Carotenoid Profile, Antioxidant Capacity, and Chromoplasts of Pink

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Carotenoid profile, antioxidant capacity, and chromoplasts of pink guava (Psidium guajava L. cv. Ćriolla´) 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. Ćriolla´) 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.

1

ACS Paragon Plus Environment


1

ABSTRACT

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Pigments of pericarp and pulp of pink guava (Psidium guajava L. cv. Ćriolla´) 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-

7

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

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pericarp and pulp could be demonstrated. The accumulation of all-trans-lycopene and all-trans-β-

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carotene coincided with the development of large crystals; the chromoplasts of pink guava belong,

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therefore, to the crystalline type.

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KEYWORDS: Psidium guajava L., carotenoids, chromoplasts, HPLC-DAD, APCI/MS,

15

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

22

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

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and MS2 fragments obtained by mass spectrometry. Spectral and mass spectrometric

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characteristics of peaks from a non-saponified extract are shown in Table 1. An HPLC

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chromatogram with UV spectra of the main carotenoids present in ripe pink guava is shown in

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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

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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

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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,

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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

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[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

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were analyzed (data not shown). This fragment was found in all-trans-β-carotene but with a low

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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

232

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-

245

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



247

spectrum. The closer the cis double bond is located to the center of the molecule, the higher is the

248

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,

260

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.

263

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

265

all-trans-lycopene, a small peak at 362 nm, indicative of a cis-isomer, was observed (Table 1).

266

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

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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

274

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

278

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

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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



298

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


Page 14 of 50

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324

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



350

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


Page 16 of 50

Page 17 of 50


376

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



402

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


Page 18 of 50

Page 19 of 50


428

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



454

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


Page 20 of 50

Page 21 of 50


480

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



506

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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Myrtaceae. Acta Hort. 2012, 959, 23–34.

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carotene and its 9-cis and 13-cis stereoisomers during postharvest ripening of nine Thai mango

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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 chlorophyll and its derivatives in the pigment mixtures: HPLC-chromatography, visible and

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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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2001; pp 1–13.

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compounds in pulps and by-products of tropical fruits from Brazil. Food Chem. 2014, 143, 398–

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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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Lipophilic and hydrophilic antioxidant capacities of common foods in the United States. J. Agric.

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patterns, localization, and substrates of polyphenol oxidase in red clover (Trifolium pratense L.).

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J. Agric. Food Chem. 2013, 63, 7421–7430.

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and vegetables: Genomics and dietetics, Chen, C., Eds.; Springer: New York, 2015; pp. 219.

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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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(49) Food and Drug Administration (FDA). Guidance for industry: A food labelling

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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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(50) Rojas-Garbanzo, C.; Pérez, A.M.; Bustos-Carmona, J.; Vaillant, F. Identification and

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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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Switzerland, 2008; Vol. 4., pp 99–118.

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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.

657 27



658

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


Page 28 of 50

Page 29 of 50


684

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).

29



Page 30 of 50

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

30


b

537

413, 455, 399, 444, 481

537

413, 455, 399, 481, 444

Page 31 of 50


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.

31



Page 32 of 50

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

Carotenoid Profile, Antioxidant Capacity, and Chromoplasts of Pink

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