Effect of sustained deficit irrigation on ...

Postharvest Biology and Technology 86 (2013) 171–180

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Effect of sustained deficit irrigation on physicochemical properties, bioactive compounds and postharvest life of pomegranate fruit (cv. ‘Mollar de Elche’) ˜ a , Francisco Artés-Hernández a,b , Encarna Aguayo a,b , María E. Pena Ginés Benito Martínez-Hernández a,b , Alejandro Galindo c , Francisco Artés a,b , Perla A. Gómez b,∗ a Postharvest and Refrigeration Group, Department of Food Engineering, Universidad Politécnica de Cartagena, Paseo Alfonso XIII, 48, 30203 Cartagena, Murcia, Spain b Institute of Plant Biotechnology, Universidad Politécnica de Cartagena, Campus Muralla del Mar, 30202 Cartagena, Murcia, Spain c Irrigation Department, Centro de Edafología y Biología Aplicada del Segura (CSIC), P.O. Box 164, E-30100 Espinardo, Murcia, Spain

a r t i c l e

i n f o

Article history: Received 18 April 2013 Accepted 30 June 2013 Keywords: Punica granatum L. Bioactive compounds Chilling injury Phenolics Punicalagins Anthocyanins

a b s t r a c t In this study, the influence of sustained deficit irrigation (SDI; 32% of reference evapotranspiration (ET0 )) on physicochemical and sensory quality and bioactive compounds of pomegranates stored for 30, 60 and 90 days in air at 5 ◦ C + 4 days at 15 ◦ C, at each storage period, was studied and compared to a control (100% ET0 ). Fruit from SDI had higher peel redness and greater firmness, soluble solids contents, vitamin C (27%), phloretin (98%) and protocatechuic acid (10%) levels, and total antioxidant capacity (TAC) (46%) than the control. Cold storage and shelf-life did not induce significant changes in soluble solids, pH, titratable acidity, and chroma and Hue. SDI fruit had retarded development of chilling injury (CI) symptoms, which appeared after 60 days of storage in comparison to 30 days in the controls. Anthocyanins, catechin, phloretin and protocatechuic, caffeic, p-coumaric and caffeic acids contents had greater increases in SDI fruit than in controls throughout the postharvest life. TAC was significantly (P < 0.05) correlated to anthocyanins, gallic acid and total vitamin C contents. Generally, after long term storage, the fruit grown under SDI showed higher sensory and nutritional quality, more health attributes and a longer shelf-life (up to 90 days at 5 ◦ C + 4 at 15 ◦ C) than fruit irrigated at 100% ET0 . © 2013 Elsevier B.V. All rights reserved.

1. Introduction Commercial orchards of pomegranate (Punica granatum L.) trees are grown in many world regions, particularly in the Mediterranean Basin, where high quality fruit are commonly obtained (Artés et al., 2000a; Stover and Mercure, 2007). Pomegranates are a wellknown source of many nutritional and bioactive compounds such as dietary fibre, organic acids, minerals (such as potassium), vitamins C, A, and K and folic acid. Nonetheless, the greatest significant added value of pomegranate is its large content of phenolic compounds, such as anthocyanins (its most important quality attribute in the red pigmentation of seeds and juice), hydrolyzable tannins (punicalagins and punicalins) condensed tannins (proanthocyanidins), catechins and phenolic acids (gallic, ellagic and chlorogenic, among others) (Gil et al., 1996a, 2000; Poyrazo˘glu et al., 2002; Mena et al., 2012a). Punicalagins, anthocyanins, phenolic acids and ascor-

∗ Corresponding author. Tel.: +34 868 071069; fax: +34 868 071079. E-mail address: [email protected] (P.A. Gómez). 0925-5214/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.postharvbio.2013.06.034

bic acid, either alone or in combination, are responsible for the antioxidant activity of pomegranates (Scalzo et al., 2004). Water scarcity in arid and semi-arid areas has led to development of new water saving techniques, such as sustained deficitary irrigation (SDI). Pomegranate possesses drought tolerance characteristics, common in xeromorphic plants, such as high leaf relative apoplastic water content and the ability to confront water stress by developing complementary stress avoidance and stress tolerance mechanisms (Rodríguez et al., 2012). Mellisho et al. (2012) reported that when taking into account the total yield during the whole season, water deficit resulted in significantly poorer marketable yield due to lesser fruit production and size. Furthermore, the same work showed higher SSC in SDI fruit from the first harvest, although SDI samples from the second harvest did not register significant SSC changes. Different results of total phenolics and anthocyanin contents and total antioxidant capacity (TAC) undergoing no change, or reductions, have been recently reported with ‘Mollar de Elche’ fruit at harvest, grown under SDI (Mena et al., 2011; Mellisho et al., 2012). Further investigation is needed to clarify these contradictory results.

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M.E. Pe˜ na et al. / Postharvest Biology and Technology 86 (2013) 171–180

Chilling alone, or combined with controlled atmosphere or modified atmosphere packaging or with heat shocks, are methods for extending postharvest life for up to 3 months (Artés et al., 1996, 2000a,b). The storage temperature must be carefully selected in order to avoid dysfunction of cell membranes, considered the primary molecular event leading to the development of chilling injuries (CI) symptoms (Zhang and Tian, 2009). Conventional storage at 5 ◦ C and 90–95% RH up to 8 weeks of ‘Mollar de Elche’ fruit reduced weight loss and decay but did not reduce the risk of CI development, such as surface pitting, husk scald, brown discoloration of both the skin and white internal membranes, arils with pale red colour and a higher sensitivity to decay (Artés et al., 1996, 2000a,b). No information is available on the influence of SDI on bioactive and nutritional compounds, and physicochemical and sensory quality of sweet pomegranates during postharvest life. In this study we investigated the effect of SDI on overall quality of ‘Mollar de Elche’ pomegranates during storage for 90 days at 5 ◦ C and after a simulated shelf-life period of 4 days at 15 ◦ C. 2. Materials and methods 2.1. Experimental conditions, plant material and treatments The experiment was carried out in 2009 at a farm located 22 km southeast of Murcia (Spain) (37◦ 57 N, 0◦ 56 W). The plant material consisted of self-rooted 10-year old pomegranate trees (P. granatum (L.) cv. Mollar de Elche). Tree spacing followed a 3 m × 5 m pattern. From day 93 (day of the year, DOY) until after the second harvest (DOY 278), pomegranate trees were daily drip-irrigated during the night, using one lateral pipe parallel to the tree row and 3 emitters per tree, each delivering 4 L water h−1 . To monitor the water supplied to each experimental unit, in-line water metres were used. Two irrigation treatments were applied. Control plants were irrigated in order to guarantee non-limiting soil water conditions (60% reference evapotranspiration (ET0 ) from the beginning of the season to the end of the first half of linear fruit growth phase, 117% ET0 during the second half of linear fruit growth phase and 99% ET0 during the end of fruit growth and ripening phase). Sustained deficit irrigation (SDI) plants were subjected to DI throughout the season, according to the criteria frequently used by the growers in the area (32% ET0 from the beginning of the season to the end of the first half of linear fruit growth phase, 74% ET0 during the second half of linear fruit growth phase, and 36% ET0 during the end of fruit growth and ripening phase). Total water applied was 711 and 382 mm for control and SDI treatments, respectively. Natural precipitation was considered as part of applied water in SDI plants. Marketable pomegranate fruit were hand-harvested by the middle of October. The fruit was placed in collapsible plastic boxes (60 cm × 40 cm × 22 cm) and transferred by car (50 km) to the pilot plant of the Universidad Politécnica de Cartagena. Neither washing nor postharvest chemical treatments were applied. Pomegranates were grouped in boxes (6 boxes for C and another 6 boxes for SDI), each box containing 24 fruit, and cold stored (CS) up to 90 d at 5 ◦ C (a theoretically safe temperature) and 90% RH. After that, a subsequent simulated shelf-life (SL) period of 4 days at 15 ◦ C (55–65% RH) was established. Three replicates of 4 fruit each were evaluated at day 0 and at each sampling day after CS (30, 60 and 90 days) and after the subsequent SL period. 2.2. Chemical and physiological analysis Chemical attributes were measured on the juice obtained by squeezing, with a hand-held juice extractor, 50 g of arils taken after

peeling 4 pomegranates from each one of the 3 replicates. Titratable acidity (TA) was determined by titration of 10 mL of aril juice plus 50 mL of water with 0.1 mol L−1 NaOH to pH 8.1 (716 DMS Titrino, Metrohm, Riverview, FL, USA) and expressed as g citric acid L−1 . SSC was determined on the juice by an Atago N1 hand-held refractometer (Tokyo, Japan) at 20 ◦ C and expressed as Brix. Nine samples were analyzed per triplicate every sampling day. For respiration rate measurements, 3 samples of 2 fruit each (415.16 g ± 22.74 g) were put into 2.5 L glass jars at 5 ◦ C and exposed to a humidified CO2 -free air stream as described by Artés et al. (1996). After closing the jars for 2 h, CO2 accumulation was monitored according to Maghoumi et al. (2013). 2.3. External colour measurements Colour measurements were made with a photocolorimeter (Minolta CR-300, Ramsey, NJ, USA) with a window aperture diameter of 8 mm, calibrated with a white plate (Y = 94.3, x = 0.3142; y = 0.3211, C light source and 2◦ observer). L*, a* and b* values were measured as averages of 9 different points: 3 around each polar zone and 3 at the equatorial area. The hue angle or colour tone [H = tan−1 b*/a*] and chroma or colour saturation [chroma = (a*2 + b*2)½] were calculated (Artés et al., 2000a). A colorimetric maturity index (CMI = L* × a* × b*−1 ) was calculated as previously described by Manera et al. (2013) for the external colour of the same pomegranates cultivar. Nine samples were analyzed every sampling day from each treatment. 2.4. Weight loss, firmness and chilling injury Weight loss of the fruit was determined by a gravimetric method and calculated as percent of the initial fresh weight (fw). The fruit firmness was measured using a flat steel plate mounted on a texture analyser (Ibertest eLib-5-W, Madrid, Spain) by the force deformation (N) perpendicularly applied on the equatorial zone at 40 mm min−1 for 3 mm after contacting the fruit. A bevelled holder prevented bruising of the opposite side. CI quantification was individually performed by applying a five point scale of damage incidence: 1 (no damage), 2 (slight), 3 (moderate, limit of marketability), 4 (severe) and 5 (extreme). Percentage of damaged fruit was calculated by counting those with a damaged diameter area of at least 5 mm (Artés et al., 2000b). Three samples of 4 pomegranates each were analyzed at every sampling day and for each treatment. 2.5. Sensory analysis Sensory analyses were performed according to international standards (Eggertj and Zook, 1986). The panel consisted of seven trained assessors (aged 25–64) screened for sensory ability (colour vision, odour detection, texture and basic taste) according to Bett (2002), as well as for the ability to communicate sensory descriptions of products (ISO 8586–1, 1993). Visual appearance, colour, dehydration, odour, arils discoloration, flavour, texture and overall quality were determined by the following 5-point hedonic scale: 1 (very poor), 2 (poor), 3 (acceptable, limit of marketability), 4 (good) and 5 (very good) (Artés-Hernández et al., 2004). 2.6. Phenolic compounds 2.6.1. Extraction Four pomegranates from each replicate and treatment were manually peeled and 80 g aril samples were frozen in liquid N2 and ground (Ika, model A11B, Germany). Frozen ground arils (2.5 g) were centrifuged in two 2.5 mL eppendorf tubes at 15,000 × g for 5 min at 4 ◦ C and the supernatant was filtered through a 0.45 ␮m


polyethersulphone filter. These extracts were stored at −80 ◦ C until phenolics (total and individuals), total anthocyanins and total antioxidant capacity determinations. 2.6.2. Individual phenolics Anthocyanins were identified and quantified according to López-Rubira et al. (2005). Filtered pomegranate extract (20 ␮L) was analyzed using an HPLC (Series 1100 Agilent Technologies, Waldbronn, Germany) equipped with a G1322A degasser, G1311A quaternary pump, G1313A autosampler, G1316A column heater and G1315B photodiode array detector. The HPLC system was controlled by the software ChemStation Agilent, v. 08.03. The stationary phase was a Gemini NX (250 mm × 4.6 mm, 5 ␮m) C18 column (Phenomenex, Torrance CA, USA). The mobile phases were water/formic acid (95:5, v/v) (A) and methanol (B) with a flow rate of 1 mL min−1 . The linear mobile phase gradient started with 2% B and followed by 32% B at 30 min, 40% B at 40 min and 98% B at 45 min, then isocratic for 5 min. For column equilibration phase B was reduced to 2% in 4 min and maintained at this concentration for 10 min. Chromatograms were recorded at 520 nm. Anthocyanins were identified by comparison of their retention times and absorption spectra with those of the following pure standards: cyanidin 3,5-di-(Cy 3,5-GLU) and 3-O-glucoside (Dp 3-GLU), delphinidin 3,5-di-(Dp 3,5-GLU) and 3-O-glucoside (Dp 3-GLU), pelargonidin 3,5-di-(Pg 3,5-GLU) and 3-O-glucoside (Pg 3-GLU). The calibration curves were made with at least six data points for each standard. The results were expressed as mg anthocyanin kg−1 fw. Each of the three replicates were analyzed by triplicate. The rest of individual phenolics were analyzed based on Artık et al. (1998), with slight modifications proposed by Poyrazo˘glu et al. (2002), as follows: filtered pomegranate extract (20 ␮L) was analyzed using the same HPLC device and stationary phase as for anthocyanin analysis. The mobile phases were water/formic acid (95:5, v/v) (A) and methanol (B) with a flow rate of 1 mL min−1 . The linear mobile phase gradient started with 10% B to reach 15% B at 5 min, 30% B at 20 min, 50% B at 35 min and 90% B at 40 min. For column equilibration phase B was reduced to 10% in 4 min and maintained at this concentration for 10 min. Chromatograms were recorded at 378 (punicalagin ˛/ˇ), 320 (phloretin, chlorogenic and p-coumaric acids), 280 (catechin, protocatechuic, caffeic and ferulic acids), 270 (gallic acid) and 254 nm (ellagic acid). Phenolics were quantified by comparison of retention time and absorption spectra with those of commercial phenolic standards (Sigma, St Louis, MO, USA). The calibration curves were made with at least seven data points for each standard. The results were expressed as mg kg−1 fw. Each of the three replicates were analyzed by triplicate. 2.6.3. Total phenolic contents Total phenolic contents was analyzed with the Folin–Ciocalteu method (Singleton and Rossi, 1965) with slight modifications (Martínez-Hernández et al., 2011). The absorbance of samples was measured at 750 nm using a Multiscan plate reader (Tecan Infinite M200, Männedorf, Switzerland). Total phenolic contents was expressed in mg gallic acid equivalent (GAE) kg−1 fw. Each of the three replicates were analyzed by triplicate. 2.7. Total antioxidant capacity (TAC) The TAC was determined by the ferric reducing antioxidant power (FRAP) (Benzie and Strain, 1999). The absorbance of extracts was measured using the same device as for total phenolic content. Results were expressed as mg ascorbic acid equivalent antioxidant capacity (AAE) kg−1 fw. Each of the three replicates were analyzed in triplicate.

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2.8. Total vitamin C The ascorbic (AA) and dehydroascorbic (DHA) acids were measured according to the methods of Zapata and Dufour (1992) and González-Molina et al. (2008) with slight modifications. Briefly, ground frozen arils (5 g) were placed into a 25 mL Falcon tube (protected from light with aluminium foil), and 10 mL of cold buffer (19.2 g L−1 citric acid, 0.5 g L−1 ethylene diamine tetraacetic acid disodium salt, 50 mL L−1 methanol and 1.68 g L−1 NaF) were added. The mixture, placed on ice, was homogenized (Ultraturrax T25 basic, IKA, Berlin, Germany) for 30 s. Then the extract was filtered through four layers of cheesecloth and pH was adjusted to 2.35–2.40 with 6 N NaOH. Subsequently, the filtrate was centrifuged at 10,500 × g for 5 min at 5 ◦ C. Afterwards, the extract was purified by solid phase extraction through a methanol-activated C18 cartridge (Sep-Pak cartridges C18, Waters, Dublin, Ireland) and filtered through a 0.45-␮m polyethersulphone filter. DHA derivatisation was carried out mixing 750 ␮L of vitamin C extract with 250 ␮L of 7.7 M 1,2-phenylenediamine in a HPLC amber vial. The mixture was allowed to react for 37 min at room temperature. Immediately after derivatisation, 20 ␮L were randomly injected on a Gemini NX (250 mm × 4.6 mm, 5 ␮m) C18 column (Phenomenex, Torrance CA, USA), using the same HPLC system previously described. The mobile phase was 5 mM hexadecyl trimethyl ammonium bromide, 50 mM KH2 PO4 and 5% methanol (pH 4.59) with isocratic flow of 1.8 mL min−1 . Chromatograms were recorded for 14 min at 261 nm (AA, Rt = 6.8 min) and 348 nm (DHA, Rt = 3.7 min). AA and DHA were quantified using commercial standards (Sigma, St Louis, MO, USA). Calibration curves were made with at least six data points for each standard. Results were expressed as mg kg−1 fw. Each of the three replicates was analyzed in triplicate. 2.9. Statistical analysis Data were subjected to analysis of variance (ANOVA), using Statgraphics Plus software (vs. 5.1, Statpoint Technologies Inc, Warrenton (VA), USA). The effect of irrigation treatment × storage period, as well as the influence of irrigation treatment, storage + shelf life period was analyzed. Values were subjected to the least significant difference test (LSD) at P < 0.05. Pearson correlation analysis was performed to corroborate relationships between TAC and bioactive compounds. 3. Results and discussion 3.1. Chemical quality attributes At harvest, control arils had a SSC of 16.0 ± 0.05 Brix (Table 1), which is lower than those early reported for this cultivar (Artés et al., 2000a,b; Mena et al., 2011). Initially, the SDI arils had 2.5% higher SSC (16.4 ± 0.06) than control samples, probably due to abiotic stress linked to the irrigation procedure, as a similar observation was reported by Mellisho et al. (2012) for such conditions. Generally, the SSC increased throughout storage and shelf-life periods with higher increments when fruit were stored at 15 ◦ C for shelf-life, compared to samples stored at 5 ◦ C, since metabolic reactions are accelerated at higher temperatures. At low storage temperatures, SDI fruit had earlier SSC increments in the arils, while higher temperatures during shelf-life period retarded these SSC increases. This might be explained by SDI-induced stress accelerating fruit metabolism. The pH of SDI treated fruit was slightly lower than that of control fruit, with values of 3.9 and 4.0, respectively (Table 1). After 30 days at 5 ◦ C, the pH of fruit of both treatments decreased by about

174


Table 1 Soluble solids content (SSC), pH and titratable acidity (TA) changes during storage (5 ◦ C up to 90 days) and shelf-life (4 days at 15 ◦ C) of whole pomegranates cv. ‘Mollar de Elche’ grown under normal (control) and sustained deficitary irrigation (SDI) conditions (n = 9). Days

Treatment

SSC (Brix)

pH

TA (g citric acid 100 mL−1 )

At harvest

Control SDI

16.0 16.4

4.00 3.94

0.26 0.25

30 Control SDI 60 Control SDI 90 Control SDI t T t×T

d 5 ◦C

+4 d 15 ◦ C

d 5 ◦C

+4 d 15 ◦ C

d 5 ◦C

+4 d 15 ◦ C

16.0 17.0

18.0 16.5

3.62 3.56

3.65 3.61

0.22 0.21

0.21 0.20

16.5 16.8

17.4 17.9

3.89 3.82

3.80 3.75

0.22 0.23

0.23 0.24

16.7 16.9

16.8 18.3

3.99 3.90

3.99 3.99

0.20 0.18

0.21 0.22

(0.05)*** (0.04)*** (0.07)***

(0.06)*** (0.04)*** (0.09)***

(0.03)*** (0.02)*** NS

(0.03)*** (0.02)*** NS

(0.04)* NS NS

NS NS NS

NS: not significant; *, **, *** significance for P ≤ 0.05. 0.01 and 0.001, respectively; t: time; T: treatment.

9.5% and then progressively increased, reaching after 90 days at 5 ◦ C similar levels to those at harvest. TA of 0.25 g citric acid L−1 was found at harvest (Table 1) regardless of the treatment and in agreement with results from Calín-Sánchez et al. (2011). During storage, no significant TA changes were found, even when SDI pomegranates showed a relatively higher respiration rate than the control fruit (5.4 ± 1.0 and 2.9 ± 0.7 mg CO2 kg−1 h−1 for control and SDI samples at 5 ◦ C, respectively). Nevertheless, these respiration rates are low, typical of a non-climacteric fruit and close to those reported by Artés et al. (1996). 3.2. External colour measurements The non-climateric nature of pomegranates highlights the importance of optimum quality at harvest, for which they should be picked when fully ripe to ensure the best flavour (Artés et al., 1996), and which can be easily determined with the CMI. Values of 37.5 and 50.0 for control and SDI irrigated samples, respectively, revealed that fruit were harvested at acceptable maturity stage, which corresponds to values over 25 (Manera et al., 2013). At harvest, skin L* and Hue of SDI were lower than those of control samples, indicating a higher redness and darkness, while chroma showed the opposite trend (Table 2), similar to what was reported

by Mellisho et al. (2012) for the same cultivar grown under SDI. L* decreased for both treatments during cold storage, although only fruit stored for 90 days showed the same L* reduction after shelflife. Slight or negligible changes in chroma and Hue values after both storage periods confirmed previous results (Artés et al., 1996, 2000a,b). 3.3. Weight loss, firmness and chilling injury Weight losses of 4.97 (control) and 3.99% (SDI) were found after 30 days at 5 ◦ C (Table 3). SDI fruit had a rather thicker cuticle than control samples and, especially, the crown area was firmly closed (data not shown), which led to the observed lower weight losses of SDI fruit during storage. Similar results have been recently reported by Laribi et al. (2013) for the same cultivar. As expected, fruit dehydration increased throughout storage, achieving weight losses of 14.7% (control) and 10.6% (SDI) after 90 days at 5 ◦ C. Losses for controls were higher than those earlier reported by Artés et al. (1996, 2000a,b) for the same cultivar under similar storage conditions (80 days at 5 ◦ C). Weight losses of SDI samples at 15 ◦ C periods were higher than those of controls. These findings emphasize the importance of low storage temperatures and high RH in order to avoid weight losses that are linked to poor fruit appearance and economic loses.

Table 2 External colour parameters (L*, Hue and chroma) during storage (5 ◦ C up to 90 days) and shelf-life (4 days at 15 ◦ C) of whole pomegranates cv. ‘Mollar de Elche’ grown under normal (control) and sustained deficitary irrigation (SDI) conditions (n = 9). Time

Treatment

L*

Hue

Chroma

At harvest

Control SDI

68.8 60.2

60.3 53.3

42.5 45.0


d 5 ◦C

+4d 15 ◦ C

d 5 ◦C

+4d 15 ◦ C

d 5 ◦C

+4d 15 ◦ C

66.7 58.0

66.9 57.5

56.7 55.5

54.1 56.9

45.2 46.4

43.4 42.9

65.2 59.6

65.2 55.6

59.7 62.1

55.9 57.7

46.6 46.0

44.4 45.3

66.3 57.6

62.0 52.8

61.8 61.7

56.1 54.0

44.9 44.7

42.8 44.1

(1.93)* (1.37)*** NS

(1.86)*** (1.32)*** NS

(4.56)* NS NS

NS NS NS

(1.51)** NS NS

NS (1.06)* NS

NS: not significant; *, **, *** significance for P ≤ 0.05, 0.01 and 0.001, respectively; t: time; T: treatment.


175

Table 3 Firmness (N) and weight loss (%) during storage (5 ◦ C up to 90 days) and shelf-life (4 days at 15 ◦ C) of whole pomegranates cv. ‘Mollar de Elche’ grown under normal (control) and sustained deficitary irrigation (SDI) conditions (n = 9). Time

Treatment

Firmness (N)

Weight loss (%)

At harvest

Control SDI

111.3 75.6

– –


d 5 ◦C

+4 d 15 ◦ C

d 5 ◦C

+4 d 15 ◦ C

107.1 88.8

68.5 64.0

4.97 3.99

1.02 1.32

78.9 82.2

71.2 67.9

9.01 9.78

1.03 1.96

68.6 70.6

69.6 64.5

14.8 10.6

0.81 1.25

(11.1)*** (7.82)*** (15.6)***

(8.05)*** (5.70)*** (11.4)***

(0.02)*** (0.02)*** NS

(0.02)*** NS (0.02)***


Firmness values of SDI samples were 47% lower than those of control fruit at harvest (Table 3). However, while SDI samples did not register significant firmness changes during storage, control fruit had a 38% firmness reduction, showing similar firmness values in both treatments after 90 days at 5 ◦ C. As was expected, the high temperature of shelf-life periods greatly reduced fruit firmness, control samples achieving the same losses after 90 days at 5 ◦ C as after the complementary shelf-life. The magnitudes of the firmness reductions during the shelf-life periods decreased throughout storage as the water losses increased. Firmness reduction of SDI samples during shelf-life was higher than that of the control. However, Sayyari et al. (2011) reported greater firmness losses of 56% for the same cultivar after 84 days at 2 ◦ C plus a shelf-life period of 4 days at 20 ◦ C. This highlights the importance of low storage temperatures for this commodity. The CI symptoms were mainly pitting and husk-scald, confirming previous reports for ‘Mollar de Elche’ fruit (Artés et al., 1996, 2000a,b). CI increased for both treatments with prolonged storage (Fig. 1). CI developed in control fruit after 30 days at 5 ◦ C. However, CI was not found in SDI fruit until day 60 at 5 ◦ C. This was in agreement with the study done by Artés et al. (1996). Several postharvest treatments, such as heat shock, polyamines, and salicylic and jasmonic acids, alleviated CI by increasing the

endogenous polymine concentration and maintaining the unsaturated/saturated fatty acid ratio which could account for high membrane integrity and fluidity (Artés et al., 1996; Sayyari et al., 2009, 2011; Mirdehghan et al., 2007a,b; Maghoumi et al., 2013). In addition, it has been reported that the stress induced by SDI leads to the genetic expression of ‘late embryogenesis abundant proteins’ which protects cell proteins and membranes (Bray, 1993). According to the latter finding, SDI could reduce cell damage as found in CI. High CI was found in SDI fruit after 90 days at 5 ◦ C plus shelf-life, possibly due to an increase in the activity of polyphenoloxidase, reaching similar levels as control samples after 90 days at 5 ◦ C. Moreover, the possibility that an oxidation process is involved in CI development has not been excluded (Defilippi et al., 2006). Here we also suggest that a high level of antioxidants, probably phenolic compounds such as punicalagins, ellagic acid and anthocyanins, may retard CI symptom development. Additionally, it seems to be that less coloured fruit, such as those coming in the controls, are relatively more sensitive to chilling injury compared to the more coloured ones from deficit irrigation. 3.4. Sensory analysis Visual appearance and overall quality scores maintained levels over the limit of marketability through storage, although the scores after the shelf-life periods following 90 days at 5 ◦ C were on or below that limit (Fig. 2). However, colour scores were below it, regardless of the treatment and storage conditions, after 60 days at 5 ◦ C. Artés et al. (2000a) found that these sensory parameters were over the limit of marketability for the same cultivar stored 90 days at 5 ◦ C. SDI samples generally showed higher visual appearance and overall quality scores than control fruit throughout storage. As expected, the high temperatures of shelf-life periods greatly decreased visual appearance and overall quality, especially for control samples. 3.5. Phenolic compounds

Fig. 1. Chilling injury (%) during storage (C) (5 ◦ C up to 90 days) and shelf-life (4 days at 15 ◦ C) (SL) of whole pomegranates cv. ‘Mollar de Elche’ grown under normal (control) and sustained deficitary irrigation (SDI) conditions (n = 9).

The initial total phenolic contents of control and SDI arils was 2402 and 1968 mg GAE kg−1 fw, respectively (Table 4). During cold storage, total phenolic contents of both treatments increased, registering a peak at day 60 and decreasing after that. During shelf-life, total phenolic contents increased for all samples. Initial total phenolic contents were in the range of values previously reported for the same cultivar (Mena et al., 2011, 2012b; Sayyari et al., 2011). However, these values were lower than the sum of

176


Table 4 Total phenolics content and total antioxidant capacity (CAT) during storage (5 ◦ C up to 90 days) and shelf-life (4 days at 15 ◦ C) of whole pomegranates cv. ‘Mollar de Elche’ grown under normal (control) and sustained deficitary irrigation (SDI) conditions (n = 5). Time

Treatment

Total phenolics (mg GAE kg−1 fw)

CAT (mg AAE kg−1 fw)

At harvest

Control SDI

2402 1968

913 1323


d 5 ◦C

+4 d 15 ◦ C

d 5 ◦C

+4 d 15 ◦ C

2007 2117

2965 2661

905 895

1228 1204

2646 2609

2678 3413

1328 1252

1288 1510

1872 2445

2190 3149

1154 1114

1496 1354

(82.8)*** NS (117.1)***

(87.7)*** (62.0)*** (124.0)***

(140.0)*** (99.0)** (198.0)*

(164.8)*** (103.8)* (207.7)*


individual phenolics of 3420 mg kg−1 fw (from Tables 5 and 6), since the large number of phenolic compounds determined by HPLC are not completely quantified by the Folin–Cicolteau method. Nonetheless, although it is as a not such a selective method, it is useful for comparison with the results from other studies. The individual phenolics found (mg kg−1 fw) from higher to lower amounts were punicalagin ˛/ˇ (931.2/693.2) > ellagic acid (588.8) > Cy 3,5-GLU (263.0) > catechin (191.0) > Cy (188) > gallic acid (128.0) > chlorogenic acid 3-GLU (125.0) > protocatechuic (77.0) > Pg 3-GLU (65) > phloretin (58.0) > p-coumaric acid (31.0) > ferulic acid (25.0) > Pg 3,5GLU (24.0) > Dp 3,5-GLU (19) > caffeic acid (8.6) (Table 5). These individual phenolics have been previously reported in several pomegranate cultivars, including ‘Mollar de Elche’ (Gil et al., 1996a,b; Poyrazo˘glu et al., 2002; López-Rubira et al., 2005; Mena et al., 2012a). However, small to negligible amounts of punicalagin and ellagic acid have been previously reported (Gil et al., 2000). Nevertheless, those measurements were not performed on water-stressed fruit. The possibility of a slight contamination with material originating from the carpellary membranes or the peel cannot be excluded. At harvest, SDI fruit had slightly lower total phenolic contents (sum of individual phenolics) of about 8% less than the controls. Similarly, Mena et al. (2012b) also reported lower phenolic contents of water-stressed ‘Mollar de Elche’ fruit. However, the influence of such stress on this fruit has not been studied in detail with regard to each individual phenolic compound at harvest, or during prolonged commercial postharvest life, as is reported here. Generally, initial anthocyanin, catechin and caffeic acid contents of SDI samples had values 11–25, 14 and 17% lower, respectively, than those of control ones (Table 6). Nevertheless, phloretin and protocatechuic acid showed the opposite behaviour, with SDIinduced stress producing increases of those values to about 98 and 10%, over the controls respectively. Other authors have indicated that the anthocyanin profile may change during different maturity stages, since the diglucoside derivatives were the prevailing pigments during the early ripening stages whereas the monoglucosides were the main pigments in the latter stages (Gil et al., 1996b). The effects of DI on phenolic composition are unclear from the scarce literature available. Some authors have suggested that it is not possible to establish a linear correlation between water stress and phenolic contents (Gobbo-Neto and Lopes, 2007). Taking into account that plant growth begins to decrease at a water potential higher than that at which stomatal closure takes place, Horner (1990) proposed that moderate water stresses, such as

were applied in these experiments, induce stomatal regulation and reduce CO2 assimilation. In this situation, carbon should be preferentially allocated to the synthesis of primary metabolites to the detriment of some secondary metabolite synthesis such as has been here found for these phenolics. However, the observed enhancement for phloretin and protocatechuic acid could be due to water stress-induced genetic regulation related to enzyme activation (Zhu et al., 2002). Ewith regard to the storage period, punicalagins (˛ and ˇ) and ellagic acid, the two major phenolic compounds reported here (56 and 20% from the sum of phenolics, respectively) showed the same trends during storage for both treatments. In this way, a progressive decrease from harvest to day 60 was found. At day 60, SDI samples registered the smallest decreases of 27 and 21%, followed by a high increase, with the greatest increases (49 and 78% for punicalagin ˛ and ellagic acid, respectively, comparing to their initial levels). Caffeic, protocatechuic and p-coumaric acids, catechin and phloretin contents of SDI samples showed a similar behaviour throughout storage, with a significant increase of 17–29% after 30–60 days, compared to their respective values at harvest, generally followed by a decrease that reached initial levels after 90 days at 5 ◦ C. In general, anthocyanin levels increased during storage SDI samples achieving the highest increases, which ranged from 45 to 65%, while anthocyanin increases in control samples were about 17–27%, compared to their respective levels at harvest. As previously reported (Miguel et al., 2004; Fawole and Opara, 2013), the observed pomegranate anthocyanin increases during storage is a result of a continued biosynthesis of these compounds favoured by the cold storage temperatures compared to the prevailing field temperature at harvest. Generally, 3,5-diglucosides had higher stability when compared with the 3-glucosides, as was earlier found in ‘Mollar’ pomegranate juice stored at 5 ◦ C (Martí et al., 2002). This can be explained if it is considered that glycoside substitution at C5 reduces the nucleophilic character of the C6 and C8 positions; thus anthocyanin 3,5-diglucosides are less prone to electrophilic attack than 3-glucosydes (Timberlake and Bridle, 1977). The trend observed for the remaining phenolics of control samples was either a reduction or stabilization of their contents throughout storage. Studies have shown that during postharvest storage of ‘Mollar de Elche’ fruit, total phenolic and total anthocyanins increased (Mirdehghan et al., 2006), while other results indicated no changes (Gil et al., 1996b). Nevertheless, it is noteworthy to state that data presented here represent the longest storage period (90 days at 5 ◦ C) in the literature. Thus there was a higher

Table 5 Phenolics compounds during storage (5 ◦ C up to 90 days) and shelf-life (4 days at 15 ◦ C) of whole pomegranates cv. ‘Mollar de Elche’ grown under normal (control) and sustained deficitary irrigation (SDI) conditions (n = 5). Time

Treatment

Punicalagin ˛

Punicalagin ˇ

Ellagic

Catechin

Gallic

Chlorogenic

Protocatechuic

Phloretin

p-Coumaric

Ferulic

Caffeic

Total

At harvest

Control SDI

931.2 921.9

693.2 696.3

588.8 420.0

191.6 159.9

128.8 135.8

125.9 48.0

77.3 85.8

59.0 115.1

31.6 26.0

25.0 31.6

8.58 7.38

2859 2646

d 5 ◦C

t T t×T

d 5 ◦C

+4d 15 ◦ C

d 5 ◦C

+4d 15 ◦ C

d 5 ◦C

+4d 15 ◦ C

d 5 ◦C

d 5 ◦C

+4 d 15 ◦ C

+4d 15 ◦ C

d 5 ◦C

+4d 15 ◦ C

d 5 ◦C

+4 d 15 ◦ C

d 5 ◦C

+4d 15 ◦ C

d 5 ◦C

+4d 15 ◦ C

d 5 ◦C

+4d 15 ◦ C

d 5 ◦C

+4d 15 ◦ C

622.4 552.7

1209 1262

669.4 686.4

644.7 739.2

338.5 406.4

831.7 817.5

192.12 133.3

264.3 197.1

170.7 139.1

276.1 293.1

40.2 43.2

42.5 54.2

47.0 107.0

62.0 61.2

66.7 148.2

91.2 63.3

62.3 84.5

18.9 43.2

25.1 24.2

31.67 32.5

9.03 9.0.1

7.21 6.99

2241 2333

3474 3568

538.4 678.8

891.9 1106

671.9 666.9

655.1 659.4

263.0 332.7

593.9 640.8

226.0 190.2

244.5 221.6

229.9 203.8

286.6 272.0

34.34 39.7

42.4 61.3

50.1 79.2

108.1 62.0

61.2 112.0

85.6 129.6

87.5 62.4

47.0 30.2

19.2 20.6

23.2 26.8

10.1 6.05

6.19 6.38

2187 2387

2979 3210

1081 1370

1029 1329

662.3 679.8

626.2 705.1

640.7 748.78

651.1 780.0

192.6 144.0

183.1 245.8

208.7 228.9

250.3 264.1

51.4 48.6

59.6 41.9

49.1 54.9

59.2 57.6

133.3 92.45

99.1 73.1

69.2 78.9

28.6 68.4

26. 30.0

27.0 33.0

6.02 6.01

6.79 7.78

3116 3476

3017 3599

(98.5)*** (69.6)** (139.3)**

(84.9)*** (60.0)*** (120.1)**

(16.0)* NS NS

NS (29.7)** NS

(43.2)*** NS (61.16)***

NS NS NS

(9.46)*** (6.69)*** (13.4)*

(30.1)** NS (42.62)**

NS NS NS

(21.7)*** NS NS

(28.1)** NS (39.7)*

(28.0)* NS (39.7)**

(10.2)*** (7.18)*** (14.4)***

(8.05)*** (5.69)** (11.4)***

NS (14.5)*** (34.9)***

(12.7)** (9.01)* (18.0)***

(4.70)*** NS (6.65)***

(2.64)*** (1.87)*** (3.73)***

(4.26)** NS NS

(4.28)* (3.02)* NS

(0.63)*** (0.45)*** (0.89)***

(0.80)** NS (1.14)**

(107.3)*** (75.9)*** (151.8)***

NS NS NS


Table 6 Anthocyanins during storage (5 ◦ C up to 90 days) and shelf-life (4 days at 15 ◦ C) of whole pomegranates cv. ‘Mollar de Elche’ grown under normal (control) and sustained deficitary irrigation (SDI) conditions (n = 5). Time

Treatment

Cy 3.5-GLC

Cy3-GLC

P 3-GLC

P 3.5-GLC

D 3.5 GLC

Total

At harvest

Control SDI

263.5 201.3

188.7 191.0

65.4 49.2

24.3 20.2

19.1 18.0

560.9 479.6


d 5 ◦C

+4 d 15 ◦ C

d 5 ◦C

+4 d 15 ◦ C

d 5 ◦C

+4 d 15 ◦ C

d 5 ◦C

+4 d 15 ◦ C

d 5 ◦C

+4 d 15 ◦ C

d 5 ◦C

+4 d 15 ◦ C

213.4 151.2

291.3 327.4

189.3 171.3

154.3 131.7

46.7 28.8

62.2 53.3

28.1 25.9

24.2 33.0

21.2 16.0

27.2 25.2

498.6 393.2

559.1 570.6

335.7 214.2

360.7 354.6

141.4 177.8

130.6 145.3

76.8 77.6

52.2 58.2

32.1 25.9

34.2 29.9

24.2 21.3

31.3 26.2

610.2 516.8

609.0 614.1

235.0 267.2

260.0 345.3

117.0 123.3

170.2 137.6

56.7 71.1

68.8 55.2

26.0 25.0

29.2 41.2

25.0 25.5

25.2 32.3

459.7 512.0

553.3 611.6

(48.4)* (34.2)*** (68.4)*

(22.1)*** NS (31.2)***

(11.8)*** (8.31)*** (16.6)***

(6.51)*** (4.61)*** (9.21)***

(4.80)*** (3.40)*** (6.79)***

(2.73)*** (1.93)*** (3.86)***

(1.32)*** (0.93)*** (1.86)*

(2.85)*** (2.01)** (4.03)***

(3.04)** (2.15)* NS

(1.16)*** NS (1.64)***

(63.1)*** (44.7)*** (89.3)***

(25.7)*** (18.2)* (36.3)***


30 Control SDI 60 Control SDI 90 Control SDI

+ 4d 15 ◦ C

NS: not significant; *, **, ***significance for P ≤ 0.05, 0.01 and 0.001, respectively; t: time; T: treatment.

177

178


Fig. 2. Sensory scores for visual appearance, colour and overall quality during storage (5 ◦ C up to 90 days) and shelf-life (4 days at 15 ◦ C) of whole pomegranates cv. ‘Mollar de Elche’ grown under normal (control) and sustained deficitary irrigation (SDI) conditions (n = 5) ±SD.

weight loss than that reported by Gil et al. (1996b), which may account for a relative increase in phenolic concentration. In other fruit like grapes, a decline in total anthocyanins (loss of red colour) has been reported (Artés-Hernández et al., 2003). After shelf-life corresponding to storage periods of 30 and 60 days at 5 ◦ C, anthocyanins, punicalagin ˛, catechin and ellagic, gallic, chlorogenic and ferulic acids contents were greatly enhanced for both irrigation treatments. Furthermore, the increases found after 30 days at 5 ◦ C plus shelf-life were higher than those after 60 days 5 ◦ C plus shelf-life. SDI samples achieved increases of up to 2–3-fold above those achieved by the latter phenolics in control samples. Caffeic and p-coumaric acids contents decreased after all of the shelf-life periods, regardless of the treatment. The considerable phenolics increases at the higher temperature at which the fruit were exposed during shelf-life are in agreement with previous reports on strawberries and coloured potatoes, that showed how higher temperatures induced higher anthocyanin contents than lower ones (Miszczak et al., 1995; Lewis et al., 1999).

Fig. 3. Ascorbic acid (A), dehydroascorbic acid (B) and total vitamin C (C) contents during storage (5 ◦ C up to 90 days) and shelf-life (4 days at 15 ◦ C) of whole pomegranates cv. ‘Mollar de Elche’ grown under normal (control) and sustained deficitary irrigation (SDI) conditions (n = 5) ±SD.

3.6. Total vitamin C At harvest, SDI samples had a 27% higher total vitamin C content than controls. High vitamin C content may serve as a protective strategy against drought injury. A similar trend has been found in broccoli grown under DI (34% less water) which showed higher vitamin C (30%) contents than controls (Toivonen et al., 1994). Vitamin C has been associated with anthocyanin stability of pomegranate (cv. ‘Mollar’) juice (Martí et al., 2002), which might explain the lower levels of these phenolics found in arils of SDI samples compared to those of control ones. The total vitamin C content for control samples of 196.4 mg kg−1 fw (Fig. 3) is higher than that reported for different ‘Mollar de Elche’ accessions (Ozgen et al., 2008), but are consistent with those previously reported for Israeli and Turkish cultivars (Schwartz et al., 2009; Mena et al., 2011). Several preharvest factors such as climatic conditions (temperature and light) and cultural practices (N fertilization, pruning, thinning,


pesticides and growth regulators) have been reported to affect the vitamin C content of several fruit and vegetables, as reviewed by Lee and Kader (2000). DHA of SDI samples was higher than that of control samples with values of 178.9 and 127.3 mg kg−1 fw, respectively. DHA itself is unstable and undergoes irreversible hydrolytic ring cleavage to 2,3-diketogulonic acid in aqueous solutions (Davey et al., 2000), and this reaction might have somehow been reduced in the water-stress samples. AA content slightly decreased (3–4%) during storage. Contrary to what was expected, since typically vitamin C stability decreases with temperature, no AA changes were found at higher temperatures during the shelf-life periods. Similarly, Izumi et al. (1984) reported that AA contents of cucumbers continuously decreased at 5 ◦ C, but no loss was observed at 20 ◦ C. This great AA stability may be explained by this compound being more stable under acidic conditions (Nagy, 1980). DHA greatly increased during shelflife periods, reaching in SDI samples the highest increases of 59% and 71% for those shelf-life periods following 30 and 60 days of cold storage, respectively. However, during the last shelf-life period (following 90 days of cold storage), control samples achieved higher DHA increases than those of SDI, 57% and 25%, respectively. The earlier DHA formation in SDI samples compared to controls might be due to higher metabolism of those water-stressed samples. 3.7. Total antioxidant capacity At harvest, TAC of control samples was 912.7 mg GAE kg−1 fw. Initial TAC values of SDI samples were 46% higher than the control (Table 4). During storage, TAC showed a similar trend for both treatments with an initial reduction (day 30) followed by a peak (day 60) and subsequently decreased until the end of storage. After 90 days at 5 ◦ C, while SDI samples registered a TAC decrease of 16%, the TAC for control was enhanced 26% over their respective values at harvest. Generally, during the shelf-life periods, TAC levels increased, ranging among 21% and 35%. It is worth mentioning the contribution of each of the analyzed bioactive compounds to TAC. The total anthocyanins, gallic acid and total vitamin C were significantly correlated with TAC, with Pearson correlation coefficients (r) of 0.59, 0.63 and 0.78, respectively, whereas no significant total punicalagins were found (Table 7). The anthocyanins Cy 3,5-GLU (the major anthocyanin found) and Dp 3,5-GLU, were also significantly correlated with TAC, with r values of 0.57 and 0.55, respectively. Different studies carried out with juices of different pomegranate cultivars have reported differences related to the phenolic compound contribution to antioxidant capacity assays (Gil et al., 2000; Ozgen et al.,

Table 7 Pearson correlation coefficient (r) between TAC and bioactive compounds in pomegranates cv. ‘Mollar de Elche’. Phenolics (0.38) Polyphenols Phloretin Catechin Anthocyanins D 3.5 GLC Cy 3.5-GLC P 3.5-GLC Cy3-GLC P 3-GLC Punicalagins ˛ ˇ Ellagicacid

Simple phenols 0.10 0.43 0.59* 0.55* 0.57* 0.35 −0.29 0.41 0.29 0.33 −0.30 0.22

Gallicacid 0.63* Chlorogenicacid −0.27 Protocatechuicacid −0.17 Ferulicacid 0.10 Caffeicacid −0.50 p-Coumaricacid −0.34

AA DHA Vitamin C

−0.19 0.78** 0.78**

The r significance (P < 0.05) is identified by one asterisk, while (P < 0.001) is identified by two asterisks. No asterisk means that no significant correlation was found.

179

2008; Çam et al., 2009), which may be a result of the juice extraction process. Similarly, Mena et al. (2011) found a total anthocyanin correlation to TAC of r = 0.69 and no significant total punicalagin correlation. However, our data show a high total vitamin C correlation of r = 0.78 (principally due to DHA contribution). Previous work with different cultivars only determined the AA content (Gil et al., 2000; Mirdehghan et al., 2006). Our findings also suggest out that punicalagins did not participate in the antioxidant capacity, as was firstly suggested by Tzulker et al. (2007). However, punicalagins have important biological activities and are involved in most pomegranate health beneficial properties (Koyama et al., 2010). In contrast, Gil et al. (2000) reported that punicalagins were the most potent antioxidants of the pomegranate phenolics. These conflicting results may be explained by the cultivar and harvest time being key factors affecting antioxidant compounds/antioxidant capacity correlations (Turfan et al., 2011). Thus, if only antioxidant capacity is analyzed in aril juices instead of the major bioactive compounds, potential benefits attributed to pomegranates may be underestimated. 4. Conclusions SDI fruit had greater sugar contents (according to SSC) and redder colour tones, and lower RR than control fruit at harvest. However, SDI samples had less initial firmness than control fruit. SDI fruit also had greater initial vitamin C, total antioxidant capacity and some individual phenolics (catechin, protocatechuic, phloretin, gallic and caffeic acids) than control samples. However, control fruit showed greater initial contents of the main phenolics (punicalagins, ellagic and anthocyanins). During cold storage, SDI had a positive effect on pomegranate quality, since lower CI symptoms, and water and firmness loss, better sensory scores and greater anthocyanin increases were found compared to control fruit. Generally, SDI samples had the highest phenolics increases during shelf-life periods. Acknowledgements The authors are grateful to the Spanish Ministry of Economy and Competitiveness – FEDER through the project AGL2010-19201C04-02-AGR for financial support and to the Spanish Cooperation Agency for International Development (AECID) for a grant to M.E. ˜ Estevez. Pena References Artés, F., Martínez, J.A., Marín, J.G., 1996. Controlled atmosphere storage of pomegranate. Z. Lebensm. Unters. Forsch. 203, 33–37. Artés, F., Tudela, J.A., Villaescusa, R., 2000b. Thermal postharvest treatments for improving pomegranate quality and shelf life. Postharvest Biol. Technol. 18, 245–251. Artés, F., Villaescusa, R., Tudela, J.A., 2000a. Modified atmosphere packaging of pomegranate. J. Food Sci. 65, 1112–1116. Artés-Hernández, F., Aguayo, E., Artés, F., 2004. Alternative atmosphere treatments for keeping quality of ‘Autumn seedless’ table grapes during long-term cold storage. Postharvest Biol. Technol. 31, 59–67. Artés-Hernández, F., Artés, F., Tomás-Barberán, F.A., 2003. Quality and enhancement of bioactive phenolics in cv. Napoleon table grapes exposed to different postharvest gaseous treatments. J. Agric. Food Chem. 51, 5290–5295. Artık, N., Murakami, H., Mori, T., 1998. Determination of phenolic compounds in pomegranate juice by using HPLC. Fruit Process. 12, 492–499. Benzie, I.F.F., Strain, J.J., 1999. Ferric reducing antioxidant power assay: direct measure of total antioxidant activity of biological fluids and modified version for simultaneous measurement of total antioxidant power and ascorbic acid concentration. Methods Enzymol. 299, 15–27. Bett, K.L., 2002. Evaluating sensory quality of fresh-cut fruits and vegetables. In: Lamikanra, O. (Ed.), Fresh-cut Fruits and Vegetables Science, Technology, and Market. CRC, Boca Ratón. Bray, E.A., 1993. Molecular responses to water deficit. Plant Physiol. 103, 1035–1040. Calín-Sánchez, A., Martínez, J.J., Vázquez-Araújo, L., Burló, F., Melgarejo, P., Carbonell-Barrachina, A.A., 2011. Volatile composition and sensory quality of Spanish pomegranates (Punica granatum L). J. Sci. Food Agric. 91, 586–592.

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