Postharvest Biology and Technology 78 (2013) 24–33

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Postharvest Biology and Technology
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Cold quarantine responses of ‘Tarocco’ oranges to short hot water and thiabendazole postharvest dip treatments
A. Palma a , S. D’Aquino a , S. Vanadia b , A. Angioni c , M. Schirra a,∗
a b c

C.N.R. Institute of Sciences of Food Production, Traversa La Crucca 3, Regione Baldinca, 07040 Li Punti, Sassari, Italy C.N.R. Institute of Sciences of Food Production, via Amendola 122/O, 70126 Bari, Italy Department of Science of Life and Environment, University of Cagliari, Via Fiorelli 1, 09126 Cagliari, Italy

a r t i c l e

i n f o

a b s t r a c t
This study investigated the effects of brief hot water and thiabendazole (TBZ) postharvest dip treatments on ultrastructural changes of fruit epicuticular wax (ECW), TBZ residues, decay development and quality traits of ‘Tarocco’ oranges [Citrus sinensis (L.) Osbek] subjected to cold quarantine, subsequent simulated transport and shelf-life. Commercially mature fruit were submerged in water at 20 ◦ C (control fruit) or TBZ at 1000 mg/L and 20 ◦ C for 60 s, or in hot water without or with TBZ at 300 mg/L and 53, 56, or 59 ◦ C for 60, 30, and 15 s respectively. Following treatments, fruit were stored for 3 weeks at 1 ◦ C (simulated quarantine conditions for fruit disinfestations against Mediterranean fruit fly, Medfly), followed by 4 days at 3 ◦ C (simulated long distance transport), and finally kept at 20 ◦ C for 3 days (shelf-life, SL). Scanning electron microscopy (SEM) analysis of ‘Tarocco’ orange surface showed that the typical wax platelets, lifting around edges of wax plates and areas free of epicuticular wax (ECW), that disappeared after hot water dips at 53–59 ◦ C for 60–15 s, become visible again after storage for 21 days at 1 ◦ C (quarantine conditions), and changes involving the appearance of rough ultrastructure, presence large curled plates, fissured wax crusts, and areas with ECW deficiencies, became much more pronounced after shelf-life. These occurrences were related to the transient effect of hot water treatment in decay control. Conversely, treatments with 300 mg/L TBZ 53 ◦ C for 60 s or 56 ◦ C for 30 s effectively reduced decay after quarantine. These treatments were as effective as standard treatment with 1000 mg/L TBZ at 20 ◦ C and produced similar TBZ residue levels in fruit, without impairing fruit quality traits such as visual appearance, weight loss, compression test, sensory attributes, juice color parameters (a*, b*, h, L*, and Chroma), and juice chemical characteristics (soluble solids content, titratable acidity, ascorbic acid, glucose, sucrose, citric acid, total phenols, total anthocyanins, and total antioxidant activity). © 2012 Elsevier B.V. All rights reserved.

Article history: Received 6 September 2012 Accepted 9 December 2012 Keywords: Citrus Decay Epicuticular wax morphology Heat treatment Thiabendazole residues Storage

1. Introduction Among the orange cultivars, ‘Moro’, ‘Sanguinello’ and ‘Tarocco’ are the most cultivated in Italy. Fruit of these cultivars are appreciated by consumers for their typical taste and peculiar characteristics of blood-red flesh and rind color, owing to the presence of phenolic compounds belonging to the class of anthocyanins (Dugo et al., 2003), some of which are known to have several health promoting properties (e.g. beneficial effects on capillary fragility and arteriosclerosis, and antiviral activity, preventing allergies and other inflammatory diseases, anti-cancer activity) (Sajia, 1994). Therefore, from a dietetic viewpoint, anthocyanins represent an added value for blood oranges due to their important therapeutic

∗ Corresponding author. Tel.: +39 0783 33224; fax: +39 0783 33959. E-mail address: mario.schirra@ispa.cnr.it (M. Schirra). 0925-5214/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.postharvbio.2012.12.002

properties, in addition to the well known health benefits of citrus fruit (Attaway and Moore, 1992). Various citrus-importing countries require quarantine security protocols to diminish the risk of accidental introduction of the Mediterranean fruit fly (Medfly, Ceratitis capitata), and fruit must be certified Medfly free. Therefore Medfly disinfestations should be performed by citrus producing-countries before exportation. Heat treatments (hot water immersion, high temperature forced air, vapor heat) (Armstrong and Mangan, 2007) and cold quarantine treatments (exposure of fruit to near-freezing temperatures for 10–16 days) (Armstrong, 1994), are technologies applied on a commercial scale for postharvest insect control of horticultural crops as alternatives to chemical fumigation (e.g. methyl bromide). Commodity response to quarantine treatment varies depending on species, cultivar, and quarantine treatment conditions. Quarantine treatments reaching a fruit core temperature of 44 ◦ C for 100 min or 46 ◦ C for 50 min did not cause damage or fruit softening in ‘Olinda’ and ‘Campbell’ oranges (Schirra et al.,

2001). The peel was weighed and its percentage with respect to the whole fruit calculated. A compression test was performed by a texture–meter interfaced with a computerized system (DO-FB0. Finally. where 0 = none.2.. host defensive responses and inhibition of pathogen development (Schirra et al. peel samples were kept frozen at −40 ◦ C until analysis. 2 = moderate. whereas fruit of the remaining group (10 fruit per replication) were used for compression test. the juice was centrifuged (Centurion Scientific Ltd. a weighted average from a chilling index was calculated. SEM was carried out with a ZEISS DSM 962 microscope at 30 kV and 250–2000× magnifications. each treatment group was divided into three subgroups of three boxes (replications) each. They were dried by washing with increasing concentrations of ethanol (20. samples were stored in a vacuum dryer. a. Briefly.4. the phases were allowed to separate and 1 mL of the organic layer was dried under a gentle nitrogen stream and the residues were dissolved with 1 mL of acetone and injected in GC–ITMS for the analysis without any clean-up step. Samples of five fruit per treatment were used for SEM analysis. 2004). Palma et al. such as development of off-flavors and off-tastes. 2.5. West Sussex. / Postharvest Biology and Technology 78 (2013) 24–33 25 2005a) but produced deleterious effects on the quality traits of blood oranges. cold quarantine can lead to chilling injury (CI) on blood oranges and the application of a hot water dip at 50 ◦ C for 180 s before cold disinfestation was able to overcome the risk of CI and decay development after quarantine. and 100%). Milan. 2. Zwick Roell.45 m acetate cellulose filter. the samples being left for 20 min before each wash. (2008).4) and then three times in deionized water.A..9% active ingredient. The dried samples were placed on aluminum stubs using silver conductive glue.4) containing 3% glutaraldehyde. Each treatment was repeated threefold (replications). fruit weight loss and compression test. according to Schirra et al.i. after simulated transport and after shelf-life. 9 = excellent. treatments and storage conditions Commercially mature blood oranges [Citrus sinensis (L. and the peel was removed. Six oranges per replication (4 replications per treatment) were weighed. ‘Tarocco’ were harvested from a farm located in southern Sardinia (Italy) and transported to the laboratory immediately after harvest. TBZ analyses were carried out on four replicates of six fruit per treatment. 2000b.. CI was evaluated as pitting or scald of the peel.3. after quarantine and after shelf-life. 70. Before analyses. 50. The mixture was agitated in a rotatory shaker for 30 min. decay and the appearance and absence of the calyx. chilling injury (CI).1.A. and 4 = severe injury. Two rind samples (2 cm × 2 cm) were excised with a razor blade from the equatorial zone of each fruit and immediately fixed in a phosphate buffer (pH 7. Germany). nutritional and functional properties of fruit. Relevant SEM micrographs at 250× magnification are shown in the figures. 95. Swiss). It has been shown that the effectiveness of postharvest hot water dip treatments at 50 ◦ C for 180 s in alleviating CI in citrus fruit. Plant material. fruit were individually weighed following treatment and after selected periods and the percentage of weight loss calculated. or in combination with 300 mg/L TBZ. The potential relevance of treatment-induced changes on TBZ residues. Materials and methods 2. 5 = fair. Reduced treatment time would be desirable to increase packinghouse output and shorten delays in fruit marketing. compression test To determine visual assessment. decreased fruit firmness and reduced fruit resistance to decay (Mulas et al. Syngenta Crop Protection S. 5 g of homogenized peel were weighed in a 40 mL screw-capped flask and 10 mL of ethyl acetate/hexane (1/1) and 2 g of NaCl were added. The fruit of the first subgroup (40 fruit per replication) were used for overall visual appearance. other methods such as cold quarantine therefore should be applied to reduce treatment impact on quality (Schirra et al. (b) water alone at 53. The concentration of TBZ in the treatments at 53–59 ◦ C was set based on preliminary trials performed in our laboratory. and a gold–palladium coating was applied with an Edwards S-150 A sputter coater. Until observation. Italy. Overall appearance was evaluated on the basis of the following hedonic scale: 1 = very poor. external and internal fruit quality attributes of blood oranges exposed to a quarantine treatment. Juice was obtained by squeezing 10 fruit per replication (3 replications per treatment) with a commercial juicer. Juice chemical analysis Chemical analyses of juice were performed following treatment and after shelf-life. TBZ residues. Visual assessment. On the other hand. no data are available in the literature on the influence of short hot water treatments used in commercial installations and its combination with TBZ on ultrastructural changes of fruit surface. and kept at 4 ◦ C until further preparation.. 2011). therefore treatment temperature should be raised to produce the heat-induced beneficial effects in terms of physical changes of ECW.) Osbek] cv. Decay incidence was assessed as percentage of rotten fruit caused by various fungi. 7 = good. Values were expressed as maximum deformation at 3 kg compression force. CI and decay control after quarantine was also evaluated and discussed. 2. The fixed tissue was rinsed three times in phosphate buffer (pH 7. after quarantine. Subsequently. The compression curves were obtained by deforming the fruits with a metal disk (15 cm diameter) at a speed of 100 mm/min. Fruit of the second subgroup (40 fruit per replication) were used for weight loss determination. Following treatments fruit were left to dry at room temperature and stored for 3 weeks at 1 ◦ C (quarantine conditions) plus 4 days of simulated transport at 3 ◦ C (quarantine + transport) and subsequent 3 days of simulated shelf-life at 20 ◦ C (quarantine + transport + SL). 3 = poor. 2. and 15 s respectively. Analysis of TBZ residues TBZ analyses were performed following treatment.. notably improve when applied in combination with the fungicide thiabendazole (TBZ) (Schirra et al. 80. 2. 56. 5 mL of juice diluted in 45 mL .. 1 = slight. or 59 ◦ C for 60. England) at 13. Then.) for 60 s (standard treatment). To determine fruit weight loss. without or with TBZ on physical. was rated using a 0–4 subjective scale. decay incidence. However.p. 2004). without impairing fruit quality (Schirra et al. 2000a). 1998. 30. Scanning Electron Microscopy (SEM) analysis SEM observations were performed following treatments. at 42. Visibly sound fruit free of defect were washed and subdivided into 8 groups corresponding to the following dip treatments: (a) water alone at 20 ◦ C (control) or in combination with 1000 mg/L TBZ (TECTO SC..5TS. The appearance and absence of the calyx were determined and the percentage of fruit showing the calyx dried and brown or detached was calculated. fruit weight loss. Titratable acidity (TA) of the juice was determined using a potentiometric titrator (Metrom 720 SM Tritino. The present investigation was therefore planned to study the cold quarantine responses of ‘Tarocco’ blood oranges to short (15–60 s) hot water dip treatments.000 × g for 15 min and the supernatant filtered through a 0.

the ECW of fruit treated with TBZ at 20 ◦ C (standard treatment) appeared more homogenous than control fruit. 3b versus Fig.. Schirra et al. cyanidin-3-malonyl-glycoside. oven 80 ◦ C. penetration and retention on the plant cuticle (Schönherr and Baur. SEM analysis 3.) was employed. results of our study suggest that the loss of details of ECW fine structure in fruit treated with TBZ may be ascribed to selective solubilization of ECW by surfactant present in the commercial formulation of TBZ fungicide mixture. b* 76 nant D65 and 10 2 Convention (McLaren. and a higher level of wax melting could be assessed at 59 ◦ C (Fig. and areas free of ECW (Fig. rough platelets. (2005) using a Bio-Rad cation guard column and a Bio-Rad Aminex HPX-87H Hydrogen form cation exchange resin-based column (300 mm × 7. cyanidin-3-glucoside. 1a). 2.d. 2000). Japan). post time 12 min. The mixture containing 3 mL of a methanol solution of 0. according to the CIE L*. Quantitative analysis of anthocyanins was carried out using the external standards method and their concentration was expressed as cyanidin-3-glucoside equivalents. t = 38 30% B. detector at 150 ◦ C.1. Noga et al. Thus. 3a) or 59 ◦ C for 15 s (Fig. CA. further statistical analyses for these parameters were pooled. Peaks of organic acids and ascorbic acid were measured at wavelengths of 210 and 245 nm respectively and were identified by comparing retention times with those of standards and quantification was carried out using external standards. Total anthocyanin contents were determined spectrophotometrically using the pH differential method (Rapisarda et al. These features were ascribed to the partial melting and redistribution of ECW layer along fruit surface. / Postharvest Biology and Technology 78 (2013) 24–33 distilled water were titrated with NaOH 0.) at 40 ◦ C. layers of ECW. However. 1965). 2004).1. The individual anthocyanins (cyanidin-3-5-diglucoside. A similar behavior involving changes in ECW ultrastructure of fruit treated with hot water was reported previously (Schirra and D’hallewin.d. 1987) presumably by selective solubilization of ECW constituents (Tamura et al. and no difference could be detected with the treatments at 20 ◦ C. fruit treated with water at 56 ◦ C for 30 s (Fig. Analysis of variance (ANOVA) was carried out according to a single factor. 2b). Phenomenex. Organic acids and ascorbic acid (AA) were determined with a Merck-Hitachi (Tokyo. D-7000 system manager.. Tokyo. (1997).005 M sulphuric acid aqueous solution and the samples were isocratically separated at 0. 2009). Then a 1 mL headspace gas sample was injected into gas chromatograph Agilent 6890 fitted with a flame ionization detector (FID) and equipped with a 5% Carbowax 20 M on 80/120 Carbograph 1 AW packed column. Japan) equipped with a L-7455 photodiode detector (DAD). Hue angle (h = arctg b*/a*) and Chroma values (Chroma = (a∗ )2 + (b∗ ) ) were calculated using illumi◦ observer. by the least significant difference test at P ≤ 0. TBZ application at 53 ◦ C resulted in a partial melting of ECW. Total phenolic content was analyzed according to the FolinCiocalteau colorimetric method (Singleton and Rossi. version XV Professional statistical program. The percentage of total soluble solids (SSC) was measured by a digital refractometer (PR-101. Palma et al. As fruit treated with water alone or in combination with TBZ did not reveal significant differences (P > 0. HPLC elution was carried out at 35 ◦ C using the following elution profile: flow rate 0. VA. All measurements were done in triplicate. 1998).. Fungicide formulation interactions with the plant cuticle are complex (Baur. Simultaneous separation and determination of organic acids and ascorbic acid were achieved according to the procedure described by Yuan and Chen (1999) and by Chinnici et al.1.. The results were expressed as percentage of anhydrous citric acid. Italy).. 1997. The chromatogram was monitored simultaneously at 360 and 520 nm. depending on the range of variation of data. raised or folded outwards around edges. L7200 autosampler and L-7100 pumps. Atago.16 mM DPPH was allowed to react for 15 min in a cuvette. Run conditions were: N2 as carrier gas at 30 mL/min. injector at 130 ◦ C. spreading. Anthocyanins were identified by LC-electrospray ionization (ESI)–MS analysis using an Agilent Technologies (Palo Alto. for acetaldehyde and ethanol determination. Antioxidant activity was assessed using the free radical DPPH.8 mm i. USA) 1100 series LC/MSD. t = 20 20% B linear gradient. Japan) liquid chromatograph with an L-7455 photodiode detector (DAD) detector. according to Bonded et al. 3b and 4b) changes in the ECW micromorphology were more evident. Mean comparisons of the effects of treatments were calculated. Color parameters of juices solution diluted 1/10 with water. t = 0 10% solvent B (acetonitrile)/90% solvent A (formic acid 1%.1. 3. A Luna C18 column (150 mm × 4. were hardly visible and the ECW platelets and granules relaxed (Fig. Percentages were subjected to the √ √ ANOVA after transformation in arcsin x or x before the ANOVA. and wax granules were observed at higher magnification (SEM micrograph not shown). irregular in shape. 2001). The mobile phase consisted of 0. Ethanol and acetaldehyde were quantified by comparison of peak area versus concentration of a calibration curve obtained from pure analytical standards subjected to the same analytical conditions (Schirra et al. the platelets tended to ‘relax’ and melt. and creating a smoother appearance.i.26 A. there is evidence that surfactants can considerably modify the ECW fine structure (Kuzyc and Meggitt. Cuticular ridges. Similarly. delphinidin-3-6-malonyl-glucoside. 1a).6 mm. 4b). Following treatment The surface of fruit dipped in water at 20 ◦ C (control fruit) displayed the typical ultrastructure of mature orange fruit (Cajuste et al. 2a and e). a*. a*. Results and discussion 3. 2005b.05) in color or chemical traits. covering the areas free of ECW. 1980). Two mL of juice were placed in a 10 mL headspace vial and incubate for 2 h in a shaking bath at 60 ◦ C. b*.6 mL/min. 1996).. Total phenols were expressed as gallic acid equivalents. USA).6.6 mm i. but wax layers were hardly visible (Fig. When treatments with TBZ were performed at 56 or 59 ◦ C (Figs.. In contrast. raised or folded outwards around edges. 1983. CIE L*..05.5 mm × 4. 4a) showed the loss of the ultrastructural features of fruit surface. when applicable.5 mL/min. increasing wetting. Dore et al. Surfactants are often added to agrochemicals to design a formulation able to enhance the effectiveness of the a. . The differences in ECW fine structure of fruit subjected to hot water at 59 ◦ C without or with TBZ were negligible. 2010) with areas covered by smooth thin layers of ECW. The inhibition percentage of the absorbance at 515 nm of DPPH solution added with sample was calculated using the following equation: Inhibition % = (Abst=0 − Abst=15 min )/Abst=0 × 100.1 N up to pH 8. cyanidin-3-dioxolane-glucoside. were measured in glass cells of 10 mm path length using a Varian Cary 50 spectrophotometer equipped with a Cary Win UV color software. acetic acid 2%). Statistical analysis Statistical analysis was performed by Statgraphics Centurion software (Herndon. Dip treatments with water at 53 ◦ C for 60 s caused the disappearance of ECW layers: the fine structures of ECW collapsed and some stomatal openings were occluded with wax (Fig. 3 m. and peonidin-3-6-malonyl-glucoside) were determined with a LaChrom Merck-Hitachi liquid chromatograph (Hitachi Ltd. BO. complete randomized block design with three replicates for each treatment. equipped with a precolumn (7. Castel Maggiore.

may be related to loss of elasticity of waxy layer and the subsequent detachment of wax plates. By contrast. partially detached from cuticle. Surface details of fine structure of TBZ treated fruit (Figs. In addition. f. TBZ). Fruit treated with TBZ at 53 ◦ C (Fig. 1c). After shelf-life The control fruit and fruit treated with water at 53 or 59 ◦ C showed changes involving the appearance of rough structures with wax deposits. 1c). 1. Build up of crystalline wax granules was also observed and areas free of ECW enlarged. Palma et al. The arrows (a and c) indicate stomata. and the lifting around edges of wax plates was increased (Fig. with large edges completely curled outwards.A. 1d versus Fig. 3f) maintained a homogeneous shape. without the appearance of ECW free zones. whereas treatments at 56 ◦ C showed less differences compared with quarantined fruits (Figs. 2d) showed very irregular wax scabs. water.1. 3d) were only small. 1–4f) were generally less visible than control and fruit treated with water at 53–59 ◦ C.1. 1c). whereas ultrastructural changes of fruit treated with TBZ at 56 ◦ C (Fig. Fruit treated with TBZ at 20 ◦ C displayed similar features as control fruit but the areas uncovered by ECW were less extensive in size (Fig. 2c. TBZ). and next subjected to 4 days of simulated transport at 3 ◦ C plus 3 days of shelf-life at 17 ◦ C (e. The appearance of fissured wax crusts along with large areas without ECW. Wax platelets of an indefinite design and stomata uncovered by wax were visible at higher magnification (SEM micrograph not shown). 3c and 4c versus Fig. d. after subsequent quarantine for 3 weeks at 1 ◦ C (c. Comparison with standard treatments (TBZ at 1000 mg/L and 20 ◦ C) showed marked differences in the shape of the wax aggregates and extension of wax deprived zones. 1–4e). 3. After quarantine The wax layer of control fruit became rough.2. fruit treated with TBZ at 59 ◦ C exhibited large irregular ECW layers. / Postharvest Biology and Technology 78 (2013) 24–33 27 Fig. . a network of large curled plates. The micromorphology of the cuticular surface of fruit treated with water at 53–59 ◦ C was similar. water.3. partially detached edges and areas without ECW (Fig. factors that may be ascribed to fruit senescence. and large areas with ECW along with skin surface. only fruit treated with TBZ at 56 ◦ C (Fig. SEM micrographs (250× magnifications) of cuticle surface of ‘Tarocco’ oranges after dip treatment at 20 ◦ C for 60 s in water (a) or thiabendazole (TBZ) at 1000 mg/L (b). and ECW deficiencies became much more pronounced than those observed after quarantine. 4d). but was different as compared to control fruit (Figs. 3.

2. and next subjected to 4 days of simulated transport at 3 ◦ C plus 3 days of shelf-life at 17 ◦ C (e. residue levels in fruit were similar (Table 1). treatment duration used in this study was short (15–60 s). TBZ). Previous studies on ‘Tarocco’ oranges (Schirra et al.. f.28 A. being the same as that employed commercially in many locations in the US. The lack of . 3..3. and e). TBZ). TBZ persistence being higher in fruit treated at 20 ◦ C with a decline after shelf-life of ca 12% versus ca 39% in fruit treated at 53–59 ◦ C. Palma et al. Influence of treatments on CI.. 2000b. 3. after subsequent quarantine for 3 weeks at 1 ◦ C (c. However. thus providing better protection to the fungicide.. whereas much longer periods (180 s) used in previous studies (Schirra et al. 1998) resulted in a greater impact of heat on ‘rearrangement’ of ECW structure and TBZ persistence. especially when fruits are returned to warm temperature (Schirra et al. and fruit quality attributes The incidence of fruit affected by CI was very small after quarantine and subsequent simulated transport (data not shown) and was relatively low also after shelf life (Table 2). Influence of treatment and storage conditions on TBZ residues in fruit Immediately after TBZ treatments at 1000 mg/L and 20 ◦ C or at 300 mg/L and 53–59 ◦ C. water. 1998) showed that fruit treated with TBZ at 50 ◦ C experienced a greater persistence of TBZ with respect to fruit treated at room temperature and this occurrence was related to the better encapsulation and coverage of the a. d. SEM micrographs (250× magnifications) of cuticle surface of ‘Tarocco’ oranges after dip treatment at 53 ◦ C for 60 s in water (a) or thiabendazole (TBZ) at 300 mg/L (b). d. the beneficial effect of hot water treatment in terms of partial melting and redistribution of ECW layer along fruit surface and subsequent closure of microwounds which represent potential gaps for conidia of wound pathogens (Schirra et al. by ECW. 2004). CI incidence was reduced in fruit treated at 53 ◦ C and remarkably reduced in the other fruit samples. It is recognized that cold quarantined blood oranges are susceptible to CI. b.2. The different effect on fruit surface of TBZ treatment at 56 ◦ C is consistent in all postharvest steps and indicates that this was the best temperature for the treatment. The arrows indicate stomata (a. 2011). water. Therefore. decay.i. are only transitory and are lost during fruit storage. Results of the present study displayed a reverse trend. / Postharvest Biology and Technology 78 (2013) 24–33 Fig.

89 b(b) a Values within columns (without brackets) or rows (in brackets) followed by unlike letters differ significantly by Fisher’s least significant difference (LSD) procedure. 3. on a whole fruit basis).96 a(a) 6.65 a(a) 6. The percentage of fruit showing the calyx dried and brown or detached was not significantly affected by treatments (data not shown). TBZ). Letters in brackets relate to comparisons of the effect of storage time within each treatment. TBZ). 4 additional days of simulated transport at 3 ◦ C (transport). without significant differences among treatments (data not shown).37 ab(ab) 5.4 ± 5.a Treatments Dip time (s) TBZ residues (mg/kg) Time 0 1000 mg/LTBZ 20 ◦ C 300 mg/L TBZ 53 ◦ C 300 mg/L TBZ 56 ◦ C 300 mg/L TBZ 59 ◦ C 60 60 30 15 6.05. after subsequent quarantine for 3 weeks at 1 ◦ C (c. water.87 a(b) 4.78 b(b) 5. .31 ab(a) Quarantine 6.72 b(b) 5.04 b(c) 3. SEM micrographs (250× magnifications) of cuticle surface of ‘Tarocco’ oranges after dip treatment at 56 ◦ C for 30 s in water (3a) or thiabendazole (TBZ) at 300 mg/L (b). d. Pooled data (mean values ± standard deviation) of this percentage after shelf life averaged 34.1–7.36 b(b) 5.26 b(c) 4.59 b(a) Transport 6. There was no Table 1 Thiabendazole (TBZ) residues (mg/kg. and subsequent 3 days of simulated shelf life.65 a(a) 5. water. / Postharvest Biology and Technology 78 (2013) 24–33 29 Fig. Overall appearance was rated as very good up to quarantine + storage (rate 8–8. in ‘Tarocco’ oranges immediately after treatment (time 0) and after 3 weeks at 1 ◦ C (quarantine).63 b(b) 5. The arrows indicate stomata (a–d and f). whereas after shelf-life all treated fruit were judged as good to very good (rate 7. Palma et al.5) and fairly good (rate 6) for the control fruit (Table 2).A. The lower score received by control fruit was related to the higher incidence of CI.4. P ≤ 0. CI found in our study was related to the relatively short period of simulated transport and shelf-life. and next subjected to 4 days of simulated transport at 3 ◦ C plus 3 days of shelf-life at 17 ◦ C (e.64 b(a) Shelf life 5. Letters without brackets relate to comparisons of the influence of treatments within each storage time.56 a(a) 6. f.5).

59 ◦ C 1000 mg/L TBZ.1 a 7.1 a 7.60 a 0. 20 ◦ C Water. treatments with hot water alone at 53–59 ◦ C significantly reduced the incidence of decay with respect to control Table 2 Influence of postharvest treatments on chilling injury index. P ≤ 0. Treatment with 300 mg/L TBZ at 53 or 56 ◦ C significantly reduced decay development with respect to control fruit.3 cd 1.30 b 0. overall appearance.07 c 0. 4 additional days of simulated transport at 3 ◦ C and subsequent 3 days of simulated shelf-life (quarantine + transport + SL). after subsequent quarantine for 3 weeks at 1 ◦ C (c. whereas the effect of the other treatments was not significant.00 c 0.05 c Overall appearance 6. rating scale 0–4.09 c 0. and next subjected to 4 days of simulated transport at 3 ◦ C plus 3 days of shelf-life at 17 ◦ C (e. overall appearance. / Postharvest Biology and Technology 78 (2013) 24–33 Fig. and decay percentage in ‘Tarocco’ oranges subjected to cold quarantine for 3 weeks at 1 ◦ C.6 b 16. Palma et al. 53 ◦ C Water.9 a 13.6 ab 3. italicum respectively (data not shown). blue mold caused by Penicillium digitatum and P. 20 ◦ C 300 mg/L TBZ. The causal agents of decay were green mold and.30 A. TBZ).3 a Decay (%) 28.7 b 23.4 c a Chilling injury index. 56 ◦ C 300 mg/L TBZ.08 c 0.05. . f.02 c 0.1 b 7. decay in untreated fruit after quarantine while after subsequent transport decay incidence averaged about 8% (data not shown). SEM micrographs (250× magnifications) of cuticle surface of ‘Tarocco’ oranges after dip treatment at 59 ◦ C for 15 s in water (a) or thiabendazole (TBZ) at 300 mg/L (b). reaching approximately 30% after shelf-life (Table 1). Values within columns followed by unlike letters differ significantly by Fisher’s least significant difference (LSD) procedure. 59 ◦ C Dip time (s) 60 60 30 15 60 60 30 15 Chilling injury 0. water.3 b 15. 53 ◦ C 300 mg/L TBZ. 56 ◦ C Water. to a lesser extent. rating scale 0–9. TBZ).a Treatments Water.2 a 7. 4. After shelf-life. d.2 a 7.5 a 7.2 a 7. The arrows indicate stomata (d and e). water.1 d 4.

71 1. 1997). a dramatic accumulation of ethanol was found in previous studies on ‘Fortune’ mandarins subjected to hot water dips at 58 ◦ C for 180 s and stored for 30 days at 6 ◦ C plus three days of simulated shelf-life at 20 ◦ C (Schirra and D’hallewin.18 b 1. and consequently lower values of the a*/b* ratio. 1990. Treatments at 53 and 56 ◦ C did not affect SSC.03 b 1. whereas lower values of fructose and malic acid were recorded in fruit treated at 56 or 59 ◦ C (Table 5). This occurrence was ascribed to the low permeability of mandarin peel to gas which favors the build-up of volatiles.37 ab 1.19 1.80 Values within columns followed by unlike letters differ significantly by Fisher’s least significant difference (LSD) procedure. which might weaken the natural defense properties of fruit.97ns 2.03 1.75 after transport and shelf-life. P ≤ 0. a*/b* ratio. with a higher h.98 b h 44.60 a 1. significantly lower AA values were recorded in fruit treated at 59 ◦ C indicating the stress conditions caused by this treatment.8 bc 77.17 a a*/b* 1.94ns 10.14 31.56 a MeCHO 0.04 b 1. 53 ◦ C Water. Fruit weight loss and fruit compression test values were not affected by treatments (data not shown). respectively.15 b b* 1.12 b 9.23 a 1.38 1. and the consequent perception of off-flavors (Shi et al. 2005).90 1. 1996). 2001) and in mature green tomatoes (Lycopersicum esculentum Mill. and with 300 mg/L TBZ at 53–56 ◦ C (0. 1980).21.71 b 32. or the SSC-TA ratio. 20 ◦ C Water. The concentrations of sucrose. After shelf-life.81 b 0.9 c 62..71 98. 59 ◦ C a Dip time (s) L* 98.5% decay).8–3.42 Values within columns followed by unlike letters differ significantly by Fisher’s least significant difference (LSD) procedure.86 b 0.12 b 8.61 a 1. SST/TA). mg/100 mL) and ethanol (EtOH.) after heat treatment at 38 ◦ C and 50% RH for 24 h (Yahia et al.14 1. with respect to control fruit (Table 3). L*..05..0 55.98 ab 45. when water was applied on ‘Tarocco’ oranges at 59 ◦ C for 15 s. An increase in the TSS/TA ratio during storage of citrus fruit was related to the accumulation of ethanol in the juice (Cohen et al.20 ab 1.94 a EtOH 21. h (hue angle) and Chroma (saturation) in ‘Tarocco’ oranges subjected to cold quarantine for 3 weeks at 1 ◦ C. respectively. Hagenmaier.77 and 7.71 and 3.83 b 0. 2007). and Chroma parameters were not significantly affected by treatments. which may cause the development of off-flavors (Cohen et al. Pooled data (mean values ± standard deviation) of weight loss averaged 2..81 ± 0. according to previous studies on ‘Fortune’ mandarins submerged in water at 50–58 ◦ C for 180 s (Schirra and D’hallewin.a Treatments Harvest Quarantine + transport + SL Water.. It is recognized that mandarins are much more prone to develop off-flavors than other citrus varieties (Shi et al. plus 4 days of simulated transport at 3 ◦ C (quarantine + transport) and subsequent 3 days of simulated shelf-life (quarantine + transport + SL).11 ± 0.43 ± 0.64 60 60 30 15 98.37 0. TA. 20 ◦ C Water. Similar adverse effects on AA levels were observed in blood oranges exposed to a fruit core temperature of 44 ◦ C for 100 min or 46 ◦ C for 50 min (Mulas et al. 2002). P ≤ 0.11 ± 0. maturity index (SST:TA ratio. Investigations on blood oranges revealed that hot water dipping for 180 s at 50 ◦ C did not significantly affect the content of AA during or after quarantine (Schirra et al. glucose. Acetaldehyde levels increased in fruit treated at 59 ◦ C. 1997). 2004). 59 ◦ C a Dip time (s) SST 11.87 10.13 c SST/TA 8.07 9.6 a 60 60 30 15 10. / Postharvest Biology and Technology 78 (2013) 24–33 31 Table 3 Influence of postharvest treatments on color parameters L* (brightness).76 10.3% decay). supporting previous studies on blood oranges (Schirra et al.1 b 100. 53 ◦ C Water. acetaldehyde (MeCHO. 1990). ns = not significant. whereas treatment at 59 ◦ C caused a decrease in red pigmentation of juice as supported by lower values of a*. However.31 ab 0. Best results in controlling decay were achieved with TBZ at 1000 mg/L and 20 ◦ C (2.79 a* 1. The lack of efficacy of TBZ at 59 ◦ C in controlling decay may be ascribed to physiological stress of the peel.a Treatments Harvest Quarantine + transport + SL Water. After shelf-life there was a relatively small decline (ca 16–22%) in AA with respect to fresh fruit.2 TA 1.A. Accordingly. .. Treatment damage and off-flavor development were reported in ‘Satsuma’ mandarins when submerged in water for 120 s at temperatures higher than 50 ◦ C (Ghasemnezhad et al.84 b 9. In blood oranges variations of free and bound hydroxycinnamic acids levels during storage were proven to be a reliable index of detrimental off-flavors development (Fallico et al. By contrast. However. results of this study showed that differences in AA content between fruit treated at 53–56 ◦ C and control fruit were not significant (Table 6).. 1997). mg/100 mL) content in ‘Tarocco’ oranges subjected to cold quarantine for 3 weeks at 1 ◦ C. TA values were lower than control fruit and SSC-TA ratio increased accordingly (Table 4).23 1. although visible symptoms of treatment damage were not detected. titratable acidity (% anhydrous citric acid). However.92ns 98. and citric acid were not significantly affected by treatments. %). higher values of b*. Various reports have shown that vitamin C loss in citrus fruit during storage at different temperatures is slight (Nagy.04 b 1. 2008). 56 ◦ C Water. fruit (Table 2).62 98. 4 additional days of simulated transport at 3 ◦ C (quarantine + transport) and subsequent 3 days of simulated shelf-life (quarantine + transport + SL). Fruit treated at 56 and 59 ◦ C registered respectively higher and notably higher values of ethanol (Table 4) and lower values of fructose and malic acid than control fruit (Table 5).. 2001).50 a Chroma* 1. a* (redness) and b* (yellowness). Palma et al. such as ethanol and acetaldehyde in the juice.05. At harvest compression test values averaged 4. 56 ◦ C Water.. compounds other than ethanol have been related with the offflavor development in citrus fruit. whereas after transport and shelf-life were 7. The low storage temperatures Table 4 Influence of postharvest treatments on soluble solids content (SSC. However..14 b 37. 2004). the present study showed that the increases of ethanol found in ‘Tarocco’ oranges treated at 56 and 59 ◦ C did not adversely affect fruit sensory quality and no visible treatment damage occurred.66.31 ± 0. Such adverse effects on fruit quality observed in ‘Fortune’ mandarins were ascribed to phytotoxic effects caused by the excessive temperature (58 ◦ C) and prolonged exposure of fruit to heat (180 s) (Schirra and D’hallewin.68 a 1. fruit appeared brown and aged and the taste was judged poor due to the off-flavor development.59 a 1. Conversely the influence of TBZ at 59 ◦ C on decay control was not significant.

a Treatments Harvest Quarantine + transport + SL 60 Water. 1998. pp.32 A. / Postharvest Biology and Technology 78 (2013) 24–33 Table 5 Influence of postharvest treatments on sucrose. Wallingford.79 4. V. 20 ◦ C Water.284ns 0. 4 additional days of simulated transport at 3 ◦ C (quarantine + transport) and subsequent 3 days of simulated shelf life (quarantine + transport + SL). Food Chem.057 0. activation of host defense responses. Wallingford.. S.092 0. R. J. Poei-Langston and Wrolstad. Italy.03 2. Heat and cold treatment.A.097 a 0.. 809–837.. 53 ◦ C 60 Water. R.1 Values within columns followed by unlike letters differ significantly by Fisher’s least significant difference (LSD) procedure.258 0.. KIotz and DeWolfe. 20 ◦ C Water.i. Lo Piero et al.5 23.33 b 2. Fruit treated at 59 ◦ C had lower values of total anthocyanins than control fruit and fruit treated at 53 ◦ C. Research supported by CNR-MiUR. ‘Sviluppo delle Esportazioni di Prodotti Agroalimentari del Mezzogiorno’. 311–340. Insect Pests and Fresh Horticultural Products: Treatments and Responses.7 ab 58. enhanced diffusion and penetration of a. 3. Armstrong. Agric. ns = not significant.036 b Cyanidin 3-glucoside 0.8 Total anthocyanins 0. Salvatore Marceddu for technical assistance in SEM analysis. the short shelf-life period. Reig-Arminana. 609–615.i. 3.16 a 1. 56 ◦ C 30 15 Water.L. Armstrong.. Berset.).4ns 102.326 b Cyanidin 3-dioxalonil glucoside 0. 2000a. In: Proceeding of 7th International Citrus Congress. Newly discovered health benefits of citrus fruits and juices. the sensitivity of fungal spores to heat treatments considerable depends by species (Sommer et al. 1981).05.79 4. Previous studies investigated the beneficial effects of hot water dips for several minutes to control green mold (Houck. in citrus fruit (Feld et al. J.080 0.06 0. L. (Eds.3 60.. E. In addition. P ≤ 0.041 a 0. as a result of heat in reducing the diffusion barrier of fruit cuticle to active ingredient penetration. 2010.. pp.W. 2011).A.).025 ab 1. J. Mangan. C.031ab 0. Baur. Heat Treatment for Postharvest Pest Control: Theory and Practice.088ns 0. treatment at 59 ◦ C cannot be applied to ‘Tarocco’ oranges being close to threshold for treatment damage.15 a 0. 1967. (2004) showed the potential of postharvest hot water dips for 180 s at 50 ◦ C to control CI and decay of blood oranges after quarantine. Kinetics and mechanism of antioxidant activity using the DPPH free radical method. F.72 Fructose 2. 2011). Dev.E. CAB International. Conversely. Individual anthocyanins were not affected by treatments. 2007). without impairing fruit quality.52 a 2.9 100. Res. P ≤ 0. ˜ Cajuste. Lafuente. blue mold (Palou et al. Mechanicistic aspects of foliar penetration of agrochemicals and the effect of adjuvants.269 Delphinidin 3-6 malonyl glucoside 0..367 ab 0.068 b 0.12 2.L.a Treatments Harvest Quarantine + transport + SL 60 Water. Castejon-Munoz and Bollen.020 c Citric acid 1. 1965).058 b 0. J. 2001.. 4 additional days of simulated transport at 3 ◦ C and subsequent 3 days of simulated shelf life (quarantine + transport + SL).. malic acid. Yet.. E.308 0. and increased permeation of a.1 ab 56.088 Values within columns followed by unlike letters differ significantly by Fisher’s least significant difference (LSD) procedure. 1136–1139. Table 6 Influence of postharvest treatments on individual anthocyanins (mg/100 mL) in ‘Tarocco’ oranges subjected to cold quarantine for 3 weeks at 1 ◦ C. In: Tang.404 a 0. better results were obtained when hot water at 53–56 ◦ C was combined with 300 mL TBZ.T.15ns 2.6 b Total phenols 105. The highest pigmentation in juice of fruit treated at 53 ◦ C and control was also confirmed by the higher values of a*/b* ratio (Table 3). 1961).29 1. Schirra et al. S.244 0.318 0. Brand-Williams. García-Breijo.. In: Paull. glucose. Differences in total phenols and antioxidant activity among treatments after shelf-life were not significant. Smoot and Melvin.014 ab 0. along with the low levels of anthocyanins and the high content of total acids.3 23. Acknowledgements The authors gratefully acknowledge Mr..038 a 0.. The beneficial effects of hot water treatment on decay control were related to heat impact on pathogens and on redistribution of ECW layer which may improve physical barriers to wound pathogen penetration and reduce disease incidence caused by green and blue mold decay in citrus fruit (Schirra et al. 59 C 15 a Dip time (s) Sucrose 5. fructose.93 2.967 b Ascorbic acid 72.. All parameters are expressed as g/100 mL).82 1.. Wang.. P. ns = not significant. 2005). 30. UK. M. and citric acid in ‘Tarocco’ oranges subjected to cold quarantine for 3 weeks at 1 ◦ C.. González-Candelas. 1993). (Eds. 1967. 1992. Commercial quarantine heat treatment. and brown rot caused by Phytophtora spp.W. References Armstrong. Bonded.042 0.90 b Antioxidant activity 31. Lebensmittel Wissenschaft Technol. 2007. that immersion time (180 s) was longer than that used in most commercial installations (15–60 s). CAB International. J. J. Various factors are known to affect the effectiveness of heat therapy against decay-causing agents.99 ab 0. into rind wounds (Schirra et al. W.032 0.086 0. L.081a 1.. 1997. Palma et al. J.2 101. 2000a.W. treatments with 300 mL TBZ at 53–56 ◦ C for 60–30 s showed a better performance than unheated TBZ as less than 1/3 of active ingredient was required in the treatment bath to produce similar TBZ residues in fruit and to achieve a similar control of decay. Attaway.16 2.03 Malic acid 0.J. 59 ◦ C a Dip time (s) Cyanidin 3-5 diglucoside 0. These include the moisture content of spores. 2001). with the exception of delphinidin-36-malonyl-glucoside that showed significantly higher values in fruit treated at 53 ◦ C and cyanidin-3-5-diglucoside and cyanidin-3malonyl-glycoside with lower values in fruit treated at 59 ◦ C.064 b Cyanidin-3-malonyl glycoside 0. After shelf-life. Veyrat. age of the inoculum and inoculum concentration and host (Fallik and Lurie. the water temperature should be increased to achieve the beneficial effects of heat in terms of inhibition of pathogens. vol. Rev.6 102. However. 1980. In addition..4 22.097 0.29 b Glucose 1... into cuticular wax. Lurie.5ns 23. 1979. 103–119.. pp. 56 C 30 ◦ Water.F.4 a 58. However. Epicuticular wax content and morphology as related to .043 a 0..077ns 0. in agreement with previous studies on red oranges stored at low temperatures (Rapisarda et al.. 53 ◦ C 60 ◦ Water. Present results have shown that hot water treatments at 53–56 ◦ C for 60–30 s effectively reduced decay development in ‘Tarocco’ oranges. during quarantine and transport. may account for the relatively small degradation rate of AA in the juice (Nagy. Acireale.41ab 2. 1994.045 a 0..097 0.024 bc 0.22 4. as revealed by the reduced efficacy of TBZ in controlling decay.403 a 0.82ns 4. Moore. Mitcham. total anthocyanin levels were higher than in freshly harvested fruit. when short (15–60 s) treatment times are applied.077 Peonydin 3-6-maloniyl glucoside 0.05. UK.

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