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1 s2.0 S096399691400194X Main PDF
1 s2.0 S096399691400194X Main PDF
1 s2.0 S096399691400194X Main PDF
a r t i c l e i n f o a b s t r a c t
Article history: Apple (Malus × domestica Borkh) has always been considered a fruit with low chlorophyll and carotenoid con-
Received 3 December 2013 tents; however these pigments contribute also to the external (peel) and internal (flesh) fruit colourations, as
Received in revised form 13 February 2014 well as to the health benefits associated with the regular consumption of this fruit. In the present work we stud-
Accepted 27 March 2014
ied the chlorophyll and carotenoid compositions of the peel and flesh of thirteen marketed apple varieties pre-
Available online 5 April 2014
senting different external colourations (green, yellow and red). All the varieties were characterised by a
Keywords:
common pigment profile comprised by chlorophylls a and b, lutein, violaxanthin, neoxanthin, β-carotene, and es-
Malus domestica terified carotenoids (mainly violaxanthin and neoxanthin) as major compounds. Total pigment content was al-
Apple cultivars ways higher in the peel (58.72–1510.77 μg/g dry wt) than in the flesh (14.80–71.57 μg/g dry wt) for each
Breeding particular cultivar. In general, the green cultivars showed the highest pigment content both in the peel and in
Carotenoids the flesh, which were followed in decreasing order by the peel of some red-skinned cultivars and the flesh of
Chlorophylls the yellow ones. In most cultivars, a characteristic chloroplastic pigment profile was found in the peel, with lutein
Xanthophyll esterification as the main free carotenoid whilst a chromoplastic profile was usually observed in the flesh, with a prevalence of
Peel
violaxanthin and neoxanthin and their corresponding acyl esters (mono- and diesters). A correlation between
Flesh
the carotenoid content and the amount of esterified xanthophylls was observed, in particular in the flesh,
which reinforce the putative role of the esterification process in the accumulation of these lipophilic compounds
within the chromoplasts.
© 2014 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodres.2014.03.025
0963-9969/© 2014 Elsevier Ltd. All rights reserved.
R. Delgado-Pelayo et al. / Food Research International 65 (2014) 272–281 273
The green colour of vegetables and most unripe fruits is due to the Hornero-Méndez, 2012) suggested a key role of the esterification of
joint occurrence of chlorophylls and carotenoids, whereas the colour xanthophyll in relation to facilitate the accumulation of these pig-
of the ripe fruit is frequently due the presence of carotenoids (Britton ments within the chromoplasts of vegetables.
& Hornero-Méndez, 1997). Both types of pigments cannot be synthe-
sized by animals and consequently it is necessary to incorporate Materials and methods
them through the diet. In green plants, the carotenoids are found in
the chloroplasts of green tissues, where their colour is masked by Plant material
the greater presence of the chlorophylls (chloroplastic pigment pro-
file) whilst in yellow, orange and red fruits, they are usually located Thirteen commercial apple cultivars were selected and obtained
in chromoplasts, and frequently present as complex mixtures of fatty from a local market, including four green-skinned varieties (Granny
acyl esters (chromoplastic pigment profile) (Britton, 1991). Smith, Green Doncella, Green Golden Delicious and Reina de Reineta),
It has been demonstrated that the chlorophyll pigments exhibit a se- three yellow-green and yellow-skinned varieties (Golden Montaña,
ries of biological properties, such as antioxidant and antimutagenic ac- Golden Delicious and Golden Rosett) and six red-skinned varieties
tivities, modulation of xenobiotic metabolising enzyme activity, and (Ariane, Fuji (I) from Italy, Fuji (F) from France, Pink Lady, Royal Gala
induction of apoptotic events in cancer cell lines, all consistent with and Starking Red Chief). Table 1 summarises the characteristics of the
the prevention of cancer and other degenerative diseases (Dashwood, studied samples.
1997; Ferruzzi & Blakeslee, 2007). Carotenoids play an important role
in attracting animals to act as pollinators and seed dispersion vehicles, Chemicals and reagents
including in this process the consumption of food by humans (Britton
& Hornero-Méndez, 1997; Howitt & Pogson, 2006). When carotenoids HPLC-grade methanol and acetone were supplied by BDH Prolabo
are ingested, they exert important biological activities: antioxidant, pro- (VWR International Eurolab, SL, Barcelona, Spain). Diethyl ether, con-
vitamin A activity (β-carotene, α-carotene, β-cryptoxanthin, etc.), inhi- taining 7 ppm butylated hydroxytoluene (BHT), was purchased from
bition of carcinogenesis, enhancement of the immune response and cell Scharlau (Scharlab, SL, Barcelona, Spain). HPLC-grade deionised water
defence against reactive oxygen species (ROS) and free radicals, and the was produced with a Milli-Q 50 system (Millipore Iberica SA, Madrid,
reduction on the risk of developing cardiovascular and other degenera- Spain). The rest of reagents were all of analytical grade.
tive diseases (reviewed by Britton, Liaaen-Jensen, & Pfander, 2009).
Recent studies have demonstrated the anti-Helicobacter pylori ac- Sample preparation
tivity of some carotenoids from Golden Delicious apples (Molnár
et al., 2010). As proposed by Liu (2003, 2004), the health benefits Fruits (4 for each cultivar) were washed and peeled, and the flesh
resulting from a diet rich in fruit and vegetables are due to a syner- and peel were separately frozen in liquid nitrogen, and subsequently
gistic and additive combination of the effects derived from complex freeze-dried (lyophilized) and ground in a mortar. The dry samples
mixture of phytochemicals present in the whole food. Most of re- were kept in plastic bags at − 80 °C until extraction and analysis.
search carried out for the phytochemical profiling of apples have The moisture contents (% water) in the flesh and peel were measured
been focused on phenolic compounds with fewer studies on chloro- in triplicate by using an Ohaus moisture balance model MB35 (Ohaus,
phyll and carotenoid pigments (Knee, 1972, 1988; Solovchenko, Switzerland).
Avertcheva, & Merzlyak, 2006), which will undoubtedly contribute
to the health benefits associated with the regular consumption of Pigment extraction
this fruit. Some early studies already investigated the chlorophyll
and carotenoid compositions of the apples (Galler & Mackinney, In accordance with an extraction procedure recently developed
1965; Gorski & Creasy, 1977; Gross & Lenz, 1979; Gross, Zachariae, for potato tubers (Fernandez-Orozco et al., 2013), the extraction
Lenz, & Eckherdt, 1978; Knee, 1972; Valadon & Mummery, 1967; of pigments was performed with N,N-dimethylformamide (DMF)
Workman, 1963) demonstrating the preferential accumulation in saturated with MgCO 3, in order to avoid the degradation and/or
the peel with respect to the pulp, and the catabolic disappearance transformation of xanthophylls containing 5,6-epoxide groups and
of chlorophylls during the ripening of the fruits. However, most of the pheophytinization (releasing of the Mg 2+ ion) of the chloro-
these studies were mainly focused in the well-known Golden Deli- phyll pigments. Briefly, an amount of the lyophilized sample (2 g
cious cultivar, and others less popular such as Grimes Golden and for flesh and 0.1 g for peel) was placed in a round-capped polypro-
Cox's Orange Pippin, and consequently the information available in pylene 50-mL centrifuge tube, and extracted with 20 mL of N,N-
the literature is very restricted taking into account the great number dimethylformamide (DMF) saturated with MgCO 3 by using an
and diversity of apple cultivars available in the markets. In the last de- Ultra-Turrax T-25 homogenizer for 1 min. Subsequently, the mixture
cade increasing efforts are being dedicated to the selection of fruits was centrifuged at 4500 ×g for 5 min at 4 °C, and the extracted chloro-
with enhanced levels of diverse phytonutrients, with special attention phylls and carotenoids, contained in the DMF, were collected. The ex-
to the carotenoids, involving multidisciplinary breeding programmes traction operation was repeated twice more with the pellet, and the
(Ampomah-Dwamena et al., 2012). Therefore, the present work was DMF fractions were pooled in a separation funnel. Pigments were trans-
aimed to study the chloroplastic and chromoplastic pigment composi- ferred to diethyl ether (80 mL) by adding 90 mL of 10% NaCl solution
tion in a wide range of commercial apple cultivars in order to provide saturated with MgCO3 at 4 °C. The mixture was vigorously shaken and
biochemical information to be used in breeding programmes directed allowed to stand until complete separation of phases. The lower phase
to improve their nutritional value, as well as to increase the understand- (aqueous DMF) was re-extracted with diethyl ether (50 mL), and the
ing of the processes governing the biosynthesis and accumulation of ca- ether fractions were pooled and washed with 50 mL 2% Na2SO4 solu-
rotenoids in apples. For this purpose, in the present study we have tion. The extract was filtered through a solid bed of anhydrous
characterised and investigated the chlorophyll and carotenoid composi- Na2SO4, and the solvent was evaporated under vacuum in a rotary evap-
tions of a wide collection of apple cultivars, distinguishing between peel orator at 30 °C. The pigments were dissolved in 1 mL of acetone (con-
and flesh, corresponding to thirteen marketed varieties presenting taining 0.1% BHT) and stored at −30 °C until analysis, being the direct
different external colouration (green, yellow and red). Especial at- extract used for the analysis of chlorophylls, individual free carotenoids
tention has been paid to determine the presence of carotenoid acyl and the esterified xanthophyll fraction (mono- and diesters).
esters, as previous work in our laboratory (Fernandez-Orozco, Samples were centrifuged at 14400 ×g for 5 min at 4 °C prior to the
Gallardo-Guerrero, & Hornero-Méndez, 2013; Mellado-Ortega & chromatographic analysis, which it was carried out in the same day of
274 R. Delgado-Pelayo et al. / Food Research International 65 (2014) 272–281
Table 1
Characteristics of the studied apple cultivars.
Cultivar Geographic origin Skin colour Flesh colour Weight (g) per fruita Fruit size (axial Moisture content (%)
diameter; mm)b
Peel Flesh
Ariane France Dark reddish Yellowish/creamed 126.5 ± 3.6 40–45 75.6 ± 0.2 83.6 ± 0.2
Fuji (F) France Reddish with a greenish background Creamed 212.6 ± 1.1 70–75 74.6 ± 0.3 80.5 ± 0.1
Fuji (I) Italy Reddish with a greenish background Creamed 227.1 ± 1.4 80–85 75.2 ± 0.2 86.0 ± 0.1
Granny Smith France Bright greenish Greenish 198.2 ± 6.1 80–85 77.4 ± 0.3 86.6 ± 0.2
Green Doncella Spain Greenish Greenish 144.2 ± 5.8 70–75 72.7 ± 0.2 83.8 ± 0.2
Green Golden Delicious France Greenish Greenish 157.8 ± 1.6 70–75 75.2 ± 0.1 83.9 ± 0.1
Golden Delicious France Yellowish Greenish/yellowish 185.0 ± 1.1 70–75 73.7 ± 0.3 83.1 ± 0.1
Golden Montaña France Yellowish Yellowish 273.9 ± 8.9 70–75 75.9 ± 0.4 83.8 ± 0.3
Golden Rosett Italy Yellowish Yellowish 247.5 ± 0.9 80–85 73.2 ± 0.3 85.1 ± 0.3
Pink Lady France Dark reddish Yellowish/creamed 198.2 ± 6.1 75–80 75.3 ± 0.1 84.3 ± 0.2
Reina de Reineta Spain Greenish Pale greenish/yellowish 193.3 ± 3.1 75–80 76.3 ± 0.2 85.6 ± 0.1
Royal Gala Spain Reddish with a greenish background Yellowish/creamed 212.5 ± 0.6 80–85 76.1 ± 0.3 85.4 ± 0.2
Starking Red Chief Italy Dark reddish Pale greenish/yellowish 228.2 ± 5.4 80–85 75.3 ± 0.3 84.3 ± 0.2
a
Weight data represent the average and standard error (n = 4).
b
As given in the marketed product label.
the preparation of the extracts. At least six independent analyses Chlorophylls and carotenoids analysis by HPLC
were performed for each sample. All operations were made under
dimmed light to prevent isomerization and photodegradation of The quantitative analysis of chlorophylls and carotenoids was
pigments. carried out by HPLC according to the method of Mínguez-Mosquera
and Hornero-Méndez (1993) with slight modifications. The HPLC
system consisted of a Waters 2695 Alliance chromatograph fitted
Pigment identification with a Waters 2998 photodiode array detector (DAD), and controlled
with Empower2 software (Waters Cromatografía, SA, Barcelona,
The identification of chlorophylls and carotenoids was carried out Spain). A reversed-phase C18 column (200 mm × 4.6 mm i.d., 3 μm,
following routine procedures consisted of: separation and isolation Mediterranea SEA18; Teknokroma, Barcelona, Spain), fitted with a
of pigments by TLC and co-chromatography (TLC and HPLC) with guard column of the same material (10 mm × 4.6 mm), was used.
pure standards, analysis of the UV–visible and mass spectra, and Separation was achieved by a binary-gradient elution using an initial
comparison with the literature values and micro-scale chemical composition of 75% acetone and 25% deionised water, which was in-
test for 5,6-epoxide groups by controlled treatment of the samples creased linearly to 95% acetone in 10 min, then hold for 7 min and
(with both the isolated pigments and the direct extracts) with dilut- raised to 100% in 3 min, and maintained constant for 10 min. Initial
ed HCl in ethanol following the analysis of the changes in the UV– conditions were reached in 5 min. The temperature of column was
visible spectrum and the chromatographic mobility (Britton, 1991, kept at 25 °C and the sample compartment was refrigerated at
1995; Britton, Liaaen-Jensen, & Pfander, 2004; Mínguez-Mosquera, 15 °C. An injection volume of 10 μL and a flow rate of 1 mL/min
Gandul-Rojas, Gallardo-Guerrero, Roca, & Jarén-Galán, 2008; Mínguez- were used. Detection was performed simultaneously at 402, 430,
Mosquera, Hornero-Méndez, & Pérez-Gálvez, 2008). Authentic carot- 450, 645 and 666 nm, and the online spectra were acquired in the
enoid and chlorophyll samples were isolated, and purified by 330–700 nm wavelength range with a resolution of 1.2 nm. Pigments
means of TLC, from natural sources: 9′-cis-neoxanthin, violaxanthin, were quantitated in the direct extracts by using calibration curves
lutein, β-carotene, chlorophyll a and chlorophyll b were obtained (up to eight concentration levels) prepared with standard stock so-
from spinach leaves (Spinacea oleracea), whereas β-cryptoxanthin lutions for each carotenoid and chlorophyll in the concentration
and zeaxanthin were obtained from red pepper (Capsicum annuum L.) range 5–100 μg/mL. Calibration curves were constructed by plotting
(Britton, 1991; Mínguez-Mosquera & Hornero-Méndez, 1993). For the peak area (at 402 nm for auroxanthin; at 450 nm for lutein, zea-
identification purposes, antheraxanthin was synthesized by the ep- xanthin, violaxanthin, neoxanthin, neochrome, β-cryptoxanthin and
oxidation of zeaxanthin with 3-chloroperoxybenzoic acid according β-carotene; at 645 nm for chlorophyll b; at 666 nm for chlorophyll a)
to the method of Barua and Olson (2001), and carotenoids contain- versus the pigment concentration. Antheraxanthin and luteoxanthin
ing 5,8-epoxide groups (auroxanthin, luteoxanthin, neochrome and were quantified by using the calibration curves of zeaxanthin and
mutatoxanthin) were prepared from the corresponding 5,6-epoxide neochrome, respectively. The concentration of xanthophyll esters
parent pigments (violaxanthin, neoxanthin and antheraxanthin) was estimated by using the calibration curve of free violaxanthin
(Britton, 1991). The identification of cis isomers was based on the due to the main occurrence of this pigment within the esterified frac-
presence and relative intensity (%AB /AII ) of the cis peak at about tion as deducted from the online UV–visible spectra (the esterification
330–340 nm in UV–visible spectrum, a reduction in the fine struc- of xanthophylls with fatty acids does not modify the chromophore
ture and a small hypsochromic shift in λmax with respect to the all- properties; Britton, 1995). The quantification of cis isomers was carried
trans counterpart, and the chromatographic behaviour in the C18 out by using the calibration curve of the corresponding all-trans coun-
HPLC column (the cis isomers show slightly longer retention times terpart. Concentration values were calculated as μg of pigment per
than the all-trans isomer) (Britton, 1995). The direct extract was sa- gramme of dry weight of sample (μg/g dry wt).
ponified in order to determine the acylated nature of some pigments.
For this purpose, an aliquot (500 μL) was evaporated under a nitro-
gen stream and the pigment residue was dissolved in 2 mL of diethyl Liquid chromatography–mass spectrometry (LC–MS (APCI+))
ether and 0.5 mL of 10% (w/v) KOH-methanol was added and left to
react during 1 h at room temperature under nitrogen atmosphere. LC–MS was performed with the same chromatographic system
After addition of water, the pigments were subsequently extracted (HPLC-DAD) described above, by coupling a Micromass ZMD4000
with diethyl ether, filtered through solid anhydrous Na2SO4, evapo- mass spectrometer equipped with a single quadrupole analyser
rated, taken up to 0.5 mL of acetone and analysed by HPLC. (Micromass Ltd, Manchester, United Kingdom) and an APCI
R. Delgado-Pelayo et al. / Food Research International 65 (2014) 272–281 275
Table 2
Chromatographic, UV–visible and mass (APCI+) spectroscopy properties of the chlorophyll and carotenoid pigments identified in apple (Malus × domestica Borkh) varieties.
Peaka Pigment Rt (min) λmax (nm) in the λmax (nm) in acetone %III/II 5,6-epoxide HPLC/MS (APCI+) fragmentation pattern
eluent solvent according to bibliographyb, c test m/z
(Atmospheric Pressure Chemical Ionisation) probe to the outlet of the high voltage lens, 0.5 kV; and cone voltage, 30 V. Nitrogen was used
DAD detector. The system was controlled with MassLynx 3.2 software as the desolvation and cone gas at 300 and 50 L/h, respectively. Mass
(Micromass Ltd, Manchester, United Kingdom). The mass spectrometer spectra were acquired within the m/z 300–1200 Da range. The chro-
condition parameters were: positive ion mode (APCI+); source tem- matographic conditions were the same as detailed for the quantitative
perature, 150 °C; probe temperature, 400 °C; corona voltage, 3.7 kV; analysis of carotenoids.
10 13
Starking
g Red Chief
16 (Red-skinned)
15 17
3 13’14 14’
2 4 6 9 11 12
1 5 7
8
Granny Smith
(Green-skinned)
0 5 10 15 20 25 30 0 5 10 15 20 25 30
Retention time (min) Retention time (min)
Fig. 1. HPLC chromatograms corresponding to the different chlorophyll and carotenoid profiles found in the peel and flesh of some representative apple cultivars. Peak identities: 1. all-
trans-Neoxanthin; 2. 9′-cis-Neoxanthin; 3. Neochrome; 4. all-trans-Violaxanthin; 5. 9-cis-Violaxanthin; 6. 13-cis-Violaxanthin; 7. Luteoxanthin; 8. Antheraxanthin; 9. all-trans-Zeaxanthin;
10. all-trans-Lutein; 11. 9-cis-Lutein; 12. 13-cis-Lutein; 13. Chlorophyll b; 13′. Chlorophyll b′; 14. Chlorophyll a; 14′. Chlorophyll a′; 15. Xanthophyll acyl esters (partially esterified); 16. all-
trans-β-Carotene; 17. Xanthophyll acyl esters (totally esterified). Detection at 450 nm.
276 R. Delgado-Pelayo et al. / Food Research International 65 (2014) 272–281
β -Carotene
f
i n
c gh j k m
l o
ab e
pq r t
d
s
u
vw x
20 25
0 5 10 15 20 25 30
Retention time (min)
Fig. 3. Mass spectra obtained by LC–MS (APCI+) for a homodiester (A; dimyristate, peak n)
Fig. 2. Peaks tentatively identified in the xanthophyll diester fraction (see Table 3 for the and a heterodiester (B; myristate palmitate, peak q) of violaxanthin. Peak references as in
compound identities). Fig. 2.
R. Delgado-Pelayo et al. / Food Research International 65 (2014) 272–281 277
Table 3
Spectroscopic (UV–visible and MS (APCI+)) properties of the main xanthophyll diesters present in the peel and flesh of apple fruits (peak letters are according to Fig .2).
a all-trans-Neoxanthin dicaprate 419, 444, 472 [96%] 909.69 737.55 [M-Capric acid + H]+, 565.40 [M-2xCapric acid + H]+
b all-trans-Violaxanthin dicaprate 418, 444, 472 [98%]
c 9-cis-Violaxanthin dicaprate 410, 434, 467 [90%]
d all-trans-Neoxanthin caprate laurate 419, 444, 472 [96%] 937.73 765.58 [M-Capric acid + H]+, 737.55 [M-Lauric acid + H]+,
e all-trans-Violaxanthin caprate laurate 418, 444, 472 [98%] 565.40 [M-Capric acid-Lauric acid + H]+
f 9-cis-Violaxanthin caprate laurate 410, 434, 467 [90%]
g all-trans-Neoxanthin dilaurate 419, 444, 472 [96%] 965.76 765.58 [M-Lauric acid + H]+, 565.40 [M-2xLauric acid + H]+
h all-trans-Violaxanthin dilaurate 418, 444, 472 [98%]
i 9-cis-Violaxanthin dilaurate 410, 434, 467 [90%]
j all-trans-Neoxanthin laurate myristate 419, 444, 472 [96%] 993.79 793.61 [M-Lauric acid + H]+, 765.58 [M-Myristic acid + H]+,
k all-trans-Violaxanthin laurate myristate 418, 444, 472 [98%] 565.40 [M-Lauric acid-Myristic acid + H]+
l 9-cis-Violaxanthin laurate myristate 410, 434, 467 [90%]
m all-trans-Neoxanthin dimyristate 419, 444, 472 [96%] 1021.82 793.61 [M-Myristic acid + H]+, 565.40 [M-2xMyristic acid + H]+
n all-trans-Violaxanthin dimyristate 418, 444, 472 [98%]
o 9-cis-Violaxanthin dimyristate 410, 434, 467 [90%]
p all-trans-Neoxanthin myristate palmitate 419, 444, 472 [96%] 1049.85 821.64 [M-Myristic acid + H]+, 793.61 [M-Palmitic acid + H]+,
q all-trans-Violaxanthin myristate palmitate 418, 444, 472 [98%] 565.40 [M-Myristic acid-Palmitic acid + H]+
r 9-cis-Violaxanthin myristate palmitate 410, 434, 467 [90%]
s all-trans-Neoxanthin dipalmitate 419, 444, 472 [96%] 1077.88 821.64 [M-Palmitic acid + H]+, 565.40 [M-2xPalmitic acid + H]+
t all-trans-Violaxanthin dipalmitate 418, 444, 472 [98%]
u 9-cis-Violaxanthin dipalmitate 410, 434, 467 [90%]
v all-trans-Neoxanthin palmitate stearate 419, 444, 472 [96%] 1105.91 849.67 [M-Palmitic acid + H]+, 821.64 [M-Stearic acid + H]+,
w all-trans-Violaxanthin palmitate stearate 418, 444, 472 [98%] 565.40 [M-Palmitic acid-Stearic acid + H]+
x 9-cis-Violaxanthin palmitate stearate 410, 434, 467 [90%]
a
Peak letters are according to Fig. 2.
278
Table 4
Chlorophyll and carotenoid compositions (μg/g dry weight)a present in flesh of thirteen commercial apple cultivars.
Golden Rosett Golden Montaña Golden Delicious Royal Gala Fuji (F) Ariane Starking Red Pink Lady Fuji (I) Reina de Reineta Green Golden Green Doncella Granny
Chief Delicious Smith
all-trans-Neoxanthin 0.32 ± 0.02 0.21 ± 0.01 0.8 ± 0.03 0.96 ± 0.13 0.33 ± 0.03 0.57 ± 0.02 0.51 ± 0.02 0.55 ± 0.02 0.51 ± 0.02 0.4 ± 0.04 0.93 ± 0.03 0.40 ± 0.01 0.99 ± 0.05
9′-cis-Neoxanthin 0.04 ± 0.00 0.04 ± 0.00 0.18 ± 0.01 0.08 ± 0.01 0.03 ± 0.00 0.06 ± 0.01 0.14 ± 0.01 0.07 ± 0.00 0.14 ± 0.01 0.14 ± 0.02 0.32 ± 0.01 0.33 ± 0.02 0.87 ± 0.05
all-trans-Violaxanthin 0.37 ± 0.02 0.47 ± 0.02 0.77 ± 0.02 0.5 ± 0.06 0.18 ± 0.01 0.49 ± 0.03 0.6 ± 0.02 0.19 ± 0.01 0.34 ± 0.02 0.46 ± 0.04 0.91 ± 0.10 0.76 ± 0.04 0.75 ± 0.04
9-cis-Violaxanthin n.d.b n.d. n.d. n.d. n.d. n.d. n.d. 0.02 ± 0.00 0.02 ± 0.00 n.d. n.d. 0.15 ± 0.01 0.11 ± 0.01
13-cis-Violaxanthin 0.01 ± 0.00 0.01 ± 0.00 n.d. n.d. n.d. n.d. n.d. 0.01 ± 0.00 0.02 ± 0.00 n.d. 0.02 ± 0.00 n.d. 0.02 ± 0.00
all-trans-Antheraxanthin 0.01 ± 0.00 0.02 ± 0.00 0.02 ± 0.00 0.01 ± 0.00 0.01 ± 0.00 0.01 ± 0.00 n.d. 0.02 ± 0.00 0.03 ± 0.00 0.02 ± 0.00 0.03 ± 0.00 0.08 ± 0.01 n.d.
all-trans-Zeaxanthin 0.01 ± 0.00 0.02 ± 0.00 n.d. 0.01 ± 0.00 0.01 ± 0.00 0.01 ± 0.00 0.02 ± 0.00 0.02 ± 0.00 0.03 ± 0.00 0.03 ± 0.00 0.02 ± 0.00 0.04 ± 0.00 n.d.
all-trans-Lutein 0.10 ± 0.01 0.19 ± 0.02 0.37 ± 0.02 0.06 ± 0.00 0.04 ± 0.00 0.14 ± 0.00 0.54 ± 0.03 0.13 ± 0.01 0.22 ± 0.01 0.32 ± 0.02 0.71 ± 0.02 1.04 ± 0.05 2.41 ± 0.10
Table 5
Chlorophyll and carotenoid compositions (μg/g dry weight)a present in peel of thirteen commercial apple Cultivars.
Golden Golden Golden Royal Gala Fuji (F) Ariane Starking Red Pink Lady Fuji (I) Reina de Green Golden Green Granny
Rosett Montaña Delicious Chief Reineta Delicious Doncella Smith
all-trans-Neoxanthin 1.54 ± 0.08 1.29 ± 0.07 1.99 ± 0.22 1.20 ± 0.05 1.38 ± 0.05 1.14 ± 0.10 1.94 ± 0.20 1.98 ± 0.12 2.06 ± 0.09 2.01 ± 0.24 2.27 ± 0.11 1.79 ± 0.02 7.11 ± 0.32
9′-cis-Neoxanthin 0.56 ± 0.06 0.56 ± 0.03 1.63 ± 0.06 0.82 ± 0.03 1.01 ± 0.03 0.59 ± 0.09 1.97 ± 0.23 2.33 ± 0.09 3.41 ± 0.15 1.52 ± 0.22 2.18 ± 0.11 2.77 ± 0.07 21.92 ± 1.55
all-trans-Violaxanthin 2.71 ± 0.28 4.92 ± 0.16 3.95 ± 0.12 4.67 ± 0.14 1.70 ± 0.10 3.55 ± 0.3 6.35 ± 0.53 3.83 ± 0.12 4.14 ± 0.16 3.95 ± 0.43 6.57 ± 0.3 6.24 ± 0.09 18.26 ± 0.89
9-cis-Violaxanthin n.d.b n.d. n.d. n.d. 0.23 ± 0.02 n.d. n.d. 0.62 ± 0.02 0.46 ± 0.04 n.d. n.d. 0.97 ± 0.08 2.37 ± 0.15
13-cis-Violaxanthin 0.19 ± 0.03 n.d. n.d. n.d. 0.10 ± 0.00 n.d. n.d. 0.22 ± 0.02 0.20 ± 0.01 n.d. n.d. 0.29 ± 0.02 0.29 ± 0.05
all-trans-Antheraxanthin 0.09 ± 0.02 0.22 ± 0.03 0.27 ± 0.02 0.13 ± 0.01 0.15 ± 0.01 0.13 ± 0.02 0.17 ± 0.05 0.37 ± 0.02 0.47 ± 0.01 n.d. 0.28 ± 0.02 0.57 ± 0.02 n.d.
all-trans-Zeaxanthin 0.28 ± 0.01 0.24 ± 0.02 n.d. 0.08 ± 0.01 0.24 ± 0.06 0.2 ± 0.02 0.16 ± 0.03 0.26 ± 0.04 0.52 ± 0.09 0.39 ± 0.02 0.1 ± 0.01 0.32 ± 0.03 n.d.
all-trans-lutein 2.63 ± 0.22 3.06 ± 0.26 5.79 ± 0.22 1.32 ± 0.06 2.48 ± 0.14 1.7 ± 0.16 7.16 ± 0.48 6.71 ± 0.22 9.59 ± 0.26 5.58 ± 0.76 6.91 ± 0.34 7.37 ± 0.17 61.53 ± 2.66
9-cis-Lutein 0.06 ± 0.01 0.13 ± 0.04 0.08 ± 0.00 0.06 ± 0.01 0.12 ± 0.01 0.04 ± 0.00 0.44 ± 0.04 0.44 ± 0.04 0.43 ± 0.03 0.41 ± 0.04 0.43 ± 0.03 0.34 ± 0.01 1.61 ± 0.08
13-cis-Lutein 0.18 ± 0.02 0.17 ± 0.02 0.23 ± 0.01 0.10 ± 0.01 0.21 ± 0.02 0.13 ± 0.01 0.41 ± 0.02 0.38 ± 0.01 0.53 ± 0.01 0.37 ± 0.04 0.45 ± 0.02 0.41 ± 0.01 2.76 ± 0.06
all-trans-β-Carotene 1.78 ± 0.11 1.62 ± 0.04 2.51 ± 0.09 2.05 ± 0.09 1.49 ± 0.27 5.09 ± 0.38 3.47 ± 0.18 5.44 ± 0.40 3.81 ± 0.21 3.35 ± 0.65 3.23 ± 0.18 5.37 ± 0.14 30.31 ± 1.56
Monoesterified 6.92 ± 0.27 8.54 ± 0.33 6.89 ± 0.67 6.96 ± 0.18 3.1 ± 0.16 10.18 ± 0.72 3.01 ± 0.2 8.07 ± 0.46 5.21 ± 0.15 3.33 ± 0.33 5.56 ± 0.66 5.58 ± 0.07 0.46 ± 0.10
xanthopyhlls
Diesterified xanthophylls 17.00 ± 0.82 23.04 ± 0.74 13.10 ± 0.42 12.09 ± 0.2 4.93 ± 0.81 38.39 ± 2.51 7.40 ± 0.41 18.52 ± 1.44 6.62 ± 0.18 12.42 ± 0.50 12.37 ± 0.91 15.83 ± 0.74 5.04 ± 0.87
Chlorophyll a 18.39 ± 2.39 18.83 ± 1.12 69.71 ± 1.54 32.45 ± 1.09 55.79 ± 2.05 36.68 ± 2.54 116.15 ± 7.25 128.66 ± 3.95 207.68 ± 6.05 124.03 ± 9.24 142.74 ± 6.53 193.24 ± 5.89 1049.26 ± 52.68
Chlorophyll b 6.29 ± 0.86 4.78 ± 0.31 20.13 ± 0.78 11.04 ± 0.33 14.7 ± 0.56 9.13 ± 0.95 32.31 ± 1.73 34.59 ± 1.40 60.79 ± 0.98 37.51 ± 3.74 44.44 ± 2.38 41.42 ± 1.04 309.86 ± 14.89
a
Data represent the average and standard error (n = 6). Violaxanthin (sum of all-trans-violaxanthin, cis-violaxanthin isomers and luteoxanthin), lutein (sum of all-trans-lutein and cis-lutein isomers), and neoxanthin (sum of all-trans-
neoxanthin, 9′-cis-neoxanthin and neochrome).
b
n.d. Not detected.
R. Delgado-Pelayo et al. / Food Research International 65 (2014) 272–281 279
carotenoids was similar in the peel and flesh of the fruit, and mark- and 29.48 ± 0.52 μg/g dry wt, in most of them. Granny Smith was the
edly higher than in the rest of the studied cultivars. Those authors cultivar with a significant higher carotenoid content in the peel
conclude that the biosynthesis and ability to accumulate carotenoids (151.65 ± 7.51 μg/g dry wt), followed by Ariane (61.14 ± 3.84 μg/g
in apple fruits must be finely controlled at genetic level, suggesting dry wt), whilst Fuji from France showed the lowest value (17.14 ±
some rate-limiting carotenogenic step as responsible for the carot- 1.36 μg/g dry wt).
enoid accumulation during fruit ripening. Regarding the flesh, the green-skinned cultivars (with the exception
In the present study, Granny Smith was the cultivar by far with of Reina de Reineta) showed the highest pigment content, with values
higher pigment content in both the peel (1510.77 ± 74.70 μg/g dry (ranging from 45.22 ± 2.25 to 71.58 ± 2.94 μg/g dry wt) similar to
wt) and the flesh (71.57 ± 2.94 μg/g dry wt), and this was mainly the peel of the yellow-skinned cultivars. This result was again due
due to their greater chlorophyll content (chlorophyll a plus chloro- to their greater presence of chlorophylls (from 27.63 ± 1.07 to
phyll b). Then, and as expected, the rest of the green cultivars 60.32 ± 2.65 μg/g dry wt), since they were greenish-fleshed apples
(Green Doncella, Green Golden Delicious and Reina de Reineta) as well. The yellow-skinned cultivars had also appreciable amounts
were those presenting the larger chlorophyll content in the peel of total pigments in the flesh (from 26.16 ± 0.99 to 37.02 ±
(234.66 ± 6.90, 187.18 ± 8.42 and 161.55 ± 12.97 μg/g dry wt, re- 0.69 μg/g dry wt), whilst the red cultivars showed in general less
spectively) although other three red-skinned cultivars (Fuji from quantity (from 14.79 ± 0.46 to 26.14 ± 0.53 μg/g dry wt). Unlike
Italy, Pink Lady, and Starking Red Chief) also showed similar values, peel, carotenoids were always present in higher quantities than
ranging between 268.46 ± 6.39 and 148.46 ± 8.60 μg/g dry wt. chlorophylls in the flesh of both yellow and red skinned apples. Nev-
Therefore the latter cultivars, characterised for presenting a reddish ertheless, chlorophylls were even quantified in the flesh of white-
colour in the peel, contained considerable amounts of chlorophylls fleshed fruits, which in general corresponded with red- and some
but they were partially masked by the anthocyanins colour. The low- yellow-skinned cultivars (Royal Gala, Pink Lady, Fuji from France,
est chlorophyll contents in the peel were found in the rest of red- Ariane and Golden Rosett).
skinned cultivars (Royal Gala, Ariane and Fuji from France) and in Qualitative (Fig. 1) and quantitative differences were also observed
the yellow ones (Golden Delicious, and especially Golden Rosett for the carotenoid composition in the peel and flesh of each cultivar.
and Golden Montaña), and all these cultivars were also those with Data of the individual pigment composition in the peel and flesh
less total pigment content in the peel. are summarised in Tables 4 and 5, respectively. In most cultivars, a
The carotenoid content of the peel was much lower than the chlo- characteristic chloroplastic pigment profile was found in the peel,
rophyll one in most of the cultivars, with the exceptions of the in which lutein (sum of all-trans-lutein and cis-lutein isomers) was
yellow-skinned Golden Rosett and Golden Montaña, and the reddish the main free carotenoid, followed by violaxanthin (sum of all-
Ariane. Moreover, there were not great differences in the carotenoid trans-violaxanthin, 9-cis-violaxanthin, 13-cis-violaxanthin and
concentration values for all cultivars, ranging between 49.17 ± 1.83 luteoxanthin), neoxanthin (sum of all-trans-neoxanthin, 9′-cis-
40
20
15.78%
0
35
Flesh
30
14.85%
25
20 Flesh 12.75%
15 (average)
10 72.40%
Fig. 5. Contribution of the free and esterified xanthophylls (mono- and diesters) to the total carotenoid content in the peel and flesh of the different commercial apple cultivars.
280 R. Delgado-Pelayo et al. / Food Research International 65 (2014) 272–281
60
neoxanthin and neochrome) and β-carotene. Exceptions to the
above pattern were the peel of the yellow-skinned varieties, Golden Peel
Montaña and Golden Rosett, and the red ones Royal Gala and Ariane, 50 R² = 0.696
in which neoxanthin and violaxanthin were the major carotenoids.
Esterified carotenoids (mainly violaxanthin and neoxanthin) were 40
also present in all cultivars, whilst antheraxanthin and zeaxanthin
were found as minor compounds or not detected in some cultivars.
The chloroplastic profile of the apple peel was replaced by a 30
Conclusions
Acknowledgement
Chlorophyll and carotenoid (free and esterified) pigments were al-
This work was supported by funding from the Ministerio de Ciencia
ways present in the peel and flesh of the apples, even in those with a
e Innovación (Spanish Government, Project AGL2010-14850/ALI) and
colourless (white) flesh. Total pigment content was always higher in
the Consejería de Economía, Innovación, Ciencia y Empleo (Junta de
the peel than in the flesh for each particular cultivar and it was mainly
Andalucía, Project P08-AGR-03477). Raúl Delgado Pelayo was the recip-
due to the chlorophyll content in most of the cultivars. In general, the
ient of a predoctoral grant (Junta de Andalucía, Proyecto de Excelencia
green, and some red-skinned, cultivars showed the highest pigment
P08-AGR-3477). Authors are members of the IBERCAROT Network,
content in the peel, whilst the lowest values were found in other red-
funded by CYTED (ref. 112RT0445).
skinned cultivars and in the yellow ones. Most of the green-skinned cul-
tivars showed also the greater concentration of pigments in the flesh,
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