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<a href=Food Chemistry 135 (2012) 1555–1562 Contents lists available at SciVerse ScienceDirect Food Chemistry journal homepage: www.elsevier .com/locate/foodchem Analytical Methods Detection of postharvest changes of ascorbic acid in fresh-cut melon, kiwi, and pineapple, by using a low cost telemetric system Antonio Barberis , Angela Fadda , Mario Schirra , GianFranco Bazzu , Pier Andrea Serra Institute of Sciences of Food Production, National Research Council, Traversa La Crucca 3, Regione Baldinca, 07040 Li Punti, Sassari, Italy Department of Clinical and Experimental Medicine, University of Sassari, v.le San Pietro 43, 07100 Sassari, Italy article info Article history: Received 8 February 2012 Received in revised form 3 May 2012 Accepted 31 May 2012 Available online 9 June 2012 Keywords: Ascorbic acid Fresh-cut fruits Electrochemical sensor Telemetry abstract The present paper deals with a novel telemetric device combined with a carbon amperometric sensor sys- tem to determine postharvest changes of ascorbic acid (AA) in fresh-cut fruits, without displacing prod- ucts out of the storage rooms. The investigation was performed on kiwi, pineapple and melon, subjected to minimal processing, packaging, cold storage, and simulated shelf life. Results demonstrated that AA content of fresh-cut fruits of all species declines differently during storage. Cold storage notably reduced the degradation rate of AA in comparison with samples stored at 20 C. The cold-chain interruption resulted in a sharp AA content reduction when the optimal storage condition was not rapidly replaced. Unpredicted results showed a high activity of oxidative enzymes, which prevented AA detection in melon samples. Our sensor system allowed us to demonstrate that both ascorbate peroxidase and ascor- bate oxidase affected the oxidative stability and the nutritional quality of fresh cut melon fruits. 2012 Elsevier Ltd. All rights reserved. 1. Introduction Fresh and minimally processed (fresh-cut) fruits and vegetables are significant sources of dietary Vitamin C (ascorbic acid, AA). A dose of 100–200 mg/day of AA has been highly recommended, since it helps to prevent many human diseases linked to oxidative stress, including cancer, diabetes mellitus, stroke ( Yokoyama et al., 2000 ) and Parkinson’s disease ( Serra, Pluchino, Marchetti, Desole, & Miele, 2008 ). AA amount in horticultural crops depends on species, variety, cultural practice, fruit age, climate and post-harvest handling. Its content usually declines during fruit storage or processing ( Lee & Kader, 2000; Schirra et al., 2008 ). Two enzymes are principally in- volved in AA oxidative metabolism, ascorbate oxidase (AO) and ascorbate peroxidase (APX). AO catalyses AA oxidation mainly dur- ing tissue growth, while APX is more related to the modification of AA content during ripening, storage or wounding, using AA to re- move the H O formed by oxidative stress ( Diallinas et al., 1997; Gomez & Lajolo, 2008 ). Due to its antioxidant activity, AA plays a crucial role in many metabolic pathways, which have a direct impact on the oxidative stability of fruits ( Lurie, 2003 ). The loss of AA is the main limiting factor of nutritional quality ( Ahvenainen, 1996 ), therefore it is re- garded as a quality indicator for fresh-cut fruits and vegetables. A large number of methods have been applied for AA determina- ⇑ Corresponding author. E-mail address: antonio.barberis@ispa.cnr.it (A. Barberis). 0308-8146/$ - see front matter 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2012.05.121 tion ( Arya, Mahajan, & Jain, 2000; Rahman Khan, Rahman, Islam, & Begum, 2006 ). Among them, titrimetric and chromatographic methods are the most commonly used since they provide a precise and accurate assessment of AA, however they can be applied only in the laboratory. In recent years, the application of sensors and biosensors tech- nology to food industry, as a powerful and low cost alternative to conventional analytical techniques, has been widely discussed by several authors ( Terry, White, & Tigwell, 2005; Velasco-Garcia & Mottram, 2003 ). Amperometric devices are the most commonly re- ported class of sensors since they are cheap and based upon the use of disposable electrodes; they are highly specific and do not need extensive sample pre-treatment or large sample volumes. Teleme- try is a well-established technique for real-time monitoring of lev- els of several biological molecules. Sophisticated telemetry devices have been developed to analyse neurochemical data and success- fully used in conjunction with micro-biosensors for the in vivo measurement of brain AA, O , glucose and lactate ( Bazzu et al., 2009; Calia et al., 2009 ). More recently, an ultralow-cost telemetric system, combined with a carbon amperometric sensor, for a rapid electrochemical detection of AA in fresh orange juice was devel- oped ( Barberis et al., 2010 ). The system, built with simple and inex- pensive components showed highly reproducible analytical performances in accordance with those obtained with traditional reference methods, thus suggesting that it could be used for a rapid monitoring of AA on several biological substrates. The aim of this work was to determine postharvest changes of ascorbic acid in kiwi, pineapple, and melon, subjected to minimal " id="pdf-obj-0-5" src="pdf-obj-0-5.jpg">

Contents lists available at SciVerse ScienceDirect

Food Chemistry

<a href=Food Chemistry 135 (2012) 1555–1562 Contents lists available at SciVerse ScienceDirect Food Chemistry journal homepage: www.elsevier .com/locate/foodchem Analytical Methods Detection of postharvest changes of ascorbic acid in fresh-cut melon, kiwi, and pineapple, by using a low cost telemetric system Antonio Barberis , Angela Fadda , Mario Schirra , GianFranco Bazzu , Pier Andrea Serra Institute of Sciences of Food Production, National Research Council, Traversa La Crucca 3, Regione Baldinca, 07040 Li Punti, Sassari, Italy Department of Clinical and Experimental Medicine, University of Sassari, v.le San Pietro 43, 07100 Sassari, Italy article info Article history: Received 8 February 2012 Received in revised form 3 May 2012 Accepted 31 May 2012 Available online 9 June 2012 Keywords: Ascorbic acid Fresh-cut fruits Electrochemical sensor Telemetry abstract The present paper deals with a novel telemetric device combined with a carbon amperometric sensor sys- tem to determine postharvest changes of ascorbic acid (AA) in fresh-cut fruits, without displacing prod- ucts out of the storage rooms. The investigation was performed on kiwi, pineapple and melon, subjected to minimal processing, packaging, cold storage, and simulated shelf life. Results demonstrated that AA content of fresh-cut fruits of all species declines differently during storage. Cold storage notably reduced the degradation rate of AA in comparison with samples stored at 20 C. The cold-chain interruption resulted in a sharp AA content reduction when the optimal storage condition was not rapidly replaced. Unpredicted results showed a high activity of oxidative enzymes, which prevented AA detection in melon samples. Our sensor system allowed us to demonstrate that both ascorbate peroxidase and ascor- bate oxidase affected the oxidative stability and the nutritional quality of fresh cut melon fruits. 2012 Elsevier Ltd. All rights reserved. 1. Introduction Fresh and minimally processed (fresh-cut) fruits and vegetables are significant sources of dietary Vitamin C (ascorbic acid, AA). A dose of 100–200 mg/day of AA has been highly recommended, since it helps to prevent many human diseases linked to oxidative stress, including cancer, diabetes mellitus, stroke ( Yokoyama et al., 2000 ) and Parkinson’s disease ( Serra, Pluchino, Marchetti, Desole, & Miele, 2008 ). AA amount in horticultural crops depends on species, variety, cultural practice, fruit age, climate and post-harvest handling. Its content usually declines during fruit storage or processing ( Lee & Kader, 2000; Schirra et al., 2008 ). Two enzymes are principally in- volved in AA oxidative metabolism, ascorbate oxidase (AO) and ascorbate peroxidase (APX). AO catalyses AA oxidation mainly dur- ing tissue growth, while APX is more related to the modification of AA content during ripening, storage or wounding, using AA to re- move the H O formed by oxidative stress ( Diallinas et al., 1997; Gomez & Lajolo, 2008 ). Due to its antioxidant activity, AA plays a crucial role in many metabolic pathways, which have a direct impact on the oxidative stability of fruits ( Lurie, 2003 ). The loss of AA is the main limiting factor of nutritional quality ( Ahvenainen, 1996 ), therefore it is re- garded as a quality indicator for fresh-cut fruits and vegetables. A large number of methods have been applied for AA determina- ⇑ Corresponding author. E-mail address: antonio.barberis@ispa.cnr.it (A. Barberis). 0308-8146/$ - see front matter 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2012.05.121 tion ( Arya, Mahajan, & Jain, 2000; Rahman Khan, Rahman, Islam, & Begum, 2006 ). Among them, titrimetric and chromatographic methods are the most commonly used since they provide a precise and accurate assessment of AA, however they can be applied only in the laboratory. In recent years, the application of sensors and biosensors tech- nology to food industry, as a powerful and low cost alternative to conventional analytical techniques, has been widely discussed by several authors ( Terry, White, & Tigwell, 2005; Velasco-Garcia & Mottram, 2003 ). Amperometric devices are the most commonly re- ported class of sensors since they are cheap and based upon the use of disposable electrodes; they are highly specific and do not need extensive sample pre-treatment or large sample volumes. Teleme- try is a well-established technique for real-time monitoring of lev- els of several biological molecules. Sophisticated telemetry devices have been developed to analyse neurochemical data and success- fully used in conjunction with micro-biosensors for the in vivo measurement of brain AA, O , glucose and lactate ( Bazzu et al., 2009; Calia et al., 2009 ). More recently, an ultralow-cost telemetric system, combined with a carbon amperometric sensor, for a rapid electrochemical detection of AA in fresh orange juice was devel- oped ( Barberis et al., 2010 ). The system, built with simple and inex- pensive components showed highly reproducible analytical performances in accordance with those obtained with traditional reference methods, thus suggesting that it could be used for a rapid monitoring of AA on several biological substrates. The aim of this work was to determine postharvest changes of ascorbic acid in kiwi, pineapple, and melon, subjected to minimal " id="pdf-obj-0-16" src="pdf-obj-0-16.jpg">

Analytical Methods

Detection of postharvest changes of ascorbic acid in fresh-cut melon, kiwi, and pineapple, by using a low cost telemetric system

Antonio Barberis a , , Angela Fadda a , Mario Schirra a , GianFranco Bazzu b , Pier Andrea Serra b

a Institute of Sciences of Food Production, National Research Council, Traversa La Crucca 3, Regione Baldinca, 07040 Li Punti, Sassari, Italy b Department of Clinical and Experimental Medicine, University of Sassari, v.le San Pietro 43, 07100 Sassari, Italy

article info

Article history:

Received 8 February 2012 Received in revised form 3 May 2012 Accepted 31 May 2012

Available online 9 June 2012

Keywords:

Ascorbic acid

Fresh-cut fruits

Electrochemical sensor

Telemetry

abstract

The present paper deals with a novel telemetric device combined with a carbon amperometric sensor sys- tem to determine postharvest changes of ascorbic acid (AA) in fresh-cut fruits, without displacing prod- ucts out of the storage rooms. The investigation was performed on kiwi, pineapple and melon, subjected to minimal processing, packaging, cold storage, and simulated shelf life. Results demonstrated that AA content of fresh-cut fruits of all species declines differently during storage. Cold storage notably reduced

the degradation rate of AA in comparison with samples stored at 20 C. The cold-chain interruption resulted in a sharp AA content reduction when the optimal storage condition was not rapidly replaced. Unpredicted results showed a high activity of oxidative enzymes, which prevented AA detection in melon samples. Our sensor system allowed us to demonstrate that both ascorbate peroxidase and ascor- bate oxidase affected the oxidative stability and the nutritional quality of fresh cut melon fruits. 2012 Elsevier Ltd. All rights reserved.

1. Introduction

Fresh and minimally processed (fresh-cut) fruits and vegetables are significant sources of dietary Vitamin C (ascorbic acid, AA). A dose of 100–200 mg/day of AA has been highly recommended, since it helps to prevent many human diseases linked to oxidative stress, including cancer, diabetes mellitus, stroke (Yokoyama et al., 2000) and Parkinson’s disease (Serra, Pluchino, Marchetti, Desole, & Miele, 2008). AA amount in horticultural crops depends on species, variety, cultural practice, fruit age, climate and post-harvest handling. Its content usually declines during fruit storage or processing (Lee & Kader, 2000; Schirra et al., 2008). Two enzymes are principally in- volved in AA oxidative metabolism, ascorbate oxidase (AO) and ascorbate peroxidase (APX). AO catalyses AA oxidation mainly dur- ing tissue growth, while APX is more related to the modification of AA content during ripening, storage or wounding, using AA to re- move the H 2 O 2 formed by oxidative stress (Diallinas et al., 1997; Gomez & Lajolo, 2008). Due to its antioxidant activity, AA plays a crucial role in many metabolic pathways, which have a direct impact on the oxidative stability of fruits (Lurie, 2003). The loss of AA is the main limiting factor of nutritional quality (Ahvenainen, 1996), therefore it is re- garded as a quality indicator for fresh-cut fruits and vegetables. A large number of methods have been applied for AA determina-

Corresponding author. E-mail address: antonio.barberis@ispa.cnr.it (A. Barberis).

0308-8146/$ - see front matter 2012 Elsevier Ltd. All rights reserved.

tion (Arya, Mahajan, & Jain, 2000; Rahman Khan, Rahman, Islam, & Begum, 2006). Among them, titrimetric and chromatographic methods are the most commonly used since they provide a precise and accurate assessment of AA, however they can be applied only in the laboratory. In recent years, the application of sensors and biosensors tech- nology to food industry, as a powerful and low cost alternative to conventional analytical techniques, has been widely discussed by several authors (Terry, White, & Tigwell, 2005; Velasco-Garcia & Mottram, 2003). Amperometric devices are the most commonly re- ported class of sensors since they are cheap and based upon the use of disposable electrodes; they are highly specific and do not need extensive sample pre-treatment or large sample volumes. Teleme- try is a well-established technique for real-time monitoring of lev- els of several biological molecules. Sophisticated telemetry devices have been developed to analyse neurochemical data and success- fully used in conjunction with micro-biosensors for the in vivo measurement of brain AA, O 2 , glucose and lactate (Bazzu et al., 2009; Calia et al., 2009). More recently, an ultralow-cost telemetric system, combined with a carbon amperometric sensor, for a rapid electrochemical detection of AA in fresh orange juice was devel- oped (Barberis et al., 2010). The system, built with simple and inex- pensive components showed highly reproducible analytical performances in accordance with those obtained with traditional reference methods, thus suggesting that it could be used for a rapid monitoring of AA on several biological substrates. The aim of this work was to determine postharvest changes of ascorbic acid in kiwi, pineapple, and melon, subjected to minimal

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processing, packaging, cold storage, and simulated shelf life by using this new technology, without displacing products out of the storage rooms.

2. Materials and methods

  • 2.1. Reagents

All chemicals were of analytical grade and used as received without any further purification. Solutions were prepared with MilliQ water (Millipore, Inc.; O = 18 MO/cm). L-ascorbic acid (99%) was purchased from Merck (Germany); stock solutions of AA were prepared daily in acetate buffer (AB) (Merck-Germany) at pH 3.7 and in phosphate buffer (PBS) at pH 6.3. The phosphate buffer saline solution was made using NaCl (137 mM), KCl (2.7 mM), Na 2 HPO 4 (8.1 mM) and KH 2 PO 4 (1.47 mM) from Sigma and then adjusted to pH 6.3. For titrimetric measurements, oxalic acid dihydrate and 2,6- dichlorophenol-indophenol sodium salt dihydrate, sodium hydro- gen carbonate (NaHCO 3 -powder P 99.5%) were purchased from Merck (Germany). Catalase from bovin liver (EC 1.11.1.6) were purchased from Sigma–Aldrich. Meta-phosphoric acid (HPO 3 ) was purchased from Merck. Hydrogen peroxide (30% solution from Merck-Germany) was diluted and used as 10% solution.

  • 2.2. Fruit samples preparation

Samples of fresh-cut kiwi fruit [Actinidia deliciosa (A. Chev.) cv Hayward], pineapple [Ananas comosus (L.) Merr.] and cantaloupe melon (Cucumis melo L. var. reticulatus Naud.) were bought from ‘‘C & G s.r.l. Company’’ (Monserrato, southern Sardinia, Italy). The produce was processed following HACCP guidelines (CAC, 1993). All raw fruits were of the highest quality class, harvested at a ripe maturity stage, cleaned before processing in a 200 ppm NaClO solution for 2 min, and rinsed with tap water. Pineapple and melon were manually peeled and sliced while kiwi fruits were mechani- cally peeled. Then fruits were cut in trapezoidal sections. After pro- cessing, pieces were washed with tap water and dried by a slight centrifugation in order to remove microorganisms and tissue flu- ids, thus reducing microbial growth and enzymatic oxidation dur- ing subsequent storage. Samples of 100 g of fruit pieces (each species separately) were packaged in see-through resealable poly- propylene trays for foodstuffs, and left overnight at 3 C. The day after processing (day 1), samples were transported at 3 C to the laboratory by a refrigerated truck early in the morning. On arrival, fruit of each species were segregated into three groups. The fruits of the first group (GROUP 1, control) were stored at 3 C for 5 days (recommended storage conditions). Fruits of the second group (GROUP 2) were stored at 20 C for 6 h at day 1 to simulate a cold chain interruption, and then re-stored at 3 C until the expiration date (day 5). Six hours was the estimated time for moving samples, covering the distance from the Company to all the markets along the scheduled itinerary. It was also the time needed to deliver the samples to our laboratory. Fruits of the third group (GROUP 3) were kept under simulated shelf life at 20 C for 5 days to favour a rapid product deterioration.

  • 2.3. Sensor description, characterization and calibration

The sensor assembly was composed of three carbon rod (length = 30 mm; Ø = 300 lm, 2H staedtler graphite pencil leads) electrodes. The working electrode was coated with a thin insulat- ing layer of epoxy resin and the active surface was ellipsoidal. Three millimetres of non-insulated carbon rod guaranteed good

electrical contact when inserted in a gold-plated socket. The pseu- do-reference and auxiliary electrodes were not insulated. The telemetric miniaturized device, weighing less than 15 g, consists of a single-supply sensor driver, a current-to-voltage (I/V) converter, a microcontroller, and a miniaturized data transmitter. It is coupled with a microsensor that generates electrical signals re- lated to electrochemical processes. The telemetric device is capable of working in oxidation mode. A biological oxidizing molecule, such as AA, can be directly detected on the surface of the amperometric sensor connected to a potentiostat. The AA oxidation currents were digitized by means of an analog-to-digital converter integrated in a peripheral interface controller (PIC) and sent to a personal com- puter by means of a miniaturized AM transmitter. The components were soldered on a single sided printed circuit board. All electronic parts used in this project were lead (Pb) free and compliant to restriction of hazardous substances (RoHS) directives. The electron- ics were calibrated and tested in vitro under different experimental conditions and exhibited high stability, low power consumption, and good linear response in the nanoampere current range (Bazzu et al., 2009; Calia et al., 2009; Serra et al., 2007). The electrochemical characterization and AA calibration were performed using a four channel system (eDAQ QuadStat, e-Corder 410 and Echem software, eDAQ Europe, Poland) placing the micro- sensors in a cell, a glass beaker containing 10 ml of AB at pH 3.7 or 10 ml of PBS at pH 6.3. AA calibration was made the day after sensor preparation, both in a 10 ml air-bubbled AB and in 10 ml air-bubbled PBS, applying a positive potential of +120 mV (vs carbon pseudo-reference elec- trode) after a stable baseline was achieved. A five-point calibration was performed by adding known amounts of AA (20 l l of a 10 mM stock solution) in order to have concentrations comprised between 0 and 100 lM in the cell. A more accurate description of sensor assembly, telemetry system and electrochemical characterization was reported previously (Barberis et al., 2010).

2.4. AA electrochemical detection in fresh-cut kiwi, pineapple and melon

AA content in fresh-cut kiwi, pineapple and melon was deter- mined immediately after processing (time 0) and daily, from day 1 until the expiration date at day 5. All measurements were carried out in triplicate (three samples from each group/day), with the telemetry system (three sensor assemblies, one for each group), at time 0 (in the C & G s.r.l. Company laboratory) and at day 1–5 (in our cold store rooms). The analysis of AA in kiwi and pineapple samples, in 10 ml of AB solution at pH 3.7, was achieved by exposing the sensors firstly to two aliquots of AA (2 20 ll of a 10 mM stock solution) and then to two aliquots (2 40 ll) of juice of each fruit species and inter- polating the resulting currents in the calibration plot obtained immediately before juice injections. The juice used for AA determi- nation was squeezed and filtered with a kitchen strainer. Based on unpredicted results, the analytical procedure for melon was modified. When the sensor was exposed to melon juice, the system registered a rapid decline of the AA content into the electro- chemical cell, probably due to the presence of AA degradative en- zymes. According to the studies of Hernández, Lobo, and Gonzáles (2006), before AA analysis it was essential to inactivate degradative enzymes and to fix the redox equilibrium between AA and dehydro- ascorbic acid (DHA). Therefore, meta-phosphoric acid (HPO 3 ), a sta- bilizer able to denature all the proteins (Hernández et al., 2006), was added to melon juice before analysis. In particular, 3 ml of 3% HPO 3 was added to 1 ml of melon juice. The solution was stirred and, after 2 min, was centrifuged at 14,515g for 10 min at 4 C with a refrigerated centrifuge (Du Pont, Thermo Sorvall Super T 21, Del- aware, USA). Two aliquots of 160 ll of supernatant were injected

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into 10 ml of PBS solution at pH 6.3 and the resulting currents were interpolated in the above-mentioned calibration plot. The addition of HPO 3 to the buffer did not affect the registered current.

  • 2.5. Kinetics analysis

Previous results demonstrated that kinetics modeling might be used to predict the influence of processing and storage on AA evo- lution (Granato, Masson, & de Freitas, 2010; Tiwari, Muthukuma- rappan, O’Donnell, & Cullen, 2008). For this reason, a first-order kinetics analysis of data of the AA content of all species was per- formed, in accordance with the model AA t = AA 0 e kt (Tiwari, Muthu- kumarappan, O’Donnell, & Cullen, 2008), where AA t is the studied parameter at any given measurement time t (time 0, day 1,

day2...day

5), AA 0 is the initial AA value of an untreated sample

and k is a rate constant. Data fitting was considered to be signifi- cant at a probability level of 95%. r 2 and relative standard deviation

of the residuals (SD) were calculated with data achieved both with the sensor and titrimetric methods.

  • 2.6. Titrimetric measurements

The AA content values obtained with the sensor method were validated by comparison with the results obtained by the standard titrimetric method (Barberis et al., 2010). AA content was ex- pressed as mg/100 g of fresh weight (FW).

  • 2.7. Influence of addiction or elimination of H 2 O 2 from melon juice

In order to accelerate the speed of the APX enzymatic oxidation of AA, two different aliquots of H 2 O 2 (in order to obtain concentra- tions of, 2.5 and 25 l m in the electrochemical cell, respectively) were added (Lamikanra & Watson, 2000) in the electrochemical cell (containing 0.5 lmol of AA) immediately after the injection of 100 ll melon juice. In a separate series of experiments the mel- on juice was substituted with 100 l l of PBS (pH = 6.3). Catalase was used to eliminate H 2 O 2 from the melon samples. One hundred and one thousand units of CAT were added, respec- tively, to two 100 ll melon juice samples and the solutions stored in the dark at room temperature for 30 min. One unit of CAT decomposes 1 lmol of H 2 O 2 min 1 at pH = 7.0 at 25 C (Unit defini- tion in Sigma–Aldrich datasheet). At the end of the reaction period the solutions were injected in the electrochemical cell.

  • 2.8. Statistical analysis

AA currents were expressed in nanoamperes and given as mean ± standard deviation (SD) of absolute oxidation currents (nA) or baseline-subtracted currents (D nA). After in vitro calibra- tions, the AA currents were plotted vs the AA concentration and the linear regression was calculated. AA-spiked data were obtained by subtracting the baseline after each injection of juice and ex- pressed as D nA. AA juice content was expressed as the molarity and then converted to mg/100 g to be comparable with reference method. In order to assure the similarity between the data ob- tained with the sensor and those obtained with the titrimetric method, a Student’s t-test to compare means was performed. As mentioned above, the AA-related amperometric signal was reduced by melon juice addiction. The mathematical model that best represented this phenomenon was the ‘‘one-phase exponen- tial decay’’ described by the following equation:

½AA ðtÞ ¼ ½AA 0 ð1=2Þ t =t 1=2

ð1Þ

where AA 0 is the initial concentration of AA (50 lM), [AA](t) is the concentration (lM) of AA that still remains and has not yet decayed

after a time t, and t 1/2 is the half-life of the decaying quantity. The half-life is the period of time it takes for a substance undergoing de- cay to decrease by half. Statistical analysis was performed by GraphPad Prism 5 for Windows software (GraphPad Software, Inc., La Jolla, CA 92037, USA). Analysis of variance (one-way ANOVA) was carried out using a unifactorial complete randomized block design. Mean compari- sons were calculated by Fisher’s least significant difference test at P 6 0.05.

3. Results and discussion

3.1. Performance of the telemetric device

The use of the telemetric system as a portable workstation al- ways allows detection of the AA content of the fresh-cut fruits in situ, without displacing samples out of the storage rooms. The electronics calibration was made with a linear distance between the transmitter and the receiver unit of about 3 m. The use of such a system and of a wireless connection ensured a low background noise, high stability and good linear response, as previously re- ported (Barberis et al., 2010; Calia et al., 2009; Serra et al., 2007). Following the same procedure of our previous work (Barberis et al., 2010), before determining AA content in the samples of fresh-cut fruits, we evaluated the specificity of our sensor investi- gating the effect of common potentially interfering compounds present in kiwi, pineapple and melon on the stability of the base- line, since the presence of potential interfering agents can alter the signal registered by the sensor. No alteration of the above- mentioned baseline was registered in the presence of the main sugars, organic acids or antioxidant components of the studied matrices, when working at a potential of +120 mV. Similar results were achieved by Akyilmaz and Dinçkaya (1999) using a biosensor based on ascorbate oxidase. They detected the O 2 consumption by a commercial dissolved oxygen probe and did not register any elec- trode response for oxalic, L-aspartic, L-glutamic, succinic, citric and glycolic acids, D(+)-glucose, and D( )-fructose. In their system, hydroquinone and catechol were the only interfering agents oxi- dized by the ascorbate oxidase, and likewise with AA. In our sys- tem, the absence of the enzyme and the low applied potential make the analysis free from the interference of the above-men- tioned molecules. A five point AA calibration was initially performed. Plots of the current response vs the concentration of AA over a dynamic range of 0–100 lM were obtained in triplicate. The linear response of the system was excellent (r 2 = 0.999, n = 5, P 6 0.05) with a sensitivity of 0.2811 ± 0.033 nA/l M). The limit of detection (LOD), which was calculated as that concentration that engendered a response three times the standard deviation of the background noise of the sensor, was 0.76 ± 0.122 l M. A further calibration with the standard addition method re- vealed that the exposition to kiwi, pineapple or melon juices did not affect the sensors performances (data not shown), in agree- ment with previous studies on orange juice (Barberis et al., 2010). Quantification of AA in the juice was achieved by interpolat- ing the response data in the calibration plot in a range from 0 to 5 mM of AA after correction of the dilution factor. The choice of the extraction medium pH was important since it affects oxidation of AA to dehydroascorbic acid (DHA) and its fur- ther degradation products. The rate of AA degradation is maximal at pH 4 and minimal at pH 2 (Gentili et al., 2008). There is neither an agreement nor a definitive answer, among authors, in order to establish which buffer solution and which pH of the buffer solution are the best to calibrate sensors for AA detection. Our choice of placing the electrodes in different buffers depended on the

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measured pH of the matrix we were working on (kiwi pH 3.6; pine- apple pH 3.7; melon pH 6.3). In some other cases it depended on the nature of the sensor (Ivanov, Tsakova, & Mirsky, 2006; O’Con- nell, Gormally, Pravda, & Guilbault, 2001). Sometimes the optimum working buffer pH for AA oxidation was reported to be near neu- trality (O’Connell et al., 2001) or at pH between 1 and 2 (Casella & Guascito, 1997) for the same type of sensor made by a conduc- tive PANI layer grown upon a carbon substrate.

3.2. AA changes in fresh-cut kiwi fruits

Sensor and reference methods showed similar pattern of AA changes over time (Figs. 1 and 2). The Student’s t-test indicated that differences between the two methods were not significant. The initial AA content in fresh-cut kiwi fruits was 65 mg/100 g, in accordance with values reported in literature for ripe kiwi fruits cv. Hayward (Gil, Aguayo, & Kader, 2006; Tavarini, Degl’Innocenti, Remorini, Massai, & Guidi, 2008). During storage a progressive AA degradation in the fruits belonging to all groups was observed. Cold storage notably reduced the degradation rate of AA up to

day 4 in comparison with samples of GROUP 2 and especially in those stored at 20 C, whereas at day 5 the reduction of AA in GROUP 2 and control samples were similar. The cold-chain inter- ruption affected the AA content of fresh-cut kiwi inducing its immediate reduction if the optimal storage conditions were not rapidly replaced. Even though the proper storage conditions were restored, kiwi was negatively affected by the interruption, losing more than 50% at day 3 when compared with the control. A fast AA degradation and a complete samples deterioration of fresh- cut kiwi fruits stored at 20 C occurred 4 days after processing. The results of kinetics analysis indicated that AA degradation of fresh-cut kiwi followed a first-order kinetics when sample are

properly stored at 3 C (r 2 = 0.964 with SD = 0.06 and r 2 =

1558 A. Barberis et al. / Food Chemistry 135 (2012) 1555–1562 measured pH of the matrix

Fig. 1. Changes in ascorbic acid (AA) content in fresh-cut kiwi fruits kept under different storage conditions, detected with the electrochemical sensors (A) and with titrimetric method (B). Values are means ± standard deviation, n = 3. Within each storage time, means followed by unlike letters differ significantly by Fisher’s least significant difference (LSD) test, P 6 0.05. No statistical difference among means was found where letters are absent. GROUP 1 = stored at 3 C; GROUP 2 = cold chain interruption at day 1 and GROUP 3 = stored at 20 C.

1558 A. Barberis et al. / Food Chemistry 135 (2012) 1555–1562 measured pH of the matrix

Fig. 2. Changes in ascorbic acid (AA) content in fresh-cut pineapple fruits under different storage conditions, detected with the electrochemical sensors (A) and with titrimetric method (B). Values are means ± standard deviation, n = 3. Within each storage, time means followed by unlike letters differ significantly by Fisher’s least significant difference (LSD) test, P 6 0.05. No statistical difference among means was found where letters are absent. GROUP 1 = stored at 3 C; GROUP 2 = cold chain interruption at day 1 and GROUP 3 = stored at 20 C.

  • 0.986 with SD = 0.05 for amperometric and titrimetric method, respec- tively), while r 2 values seemed ambiguous when a cold chain inter- ruption or a storage at 20 C occurred (data not shown). Our results are in accordance with those of Kalt (2005) who examined how AA content changes in relation with the stage of maturity, concluding that the more mature the fruit the lower AA content. The authors found that AA content declines concur- rently with the degradation of fruit tissues. In our work, kiwi fruits were harvested at full maturity and they were subjected to severe wounding stress, which strongly accelerated tissue deterioration. 3.3. AA changes in fresh-cut pineapple fruits AA content in fresh-cut pineapple, measured with the telemet- ric system was 37 mg/100 g at time 0 (Fig. 2A). A slight decrease of AA was observed in the control samples with a 6% and 30% AA loss registered after processing and at the expiration date, respectively. Differently from kiwi, pineapple showed a higher tolerance to- wards the cold chain interruption and, when the proper storage conditions were replaced, the AA content remained almost stable until the expiration date. The change in temperature did not imme- diately affect the AA content of pineapple belonging to GROUP 2; only at day 3 were AA values of GROUP 2 significantly different than control. Our results are similar to those of Gil et al. (2006) which measured a significant but moderate (22%) loss of AA during 6 days of storage of fresh-cut pineapple samples. Fresh-cut pineap- ple pieces stored at 20 C were judged to be under the limit of mar- ketability 4 days after processing, when more than 75% of AA had degraded. The results of kinetics analysis indicated that AA degra- dation of fresh-cut pineapple followed a first-order kinetics only when samples were properly stored at 3 C (r 2 = 0.977 with SD = 0.05 and r 2 = 0.991 and SD = 0.01 for the amperometric and titrimetric methods, respectively) whereas, when a cold chain

A. Barberis et al. / Food Chemistry 135 (2012) 1555–1562

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interruption occurred, the data no longer fitted to the model. Re- sults obtained with the telemetric system were confirmed by refer- ence method (Fig. 2B). Also in this case, AA amounts detected with the sensor were not significantly different from those achieved with the reference one.

3.4. AA changes in fresh-cut melon fruits

The results obtained on the fresh-cut melon samples were to- tally unpredicted. A first attempt to determine AA content in the samples was made by exposing the sensors to two aliquots of AA and then to two 40 l l aliquots of juice, following the same proce- dure used for kiwi and pineapple. A current increase corresponding to the added amount of melon was expected. Surprisingly a rapid decline of the AA content into the cell was observed. The experi- ment was repeated as reported in Fig. 3, where two aliquots of 25 l l of a solution of AA 10 mM and a 100 ll of melon juice were injected in 10 ml of PBS solution. The curve in the graph indicated that the entire AA amount into the electrochemical cell (0.5 l mol) was completely consumed in about 50 min, when the current val- ues reached the baseline level. On the other hand, when AA was not previously injected in the cell, no current shift (neither increase nor decrease) from the baseline was recorded, not even after sev- eral aliquots of melon juice were added (inset of Fig. 3). This result was confirmed by the titrimetric method, since repeated measures, with a time interval of 5 min, on the same sample registered decreasing value of AA content. The most plausible hypothesis, supported by Lamikanra and Watson (2000), is that fresh-cut pro- cessing resulted in a loss of cellular compartmentation at the cut surface with a consequent increase in interaction between AA oxi- dative enzymes and their substrate. In our study this interaction was enhanced when samples were squeezed to be analysed. A combination of a, not better-defined, enzymatic activity with H 2 O 2 and free radicals, was supposed to be actively degrading the AA amount into the electrochemical cell. The enzymatic activity could be consistent with that of ascor- bate oxidase (AO) or ascorbate peroxidase (APX). According to Be- litz, Grosch, and Schieberle (2004) and Gentili et al. (2008) AA in foodstuffs is probably bound to proteins. Studies on AA biosynthe- sis in fresh cut fruits are limited so that the influence of enzymes involved in the oxidation, AO and APX, on changes of AA content, during storage and in response to wounding, is still a moot point. In order to explain this phenomenon we kept investigating in two directions: on the one hand we concentrated on the role of enzymatic activity as a causative factor of AA changes in melon samples and on how these changes were influenced by date and/

or treatments; on the other hand we tried to understand which en- zyme was responsible for such a rapid AA degradation. The half life of exponential decay of the curve of AA degradation was used as an indicator of the combined action of oxidative enzy- matic activity and Reactive Oxygen Species (ROS) (Fig. 4). The fas- ter the AA degradation the lower the half-life. Half-life values registered at time = 0 were high (about 12 min), thus suggesting that the enzymes involved in the oxidation were not immediately activated by wounding. The expression of the activity of the pool of enzymes involved in the oxidation, activated by wounding, was investigated as an effect of slicing on melon (Diallinas et al., 1997), or as a consequence of flavedo injuries caused by chilling on oranges, apricot and cantaloupe melon (Ben-Amor et al., 1999; Imahori, Takemura, & Bai, 2008; Sala & Lafuente, 2004). Nev- ertheless there still is a lack of information on how long the melon takes to activate its defensive mechanisms against oxidative stress. Studies on wounded sweet potato leaves indicate that APX requires 6–12 h to be expressed under stress conditions (Lin et al., 2011). After one night of storage at 3 C, an increase in the speed of AA degradation was registered in all samples. At day 2, a significant decrease of half-life values, compared to those of day 1, was regis- tered, but only for fruits of GROUP 2 and GROUP 3 (8 and 7.5 min, respectively), apparently influenced by the increase of the storage temperature. It must be noticed that, when the recommended stor- age conditions were applied, the half-life values showed a slight (but not significant) decrease until day 3 and a new increase for the last 2 days of storage. This suggested that AA level decreased as a consequence of processing, but its degradation rate was re- duced by the low storage temperature. The melon samples sub- jected to the cold-chain interruption showed a significant increase of AA degradation at day 2, but later, when the recom- mended storage conditions were replaced, the half-life values rose up again (16.5 and 17.6 min at day 4 and day 5, respectively). We cannot explain if the enzymatic activity slowed down as a conse- quence of the re-established proper storage conditions and/or be- cause it was compromised earlier by the previous cold chain interruption. Finally a continuous decrease of half-life values (from 12 min at day 1 to about 3 min at day 3) was observed in melon samples stored at 20 C until day 3, associated with a rapid deteri- oration of fruit pieces. Fruits of GROUP 3 reached the limit of mar- ketability 2 days before the expiration date when the highest values of half-life were registered. Studies on tomato and bell pepper fruits revealed that the de- crease of AA coincided with the beginning of ripening and with an increase of AO activity (Yahia, Contreras-Padilla, & Gonzales- Aguilar, 2001). AA oxidation is catalysed by AO, which might be

A. Barberis et al. / Food Chemistry 135 (2012) 1555–1562 1559 interruption occurred, the data no

Fig. 3. Amperometric response after adding melon juice to a solution of AA 50 lM. Arrows with ‘‘A’’ correspond to two subsequent injections of AA (25 lM) in the electrochemical cell. Arrow with ‘‘B’’ corresponds to an injection of 100 ll of melon juice. The addition of spiked melon juice to the buffer did not determine any long- lasting increase of the registered currents (inset).

A. Barberis et al. / Food Chemistry 135 (2012) 1555–1562 1559 interruption occurred, the data no

Fig. 4. Half life of exponential decay of the curve of AA degradation of melon samples, under different storage conditions, immediately after processing (time 0) up to the expiration date (day 5). Half life values are means ± standard deviation, n = 3. Within each storage condition, means followed by unlike letters differ significantly by Fisher’s least significant difference (LSD) test, P 6 0.05.

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related to tissues growth, even though studies on different melon tissues evidenced a specific activity of AO in mature and ripe melon fruits (Gomez & Lajolo, 2008). AO activity was detected at high lev- els in ovaries and in the growing shoot apex and, to a lower extent, in other tissues suggesting that AO possesses a specific function during fruit development, ripening and softening although its mode of action is unclear (Al-Madhoun et al., 2003). In addition, during fruit development, a progressive decrease in the soluble ba- sic AO isoenzymes, followed by the predominance of the apoplastic acidic AO isoenzymes, was observed (Al-Madhoun, Sanmartin, & Kanellis, 2003). On the other hand, Diallinas et al. (1997) reported that melon AO is not expressed in late stages of fruit ripening and was repressed in wounded fruits. Additionally, AO is not expressed either under stress conditions or in a specific developmental stage, which leads to massive ethylene production. In transgenic melon fruit, which do not produce ethylene, AO expression is not re- pressed by wounding. It seems that endogenous ethylene, pro- duced in response to wounding, might be the cause of AO repression (Diallinas et al., 1997). APX is part of an efficient antioxidant defensive system evolved by plants, which prevents the accumulation of ROS and repairs oxi- dative damage. In this complex mechanism, SOD (superoxide dis- mutase) dismutates superoxide radicals to H 2 O 2 and O 2 in a spontaneous and rapid reaction, in order to protect the cells from damage by superoxide radical reaction. H 2 O 2 , which is a potentially toxic compound, is then reduced to water by a number of enzymes such as catalase (CAT) and APX (Imahori et al., 2008). Studies on cherry tomato revealed an intense activity of APX and AO due to the exposition of exocarp to very high temperature and strong so- lar radiation (Rosales et al., 2006), thus revealing the primary role of these two enzymes under stress conditions. According to Lamikanra and Watson (2000), the peroxidase activity in wounded and fresh-cut cantaloupe melon fruits exhibits a high affinity for ascorbate. The studies of Gomez and Lajolo (2008) and of Diallinas et al. (1997) on mango and guava, sug- gested APX is related more to the modification of AA content dur- ing ripening, storage or wounding, using AA to remove the H 2 O 2 formed by oxidative stress. Similar results were obtained on pear fruits (Lentheric, Pinto, Vendrell, & Larrigaudiere, 1999) and Capsi- cum annuum L. (Schantz, Schreiber, Guillemaut, & Schantz, 1995). In order to better understand which enzymes, AO, APX or both, were responsible for AA degradation in our experimental condi- tions, we formulated two hypotheses, starting from the fact that APX reduces hydrogen peroxide using AA as a substrate. APX catal- yses the transfer of electrons from AA to hydrogen peroxide, pro- ducing dehydroascorbic acid and water as follows:

AA þ H 2 O 2 ! Dehydroascorbate þ H 2 O:

The first assumption we made was that if APX was responsible for AA degradation, adding increasing amounts of H 2 O 2 to the mel- on samples would accelerate the enzymatic activity in the samples and increase the speed of AA degradation. In order to validate this hypothesis, using the sensor system, we followed the same procedure applied for the experiment in Fig. 3. We previously evaluated the AA oxidation capacity of two different concentrations of H 2 O 2 , 2.5 lM and 25 lM, by separately injecting the appropriate amount of H 2 O 2 , in a 10 ml electrochemical cell containing AA 50 lM and monitoring the correspondent current decreasing. The experiment was then repeated by adding the same different aliquots of H 2 O 2 in the electrochemical cell immediately after the injection of 100 ll melon juice. Both the oxidation capac- ity of H 2 O 2 and the enzymatic activity of ‘‘melon + H 2 O 2 ’’ were ex- pressed as half life for the exponential decay of the curve of AA degradation. The results were compared with the control (100 l l of melon juice added to a 50 lM AA solution) and reported in Fig. 5. The addition of H 2 O 2 at 2.5 lM to a solution of AA at

50 l M resulted in a similar exponential decay to the control, with the difference among half-life values not being significant. On the other hand, the half-life of the curve of ‘‘melon + 2.5 l M H 2 O 2 ’’ was significantly lower (5.8 min) than the control (9.9 min), indi- cating that the enzymatic activity increased, probably due to a syn- ergic effect among the added H 2 O 2 and the enzymes present in the melon samples. A further confirmation of this hypothesis came from the half life values of curves of ‘‘25 lM H 2 O 2 ’’, which showed a very sharp (3.5 min) exponential decay, while ‘‘melon + 25 l M H 2 O 2 ’’ degraded all AA in the electrochemical cell almost instantaneously. The second assumption we made was that, eliminating all H 2 O 2 from the melon samples, the APX activity should stop. In order to eliminate H 2 O 2 we added 100 U and 1000 U of CAT to two 100 ll samples of melon juice, as described in the materials and methods section. At the end of the reaction period, the obtained solutions were injected into a 10 ml electrochemical cell containing AA 50 l M and the correspondent current decreases were monitored. If APX was the only enzyme responsible for AA degradation in the melon samples, the sensors should not be able to register any current decrease. On the contrary, in the case that a further decreasing of AA current was observed, the residual activity responsible for AA degradation should be related to a combined ac- tion of AO and free radicals. The half life values when 100 U or 1000 U of CAT were added were 29 and 50 min, respectively, indi- cating that the enzymatic oxidation reactions in the samples were, respectively, very high and almost null when compared with the control one (pure melon juice). Despite APX was stopped by the absence of H 2 O 2 , the sensors system was not able to detect any increase of the current, which might be ascribed to an increase of AA in the electrochemical cell. This is a very relevant fact because it indicates a residual enzymatic activity not linked to the presence of H 2 O 2, not imputable to APX, but probably due to AO and some free radicals. This last hypothesis was confirmed by the experiment carried out on melon samples added of HPO 3 . Meta-phosphoric acid is a well-established stabi- lizer in order to prevent AA oxidation (Gentili et al., 2008; Hernán- dez et al., 2006). It denatures all the proteins and enzymes present in the samples, enabling the detection of AA (Fig. 6). No current

1560 A. Barberis et al. / Food Chemistry 135 (2012) 1555–1562 related to tissues growth, even

Fig. 5. Half life of exponential decay of the curve of AA degradation of: pure melon juice; two different concentrations (2.5 and 25 lM) of H 2 O 2 ; melon juice added of two different concentrations (2.5 and 25 lM) of H 2 O 2 ; melon juice added of two doses of CAT (100 and 1000 U). Half life values are means ± standard deviation, n = 3. Means followed by unlike letters differ significantly by Fisher’s least significant difference (LSD) test, P 6 0.05.

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shift from the baseline was observed even after several injections of HPO 3 since it did not affect the signal registered by the sensors. Fig. 6A shows the AA values in melon samples registered after stopping the enzymatic activity. AA content varied from the initial 35 to 23 mg/100 g at expiration date when stored at 3 C (a loss of 33% of the initial content), in accordance with the results obtained by Gil et al. (2006). A 54% loss of the initial AA content was regis- tered at the end of the storage period as a consequence of the cold chain interruption, but a significant reduction of AA values, com- pared with the control, started only from day 3. After that, AA con- tent remained significantly lower than the control up until the expiration date, even though the optimal storage conditions were replaced. Differently, the AA content of melon samples continu- ously kept at 20 C significantly decreased from day 2 to day 4 and remained unchanged up to the expiration date. These results are consistent with those showed in Fig. 4. Indeed, when the enzymatic activity was low (Fig. 4, GROUP 1) a slight de- crease of AA was registered (Fig. 6, GROUP 1); the 6 h interruption of the cold-chain determined a temporary, but significant, increase of the enzymatic activity (Fig. 4, GROUP 2), able to significantly re- duce the AA content if compared with the control (Fig. 6, GROUP 2). The residual AA content was preserved when the optimal storage conditions were replaced; a definitive interruption of the cold- chain resulted in a dramatic increase of the enzymatic activity (Fig. 4, GROUP 3) and a rapid degradation of AA content in the mel- on samples (Fig. 6, GROUP 3). The results of the kinetics analysis indicated that AA degrada- tion of fresh-cut melon followed a first-order kinetics, not only when sample are properly stored at 3 C (r 2 = 0.975 with SD = 0.03 and r 2 = 0.996 and SD = 0.03 for the amperometric and titri- metric methods, respectively), but also when a cold chain interrup- tion (r 2 = 0.969 with SD = 0.05 and r 2 = 0.987 with SD = 0.02 for the

A. Barberis et al. / Food Chemistry 135 (2012) 1555–1562 1561 shift from the baseline was

Fig. 6. Changes in ascorbic acid (AA) content in fresh-cut melon fruits, when juice was added of HPO 3 , under different storage conditions, detected with the electrochemical sensors (A) and with titrimetric method (B). Values are means ± standard deviation, n = 3. Within each storage time, means followed by unlike letters differ significantly by Fisher’s least significant difference (LSD) test, P 6 0.05. No statistical difference among means was found where letters are absent. GROUP 1 = stored at 3 C; GROUP 2 = cold chain interruption at day 1 and GROUP 3 = stored at 20 C.

amperometric and titrimetric methods, respectively) or a storage at 20 C occurred (r 2 = 0.964 with SD = 0.11 and r 2 = 0.971 with SD = 0.09 for the amperometric and titrimetric methods, respec- tively), thus confirming that this species has a completely different oxidative metabolism when compared with kiwi and pineapple. When the cold chain was interrupted the AA degradation rate in- creased, but still followed a first order kinetics. When samples were stored at 20 C the degradation rate increased more but, also in this case, followed a first order kinetics. These results indicate that AA degradation in fresh-cut melon samples was affected by the different storage treatments but, more than this, by the inter- action at a cut surface between AA oxidative enzymes and their substrate at the moment of cutting. As can be well observed from Fig. 1, after the cold chain interruption the AA content of kiwi fell immediately but then, when the proper storage conditions were restored, AA degradation continues gradually. In pineapple, we ob- served a similar collapse of AA content but only after 24 h from the cold chain interruption (Fig. 2). In melon samples there were not any crush of the system and the AA content degrade gradually fol- lowing a first-order kinetics (Fig. 6). The new obtained results are partially in accordance with those obtained by Granato et al. (2010), which found AA degradation to follow first-order kinetics when the combined action of cold stor- age temperature and the use of sodium benzoate reduced the AA oxidative enzymatic reactions rate, thus improving the stability of the dessert. Similarly, in our work, the AA degradation rate fol- lowed first-order kinetics only when the proper storage conditions (3 C) implement the stability of the produce.

3.5. Comparison between methods: Sensors vs titrimetric

In this study we presented an application of a highly innovative telemetric system for the real-time detection of AA in fresh-cut fruits. The system, built with simple an inexpensive disposable components, showed highly reproducible analytical performances, absolutely consistent with those obtained with the official method. It was demonstrated that the device can be used for a rapid mon- itoring of AA during every step of post-harvest life of fresh-cut fruits, without displacing the products out of storage and refriger- ating rooms. It can be considered as a portable workstation, which can be used in situ by non-specialized personnel. The results of the Student’s t-test did not revealed statistical dif- ferences between the sensor and the reference methods. Differ- ences within 20% between values registered by sensors and by redox titration were reported as tolerant in most applications (Iva- nov et al., 2006). Moreover, en error of this magnitude is acceptable since it is free from any interference. Other studies carried out with constant potential amperometric techniques, showed an over-esti- mated level of AA in comparison with the reference method (Civit, Nassef, Fragoso, & O’Sullivan, 2008); such a result did not suggest the presence of interferents, but a presumed higher sensitivity of the sensor, especially in function of the materials employed to build the working electrode.

4. Conclusions

The use of the telemetry system in the postharvest sector of fresh cut fruits is a novelty. This technology is low cost, user friendly, and enables analysis to be performed without displacing the products out of the store rooms. The results went beyond expectations since the simple device composed of three graphite rods, built to detect molecules with a strong impact on the oxida- tive stability and the nutritional quality of fresh cut fruits, also en- ables investigation of the activity of complex enzymatic systems such as APX and AO.

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Acknowledgments

This work was partially supported by MiEF-CNR, Project ‘‘Innov- azione e Sviluppo del Mezzogiorno - Conoscenze Integrate per Sos- tenibilità ed Innovazione del Made in Italy Agroalimentare – Legge n. 191/2009’’ and by Fondazione Banco di Sardegna. The authors are in debt to Dr. M.T. Lafuente, Instituto de Agroquímica y Tec- nología de Alimentos, Consejo Superior de Investigaciones Científ- icas (CSIC), Burjassot (Valencia), Spain for critical review of the paper. They also thank Mr. D. Mura and Mr. A. Petretto for technical assistance in chemical analyses.

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