Journal of Food Composition and Analysis 22 (2009) 295–302

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Journal of Food Composition and Analysis

journal homepage: www.elsevier.com/locate/jfca

Original Article

Effect of ascorbic acid on deterioration of carotenoids and colour in

ultrafrozen orange juice
Antonio J. Meléndez-Martı́nez, Isabel M. Vicario, Francisco J. Heredia *
Laboratory of Food Colour & Quality, Department of Nutrition and Food Science, Faculty of Pharmacy, University of Seville, 41012 Seville, Spain

A R T I C L E I N F O A B S T R A C T

Article history: The influence of externally added ascorbic acid (AA) on the deterioration of carotenoid pattern and
Received 29 April 2008 colour of orange juices has been assessed. Regardless of the enrichment of the samples with ascorbic
Received in revised form 14 November 2008 acid, the changes in their carotenoid profile were analogous and involved mainly the epoxycarotenoids.
Accepted 1 December 2008
The decreases in the levels of the two major carotenoids, namely (9Z)-violaxanthin and (9Z)- or (90 Z)-
antheraxanthin, due to acid-elicited 5,6-epoxide to 5,8-furanoxide isomerizations, were markedly
Keywords: distinct, with the levels of the former dropping more markedly. It was observed, in any event, that the
5,6-Epoxides
decreases were higher in the sample spiked with the higher amount of ascorbic. In view of these results it
5,8-Furanoxides
Ascorbic acid
has been hypothesized that the location of (9Z)-violaxanthin in orange cloud particles could be more
Carotenoids accessible to the juice acids to that of (9Z)- or (90 Z)-antheraxanthin. In addition, the enrichment of the
CIELAB juices seems to promote the contact of the carotenoids and the acids during their deterioration. In terms
Colour of colour, it has been concluded that these changes in the carotenoids led to colour differences that can be
Deterioration discerned visually and that the use of a black background for the measurements yielded a better
Epoxycarotenoids differentiation among the samples.
Orange juice ß 2009 Elsevier Inc. All rights reserved.
Food analysis
Food composition

1. Introduction
The study of the carotenoid profile of citrus products in general
(Dhuique-Mayer et al., 2005; Fanciullino et al., 2006; Agocs et al.,
2007; Matsumoto et al., 2007; Wang et al., 2008; Beltrán González
et al., 2008) and orange juices in particular (Lee and Castle, 2001;
Gama and Sylos, 2005; Meléndez-Martı́nez et al., 2005a, 2008b)
continues to attract the interest of researchers from diverse
geographical areas. The motives for this interest can be summar-
ized under two broad headings: first, citrus fruits and their derived
products are food commodities in much of the world (United
States, Brazil, China, Korea, the Mediterranean Basin, etc.). It is
therefore important to characterize them to cater for the
consumers’ demands, above all because colour plays a key role
in their acceptability (reviewed in (Meléndez-Martı́nez et al.,
2005)). Thus, a myriad of studies undertaken to study that property
on its own (Meléndez-Martı́nez et al., 2004, 2006; Pérez-López
et al., 2006) or in relation to the pigment content (Arena et al.,
* Tel.: +34 954556761; fax: +34 95455 7017.
E-mail address: heredia@us.es (F.J. Heredia).
2000; Lee, 2002; Meléndez-Martı́nez et al., 2003, 2007c) have been
conducted in recent years.
Secondly, the study of citrus carotenoids has been considerably
boosted in the context of renewed interest in these pigments
because of likely health benefits attributed to them (Olson, 1999;
Stahl and Sies, 2003). This in turn is related to general
recommendations of increasing the intake of fruits and vegetables
(Steinmetz and Potter, 1996; Southon, 2000). These studies are
especially important in the case of citrus as certain of them,
notably oranges, are considered to be among the fruits with the
most complex carotenoid pattern (Gross, 1987; Meléndez-
Martı́nez et al., 2007d). This is one of the reasons why the
molecular basis underlying their complex carotenoid deposition is
also receiving much attention (Rodrigo et al., 2004; Kato et al.,
2004, 2006; Rodrigo and Zacarı́as, 2007; Fanciullino et al., 2007).
In either case, and in connection to general investigations
addressing the effect of different practices in the characteristics of
orange juices (Farnworth et al., 2001; Luckow and Delahunty,
2004), it is important to assess the impact of different factors in the
carotenoid content of these products. These studies are unques-
tionably relevant, since, as mentioned earlier on, they could alter
consumer acceptability of these fruits as well as their nutritional
value. In this regard, the changes undergone by the carotenoids as a

0889-1575/$ – see front matter ß 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.jfca.2008.12.005
296 A.J. Meléndez-Martı´nez et al. / Journal of Food Composition and Analysis 22 (2009) 295–302
consequence of industrial treatments of the orange juice to
increase shelf-life have been well-studied (Lee and Coates, 2003;
Sánchez-Moreno et al., 2005; Cortés et al., 2006; Torres Gama and
de Sylos, 2007; Meléndez-Martı́nez et al., 2008b). Nonetheless,
very little is known to date concerning the influence of ascorbic
acid in the pattern of orange juice carotenoids; ascorbic acid is one
of the major citrus micronutrients and is also reported to be related
to quality changes during the industrial processing and storage of
the product (Nagy, 1980; Lee and Coates, 1997). Since the lack of
information on this matter is surprising this study was undertaken
to shed some light on it. More specifically, this study is aimed at
assessing the effect of externally added ascorbic acid on the typical
changes in individual carotenoids and colour observed during the
shelf-life of orange juice.
2. Materials and methods
2.1. Samples
Three 1-L bottles of ultrafrozen orange juice (UFOJ) from the
same batch were used for the study. The bottles were made of
high-density polyethylene and had headspace. Their thawing was
performed at room temperature overnight, after which one of the
samples (UA0) was not enriched with L-ascorbic acid (AA), 200 and
400 mg of the vitamin were added to the other two (samples UA2
and UA4, respectively). After vitamin addition, the bottles were
thoroughly shaken to ensure complete dissolution of the vitamin.
The amounts of ascorbic acid were chosen by taking into
consideration reports of a wide variation range in content in
orange juices (Nagy, 1980; Meléndez-Martı́nez et al., 2007b). The
samples were kept at room temperature and analysed at 48-h
intervals (0, 48 and 96 h).
2.2. HPLC analysis of carotenoids
The HPLC analysis, sample preparation, identification and
quantitation of orange juice carotenoids were carried out as
explained in a previous work (Meléndez-Martı́nez et al., 2007a). All
the operations involving carotenoid extracts were performed
under dim light. In short, 10-mL aliquots of juice were mixed with
50 mL of the extracting solvent (methanol/acetone/hexane,
25:25:50, containing 0.1% butylated hydroxytoluene) and the
coloured hexane phase subsequently saponified with 10% etha-
nolic potassium hydroxide. For liquid chromatography analysis, a
C30 column (5 mm, 250 mm  4.6 mm) (YMC, Wilmington, NC)
kept at 17 8C was used as stationary phase and methanol (MeOH),
tert-butyl methyl ether (TBME) (both containing 0.1% of butylated
hydroxytoluene and 0.05% of triethylamine) and water were used
in the mobile phase, according to the following linear gradient: 90%
MeOH + 5% TBME + 5% water; 12 min: 95% MeOH + 5% TBME;
25 min: 89% MeOH + 11% TBME; 40 min: 75% MeOH + 25% TBME;
60 min: 50% MeOH + 50% TBME; 62 min: 90% MeOH + 5%
TBME + 5% water. Chromatograms were monitored at 430, 450
and 486 nm, and the flow was set at 1 mL/min.
For the identification of most carotenoids, the chromatographic
and spectroscopic features were compared to those of the
corresponding standards, which were isolated according to
standard procedures or semi-synthesized. Other carotenoids were
isolated from orange juice and identified on the basis of their UV/
vis and mass spectra, chromatographic features, chemical tests and
co-chromatography with mixtures of geometrical isomers of
appropriated standards (Meléndez-Martı́nez et al., 2005b,c,
2007e). Minor peaks 2, 3 and 4 were tentatively identified as
latoxanthin (50 ,60 -epoxy-5,6,50 ,60 -tetrahydro-b,b-carotene-3,5,6,-
30 -tetrol) (peak 2) and latochrome (50 ,80 -epoxy-5,6,50 ,80 -tetrahy-
dro-b,b-carotene-3,5,6,30 -tetrol) isomers (peaks 3 and 4) (Melén-
dez-Martı́nez et al., 2008a). To help identify Z isomers, the
corresponding all-E-carotenoids were stereo-mutated and tenta-
tively identified on the basis of the shifts of their absorption
maxima and the intensity of their absorption maxima in the UV
region were taken into consideration (Zechmeister, 1962).
The pigments were quantified by external calibration from
four-level calibration curves constructed with the corresponding
all-E-standards. The concentration of zeinoxanthin (b,e-caroten-3-
ol) was calculated out of the calibration curve of lutein (b,e-
carotene-3,30 -diol), because both have virtually identical spectra.
The total contents of carotenoids were assessed as the sum of the
content of the individual pigments.
2.3. Objective colour measurement by tristimulus colorimetry
The reflectance visible spectra (380-770 nm) were obtained at
1 nm intervals by means of a CAS 140 B spectroradiometer
(Instrument Systems, Munich, Germany) equipped with an
external incandescent lamp, a Top 100 telescope optical probe
(Instrument Systems, Munich, Germany) and a Tamron zoom mod.
SP 23A (Tamron USA, Inc., Commack, NY, USA). The apparatus was
set to make three consecutive readings of each sample and the
colour coordinates obtained were their averages. The orange juices
were shaken vigorously to re-suspend the pulp particles and
immediately placed into transparent plastic cuvettes (475 mm
 350 mm  10 mm) to take the colour readings, which were
acquire in a dim ambient illumination to avoid possible
interferences from other external light sources. Furthermore, the
cuvettes containing the samples were placed inside a box lined
with homogeneous grey cardboard, to which the external
illumination source was attached. The zoom, to which the probe
was attached, was placed in a straight line from the sample,
specifically 50 cm apart. Concerning the geometry of presentation,
458 incident illumination was used.
The blank reference measurements were made with the cuvette
filled with distilled water against a reference BaSO4 pressed plate
(USRS-99-010, Labsphere Inc. North Sutton, NH, USA). The colour
of the juices was assessed against a white background (reference
BaSO4 plate) and a black background (round homogeneous plastic
piece).
The colour parameters corresponding to the uniform colour
space CIELAB (CIE, 1978) were obtained directly from the
apparatus considering the 108 Observer and Illuminant D65 as
references.
2.4. Titratable acidity and pH
The titratable acidity expressed to citric acid was assessed by
standard procedures. In brief, the samples of orange juice were
diluted with distilled water, a few drops of ethanolic phe-
nolphtalein (1%) were added, and the juice mixture was
subsequently titrated with aqueous NaOH 0.1 M until colour
change. A model 450A Orion pHmeter was used for the pH
measurements.
3. Results and discussion
3.1. Changes in the carotenoid profile
The chromatograms obtained for the non-enriched sample
(UA0) at 48-h intervals are shown in Fig. 1 (peak annotation in
Table 1). Regardless of the enrichment of the samples with
ascorbic acid, it was observed that the changes in the carotenoid
profile of the samples were analogous. Such changes involved
mainly the epoxycarotenoids represented in Fig. 2 and were
already noticeable when the samples were analysed at t = 48 h.
 A.J. Meléndez-Martı´nez et al. / Journal of Food Composition and Analysis 22 (2009) 295–302 297

Fig. 1. Chromatograms at 430 nm of the carotenoid extract of the non-enriched sample (UA0) at 48-h intervals. For peak identification see Table 1.

Thus, auroxanthin (5,8:50 ,80 -diepoxy-5,8,50 ,80 -tetrahydro-b,b-
carotene-3,30 -diol), which was not detected in the ultrafrozen
orange juices (UFOJ) surveyed when freshly thawed (Meléndez-
Martı́nez et al., 2007a, 2008b), became clearly detectable. In
addition, a marked increase in the levels of a (Z)-luteoxanthin
(5,6:50 ,80 -diepoxy-5,6,50 ,80 -tetrahydro-b,b-carotene-3,30 -diol)
isomer (peak 12) as well as the appearance of another
(Z)-luteoxanthin isomer (peak 11) that was not clearly detected
in freshly thawed UFOJ was also observed.
These increases in the levels of the diepoxycarotenoids
luteoxanthin and auroxanthin were attributed to the isomerization
of their 5,6:50 ,60 -diepoxy isomer violaxanthin (5,6:50 ,60 -diepoxy-
298 A.J. Meléndez-Martı´nez et al. / Journal of Food Composition and Analysis 22 (2009) 295–302

Fig. 2. Chemical structures of antheraxanthin, auroxanthin, luteoxanthin, mutatoxanthin and violaxanthin.

5,6,50 ,60 -tetrahydro-b,b-carotene-3,30 -diol) (Fig. 1), which is this point it is opportune to remark that the eventual total
known to take place in presence of even traces of acid. In the conversion of the different geometrical isomers of violaxanthin
case of orange juices, isomerization is therefore favoured by the occurring in orange juice into auroxanthin leads to the formation of
loss of compartmentation brought about by the squeezing of the not fewer than 12 isomers of the latter carotenoid (Meléndez-
oranges, which brings together organic acids and carotenoids. At Martı́nez et al., 2007e), since the acidification of violaxanthin
results in the formation of up to three auroxanthin optical isomers,
Table 1 namely (8R, 80 R)-, (8R, 80 S)- and (8S, 80 S)-auroxanthin (Britton
Peak identification. et al., 2004).
Peak Carotenoid (code in Table 2) This fact is to be taken into consideration in connection to the
efficiency of C30 stationary phases in the separation of stereo-
1 Unidentified (maxima at 372, 392 and 414 nm)
2 Unidentified (latoxanthin?)
isomers of carotenoids, because they can eventually make much
3 Unidentified (latochrome isomer?) more intricate the already complex orange juice carotenoid profile
4 Unidentified (latochrome isomer?) by increasing the probability of co-elutions (Meléndez-Martı́nez
5 (All-E)-violaxanthin + (Z)-violaxanthin isomers (ctV) et al., 2007a, 2008b). The acid-promoted 5,6-epoxycarotenoids to
6 Luteoxanthin + (Z)-anteraxanthin isomer (LX+cAN)
5,8-epoxycarotenoids isomerizations (Fig. 3) explain the drop in
7 Auroxanthin (AU_A)
8 (Z)-Anteraxanthin isomer (cAN) the levels of antheraxanthin (5,6-epoxy-5,6-dihydro-b,b-caro-
9 (Z)-Anteraxanthin isomer + auroxanthin (cAN+AU_B) tene-3,30 -diol) geometrical isomers and the concomitant rise in the
10 (9Z)-Violaxanthin (9cV) levels of the mutatoxanthin (5,8-epoxy-5,8-dihydro-b,b-caro-
11 (Z)-Luteoxanthin isomer (cLX_A) tene-3,30 -diol) isomers (Fig. 2). Likewise, they may also account
12 (Z)-Luteoxanthin Isomer (cLX_B)
13 Mutatoxanthin epimer (MX_A)
for the formation of the minor compounds tentatively identified as
14 Lutein (LT) latochrome isomers (peaks 3 and 4) which were already detectable
15 Mutatoxanthin epimer (MX_B) at 48 h from the commencement of the study but not in freshly
16 Zeaxanthin (ZX)
17 (9Z) or (90 Z)-antheraxanthin (9cAN)
18 Zeinoxanthin (ZIX)
19 b-cryptoxanthin (BCR)
20 (Z)-z-carotene isomer (cZC)
21 a-carotene (AC)
22 b-carotene + z-carotene (BC+cZC)
For spectroscopic and chromatographic details see references (Meléndez-Martı́nez
et al., 2005b,c, 2007a,e, 2008b). Fig. 3. Scheme of the isomerization of a 5,6-epoxy group into a 5,8-epoxy group.
 A.J. Meléndez-Martı´nez et al. / Journal of Food Composition and Analysis 22 (2009) 295–302 299

thawed juices (Fig. 1). These peaks may well correspond to the 5,8-
epoxyderivatives of the compound tentatively identified as
latoxanthin (peak 2). Analogous 5,6-epoxy to 5,8-epoxycarote-
noids isomerizations upon the processing of other acidic fruit
products, specifically mango derivatives have been described
(Mercadante and Rodriguez-Amaya, 1998).
In spite of the fact that the evaluation of the changes in every
individual 5,6-epoxycarotenoid during the study was impaired by
inevitable co-elutions of some of them, the decreases in the levels of
the major compounds, namely (9Z)-violaxanthin and (9Z) or (90 Z)-
antheraxanthin, could be followed with clarity. This fact was
important because such compounds also happen to be the
predominant carotenoids in ultrafrozen orange juices and are hence
presumed to have a prominent role in their colour. Thus, it was
noticed that (9Z)-violaxanthin accounted for 31–34% of the total
carotenoid in the orange juices analysed at t = 0, whereas (9Z)- or
(90 Z)-antheraxanthin accounted for a further 12-13%. That is, both
pigments on their own made up half the total carotenoid content in
the freshly thawed juices. As far as (9Z)-violaxanthin was concerned,
a clear decrease in its concentration, specifically 4.64 mg/L (67.4%),
was observed in the non-enriched juice (UA0) at the end of the study
(Table 2). As for the effect of the AA concentration on the level of this
carotenoid, it was seen that the losses in the enriched samples were
higher, more specifically 5.18 (77.2%) and 6.73 mg/L (85.2%) for the
samples UA2 (enriched with 200 mg/L of ascorbic acid) and UA4
(enriched with 400 mg/L of ascorbic acid) (Table 2). These
observations seem to indicate that the higher the ascorbic acid
content the greater the rate of 5,6-epoxy to 5,8-epoxycarotenoid
isomerization of (9Z)-violaxanthin. Concerning (9Z)- or (90 Z)-
antheraxanthin, slight decreases were observed in the samples
UA0 and UA2 (15.2% and 16.3%, respectively), and a much higher
drop in the case of the sample UA4, specifically 35.8%. After
performing the corresponding ANOVA analyses, it was seen that the
levels of (9Z)-violaxanthin at the end of the study were statistically
different (p < 0.05) as a function of the AA added, which was not true
in the case of (9Z)- or (90 Z)-antheraxanthin.
These findings seem to point to that the enrichment with AA
favours 5,6-epoxy to 5,8-furanoxide isomerizations in orange juice,
because neither the titratable acidity (ranging from 1.01 to 1.10 g
citric acid/100 mL) nor the pH (varying from 3.53 to 3.63 units)
changed considerably along the study. More importantly, it can be
concluded that (9Z)-violaxanthin and (9Z)- or (90 Z)-antherax-
anthin exhibit a markedly different susceptibility to such
isomerization in orange juices, independently of their enrichment
with AA, the isomerization rate of the former being 2.4–4.7-fold
higher in this study. In this respect, it is to be stressed that both
carotenoids show different susceptibility to the acid-elicited
isomerization in orange juice, as their isomerization when they
are in solution and this is acidified is extremely rapid (Eugster,
1995; Rodriguez-Amaya, 2001). It is also to be noted that
carotenoids are not dissolved in orange juice, but forming part
of the pulp particles which are suspended in the aqueous phase of
the juice. Thus, the topology of such particles could play a
preponderant role in the isomerization reactions.
Taking all these observations together, these findings seem to
indicate that, firstly, the location of (9Z)-violaxanthin within the
pulp particles make it more accessible to the orange acids in
comparison to that of (9Z)- or (90 Z)-antheraxanthin. Considering
that size, polarity and shape are important features determining
the location of organic molecules in their milieus, the different rate
of isomerization observed may be due ultimately to the presence of
one additional epoxy group in violaxanthin (Fig. 2). The different
rate of isomerization could also be due to both carotenoids being
differently esterified, since citrus xanthophylls are known to occur
both free and esterified with fatty acids (Wingerath et al., 1996;
Breithaupt and Bamedi, 2001). Secondly, the enrichment of the
juices with ascorbic acid seems to favour the putting into contact of
the carotenoids and the acids since the pH does not change
considerably, as it has been remarked earlier. In this regard, it is to
be considered that the ultrafrozen orange juice studied is not
subjected to thermal treatments, so its colloidal stability is shorter
as compared to other juices. Consequently, changes in its
suspended particles, the so-called orange juice cloud, became
apparent during the study. In relation to this fact, it is well-known
that the clarification observed in not thermally-treated orange
juices is largely due to the modifications of pectin, a complex group
of polysaccharides that are essential constituents of the cell walls
of plants. Chemically these anionic heteropolysaccharides are
mainly composed of D-galacturonic acid units that are esterified at
certain positions along their backbone. Its methyl ester groups are
deesterified by the action of pectinmethylesterases (PMEs), with
what charged areas are created that lead to the formation of
calcium pectate gels that ultimately precipitate clarifying the juice
(Wicker et al., 2003; Croak and Corredig, 2006). Although the

Table 2
Carotenoid levels (mg/L) of the samples surveyed.

Sample t (h) Added Total ctV LX+cAN AU_A cAN cAN+AU_B 9cV cLX_A cLX_B MX_A
ascorbic carotenoids

UA0 0 0 mg 22.18 3.24  0.12 1.06  0.03 – 0.16  0.01 – 6.88  0.18 – 0.70  0.03 0.72  0.02
UA0 48 0 mg 25.01 3.58  0.46 1.41  0.03 0.88  0.05 – 0.76  0.04 4.90  0.02y – 1.44  0.02 1.47  0.04
UA0 96 0 mg 26.16 3.62  0.35 1.47  0.12 1.09  0.07 – 1.00  0.08 2.24  0.20 1.38  0.12 1.63  0.12 2.01  0.16
UA2 0 200 mg 21.67 3.23  0.03 1.07  0.01 – 0.16  0.00 – 6.71  0.11 – 0.66  0.00 0.69  0.00
UA2 48 200 mg 27.40 3.72  0.62 1.64  0.09 0.96  0.16 – 0.77  0.14 3.90  0.19 1.24  0.11 1.66  0.09 1.58  0.04
UA2 96 200 mg 25.36 3.18  0.07 1.42  0.04 1.20  0.09 – 0.97  0.09 1.53  0.06 1.35  0.12 1.84  0.10 2.06  0.07
UA4 0 400 mg 25.34 3.95  0.06 1.23  0.02 – 0.21  0.05 – 7.90  0.13 – 0.78  0.03 0.77  0.00
UA4 48 400 mg 27.58 4.07  0.19 1.57  0.17 0.75  0.06 – 0.93  0.15 4.11  0.18 1.35  0.18 1.53  0.16 1.62  0.15
UA4 96 400 mg 22.22 2.51  0.08 1.15  0.10 1.02  0.13 – 0.82  0.13 1.17  0.13 1.14  0.16 1.59  0.18 1.86  0.19

Sample t (h) Added Total LT MX_B ZX 9cAN ZIX BCR cZC AC BC+cZC
ascorbic carotenoids

UA0 0 0 mg 22.18 0.79  0.01 1.32  0.03 1.96  0.05 2.64  0.05 0.47  0.02 1.18  0.02 0.42  0.03 0.15  0.01 0.48  0.01
UA0 48 0 mg 25.01 0.91  0.03 1.96  0.06 2.13  0.06 2.41  0.04 0.53  0.01 1.36  0.07 0.48  0.06 0.17  0.01 0.60  0.03
UA0 96 0 mg 26.16 1.08  0.10 2.74  0.32 2.34  0.18 2.24  0.20 0.57  0.05 1.42  0.14 0.44  0.01 0.19  0.01 0.71  0.06
UA2 0 200 mg 21.67 0.77  0.00 1.27  0.02 1.89  0.03 2.57  0.06 0.46  0.01 1.15  0.00 0.42  0.02 0.14  0.01 0.48  0.00
UA2 48 200 mg 27.40 1.01  0.05 2.15  0.04 2.39  0.01 2.75  0.03 0.62  0.00 1.53  0.01 0.59  0.05 0.21  0.01 0.68  0.01
UA2 96 200 mg 25.36 1.09  0.05 2.92  0.39 2.34  0.05 2.15  0.05 0.59  0.00 1.42  0.01 0.43  0.00 0.16  0.00 0.71  0.02
UA4 0 400 mg 25.34 0.85  0.04 1.43  0.08 2.16  0.09 3.07  0.13 0.53  0.01 1.32  0.08 0.47  0.02 0.17  0.01 0.50  0.07
UA4 48 400 mg 27.58 1.09  0.08 2.27  0.37 2.37  0.18 2.67  0.12 0.58  0.03 1.45  0.09 0.45  0.00 0.17  0.01 0.61  0.01
UA4 96 400 mg 22.22 0.94  0.09 2.57  0.48 2.16  0.17 1.97  0.16 0.55  0.02 1.34  0.10 0.42  0.08 0.20  0.05 0.81  0.12
300 A.J. Meléndez-Martı´nez et al. / Journal of Food Composition and Analysis 22 (2009) 295–302

changes in the juice cloud were apparent in all the samples, the
marked effect of the addition of AA in the isomerization of
epoxycarotenoids may be due to the fact that this favours the
structural changes in the cloud particles. This hypothesis is
supported by a study on the effect of AA and other agents on the
viscosity of orange peel pectin solutions, in which it was concluded
that the higher the levels of AA, the greater the increase in specific
fluidity (Kar and Arslan, 1999).
With respect to the carotenes fraction, it was observed that the
levels of (Z)-z-carotene and a-carotene (peaks 20 and 21,
respectively) did not change appreciably. As for the mixture of
b-carotene and a (Z)-z-carotene isomer (peak 22), a clear increase
was noticed. The evaluation of the average spectrum at the
beginning and the end of the study (Fig. 4) revealed an increase in
the levels of the latter.

3.2. Changes in the colour

Theoretically, it would be sensible to expect that the

isomerization of the 5,6-epoxycarotenoids would bring about
easily discernible changes in the visible spectra of the juices,
because such rearrangements lead to hypsochromic displacements
of around 20 nm when only one epoxy group is involved
(mutatoxanthin, luteoxanthin) or 40 nm, when two 5,6-epoxy
groups are isomerized into 5,8-furanoxides (auroxanthin) (Fig. 5).
However, as it can be observed in Fig. 6, the changes in the visible
reflection spectra of the juices are not as conspicuous as expected,
above all in the case of juices at t = 0 and 48 h and in the 400–
500 nm interval, which is the region of maximum absorbance of
the orange juice carotenoids studied. This observation illustrates
by itself the intricacy of the relationships between the carotenoid
pattern of orange juices and their colour. In the case of juices at
t = 72 h, higher reflectance was observed from roughly 500 nm
onwards, above all when the black background was used for the

Fig. 5. Depiction of the acid-elicited hypsochromic shifts in the absorption maxima

of violaxanthin and antheraxanthin.

colour assessments, although the shape of the spectra was virtually

Fig. 4. Averaged spectra of peak 22 at beginning (up) and end (down) of the study.
identical as compared to the remaining juices.
The hypsochromic shifts referred to above have been studied in
terms of CIELAB colour coordinates previously by our research
group. In this sense it was concluded that the isomerization of one
5,6-epoxy group in violaxanthin to give luteoxanthin involved a
slight rise in the value of a* and a decrease in the value of b*, while
the re-arrangement of the remaining 5,6-epoxide group in
luteoxanthin to form auroxanthin, was accompanied by a sharp
rise in a* and a marked drop in b* (Meléndez-Martı́nez et al., 2007).
Those observations were obtained from pure solutions of the
pigments, so that in the samples analysed for this study such
changes were expected to be somewhat masked by the complexity
of their pigment pattern. Specifically, it was noticed that, for
measurements carried out with white background, L*, the
psychometric index of lightness in the CIELAB space, augmented
from the beginning to the conclusion of the study, whereas a*
decreased. The coordinates b* and C*ab, also diminished along the
study, more noticeably in the orange juice spiked with 400 mg of
ascorbic acid. As for the angular coordinate hue, very slight rises
were observed between the commencement and the end of the
study (Table 3). The colour differences between the samples at the
beginning and the end of the study were 3.09, 2.78 and 7.89 CIELAB
units for the samples UA0, UA2 and UA4, respectively. These
results indicated that those colour differences could be distin-
guished visually by people with rigorous colour tolerances, as they
are very close or over 2.8 CIELAB units, which has been long
 A.J. Meléndez-Martı´nez et al. / Journal of Food Composition and Analysis 22 (2009) 295–302 301

somewhat more clear. As for the changes in the coordinate a*, it

was noticed that its trend along the study differed from that
observed in the measurements made with white background.
Thus, its values decreased from the commencement of the study to
the middle of it to increase eventually, even above the initial value
in the case of the samples UA0 and UA2. The variation of the values
of b* and C*ab over the study, it was also different relative to the
values obtained with the white background, such that their values
increased from the beginning to the conclusion of the study. As for
hue, slight increases were observed between the commencement
and the end of the study (Table 3). The colour differences between
the samples at the beginning and the end of the study were 7.52,
7.51 and 5.28 CIELAB units for the samples UA0, UA2 and UA4,
respectively, which taken together indicated that the use of the
black background led to a greater differentiation between samples
relative to the white background. In any case, the colour
differences were over the threshold of 2.8 CIELAB units mentioned
earlier on.

Acknowledgements

The authors wish to express their gratitude to the firm Zumos

Vitafresh (Almonte, Spain) for the provision of the ultrafrozen
orange juice samples analysed in this study.

References

Agocs, A., Nagy, V., Szab, Z., Mdrk, L., Ohmacht, R., Deli, J., 2007. Comparative study
on the carotenoid composition of the peel and the pulp of different citrus
species. Innovative Food Science & Emerging Technologies 8, 390–394.
Arena, E., Fallico, B., Maccarone, E., 2000. Influence of carotenoids and pulps on the
color modification of blood orange juice. Journal of Food Science 65 (3), 458–
460.
Beltrán González, F., Pérez-López, A.J., López-Nicolás, J.M., Carbonell-Barrachina,
A.A., 2008. Effects of agricultural practices on instrumental colour, mineral
content, carotenoid composition, and sensory quality of mandarin orange juice,
cv. Hernandina. SO. Journal of the Science of Food and Agriculture 88, 1731–
1738.
Breithaupt, D.E., Bamedi, A., 2001. Carotenoid esters in vegetables and fruits: a
screening with emphasis on b-cryptoxanthin esters. Journal of Agricultural and
Fig. 6. Reflection spectra of the non-enriched sample during the study (t = 0, 48 and
Food Chemistry 49, 2064–2070.
96 h). Britton, G., Liaaen-Jensen, S., Pfander, H., 2004. Carotenoids Handbook. Birkhäuser,
Basel, Switzerland.
considered as one of the thresholds to test the ability of people to CIE, 1978. Recommendations on Uniform Color Spaces, Color-Difference Equations,
discriminate colours (Lozano, 1978; Melgosa et al., 2001). Psychometric Color Terms. CIE Publication No. 15 (E-1.3.1) 1971, Supplement 2.
Central Bureau of the CIE, Vienna, Austria.
In the case of the measurements taken against the black Cortés, C., Torregrosa, F., Esteve, M.J., Frı́gola, A., 2006. Carotenoid profile modifica-
background, the rise in the values of L* along the study was tion during refrigerated storage in untreated and pasteurized orange juice and
orange juice treated with high-intensity pulsed electric fields. Journal of
Agricultural and Food Chemistry 54, 6247–6254.
Table 3
Croak, S., Corredig, M., 2006. The role of pectin in orange juice stabilization: effect of
Colour coordinates of the samples studied during the study.
pectin methylesterase and pectinase activity on the size of cloud particles. Food
Code t (h) Added L* a* b* C*ab hab Hydrocolloids 20, 961–965.
Dhuique-Mayer, C., Caris-Veyrat, C., Ollitrault, P., Curk, F., Amiot, M.J., 2005.
ascorbic (mg)
Varietal and interspecific influence on micronutrient contents in citrus from
White background the Mediterranean area. Journal of Agricultural and Food Chemistry 53, 2140–
UA0 0 0 72.36 13.95 70.08 71.46 78.75 2145.
UA0 48 0 72.52 13.15 66.41 67.70 78.80 Eugster, C.H., 1995. Chemical derivatization: microscale tests for the presence of
common functional groups in carotenoids. In: Britton, S., Liaaen-Jensen, H., P-
UA0 96 0 74.33 13.02 67.89 69.12 79.14
fander, E. (Eds.), Carotenoids, vol. 1A: Isolation and Analysis. Birkhäuser, Basel,
UA2 0 200 72.85 13.07 70.21 71.42 79.45
Switzerland, pp. 71–80.
UA2 48 200 72.98 12.58 68.93 70.07 79.65 Fanciullino, A.L., Dhuique-Mayer, C., Luro, F., Casanova, J., Morillon, R., Ollitrault, P.,
UA2 96 200 74.96 12.55 68.48 69.62 79.61 2006. Carotenoid diversity in cultivated citrus is highly influenced by genetic
UA4 0 400 73.71 13.92 70.98 72.33 78.91 factors. Journal of Agricultural and Food Chemistry 54, 4397–4406.
UA4 48 400 71.93 13.58 71.21 72.49 79.21 Fanciullino, A.L., Dhuique-Mayer, C., Luro, F., Morillon, R., Ollitrault, P., 2007.
UA4 96 400 74.25 12.37 63.26 64.45 78.94 Carotenoid biosynthetic pathway in the citrus genus: number of copies and
phylogenetic diversity of seven genes. Journal of Agricultural and Food Chem-
Black background istry 55, 7405–7417.
UA0 0 0 60.19 7.94 56.34 56.89 81.98 Farnworth, E.R., Lagacé, M., Couture, R., Yaylayan, V., Stewart, B., 2001. Thermal
UA0 48 0 60.85 7.08 53.42 53.89 82.45 processing, storage conditions, and the composition and physical properties of
UA0 96 0 66.48 8.31 60.44 61.01 82.17 orange juice. Food Research International 34, 25–30.
UA2 0 200 60.59 7.80 56.43 56.97 82.13 Gama, J.J.T., Sylos, C.M., 2005. Major carotenoid composition of Brazilian Valencia
UA2 48 200 61.83 6.77 53.72 54.14 82.82 orange juice: identification and quantification by HPLC. Food Research Inter-
UA2 96 200 67.14 7.91 60.11 60.63 82.51 national 38, 899–903.
UA4 0 400 61.27 8.46 55.80 56.44 81.38 Gross, J., 1987. Pigments in Fruits. Academic Press, Inc, Orlando, FL.
UA4 48 400 60.42 7.14 61.49 61.90 83.38 Kar, F., Arslan, N., 1999. Characterization of orange peel pectin and effect of sugars,
UA4 96 400 66.41 8.11 56.95 57.52 81.89 ascorbic acid, ammonium persulfate, salts on viscosity of orange peel pectin
solutions. Carbohydrate Polymers 40, 285–291.
302 A.J. Meléndez-Martı´nez et al. / Journal of Food Composition and Analysis 22 (2009) 295–302
Kato, M., Ikoma, Y., Matsumoto, H., Sugiura, M., Hyodo, H., Yano, M., 2004. Accu-
mulation of carotenoids and expression of carotenoid biosynthetic genes during
maturation in citrus fruits. Plant Physiology 134, 824–837.
Kato, M., Matsumoto, H., Ikoma, Y., Okuda, H., Yano, M., 2006. The role of carotenoid
cleavage dioxygenases in the regulation of carotenoid profiles during matura-
tion in citrus fruit. Journal of Experimental Botany 57, 2153–2164.
Lee, H.S., Coates, G.A., 2003. Effect of thermal pasteurization on Valencia orange
juice color and pigments. Lebensmittel-Wissenschaft und Technnologie-Food
Science and Technology 36, 153–156.
Lee, H.S., 2002. Characterization of major anthocyanins and the color of red-fleshed
budd blood orange (Citrus sinensis). Journal of Agricultural and Food Chemistry
50, 1243–1246.
Lee, H.S., Castle, W.S., 2001. Seasonal changes of carotenoid pigments and color in
Hamlin, Earlygold, and Budd Blood orange juices. Journal of Agricultural and
Food Chemistry 49, 877–882.
Lee, H.S., Coates, G.A., 1997. Vitamin C contents in processed Florida citrus juice
products from 1986 to 1995 survey. Journal of Agricultural and Food Chemistry
45, 2550–2555.
Lozano, R.D., 1978. El color y su medición. Buenos Aires, Americalee.
Luckow, T., Delahunty, C., 2004. Consumer acceptance of orange juice containing
functional ingredients. Food Research International 37, 805–814.
Matsumoto, H., Ikoma, Y., Kato, M., Kuniga, T., Nakajima, N., Yoshida, T., 2007.
Quantification of carotenoids in citrus fruit by LC-MS and comparison of
patterns of seasonal changes for carotenoids among citrus varieties. Journal
of Agricultural and Food Chemistry 55, 2356–2368.
Meléndez-Martı́nez, A.J., Britton, G., Vicario, I.M., Heredia, F.J., 2005a. Color and
carotenoid profile of Spanish Valencia late ultrafrozen orange juices. Food
Research International 38, 931–936.
Meléndez-Martı́nez, A.J., Britton, G., Vicario, I.M., Heredia, F.J., 2005b. Identification
of isolutein (lutein epoxide) as cis-antheraxanthin in orange juice. Journal of
Agricultural and Food Chemistry 53, 9369–9373.
Meléndez-Martı́nez, A.J., Britton, G., Vicario, I.M., Heredia, F.J., 2005c. Identification
of zeinoxanthin in orange juices. Journal of Agricultural and Food Chemistry 53,
6362–6367.
Meléndez-Martı́nez, A.J., Britton, G., Vicario, I.M., Heredia, F.J., 2007. Relationship
between the colour and the chemical structure of carotenoid pigments. Food
Chemistry 101, 1145–1150.
Meléndez-Martı́nez, A.J., Britton, G., Vicario, I.M., Heredia, F.J., 2008a. Does the
carotenoid neoxanthin occur in orange juice? Food Chemistry 107, 49–54.
Meléndez-Martı́nez, A.J., Britton, G., Vicario, I.M., Heredia, F.J., 2008b. The complex
carotenoid pattern of orange juices from concentrate. Food Chemistry 109, 546–
553.
Meléndez-Martı́nez, A.J., Vicario, I.M., Heredia, F.J., 2007a. Carotenoids, color and
ascorbic acid content of a novel frozen-marketed orange juice. Journal of
Agricultural and Food Chemistry 55, 1347–1355.
Meléndez-Martı́nez, A.J., Vicario, I.M., Heredia, F.J., 2007b. Provitamin A carotenoids
and ascorbic acid contents of the different types of orange juices marketed in
Spain. Food Chemistry 101, 177–184.
Meléndez-Martı́nez, A.J., Vicario, I.M., Heredia, F.J., 2007c. Rapid assessment of
vitamin A activity through objective color measurements for the quality control
of orange juices with diverse carotenoid profiles. Journal of Agricultural and
Food Chemistry 55, 2808–2815.
Meléndez-Martı́nez, A.J., Vicario, I.M., Heredia, F.J., 2007d. Review: analysis of car-
otenoids in orange juice. Journal of Food Composition and Analysis 20, 638–649.
Meléndez-Martı́nez, A.J., Vicario, I.M., Heredia, F.J., 2003. Application of Tristimulus
Colorimetry to estimate the carotenoids content in ultrafrozen orange juices.
Journal of Agricultural and Food Chemistry 51, 7266–7270.
Meléndez-Martı́nez, A.J., Vicario, I.M., Heredia, F.J., 2007e. Geometrical isomers of
violaxanthin in orange juice. Food Chemistry 104, 169–175.
Meléndez-Martı́nez, A.J., Vicario, I.M., Heredia, F.J., 2004. Correlation between visual
and instrumental colour measurements of orange juice dilutions. Effect of the
background. Food Quality and Preference 16, 471–478.
Meléndez-Martı́nez, A.J., Vicario, I.M., Heredia, F.J., 2005. Instrumental measure-
ment of orange juice colour: a review. Journal of the Science of Food and
Agriculture 85, 894–901.
Meléndez-Martı́nez, A.J., Vicario, I.M., Heredia, F.J., 2006. Influence of white refer-
ence measurement and background on the colour specification of orange juices
by means of diffuse reflectance spectrophotometry. Journal of AOAC Interna-
tional 89, 452–457.
Melgosa, M., Pérez, M.M., Yebra, A., Huertas, R., Hita, E., 2001. Algunas reflexiones y
recientes recomendaciones internacionales sobre evaluación de diferencias de
color. Óptica Pura y Aplicada 34, 1–10.
Mercadante, A.Z., Rodriguez-Amaya, D.B., 1998. Effects of ripening, cultivar differ-
ences, and processing on the carotenoid composition of mango. Journal of
Agricultural and Food Chemistry 46, 128–130.
Nagy, S., 1980. Vitamin C contens of citrus fruit and their products: a review. Journal
of Agricultural and Food Chemistry 28, 8–18.
Olson, J.A., 1999. Carotenoids and human health. Archivos Latinoamericanos de
Nutrición 49, 7–11.
Pérez-López, A.J., Beltran, F., Serrano-Megı́as, M., Saura López, D., Carbonell-Barra-
china, Á.A., 2006. Changes in orange juice color by addition of mandarin juice.
European Food Research and Technology 222, 516–520.
Rodrigo, M.J., Marcos, J.F., Zacarı́as, L., 2004. Biochemical and molecular analysis of
carotenoid biosynthesis in flavedo of orange (Citrus sinensis L.) during fruit
development and maturation. Journal of Agricultural and Food Chemistry 52,
6724–6731.
Rodrigo, M.J., Zacarı́as, L., 2007. Effect of postharvest ethylene treatment on car-
otenoid accumulation and the expression of carotenoid biosynthetic genes in
the flavedo of orange (Citrus sinensis L. Osbeck) fruit. Postharvest Biology and
Technology 43, 14–22.
Rodriguez-Amaya, D.B., 2001. A Guide to Carotenoid Analysis in Foods. ILSI Press,
Washington, DC.
Sánchez-Moreno, C., Plaza, L., Elez-Martı́nez, P., De Ancos, B., Martı́n-Belloso, O.,
Cano, M.P., 2005. Impact of high pressure and pulsed electric fields on bioactive
compounds and antioxidant activity of orange juice in comparison with tradi-
tional thermal processing. Journal of Agricultural and Food Chemistry 53, 4403–
4409.
Southon, S., 2000. Increased fruit and vegetable consumption within the EU:
potential health benefits. Food Research International 33, 211–217.
Stahl, W., Sies, H., 2003. Antioxidant activity of carotenoids. Molecular Aspects of
Medicine 24, 345–351.
Steinmetz, K.A., Potter, J.D., 1996. Vegetables, fruit, and cancer prevention: a review.
Journal of the American Dietetic Association 96, 1027–1039.
Torres Gama, J.J., de Sylos, C.M., 2007. Effect of thermal pasteurization and con-
centration on carotenoid composition of Brazilian Valencia orange juice. Food
Chemistry 100, 1686–1690.
Wang, Y.C., Chuang, Y.C., Hsu, H.W., 2008. The flavonoid, carotenoid and pectin
content in peels of citrus cultivated in Taiwan. Food Chemistry 106, 277–
284.
Wicker, L., Ackerley, J.L., Hunter, J.L., 2003. Modification of pectin by pectinmethy-
lesterase and the role in stability of juice beverages. Food Hydrocolloids 17,
809–814.
Wingerath, T., Stahl, W., Kirsch, D., Kaufmann, R., Sies, H., 1996. Fruit juice carotenol
fatty acid esters and carotenoids as identified by matrix-assisted laser deso-
rption ionization (MALDI) mass spectrometry. Journal of Agricultural and Food
Chemistry 44, 2006–2013.
Zechmeister, L., 1962. Cis-trans Isomeric Carotenoids Vitamins A and Arylpolyenes.
Springer Verlag, Vienna, Austria.