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LWT - Food Science and Technology 64 (2015) 259e263

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LWT - Food Science and Technology

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Carotenoids, and ascorbic acid content in yellow passion fruit (Passiflora edulis) grown under different cultivation systems

* Paula Becker Pertuzatti a, , Marla Sganzerla b, Andressa Carolina Jacques c, Milene Teixeira Barcia b, Rui Carlos Zambiazi d

a Universidade Federal de Mato Grosso, Campus Universitario do Araguaia, Barra do Garças 78600-000, Mato Grosso, Brazil b Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos, Campinas 13083-862, Sao~ Paulo, Brazil c Universidade Federal do Pampa, Campus Bage, Bage 96413170, Rio Grande do Sul, Brazil d Universidade Federal de Pelotas, Centro de Ci^encias Químicas, Farmac^euticas e de Alimentos, Pelotas 96010-900, Rio Grande do Sul, Brazil

article info abstract

Article history: The present study aimed to identify and compare the content of tocopherols, ascorbic acid and carot- Received 28 February 2015 enoids in yellow passion fruit grown under different cultivation systems. The passion fruits were ob- Received in revised form tained from two systems: an organic system and a conventional system. Samples from both systems 13 May 2015 were obtained in 2008. The tocopherols, carotenoids, and ascorbic acid analysis were carried out using Accepted 15 May 2015 high-performance liquid chromatography. The results showed that the passion fruit grown under the Available online 30 May 2015 organic cultivation approach contained a higher content of tocopherols than those cultivated using conventional methods (0.061 and 0.052 mg of tocopherols.100 g 1 of fresh fruit, respectively). The major Keywords: Phytochemicals component in the samples produced by both systems was g-tocopherols, which were found at a con- $ 1 $ 1 HPLC centration of 0.045 mg 100 g in the organic fruits and 0.042 mg 100 g in the conventional fruits. The 2 2 1 Yellow passion fruit amount of total ascorbic acid was 2.3 10 and 1.9 10 mg$100 g in the samples from the organic and conventional systems respectively. The quantification of individual carotenoids in both the organic and conventional passion fruit was 13.99 mg$100 g 1and 25.10 mg$100 g 1 respectively. The conven- tional passion fruit contained double the content of the carotenoids present in the organic fruits. b- Criptoxanthin was the main carotenoid found in both fruits. © 2015 Elsevier Ltd. All rights reserved.

1. Introduction result from intake of the antioxidants that are present in fruit and vegetables, an increasing amount of attention is being placed on Fruits and vegetables containing C, (tocoph- identifying potentially antioxidant substances, such as carotenoids erols), and carotenoids (a-carotene, b-carotene, b-cryptoxanthin, and (Fraser & Bramley, 2004; Halliwell, 1997; Matheus- lutein, zeaxanthin, and lycopene) are believed to provide a natural Roth, 1991; Melendez-Martínez, Vicario, & Heredia, 2004; Pertu- source of antioxidants (Kchaou, Abbes, Attia, & Besbes, 2014; zatti et al., 2014; Rock, Fada, Jacob, & Bowen, 1996). The prevention Pertuzatti et al., 2014; Romojaro, Sanchez-Bel, Serrano, & Pretel, of diseases that is provided by carotenoids is directly associated 2013). Antioxidant activity is associated with decreases in DNA with the antioxidant activity offered by these substances due to damage, a reduction in lipid peroxidation, maintenance of the singlet oxygen scavenging and the way in which they reaction with immune function, and prevention of the development of some free radicals (Fiedor & Burda, 2014; Sentanin & Rodriguez-Amaya, diseases (Gropper, Smith, & Groff, 2005). Nowadays, it is commonly 2007). accepted that diets that are high in fruits and vegetables protect Carotenoids are natural pigments that are found throughout the against several human diseases, some of which are especially flowering plant kingdom as a pigment. They are mostly responsible serious such as cardiovascular diseases and cancer. Given the fact for the yellow, orange and red color of fruits, and some of them are that many existing studies indicate that protective effects may important precursors (da Silva et al., 2014). In one study, thirteen carotenoids were identified and separated in passion fruit using HPLC, with z-carotene identified as the predominant com- * Corresponding author. pound. These compounds give visual appeal to juices and are E-mail address: [email protected] (P.B. Pertuzatti).

http://dx.doi.org/10.1016/j.lwt.2015.05.031 0023-6438/© 2015 Elsevier Ltd. All rights reserved. 260 P.B. Pertuzatti et al. / LWT - Food Science and Technology 64 (2015) 259e263 important for their provitamin A and antioxidant activity 2. Materials and methods (Mercadante, Britton, & Rodriguez-Amaya, 1998; Talcott, Percival, Pittet-Moore, & Celoria, 2003). The b-carotene content in passion 2.1. Materials fruit pulp is 1362 mg.100 g 1and the total carotenoid content ranges between 27,600 and 35,400 mg.100 g 1 (Cavalcante, Dias, The passion fruits (P. edulis Sims f. flavicarpa, Degener) of the Nascimento, & Freire, 2011; da Silva et al., 2014). However, carot- organic and conventional system were obtained from the same enoids and vitamin accumulation in passion fruit is variable and private producer in the city of Florianopolis (SC/Brazil). The ripened depends, among many factors, on the maturity stage of the fruit fruits (surface totally yellow, pH 2.8 and 11e13Brix) were collected and the cultivation systems used to grow it (Rotili, Vorpagel, Braga, from a 2008 harvest transported at 10 C in thermos boxes refrig- Kuhn, & Salibe, 2013). erated with ice blocks, and stored in an ultra-freezer at 80 C. The Vitamin C, or merely ascorbic acid (AA), is a hydrosoluble and analyses were carried out after three days of storage in the chro- thermolabil vitamin. AA is widely distributed in products of vege- matography laboratory/DCTA-UFPEL in Pelotas (RS/Brazil). Five table origin and is mainly found in citric fruits and green leaves batches of passion fruits from each cultivation system were eval- (Zhang & Hamauzu, 2004). The Association of Official Analytical uated. Each one was composed of 20 units. The samples were Chemists (AOAC) standard methodology for determination of immediately evaluated in triplicate following preparation. The Vitamin C employs a titration method; however, ascorbic acid can seeds were separated, and the pulp was homogenized in a mixer also be determined by spectrophotometric, enzymatic and chro- using approximately 200 g of fruit. The standards of g- and d-to- matographic methods (Spínola, Mendes, Camara,^ & Castilho, 2013). copherols were obtained from Sigma Aldrich (Steinheim, Germany) Liquid chromatographic methods have the potential for the with 96 and 90% of purity, respectively; a- was from simultaneous determination of other metabolites, as dehy- Sigma Aldrich (Steinheim, Germany) with 99% of purity; the L- droascorbic acid. Either reduced or oxidized (dehydroascorbic acid) (þ)-ascorbic acid was from Synth (Diadema, Brazil) (99% purity); forms of AA are equally active; though the oxidized form is reduced and the L-(þ)-dehydroascorbic acid from Fluka (Saint Louis, USA) in natural sources. The transformation of AA into dehydroascorbic with purity higher than 80%. The standards of the carotenoids b- generally is reversible, allowing either substance to be transformed cryptoxanthin, lycopene, lutein, and zeaxanthin were obtained into another. This capacity of transformation works as an oxy- from Chromadex (Irvine, USA); and b-carotene was from Fluka reduction system, carrying hydrogen in the respiration process at (Saint Louis, USA) at 97% of purity. All standards were dissolved in a the cellular level (Welch, 1995). Natural ascorbic acid levels have mobile phase used in each method, and a calibration curve was been reported at 40e65 mg/100 g for fresh passion fruit (Spínola prepared. et al., 2013; Talcott et al., 2003). Vitamin E consists of four tocopherols: a, b, g and d, and the 2.2. Tocopherols and carotenoids analyses corresponding : a-, b, g and d, which contain unsatu- rated side chains. The a-tocopherol is the most biologically active The extraction of tocopherols was performed in accordance with form. Most plant-derived foods, especially fruits and vegetables, the methodology described by Burns, Fraser, and Bramley (2003), contain low-to-moderate levels of vitamin E activity; however, due with a number of minor modifications. The fruit extracts were to the abundance of plant-derived foods in our diets, they provide a obtained using cold acetone and mixed with petroleum ether. The significant and consistent source of vitamin E (Eintemiller & Lee, ethereal phase was separated and centrifuged at 9000 rpm for 2004). Although some studies have reported the presence of 10 min (microcentrifugue NT800 Nova Tecnica - Sao~ Paulo, Brazil) tocopherol in oil extracted from the seeds of passion fruit (499.3 mg and then transferred to a 1.5 mL vial for tocopherols analysis. total tocopherol kg 1)(Malacrida, 2012), no study has been carried The identification and quantification of tocopherols was per- out on the tocopherol content of passion fruit pulp. formed using a high-performance liquid chromatography system Around 120 species of Passiflora are native to Brazil, sixty of (SHIMADZU e Kyoto, Japan), which consisted of a LC-10ATVP sol- which produce fruits that could be direct or indirectly used as food vent delivery pump, FCV-10ALVPc degasser, DGU-14A rheodyne sources. Yellow passion fruit (Passiflora edulis Flavicarpa) is the pump, SCL-10AVP, CTO-10ASVP, column oven and an autosampler most cultivated fruit in Brazil, and it is predominantly utilized in SIL-10AF. The chromatographic separation was performed in an juice processing (Meletti, 2011). In 2012, passion fruit production in analytical reversed-phase column Shim-Pak CLC-ODS Brazil was higher than guava and melon production, and produc- (4.6 mm 150 mm 4 mm) with octadecil groups as the stationary tion of this fruit is increasing due to the technological progress that phase. A fluorescence detector RF e 10AXL was used at an excitation is being witnessed in all regions of Brazil. This makes Brazil an wavelength of 290 nm and emission wavelength of 330 nm. 10 mLof important world exporter of passion fruit juice (IBGE, 2012; Meletti, the sample was injected using an initial mobile phase acetonitrile/ Oliveira, & Ruggiero, 2010). A number of studies have attempted to methanol/isopropanol (50:40:10, v/v/v) for 10 min. The mobile characterize passion fruit composition; however, none of them phase was linearly altered to acetonitrile/methanol/isopropanol have used HPLC analyses (carotenoids, tocopherols and vitamin C) (30:65:5, v/v/v) until 12 min passed; and then linearly returned to to evaluate the quality traits of organic and conventional passion the initial mobile phase until 15 min of analyze. A constant flow of fruit. 1 mL min 1 (Barcia, Jacques, Pertuzatti, & Zambiazi, 2010)was The present demand for health foods that are produced without used. environmental damage and with respect to biological diversity for The peaks were identified by comparing the standards of to- not using chemical fertilizers and pesticides, is a tendency that copherols, and the quantification was performed according to the favors the creation of new opportunities, especially those for small external standard calibration curve (d-tocopherol, g-tocopherol producers (Fischer et al., 2004). and a-tocopherol). The results were expressed in mg tocopherol The present study aimed to compare the content of tocopherols g 1of fruit. (g, d and a), ascorbic acid (L-ascorbic and L-dehydroascorbic) and For the purposes of the carotenoids analysis the same extract carotenoids (b-carotene, b-cryptoxanthin, lutein, lycopene and used for tocopherols was used and a 25 mL-aliquot of the extract zeaxanthin) in yellow passion fruit grown under two different was saponified with 25 mL of KOH 1.5 N solution in ethanol, cultivation systems. following the procedure of Pertuzatti et al., 2012. The mixture was left for 18 h in the dark, after which it was transferred to a P.B. Pertuzatti et al. / LWT - Food Science and Technology 64 (2015) 259e263 261 separatory funnel with 40 mL of water and 40 mL of petroleum 3. Results and discussion ether. The lower phase was transferred to another separatory funnel (aqueous phase) and washed with 20 mL of petroleum ether. Post-harvest characterization aims to facilitate decision making The upper phase was washed with 20 mL of water. The lower phase for producers by providing them with the information they need to of both separatory funnels was discarded and the upper phases develop a strategy that minimizes the development of poor-quality were put together, before being concentrated in a rotary evaporator fruit and helps them to control the production chain. at 35 C and finally re-dissolved in 5 mL of methanol:acetonitrile, The initial moisture content of passion fruits cultivated in the 30:70 v/v. The extract was centrifuged at 9000 rpm for 10 min conventional and organic system was 79.65 mg.100 g 1 and (microcentrifugue NT800 Nova Tecnica e Sao~ Paulo, Brazil) and 80.15 mg.100 g 1 respectively. The fruit from the conventional then transferred to a 1.5 mL vial for the analysis of carotenoids. A system exhibited several chromatographic peaks under the HPLC 25 mL aliquot of the resulting sample was injected into the system of analysis that had not previously been identified. Five carotenoids high-performance liquid chromatography that consisted of the standards were used in this study. In a previous study by modules previously described, coupled to spectrophotometric de- Mercadante et al. (1998), thirteen carotenoids were conclusively tector UV/V SPD-10AVVP with wavelength at 450 nm. Where was identified in yellow passion fruit (P. edulis) using electron impact used as initial mobile phase methanol/acetonitrile, 30/70 (v/v), mass spectrometry, complemented by UV-visible spectropho- which was altered to methanol/acetonitrile/ethyl acetate (10:80:10, tometry and co-chromatography. Of these thirteen carotenoids, v/v/v) for 10 min and then kept constant for a further 35 min. seven were identified for the first time. Also, the study did not Following this, it was turned to methanol/acetonitrile/ethyl acetate report the variety and original location of where the fruits were (5:80:15, v/v/v) and maintained in this state for 40 min before it cultivated. was finally returned to the initial mobile phase for 45 min so that it According to the results presented in Table 1, both the cultiva- could be analyzed. A constant flow of 1 mL min 1 was used. The tion systems used to grow passion fruits displayed significant dif- data was acquired and processed using the Class-VP software. ference for all carotenoids analyzed. The highest amount of The peaks were identified by comparing the retention times of carotenoid identified in both organic and conventional cultivation the standards, and the quantification was evaluated according to was b-criptoxanthin; however, the content of b-criptoxanthin in the external standard calibration curve of b-cryptoxanthin, lyco- the fruit from the conventional crop was almost twice that of the pene, lutein, zeaxanthin and b-carotene. The results were organic crop. Lutein and zeaxanthin were present in the lowest expressed in mg.100 g 1of fruit. concentrations in the analyzed fruits. The total concentration of carotenoids present in the conventional crop was twice that of the 2.3. Ascorbic acid analysis carotenoid content found in the fruits from the organic crop. Several existing studies indicate that organic cultivation results in The ascorbic acid was analyzed according to the methodology higher phytochemical content (Cavalcante et al., 2011). However described by Vinci, Botre, and Mele (1995), with few modifications, Borguini, Bastos, Moita-Neto, Capasso, and Torres (2013) analyzed as described by Jacques, Pertuzatti, Barcia, Zambiazi, and Chim the lycopene content of organic and conventional tomatoes and did (2010). Ten grams of ground sample was weighed, and 30 mL of not find any significant difference between the organic and con- metaphosphoric acid (4.5% in ultrapure water) solution was added, ventional systems. The carotenoid content in fruits depends on before the solution was left to rest for an hour in an amber flask. The many variables, including the process of cultivation; however, some solution was then transferred to a 50 mL volumetric flask, and the authors have observed that additional factors, such as exposure to volume was completed with ultrapure water. The sample was light, temperature and degree of fruit ripeness, also have an impact filtered through filter paper, and the supernatant was centrifuged on the production of carotenoids (Abushita, Daood, & Biacs, 2000; at 7000 rpm for 10 min (microcentrifugue NT800 Nova Tecnica e Borguini et al., 2013). Therefore, the highest carotenoid content in Sao~ Paulo, Brazil). After that, the sample was transferred to 1.5 mL conventional passion fruit could be due to different light incidences vial. or control over the fruit ripening. 10 mL of the sample, which consisted of the modules described The content of b-carotene (0.582 mg.100 g 1) found in sweet previously, was injected into the system of high-performance liquid passion fruit by Souza, Ferreira, Chaves, and Teixeira (2008) was chromatography. A spectrophotometric detector UV/V SPD-10AVVP much higher than the concentration found in this study with wavelength at 254 nm was employed. Ultrapure water with (0.056e0.077 mg.100 g 1). These authors attribute this variation 0.1% of acetic acid as a mobile phase was employed with a flow of not just to the geographic effect, but also to the maturation stage of 0.8 mLmin 1. The data were acquired and processed using the the fruits at the point of harvest, crop year, variety and storage Class-VP software. conditions. The content of carotenoids in vegetables cannot be The peaks were identified by comparing the retention time of considered to be an absolute value, and it may be affected by the standards of L-ascorbic and dehydroascorbic acid. The vitamin C several factors. As a result of the many functions or properties quantification was evaluated according to the external standard attributed to the production of carotenoids, there is a worldwide calibration curve of L-(þ)-ascorbic acid and the L-(þ)-dehy- effort to obtain reliable analytical data that provides deeper in- droascorbic acid. The results were expressed in mg.g 1of fruit. sights into the development of these compounds. Since there is high number of natural carotenoids, conclusive identification is 2.4. Moisture difficult. The information about the carotenoid composition of fruits that is presented in existing literature is frequently incom- The moisture determination was performed in triplicate in plete or conflicting (Mercadante et al., 1998). accordance with the AOAC method (AOAC, 2006). Three distinct peaks of the d-, (bþg)- and a-tocopherols, were identified by comparing the retention times between the sample 2.5. Statistical analysis and the corresponding standards. The second peak in the middle location corresponded to the sum of the b- and g-tocopherols, since The data were submitted to analyze the variance and compare these isomers were not resolved using RP-HPLC. The calibration means using the Tukey test at 5% level of significance. The statistical curves exhibited excellent linearity (r2 > 0.997, n ¼ 6 for all curves). analysis was performed using Version 7.0 of the Statistica program. Using a-tocopherol as a reference, the response factors (relative 262 P.B. Pertuzatti et al. / LWT - Food Science and Technology 64 (2015) 259e263

Table 1 Content of carotenoids in conventional and organic passion fruit (mg.100 g 1), limit of detection (LOD) and limit of quantification (LOQ) to carotenoids analysis.

Passion fruit Lutein þ Zeaxanthin b-Criptoxantin Licopene b-Carotene Total carotenoids

Organic crop 0.001a 13.94b 0.002b 0.056b 13.99b Conventional crop 0.001b 24.99a 0.028a 0.077a 25.10a LOD 6.50 10 4 0.07 9.89 10 3 8.62 10 3 e LOQ 2.17 10 3 0.24 30.00 10 3 2.87 10 2 e

Averages followed by different letters on column are significantly different at a confidence level of 95% by Tukey test, for the samples containing carotenoids contents. Coefficient of variation <8.0%.

Table 2 Content of tocopherols in conventional and organic passion fruit (mg.100 g 1).

d-Tocopherol (bþg)-Tocopherol a-Tocopherol Total (aþbþgþd)

Organic crop 0.016a 0.045a nd 0.061a Conventional crop 0.010b 0.042b nd 0.052b

Averages followed by different letters on column are significantly different at a confidence level of 95% by Tukey test, for the samples containing tocopherols contents. nd e not detected; Coefficient of variation <6.0%.

Fig. 1. Vitamin C peaks of passion fruits from conventional (a) and organic (b) cultivation, obtained by HPLC analysis using a reverse phase column (C18) and UV detector at 254 nm. Peak identification: (1) dehydro ascorbic acid; (2) L-ascorbic acid.

fluorescence sensitivities) were 4.6 and 3.5 for g- and d-tocopherol, Fig. 1 provides the typical chromatogram referent to the sepa- respectively. ration of ascorbic acid using HPLC. Peak 1 corresponds to L-dehy- Both systems used to cultivate passion fruit exhibited significant droascorbic acid with a retention time of 2.63 min, and Peak 2 to L- differences in terms of the contents of the tocopherols analyzed, as ascorbic acid in 3.20 min. In terms of the total ascorbic acid content shown in Table 2. The contents of tocopherols in the fruits grown (Table 3), the fruit from the organic cultivation contained the via organic cultivation were superior to those developed using highest concentration of ascorbic acid. Considering individually the 1 conventional methods: 0.061 mg of tocopherols per 100 g of two forms of ascorbic acid (L-ascorbic acid and L-dehydroascorbic fresh fruit and 0.052 mg of tocopherols per 100 g 1 of fresh fruit, acid) the fruit from the organic cultivation contained a higher respectively. This could be due to the stressful conditions that are concentration of L-ascorbic acid. As with the tocopherols, the total inherent in the organic cultivation system, which favor the syn- vitamin C content of the organic fruits was more expressive. It is thesis of bioactive compounds from the secondary metabolism widely accepted that vitamin C levels in fruits are subject to a wide (Macoris, De Marchi, Janzantti, & Monteiro, 2012). In both cultiva- variety of environmental factors (Genovese, Pinto, Gonçalves, & tion systems, the major component was g-tocopherol, 0.045 mg of Lajolo, 2008). These variables include light, temperature, salts, g-tocopherol.100 g 1 in the organic system and 0.042 mg of g- and the presence of atmospheric pollutants, metals, and herbicides. tocopherol.100 g 1 in the conventional system. The g-tocopherol This variable nature could explain the differences between the fruit also was the main tocopherol found in passion fruit seed oil produced via the two evaluated cultivation systems assessed in this (Malacrida & Jorge, 2012). In terms of the content of d-tocopherol, work. Independently, the content of vitamin C found in this 0.016 mg d-tocopherol.100 g 1 was present in the organic cultivar experiment was superior to the concentrations identified by Vinci and 0.010 mg d-tocopherol.100 g 1 in the conventional cultivar. The presence of a etocopherol was not confirmed in the passion fruits. Although the passion fruits only contained a small amount of to- Table 3 copherols, the tocopherols present in the organic passion fruit were Content of ascorbic acid in conventional and organic passion fruit (mg.100 g 1). superior to those in the conventionally cultivated fruit. Data on the Passion fruit L-ascorbic acid L-dehydroascorbic acid Ascorbic acid total tocopherol content of fruits in existing literature is rare. In one Organic crop 41.00a 2.3 102a 2.3 102a research study that examined passion fruit pulp (Barcia et al., 2010), b 2b 2b Conventional 18.00 1.8 10 1.9 10 a total of 0.043 mg.100 g 1 tocopherol was identified and this content was lower than that present in the other fruits analyzed by Averages followed by different letters on column are significantly different at a confidence level of 95% by Tukey test, for the samples containing ascorbic acid the authors (blueberries, peaches, blackberries, pears). contents; Coefficient of variation <9.0%. P.B. Pertuzatti et al. / LWT - Food Science and Technology 64 (2015) 259e263 263

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