Impact of atmospheric ozone-enrichment on quality-related attributes of tomato fruit

Postharvest Biology and Technology 45 (2007) 317–325

Impact of atmospheric ozone-enrichment on quality-related attributes of tomato fruit Nikos Tzortzakis, Anne Borland, Ian Singleton, Jeremy Barnes ∗ Environmental and Molecular Plant Physiology, Institute for Research on the Environment and Sustainability, School of Biology and Psychology, Newcastle University, Newcastle upon Tyne NE1 7RU, UK Received 17 May 2006; accepted 11 March 2007

Abstract Tomato fruit (Lycopersicon esculentum L. cv. Carousel) were exposed to ozone concentrations ranging between 0.005 (controls) and 1.0 ␮mol mol−1 at 13 ◦ C and 95% RH. Quality-related attributes and organoleptic characteristics were examined during and following ozone treatment. Levels of soluble sugars (glucose, fructose) were maintained in ozone-treated fruit following transfer to ‘clean air’, and a transient increase in ␤-carotene, lutein and lycopene content was observed in ozone-treated fruit, though the effect was not sustained. Ozone-enrichment also maintained fruit firmness in comparison with fruit stored in ‘clean air’. Ozone-treatment did not affect fruit weight loss, antioxidant status, CO2 /H2 O exchange, ethylene production or organic acid, vitamin C (pulp and seed) and total phenolic content. Panel trials (employing choice tests, based on both appearance and sensory evaluation) revealed an overwhelming preference for fruit subject to low-level ozone-enrichment (0.15 ␮mol mol−1 ), with the effect persisting following packaging. © 2007 Elsevier B.V. All rights reserved. Keywords: Fruit storage; Preservation; Organoleptics; Sensory evaluation; Tomato

1. Introduction The growth in the consumption of fresh produce over the last century has driven commercial demand for improved storage/transit conditions to manage postharvest disease proliferation and maintain fruit quality (i.e. flavour, colour, nutritional aspects, firmness, ‘shelf-life’ and processing attributes) (Brummell and Harpster, 2001). Currently, an array of chemical treatments are used to preserve fresh produce. However, there are growing health and environmental concerns over current practices, due mainly to the risk of generating potentially harmful by-products and residues (Spotts and Peters, 1980). There are also growing practical concerns over the increasingly poor control achieved over a spectrum of spoilage organisms (Sapers, 1998). As a consequence, there is considerable interest in alternative, safe, but effective, sanitizing agents for use in the fresh produce industry (see Rice et al., 1982; Perez et al.,

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Corresponding author at: Environmental and Molecular Plant Physiology, Institute for Research on the Environment and Sustainability, School of Biology and Psychology: Division of Biology, Devonshire Building, Newcastle University, Newcastle Upon Tyne NE1 7RU, UK. Tel.: +44 191 246 4837. E-mail address: [email protected] (J. Barnes). 0925-5214/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.postharvbio.2007.03.004

1999). One candidate is ozone (Rice, 2002; Palou et al., 2006), a gas awarded GRASS1 status by an expert panel in conjunction with the US Food and Drug Administration in 1997 (Graham et al., 1997), and which has since received formal US-FDA approval (in 2003) as a Food Additive (Karaca and Velioglu, 2007). Legislation facilitating the deployment of ozone in the food services sector has driven growing interest in the potential afforded by the gas for a variety of applications in the fruit and vegetable supply chain. Recent work highlights the potential of the gas to curb microbial spoilage of fruit and vegetables in cold-storage (Liew and Prange, 1994; Barth et al., 1995; Sarig et al., 1996; Perez et al., 1999; Palou et al., 2002; Aguayo et al., 2006; Tzortzakis et al., 2007) and to preserve the condition of ethylene-sensitive commodities (Skog and Chu, 2001). However, rather less attention has been paid to the impacts of ozone-enrichment on the quality of treated fruit and vegetables. Skog and Chu (2001) reported that ozone (0.4 ␮mol mol−1 )2 significantly improved the quality and storage life of cold-stored broccoli and cucumber. Barth et al. (1995) and Salvador et al. (2006) report similar findings for ozone-treated blackber1 2

‘Generally recognised as safe’ status (GRASS). 1 ␮mol mol−1 (SI convention) is equivalent to 1 ppm or 1 ␮L L−1 .

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ries and persimmon, respectively. Other studies, for example on strawberries, reveal ozone-induced delays in fruit softening (Perez et al., 1999; Nadas et al., 2003) and potentially beneficial effects on key nutritional components such as the levels of non-soluble carbohydrates (Kute et al., 1995; Aguayo et al., 2006) and antioxidants, e.g. ascorbic and foumaric acid (Perez et al., 1999; Aguayo et al., 2006). However, there may be tradeoffs, in the case of treatment of strawberries, against a loss in fruit aroma (Perez et al., 1999). Low-level ozone-enrichment has been reported to have no effects on organic acid composition of treated tomatoes and citrus (Smilanick, 2003), nor on shelf-life determinants (i.e. rates of respiration and ethylene production) in grapes and peaches. However, some investigations have reported increased rates of water loss in ozone-treated fruit (Palou et al., 2002). At the market interface, only produce that corresponds to the expectations of the consumer is acceptable. Thus, it is vital to assess the effects of potentially innovative practices on sensory and organoleptic properties of fruit and vegetables. This is particularly so in the case of ozone-enrichment, as effects on quality-related characteristics seem highly dependent on the commodity and storage conditions (Liew and Prange, 1994). The present study was driven by commercial interests in the benefits of storing tomato fruit in an ozone-enriched environment, and the lack of information about the effects of such treatments on quality-related characteristics. The study reports the effects of sustained exposure to 0.05 ␮mol mol−1 or 1.0 ␮mol mol−1 ozone-enriched air on quality-related attributes of cold-stored tomato fruit: (i) physiological parameters (including weight loss, fruit firmness, and rates of respiration/transpiration and ethylene production), (ii) fruit chemical composition (e.g. vitamin C content, antioxidant capacity, organic acid content (citrate, malate), soluble sugar profile, carotenoid (lycopene, ␤-carotene, lutein) and total phenolic content) and (iii) sensory properties as evaluated by a discerning consumer panel under controlled conditions.

Advanced Pollution Instrumentation Inc., San Diego, CA) via a multi-channel PTFE® -sample/logging system (ICAM Ltd., Worthing, Sussex, UK). The photometric analyser was calibrated at monthly intervals against a Dasibi 1008PC unit meeting with US-EPA internal calibration standards, that was calibrated at 6-monthly intervals against an absolute external standard. Chambers were maintained at 13 ◦ C, 95% relative humidity (RH), with both temperature and RH monitored and logged during experiments with the aid of cross-calibrated temperature/humidity sensors (Vaisala HMI 32, Vaisala OY, Helsinki, Finland). Full-ripe tomato fruit (cv. Carousel; 50–60 mm diameter) were exposed to CFA or ozone (0.05 or 1.0 ␮mol mol−1 ) for between 1 and 6 days. After 6 days, a batch of tomatoes were transferred to duplicate controlled environment chambers ventilated with CFA for a further 6 days at 13 ◦ C and 95% RH. Experiments were conducted on a minimum of six independent replicate fruit exposed in duplicate chambers at each time point for every treatment.

2. Materials and methods

2.4. CO2 /H2 O exchange of tomato fruit

2.1. Fumigation system and ozone treatments

Gas exchange was measured with a Walz CMS-400 compact mini-cuvette system equipped with a twin channel BINOS-100 infrared gas (CO2 /H2 O) analyser (IRGA) (H. Walz GmbH, Effeltrich, Germany). Fruit were enclosed in a well-stirred cuvette, and allowed to equilibrate for 10 min prior to data collection. Relative humidity of air entering the cuvette was adjusted to ca. 55% and air was introduced at 500 mL min−1 . Data were logged continuously over 10 min. Gas exchange parameters were calculated using the DIAGAS software package (H. Walz GmbH) based on the equations defined by Von Caemmerer and Farquhar (1981). Results reported represent duplicate measurements made on no less than six independent fruit per treatment at each time point.

Ozone treatments were administered in a system comprising eight Perspex® chambers (each with an internal volume of 0.28 m3 ), housed in a walk-in controlled environment chamber (see Tzortzakis et al., 2007 for details). Each chamber was ventilated with charcoal/particulate-filtered air (‘clean air’ [CFA <0.005 ␮mol mol−1 ozone]) at a flow rate sufficient to achieve two complete air changes per minute in each chamber, and turbulence within each chamber was created by an integral 12 V fan. Ozone was generated by electric discharge from pure oxygen (model SGA01 Pacific Ozone Technology Inc., Brentwood, CA, USA), to avoid the generation of impurities, was introduced into the CFA airstream entering four of the chambers. The introduction of ozone to each chamber was controlled via stainless steel needle-valved gap flow meters. The ozone concentration in each chamber was recorded every 6 min by a photometric analyser (model 450, manufactured by

2.2. Water loss of tomato fruit Fruit were selected for uniformity and absence of any damage, labelled and then weighed prior to exposure to CFA (<0.005 ozone) or ozone-enriched air (0.05 ␮mol mol−1 or 1.0 ␮mol mol−1 ) at 13 ◦ C and 95% RH. Fruit were weighed at intervals and weight loss computed. 2.3. Tomato fruit firmness Firmness was measured at the furthest two points apart on the equator of each fruit (i.e. equidistant between the top and bottom of each fruit) with a Texturometer (model FT 011, supplied by TR Scientific Instruments, Forli, Italy) using a plunger of 8 mm diameter. Results were expressed in terms of the force (in newtons (N)) required to break the radial pericarp (i.e. skin/surface) of each tomato. Measurements were made at room temperature.

2.5. Ethylene production by tomato fruit Fruit were weighed and measured, then placed in air-tight glass jars (0.54 dm3 ) for 10 min (to allow equilibrium at room

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temperature). After 10 min equilibration, the flask was sealed. Head-space samples for ethylene determination were taken after 1 h incubation in the dark at room temperature. Air samples were introduced into a gas chromatograph equipped with a flame ionisation detector (Shimadzu GC-14B, supplied and maintained by Dyson Instruments Ltd., Houghton-le-Spring, Tyne and Wear, UK). All measurements were duplicated and flow rates recorded. Samples of room air were tested for ethylene prior to and during experimentation. Quantification of ethylene was achieved using a 10 ␮mol mol−1 standard (Supelco, Supelco Park, Bellafonte, PA). Data were presented in terms of fruit fresh weight (g). Ethylene production was calculated according to the following equation: rate of ethylene production = [(F − A)/S] × (V1 − V2 ) × (1/w) × (1/t); where F represents area of peak in presence of fruit, A represents area of ethylene peak in absence of fruit, S represents ethylene peak area of 10 ␮mol mol−1 standard, V1 , V2 represent jar and fruit volume (mL), respectively, w represents fruit weight (g) and t represents incubation time (h). 2.6. Organic acid content of tomato fruit Methanol (80%, v/v) extracts of tomato (ca. 1 g) were assayed for malate employing an NADH-linked assay (Hohorst, 1965). Malate content was expressed as ␮mol malic acid g−1 fresh weight of tomato tissue. Titratable acidity (TA) was determined using a method recommended by the Association of Official Analytical Chemistry (AOAC), involving the titration of blended tomato tissue (5 g) with 0.1 M NaOH, and the formation of pink precipitate monitored using phenolphthalein. Titratable acidity was expressed in terms of citric acid concentration (␮mol g−1 fresh weight). 2.7. Soluble sugar composition of tomato fruit Soluble sugars were extracted from tomato fruit (ca. 0.5 g) in 80% (v/v) methanol. Extracts were passed through ion exchange columns (Dowex AG50W and Amberlite IRA-67 in series), then sugar content profiled by HPLC employing a pulsed amperometric detector (ED40 electrochemical detector, Dionex, Cambridge, UK) as described in greater detail elsewhere (Borland et al., 2006). Sample components were eluted isocratically using 150 mM NaOH at a flow rate of 1 mL min−1 (Adams et al., 1992). Peak area was recorded and results expressed as mmol sugars g−1 fresh weight, based on the integrated peak area resulting from the introduction of the appropriate analytical grade standards at regular intervals during the analytical procedure. 2.8. Ascorbic acid (vitamin C) content and redox status in tomato fruit The content of ascorbate (ASC) and dehydroascorbate (DHA) was determined using the spectrophotometric method of Takahama and Oniki (1992). Individual fruit were separated into pericarp (flesh and skin) and pulp (placenta and locular tissue including seed) tissue, prior to homogenisation in ice-cold

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extraction buffer (metaphosphoric acid (6%, w/v) containing 0.2 mM DTPA). Extracts were then subject to centrifugation at 4 ◦ C at 17,900 × g. Assays were performed in matched quartz cuvettes employing 50 ␮L of extract in 945 ␮L phosphate buffer (1 M K2 HPO4 /KH2 PO4 ; pH 6.1) at 25 ◦ C. Absorbance was read at 265 nm, then 5 ␮L ascorbate oxidase (AO, 1 unit; EC 232852-6; Sigma Aldrich, Poole, UK) added and a reading taken following the complete oxidation of ASC to DHA (<2 min). The redox status of ascorbate was calculated as percentage (%) ASC = (ASC/(ASC + DHA)) × 100. Six measurements on independent fruit were made for each time × treatment combination for pericarp or pulp. Ascorbate concentrations were computed and an extinction coefficient for ascorbic acid of 14 mm−1 cm−1 at 265 nm was used in calculations (Nakano and Asada, 1981). Recovery experiments were performed by spiking samples with known amounts of ASC. 2.9. Antioxidant levels in tomato fruit A chemiluminescent assay (ABEL® hydroxyl radical antioxidant test; Knight Scientific Ltd., Plymouth, UK) was adapted for measuring the capacity of tomato juice to scavenge hydroxyl radicals. Tomato fruit were separated into pericarp and pulp tissue. Hydroxyl scavenging capacity was determined for replicate extracts using a luminometer (LKB-1251, Wallac, Abo, Finland) fitted with an automatic dispenser (LKB-1291 Dispenser, Wallac, Abo, Finland). Assays were performed at room temperature in tubes containing 10 ␮L aliquots of tomato extract in 340 ␮L of Reconstitution and Assay (R&A) buffer, 50 ␮L antioxidant Pholasin® and 500 ␮L of solution-A. In the carousel of the luminometer, samples were made up to 1 mL by adding 100 ␮L of solution-B, using an automated injection system to avoid exposure to light. Each assay was run for 30 s, with peak luminescence attained within 5 s. Six independent measurements (i.e. on independent replicate fruit) were made on pericarp and pulp from independent fruit for each treatment. The reduction in peak luminescence was expressed in d-mannitol equivalents (0–10 mM). Calibration curves were run as part of the assay (y = −2639.9 ln(x) + 5588.5; r2 = 0.81; P < 0.05, where y represents relative light units and x represents d-mannitol concentration (mmol L−1 )). 2.10. Carotenoid composition in tomato fruit Homogenised tomato tissue (ca. 5 g) was added to a roundbottomed flask with 4 g silica gel and subjected to rotary evaporation (IKA RVO5 basic IB, ESSLAB, Essex, UK) under vacuum at a maximum of 35 ◦ C. Methanol (10 mL) was added to capture remaining water and assist in the transfer of lipophilic carotenoids, prior to ultrasonication (Decon FSMinor, Decon Ultrasonics Ltd., UK) and evaporation under vacuum. A mixture of 10 mL tetrahydrofuran:10 mL acetone (1:1) was added, and the solution subjected to ultrasonication. This procedure was repeated three times, until all colour was extracted from the tissue. Extracts were then bulked together and subject to evaporation under vacuum prior to re-suspension in ca. 40 mL ethyl acetate and dH2 O. Employing a separation funnel, the organic

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phase (coloured layer) was collected and dried, using anhydrous Na2 SO4 , prior to evaporating the solvent under vacuum in a fresh flask. The residue was re-extracted in 10 mL ethyl acetate, and stored at −20 ◦ C prior to analysis. Standard precautions were taken throughout to prevent exposure of carotenoids to light, oxygen, acid and heat. Carotenoids were analysed using HPLC (Kontron Instrument Ltd., Bletchley, UK) employing UV detection at 455 nm. Samples (80 ␮L) were injected on to a C18 5 ␮m reverse-phase column (Fison chromatography). The mobile phase (1 mL min−1 ) consisted of solution A (acetonitrile:tetrahydrofuran [ACN:THF] 75:25% (v/v)) and solution B (triethylamine [TEA:H2 O] 0.1% (v/v)), and was introduced starting with 70% solution A and 30% solution B, followed by a linear gradient to 100% of solution A over 30 min, then holding at 100% of solution A for 10 min. Conditions were returned to 70% solution A:30% solution B by linear gradient within 3 min, and held on the washing step for 7 min. Retention times were 18.2, 29.5 and 35.4 min for lutein, lycopene and ␤-carotene, respectively. Calibration curves were prepared in the range 0–186 nmol mL−1 lutein, lycopene and ␤-carotene employing off-the-shelf standards (Sigma Aldrich, Poole, UK) prepared for analysis in the same manner as samples. Spiking of samples revealed no evidence of treatment interferences in the analysis of carotenoids. 2.11. Phenolic content of tomato juice Phenolics were extracted from carotenoid extracts as described by Cano et al. (2003) and assayed using FolinCiocalteu reagent (Sigma Aldrich, Poole, UK). A 3 mL aliquot of the extract prepared for the analysis of carotenoids (see Section 2.10) was dried under nitrogen and washed with 1 mL of methanol (70%, v/v). A 500 ␮L aliquot of the sample was placed in a test tube, and 1 mL of water (Milli-Q) and 2.5 mL of FolinCiocalteu reagent (10-fold diluted; Sigma Aldrich, Poole, UK) added. The sample (4 mL) was incubated in the dark for 1 h, and absorbance at 755 nm recorded (Genesys 10 Vis, ThermoSpectronic, Rochester, USA). The results were expressed in terms of gallic acid equivalents (GAE; Sigma Aldrich, Poole, UK) per 100 g fresh weight of tissue based on standard calibration curves prepared using analytical grade reagent (y = 1.971x + 0.047; r2 = 0.99; P < 0.001, where y represents absorbance at 755 nm and x represents gallic acid equivalent (␮mols 100 g f.wt.)). Total phenolic content was determined according to Dewanto et al. (2002) in extracts prepared from 5 g blended tomato tissue following repeated (four-fold) addition of 2.5 mL 50% (v/v) methanol under ultrasonication (MSE Sonicprep 150 ultrasonic disintegrator; Sanyo, Loughborough, UK). Aliquots were collected and centrifuged for 5 min at 4 ◦ C at 3000 × g (Harrier 18/80, Sanyo Scientific, USA). The supernatant was transferred to a fresh Eppendorf tube and 125 ␮L pipetted into a fresh test tube, to which 1.5 mL of water (Milli-Q), 125 ␮L of FolinCiocalteu reagent (Sigma Aldrich, Poole, UK) and 1.25 mL of 7% (w/v) sodium carbonate were added. The reaction mix (3 mL) was incubated in the dark for 1.5 h, prior to reading the absorbance at 755 nm (Genesys 10 Vis, ThermoSpectronic,

Rochester, USA). Results were expressed in terms of gallic acid equivalents (GAE; Sigma Aldrich, Poole, UK) per 100 g fresh weight of tissue. 2.12. Sensory evaluation For the sensory evaluation, 23 experienced panellists (employed at Paradise Foods Ltd. and Kavli Ltd., Gateshead, UK; aged 25–40 years old) were employed to assess fruit subject to storage for 7 days in ‘clean air’ (CFA) or ozone-enriched air (0.15 ␮mol mol−1 or 1.0 ␮mol mol−1 ). The panel initially assessed treatment preferences, for which individual panellists were challenged with more than one fruit from each sample to ensure representative results. Panellists were subsequently challenged with fresh fruit from each treatment and asked to assess appearance, aroma, sweetness and texture using scales (values of acceptance) with anchor points 1: ‘Poor/unsweet/soft’ and 5: ‘excellent/very sweet/firm’. Scales were converted to percentage values. Individual panellists were presented with three plates (one representing each treatment) containing three whole tomato fruit and three halved-fruit for the sensory analysis, with all tests conducted under the same conditions and without time limit. Panel tests were conducted in isolation in booths in the same room to prevent interchange between panel members. Sensory evaluation tests were performed on fruit exposed to CFA or ozone-enrichment, then packed on-site at the premises of a commercial partner (employing standard practices at Paradise Foods Ltd., Team Valley, Gateshead, Tyne & Wear, U.K.) following sanitation using a spinning disc aspirator. Fruit were packed using a flow-wrapper form-sealing machine. All samples were assessed prior to packaging and following an astringency treatment where packaged product was kept at 1–5 ◦ C for 3 days, then 8/9 ◦ C prior to analysis. Assessments were continued until fruit condition was considered unacceptable in terms of appearance, odour, flavour and/or texture. 2.13. Statistical analysis Statistical analyses were performed using SPSS (SPSS Inc., Chicago, USA) and graphs were produced using Prism v.2.0 (Graph Pad Inc., San Diego, USA) and Microsoft Excel 2002. Data were first tested for normality, then subject to analysis of variance (ANOVA) or repeated measures analysis of variance (RM-ANOVA). Significant differences between mean values were determined using the least significant difference LSD (P = 0.05) following one-way ANOVA. Percentage values (weight loss and sensory attributes) were log-transformed prior to subjecting data to ANOVA or non-parametric tests (Kruskal–Wallis and Mann–Whitney). 3. Results 3.1. Effects of ozone-enrichment on water loss from fruit Fruit weight loss (%) was significantly (P < 0.05) increased in the ‘high’ ozone treatment (1.0 ␮mol mol−1 ); fruit stored at

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Fig. 1. Impacts of ozone-enrichment on firmness of tomato fruit exposed to charcoal-filtered ‘clean air’ (CFA: ) or ozone; 0.05 ␮mol mol−1 ( ) or 1.0 ␮mol mol−1 () for 6 days and then transferred to ‘clean air’ for an additional 6 days. Fruit were maintained throughout at 13 ◦ C and 95% RH. Values represent mean (±S.E.) of measurements made on six independent fruit (two measurements per fruit) per treatment.

1.0 ␮mol mol−1 ozone loosing 0.5% more weight than their counterparts maintained throughout in CFA. However, the effect did not persist when fruit were removed from ozone, so that the weight loss of fruit 6 days after the end of the ozone treatment was not significantly different from that of control fruit. There were no significant effects of ‘low-level’ ozone-enrichment on fruit weight, during or following treatment. 3.2. Effects of ozone-enrichment on fruit firmness The effect of ozone on the firmness of tomato fruit is shown in Fig. 1. Six days storage in an ozone-enriched atmosphere resulted in no change in fruit firmness. However, when treated fruit were subsequently transferred to CFA, fruit previously exposed to ozone remained substantially (P = 0.001) firmer than fruit subjected to traditional storage conditions throughout. 3.3. Impacts of ozone-enrichment on the organic acid composition Both citrate and malate concentrations declined (P < 0.001) as fruit ripened (Fig. 2). Ozone-enrichment resulted in no changes in citrate or malate levels, but citrate levels declined more rapidly (P < 0.05) in fruit previously subjected to low-level ozone-enrichment (0.05 ␮mol mol−1 ) than their counterparts maintained throughout in ‘clean air’. Malate concentrations were unaffected by ozone-enrichment. 3.4. Impacts of ozone-enrichment on soluble sugar composition

Fig. 2. Impacts of ozone-enrichment on citric acid and malic acid content of tomato fruit. Fruit were exposed to ‘clean air’ (CFA: ) or ozone-enriched air; 0.05 ␮mol mol−1 ( ) or 1.0 ␮mol mol−1 () for 6 days and then transferred to ‘clean air’ for an additional 6 days. Fruit maintained throughout at 13 ◦ C and 95% RH. Values represent mean (±S.E.) of measurements made on six independent fruit per treatment.

dominant soluble carbohydrate fractions) remained high in ozone-treated fruit following treatment, so that 6 days after treated fruit were transferred to ‘clean air’ the soluble sugar content of fruit previously subjected to ozone-enrichment was significantly (P < 0.01) greater than that of ‘control’ fruit maintained throughout in CFA. 3.5. Ascorbic acid (vitamin C) content, redox status and antioxidant levels Ozone-enrichment resulted in no significant changes in ascorbic acid (AA; vitamin C) content or AA redox status in tomato pericarp or pulp tissue during or following ozone-enrichment. Fruit pericarp exhibited a transient increase in antioxidative activity (ca. 40%; P < 0.05) after 1-day exposure to ozone, but this effect did not persist (data not presented). Ozone-treatment resulted in no significant changes in antioxidant properties of pulp tissue. 3.6. Effects of ozone-enrichment on carotenoid composition

Soluble sugar levels attained a maximum after six days storage and declined (P < 0.01) thereafter (Fig. 3). Six days storage in an ozone-enriched atmosphere resulted in no significant changes in fruit soluble sugar content. However, soluble sugar levels (especially fructose and glucose content—the pre-

Ozone-enrichment resulted in a two-to-three-fold increase in ␤-carotene, lutein and lycopene content after 1 day of exposure to ozone (Fig. 4). However, the effect did not persist, so after six days exposure to ozone there was no significant

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Fig. 3. Impacts of ozone-enrichment on soluble carbohydrate content of tomato fruit exposed to ‘clean air’ (CFA: ) or ozone-enriched air; 0.05 ␮mol mol−1 ( ) or 1.0 ␮mol mol−1 () for 6 days and then transferred to ‘clean air’ for an additional 6 days. Fruit maintained throughout at 13 ◦ C and 95% RH. Values represent mean (±S.E.) of measurements made on six independent fruit per treatment.

difference in the carotenoid composition between fruit subjected to ozone-enrichment and those maintained throughout in ‘clean air’. 3.7. Effects of ozone-enrichment on phenolic content Ozone-treatment tended to increase fruit (total) phenol content, but the effects did not attain statistical significance (Fig. 5). The phenol content of the organic phase tended to be decreased by ozone-treatment, but effects were not statistically significant. 3.8. Effects of ozone-enrichment on CO2 /H2 O exchange and ethylene production Ozone-enrichment resulted in no significant change in the rate of fruit respiration, transpiration or ethylene production dur-

Fig. 4. Impacts of ozone-enrichment on carotenoid composition of tomato fruit exposed to ‘clean air’ (CFA: ) or ozone-enriched air; 0.05 ␮mol mol−1 ( ) or 1.0 ␮mol mol−1 () for 6 days and then transferred to ‘clean air’ for an additional 6 days. Fruit maintained throughout at 13 ◦ C and 95% RH. Values represent mean (±S.E.) of measurements made on six independent fruit per treatment.

ing and/or following storage in an ozone-enriched atmosphere (data not presented). 3.9. Sensory evaluation Sensory evaluation revealed substantial differences between treatments, with up to 96% of individuals expressing a preference (Table 1). Of those expressing a preference, the majority (70% of jurors) of taste panelists preferred fruit subjected to low-level ozone-enrichment (0.15 ␮mol mol−1 ), and this effect

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Fig. 5. Impacts of ozone-enrichment on total phenolic content and phenolic content of the organic phase; on tomato fruit exposed to ‘clean air’ (CFA: ) or ozone-enriched air; 0.05 ␮mol mol−1 ( ) or 1.0 ␮mol mol−1 () for 6 days and then transferred to ‘clean air’ for an additional 6 days. Fruit maintained throughout at 13 ◦ C and 95% RH. Values represent mean (±S.E.). of measurements made on six independent fruit per treatment. Table 1 Panel evaluation of differences (%) between fresh tomatoes exposed to ‘clean air’ (CFA) or ozone-enriched air (0.15 or 1.0 ␮mol mol−1 ) for 7 days then packed and assessed after 5 days Difference (%)

Yes No No opinion

‘Carousel’ tomato Before packing

Following packing

80 15 5

82 14 4

Fruit maintained throughout at 13 ◦ C and 95% RH. Values represent mean of the opinion recorded by 23 panellists.

was mirrored by trials conducted following astringency tests on packed produce (Fig. 6). Non-parametric analysis of panel judgements revealed tomato fruit to be significantly sweeter (P < 0.001) following storage in an ozone-enriched atmosphere (Fig. 6) and this effect appeared to be sustained, as a similar finding was recorded when taste panellists were challenged with fruit following astringency tests (Fig. 6).

4. Discussion Measurements revealed tomato fruit subject to ozoneenrichment were perceptibly sweeter and retained their firmness in comparison with fruit subject to traditional storage/transit practice. Moreover, taste panel trials revealed an overwhelming preference for ozone-treated fruit during choice tests, based on both appearance and sensory evaluation. Ozone-induced enhancement of fruit quality was not associated with significant effects on weight loss, organic acid, phenolic and vitamin C content, antioxidant status, CO2 /H2 O exchange or ethylene production both during, and following, sustained exposure to ozone-enriched air. Preference for ozone-treated tomato fruit, in which levels of the dominant non-structural carbohydrate fractions (fructose and glucose) were sustained following ozone-treatment, is consistent with the recognised influence of non-structural carbohydrate:organic acid balance on the taste (i.e. degree of sweetness and sourness) and flavour of tomatoes as perceived by trained sensory judges (Hobson and Bedford, 1989; Malundo

Fig. 6. Quantitative analysis of the impacts of ozone-enrichment on sensory attributes of ‘Carousel’ tomato fruit exposed to ‘clean air’ (CFA: --) or ozone-enriched air; 0.15 ␮mol mol−1 (--) or 1.0 ␮mol mol−1 (-䊉-) (A) following exposure for 7 days and (B) following packaging (5 days after packaging). Fruit maintained throughout at 13 ◦ C and 95% RH. Values represent mean of assessments made by 23 panellists per treatment.

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et al., 1995). Findings were consistent with the reported increase in fructose and glucose content reported in strawberry (Kute et al., 1995) and tomato (Aguayo et al., 2006) fruit in response to low-level atmospheric ozone-enrichment. In contrast, no change in organic acid composition was observed in ozone-treated tomato fruit, a finding at odds with the study of Aguayo et al. (2006), but consistent with the reported effects of ozonetreatment on citrus (0.1 ␮mol mol−1 ; Smilanick, 2003), pear and apple (1.7 ␮mol mol−1 ; Skog and Chu, 2001). Ozone-treated fruit retained their firmness when removed from the treatment in comparison with traditionally stored fruit which softened over the same period. This finding was unexpected given the ethylene-insensitivity of full-ripe tomato fruit, but is of considerable significance from a commercial perspective as the observation suggests that ozone-treatment may improve the shelf-life of tomato fruit. Aguayo et al. (2006) reported similar findings. The mechanisms underlying the effects of ozone on fruit firmness remain to be ascertained, but it is known that cell wall matrices, especially pectins, undergo disruption during fruit ripening and it is these modifications that are believed responsible for the decrease in tissue firmness that accompanies ripening (Tucker and Greison, 1987). Pectins that are degraded during ripening, undergo both solubilization and depolymerization (Seymour et al., 1990). The role of ozone–ethylene interactions in mediating the observed effects remains to be ascertained. Ozone reacts rapidly with ethylene, and for those commodities that benefit from ethylene removal during storage (such as bananas, persimmon and top-fruit), ozone is considered a potential tool to extend storage life (i.e. fruit ripening/firmness) with the added advantage of controlling disease proliferation (Jin et al., 1989; Aguayo et al., 2006; Salvador et al., 2006; Tzortzakis et al., 2007). Interestingly, ozone-treatment resulted in no significant change in the antioxidant status of tomato fruit. Similar findings are reported by Kute et al. (1995) who exposed strawberry fruit to ozone concentrations between 0.3 and 0.7 ␮mol mol−1 for up to 1 week. In contrast, Perez et al. (1999) reported a three-fold increase in the ascorbate content of ozone-treated (0.35 ␮mol mol−1 ) strawberry fruit after 3 days of cold storage. Antioxidants prevent the accumulation of potentially damaging reactive oxygen species (ROS), which occur as a product of cellular metabolism and act as secondary messengers in hormone signal transduction (Andrews et al., 2004). Impacts of storage treatments on the antioxidative properties of tomato fruit and tomato products are of concern because tomato fruit are recognised to be particularly rich in several antioxidants (Tonucci et al., 1995; Sharma and Le Maguer, 1996; Beecher, 1998) including vitamin C, carotenoids (especially lycopene; Beecher, 1998; Clinton, 1998) and vitamin A (Scalfi et al., 2000). Indeed, tomato fruit are a key nutritive source of many of these compounds which are important in the prevention of chronic illnesses such as cardiovascular disease and cancer (Jang et al., 1997; Arai et al., 2000). In this regard, it is worthy to note the transient increase in carotenoid content (including lycopene) after 24 h exposure to ozone-enriched air. A similar transient increase in anthocyanin content and colour intensity has been observed in blackberry

and strawberry fruit subject to storage in an ozone-enriched atmosphere (Barth et al., 1995; Perez et al., 1999). Weight loss of tomato fruit was found to be unaffected by low-level ozone-treatment, but was increased by exposure to higher concentrations of ozone. This finding is consistent with effects observed on a range of other fresh commodities including ‘Howes’ cranberries (Norton et al., 1968), ‘Casselman’ plums (Crisoto et al., 1993), ‘Zee Lady” peaches (Palou et al., 2001, 2002), onions (Song et al., 2000) and tomatoes (Shah et al., 2004). It has been suggested that higher levels of ozone may result in damage to the cuticle and/or epidermal tissues (Palou et al., 2002). Some researchers (e.g. Crisoto et al., 1993) attribute the observed effects to reactions between ozone and surface wax constituents, but this seems unlikely as epicuticular waxes have been shown not to react with ozone per se (Barnes and Brown, 1990). Low-level atmospheric ozone-enrichment during cold storage is capable of controlling not only disease development and proliferation (Aguayo et al., 2006; Tzortzakis et al., 2007), but also maintains fruit quality in terms of taste and firmness. Panel trials revealed an overwhelming preference for ozone-treated fruit during choice tests, based on both appearance and sensory evaluation. Preference for ozone-treated fruit was not associated with significant changes in fruit weight loss, organic acid and vitamin C content, antioxidant status, phenolic content, CO2 /H2 O exchange or ethylene production, during or following sustained exposure to ozone-enriched air. It is worthy of note that the lowest-level of ozone adopted in the present study falls below levels likely to damage store components as well as the occupational exposure limit in many European countries, and associated member states, for the protection of human health. Trials instigated with a variety of European partners, employing a range of produce packed and stored under commercial conditions, suggest laboratory-identified benefits associated with low-level ozone-treatment maybe transferable to the commercial environment. Acknowledgements This study was supported by the National Research Foundation of Greece (IKY) via the award of a scholarship to NT. We thank Dr. E. Mutch and Dr. E. Okello for analytical assistance with carotenoid and ethylene measurements, also M. Davenport and A. Smith of Paradise Foods Ltd. for assistance with panel trials. All fruit were kindly provided by MBMG Produce Ltd. We are indebted to BioFresh (http://www.bio-fresh.co.uk) for supporting commercial trials of the technology, covered (in the U.K.) by a trials license from the U.K. Pesticide Safety Directorate (COP 2005/00578 PP). References Adams, P., Thomas, J.G., Vernon, D.M., Bohnert, H.J., Jensen, R.G., 1992. Distinct cellular and organismic responses to salt stress. Plant Cell Physiol. 33, 1215–1223. Aguayo, E., Escalona, V.H., Art´es, F., 2006. Effect of cyclic exposure to ozone gas on physicochemical, sensorial and microbial quality of whole and sliced tomatoes. Postharvest Biol. Technol. 39, 169–177.

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