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Carotenoid profile, antioxidant capacity, and chromoplasts of pink guava (Psidium guajava L. cv. ´Criolla´) during fruit ripening Carolina Rojas-Garbanzo, Maike Gleichenhagen, Annerose Heller, Patricia Esquivel, Nadine Schulze-Kaysers, and Andreas Schieber J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b04560 • Publication Date (Web): 16 Mar 2017 Downloaded from http://pubs.acs.org on March 18, 2017
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Journal of Agricultural and Food Chemistry
Carotenoid profile, antioxidant capacity, and chromoplasts of pink guava (Psidium guajava L. cv. ´Criolla´) during fruit ripening
Carolina Rojas-Garbanzo 1,2, Maike Gleichenhagen 1, Annerose Heller 3, Patricia Esquivel 4, Nadine Schulze-Kaysers 1*, Andreas Schieber 1
1
Institute of Nutritional and Food Sciences; Molecular Food Technology, University of Bonn,
Römerstraße 164, D-53117 Bonn, Germany.
[email protected],
[email protected],
[email protected] 2
National Center for Food Science and Technology (CITA), University of Costa Rica, Postal
address 11501-2060 San José, Costa Rica.
[email protected] 3
Institute of Botany (210), University of Hohenheim, Garbenstraße 30, D-70599 Stuttgart,
Germany.
[email protected] 4
School of Food Technology, University of Costa Rica, 11501-2060 San José, Costa Rica.
[email protected] *
Corresponding author:
[email protected]; Phone: (+49) 228-734216; Fax: (+49) 228-
734429; Römerstraße 164, D-53117 Bonn, Germany.
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ABSTRACT
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Pigments of pericarp and pulp of pink guava (Psidium guajava L. cv. ´Criolla´) were investigated
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to elucidate the profile and the accumulation of main carotenoids during four stages of fruit
4
ripening by using HPLC-DAD and APCI-MS/MS analysis. Seventeen carotenoids were identified
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and changes in their profile during fruit ripening were observed. The carotenoids all-trans-β-
6
carotene, 15-cis-lycopene, and all-trans-lycopene were present in all ripening stages, but all-
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trans-lycopene was found to be predominant (from 63 % to 92 % of total carotenoids) and
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responsible for the high lipophilic antioxidant capacity determined by spectrophotometric assays.
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By using light- and transmission electron microscopy, the development of chromoplasts in
10
pericarp and pulp could be demonstrated. The accumulation of all-trans-lycopene and all-trans-β-
11
carotene coincided with the development of large crystals; the chromoplasts of pink guava belong,
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therefore, to the crystalline type.
13 14
KEYWORDS: Psidium guajava L., carotenoids, chromoplasts, HPLC-DAD, APCI/MS,
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microscopy
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1. INTRODUCTION
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Guava (Psidium guajava L.) is a sweet, aromatic fruit native to Central America and belongs
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to the Myrtaceae family. It has been consumed for more than 2000 years, and nowadays, it grows
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wild throughout the tropical and subtropical regions of the world.1 The popular cultivars suitable
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for fresh consumption are those having an intense aroma and a pink color. During guava season,
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the fruit is consumed fresh or processed into juice and jam.1 This fruit is of high economic
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importance to countries in Latin America (Brazil, Mexico and Colombia), Africa (Egypt) and
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Asia (India, Pakistan, Malaysia and Thailand), of which India is considered the largest guava
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producer worldwide, followed by China and Thailand.1,2
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The guava plant has also been used as a traditional medicine in countries such as Taiwan,
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Japan, China and Korea.3 Several investigations have demonstrated its potential as a source of
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bioactive compounds such as carotenoids and polyphenols.3–5 Carotenoids have become of
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interest because of their association with the prevention of atherosclerosis, the maintenance of
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immune functions, and the risk reduction for age-related macular degeneration, cataract, cancer,
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cardiovascular and degenerative diseases.6 Some carotenoids also have provitamin A activity.
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They are transformed into vitamin A, which can prevent serious eye diseases such as night
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blindness, susceptibility to infection, rough, scaly skin, and retarded tooth and bone
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development.7 This fact has been given particular attention because vitamin A deficiency today is
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a worldwide public health issue in 122 countries.8
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Carotenoids are synthetized in all types of plastids, among them chromoplasts accumulate
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carotenoids in high concentration.9 According to the carotenoid-bearing fine structural elements in
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the final state of chromoplast development, Sitte et al.10 classified chromoplasts as globulous,
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tubulous, membraneous, and crystalline. The different chromoplast types are closely related to the
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physicochemical characteristics of their carotenoids. Also, the stability and bioavailability of
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carotenoids are linked to the fine structure of chromoplasts.11-12 Carotenoids with globular and
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tubular structure are more prone to trans-cis isomerization than those present with crystal 3
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structure.12 Besides, it has been demonstrated that carotenoids are absorbed better when they are
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present of globular type chromoplasts compared to the crystal type.11
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The pigments of pink guava were investigated previously.5, 13-16 According to these studies,
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the predominant carotenoid in ripe guava is lycopene, followed by β-carotene and traces of other
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pigments.13-16 Mercadante et al.5 and Padula and Rodríguez-Amaya13 reported a carotenoid profile
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of pink guava from Brazil, while González et al.14 focused on the identification and quantification
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of four carotenoids present in three cultivars of guava grown in Colombia at three ripening stages.
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Nevertheless, details on the profile and accumulation of carotenoids during the ripening process,
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and the ultrastructure of chromoplasts of pink guava are still missing. Because guava may be an
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important dietary source of carotenoids in tropical and subtropical countries, more information
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about carotenoid accumulation and chromoplast development is necessary.
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2. MATERIAL AND METHODS
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2.1 Plant material
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Pink guava (Psidium guajava L.) fruits of the commercial Costa Rican cultivar ‘Criolla’ were
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obtained from San Vicente agricultural farm (Turrialba, Costa Rica) in October 2014 and July
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2015. Two days after harvest the fruit were prepared for microscopy and physicochemical
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analysis. Fruits of four post-harvest ripening stages (RG1, RG2, RG3, RG4) were studied (Figure
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1). RG1 was defined as fruit with dark green color and hard texture, an overall whitish pericarp
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and light pink pulp (fleshy inner part). Its total soluble solids (TSS) was 6.93 ± 0.12 °Brix. At
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RG2, the peel got a lighter green tone and the fruit pericarp took a slightly pink tone. TSS
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increased at 8.47 ± 0.12 °Brix. The external color change at RG3 was associated with the
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appearance of light green to light yellow color of the peel. At this stage, TSS reached 9.07 ± 0.12°
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Brix, and pericarp and pulp were completely pink. At RG4, the TSS was 10.27 ± 0.12 °Brix and
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the peel was yellow, whereas pericarp and pulp remained pink.
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2.2 Chemical reagents
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All reagents and solvents used were of analytical, HPLC or MS grade. Methyl-tert-buthyl-
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ether (MTBE) and butylhydroxytoluene (BHT), acetone and Celite® 525 were obtained from Carl
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Roth (Karlsruhe, Germany). Butylhydroxyanysol (BHA), ammonium formate, 6-hydroxy-2,5,7,8-
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tetramethylchroman-2-carboxylic acid (Trolox), and 2,2' azobis (2 amidinopropane)
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dihydrochloride (AAPH) were purchased from Sigma-Aldrich (Steinheim, Germany). Methanol
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(MeOH) and light petroleum (LP) were sourced from Chemsolute (Renningen, Germany). Ethyl
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acetate was supplied by Fischer Chemical (Loughborough, UK), and fluorescein was obtained
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from Alfa Aesar GmbH (Karlsruhe, Germany). The standards lycopene and β-carotene (purity
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99%) were purchased from Extrasynthese (Genay, France). Glutaraldehyde, tannic acid, osmium
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tetroxide, uranyl acetate and LR-White were sourced from Science Service (Munich, Germany)
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and Epon from Plano GmbH (Wetzlar, Germany).
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2.3 Physicochemical analysis
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2.3.1 Total soluble solids and moisture content
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After removing the green peel using a knife, fresh guava pericarp and pulp were cut into small
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pieces and homogenized separately. Seeds were removed with a sieve. Total soluble solids (TSS)
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were determined at 20 °C using a handheld refractometer (Krüss, Hamburg, Germany).
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Homogenized samples of pericarp and pulp together, as well as pericarp and pulp separately, were
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used to determine moisture content according to the AOAC official method (920 151).17 Then the
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samples were frozen, lyophilized, ground, packed in brown bottles under a nitrogen atmosphere,
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and stored at -80 °C until carotenoid extraction. All chemical parameters of the fruits investigated
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were determined at least in triplicate.
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2.3.2 Extraction of carotenoids
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The extraction of carotenoids of pericarp and pulp was carried out under dim red light
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according to Schweiggert et al.8 with some modifications. For this purpose, 1.0 g of lyophilized
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sample was weighed and 0.1 g Celite® 545 was added. A first extraction with 10 mL of acetone
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for one minute was followed by an extraction with a mixture of MeOH, ethyl acetate and LP
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(1:1:1, v/v/v) containing 0.1 g/L of BHT and 0.1 g/L of BHA until the residue was colorless. The
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combined supernatants were again extracted with distilled water and LP (with 0.1 g/L of
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BHT/BHA, 1:1, v/v). The aqueous phase was then extracted with LP (0.1 g/L of BHT/BHA) until
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it was colorless. The organic phase was washed with water. The upper layer was transferred to a
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new tube and the lower aqueous phase was extracted once with water and then once with LP
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(containing 0.1 g/L of BHT/BHA) and ethyl acetate (1:1). The combined upper fractions were
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filtered and dried under a stream of nitrogen. The remaining residue was made up to 3 mL with
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MTBE/MeOH (1:1 v/v). Samples were filtered using 0.22 µm pore size Chromafil filter made of
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regenerated cellulose (Macherey-Nagel, Düren, Germany) before being used for HPLC and LC-
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MS analysis.
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2.3.3 Identification of carotenoids
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HPLC-DAD analysis of carotenoids was performed on a Waters (Milford, MA) liquid
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chromatography system coupled to a 996 photodiode-array-detector (PDA). Waters Empower
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software (version 5.0) was used for data acquisition and processing. Carotenoids were analyzed
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using a C30 reversed phase column (150 × 2.1 mm, 3 µm), protected by a 10 × 2.1 mm, 3 µm
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particle size C30 guard column (YMC Europe GmbH, Dinslaken, Germany). The mobile phase
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consisted of methanol/MTBE/water (81:17:2, v/v/v; eluent A) and methanol/MTBE/water
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(10:88:2, v/v/v; eluent B) using the following gradient elution: 100 % A (10 min), 100 % A to 0
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% A (30 min), 0 % A (5 min), 0 % A to 100 % A (1 min), and 100 % A (4 min). Total run time
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was 50 min at a flow rate of 0.35 mL/min. The injection volume was 10 µL and the column oven
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temperature was set at 25 °C. Carotenoids were monitored at 450 nm and spectra were recorded
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from 200–600 nm.
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For LC-MS analysis, an Acquity UPLC I-class system from Waters (Milford, MA) consisting
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of a binary pump (BSM), an autosampler (SM-FL, cooled at 10 °C), column oven (CM set at 25
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°C), and a diode array detector (eλ PDA) scanning from 250 to 700 nm was coupled to a LTQ XL
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linear ion trap (Thermo Scientific, Waltham, MA) with an atmospheric pressure chemical
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ionization interface operating in the positive ion mode. The same conditions of HPLC analysis
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were used except that ammonium formate (5 mM) was added to the eluents to improve ionization
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of the target compounds. The standards all-trans-lycopene and all-trans-β-carotene (20 mg/L,
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injection volume 8 µL) were used for the optimization of the ionization and fragmentation
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parameters. Characteristic ions of all-trans-β-carotene and all-trans-lycopene were detected at m/z
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537 and compared with standards. Positive ion mass spectra were recorded in the range of m/z
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200−1000. Nitrogen was used as both drying and nebulizing gas. The resulting flow rates were as
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follows: sheat gas flow 60 arb, auxiliary gas flow 5 arb, and sweep gas flow 5 arb. Capillary
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temperature was set at 275 °C and a potential of 11.05 V was applied on the capillary. Vaporizer
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temperature and source current were set at 300 °C and 2 µA, respectively. Normalized collision
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energy was 20 %. Data acquisition and processing were performed using Thermo Xcalibur 2.2
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SP1.48 Software (Thermo Scientific, Waltham, MA).
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2.3.4 Quantification of carotenoids and provitamin A value Carotenoids were quantified by HPLC using three external calibration curves, one of all-trans-
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β-carotene (1.00 to 25.00 µg mL−1) and two of all-trans-lycopene (1.00 to 25.00 µg mL−1 and
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10.00 to 100.00 µg mL−1) at a minimum of seven levels of concentration. Three concentrations of
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all-trans-lycopene and all-trans-β-carotene were prepared for recovery determination. The
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percentage recoveries for all-trans-lycopene and all-trans-β-carotene ranged from 90 % to 93 %
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The provitamin A value was calculated by using the National Academy of Sciences conversion
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factor (1 Retinol Equivalent = 6 β –carotene; 1 International Unit = 3 β –carotene)18 and the
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relative percentage of bioactivity.19
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2.3.5 Antioxidant capacity Lipophilic and hydrophilic oxygen radical absorbance capacity (L-ORAC and H-ORAC
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respectively) were determined in pericarp and pulp together after separation of the peel, according
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to the method of Huang et al.20–21 The fluorescence was recorded every 2 min after the addition of
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AAPH during one hour using a microplate reader Synergy HT (Bio-Tek Instruments, Winooski,
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VT) set at a constant temperature of 37 °C. L-ORAC and H-ORAC were expressed as micromoles
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(µmol) of Trolox equivalents (TE) per gram dry weight using an external calibration curve of
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Trolox (10.0-200.0 µmol L-1 for L-ORAC and 20.0-100.0 µmol L-1 for H-ORAC).
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2.4 Light and transmission electron microscopy For light microscopy, fruits of all ripening stages were cut into halves and fresh, freehand
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sections of the outer peel, the pericarp and the pulp were used to characterize chromoplast
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development in bright field and differential interference contrast (DIC). A light microscopic
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Axioplan (Zeiss, Göttingen, Germany) coupled to a digital camera DMC 2900 (Leica, Wetzlar,
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Germany) was used. Length and width of about 50 carotenoid crystals were determined on
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micrographs (magnification 100 x) using the measurement tool of Photoshop CS6 (Adobe
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Systems, San José, CA).
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For transmission electron microscopy, samples of stages RG2 and RG3 from the inner,
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already lightly pink stained part of the pericarp were cut to sizes of 0.5 mm x 1 mm x 2 mm with a
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razor blade and immediately fixed in 3 % (v/v) glutaraldehyde and 1 % tannic acid buffered in 0.1
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M sodium phosphate buffer (pH 7.4) for 2 h. After three washing steps in buffer, a second fixation
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in 1 % osmium tetroxide, buffered with 0.1 sodium phosphate buffer (pH 7.4) for 2 h and three 8
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washing steps in double-distilled water followed. For dehydration, the progressive lowering of
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temperature method was applied (1 h in 30 % ethanol at 0 °C; 1 h in 50 % ethanol at −20 °C;
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overnight in 70 % ethanol at −35 °C; 1 h in 90 % ethanol at −35 °C, 1 h in 100 % ethanol at −35
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°C). After warming to room temperature, one part of the samples was embedded in LR-White
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Resin (Science Service, Munich, Germany), another part was completely dehydrated in four steps
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in dried 100 % ethanol (molecular sieve) and embedded in Epon (Plano, Wetzlar, Germany). The
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infiltrated samples were transferred to gelatine capsules (LR-White) and flat embedding moulds
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(Epon), and polymerized at 60 °C for 24 h and 48 h, respectively. Ultrathin sections were
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prepared using a diamond knife (Drukker International, Cuijk, The Netherlands) and the Ultracut
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UCT Microtome (Leica, Wetzlar, Germany). Sections were collected on Pioloform-carbon-coated
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copper grids, stained with uranyl acetate, followed by lead citrate, and investigated in a
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transmission electron microscope (EM10, Zeiss, Oberkochen, Germany) at 60 kV. EM negatives
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were scanned with an Epson Perfection 2450 scanner. Brightness and contrast were adjusted using
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Photoshop CS2 or CS6.
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2.5 Statistical analysis
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To evaluate the changes in main carotenoid contents and in lipophilic and hydrophilic
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antioxidant capacity during ripening, significant differences between the RG1, RG2, RG3, and
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RG4 were determined by performing an ANOVA (α=0.05) and Tukey's test using JMP 4.1
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software (JMP Corporate, USA). Data were expressed as the mean ± standard deviation.
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3. RESULTS AND DISCUSSION
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The carotenoid profile and content, and the antioxidant capacity of pericarp and pulp from
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RG1 to RG4 as well as the chromoplast development were investigated to track the accumulation
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of the main carotenoids during ripening of pink guava.
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3.1 Identification of carotenoids and other non-polar compounds
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The characterization of carotenoids was based on retention time, shape of UV absorption
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spectra (from 200–600 nm), % III/II at 450 nm, presence of cis-peak, Q ratio, and on the m/z ion
198
and MS2 fragments obtained by mass spectrometry. Spectral and mass spectrometric
199
characteristics of peaks from a non-saponified extract are shown in Table 1. An HPLC
200
chromatogram with UV spectra of the main carotenoids present in ripe pink guava is shown in
201
Figure 2. In total, 22 compounds were detected, among them 17 carotenoids, two chlorophylls and one
202 203
pheophytin. Two compounds remained unknown (peaks 14 and 18), although a distinct UV
204
spectrum was obtained, but it did not correspond to any known carotenoid. Three compounds
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(peaks 2, 6, and 10) showed a loss of 18 Da, i.e., a H2O moiety, which is indicative of compounds
206
belonging to the xanthophyll group (with at least one OH group); the other 14 carotenoids were
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carotenes. Carotenoids corresponding to peaks 2, 5, 6, 7, 9, 10, 13, and 21 presented trans-
208
configuration, whereas the carotenoids corresponding to peaks 4, 11, 12, 15, 16, 17, 19, 20, and
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22 were cis-isomers. The xanthopylls cryptoxanthin, rubixanthin, neochrome, 5,6,5',6'-diepoxy-β-
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carotene, 5,8-epoxy-3,3',4-trihydroxy-β-carotene, as well as some isomers of β-carotene
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previously reported in pink guava from Brazil5 were not found in our samples, but Mercadante et
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al.5 and Padula and Rodríguez-Amaya13 investigated the Brazilian cultivar (IAC-4) using a
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different method of extraction, as well as different parameters in the LC-MS analysis.
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Carotenoids 9 and 21 (Figure 2) were identified as all-trans-β-carotene and all-trans-lycopene,
215
respectively, by comparison with their standards. Both carotenoids generated the parent ion at m/z
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537 and resulted in the product ions at m/z 481, 413, and 399 (Table 1), corresponding to bi-
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allylic cleavage of the C-3,4, C-4,5, C-7,8, and C-8,9 single bond adjacent to the chromophore.22–
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23
219
[M+H−C6H10]⁺ at m/z 455 and [M+H−C7H8]⁺ at m/z 444, shown in our study only by all-trans-
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lycopene (Table 1 in the Supporting Information). However, the m/z ion 444 was already reported
A difference between all-trans-lycopene and all-trans-β-carotene was given by the product ions
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for all-trans-β-carotene detected in pink guava from Colombia14 and in papaya.24 In order to
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confirm the product ion at m/z 455 as a unique fragment of all-trans-lycopene, both standards
223
were analyzed (data not shown). This fragment was found in all-trans-β-carotene but with a low
224
intensity (14), whereas the intensity for all-trans-lycopene was 86 % relative to the base peak.
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Conclusively, no unique fragment ions for definitive differentiation of lycopene and β-carotene
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were found, that is why differentiation is still related to retention time, UV-spectra and % III/II.
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All-trans-β-carotene and all-trans-lycopene (peaks 9 and 21) were previously identified by
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MS/MS in pink guava from Brazil and from Colombia.5, 13-14
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In addition to high levels of all-trans-lycopene, several cis-isomers of lycopene (compounds
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15, 16, 17, 19, 20, and 22) were found in guava. In good agreement with the fragmentation of the
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authentic all-trans-lycopene standard, the MS/MS experiment of cis-lycopene at m/z 537 revealed
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characteristic losses of 56, 70, 82, 92 and 124 Da (Table 1 in the Supporting Information).
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Unfortunately, differentiation of cis-isomers by their MS/MS spectra was not possible as no
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pattern of fragmentation could be established. However, five out of six isomers showed the
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product ion [M+H−124]+ at m/z 413 as their major fragment. The peak intensity of all fragments
236
varied for the cis-isomers. Thus, tentative identification was based on their UV-Vis characteristics
237
(Table 2 in the Supporting Information). The identification of cis-isomers from lycopene based on
238
the spectroscopic characteristics is more complicated than that of cyclic carotenoids.25 However,
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their chromatographic behavior helps to distinguish the cis-isomers.
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According to Britton,26 the UV-Vis spectrum of a cis-isomer shows the appearance of the cis-
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peak (λcis between 330 and 350 nm), located 142 nm below λIII in the spectrum of the
242
corresponding all-trans-carotenoid. In the case of all-trans-lycopene found in guava, λIII
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corresponds to 504 nm (Table 1) and the difference with their respective cis-isomers (∆λIII trans–cis)
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was found to be in a range from 136 to 145 nm (Table 3). Another distinctive characteristic of cis-
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isomers is the intensity of the cis-peak (Q-ratio), indicated by the ratio between the height of the
246
cis-peak and that of the middle main absorption peak (II), and measured from the baseline of the 11
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spectrum. The closer the cis double bond is located to the center of the molecule, the higher is the
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Q-ratio.26 Based on these considerations, peak 16 was tentatively identified as 15-cis-lycopene as
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its Q-ratio showed the highest value, and peak 22 was tentatively identified as 5-cis-lycopene
250
presenting the lowest Q-ratio (Table 2 in Supporting Information).
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In order to characterize the remaining cis-isomers of lycopene, the intensity of the
252
hypsochromic shift (∆λmax trans-cis) was also analyzed (Figure 1 and Table 2 in the Supporting
253
Information). Isomer identification is based on the facts that: (1) mono-cis-isomers usually show a
254
shift of 0–6 nm, whereas di-cis-isomers have a shift higher than 12 nm compared to the all-trans
255
form; (2) di-cis-isomers may be shifted further to shorter wavelengths compared to their mono-cis
256
form; and (3) the central cis-isomers of lycopene such as 13-cis, 13'-cis, 15-cis and 15'-cis have an
257
intense peak in the UV region at about 340 nm.6 Considering these facts, as well as the order of
258
elution reported previously using C30 columns, 25, 27–28 peaks 15, 17, 19, and 20 were tentatively
259
identified as 13,15-di-cis-lycopene, 9,13-di-cis-lycopene, 13-cis-lycopene, and 9-cis-lycopene,
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respectively.
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Based on the UV-Vis spectral data taken at the end of the main peak of all-trans-lycopene
262
with typical wavelengths of this cis-isomer, peak 22 was tentatively identified as 5-cis-lycopene.
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As this isomer and all-trans-lycopene are not completely separated, an overlap of both UV spectra
264
occurred. This overlap would be the reason, why on closer examination of the UV-Vis spectra of
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all-trans-lycopene, a small peak at 362 nm, indicative of a cis-isomer, was observed (Table 1).
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This initially complicated the identification of peak 21 as all-trans-lycopene, but the presence of a
267
cis-peak was also previously reported by Melendez-Martínez et al.25
268
Several authors reported that the 5-cis-isomer and 5'-cis-isomer eluate immediately before or
269
after all-trans-lycopene. There was evidence from its chromatographic characteristics and the
270
molecular ion at m/z 537 that peak 18 could be 5'-cis-lycopene, but its identity is still uncertain
271
since the order of elution does not correspond to that previously reported for cis-isomers of
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lycopene.25, 26–28 Concerning cis-isomers of β-carotene, the identification of 15-cis-β-carotene 12
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(peak 11) was based on the same spectroscopic criteria and its main m/z ion in comparison to the
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all-trans-β-carotene standard. The identification of cis-trans-isomers is important for the
275
assessment of their biological activities, especially regarding their bioavailability, transport and
276
distribution in tissues.6
277
Among the xanthophylls detected, peak 2 was tentatively identified as lutein. The MS
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experiment showed a molecular ion at m/z 569. Moreover, the MS/MS exhibited product ions at
279
m/z 551 and 459, resulting from the loss of water [M+H−18]+ and a further in-chain elimination of
280
C7H8 [M+H−92−H2O]+ from the polyene chain (Table 1 in the Supporting Information). This loss
281
is characteristic for carotenoids having hydroxyl groups.22, 29 In this study, MS/MS fragmentation
282
of lutein also showed a product ion at m/z 495 formed by the loss of water from the protonated
283
molecule and retro-Diels-Alder fragmentation of the α-ionone ring, [M+H−18−C4H8]+. This
284
fragment was previously explained by van Breemen et al.23 as a unique fragment of lutein when
285
positive ionization was applied. Nevertheless, lutein may easily be confused with zeaxanthin,
286
which also yields a parent ion at m/z 569 and product ions at m/z 459 and 477.25 However, the
287
product ion at m/z 495 as a unique fragment of lutein, a higher % III/II ratio than zeaxanthin, and
288
the UV-spectra of lutein are some characteristics that helped to distinguish lutein from
289
zeaxanthin.23, 30 Besides, comparison of the MS data reported for pink guava fruits from
290
Colombia14 allowed the tentative identification of peak 2 as lutein.
291
Peak 4 showed a parent ion at m/z 545 [M+H]+, corresponding to the molecular mass of
292
phytoene. Based on the results reported by Meléndez-Martínez et al.,25 the UV-Vis spectrum of
293
peak 4 showed a lower fine structure (Figure 2) compared to the UV-Vis spectrum of all-trans-
294
phytoene, showing more concordance with the UV-Vis spectrum of 15-cis-phytoene. Besides,
295
Fraser et al.31 showed that this cis-isomer is the major naturally occurring phytoene in tomatoes,
296
which is in good agreement with the fact that the synthesis of carotenoids begins directly from 15-
297
cis-phytoene. Thus, peak 4 was tentatively assigned to 15-cis-phytoene. 13
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Fragmentation of the parent ion (m/z 543) of peak 5 exhibited a characteristic loss of 82 Da
299
(Table 1 in the Supporting Information), resulting in a product ion at m/z 461. As Enzell and
300
Back22 and Schweiggert et al.24 indicated, this loss corresponds to a C6H10-group caused by the
301
cleavage of the C4-5 or the C4'-5' phytofluene single bond, followed by a hydrogen atom transfer.
302
Peak 5 was therefore assigned to phytofluene. An analogous elimination of a C6H10-group during
303
fragmentation of a compound with m/z 541 resulted in a fragment ion [M+H−82]+, generating the
304
typical m/z at 459 of ζ-carotene (peak 7).22
305
Peak 6 was tentatively identified as auroxanthin, based on comparison of UV-Vis
306
characteristics with those previously reported by Pinto de Abreu et al.,32 who identified
307
auroxanthin in cashew apple (Anacardium occidentale L.). Its identity was corroborated by the
308
MS data, showing a parent ion at m/z 601 [M+H] +. The main fragmentation of peak 6
309
corresponded to the loss of water, resulting in a product ion at m/z 583. As auroxanthin has a 3-
310
hydroxy-5,8-expoxy end group on both sites of the polyene chain, it has been characterized
311
elsewhere not only by the water loss but also by a fragment at m/z 221. This product ion results
312
from the cleavage of the C10-11 bond in the polyene chain, from the epoxy end group.33
313
However, due to the low intensity of peak 6 in guava, this fragment was not detected in our study.
314
The identity of this carotenoid as auroxanthin remains, therefore, tentative.
315
The MS experiment of peak 10 showed a parent ion at m/z 569 (Table 1). Comparison of the
316
mass spectrometric data as well as the chromatographic behavior with those previously reported
317
for guava fruits from Brazil allowed its identification as cryptoflavin.5, 14, 30 Likewise, compound
318
12 was deduced to be 7-cis, 9-cis, 7'-cis, 9'-cis tetra-cis-lycopene (prolycopene),26, 34 based on λ
319
(414, 432, and 461 nm) and compositional formula (C40H56) derived from a parent ion at m/z 537
320
[M+H]+ (Table 1).
321
In the case of ƴ-carotene (peak 13), this carotenoid showed a similar fragmentation pathway as
322
lycopene and β-carotene (m/z ion 455 and 467, respectively), due to the β-ionone moiety and an
323
acyclic terminus such as β-carotene and lycopene, respectively.23 This carotenoid may be easily 14
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confused with a cis-lycopene, but its % III/II value allowed the identification of peak 13 as ƴ-
325
carotene.26
326
Also chlorophyll pigments were detected. Chlorophyll b (peak 1) and chlorophyll a (peak 3)
327
were identified by their order of elution, UV-Vis spectra and their parent ions at m/z 907 and 893,
328
respectively.35 Peak 8 was characterized as pheophytin a, a derivative of chlorophyll a, with a
329
parent ion at m/z 871.36-37 These compounds were found only in the pericarp at RG1 (Figure 2 in
330
the Supporting Information) when the pericarp was whitish and the fruit hard. The presence of
331
chlorophyll in the pericarp at RG1 might be due to the difficulty to remove completely the green
332
peel from the pericarp.
333 334
3.2 Accumulation of carotenoids in guava pericarp and pulp
335
The level of carotenoids in fruits is influenced by the ripening time, genotype, and type of
336
tissue.7 Therefore, we characterized the carotenoid profile in pericarp and pulp of pink guava cv.
337
“Criolla” at four ripening stages (Table 2 and Figure 2 in the Supporting Information).
338
Already at RG1, an incipient carotenoid biosynthesis was observed, although the pericarp was
339
whitish (Figure 1). Lutein (peak 2) was present in pericarp and pulp at RG1, whereas auroxanthin
340
(peak 6) appeared at RG4 and at RG3, respectively (Table 2). Also cryptoflavin (peak 9) was
341
found at different ripening stages (RG3 and RG2, respectively). According to Rodríguez-
342
Amaya38, auroxanthin and cryptoflavin appear as a result of the hydroxylation, epoxidation and
343
epoxide-furanoxide rearrangement of β-carotene when it is exposed to light or heat. Nevertheless,
344
our results suggested that these carotenoids were derived from biosynthesis because no heat
345
treatment or light exposure was applied in our experimental approach. Besides, these carotenoids
346
were detected in non-saponified extracts of guava.
347
At RG1 the colorless carotenoids 15-cis-phytoene (three conjugated double bonds) and
348
phytofluene (five conjugated double bonds) were present in pericarp and pulp of guava. In order
349
for carotenoids to be colored, at least seven conjugated double bonds are required.26 ζ-Carotene, 15
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which is light yellow, was found at RG3 but below the limit of quantification. The increased
351
levels of carotenoids with nine conjugated double bonds such as lutein (peak 2), cryptoflavin
352
(peak 10), ƴ-carotene (peak 13), and all-trans-β-carotene (peak 9), as well as carotenoids with
353
eleven double bonds such as all-trans-lycopene (peak 21) and its isomers leaded to the more
354
intense color of pericarp and pulp at all ripening stages.26 In the course of fruit ripening, a
355
pronounced increase in all-trans-β-carotene, all-trans-lycopene, ƴ-carotene as well as in the
356
isomer 15-cis-lycopene (16) was observed (Figure 4). Most of the cis-isomers of lycopene were
357
not detectable in pericarp at RG1, whereas in the pulp, all the cis-isomers were detectable at RG2
358
(Table 4). In general the accumulation of carotenoids in the pulp was faster than in the pericarp.
359
All-trans-lycopene was found to be the main carotenoid in both pericarp and pulp during the
360
ripening process. It showed a 13-fold rise in pericarp from RG1 to RG4, whereas in pulp it
361
showed a 3-fold rise (Table 2). Nevertheless, all-trans-lycopene content in the pericarp and in the
362
pulp at RG3 was higher than at RG4. The increase observed for the cis-isomers 9,13 di-cis-
363
lycopene and 15-cis-lycopene at RG4 demonstrated a higher rate of cyclization and isomerization
364
than the rate of all-trans-lycopene biosynthesis. At all ripening stages, in pericarp and pulp, carotenoids with trans-configuration made up
365 366
more than 82 % of the total carotenoid content (Table 2). As major carotenoid with trans-
367
configuration, all-trans-lycopene represented 65 % (RG4) to 100 % (RG1) of the total carotenoid
368
content in the pericarp, whereas in the pulp it ranged from 63 to 91 % (Table 2). The total
369
carotenoid content showed an increase higher than 19-fold in pericarp and 5-fold in pulp (Table
370
2).
371
To the best of our knowledge, the most recent report about carotenoid content in guava was
372
published by Ribeiro da Silva et al.,39 who reported a lycopene content in ripe pink guava
373
(cultivar not mentioned) of 35 µg/100 g dw. This value is 830 times lower than the lycopene
374
content of the pink guava cv. “Criolla” at RG4. The ripe pink guava also presented higher
375
lycopene contents than papaya and Surinam cherry (2.4-fold and 3.5-fold, respectively),39 but 1.216
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fold lower than tomato11, in which all-trans-lycopene was also found to be the main carotenoid in
377
crystalline chromoplasts.40 Ripe pink guava cv. “Criolla” exhibited a lower β-carotene content
378
than acerola, mombin, cashew apple, and mango.39
379 380 381
3.3 Lipophilic and hydrophilic antioxidant capacity During ripening of fruits, antioxidative substances including hydrophilic and lipophilic
382
compounds such as carotenoids, polyphenols, and vitamin C may either be generated or lost. The
383
antioxidant capacity of a fruit can be assessed by different methods, among them ORAC allows
384
the determination of the lipophilic and hydrophilic antioxidant capacities.41 The lipophilic oxygen
385
radical absorbance capacity (L-ORAC) of pink guava increased significantly during the ripening
386
process (Table 3). Considering the content of the main carotenoids, its L-ORAC value is mainly
387
based on all-trans-lycopene, its isomers and all-trans-β-carotene. Based on literature reports, it
388
can be stated that L-ORAC of ripe guava at RG4 (32 µmol TE equiv/100g fw) is lower than that
389
of fruits such as apple (41 µmol TE equiv/100g fw), banana (66 µmol TE equiv/100g fw),
390
blueberries (103 µmol TE equiv/100g fw) or cranberries (202 µmol TE equiv/100g fw), but higher
391
than melon (14 µmol TE equiv/100g fw), kiwi (24 µmol TE equiv/100g fw), nectarine (29 µmol
392
TE equiv/100g fw) or cherries (17 µmol TE equiv/100g fw).41
393
For all ripening stages, total oxygen radical absorbance capacity is underestimated because L-
394
ORAC includes only lipophilic components, i.e., carotenoids. It does not take into account the
395
absorbance activity of hydrophilic compounds such as polyphenols and vitamin C.42 Thus, the
396
hydrophilic oxygen radical absorbance capacity (H-ORAC) was also determined, which ranged
397
from 10.1 (RG1) to 16.3 µmol TE/g (RG4) (Table 3). The value of H-ORAC obtained for ripe
398
pink guava (RG4) was lower than the oxygen radical absorbance capacity reported for three
399
varieties of pink guava cultivated in Texas, USA.43 The authors did not determined L-ORAC.
400
This difference is explained due to different cultivars analyzed as well as different conditions
401
during the extraction. 17
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Nevertheless, L-ORAC is higher than H-ORAC. Therefore, the total oxygen radical
403
absorbance capacity of pink guava might arise from lycopene, the main carotenoid in guava with
404
the longest conjugated double bond system responsible for its antioxidant activity.6
405 406 407
3.4 Fruit and chromoplast development Light microscopy was used to follow the fruit ripening process and the development of
408
chromoplasts in pericarp and pulp of pink guava (Figures 3–5). The fruit is a special berry in that
409
the outer green part (peel) develops from the receptacle of the flower. Therefore, the peel of the
410
guava is not part of the pericarp. The pericarp of the pink guava investigated was uniform, soft,
411
and white to pink colored tissue surrounding the pink inner juicy fruit (pulp), which develops
412
from the seed-bearing placenta (Figure 1). Cross sections revealed the parenchymatic character of
413
all fruit layers. Under the epidermal layer, some denser hypodermal layers of parenchyma with
414
chloroplasts were found (the green peel) (Figure 3A). Chlorophyll a and b might arise from these
415
chloroplasts. Adjacent to the green peel was the pericarp (Figure 5A) with large parenchyma cells
416
around clusters of sclereids (stone cells) (Figure 3B). These stone cells are responsible for the
417
gritty texture of this fruit. A very soft parenchyma built the inner juicy part, which was the major
418
part of the fruit.
419
During fruit ripening (RG1 to RG4), chromoplasts developed in the parenchyma of pericarp
420
and pulp. At ripening stage RG1, when the pericarp appeared whitish (Figure 1), chromoplasts
421
were not detectable in the cells (Figure 4A), whereas at ripening stage RG2, when the pericarp
422
was slightly pink, chromoplasts became visible (Figure 4B). Pink chromoplasts were obvious in
423
the pericarp at ripening stages RG3 (not shown) and RG4 (Figure 4C) as well as in pulp cells
424
(Figure 4D). At these advanced ripening stages hardly any cell walls were visible by light
425
microscopy.
426 427
In the chromoplasts, the carotenoids accumulate and built large flat crystals in form of triangles, squares, rectangles or polygons in the chromoplasts (Figure 5). Their size ranged from 3 18
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µm x 3 µm up to 7 µm x 20 µm. Therefore, the chromoplasts of the pink guava ‘Criolla’ belong to
429
the crystalline type. In comparison with the chromoplasts of tomato, the carotenoid crystals are
430
smaller but larger than of red papaya.11
431
Transmission electron microscopy of chromoplasts at ripening stage RG2 revealed that the
432
chromoplasts were quite variable, not only in size but also in ultrastructure. They were small
433
compared with the size of mitochondria (Figure 6A). The size of these plastids, the absence of
434
typical chloroplast, thylakoids and starch, and the presence of typical chromoplast fine structures
435
indicate that chromoplast development in pink guava started from proplastids and was already in
436
progress at ripening stage RG2.
437
The developing chromoplasts showed carotenoid crystals, some large plastoglobuli in variable
438
sizes, vesicles, and long strands of stacked membranes, but no grana thylakoids and starch grains
439
(Fig. 6 and 7). The long stacked membranes were no more prominent at ripening stage RG3. It
440
might be that these membranes are involved in the biosynthesis of carotenoid crystals, because
441
membranes always envelop the crystals. During chromoplast development, especially the
442
carotenoid crystals grew, but number and size of plastoglobuli did not change. The crystalline
443
structure of carotenoids is only visible by the pleated membranes in the space left by the
444
carotenoids that are dissolved in the lipophilic resin during preparation for transmission electron
445
microscopy. At this stage chromoplasts carrying large crystals looked distorted as the carotenoid
446
crystals were dissolved during preparation (Figure 7). The dissociation of the cell walls,
447
particularly the loosening of the middle lamellae, demonstrated that the ripening process of the
448
pericarp tissue was advanced already at ripening stage RG2 (Figure 6B), but especially at ripening
449
stage RG3 (Figure 7).
450
The development of chromoplasts and accumulation of crystals coincided with the increasing
451
carotenoid content from ripening stage RG1 to RG4. At RG1 only negligible amounts of
452
pigmented carotenoids such as lycopene and β-carotene were detected by HPLC-APCI/MS in
453
pericarp and pulp of pink guava, whereas from RG2 to RG4 a striking increase, especially in the 19
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all-trans-lycopene content (peak 21) was observed (Table 2). It is well known that all-trans-
455
lycopene and all-trans-β-carotene are carotenoids accumulating in crystalline chromoplasts.
456
Carotenoids such as 15-cis-phytone (peak 4), phytofluene (peak 5), and the cis-isomers might be
457
present in the plastoglobuli of the guava chromoplasts. Normally, they are dominant in
458
chromoplasts of the globulous type.44–46
459
Determination of the structure of carotenoids in chromoplasts, their profile and accumulation
460
in the fruit helps to understand why pink guava can be considered as a functional food. From a
461
nutritional point of view, ripe pink guava cv. “Criolla” can be classified as a very good source of
462
all-trans-lycopene and all-trans-β-carotene, since this is defined by a concentration of >2 mg/100
463
g fw.6
464
Also ripe pink guava can be considered as a source of vitamin A. Of the 17 carotenoids
465
identified in ripe pink guava cv. “Criolla”, all-trans-β- carotene and γ-carotene contribute to the
466
biosynthesis of provitamin A. The relative bioactivity reported for these carotenoids is 100 % and
467
50 %, respectively.19 This bioactivity means the ability of the carotenoids to generate one or two
468
molecules of vitamin A, or to act as intermediate for the synthesis of carotenoids with provitamin
469
A activity.19 The provitamin A value of ripe pink guava in pericarp was 203 RE 100 g-1 (fw),
470
whereas in pulp was 195 RE 100 g-1 (fw). Compared with other foods with all-trans-lycopene and
471
all-trans-β- carotene as the main carotenoids in chromoplasts of crystalline type, ripe pink guava
472
presented a higher provitamin A value than papaya (74 RE 100 g-1, fw)47 but lower than tomato
473
(750 RE 100 g-1, fw)48. The provitamin A value in 100 g of ripe pink guava (406 IU in pericarp
474
and 392 IU in pulp) corresponds to 7.5 % of the recommended daily intake (RDI) of vitamin A as
475
suggested by the FDA49, which is 5000 IU.
476
Nevertheless, not only the concentration but also the physico-chemical characteristics of
477
carotenoids need to be considered because they may affect the chemical stability and the
478
bioavailability of the deposited carotenoids in food.12 The main carotenoids in ripe pink guava cv.
479
“Criolla”, lycopene and β-carotene are present in trans-configuration, which is the 20
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thermodynamically more stable configuration of carotenoids.50 These main carotenoids were
481
accumulating in the crystalline chromoplasts, where the carotenoids are less prone to trans-cis
482
isomerization than in globulous or tubulous types.51 This issue is an advantage for the chemical
483
stability of trans-carotenoids. Hence, carotenoids presenting crystal structure would help to delay
484
the deterioration of products made from guava, as their capacity as antioxidant is not affected.
485
Carotenoids of crystalline chromoplasts seem to have a lower bioavailability than of other
486
chromoplast types.40 Besides the physicochemical characteristics of carotenoids, there are other
487
important factors that influence bioavailability, such as the interaction of carotenoids with
488
lipoproteins responsible for the transport of carotenoids in the blood,52 or components of food,
489
e.g., the interaction with pectin. The pink guava ‘Criolla’ is rich in pectin (about 5.7 % was
490
found), that may affect the bioavailability of crystalloid carotenoids.11, 53 In vivo analysis are
491
required in order to determine the bioavailability of carotenoids of pink guava. Also the
492
interactions of food components should be considered in these tests.
493 494
ABBREVIATIONS
495
AAPH, 2,2' azobis (2 amidinopropane) dihydrochloride; APCI, atmospheric pressure
496
chemical ionization; BHA, butylhydroxyanisol; BHT, butylhydroxytoluene; DIC, differential
497
interference contrast; DAD, diode array detector; dw, dry weight; fw, fresh weight; EM, electro
498
micrographs; HPLC, high-performance liquid chromatography; LP, light petroleum; MS, mass
499
spectrometry; MeOH, methanol; MTBE, methyl-tert.-butyl-ether; PSY, phytoene synthase; RG1,
500
ripening stage 1; RG2, ripening stage 2; RG3, ripening stage 3; RG4, ripening stage 4; TSS, total
501
soluble solids.
502 503 504 505
ACKNOWLEDGMENTS Joany González from the Farm San Vicente (Turrialba, Costa Rica) is acknowledged for his support by harvesting and donating guava fruits. We thank Erika Rücker (Institut of Botany, 21
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Hohenheim University) for supporting transmission electron microscopy studies. The University
507
of Costa Rica, San José, Costa Rica, and the German Academy Exchange Service (DAAD), Bonn,
508
Germany, are acknowledged for granting the scholarship to the Ph.D. student (C. R-G).
509 510
SUPPORTING INFORMATION. Specific APCI-MS/MS product ions obtained from the
511
[M+H]+ ions of carotenoids; UV-Vis characteristics of cis-isomers of lycopene and β-carotene;
512
comparison of the Q-ratio; UV-spectra of cis-isomers of lycopene; UV-chromatograms of
513
pericarp and flesh at four ripening stages (PDF).
514 515
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516
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characterization of Costa Rican papaya (Carica papaya L.) hybrids and lines with particular focus
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on their genuine carotenoid profile. J. Agric. Food Chem. 2012, 60, 2577–2585.
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comprehensive analysis of cis/trans geometrical isomers of carotenoids for nutritional studies.
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S., Pfander, H., Eds.; Birkhäuser Verlag: Basel, Switzerland, 1995; Vol. 1B., pp 21–25.c
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(27) Li, J.; Deng, Z.; Liu, R.; Loewen, S.; Tsao, R. Ultra-performance liquid chromatographic
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separation of geometric isomers of carotenoids and antioxidant activities of 20 tomato cultivars
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and breeding lines. Food Chem. 2012, 138, 1341–1350.
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Jensen, S., Pfander, H., Eds.; Birkhäuser Verlag: Basel, Switzerland, 2004; pp 0.1–563.
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biosynthesis during tomato fruit development. Plant Physiol. 1994, 105, 405–413.
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structural and geometric isomers of carotenoids in mature grapes. J. Mass Spectrom. 2010, 45,
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of flavonoids, carotenoids and chlorophyll concentration in Cynodon dactylon (L.) by HPLC
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(36) Aparicio-Ruiz, R.; Riedl, K.M.; Schwartz, S.J. Identification and quantification of metallo –
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mass spectroscopy studies. Adv. Technol. 2012, 1(1), 16-24.
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(38) Rodríguez-Amaya, D. A guide to carotenoid analysis in foods; JLSI Press: Washington DC,
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(40) Schweiggert, R.; Steingass, C.B.; Heller, A.; Esquivel, P.; Carle, R. Characterization of
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chromoplasts and carotenoids of red- and yellow-fleshed papaya (Carica papaya L.). Planta
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2011, 234, 1031–1044.
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(46) Stange-Klein, C.; Rodríguez-Concepcion, M. Carotenoids in carrots. In Pigments in fruits
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papaya (Carica papaya) cultivars grown in Hawaii. J. Food Comp. Anal. 2006, 19, 434−445.
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(48) Rodriguez-Amaya, D.B.; Bobbio, P.A; Bobbio, F.O. Carotenoid composition and vitamin A
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value of the Brasilian fruit Cyphomandra betacea. Food Chem. 1983. 12, 61−65.
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Guide (14. Appendix F: Calculate the Percent Daily Value for the Appropriate Nutrients). URL
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(http://www.fda.gov/Food/GuidanceRegulation/GuidanceDocumentsRegulatoryInformation/Labe
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lingNutrition/ucm064928.htm) (26.08.16).
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quantification of carotenoids by HPLC-DAD during the process of peach palm (Bactris gasipaes
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H.B.K) flour. Food Res. Int. 2011, 44, 2377–2384.
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(51) Vásquez-Caicedo, A.L.; Heller, A.; Neidhart, S.; Carle, R. Chromoplasts morphology and β-
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carotene accumulation during postharvest ripening of mango Cv. ‘Tommy Atkins’. J. Agric. Food
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Chem. 2006, 54, 5769–5776.
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(52) Britton, G.; Helliwell, J.R. Carotenoid-protein interactions. In Carotenoids: Natural
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functions; Britton, G., Liaaen-Jensen, S., Pfander, H., Eds.; Birkhäuser Verlag: Basel,
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(53) Sidhu, J. Tropical Fruit III: Production, processing and quality of guava, lychee, and papaya.
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J.S.B., Cano, M.P., Eds.; John Wiley & Sons, Ltd: Iowa, 2012; pp 125–563.
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Figure captions
659
Figure 1. (A) Pink guava (Psidium guajava L. cv. ‘Criolla’). Habitus/appearance of whole fruits
660
and cross sections at the different ripening stages RG1 to RG4. (B) Description of the fruit parts.
661 662
Figure 2. HPLC chromatograms (450 nm) of carotenoid extract from pink guava at RG4 with the
663
main respective UV-Vis spectra of selected peaks.
664 665
Figure 3. Anatomy of the green peel and the pericarp of pink guava (light micrographs, bright
666
field). (A) RG3, cross section of the green peel. Between a single epidermal layer (e) and the
667
pericarp (pc) are some parenchymatic cell layers with chloroplasts (pr). (B) RG2, cross section of
668
pericarp tissue. Clusters of stone cells (sc) are surrounded by parenchymatic cells (p).
669 670
Figure 4. Chromoplast development in pericarp and pulp of pink guava (light micrographs, bright
671
field). (A) RG1, hardly any plastids are visible at this stage in the parenchymatic cells (p) of the
672
pericarp; stone cells (sc). (B) RG2, some chromoplasts with carotenoid crystals (arrows) appear in
673
the parenchymatic cells (p) of the pericarp. (C) RG3, chromoplasts with large carotenoid crystals
674
(arrows) accumulate in the pericarp; stone cells (sc). (D) RG4, chromoplasts with large carotenoid
675
crystals (arrows) accumulate in the pulp. In RG4, due to the advanced ripening stage of the fruit,
676
cell walls of the parenchymatic cells in pericarp and pulp are hardly visible.
677 678
Figure 5. Light micrographs demonstrating color and form of carotenoid crystals in pink guava at
679
RG 3. (A) Large carotenoid crystals (c) in variable forms and typical pink color in bright field. (B)
680
Differential interference contrast demonstrating the crystalline character.
681 682
Figure 6. Transmission electron micrographs of chromoplasts in pericarp parenchyma cells of
683
pink guava at RG 2. (A) Small chromoplasts with large plastoglobuli (pg), vesicles (arrow heads), 28
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and long strands of stacked membranes (mb); mitochondrium (m), vacuole (v). (B) Small
685
chromoplasts with plastoglobuli (pg), vesicles (arrow heads), and small carotenoid crystals
686
(arrows); cell wall (cw), mitochondrium (m), vacuole (v). The loosening of the cell wall during
687
fruit ripening is visible by the separation of the cell wall (cw) at the middle lamella (ml).
688 689
Figure 7. Transmission electron micrograph of a chromoplast in a pericarp parenchyma cell of
690
pink guava at RG 3. Typical long, shrunken, and distorted chromoplast with plastoglobuli (pg)
691
variable in size and vesicles (arrow head). The spaces with pleated membranes (arrows) mark the
692
positions of the large carotenoid crystals (c) that were dissolved in the resin during preparation;
693
cell wall (cw).
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Table 1. UV/Vis and MS data of major nonpolar compounds of pink guava (Psidium guajava L. cv. ‘Criolla’). Fragments are given in order of intensity.
retention peak-number time (min) chlorophylls and derivative
HPLC-DAD UV/Vis spectrum (nm) cis-Peak
I
II
III
% III/II
tentative identification
[M+H]⁺⁺ m/z
MS2 m/z
1
3.2
--
--
465
650
--
chlorophyll b
907
--
3
4.2
--
--
434
665
--
chlorophyll a
893
--
8
15.3
--
--
409
666
--
pheophytin a
871
593, 533
2
3.9
--
417
443
474
86
lutein
569
551, 459, 463, 477, 495
6
10.1
--
-
399
42
79
auroxanthin
601
583, 487, 572, 545, 526
10
16.6
342
405
429
451
26
cryptoflavin
569
551
4
5.8
--
--
286
--
--
15-cis-phytoene
545
463, 489, 393, 439, 383
5
6.9
--
--
348
366
83
phytofluene
543
524, 295, 461, 321, 487
7
15.0
--
382
402
426
100
ζ-carotene
541
459, 489, 393, 439, 383
9
15.7
--
428
451
472
37
all-trans-β-carotene b
537
413, 467, 481, 399, 401
11
17.5
342
414
431
467
31
15-cis-β-carotene
537
399, 413, 519, 495, 439
12
18.8
342
--
437
463
56
prolycopene
537
518, 481, 455, 413, 444
13
21.6
348
432
457
489
78
ƴ-carotene
537
455, 467, 403, 425, 345
15
24.2
360
444
467
495
47
13,15-cis-lycopene
537
444, 518, 455, 448, 413
16
25.4
361
440
466
497
52
15-cis-lycopene
537
467, 481, 455, 444, 399
17
26.5
361
--
464
496
40
9,13-di-cis-lycopene
537
413, 455, 467, 453, 481
19
28.5
361
439
468
498
66
13-cis-lycopene
537
399, 494, 467, 401, 413
20
29.0
361
440
467
499
63
9-cis-lycopene
537
413, 536, 427, 518, 399
xanthophylls
carotenes
21
32.0
362
446
472
504
74
all-trans-lycopene
22
32.3
368
445
472
503
72
5-cis-lycopene
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b
537
413, 455, 399, 444, 481
537
413, 455, 399, 481, 444
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unknown
a
14
23.4
--
--
432
455
100
--
801
--
18
28.0
--
447
472
503
64
--
537
413, 455, 518, 467, 399
Fragments for each carotenoid are written in order of intensity. b Compared with standards.
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Table 2. Content of major carotenoids in pericarp and pulp of pink guava (Psidium guajava L. cv. ‘Criolla’) at four ripening stages (RG1–RG4). Values for pericarp or for pulp in the same line with different letters are significantly different (p