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Eur. J. Hortic. Sci. 81(4), 197–203 | ISSN 1611-4426 print, 1611-4434 online | http://dx.doi.org/10.17660/eJHS.2016/81.4.2 | © ISHS 2016

High CO2-modified atmosphere to preserve sensory and nutritional quality of organic table grape (cv. ‘Italia’) during storage and shelf-life M. Cefola and B. PaceInstitute of Sciences of Food Production, CNR National Research Council of Italy, Bari, Italy

Original article

fruit and causes decay to rachis and berries if used excessive-ly (Teles et al., 2014). Moreover, SO2 residues are dangerous to people allergic to sulphites; 10 µL L-1 is the maximum tol-erance to sulphite residues in fruit, established by the US Food and Drug Administration (Guillén et al., 2007), while the European Union has forbidden the use of SO2 (Directive 95/2/CE, 1995). Nowadays, some alternative techniques to SO2 seem to be useful to prevent decay in grapes, such as controlled atmospheres (CA) (Eris et al., 1993; Kader et al., 1997; Basiouny, 1998; Crisosto et al., 2002; Retamales et al., 2003), modified atmosphere (MA) packaging (Artés-Hernan-dez et al., 2000; Yamashita et al., 2000; Artés-Hernandez et al., 2006; Valero et al., 2006) and the use of O3 (Artés-Her-nandez et al., 2002; Palou et al., 2002; Feliziani et al., 1999). Controlled atmospheres (CA), with low O2 and/or high CO2, contributed to the maintenance of storage quality and the ex-tension of shelf life of fruits and vegetables (Beaudry, 1999). The effects of CA conditions with high CO2 on the physiology and quality of many table grape varieties have been evaluat-ed (Basiouny, 1998; Crisosto et al., 2002; Artés-Hernandez et al., 2004). These results have shown that, in table grapes, the application of CA could retard senescence, reduce stem and berry respiration, limit rachis browning and decay and pre-serve berry firmness. On the other hand, the use of high CO2 concentrations may contribute to off-flavour development and rachis browning (Ahumada et al., 1996; Crisosto et al., 2002).

German Society for Horticultural Science

SummaryTable grape is a non-climacteric fruit subject to

serious water and quality loss during postharvest handling. Gentle handling, careful bunch cleaning, fast cooling, low temperature, and sulfur dioxide (SO2) pad application during storage are generally used to improve storage. In this study, the effect of cold storage in modified atmosphere (MA) in different CO2 concentrations (0-20%) on the quality evaluated after a shelf-life period at ambient temperature, was studied. Organic table grape bunches (cv. ‘Italia’), were packaged in MA bags and stored at 2°C (± 1.0) for 14 days, using MA with 0% (air), 10% and 20% initial CO2 concentrations. Unpacked samples were used as control. The gas composition inside packages was periodically measured. After 7 and 14 days, all packages were opened and transferred in air at 20°C for 3 days: sensory, physical and chemical-nutritional parameters were measured. A significant effect of high CO2 atmosphere on delaying visual quality and decay was shown. The best results in terms of preservation of sensory as well as nutritional quality were obtained using a 10% CO2 as initial concentration. Actually, this treatment was able to ensure a suitable atmosphere composition during all storage period: the O2 and CO2 concentrations after 14 days of MA storage, were found to be about 7% and 15%, respectively, i.e., a CO2

level enough to control the decay development and an O2 concentration sufficiently far from the anoxia threshold, as detected by acetaldehyde and ethanol measurement.

Keywordsacetaldehyde, ethanol, high carbon dioxide, total phenols, visual quality, Vitis vinifera L.

Significance of this studyWhat is already known on this subject?• MA preserves table grape quality; information about

the effect of cold storage in MA followed by a period at ambient temperature in air on sensory-nutritional traits are needed.

What are the new findings?• It was found the optimal initial atmosphere

composition (20% O2 plus 10% CO2) to be used during cold storage or transport in order to preserve sensory as well as nutritional quality of organic table grape during shelf-life.

What is the expected impact on horticulture?• Results are of practical application to improve the

storage and transport of organic table grape of a cultivar highly appreciated and commercialized in many European countries.

Introduction Organic table grape is highly perishable and its quality

deteriorates rapidly after harvest, due mainly to water loss, which cause stem browning and sensitivity to microbial de-cay. Low temperature storage is one of the most effective technologies to extend the postharvest life of fruit and veg-etables. However, in table grapes low temperature storage life is limited by high sensitivity to fungal attack, mainly from Botrytis cinerea. The most common method to control decay during cold storage of bunches is fumigation with sulfur di-oxide (SO2) (Droby et al., 2004; Carter et al., 2015). However, SO2 is highly corrosive to metals, injurious to most other fresh

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Cefola and Pace | High CO2-modified atmosphere for organic table grape

Among the packaging technologies commercially avail-able for fresh produce, there are modified atmosphere-pack-aging systems for small and large quantities of produce (Sandhya, 2010). In these systems the pallet box, which con-tain the fresh fruits or vegetables, is packaged in a big plastic bag, in which it is possible to create a modified atmosphere. Commonly these systems are used in cold rooms or mainly during transport in refrigerated container and are opened before the retail period.

The aim of this paper is to select the proper modified at-mosphere composition to cold store table grape with the aim to obtain a product with a good quality for the retail period (shelf-life). Thus organic table grapes were stored in differ-ent modified atmosphere concentrations by simulating, in small packages, the same conditions of the modified atmo-sphere-packaging systems. Table grape quality traits were evaluated after a period at low temperature in MA followed by a period at ambient temperature in air, reproducing a cold storage or transport period in refrigerated room or contain-er, followed by a shelf-life period during retail sales.

Materials and methods

Fruit material and experimental set-upOrganically-grown table grapes (Vitis vinifera L.) cv.

‘Italia’, were harvested from a local farm and transported within 1 h to the postharvest laboratory of Institute of Sci-ences of Food Production. Harvested bunches were selected on the basis of absence of evident defects or diseases, and were randomly distributed into four clusters, representative of each packaging treatment. Four packaging treatments were applied as reported in Table 1. For each treatment 10 packages (5 replicates × 2 storage times) were prepared, by placing about 1 kg of table grape inside polyethylene tere-phthalate (PET, model CL1/135, Carton Pack, Italy) trays, sealed (model Boxer 50, Lavezzini Vacuum Packaging Sys-tem, Italy) in 30 × 40 cm polyamide/polyethylene (PA/PE) plastic bags (pCO2 40 cm3 m2 d-1 bar-1, 140 µm thick, Orved, Italy) using the following initial percentage concentrations of O2/CO2: 20/0.03 (passive modified atmosphere - PMA), 20/10 (MA-10%), and 20/20 (MA-20%). Samples in un-sealed bags were used as control. The following periods for cold storage (CS) and shelf life (SL) were applied: 7 and 14 days at 2°C (± 1.0), simulating the cold storage or transport period, followed by a SL of 3 days at 20°C as a retail sale peri-od. Thus, at the end of each CS period (7 or 14 days) packages were opened and stored in air for 3 days. Samples were ana-lyzed at harvest, after 7 days of CS plus 3 days SL and after 14 days CS plus 3 days SL for changes in sensory (visual quality and decay), physical (total soluble solids, pH, color, firmness and weight loss) and chemical-nutritional (antioxidant ac-tivity, total phenolic content, total sugars, titratable acidity, acetaldehyde and ethanol content) parameters as below de-scribed.

Atmosphere composition analysis inside packagesAtmosphere analysis in PMA, MA-10% and MA-20%

packages was carried out initially (just after packaging) and periodically during CS by using a gas analyzer (CheckPoint, PBI Dansensor, Ringsted, Denmark).

Sensory and physical analysis Visual quality was evaluated by a group of ten trained

people, on a subjective 5 to 1 scale, with 5 = excellent, no defects; 4 = very good, minor defects; 3 = fair, moderate de-fects; 2 = poor, major defects; 1 = inedible. A score of 3 was considered to be the limit of marketability, while a score of 2 the limit of edibility. Decay was scored on 1 to 5 scale where 1 = no decay; 2 = slight decay, but product saleable, <2% af-fected; 3 = moderate decay, product useable but not saleable, <5% affected; 4 = moderately severe, <15% affected; and 5 = severe, unusable (Cefola et al., 2011).

Total soluble solid (TSS) content expressed in °Brix, was measured using a refract-meter (model DBR35, XS Instru-ments, Italy) on a liquid extract obtained by whisking in a blender (1 min; 14,000 rev. min-1) 10 berries from each rep-licate and then filtering the juice.

The pH of the berries juice was measured with a pH-me-ter. Colour parameters (L*, a* and b*) were measured on 3 random points on peel surface of 10 berries for each repli-cate with a colorimeter (CR-400, Konica Minolta, Osaka, Ja-pan) in the reflectance mode and in the CIE L* a* b* colour scale. The colorimeter was calibrated with a standard refer-ence having values of L*, a* and b* corresponding to 97.55, 1.32 and 1.41, respectively.

Berry firmness was measured using a firmness tester (ZwickLine Z0.5; Zwick/Roell, Ulm, Germany) as the relative deformation of the berry fruits up to a 10 N load (deforma-tion method), by using a plate of 100 mm of diameter, and was expressed in percentage (Cefola et al., 2011).

Samples were weighed individually and weight loss was expressed as g per 100 g of fresh weight (fw).

Chemical-nutritional analysis The following extraction procedure was used for both

antioxidant activity and total phenols determinations. Five grams of berries tissues, without seeds, were homogenised in methanol: water solution (80:20) for 1 min, and then centrifuged at 5°C and 6,440× g for 5 min. Antioxidant as-say was performed following the procedure described by Brand-William et al. (1995) with minor modifications. The diluted sample, 50 μL, was pipetted into 0.95 mL of diphen-ylpicrylhydrazyl (DPPH) solution to initiate the reaction. The absorbance was read after 40 min at 515 nm. Trolox was used as a standard and the antioxidant activity was report-ed in milligrams of Trolox equivalents per 100 grams of fw. Total phenols were determined according to the method of Singleton et al. (1965). Each extract (100 μL) was mixed with 1.58 mL water, 100 μL of Folin-Ciocalteu reagent and 300 μL

Table 1. Packaging treatments, abbreviations, and percentage of initial oxygen (O2) and carbon dioxide (CO2) inside cv. ‘Italia’ organic table grape bags.

Treatments Abbreviations Initial O2/CO2 (%) RemarksControl CTRL 20.0/0.03 Unsealed open bagsPassive modified atmosphere PMA 20.0/0.03

Sealed bagsModified atmosphere 10% CO2 MA-10% 20.0/10.0Modified atmosphere 20% CO2 MA-20% 20.0/20.0

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of sodium carbonate solution (200 g L-1). After the solution had been standing for two hours, the absorbance was read at 765 nm against a blank using a spectrophotometer (UV-1800, Shimadzu, Kyoto, Japan). The content of total phenols was calculated on the basis of the calibration curve of caffeic acid and was expressed as milligrams of caffeic acid per 100 grams of fw.

Total sugars content was analyzed by a phenol-sulfu-ric colorimetric method (Buysse and Merckx, 1993). 5 g of chopped berry were homogenized with 15 mL of 95% etha-nol for 2 min and centrifuged for 5 min at 6,440 xg. The ex-tract proper diluted was used for the determination. Color development was determined at 490 nm using a spectropho-tometer and glucose as standard.

Titratable acidity was determined by potentiometric ti-tration (Technotrate, Kartell, Noviglio, Italy) with 0.1 mol L-1 NaOH to pH 8.1 using 10 mL of juice. Results were expressed as g tartaric acid per 100 g fw.

For acetaldehyde and ethanol analysis, 5 gram of chopped berry sample were put into 22 mL glass test tube, sealed with rubber stopper and stored at -20°C until analysis (Mateos et al., 1993). After 1 hour incubation at 65°C water bath, a 1 mL headspace gas sample was taken and injected into gas chromatograph (Varian model CP 3800, Palo Alto, CA, USA) equipped with a capillary column Phenomenex Zebron ZB-WAX Plus (Torrance, CA, USA), 30 m length, 0.32 mm internal diameter, 0.50 µm film thickness and a flame ionization de-tector. A series of ethanol and acetaldehyde standard solu-tions at different concentration was used to make calibration curves.

Statistical analysisA one way ANOVA for fresh product and packaging treat-

ment (Table 1) at each sampling day (7 days of CS plus 3 days SL and 14 days CS plus 3 days SL) was performed. Mean val-ues for modified atmosphere treatments were separated us-ing the Student-Newman-Keuls (SNK) test.

Results and discussion

Atmosphere composition analysis inside packagesDuring the cold storage (CS) inside packages, an O2 re-

duction accompanied by an increase in CO2 was detected

due to the table grape respiration activity (Figure 1). During the first 3 days, O2 and CO2 concentration inside all packages changed significantly due to the aerobic metabolism of the table grape. Subsequently, the values remained almost stable until the 9th day; after that a decrease in O2 and an increase in CO2 was measured, probably associated to the enhancement of respiration activity (Figure 1). After 14 days of CS the O2/CO2 concentration (%) in PMA, MA-10% and MA-20% was about 10/8, 7/15 and 3/26 respectively (Figure 1). During all storage, in samples stored in PMA the highest CO2 accu-mulation was detected (from 0 to 8%). This, probably was related to the well-known inhibitory action performed by the high CO2 (Kader, 2002) as occurred in MA-10% and MA-20% packages.

Changes in sensory and physical parametersThe CS did not affect the visual quality, moreover, no de-

cay was scored on table grapes stored in all treatments after the CS (data not shown). On the other hand, the storage in different CO2 concentrations significantly affected the visual quality of table grape evaluated after 7 and 14 days of CS plus 3 days of SL (Figure 2, Table 2). In particular, samples stored in MA-10% and MA-20% showed a visual quality similar to fresh harvested samples after 7 days of CS plus 3 days of SL and resulted above the limit of marketability at the end of the trial (14 d CS + 3 d SL). Whereas, samples stored in PMA and unsealed bags (control) showed a more evident reduction in visual quality at each sampling day, resulting respectively at the marketable limit or not marketable just after 14 days of CS plus 3 days of SL. As for decay, at the end of the trial, the same samples which reported the lowest visual quality scores (PMA and control) showed respectively a moderate severe and severe deterioration; whereas samples stored in MA-10% and MA-20% showed a slight and moderate decay respectively (Table 2). These results suggested an effect of high CO2 on decay control as previously reported by different authors (Retamales et al., 2003; Valero et al., 2006). Similar-ly to our results, Crisosto et al. (2002) showed that on ‘Red Globe’ cultivar, 10% CO2 combined with 3, 6 or 12% O2 limits Botrytis decay development during 12 weeks of cold storage.

No significant changes in colour parameters among treatments were detected during the CS and in SL (data not shown). Our results agree with those reported by

Figure 1. Oxygen (A) and carbon dioxide (B) gas concentration (%) inside packages during storage time at 2°C (± 1°C). Mean ± standard deviation. PMA: Passive modified atmosphere; MA-10%: Modified atmosphere 10% CO2; MA-20%: Modified atmosphere 20% CO2.

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Storage time (days)

PMAMA-10%MA-20%

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2 (%

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Storage time (days)

A B

FIGURE 1. Oxygen (A) and carbon dioxide (B) gas concentration (%) inside packages during storage time at 2°C (±1°C). Mean ± standard deviation. PMA: Passive modified atmosphere; MA-10%: Modified atmosphere 10% CO2; MA-20%: Modified atmosphere 20% CO2.

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FIGURE 1. Oxygen (A) and carbon dioxide (B) gas concentration (%) inside packages during storage time at 2°C (±1°C). Mean ± standard deviation. PMA: Passive modified atmosphere; MA-10%: Modified atmosphere 10% CO2; MA-20%: Modified atmosphere 20% CO2.

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Artés-Hernández et al. (2006) in ‘Superior seedless’ cultivar stored for 7 days at 0°C followed by 4 days at 8°C plus 2 days at 20°C under modified atmosphere packaging and in ‘Car-dinal’ cultivar stored for 33 days at 0°C previously treated under a gas mixture containing 20/20/60 O2/CO2/N2 for 3 days (Sanchez-Ballesta et al., 2006).

In addition, no significant changes in TSS, pH and rela-tive deformation were measured in all samples during CS and SL in comparison with data measured at harvest (°Brix: 13.7 ±0.3; pH: 3.6 ±0.12; berry deformation: 15.9 ±0.03%), these data are in agreement with information reported by Cefola et al. (2011) on table grape. Similarly, other authors

10

FIGURE 2. Effect of packaging treatments on organic table grape cv. ‘Italia’ after 14 days of cold storage at 2°C and 3 days of shelf-life at 20°C respect to fresh samples. Control:sample stored in unsealed open bags; PMA: Passive Modified Atmosphere; MA-10%: Modified Atmosphere, 10% CO2; MA-20%: Modified Atmosphere, 20% CO2 (Table 1).

Figure 2. Effect of packaging treatments on organic table grape cv. ‘Italia’ after 14 days of cold storage at 2°C and 3 days of shelf-life at 20°C respect to fresh samples. Control: sample stored in unsealed open bags; PMA: Passive Modified Atmosphere; MA-10%: Modified Atmosphere, 10% CO2; MA-20%: Modified Atmosphere, 20% CO2 (Table 1).

Table 2. Effect of packaging treatments on visual quality and decay of organic table grape (cv. ‘Italia’) evaluated after 7 days of cold storage (CS) at 2°C and 3 days of shelf-life (SL) at 20°C, or after 14 days of CS and 3 days of SL.

Visual quality (5-1) Decay

(1-5)At harvest 5.0 a 5.0 a 1.0 1.0 dTreatments 7CS + 3SL 14CS + 3SL 7CS + 3SL 14CS + 3SL Control 4.3 b 2.3 c 1.7 4.7 aPMA 4.7 ab 3.0 bc 1.3 3.7 abMA-10% 4.8 a 3.8 b 1.3 2.3 cMA-20% 5.0 a 3.5 b 1.0 3.0 bcSignificance * * ns **

ns, not significant; * P≤0.05; ** P≤0.01.Visual quality score: 5 = excellent, no defects, 4 = very good, minor defects, 3 = fair, moderate defects, 2 = poor, major defects, 1 = inedible. Decay score: 1 = no decay, 2 = slight decay, but product salable, <2% affected; 3 = moderate decay, product useable but not salable, <5% affected, 4 = moderately severe, <15% affected and 5 = severe, unusable.

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have shown low variations in these attributes during the CS in other non-climacteric fruits (Cantwell, 1995). As for weight loss, no significant changes among treatments were measured after the CS, whereas a slight increase (about 2%) were recorded after 3 days at 20°C in all treatments.

Changes in chemical-nutritional parametersThe antioxidant activity remained almost unchanged af-

ter each sampling day in all samples stored in PMA respect to fresh samples, whereas a significant reduction respect to fresh samples in MA-20% and in control table grapes was measured, mainly at the end of the trial. Inversely, a signif-icant increase in antioxidant activity in MA-10% was reg-istered after 14 d of CS plus 3 d of SL (Table 3). Similarly results were obtained by (Sanchez-Ballesta et al., 2007) on table grape treated with 20% of CO2, suggesting the ability of MAP with high CO2 to prevent the formation of ROS (Reactive oxygen species) rather than their inactivation once formed (Romero et al., 2008). Total phenols increased in all treat-ments, respect to fresh samples, at each sampling day; in par-ticular the highest values were detected in MA-10% samples. The increase in total phenols is probably due to the stress associated to the storage condition applied. In agreement, Siriphanich and Kader (1985), and Ke and Saltveit (1989) observed that under high CO2 atmospheres phenylalanine ammonia lyase (PAL) activity was induced and the transfer in air (bags opening), was associated with a rapid increase in soluble phenolic content.

The total sugar content remained almost the same in all treatments (Table 3). This last result is in agreement with data reported on TSS, and is associated to the no-climacter-ic postharvest physiology of table grape as above discussed. Related to this result, no changes in titratable acidity was registered during CS and SL in all treatments: mean values similar to initial data (0.36 ±0.01 g tartaric acid 100 g-1 fw) were measured at each sampling day.

Packaging treatments affected significantly the acetalde-hyde and ethanol concentration during storage. In particular acetaldehyde and ethanol significantly accumulated in MA-20% samples, mainly at the end of the storage when a MA composition of about 3% O2 and 26% CO2 was established (Figure 1, Table 4). In addition ethanol accumulated, al-though in lower concentration, also in control and PMA sam-ples at the end of the trial. Only samples packaged in 10% CO2 showed the lowest accumulation of acetaldehyde and ethanol (Table 4), especially at the end of storage and shelf life. The postharvest storage of table grape in high CO2 and/or low O2 may be responsible for anaerobic fermentation, with acetaldehyde and ethanol accumulation (Kader, 1987; Ke et al., 1993) and off-flavour development (Mattheis and Fellman, 2000). Several authors observed a good correlation between the accumulation of ethanol and the development of off-flavour in several products stored in low O2 and/or high CO2 (Ke et al., 1991). Similarly, Crisosto et al. (2002) showed that in ‘Red Globe’ table grapes, CO2 concentration above 10% accelerated stem browning and ‘off-flavor’ devel-opment.

Table 3. Effect of packaging treatments on antioxidant activity, total phenols and total sugar content of organic table grape (cv. ‘Italia’) evaluated after 7 days of cold storage (CS) at 2°C and 3 days of shelf-life (SL) at 20°C, or 14 days of CS and 3 days of SL.

Antioxidant activity (mg Trolox 100 g-1 fw) Total phenols

(mg caffeic acid 100 g-1 fw) Total sugars content (g 100 g-1 fw)

At harvest 68.0 a 68.0 b 6.5 c 6.5 d 12.0 b 12.0 bTreatments 7CS + 3SL 14CS + 3SL 7CS + 3SL 14CS + 3SL 7CS + 3SL 14CS + 3SL Control 56.0 ab 50.0 c 10.5 b 10.6 b 12.0 b 12.1 bPMA 63.0 a 60.0 bc 17.1 a 19.4 a 11.8 b 11.7 bMA-10% 67.0 a 88.0 a 15.6 a 26.4 a 14.4 a 12.2 bMA-20% 45.0 b 52.0 c 11.8 b 17.1 b 10.1 c 13.2 aSignificance * ** *** **** *** ***

* P≤0.05; ** P≤0.01;*** P≤0.001; **** P≤0.0001.

Table 4. Effect of packaging treatments on acetaldehyde and ethanol concentrations of organic table grape (cv. ‘Italia’) evaluated after 7 days of cold storage (CS) at 2°C and 3 days of shelf-life (SL) at 20°C, or after 14 days of CS and 3 days of SL.

Acetaldehyde (nl g-1 fw)

Ethanol (nl g-1 fw)

At harvest 1.17 c 1.17 b 15.30 b 15.30 dTreatments 7CS + 3SL 14CS + 3SL 7CS + 3SL 14CS + 3SLControl 0.92 bc 2.25 b 78.97 b 211.79 bPMA 5.63 a 2.26 b 24.48 b 137.29 bcMA-10% 0.67 bc 1.74 b 43.41 b 54.70 cdMA-20% 3.65 ab 12.5 a 303.23 a 1048.60 aSignificance * *** * ****

* P≤0.05; *** P≤0.001; **** P≤0.0001.

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Cefola and Pace | High CO2-modified atmosphere for organic table grape

ConclusionsResults showed a significant effect of high CO2-modified

atmosphere on preserving visual quality and delaying decay of organic table grape. The best results in terms of preserva-tion of sensory as well as nutritional quality were obtained using as initial atmosphere concentration 20% O2 plus 10% CO2. Actually, this treatment was able to ensure a suitable at-mosphere composition during the whole storage period; the O2 and CO2 concentrations after 14 days of MA storage were found to be about 7% and 15%, respectively, i.e., a CO2 level enough to control the decay development and an O2 concen-tration sufficiently far from the anoxia, as showed by mea-suring acetaldehyde and ethanol. On the other hand, the gas initial mixture with 20% CO2 seemed also appropriate to im-prove the shelf-life of table grape, even if in these conditions a fermentative metabolism may occur, leading to significant higher contents in ethanol and acetaldehyde, with a possi-ble negative influence on sensorial quality. Future research will be aimed to find for each table grape cultivar the opti-mal initial CO2 concentration able to guarantee sensory and nutritional quality during CS and SL, without fermentative phenomena.

AcknowledgmentsThis work was supported by the following research

projects: Sviluppo di prodotti alimentari innovativi mediante soluzioni biotecnologiche, impiantistiche e tecnologiche (PROINNO_BIT – PON02_00657_00186_3417037/1); Apulian Regional Project – Container innovativo isotermico intermodale equipaggiato con atmosfera controllata per il trasporto di prodotti ortofrutticoli freschi (CONTINNOVA - VFQA3D0). The authors thank Vito Linsalata for the analysis of acetaldehyde and ethanol.

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Received: Apr. 4, 2016Accepted: May 25, 2016

Addresses of authors: Maria Cefola and Bernardo Pace*Institute of Sciences of Food Production, CNR National Research Council of Italy, via Amendola 122/O, 70125 Bari, ItalyPhone: 0039-0805929304, Fax: 0039-0805929373* Corresponding author; E-mail: bernardo.pace@ispa.cnr.it