was used in all the samples. The reason for using this specific amountis based on previous investigations stating that an increasing amountof olive oil (N5%) present during digestion does not result in a furtherenhancement of the carotenoid in vitro bioaccessibility (Colle, VanBuggenhout, Lemmens, Van Loey, & Hendrickx, 2012). These authorssuggested that when the lipid load (olive oil) is low (5%), the lipid hy-drolysis is complete. However, when the lipid load is higher (10%), thehydrolysis of long chain triglycerides is incomplete resulting in no sig-nificant changes in the carotenoid micellarization compared to lowerlipid loads.

A microscopic characterization of the isolated fractions was per-formed. The obtained micrographs of the chromoplast fraction weresimilar to the presented in our previous investigations (Palmero et al.,2013, 2014). In these micrographs it was possible to observe intactchromoplasts and clusters of subcellular material with the inclusion ofcarotenoid crystals (Fig. 1-A and B). According to Jeffery, Turner, andKing (2012), the chromoplast membrane consists of thin lipid layersthat are easily destroyed during processing and/or the digestion pro-cess. By this means, the chromoplast substructure (chromoplast mor-phology and carotenoid location/association within the chromoplast)was established as the main structural barrier hindering carotenoidmicellarization. We considered of significance to evaluate this fractionas it represents an intracellular level of structural barriers (betweenthe cell and the free form). In regard to the isolated cell cluster fractions,the cells in both matrices (i.e. tomatoes and red carrots) appear intactaccording to the micrographs (Fig. 1-C and D). Therefore, the cell wallwas confirmed to be the main natural structural barrier hindering ca-rotenoid release in this fraction.

Fig. 2 shows the results for the amounts of lycopene and β-carotenein the phases after the in vitro digestion as a function of the initial ca-rotenoid amount in the chromoplast fractions from tomatoes (A) andred carrots (B). According to the corresponding parameter estimates(Table 1), a significantly higher (P b 0.05) amount of carotenoids incor-porated in each phasewas foundwith an increasing amount of lycopeneand β-carotene in the starting material. This indicates that no oil andmicellar saturation occurred in the present conditions.

For both matrices, i.e. tomatoes and red carrots, the highest amountof carotenoids remained within the non-digested oil phase, suggestingthat a considerable proportion of the carotenoids (64.2–51.3% and44.8–55.3% of lycopene–β-carotene in tomatoes and red carrots, respec-tively, at the highest initial amount) can be released from the chromo-plast fraction but is not transferred to the micellar phase. However,part of the carotenoids remains within the insoluble phase (approxi-mately 25% for lycopene andβ-carotene in the twomatrices), indicatingthat the chromoplast sub-structure is acting as a structural barrier forcarotenoid release.

Furthermore, these results show that an important limitation existsin the transfer of the carotenoids from the oil to the micellar phase.The slope values obtained for the micellar phase (Table 1) are indeed

Fig. 1. Isolated chromoplast fractions from tomatoes (A) and red carrots (B). Scale bars 100 μm. Isolated cell cluster fractions from tomatoes (C) and red carrots (D). Scale bars 500 μm.

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reflecting this limitation, which is interestingly more evident for lyco-pene (the slope value of lycopene in the micellar phase is lower thanthat of β-carotene). The results clearly suggest that β-carotene showsa higher micellarization efficiency compared to lycopene. This observa-tion is in agreement with literature information indicating that micellarincorporation of β-carotene is higher than lycopene due to their dif-ferent hydrophobicity (Ryan et al., 2008; Sy et al., 2012; Tyssandier,Lyan, & Borel, 2001; Van het Hof, West, Weststrate, & Hautvast,2000). In fact, lycopene having a linear structure is more lipophilicthan β-carotene containing cyclic end moieties (Britton, 1995).

In addition, carotenoid interactions during micellar incorporationshould be considered. Competition between lycopene and β-carotene

Fig. 2.Amount of lycopene in the non-digested oil phase (♦), of lycopene in themicellar phase (ofβ-carotene in themicellar phase (+) and of β-carotene in the insoluble phase (●) after the inred carrots (B). The full lines represent the linear models.

for micellar incorporation has been reported (Van den Berg, 1999).According to Fernández-García, Mínguez-Mosquera, and Pérez-Gálvez(2007) the initial specific localization of the carotenoids within theoil phase could explain the possible interactions between them duringmicellar incorporation. More apolar carotenoids are located in the coreof the oil droplets while the less apolar carotenoids are found in theouter layers (Borel et al., 1996). Thereby, β-carotene transfer into mi-celles would be facilitated by being positioned in a more external partof the oil droplets. As a consequence, the favored β-carotene transferinto the micelles could also hinder lycopene incorporation.

The slope values for the insoluble phase for both matrices (Table 1)are higher for lycopene compared to β-carotene. This suggests that

■), of lycopene in the insoluble phase (▲), of β-carotene in the non-digested oil phase (x),vitro digestion of the different concentrated chromoplast fractions from tomatoes (A) and

Table 1Parameter estimates of the linear model describing the amounts of carotenoids remaining in the non-digested oil, micellar and in the insoluble phases after the in vitro digestion of thechromoplast or cell cluster fractions from tomatoes and red carrots. The parameter estimates smaller than 0.05 represent significant differences.

Matrix Carotenoid Phases Slope P Slope P

Chromoplast fraction Cell cluster fraction

Tomato Lycopene Non-digested oil 0.35 ± 0.06 0.0002 0.53 ± 0.03 b0.0001Micellar 0.11 ± 0.01 b0.0001 0.011 ± 0.002 0.0002Insoluble 0.54 ± 0.03 b0.0001 0.45 ± 0.03 b0.0001

β-Carotene Non-digested oil 0.37 ± 0.06 0.0002 0.58 ± 0.05 b0.0001Micellar 0.32 ± 0.04 b0.0001 0.05 ± 0.01 0.0059Insoluble 0.30 ± 0.03 b0.0001 0.37 ± 0.05 b0.0001

Red carrot Lycopene Non-digested oil 0.54 ± 0.03 b0.0001 0.56 ± 0.03 b0.0001Micellar 0.04 ± 0.003 b0.0001 0.03 ± 0.003 b0.0001Insoluble 0.41 ± 0.03 b0.0001 0.41 ± 0.03 b0.0001

β-Carotene Non-digested oil 0.52 ± 0.03 b0.0001 0.62 ± 0.04 b0.0001Micellar 0.29 ± 0.02 b0.0001 0.05 ± 0.006 b0.0001Insoluble 0.18 ± 0.008 b0.0001 0.33 ± 0.033 b0.0001

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lycopene release from the matrix is also more limited. The differencesin lycopene and β-carotene transfer from the matrix into the oil phasemight be on one side determined by their hydrophobicity, in whereβ-carotene becomes more easily dissolved into the oil phase. On theother side, matrix related effects might be playing a role. In previousinvestigations it was hypothesized that lycopene might be morestrongly bound with other components than β-carotene resulting ina limited release from the matrix (Palmero et al., 2014). In the presentexperiments, lycopene and β-carotene were evaluated within the samematrix. Therefore, both types of carotenoids are locatedwithin the samechromoplast organelle, however, they could be sub-located or associ-ated differently with other molecules influencing their different re-lease efficiencies (Clinton, 1998).

For a given carotenoid, lycopene and β-carotene respective distribu-tions over the three phases were interestingly similar in both typesof matrices. This observation results from comparing similar initialamounts of each type of carotenoid in the twomatrices and thedistribu-tion percent of the carotenoids in each phase. At an initial amount of ly-copene of 35.9 μg and of 15.3 μg of β-carotene in the chromoplastfraction from tomatoes, 64.2% of the lycopene was transferred into theoil, 7.6% was incorporated into the micelles while 28.2% remained inthe insoluble phase. In the case of β-carotene, 52.3% was transferredinto the oil phase, 33.6% was incorporated into the micellar phase and14.2% remained in the insoluble phase. When the starting amount oflycopene corresponded to 40.4 μg and 12.4 μg for β-carotene in thechromoplast fraction from red carrots, their final distribution was 66.6and 56.6% in the non-digested oil phase, 7.8 and 25.7% in the micellar

Fig. 3.Amount of lycopene in the non-digested oil phase (♦), of lycopene in themicellar phase (of β-carotene in the micellar phase (+) and of β-carotene in the insoluble phase (●) after the ired carrots (B). The full lines represent the linear models.

phase and 25.6 and 17.7% in the insoluble phase, respectively. Similardistributions of the carotenoids in the different phases after digestionsuggest that in this case the effect of the type of matrix on carotenoidtransfer was limited. Therefore, the differences in the structural charac-teristics of the chromoplasts sub-structures between tomatoes and redcarrots did not seem to affect carotenoid release from the matrix andsubsequent transfers into the oil and micellar phases.

Fig. 3 shows the amount of lycopene and β-carotene in the differentphases as a function of initial carotenoid amount in the cell cluster frac-tions from tomatoes (A) and red carrots (B). As for the chromoplastfractions, a significant increase (P b 0.05) (Table 1) in the amount of ca-rotenoids in each phase was found with an increasing initial amount.However, some important differences between the cell cluster and thechromoplast fractions become evident. In the case of the cell clusterfraction, the most remarkable observation is that for the two types ofmatrices, lycopene and β-carotene remained mainly within the insolu-ble phase. This confirms that the cell wall is acting as an importantstructural barrier limiting carotenoid release from thematrix. Similarly,Rich, Bailey, et al. (2003) also found thatmost part of the carotenoids re-mains within the non-digested carrot and spinach matrices, remarkingthat a low transfer rate to the oil phase occurs during the gastricdigestion.

An unexpected result was obtained at higher initial amounts ofcarotenoids. The final amount of lycopene and β-carotene in the oilphase was similar to the amount remaining within the insolublephase. This would suggest that when a higher amount of carotenoidsis released from the matrix, they accumulate in the oil phase due to

■), of lycopene in the insoluble phase (▲), of β-carotene in the non-digested oil phase (x),n vitro digestion of the different concentrated cell cluster fractions from tomatoes (A) and

836 P. Palmero et al. / Food Research International 64 (2014) 831–838

the limited transfer into the micelles. The slopes of the regression linesfor the cell cluster fraction (Table 1) confirm again that a limited micel-lar incorporation of the two types of carotenoids is occurring. Thismeans that even when a higher amount of the carotenoids solubilizeinto the oil phase, the transfer efficiency into the micelles remainslow. In fact, besides carotenoid release from the matrix and solubiliza-tion into the oil phase, the most predominant factor in determining ca-rotenoid absorption is their limited transfer rates from the oil into themicelles. Tyssandier et al. (2003) already mentioned that the limitedtransfer of carotenoids from the matrix into the micelles explains thelow bioavailability of these components. Similar to the results fromthe chromoplast fractions, higher β-carotene slope values for themicel-lar phase compared to lycopene (Table 1)were obtained. This highlightsthe influence of the carotenoid hydrophobicity on the transfer efficiencyfrom the oil to the micellar phase. The polarity of the molecule deter-mines the extent to which carotenoids are transferred into the mixedmicelles (Canene-Adams & Erdman, 2009). Particularly, the significantrestriction of lycopene transfer into the micelles is evident for all thecases studied. This is in accordance with the limited lycopene bioacces-sibility found throughout several studies (Colle, Lemmens, et al., 2010;Moelants et al., 2012; Reboul et al., 2006; Ryan et al., 2008).

Contrarily towhatwas observed for the chromoplast fraction, differ-ences in the carotenoid transfer efficiencies between the cell clusterfractions of tomatoes and red carrots were found. When the startingamount of lycopene corresponded to 27.8 μg and 3.5 μg for β-carotenein the cell cluster fraction from red tomatoes, their final distributionwithin the three phases corresponded to 18.9% and 12.3% in the non-digested oil phase, 0.7 and 6.2% in the micellar phase and 80.3 and81.5% in the insoluble phase, respectively. For the cell cluster fractionfrom red carrots, when the initial amount subjected to the in vitrodigestion procedure corresponded to 29.6 μg of lycopene and 6.1 μg of

Fig. 4. β-Carotene and lycopene distribution (%)± standard deviation in the oil, micellar and in(■) chromoplast fractions from tomatoes (A) and red carrots (B), and the cell cluster fractionsconfidence level of 0.05 between the carotenoid distribution of the non-treated and the therm

β-carotene, the final distribution in the three phases corresponded to46.4 and 48% in the non-digested oil phase, 3.2 and 6.9% in the micellarphase and 50.5 and 45.1% in the insoluble phase, respectively. These re-sults indicate that carotenoids can be more easily released from carrotthan from tomatoes. Differences in cell wall composition and character-istics such as thickness and size between tomato and carrot mightexplain the obtained results (Jeffery et al., 2012).

3.2. Lycopene and β-carotene transfer efficiency from thematrix into the oiland micellar phases upon thermal treatments

The chromoplast fractions from tomatoes and red carrots werethermally treated at 95 °C for 10 min and subjected to the in vitro di-gestion procedure. The intensity of the treatment was selected basedon previous investigations mentioning that organelle structure aswell as carotenoid–protein disruption occur at that temperature (DeBelie et al., 2000). The distribution of the carotenoids over the micellar,oil and insoluble phases for both the control and thermally treatedsamples was quantified. After the thermal treatment, the amount of ly-copene and β-carotene in the insoluble phase from the tomato chromo-plast fractionwas significantly higher (P b 0.05) than in the non-treatedsample (Fig. 4-A). Accordingly, a proportional decrease (P b 0.05) ofboth lycopene and β-carotene in the oil phase was obtained. Thiswould mean that thermal treatments might induce matrix structuralchanges and/or denaturation of heat sensitive compounds that led toan enhanced encapsulation of the carotenoids thereby limiting their re-lease from thematrix and subsequent transfer into the oil. Since thepre-pared chromoplast fraction is depleted from cell wall material, it can beexcluded that carotenoids became entrapped by a cell wall polysaccha-ride network. In contrast, an irreversible complex formation betweendenatured proteins and carotenoids could be formed during heating

soluble phases after the in vitro digestion of the non-treated ( ) and the thermally treatedfrom tomatoes (C) and red carrots (D). The symbol * represents significant differences at aally treated fraction in the respective phase.

837P. Palmero et al. / Food Research International 64 (2014) 831–838

(Takada & Nelson, 1983) which might result in an entrapment of thecarotenoids.

With regard to the chromoplast fraction from red carrots, no signif-icant differences in the distribution of the carotenoids were found uponthermal treatments (Fig. 4-B). Differences in the carotenoid distribu-tions after digestion between tomato and red carrot chromoplast frac-tions indicate that the type of the chromoplast sub-structure has asignificant influence on the carotenoid release upon thermal treat-ments. The tomato and carrot chromoplast sub-structures differ in char-acteristics such as carotenoid localization and carotenoid associationwith other components (e.g. proteins) which determine a different re-lease of the carotenoids from the matrix upon thermal treatments(Palmero et al., 2014). Interestingly, this was not observed in theuntreated samples (Fig. 2) indicating that thermal processing can affectcarotenoid release differently depending on the chromoplast sub-structure characteristics.

Confirming previous results (Fig. 2), for both types of matricesthe highest percentage of carotenoids remained within the oil phase,indicating that though a significant amount of the carotenoids can bereleased from the chromoplast fraction, their incorporation into themi-celles is limited. In fact, also in this case and in particular for lycopene,the transfer into the micelles was not affected by the applied thermaltreatments.

The cell cluster fractions from tomato and red carrot were thermallytreated at 125 °C for 25min. According to Sila, Smout, Vu, Van Loey, andHendrickx (2005) pectin depolymerisation in carrots occurs at this tem-perature, potentially assisting carotenoid liberation from the matrixand enhancing carotenoid bioaccessibility (Lemmens et al., 2009).Fig. 4 also shows the carotenoid distribution over the oil, micellar andinsoluble phases of the control and the thermally treated cell clusterfractions from tomatoes (C) and from red carrots (D). For both typesof matrices, carotenoids predominantly remained within the insolublephase highlighting the significance of the cell wall as a structural barrierfor carotenoid release. No significant differences (P N 0.05) of lycopenedistribution over the three phases after the thermal treatments for thetwo types of matrices were found. However, the trend shows that ahigher amount of lycopene remained within the insoluble phase afterthe thermal treatments with a consequently lower amount in theoil phase. This would indicate that depolymerized cell wall materialmight be hindering lycopene release. Similar observations have beenpreviously made by Svelander et al. (2010), Colle, Van Buggenhout,Van Loey, and Hendrickx (2010) and López‐Sánchez et al. (2011)in thermally treated tomato puree. However, interesting differenceswere obtained for β-carotene. The relative amount of β-carotene inthe insoluble phase of the cell cluster fraction from tomatoes after thethermal treatment was significantly lower (P b 0.05) than the non-treated. Equivalently, β-carotene relative amount was significantlyhigher (P b 0.05) in the micellar phase. Although not significantly dif-ferent (P N 0.05), similar trends for β-carotene in the insoluble and mi-cellar phases in the cell cluster fraction from red carrots after thethermal treatments were found. It may be suggested that both typesof carotenoids could become entrapped after the thermal treatment.The entrapment of carotenoids upon thermal treatment can occur atthe chromoplast level, as previously mentioned, but in this case thepresence of depolymerized cell wall material surrounding the caroten-oids could also have an influence. However, due to differences betweenlycopene and β-carotene molecular structures, β-carotene could bereleased from the matrix at the applied thermal treatments whilelycopene remained embedded. These results are in line with previousinvestigations studying the effect of thermal treatments and high pres-sure homogenization on lycopene and β-carotene bioaccessibility fromthe same matrix (Gupta, Kopec, Schwartz, & Balasubramaniam, 2011;Ryan et al., 2008; Sánchez-Moreno, Plaza, de Ancos, & Cano, 2004). Inall cases it was suggested that lycopene was retained in the matrixwhile β-carotene could be released. This once more supports the sig-nificance of the type of carotenoid in determining the effect of thermal

processing in their bioaccessibility (Palmero et al., 2014). Underliningoverall results, the molecular structure of the carotenoid has an impor-tant role in determining its release from the matrix, transfer into theoil and micellar phases and the effect of thermal processing.

Based on the results, it can be assumed that the limited enhance-ments of lycopene bioaccessibility upon different kinds of processingfound in literature (Colle, Lemmens, et al., 2010; Knockaert et al.,2012; Panozzo et al., 2013; Svelander et al., 2010; Tibäck et al., 2009)can be related to its particular molecular characteristics that comprisean obstacle for micellar incorporation. This means that even when ahigh amount of lycopene can be released from the matrix into the oilphase (expressed in the first set of experiments as higher initial amountof lycopene in the starting material or by studying the chromoplastfractions), a low subsequent micellar incorporation occurs.

4. Conclusions

Carotenoid release from thematrix and subsequent transfer into theoil and micellar phases constitute significant steps during digestion inorder to becomeavailable for absorption. Several factors such as thenat-ural structural barriers and carotenoid hydrophobicity play an impor-tant role in determining these steps. The present study gave an insightin the effect of these two parameters on carotenoid transfer efficiency.In particular, the transfer of the carotenoids from the matrix into theoil phasewas highly determined by the level of structural barriers. How-ever, the transfer efficiency from the oil phase into the micelles washighly determined by the carotenoid molecular structure. β-Carotenemicellarization efficiencywas found to be higher compared to lycopene,indicating that the oil solubility and micellarization of the less apolarcarotenoid are favored. Notwithstanding, for the two types of caroten-oids, micellarization levels were low indicating that by nature thesemicronutrients are poorly absorbed during digestion because of theirmolecular characteristics that limit their incorporation into themicelles.

The type of carotenoid was also a determinant for the effect of theapplied thermal treatments on carotenoid transfer efficiencies. Higherratios of lycopene remained within the insoluble phase after thermaltreatment. This would suggest that lycopene became entrapped bythe matrix upon thermal treatment. Additionally, the low transfer effi-ciency of lycopene into the micelles accounts for a null effect of thethermal treatments on lycopene micellarization. On the contrary, forβ-carotene, a thermal treatment of the cell cluster fraction contributesto an enhanced release from the cell clusters and increased micellar in-corporation. Therefore, themolecular structure of the carotenoid plays asignificant role in its release from thematrix andmicellar incorporationupon thermal treatments. It is at the same time the main cause of thepoor transfer efficiency into the micelles. Even when a higher amountof carotenoids can be released from the matrix (elimination of the cellwall and/or processing) and solubilized into the oil phase, a poor trans-fer into themicelles follows,whichmight explain the limited carotenoidbioaccessibility found throughout several studies.

Acknowledgments

This research was financially supported by the Seventh FrameworkProgramme (FP7) of the European Union under the Marie Curie InitialTraining Network ‘HST FoodTrain’ (Grant agreement 264470).

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