Food Chemistry 138 (2013) 1515–1520

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Food Chemistry

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Isomerisation of carrot b-carotene in presence of oil during thermaland combined thermal/high pressure processing

Griet Knockaert, Sudheer K. Pulissery, Lien Lemmens, Sandy Van Buggenhout, Marc Hendrickx,Ann Van Loey ⇑Laboratory of Food Technology and Leuven Food Science and Nutrition Research Centre (LFoRCe), Department of Microbial and Molecular Systems (M2S),Katholieke Universiteit Leuven, Kasteelpark Arenberg 22, 3001 Leuven, Belgium

a r t i c l e i n f o

Article history:Received 13 June 2012Received in revised form 31 August 2012Accepted 1 October 2012Available online 10 November 2012

Keywords:b-CaroteneCarrotOlive oilIsomerisationThermal processingHigh pressure processing

0308-8146/$ - see front matter � 2012 Elsevier Ltd. Ahttp://dx.doi.org/10.1016/j.foodchem.2012.10.080

⇑ Corresponding author. Tel.: +32 16 32 15 67; fax:E-mail address: ann.vanloey@biw.kuleuven.be (A.

a b s t r a c t

The effect of thermal processing (85–130 �C) and combined thermal/high pressure processing (100 �Ccombined with 0.1 to 700 MPa and 700 MPa combined with 85–115 �C) on b-carotene isomerisation inan olive oil/carrot emulsion and pure olive oil phase enriched with carrot b-carotene was investigated.Thermal processing always resulted in an increase in the contribution of the cis-isomers, with theincrease being more pronounced at higher temperatures. In the oil/carrot emulsion, less b-carotene isom-erisation was observed during combined thermal/high pressure processing compared to thermal process-ing. This effect was attributed to strengthening of the carrot cell walls under high pressure, therebyhindering the transfer of b-carotene to the oil phase and lowering its susceptibility to isomerisation. Inan oil phase enriched with b-carotene, b-carotene isomerisation was not influenced by the applied pres-sure at 100 �C and became almost temperature insensitive at 700 MPa.

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1. Introduction

Food products are pasteurised or sterilised in order to destroypathogenic and spoilage microorganisms and enhance the shelflife. Thermal processing is the conventional method for food pres-ervation and both vegetative microorganisms and bacterial endo-spores can be inactivated by high temperatures. Nowadays,interest in high pressure processing as a new preservation tech-nique is growing (Balasubramaniam & Farkas, 2008). High pressurecan inactivate vegetative microorganisms by damage to cell mem-branes, enzyme denaturation, decrease of intracellular pH, etc.Consequently, high pressure processing can be used for food pas-teurisation (Smelt, 1998). Bacterial endospores on the other handcannot be inactivated by high pressure alone as they can survivehigh pressure treatments of more than 1000 MPa at room temper-ature (Buckow & Heinz, 2008; Cheftel, 1995). Therefore, a combi-nation of high pressure and elevated temperature is necessary forhigh pressure sterilisation (Balasubramaniam & Farkas, 2008;Ramaswamy, 2011). As a result of adiabatic heating during pres-sure build up, the desired process temperature is reached fasterduring high pressure sterilisation and shorter process times arethus possible compared to thermal sterilisation (Matser, Krebbers,van den Berg, & Bartels, 2004).

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+32 16 32 19 60.Van Loey).

However, processing also affects the nutritional quality of foodproducts. In the specific case of carrots, the most important micro-nutrient is b-carotene, which is a lipid-soluble carotenoid. Its typ-ical chemical structure, consisting of a polyene chain with 11conjugated double bonds and a b-ring at each end of the chain,gives b-carotene some health related properties (Britton, 1995).Firstly, b-carotene has provitamin A activity. In theory, each mole-cule of b-carotene may be converted into 2 molecules of retinol(=vitamin A), which plays an important role in the human bodyin vision, cell differentiation, and organ development (Weber &Grune, 2012). Besides this, b-carotene has antioxidant activity. b-Carotene is able to quench singlet oxygen or to interact with freeradicals and may in this way protect human cells and tissuesagainst oxidative damage, which can normally lead to the develop-ment of cancers or cardiovascular diseases (Dutta, Chaudhuri, &Chakraborty, 2005; Krinsky, 1994). Unfortunately, the system ofconjugated double bonds makes b-carotene also susceptible toisomerisation. In nature, b-carotene is mostly present as all-trans-b-carotene, which is thermodynamically the most stableform. During processing, some b-carotene cis-isomers, which arerelatively thermodynamically stable, may be formed (Britton,1995). Formation of cis-isomers is undesirable, because it resultsin a decrease of the health related properties (Schieber & Carle,2005). Moreover, some studies reported a lower bioavailability ofb-carotene cis-isomers compared to the all-trans-form (Deming,Teixeira, & Erdman, 2002; Stahl, Schwarz, von Laar, & Sies, 1995).

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The formation of cis-isomers from all-trans-b-carotene has alreadybeen shown during thermal sterilisation of carrot products (Chen,Peng, & Chen, 1995; Knockaert et al., 2011; Lemmens et al.,2010). On the other hand, little information on the effect of com-bined thermal/high pressure processing on b-carotene isomerisa-tion in carrot products is available. Besides processing, otherfactors may influence the formation of b-carotene cis-isomers. Animportant factor in this context is the presence of oil because sol-ubilisation of b-carotene crystals in an oil phase may enhance itssusceptibility to isomerisation (Marx, Stuparic, Schieber, & Carle,2003). The effect of oil on b-carotene isomerisation during thermalprocessing has already been described (Achir, Pénicaud, Avallone,& Bohuon, 2011; Achir, Randrianatoandro, Bohuon, Laffargue, &Avallone, 2010), whereas b-carotene isomerisation during com-bined thermal/high pressure processing in presence of oil has notcurrently been described in the literature.

In the present study, b-carotene isomerisation during thermaland combined thermal/high pressure processing in presence ofoil was investigated. Both an oil/carrot emulsion, in which carrotcells are still present, and an oil phase enriched with carrot b-carotene, in which no carrot cells are present, were used.

2. Materials and methods

2.1. Sample preparation

Fresh carrots (Daucus carota cv Nerac) were purchased in a localshop in Belgium and were stored at 4 �C. Carrots were peeled, cutinto pieces and a carrot puree was prepared by blending the carrotpieces with water (1:1) for 1 min in a kitchen blender.

For the preparation of a stable olive oil/carrot emulsion, carrotpuree was high pressure homogenised (Panda 2K, Gea Niro Soavi,Mechelen, Belgium) at 10 MPa in presence of 5% (w/w) extra virginolive oil.

To obtain an oil phase enriched with carrot b-carotene, b-car-otene was transferred from the carrot puree to the oil phase.Firstly, carrot puree was high pressure homogenised (Panda 2K,Gea Niro Soavi, Mechelen, Belgium) at 100 MPa to break thecarrot cells and facilitate the release of b-carotene. After additionof 16% (w/w) extra virgin olive oil to the homogenised carrotpuree, the mixture was incubated for 5 h at room temperaturewhile rotating end-over-end. Finally, the oil phase enriched withb-carotene was separated by centrifugation for 15 min at 18,900gand 4 �C.

2.2. Thermal treatments

For the thermal treatments, which were carried out in a temper-ature-controlled oil bath, closed stainless steel reactor tubes(external diameter = 12 mm, internal diameter = 5 mm, length =100 mm) were completely filled with oil/carrot emulsion orenriched oil to make the headspace negligible and minimise thecontact with oxygen. During the treatments, the temperatureprofile in one tube was measured using a type T thermocouple con-nected to a thermocouple box (TR9216, Ellab, Hilleroed, Denmark)and a CMC-92 data acquisition system (Ellab, Hilleroed, Denmark).All tubes were first equilibrated for 10 min at 40 �C. The tubeswere then placed in the oil bath at the preset process temperature(85–130 �C) and were cooled in ice water after a certain holdingtime. One sample, representing the sample of time point 0 min,was immediately cooled after equilibration at 40 �C. After cooling,the samples were removed from the reactor tubes, frozen in liquidnitrogen and stored at �80 �C until b-carotene analysis.

2.3. Combined thermal/high pressure treatments

Combined thermal/high pressure treatments were carried out ina laboratory scale 6-vessel high pressure equipment (HPIU-10.000,serial No. 95/1994, Resato, Roden, The Netherlands). A propyleneglycol fluid (PG fluid, Resato, Roden, The Netherlands) was usedas the pressure medium. Polyoxymethylene acetal cylindrical tubes(12 mm internal diameter, 4 mm thickness, 85 mm length) werecompletely filled with oil/carrot emulsion or enriched oil. The tubeswere placed in the pressure vessels already equilibrated at the pre-set process temperature. The temperature in one tube was regis-tered using a type J thermocouple (Ellab, Denmark). When aninitial temperature in the samples was reached, required to obtainthe preset process temperature in the samples after pressure buildup, pressure was built up to the preset pressure (initial step of150 MPa followed by a pressure increase at a rate of 10 MPa/s).After the holding time, the vessels were decompressed. One samplewas removed immediately after pressure build up, representing thesample of time point 0 min. After decompression, the samples wereremoved from the vessels and were immediately cooled in icewater. The samples were removed from the reactor tubes, frozenin liquid nitrogen and stored at �80 �C until b-carotene analysis.

2.4. Determination of the concentration of b-carotene isomers

In a first step, carotenoids were extracted from the oil/carrotemulsion or enriched oil phase using an extraction procedurebased on the method described by Sadler, Davis, and Dezman(1990) with some modifications. One gram of CaCl2�2H2O and50 ml extraction solution (50% hexane, 25% acetone, 25% ethanol,0.1% BHT) were added to 1 g emulsion or enriched oil phase andthe mixture was stirred for 20 min at 4 �C. Afterwards, 15 ml re-agent grade water was added and the mixture was stirred for an-other 10 min at 4 �C. The organic phase, containing thecarotenoids, was separated from the water layer using a separationfunnel and was filtered (Chromafil PET filters, 0.20 lm pore size –25 mm diameter).

In a second step, all-trans-b-carotene and its cis-isomers wereseparated using an HPLC system equipped with a reversed phaseC30-column (5 lm � 250 mm � 4.6 mm, YMC Europe, Dinslaken,Germany) and a diode array detector (Agilent Technologies 1200Series, Diegem, Belgium). During analysis, the autosampler andcolumn were kept at 4 and 25 �C respectively. To get a goodseparation of all-trans-b-carotene and its cis-isomers, lineargradient elution was applied. The gradient was built up over20 min from 81% MeOH, 15% methyl-t-butyl-ether and 4% reagentgrade water to 41% MeOH, 55% methyl-t-butyl-ether and 4%reagent grade water at a flow rate of 1 ml/min. Identification ofthe b-carotene isomers was based on comparison of their retentiontimes and spectral characteristics with those of standards ofthe different b-carotene isomers. The absorbance of the differ-ent isomers was measured at 450 nm (=maximal absorbance ofall-trans-b-carotene). The concentration of all-trans-b-caroteneand its cis-isomers was quantified using calibration curves ofall-trans-b-carotene, 15-cis-b-carotene, 13-cis-b-carotene and9-cis-b-carotene standards, respectively (CaroteNature, Lupsingen,Switzerland). During the whole procedure, light was excluded asmuch as possible to avoid b-carotene degradation due to contactwith light. Extractions were performed in duplicate.

3. Results and discussion

3.1. b-Carotene isomerisation during thermal treatments

In a first experiment, b-carotene isomerisation during thermaltreatments of both an oil/carrot emulsion and an oil phase

G. Knockaert et al. / Food Chemistry 138 (2013) 1515–1520 1517

enriched with b-carotene was investigated. Thermal treatmentswere performed at 85, 100, 115 and 130 �C and treatment timesbetween 0 and 120 min (85 �C) or 0 and 60 min (other tempera-tures) were applied.

To rule out the problem of changing b-carotene extractabilityas a function of temperature and treatment time, the results ofall-trans-b-carotene and total cis-isomers were expressed ascontributions to the total b-carotene concentration as suggestedby Lemmens et al. (2010) using following equations:

Cb-carotene;t

Cðb-caroteneþcis-isomersÞ;t

Csum cis-isomers;t

Cðb-caroteneþcis-isomersÞ;t

In the equations, b-carotene represents all-trans-b-carotene. Tocalculate the concentration of total cis-isomers, only the concentra-tions of 9-cis-, 13-cis- and 15-cis-b-carotene were taken into accountas these were the only cis-isomers that could be identified andquantified. These isomers are described in the literature to be themain b-carotene cis-isomers (Rodriguez-Amaya & Kimura, 2004).

In Fig. 1, the results for the thermal treatments of both matricesare given. For each treatment, a new oil/carrot emulsion or en-riched oil phase was prepared. As the initial contribution of thecis-isomers differed from preparation to preparation, the absolutedecrease in the contribution of all-trans-b-carotene in relation totime point zero and the associated increase in the contribution ofthe cis-isomers are given as a function of time.

In both the oil/carrot emulsion and the enriched oil phase, thecontribution of all-trans-b-carotene decreased with increasingtreatment time as a result of isomerisation. As isomerisation isa reversible reaction (Dugave, 2006), a plateau value wasreached after some time. At that moment, the conversion of

A

C

Fig. 1. Isomerisation of all-trans-b-carotene (A) and formation of total cis-isomers (B) information of total cis-isomers (D) in an olive oil phase enriched with carrot b-carotene

all-trans-b-carotene to the cis-isomers is in equilibrium with thereverse reaction. As the temperature increased, the level ofisomerisation was also increased. In the oil/carrot emulsion forexample, the contribution of the total cis-isomers increased with15% after a treatment of 60 min at 85 �C. At 100 and 115 �C onthe other hand, a similar increase was already reached after 20and 8 min, respectively. During the treatment at 130 �C, thisincrease was even reached after less than 5 min and an increaseof 45% was observed after 60 min. As the initial contribution ofthe cis-isomers in the oil/carrot emulsion was approximately 5%,b-carotene was present as a 50:50 mixture of all-trans-b-caroteneand cis-b-carotene in the equilibrium state. Colle et al. (2010)found a similar result for lycopene isomerisation in an olive oil/tomato emulsion after a treatment of 60 min at 130 �C.

In the enriched oil phase, the increase in the contribution oftotal cis-isomers was slightly lower compared to the oil/carrotemulsion. As isomerisation is a reversible reaction, the differentcis-isomers might return to the all-trans-form. As no protectivecarrot matrix is present in the enriched oil phase, the b-carotenecis-isomers were possibly more sensitive to back-isomerisationcompared to the oil/carrot emulsion, resulting in a lower increasein the contribution of total cis-isomers.

3.2. b-Carotene isomerisation during combined thermal/high pressuretreatments

In a second experiment, b-carotene isomerisation in both theoil/carrot emulsion and enriched oil phase during combined ther-mal/high pressure treatments was investigated. Combined ther-mal/high pressure treatments were carried out at a singletemperature (100 �C) and different pressures (300, 500 and700 MPa) for 0–60 min on the one hand and at a single pressure(700 MPa) and different temperatures (85, 100 and 115 �C) for

B

D

an olive oil/carrot emulsion as well as isomerisation of all-trans-b-carotene (C) andduring thermal treatments at 85 �C ( ), 100 �C ( ), 115 �C ( ) and 130 �C (�).

A

B

Fig. 2. Temperature evolution of an olive oil/carrot emulsion (A) and of an olive oilsample (B) during treatments at 100 �C combined with 0.1 MPa (black line) and500 MPa (grey line).

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0–60 min on the other hand. As a new batch of carrots (of the samevariety) was used for each group of treatments, the thermal treat-ment at 100 �C and atmospheric pressure was repeated each timeas a reference.

The temperature evolutions of an oil/carrot emulsion and of anoil sample during a thermal treatment at 100 �C and during a com-bined thermal/high pressure treatment at 100 �C and 500 MPa areshown in Fig. 2. For thermal treatments at atmospheric pressure,almost 15 min were required to heat the oil/carrot emulsion oroil phase up to the process temperature (100 �C). At atmosphericpressure, heat transfer occurs by conduction and/or convection,which are rather slow processes, and this explains the long dy-namic heating phase. For high pressure treatments on the otherhand, the temperature of the samples increases very fast duringpressure build up as a result of compression, which is also knownas adiabatic heating. The magnitude of this temperature increase isdetermined by the initial product temperature, the applied pres-sure and the material properties of the product. In the specific caseof the treatment of olive oil at 500 MPa, the temperature increasedat a rate of 7.44 �C/100 MPa. Rasanayagam et al. (2003) reportedthe same temperature increase for pressurisation of pure olive oilto 500 MPa. Directly after pressure build up, the temperature ofthe samples increased slightly above the final process temperature,that was reached afterwards. As a result of the fast temperature in-crease during pressure build up, the process temperature in the oilphase was reached after 2.5 min. In the oil/carrot emulsion, thetemperature increased at a lower rate (4.92 �C/100 MPa) duringpressure build up compared to the pure oil phase and the processtemperature was reached after 6 min, which is still substantiallyfaster compared to atmospheric pressure.

In Fig. 3, the results for all-trans-b-carotene isomerisation and forthe formation of total cis-isomers with time are given for the highpressure treatments at 100 �C and varying pressures for both the

enriched oil phase and the oil/carrot emulsion. Similar to the resultsof the thermal treatments, the absolute decrease or increase in con-tribution, in relation to time point zero is given. In the pure oil phaseenriched with b-carotene, an increase in the percentage of b-carotene cis-isomers of 15% was observed after the thermal treat-ment at 100 �C, which is similar to the first experiment. Moreover,b-carotene isomerisation in oil at 100 �C seems to be independenton the pressure that is applied as the curves of formation of cis-isomers at all different pressures coincide. In other words, for treat-ments at 100 �C, high pressure seems not to influence b-caroteneisomerisation in the oil phase. Nevertheless, clearly less isomerisa-tion occurred in the oil/carrot emulsion during high pressure treat-ments compared to the thermal treatment, and the degree ofisomerisation was independent on the pressure level in case highpressure was applied. Whereas the percentage of the total cis-isomers increased by 25% during the thermal treatment at 100 �C,which is comparable with the result of the first experiment, an in-crease of only 10% was observed under high pressure. Solubilisationof carotenoids is believed to be a prerequisite for isomerisation(Marx et al., 2003) and as b-carotene is lipophilic, solubilisation inan oil phase is necessary. Therefore, it is hypothesised that thetransfer of b-carotene from the carrot cells to the oil phase in theoil/carrot emulsion was hindered under high pressure. In carrotcells, which are surrounded by a cell membrane and a cell wall,b-carotene is present in crystalline form in the chromoplasts,which are also surrounded by a double bilayer membrane(Hornero-Méndez & Mínguez-Mosquera, 2007). Consequently,several physical barriers need to be broken before b-carotene canreach the oil phase. As temperature and high pressure affect thesebarriers differently, they may consequently have a differentinfluence on the ability of b-carotene to be transferred to the oilphase. Cell membranes are known to be easily destroyed by hightemperatures (Van Buggenhout et al., 2010). Even so, the destruc-tive effect of high pressure on cell membranes has also been demon-strated. For example, a total loss of membrane integrity in onioncells after high pressure treatments at pressures of 300 MPa andhigher was observed by Gonzalez, Jernstedt, Slaughter, and Barrett(2010). Cell walls on the other hand behave quite differently underthermal or combined thermal/high pressure conditions. The plantcell wall is built up of different polysaccharides of which pectinplays an important role in cell integrity. Pectin mainly consists ofa-(1,4)-linked galacturonic acids, which can be methoxylated(Ridley, O’Neill, & Mohnen, 2001). At elevated temperature, highmethoxylated pectin may undergo b-eliminative depolymerisationwhich results in pectin solubilisation and is responsible forweakening of the cell walls. The b-eliminative depolymerisationreaction mainly occurs at higher pH values. As carrots have a pHof 6.5, the softening of carrot cell walls as a result of b-eliminativedepolymerisation during thermal treatments has been shownrepeatedly (De Roeck, Mols, Duvetter, Van Loey, & Hendrickx,2010; Sila, Smout, Elliot, Van Loey, & Hendrickx, 2006). Nextto b-eliminative depolymerisation, demethoxylation of highmethoxylated pectin may also occur at elevated temperature whichdecreases the susceptibility to b-eliminative depolymerisation. DeRoeck et al. (2009) showed that pectin demethoxylation is highlyaccelerated under combined thermal/high pressure conditions,which is accompanied by a strong retardation of the b-eliminativedepolymerisation of pectin. Furthermore, demethoxylation ofpectin may enhance the formation of calcium bridges betweenlow methoxylated pectin and calcium ions naturally present inthe plant tissue resulting in a strong pectin network (Van Buren,1979). Less pectin solubilisation as a result of limited b-eliminativedepolymerisation combined with the formation of a pectin networkprobably strengthened the cell walls during combined thermal/highpressure treatments of the oil/carrot emulsion. In this way, thetransport of b-carotene from the carrot cells to the oil phase was

BA

DC

Fig. 3. Isomerisation of all-trans-b-carotene (A) and formation of total cis-isomers (B) in an olive oil/carrot emulsion and isomerisation of all-trans-b-carotene (C) andformation of total cis-isomers (D) in an olive oil phase enriched with carrot b-carotene during combined thermal/high pressure treatments at 100 �C and 300 MPa ( ),500 MPa ( ) and 700 MPa ( ) and during a thermal treatment at 100 �C (�).

BA

DC

Fig. 4. Isomerisation of all-trans-b-carotene (A) and formation of total cis-isomers (B) in an olive oil/carrot emulsion and isomerisation of all-trans-b-carotene (C) andformation of total cis-isomers (D) in an olive oil phase enriched with carrot b-carotene during combined thermal/high pressure treatments at 700 MPa and 85 �C ( ), 100 �C( ) and 115 �C ( ) and during a thermal treatment at 100 �C (�).

G. Knockaert et al. / Food Chemistry 138 (2013) 1515–1520 1519

probably hindered, thereby limiting the possibility of b-carotene toisomerise. In a previous study, less b-carotene isomerisation was

also observed in high pressure sterilised carrot pieces comparedto thermally sterilised carrot pieces (Knockaert et al., 2011).

1520 G. Knockaert et al. / Food Chemistry 138 (2013) 1515–1520

The results for b-carotene isomerisation in an oil/carrot emul-sion or enriched oil phase during combined thermal/high pressuretreatments at 700 MPa and varying temperatures are given inFig. 4. In the oil/carrot emulsion, the percentage of total cis-isomersonly increased by 10% under high pressure whereas an increase of25% was obtained at atmospheric pressure. This result is similar tothe one of the combined thermal/high pressure treatments of theoil/carrot emulsion at 100 �C and varying pressures (see Fig. 3).The cell walls were probably also strengthened under high pres-sure, as explained in the previous paragraph, thereby limiting thetransfer of b-carotene from the chromoplasts to the oil phase. Inthe enriched oil phase, no difference between all conditions testedwas observed. In other words, the increase in percentage of the cis-isomers at 700 MPa was independent on the temperature that wasapplied. This is in contrast with the results of the thermal treat-ments at atmospheric pressure where the degree of isomerisationincreased with increasing temperature (see Fig. 1). These resultsindicate that the reaction of b-carotene isomerisation becomes al-most temperature insensitive at 700 MPa. No clear explanation forthis observation could be found.

4. Conclusion

The effect of thermal and combined thermal/high pressure pro-cessing on b-carotene isomerisation was investigated in an oliveoil/carrot emulsion and in a pure olive oil phase enriched with car-rot b-carotene. During thermal processing of both the oil/carrotemulsion and the enriched oil phase, the amount of cis-isomers in-creased as a function of time and the increase was more pro-nounced at higher temperatures. During combined thermal/highpressure processing on the other hand, b-carotene isomerisationoccurred quite differently in both matrices. In the oil/carrot emul-sion, an average increase of only 10% was observed at all combinedthermal/high pressure treatments that were performed in thisstudy, compared to an increase of 25% during a treatment at100 �C and atmospheric pressure. The lower degree of b-caroteneisomerisation during combined thermal/high pressure processingof an oil/carrot emulsion was suggested to be related to thestrengthening of the carrot cell walls under high pressure. Thisstrengthening probably hindered the transfer of b-carotene to theoil phase thereby lowering its susceptibility to isomerisation. Inthe pure oil phase enriched with b-carotene, an average increasein the contribution of the cis-isomers of 15% was observed after60 min, both at 100 �C and varying pressures and at 700 MPa andvarying temperatures. From this result, it was concluded that highpressure does not influence b-carotene isomerisation in an oilphase at 100 �C and that the reaction in the oil phase becomes al-most temperature insensitive at 700 MPa.

Acknowledgments

This research was financially supported by the Research Foun-dation Flanders (FWO) (Griet Knockaert and Sandy Van Buggen-hout and project G.0626.11), by the Research Council KU Leuven(Lien Lemmens) and by an Erasmus Mundus Fellowship (SudheerK. Pulissery).

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