Innovative Food Science and Emerging Technologies 7 (2006) 132 139 www.elsevier.

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Antioxidant properties of natural carotenoid extracts against the AAPH-initiated oxidation of food emulsions
Sotirios Kiokias, Vassiliki Oreopoulou
Laboratory of Food Chemistry and Technology, School of Chemical Engineering, National Technical University of Athens, Iroon Politechniou, 9, 15780, Athens, Greece Received 30 April 2005; accepted 23 December 2005

Abstract The antioxidant effects of natural carotenoid extracts on the oxidation of oil-in-water emulsions (initiated by a water-soluble radical initiator at 60 C) were spectrophotometrically assessed at 233 nm. Headspace volatiles were also monitored for selected samples. At concentration of 1 g l 1 the tested carotenoids significantly reduced the oxidative deterioration of sunflower oil-in-water emulsions. The presence of polar groups was found to modulate the carotenoid activity. Annatto extracts (orbiting and bixin) containing carotenoids with carboxylic and esters groups, were more effective than marigold extract containing lutein (with two hydroxyl groups). -Carotene and a tomato (lycopene-rich) extract, (unsubstituted carotenes), were the least effective. For polar carotenoids, the antioxidant activity did not increase with concentration above a specific level. The tested carotenoids strongly inhibited the formation of volatile aldehydes. Mixtures of lutein or -carotene with tocopherol isomers increased the antioxidant activity of individual carotenoids. Mixtures of carotenoids with ascorbic acid at 0.1 g l 1 exerted a tendency, not significant though, for increasing the activity of each individual carotenoid. 2006 Elsevier Ltd. All rights reserved.
Keywords: Carotenoids; Antioxidant; Oil-in-water emulsions; Azo-initiated oxidation Industrial relevance: This paper is especially interesting since it concentrates on the antioxidative potential of several natural carotenoid preperations which have not been widely investigated so far. One aspect of specific relevance of this work is the interaction of carotenoids with other antioxidants especially ascorbic acid as frequently present in plant food products. Interestingly, combinations of all the carotenoids tested with ascorbic acid showed a tendency for enhanced antioxidant activity; although their effect was not significantly stronger than the effect of individual compounds.

1. Introduction Carotenoids comprise a widespread class of natural pigments, which are primarily used by industry as colorants in various manufactured food and drinks (Faure, Galabert, Le Moel, & Nabet, 1999). The best-documented function of carotenoids is their provitamin A activity, especially that of carotene and to a lesser extent of -cryptoxanthin and lutein (Van den Berg et al., 2000). Although close to 600 carotenoids have been identified in nature, only 50 possess provitamin A activity and about 40 are present in a typical human diet that

Corresponding author. Tel.: +30 210 7723166; fax: +30 210 7723163. E-mail address: vasor@chemeng.ntua.gr (V. Oreopoulou). 1466-8564/$ - see front matter 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.ifset.2005.12.004

provides 10004000 g of carotenoids daily (Mangels, Holden, Beecher, & Lanza, 1993). In literature, carotenoids have been reported to act as chain breaking antioxidants under specific conditions in vitro (Bast, Haanen, & VandenBerg, 1998; Haila, Lievonen, & Heinonen, 1996) or in vivo (Kiokias & Gordon, 2003b; Rao & Agarwal, 1998). The antioxidant potential of certain carotenoids has been summarized by several authors (Kritchevski, 1999; Pryor, Stahl, & Rock, 2000; Kiokias & Gordon, 2004). However, whether the structure of carotenoids may determine their functionality is a matter of scientific importance, which still has not been elucidated. A certain body of recent research (Dickinson, 2001; Kiokias & Bot, 2005; Kiokias, Reiffers-Magnani, & Bot, 2004) has focused on the microstructural stability of oil-in-

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water emulsions, which are structurally similar to many common foodstuffs (e.g. milk, mayonnaise, fresh cheese type products etc.). However, not much research has been done yet on the oxidative destabilisation of these emulsion systems. A better understanding of the factors monitoring the oxidative deterioration of emulsions would offer strategies to improve the organoleptic and nutritional value of the related products. Moreover, apart from their technological importance, emulsion systems generally mimic the amphiphilic nature and the basic structural characteristics of important biological membranes, which are also prone to in vivo oxidative degradation when attacked by singlet oxygen and free radicals (Halliwell & Gutteridge, 1995; Rice-Evans, 2000). In that aspect, in vitro research on the oxidative stability and antioxidation of model emulsions could provide with useful information of nutritional interest and thereby serve as pilot studies for in vivo clinical trials. So far, little research on the antioxidant activity of carotenoids in multicomponent systems has been reported in the literature (Heinonen, Haila, Lampi, & Piironen, 1997). This paper initially focuses on the antioxidant potential of several natural carotenoid preparations, such as annatto, paprika, marigold and tomato extracts, which have not been widely investigated so far with regard to their antioxidant potential. An alternative (to auto- or metal-catalysed oxidation) model of oxidation was used in this study, with AAPH (2,2azobis-amidinopropane dihydrochloride) as a water-soluble initiator in the water phase of the emulsion. The effect of concentration and structure on the antioxidant potential of carotenoids is also determined and discussed under these experimental conditions. 2. Materials and methods 2.1. Materials Refined sunflower oil was purchased at a retail outlet. -, and -tocopherols, ascorbic acid as well as the carotenoid and aldehyde standards (hexanal, 2-heptenal, 2,6 nonadienal) were purchased from Sigma-Aldrich Co. Ltd., Poole, UK. The natural carotenoids, marigold (lutein-rich), tomato (lycopene-rich), and annatto (bixin, norbixin) extracts were kindly donated by Overseal Foods Ltd., Swadlincote, UK. AAPH (2,2-azobisamidinopropane dihydrochloride) was purchased from Aldrich Chemical Co., Gillingham, UK. Other chemicals were of analytical grade. 2.2. Preparation and oxidation of oil-in-water emulsions Tocopherols were removed from sunflower oil by open column chromatography (Kiokias & Gordon, 2003a). Sunflower oil was passed twice under vacuum through the column (wrapped in aluminium foil and connected to a Buchner flask) containing aluminium oxide (dried overnight at 200 C). Each oil-in-water emulsion (10 g) was prepared by cooling a solution of distilled water (9 g) containing Tween 20 (0.1 g) as emulsifier

and 0.1 ml of AAPH as a chemical initiator. A VC-50 sonicator (Sonics and Materials Inc., Danbury, USA) was immersed in the solution (placed in an ice bath) and 1 g of purified sunflower oil (containing carotenoids at the required concentration level) was added dropwise. Sonication was continued for 5 min after the oil had been added. Droplet size distributions of the emulsions were measured by a static light-scattering technique, (Gelin, Poyen, Courthaudon, Le Meste, & Lorient, 1994), with a Malvern Instruments particle and droplet sizer (Series 2600, focal length 63 mm, beam length 14.3 mm). The mean droplet diameter d3.2 (m) of the emulsions was calculated as following: d3.2 = ni di3 / ni di2, where ni is the number of droplets with diameter di. It was found that the mean d3.2 value for the emulsions was 1.21 0.08 m. After their preparation, emulsion samples were transferred to screw-capped sample vials and held in a shaking water bath (Haake, SWB 29, Fisons, Karslruhe, Germany) where allowed to oxidise at 60 C for 4 h. 2.3. Analytical methods 2.3.1. Evaluation of oxidative stability The oxidative stability of the emulsion was determined by monitoring the formation of conjugated dienes (Kiokias & Gordon, 2003a). Emulsion samples (0.1 ml) were periodically removed (every 30 min) during the period of oxidation, diluted to 10 ml in ethanol and their absorbance at 233 nm was determined with a UVVis Spectrometer (Lambda Bio 20, Perkin Elmer, Basingstoke, UK) as an oxidative indicator. A static headspace solid phase microextraction (SPME) method was used for the analysis of the produced volatiles in the oxidised samples (Zabaras & Wyllie, 2002). Emulsion sample (1 g) was weighed into a vial that was then purged with nitrogen for 2 min and sealed with a septum secured by an aluminium cap. A SPME fibre (75 nm, Carboxen-PDMS) was introduced through the septum into the headspace and was retained in the vial, held at 60 C in the dark for 30 min. The fibre was then removed from the vial and inserted directly into the injection port of a Hewlett Packard (HP 6890) GC, equipped with a flame ionisation detector and an integrator. A WCOT CP-SIL 8CB fused silica column, 60 0.25 mm i. d., df = 0.25 (Chrompack UK Ltd., London, UK) was used, with helium as the carrier gas. The column temperature was programmed to increase from 40 to 90 C in 12 min, and then to 250 C in 15 min. The volatiles were identified by the retention times of their pure compounds, which were separately analysed under these experimental conditions. For each analysis, 1 l of 1,2 dichlorobenzene (100 ng l 1 in methanol) was injected as a standard just before the insertion of the SPME fiber in the GC-port. The resulting dichlorobenzene peak area was compared to the peaks of volatile aldehydes for their subsequent quantification.

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2.3.2. Carotenoid pigment analysis by HPLC A Hewlett Packard 1050 liquid chromatograph, equipped with a diode array detector, was used for the analysis of the various carotenoids, with data collected by a Chemstation 7. Sample (20 l) was injected onto a Spherisorb ODS-1 reversed phase column (250 4.6 mm i.d., 5 m particle size), protected by a guard column (ODS-1). For -carotene as well as for lycopene and annatto extracts, an isocratic elution with a mobile phase of acetonitrilemethanoldichloromethane (75 : 20 : 5) was used (Scott, Finglas, Seale, Hart, & Gortz, 1996). For the lutein-rich extract (marigold), the following conditions were used: flow rate: 2 ml/min, linear gradient: methanolethyl acetate (3 : 2 v/v) changing in 20 min to methanolethyl acetate (2 : 3 v/v). Prior to the analysis of the marigold extract a saponification procedure (methanolic KOH, under nitrogen in the dark) was applied to remove interfering pigments and hydrolyse the present lutein esters as described by Scott et al. (1996). Carotenoid samples were examined for the presence of tocopherols by a normal phase HPLC analysis as described by the AOCS official method Ce 8-89 (AOCS, 1989). 2.4. Model of statistical analysis To assess more precisely the effect of each carotenoid, the results were expressed in terms of a protective factor (R), which was calculated as following: R tCAR =tCNT where tCAR: time (h) for the carotenoid containing emulsion to reach the absorbance value (A233) of 0.80 and tCNT: time (h) for the control emulsion to undergo the same level of deterioration. Times were calculated form the plots of absorbance change (at 233 nm) against time of oxidation (h). In these experiments, the A233: 0.80 was arbitrary used as a sufficient measure of oxidative deterioration (which represented a 200300% increase of the initial absorbance) indicating that AAPH-initiated oxidation had clearly proceeded. The average values (R value of CD measurements or volatile concentrations) for each treatment (control or emulsion with added antioxidants) were calculated from the replicate experiments (n = 5). Data were analysed by one-way ANOVA test and least significant differences (LSD) were used to assess whether the values of the carotenoid emulsions significantly differed (p b 0.05) from the control or between each other. Genstat 5.0 was the statistical program used. 3. Results and discussion 3.1. Analysis of carotenoid samples Carotenoid samples were analyzed by Reversed Phase HPLC for the identification of the containing pigments. The unsaponified sample of marigold extract contained lutein esters (mainly palmitate and dipalmitate) together with free lutein, so that quantification was not possible. In the saponified sample, after the removal of unwanted pigments and lipids and the hydro-

lisation of esters, the following isomers of lutein were identified: all-trans lutein (38.9%), 13-cis lutein (28.6%), 9-cis lutein (20.3%) and zeaxanthin (12.2%). Annatto water-soluble preparation contained mainly norbixin and traces of bixin, whereas lipid-soluble annatto was found to be pure in bixin. In the tomato extract, lycopene was the major component (87%) but a considerable proportion of -carotene (11.5%) and traces of carotene (1.5%), were also present. The total active carotenoid concentration of each tested extract, expressed as a percentage of the major identified carotenoid, was determined by visible absorption spectroscopy, using absorptivity values reported in the literature (Scott et al., 1996). The unsaponified marigold extract (which was used in the oxidation experiments) contained 55% lutein as esters, lipid-soluble annatto contained 95% bixin, and all-trans -carotene was 98% pure. No tocopherols were traced in carotenoid samples, with the exception of lycopene that contained 1.5% -tocopherol. 3.2. Effect of carotenoid structure on radical scavenging activity In the initial experiments, it was checked whether the various carotenoid preparations, added to the emulsion at an active concentration of 1 g l 1 in oil, can inhibit the AAPH-initiated oxidative deterioration. Carotenoids with different polarity (structure indicated in Fig. 1a) were used to elucidate the effect of polar groups on the stabilisation of emulsions. The oxidation parameters (temperature, time and AAPH concentration) were selected after some preliminary experiments as appropriate for promoting lipid oxidation. Measurement of conjugated dienes at 233 nm was the main method of monitoring lipid oxidation. The appearance of conjugated dienes in oxidised lipids is due to the double bond shift following free radical attack on hydrogen atoms of methylene groups separating double bonds. The method was preferred over the iodometric Peroxide Value determination, for its direct application in emulsion systems, (Kiokias & Gordon, 2003a), whereas it is also much simpler and faster, does not depend on chemical reactions or colour development, and requires a smaller sample size. However, the presence of compounds absorbing in the region of conjugated dienes (also including carotenoids) may interfere with the measurements. To minimise such interference, the initial absorbance at 233 nm (reflecting the level of baseline oxidation or even a small contribution of the carotenoid conjugated structure) was subtracted from the subsequent values to assess more accurately the formation of conjugated diene hydroperoxides, which are produced as the methyleneinterrupted polyunsaturated fatty acids are oxidised in time. As clearly shown in Fig. 2, the control emulsion presented a faster increase of absorbance at 233 nm, with comparison to the carotenoid-containing emulsions. Therefore, the tested carotenoids seem to inhibit the AAPH-initiated oxidation and exert an antioxidant character. At a constant temperature of 60 C, thermal decomposition of the AAPH initiator generates free radicals, which then react with oxygen to produce the corresponding peroxyl radicals. Under these conditions, carotenoid molecules

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(a) bixin
COOH

COOCH3

COOH

norbixin
COOH

OH

lutein
HO

-carotene

lycopene

(b)

+ROO.

R
O O

Fig. 1. (a) Structure of the carotenoid pigments tested in the current study. (b) Addition of a peroxyl radical to the carotenoid molecule (formation of a resonance stabilized carbon centered radical).

compete with the linoleyl group of triglycerides (linoleic acid is the major fatty acid of sunflower oil) for reaction with the azo peroxyl radicals. Interestingly, probably due to the fact that the rate of reaction of carotenoids and linoleyl TGs with the radical species is sufficiently large from the beginning, no lag phase was observed in all oxidation experiments. Carotenoids have been reported (Burton & Ingold, 1984; Edge, Truscoot, & McGarvey, 1997) to act as radical scavengers due to the extensive system of conjugated double bonds in their molecule that makes them very susceptible to radical addition. According to this particular mechanism, -carotene (as an example) is capable of scavenging peroxyl radicals (reaction 1, Fig. 1b). The resulting carbon centred radical (ROO--CAR) reacts rapidly and reversibly with oxygen to form a new, chaincarrying peroxyl radical (ROO--CAR-OO, reaction 2). The carbon centred radical is resonance stabilized to such an extent, that when the oxygen pressure is lowered the equilibrium of reaction 2 shifts sufficiently to the left, to effectively lower the

concentration of peroxyl radicals and hence reduce the amount of autoxidation in the system (Burton, 1988). Furthermore, the
1,00 0,75 0,50 0,25 0,00 0 1 2 Time (h) 3 4

Fig. 2. Effect of carotenoids (at 1 g l 1 in oil) on the oxidative stability of 10% o/ w emulsions, as expressed by absorption at 233 nm (reflecting the formation of conjugated dienes) after 4 h of AAPH-initiated oxidation at 60 C. Control (), -carotene (), lutein-rich extract (), bixin (+) and norbixin ().

Abs-233 nm

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-carotene radical adduct can also undergo termination by reaction with another peroxyl radical (reaction 3). Reactions: CAR ROO ROO CAR

ROO CAR ROOinactive products:

ROO CAR O2 ROO CAR OO

2 3

The direct antioxidant effects of carotenoids in emulsions, as observed in this study, is in contrast to the findings of Heinonen et al. (1997) who concluded that carotenoids were not effective as antioxidants in food emulsions, in the absence of tocopherols. A basic difference refers to the oxidation model; the earlier study relied on aerial oxidation, whereas AAPH was added as a radical initiator in the current work. As the AAPH radical combines very fast with oxygen (pre-existing in the headspace of the sample vial), it is likely to reduce the oxygen content of the solution enhancing the antioxidant character of carotenoids, which is thereby favoured under these relatively low oxygen pressure conditions. Secondly, the carotenoid concentration in the current study (1 g l 1) is quite higher than the concentration used in the earlier work (1050 g g 1), a factor that probably contributes to the observed antioxidant effect. It must be stressed that since the present study served as a pilot for a subsequent human trial, relatively high carotenoid levels have been tested, (similar to daily dose levels of a normal diet). Statistical analysis of data (p b 0.05) concluded evident differences in the antioxidant activity of the various carotenoids, as expressed by the protective factor (R) in 5 replicate experiments (Table 1). The following antioxidant hierarchy was established: bixind = norbixind (annatto extracts) N lutein-rich extractc N all-trans pure -caroteneb = lycopene-rich extractb N controla. Therefore, the annatto extracts (containing the most polar carotenoids with carboxylic acid and esters groups) were stronger antioxidants than marigold extract containing lutein (with two hydroxyl groups). -Carotene and lycopene samples (carotenoids lacking polar substitutes) were less effective but still clear antioxidants compared with the control. This tendency reveals an association between structure and carotenoid activity,
Table 1 Parameters related to carotenoid activity (R) or degradation (DA), after 4 h of AAPH-initiated oxidation of 10% O/W emulsions at 60 C Tested carotenoids (at 1 g l 1) Control -carotene Lycopene Lutein Bixin Norbixin Protective factor (R) 233 nm (mean SD, n = 5, p b 0.05) 1.00a 0.00 1.46b 0.20 1.42b 0.18 1.85c 0.33 3.47d 0.20 3.36d 0.21 % loss of absorption (DA) max (mean SD, n = 3, p b 0.05) 0.13a 0.00 18.47b 3.56 21.52b 2.53 31.90c 1.13 45.74d 1.30 49.60d 1.22

which somehow depends on the nature of the end group. It seems that the antioxidant potential of carotenoids against AAPH-derived radicals is enhanced with increasing polarity in the carotenoid molecule. Woodall, Britton, Jackson, and Weesie (1997) suggested that the different reactivities of carotenoids against free radicals can be partly attributed to variations in the electron distribution along the polyene chain of different chromophores, which would alter the susceptibility of free radical addition to the conjugated double bond system. Therefore, this structural feature is mainly responsible for the chemical reactivity of carotenoids towards oxidizing agents and free radicals, and consequently, for any antioxidant role. In the present study, substitution of the hydrogens in the carotenoid molecule with polar oxo-groups (such as in bixin and norbixin) was found to increase the overall peroxyl radical trapping ability. This may happen due to the electron withdrawing character of the oxygen atoms, which substantially reduces the unpaired electron density of the carbon centred radical and thereby its reactivity with AAPH-derived radicals, leading to a more evident antioxidant character of the very polar carotenoids. An alternative hypothesis, for the superior effect of polar carotenoids, refers to their distribution in an emulsion system. In the current study, -carotene is likely to be homogeneously dispersed in the oil droplets, whereas the more polar xanthophylls (lutein, and annatto carotenoids) may be located near the oilwater interface, where free radical attack from AAPH-derived radicals first occurs. Therefore, if the more polar carotenoids are preferentially distributed at the droplet's surface they may exert a higher antioxidant activity (compared to the hydrophobic carotenoids -carotene and lycopene) in the emulsions, since they can react more effectively with AAPHradicals generated in the aqueous phase. 3.3. Oxidative degradation of carotenoids Understanding the fundamental chemistry of the reactions of carotenoids with oxidising agents is essential in order to assess their real value as protective antioxidants. In literature, relatively low activation energy for the oxidation of -carotene has been reported, indicating that carotenoid reaction with free radicals is quite fast as compared, for instance, to the autoxidation reaction of linoleic acid (Mortensen, Skibsted, Sampson, Rice-Evans, & Everett, 1997). This difference in reactivity can be attributed to the greater stability of the allylic radical produced from the -carotene molecule, which is stabilised over 11 double bonds, compared with 2 double bonds in the linoleic acid. Both spontaneous and radical initiated oxidation of carotenoids can be followed by observing the loss of colour (or bleaching) in the visible spectrum, as AAPH-initiated oxidation proceeds. According to Miller, Sampson, Bramley, and RiceEvans (1996) bleaching is accompanied by loss of carotenoids with time and the reaction rate increases with temperature. In the current study, this hypothesis was checked by measuring the absorbance at max, which slightly differs between the

Values having a different letter in the same column are significantly different (in the order of antioxidant activity, a b b b c b d). DA (%) = [Astart Afin] 100 / Astart, % loss of absorbance at max.

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Table 2 Effect of carotenoid extracts (at 1 g l 1 in oil) on volatile aldehydes formation after 4 h of AAPH-initiated oxidation of 10% o/w emulsions at 60 C Major volatile aldehydes Retention Concentration of aldehydes (ppm) (mean SD, time n = 5) (GC-Static Headspace Analysis) (min) Control -carotene Lutein Bixin 87.7a 13.3 24.1b 7.4 27.3b 9.4 15.4c 5.9 107.4a 22.3 47.0b 11.7 30.3c 6.9 34.4c 5.18 26.9a 7.2 21.1b 4.5 13.3c 4.5 13.2c 4.4

1,5

Hexanal 12.0 2-heptenal 17.8 2,6 nonadienal 25.4

Values having a different letter in the same row are significantly different (in the order of antioxidant activity a b b b c).

0,5

0 0 1 2 3 4 5 Concentration (g l-1)
Fig. 3. Effect of concentration (in the range of 0.55 g l 1 in oil) on the antioxidant activity in 10% o/w emulsions subjected to AAPH-initiated oxidation at 60 C (expressed as average R value, n = 5) of selected carotenoids: -carotene () lutein () and bixin (+).

most stable with time. It is therefore clear that the higher the rate of carotenoid degradation, the faster the carotenoids react with AAPH-derived peroxy radicals and consequently the more effectively they act as antioxidants under these conditions. 3.4. Effect of concentration on carotenoid antioxidant activity In another series of experiments, the effect of concentration (in the range of 0.55 g l 1 in oil) on the carotenoid activity was examined. An overall plot presenting the change of antioxidant activity with concentration is given in Fig. 3. -Carotene showed a clear increase in antioxidant activity with concentration up to 1 g l 1 (1.9 10 3 M) whereas a smaller but still clear change occurred in the whole tested range above this value. Bixin showed no significant increase in activity above 1 g l 1 carotenoid in oil (2.6 10 3 M). For the marigold extract, the activity increased significantly with concentration up to 3 g l 1 (5.3 10 3 M) and then stabilized. The relatively high activity of xanthophylls (bixin, lutein) samples at low concentrations with no marked increase in activity above a certain level may reflect the increased concentration of these carotenoids at the oilwater interface as well as its more effective radical scavenging ability. Possibly carotenoid molecules can no longer pack into the surface if the carotenoid concentration is N 1 g l 1 for bixin or N 3 g l 1 for lutein.

pigments as following: -carotene = 450 nm; lutein = 445 nm; bixin, norbixin = 425 nm; lycopene = 472 nm. Results are presented in Table 1, as % loss of absorbance at max after 4 h of oxidation, expressed as DA (%): DA% Astart Afin 100=Astart : All carotenoids showed a steady decrease as AAPH-initiated oxidation proceeded with time, reflecting a partial disruption of their polyene chromophore, during the reaction of the compounds with the produced radical species. It must be stressed that annatto carotenoids (which were previously mentioned as the most effective antioxidants) were most rapidly consumed followed by lutein sample, whereas the carotene extracts, were

2-heptenal (Rt=17.8)
17.786 50 40
Peak Intensity

2-6 nonadienal
(Rt=25.8)

12.031

30

(Rt=20.5)

20

10

15
Retention Time (Rt-min)

20

20.512

25

Fig. 4. Chromatogram of the major volatile aldehydes formed by decomposition of linoleyl-hydroperoxides during AAPH initiated oxidation of emulsions at 60 C (GC-Static-Headspace Analysis).

25.383

Hexanal (Rt=12.0)

1,2 DCB

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Table 3 Effect of - or -tocopherols (at 0.1 or 0.2 mM) on the antioxidant activity of carotenoids at 1 g l 1 as expressed by average R values (n = 5) after 4 h of AAPH-initiated oxidation of 10% o/w emulsions at 60 C Treatment (carotenoid + 0.1 or 0.2 mM tocopherol) Lutein Lutein + 0.1 Lutein + 0.2 -carotene -carotene + 0.1 -carotene + 0.2 Bixin Bixin + 0.1 Bixin + 0.2 R values (mean, n = 5) -tocopherol 1.11a 0.08 1.28b 0.10 1.30b 0.06 1.16a 0.07 1.25b 0.09 1.39c 0.14 1.24a 0.09 1.30a 0.06 1.27a 0.08 For mixtures with: -tocopherol 1.51a 0.24 1.72b 0.29 2.14c 0.29 1.41a 0.25 1.84b 0.37 2.11c 0.37 1.96a 0.17 2.02a 0.25 2.06a 0.23

Values having a different letter in the same column are significantly different (in the order of antioxidant activity a b b b c).

3.5. Effect of carotenoids on the formation of volatile aldehydes Under the experimental conditions used in this study, some decomposition of hydroperoxides also occurred. Static headspace SPME extraction was used to identify the volatiles formed in the chemically oxidized emulsions. SPME has been widely accepted in recent years as a useful analytical tool to measure volatile components, having the advantages of being a relatively quick, one step method of extracting volatiles from the headspace that does not require solvents or costly equipment (Song, Gardner, Holland, & Beudry, 1997). In the current work, it was found that hexanal, 2-heptenal and 2,6-nonadienal were the major volatiles present in the oxidized emulsion samples (Fig. 4). The finding generally agrees with similar studies identifying these certain aldehydes among the main products of linoleic acid hydroperoxide decomposition (Snyder, Frankel, & Selke, 1985). Interestingly, all the tested carotenoids (added at 1 g l 1) inhibited markedly the formation of all the volatile aldehydes when compared with the control as indicated in Table 2. Bixin and lutein were generally found to be more effective than carotene, a finding which is consistent with their efficiency in inhibiting the formation of hydroperoxides. In the literature, there is very limited data about the effect of carotenoids on the formation of secondary oxidation products. Heinonen et al. (1997), by measuring the formation of hexanal and 2-heptenal in emulsions, did not observe any significant inhibitory effect of carotene. However, Warner and Frankel (1987) reported that twice as much 2-heptenal was formed in the control sample compared to soybean oil containing 200 ppm of -carotene, a conclusion closer to the finding of the present study. 3.6. Interaction of carotenoids with other antioxidants The antioxidant properties of mixtures of carotenoids (1 g l ) with - or -tocopherols (at 0.1 or 0.2 mM levels in oil) were also studied in sunflower oil-in-water emulsions and the results are
1

presented in Table 3. The antioxidant activity of -carotene was increased by the addition of both tocopherol isomers in a dose dependent effect. The same tendency was observed for lutein tocopherol interactions, though increase of -tocopherol level to 0.2 mM did not improve the antioxidant efficiency of the mixture. Bixin was the only carotenoid that did not exhibit an enhanced antioxidant effect in combination with or tocopherol at both concentration levels. The fact that bixin exerted alone a very strong antioxidant potential may associate with no further improvement of its activity as mixtured with tocopherols. The increased antioxidant activity of carotenoid combinations with -tocopherol is consistent with findings in other lipid systems (Haila et al., 1996; Henry, Gatignani, & Scwharz, 1998). Also, it is proposed that a combination of lutein and -tocopherol was more efficient than -tocopherol alone, in inhibiting the hydroperoxide formation of autoxidised triglycerides (Palozza & Krinsky, 1992). Recent photolysis studies demonstrated that carotenoid radicals are reduced by - or tocopherols by an electron transfer mechanism (Bohm, Edge, Lange, McGarvey, & Truscott, 1997). Tocopherols may also quench radicals directly, retard the formation of the carotenoid radical and inhibit its further degradation. There is very little evidence in literature about the interactions of carotenoids with ascorbic acid. In the present study, the antioxidant activity of the carotenoids (1 g l 1) was studied in mixtures with ascorbic acid (0.1 g l 1), and the activity was compared with that of the individual antioxidants. Combinations of all the carotenoids with ascorbic acid exhibited a tendency for an enhanced antioxidant activity, however their effect was not significantly stronger than the effect of the individual compounds. Mortensen et al. (1997) proposed that dietary carotenoids react with a wide range of radical species to produce radical cations by electron transfer, which in turn interact with vitamin C to regenerate the carotenoid. In the current study, the ascorbic acid in the aqueous phase is likely to interact directly with azoperoxy radicals generated from AAPH, and a reduction in the azo peroxy-radical concentration before reaction with lipid molecules appears to be the dominant effect of the ascorbic acid. Acknowledgements We thank Dr. Mike Gordon (School of Food Biosciences, The University of Reading) for his constant support and technical guidance during this research project. Also we thank Overseal Foods Ltd. for providing the carotenoid extracts for this study. The project was financed by a European Marie-Cure Reintegration Program (6th Framework, Contract nr: 513675).

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