Effect of temperature abuse and improper atmosphere packaging on volatile profile and quality of rocket leaves

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Effect of temperature abuse and improper atmosphere packaging on volatile profile and quality of rocket leaves ⁎

Leonarda Mastrandrea, Maria Luisa Amodio , Maria Lucia V. de Chiara, Sandra Pati, Giancarlo Colelli Dip.to di Scienze Agrarie degli Alimenti e dell’Ambiente, Università di Foggia, Via Napoli 25, 71122, Italy

A R T I C L E I N F O

A B S T R A C T

Keywords: Off-odors Vitamin C Shelf-life Appearance

This study aimed to investigate the effect of temperature abuse and improper packaging on volatiles profile, vitamin C and sensorial attributes of rocket leaves packaged in modified atmosphere. Leaves packed in suboptimal conditions (high ratio of product weight/bag surface) were stored for 10 days at 0 and 5 °C, and for 8 days at 15 °C. Rocket leaves were kept in macroperforated bags in order to prevent modification of atmosphere within the headspace (as control). The packed rockets at 0 °C retained ascorbic acid content while it decreased during storage at higher temperatures. The main losses in the appearance and vitamin C content were observed when the O2 level reached about 0 kPa corresponding to the highest CO2 accumulation in the bag (25 kPa). The off-odors from dimethyl sulfide (DMS), dimethyl disulfides (DMDS) and other volatiles were produced at 5° and 15 °C, changing the initial headspace fingerprint, which was best preserved at 0 °C. Results of this work showed that improper packaging condition may decrease the shelf-life of rocket leaves compared to storage in air, inducing loss of appearance score, the production of off-odors and the degradation of Vitamin C. No additional benefit was obtained by optimal gas composition when bags were stored at 0 °C, indicating that the use of low temperature was effective to slowing down degradation reactions.

1. Introduction Wild rocket (Diplotaxis tenuifolia) is one of the most popular leafy green vegetables in Europe. It is most appreciated for its sulfurous odor and characteristic bitter and pungent taste due to the presence of glucosinolates, typical compounds of Brassicacea family (Blažević & Mastelić, 2008), and their breakdown products isothiocyanates (Bennett, Rosa, Mellon, & Kroon, 2006; Bennett et al., 2002; D'Antuono, Elementi, & Neri, 2009; Pasini, Verardo, Cerratani, Caboni, & D'Antuono, 2011). Rocket is available in the market as raw produce or washed and packed in polypropylene (PP) film bags (Løkke, Seefeldt, & Edelenbos, 2012). The respiration rate (RR) of the product determines the extent of the changes in the internal gas composition of a package, depending also on the barrier properties of the packaging material, and the temperature under which the product is stored. Optimal conditions occur when MAP design results in the optimal gas concentrations at the equilibrium. On the contrary, improper MAP design and temperature abuse may lead to loss of quality, production of off-odor and increased product metabolism, which will in turn affect final gas composition inside the package. For MAP design, once the bag material and dimensions are fixed the atmosphere at the equilibrium

⁎

will be strictly related to produce RR (Sivertsvik, Rosnes, & Bergslien, 2002). In rocket leaves, RR has been shown to be highly variable according to the cultivar, the season, and the number of cutting (i.e. first, second, third, etc.). A variation from 6.95 to 3.92O2 mmol kg−1 h−1 at 20 °C was reported by Seefeldt, Løkke, and Edelenbos (2012) passing from spring to late summer, and some differences are due to the number of plant cuttings (Martínez-Sánchez, Allende, Cortes-Galera, & Gil, 2008). Koukounaras et al. (2007) reported a decrease in RR with increase in maturity, from 500 to 300 mg CO2 kg−1h−1 at 10 °C. The same authors indicated a potential storage of 16 days for fresh rocket leaves stored in air at 0 °C, and 13 days at 5 °C, whereas, Amodio et al. (2015) reported a shelf-life of about 6 days for packed rocket stored at 5 °C, limited by appearance degradation, when an estimated atmosphere with about 8.5% of O2 and 8 of CO2 was reached. MartínezSánchez, Marin, Llorach, Ferreres, and Gil (2006) showed that a controlled atmosphere with 5 kPa O2 and 10 kPa CO2 was effective in preserving a good appearance of the leaves (still commercially acceptable after 14 days of storage) if compared to the storage in air (not commercially acceptable after 10 days) at 4 °C. A more recent work suggested that an atmosphere with O2 higher than 2 kPa and CO2 lower than 15 kPa can be optimal (Rux, Caleb, Geyer, & Mahajan, 2017). As

Corresponding author. E-mail address: [email protected] (M.L. Amodio).

http://dx.doi.org/10.1016/j.fpsl.2017.08.004 Received 19 April 2017; Received in revised form 5 July 2017; Accepted 8 August 2017 2214-2894/ © 2017 Elsevier Ltd. All rights reserved.

Please cite this article as: Mastrandrea, L., Food Packaging and Shelf Life (2017), http://dx.doi.org/10.1016/j.fpsl.2017.08.004

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2.2. Packaging gas composition

for volatiles, an accumulation of ethanol, dimethyl disulfide, aromatic aldehydes (benzaldehyde and benzeneacetaldehyde) and isopropyl isothiocyanate was observed when oxygen dropped below 2 kPa and particularly after washing; volatiles were extracted using the static headspace sampling technique after homogenization and pestling with a mortar for 60 s. According to various studies, extraction methods of volatile organic compounds (VOCs) including sample homogenization, manipulation, the eventual sample heating, and sampling technique, i.e. static (Rux et al., 2017) or dynamic headspace (Spadafora et al., 2016), SPME (Luca, Mahajan, & Edelenbos, 2016), hydrodistillation (Blažević & Mastelić, 2008), significantly affect the volatile composition since they may enhance enzymatic and oxidation reactions yielding to odor formation and degradation. Luca et al. (2016) investigated VOCs of wild rocket by SPME technique, from both intact and wounded leaves, stored in MA in closed jars to mimic packaging in low and high OTR films, at two different storage temperatures (5 and 10 °C). It was concluded that acetone, carbon disulfide, Dimethyl sulfide (DMS), dimethyl disulfide (DMDS), nitromethane, pentane, 3-methylfuran, 2ethylfuran, and DMDS were released in high concentrations under moderate O2 conditions (O2≥ 2.1 kPa). Spadafora et al. (2016) recorded VOC profile in fresh rocket packed in PP bags by using thermal desorption as VOC sampling technique and discriminated well between days and storage temperature. In this work, leaves were lightly manipulated (soft crushing) in order to enhance volatile release. The research found a close correlation of a group of aldehydes to the drop in vitamin C content, the fall in isothiocyanates and the increase in DMS and DMDS at 5° and 10 °C, compared with conditions at 0 °C. Loss of Vitamin C has also been associated with gas and temperature conditions over storage. Martínez-Sánchez et al. (2006) reported that ascorbic acid was converted into dehydroascorbic acid during storage and that vitamin C content was higher in rocket leaves stored in controlled atmosphere with the composition of 5 kPa O2 + 5 kPa CO2 and 5 kPa O2 + 10 kPa CO2, compared to air-stored samples. Amodio, Derossi, Mastrandrea, and Colelli (2015) found that ascorbic acid degradation was a critical factor in non-isothermal storage. Beneficial effect of MAP on rocket are amply discussed in literature, but the information on detrimental effect of suboptimal storage condition on shelf-life are seldom reported. Objective of this work was to assess the effect of improper MAP on ascorbic acid, appearance and volatile profile of washed rocket packed in PP bags and stored at constant temperature of 0, 5 and 15 °C. Moreover a second objective was to discriminate the effect of gas composition at 5 °C, by sampling volatiles in rocket leaves stored on macroperforated bags, without any sample manipulation.

Concentrations of O2 and CO2 inside the packages were monitored with a gas analyzer WITT Mapy 4.0 (Witten, Germany). Test probe of gas analyzer was inserted into each package through an adhesive rubber septum to prevent air leaking from the package. 2.3. Volatile extraction and headspace SPME GC–MS analysis Before sampling, bags were equilibrated at 15 °C for 30 min. Before sampling the control samples stored in macroperforated bags in air at 5 °C were transferred in non-perforated bags before sampling. Volatiles were collected in the bag headspace, introducing SPME fibre inside the package through a rubber septum. A carboxen/polydimethylsiloxane (CAR/PDMS) fibre of 85 μm was exposed for 30 min to the bag headspace and introduced into the GC injector port at 250 °C, for a desorption time of 4 min, using the split injection mode (1:20). An Agilent gas chromatograph model 6890 Series coupled to an Agilent 5975C network mass selective detector was used. Analytes were separated on a HP–5 ms capillary column (60 m × 250 μm × 0.25 μm) by applying the following temperature program: 40 °C for 4 min, up to 140 °C at 3 °C/min, with a final holding time of 10 min. The temperature program was optimized selecting temperature and time conditions providing the shortest run and the maximum number of volatile compounds. Transfer line temperature was 280 °C. Mass detector conditions were: electronic impact mode at 70 eV; source temperature at 230 °C; scanning rate at 2.88 scan/s; mass scanning range of m/z 30–400. The carrier gas was helium at 1.0 mL/min. Compounds were identified by comparing their retention times and mass spectra with those of pure compounds (Sigma-Aldrich, Milan, Italy), when available, or putatively assigned by comparing the mass spectra with the data of a system library (NIST 02, p > 80). 2.4. Vitamin C content Five grams of fresh rocket tissue were homogenized with 10 mL of MeOH/H2O (5:95) plus citric acid (21 g L−1) with EDTA (0.5 g L−1). The homogenate was filtered through cheesecloth and a C18 Bakerbond SPE column (Waters, Milford, MA, USA). Ascorbic acid (AA) and dehydroascorbic acid (DHAA) contents were determined as described by Zapata and Dufour (1992), with some modifications. The HPLC analysis was achieved after derivatization of DHAA into the fluorophore 3-(1,2dihydroxyethyl) furol [3,4-b]quinoxaline-1-one (DFQ), with 1,2-phenylenediamine dihydrochloride (OPDA). Samples of 20 μL were analyzed with an Agilent 1200 Series HPLC. The HPLC system consisted of a G1312A binary pump, a G1329A autosampler, a G1315 B photodiode array detector from Agilent Technologies (Waldbronn, Germany). Separations of DFQ and AA were achieved on a Zorbax Eclipse XDB- C18 column (150 mm × 4.6 mm; 5 μm particle size; Agilent Technologies, Santa Clara, CA, USA). The mobile phase was MeOH/H2O (5:95 v/v) containing 5 mM cetrimide and 50 mM potassium dihydrogen phosphate at pH 4.5. The flow rate was 1 mL/min. AA and DHAA contents were expressed as mg of ascorbic or dehydroascorbic acid per 100 g of fresh weight (mg 100 g−1).

2. Materials and methods 2.1. Plant material and processing Fresh rocket leaves (Diplotaxis tenuifolia) were harvested in Salento (Apulia, Italy). To ensure minimal processing phase the leaves were washed in chlorine solution (0.01% v/v) before being drained, portioned into 50 g samples and packaged in PP bags (17.5 × 17.5 cm2, OTR = 1800 cm3m2d−1, WVTR = 6gm2d−1 at standard temperate conditions). Fifty-four bags (3 replicates × 3 temperatures × 6 sampling times) were stored at 0, 5 and 15 °C for vitamin C determination and sensorial analysis and 54 bags were also arranged for the analysis of headspace volatiles. Similarly, 72 samples were placed in macroperforated bags of the same material, in order to prevent the modification of the atmosphere (control samples). Vitamin C and sensorial attributes were evaluated at day 0 and after 1, 2, 3, 6, 8 and 10 storage days for samples at 0 and 5 °C, while for samples kept at 15 °C sampling was interrupted on day eight. Volatiles analysis was carried out at day 0 and after 2, 3, 6, 7, 8 and 10 storage days for samples at 0 and 5 °C and after 8 storage days for samples at 15 °C.

2.5. Sensorial analysis The appearance and off-odor scores of all samples were evaluated by a 5-member trained panel. Appearance was subjectively scored on a 5 to 1 scale, where 5 = excellent (fresh and turgid appearance, bright and uniform green color), 4 = good (slight loss of turgidity and fresh appearance), 3 = fair (noticeable loss of turgidity and possible slight loss of green color), 2 = poor (severe loss of turgidity, wrinkling and yellowing of leafy blades), 1 = very poor (severe yellowing of leafy blades and wilting, possible appearance of decay). A score of 3 was considered as the limit of marketability. Off-odor was scored on a 5 to 1 2

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Fig. 1. Gas composition variations during the rocket storage at 0, 5 and 15 °C. Values are expressed as mean values of 3 replicates ± standard deviation.

scale, where 1 = no off-odor, 2 = slightly off-odor, 3 = moderate offodor (limit of marketability), 4 = strong off-odor and 5 = very strong off-odor, sulfur compounds and rotten cabbage taste. A score of 3 was considered as the limit of off-odor acceptability; over this limit the product was considered not marketable. 2.6. Statistical analysis Data represents the mean of three replicates for treatment (standard deviation is calculated). Vitamin C content, sensorial attributes and volatiles data were subjected to analysis of variance, using Statgraphics Centurion XVI software (Statpoint Technologies, Inc. |Virginia, US). Mean separation among treatments at each storage time was performed using the Tuckey test (p < 0.05) 3. Results and discussion 3.1. Atmosphere composition Fig. 3. Shelf-life evaluation for different quality parameters in rocket stored in air and MA condition at 0, 5 and 15 °C: appearance (a); off-odor production (b) and ascorbic acid (C).Different letters indicate significant differences among treatments (P < 0.05).

The variation in gas composition inside packages of rocket leaves stored at 0, 5 and 15 °C is shown in Fig. 1. From this figure it can be observed as O2 consumption and CO2 production rates were related to the storage temperature. Packaging conditions leaded to reach suboptimal gas composition inducing anoxia conditions after 3 days at 15 °C and 6 at 5 °C with CO2 peaks of 25 kPa. Gas composition inside the MA package at 0 °C accounted for 8.12 ± 4.05 kPa O2 and 13.67 ± 4.04 kPa CO2 in 10 storage days.

score (Fig. 2a) already below the limit of marketability (3), showing a complete oxidation of AA to DHAA (Fig. 2c,d). At the same time, offodor score (Fig. 2b) started to rise reaching already the limit of 3. As for samples stored in MAP at 5 °C, the consequences of anoxia were more visible after 8 days of storage, when samples reached an appearance score of 2, and a complete degradation of AA to DHAA was observed; off-odor score slightly increased from 6 to 8 days and then was suddenly beyond the marketability limit after 10 days. Samples stored in air at 15 °C showed an intermediate behavior between MAP stored

3.2. Vitamin C and sensory evaluations Fig. 2 illustrates the results of vitamin C and sensory evaluations. After 3 days, samples stored in MAP at 15 °C received an appearance

Fig. 2. Sensorial and quality attributes variation in rocket leaves stored in MA condition and in air at 0, 5 and 15 °C: (a) appearance score; (b) off-odor production, (c) ascorbic acid (AA), (d) dehydroascorbic acid (DHAA). Mean values of 3 replicates. Different letters at different times of storage indicate significant differences among treatments (P < 0.05).

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Table 1 Effect of modified atmosphere at 0°, 5° and 15 °C on volatile composition of rocket package headspace during the storage. Data are referred to the initial time (t0),and to 8 days of MAP storage (MAP t8) and air storage (AIR t8). tR, retention time. Values of peak area are divided for a factor of 106; different lowercase letters after peak area values indicate significant differences among 0°MAPt8, 15°MAPt8 and 5°MAPt8 samples (P < 0.05); different capital letters indicatesignificant differences between 5°MAPt8 and 5°AIRt8 samples (P < 0.05). 15° MAP t8

5° MAP t8

5° AIR t8

Odor descriptorc

574 ± 180 a 5440 ± 3000 a 9 ± 6 475 ± 170 a 8.47 ± 0.33 a 43 ± 4 a

11.0 ± 2.2 bA 2.6 ± 1.7 bB 17 ± 7 A 0.74 ± 0.03 b 0.27 ± 0.01 b 0.36 ± 0.04 b

1.03 ± 0.40 B 10.0 ± 2.7 A 1.9 ± 1.4 B 0.73 ± 0.02 0.25 ± 0.05 0.36 ± 0.03

sulfurous sulfurous alliaceous, garlic sulfurous, cabbage-like green pungent, horseradish

3.38 ± 1.14 b 0.54 ± 0.04 b 0.96 ± 0.04 b

482 ± 94 a 1160 ± 250 a 20.8 ± 6.5 a

7.8 ± 3.4 b 0.48 ± 0.06 b 0.92 ± 0.02 b

2.1 ± 1.5 0.54 ± 0.05 0.94 ± 0.04

ethereal, acetone, chocolate chemical green, bean-like

0.44 ± 0.17 19.4 ± 11.8

0.57 ± 0.02 b 6.3 ± 2.2

7.1 ± 4.3 a 6.5 ± 2.4

0.57 ± 0.03 b 7.3 ± 1.7 B

0.65 ± 0.08 22.83 ± 1.01 A

fatty aldehydic

7.3 ± 1.0 126 ± 30

2.55 ± 0.76 60 ± 28

5.7 ± 4.4 421 ± 417

3.6 ± 1.4 B 86 ± 47 B

11.8 ± 1.3 A 482 ± 55 A

herbal citrus, sweet, lemon

Volatile compounds

tR

t0

0° MAP t8

Sulfur compounds dimethyl sulfide d,e dimethyl disulfide d,e thiophene d,e tetrahydrothiophene d,e n-pentyl isothiocyanate e 4-methylpentyl isothiocyanate

3.69 8 5.78 10.44 24.91 27.9

0.54 ± 0.03 0.86 ± 0.01 35 ± 2 0.75 ± 0.06 0.237 ± 0.003 0.36 ± 0.01

0.53 0.74 23.6 0.71 0.24 0.32

4.68 6.59 19.54

3.64 ± 0.32 0.61 ± 0.10 0.96 ± 0.05

20.5 25.14 16.58 21.4

Furans 3-methyl furan d,e 2-ethyl furan e 2-pentyl furan d,e Aldehydes (E,E) 2,4-heptadienal nonanal d,e Terpenes α-Pinene d,e D-limonene d,e

d,e

e

± ± ± ± ± ±

0.03 0.06 1.6 0.01 0.03 0.01

b b b b b

a,b; Tukey HSD significant differences among MAP treatments at 0°, 5° an 15 °C. A,B; Tukey HSD significant differences between 5°MAPt8 and control in air. c Odor descriptors using published data (Jirovetz et al., 2002 and references therein; Sigma-Aldrich, 2001). d Compounds identified through retention times and mass spectra of pure compounds. e Compounds putatively identified by mass spectra comparison with Wiley library.

(Fig. 2c). The oxidized form, the DHAA decreased over time for samples stored at 0 °C and was significantly higher in leaves stored in air until the 3th storage day (Fig. 2d); the low amount of DHAA had a minimal contribution to the total Vitamin C content (data not shown), which was present especially in the form of ascorbic acid. A significant decrease was observed in the AA content in MA-stored rocket at 5 °C from the 6th storage days which was totally oxidized on the 8th day of storage (Fig. 2c); conversely, the amount of DHAA increased in packed leaves and was constant during the storage in air with significant differences from the 6th storage days (Fig. 2d). This data indicated a high antioxidant activity as response to stress. AA content rapidly decreased during the MA storage at 15 °C with significant differences respectively starting from the 2th day of storage (gas composition: 2.9 ± 1.3 kPa O2 and 23.8 ± 2.6 kPa CO2) as indicated in Fig. 2c; the third storage day resulted in the loss of AA, which was most likely converted to DHAA which, also in this case, was the predominant form (Fig. 2d). When considering loss in ascorbic acid, shelf-life was calculated as the number of days needed to reach a content of 20 mg 100 g−1, which corresponds to 50% of the recommended daily intake, as suggested by Australia and New Zealand Food Authority (2001). Shelf-life estimated for rocket stored in air at 0 and 5 °C and MAP at 0 °C was higher than 12 days and decreased to 8 days when samples were stored in MAP at 5 °C. Samples stored in air at 15 °C showed a shelf-life of about 6 days, which was reduced to 3 days when samples were stored in MAP (Fig. 3c) indicating an interaction with temperature and gas composition also at 15 °C. These data suggested that storage in MAP at 0 °C did not add any beneficial effect to rocket as the appearance score, ascorbic acid content were preserved and off-odor production was limited with no differences with the storage in air. Moreover, quality losses were much pronounced in MAP at 5 °C and 15 °C, starting from the 8th and 3th storage day, respectively, corresponding to an high accumulation of CO2 inside the bags and the complete depletion of O2.

samples at 15 and 5 °C, whereas sample stored in air at 5 °C were judged still marketable after 10 days of storage, showing no off-odor development, and a good retention of AA. As for samples stored at 0 °C, no differences were observed between MAP and air condition, not only for AA retention, but also for appearance score, and off odor. Fig. 3(a) shows the e shelf-life based on appearance score, calculated as the number of days to reach a score 3 (limit of marketability), being 11 days for MAP and air-stored samples at 0 °C, 10 and 4 days for samples stored in air at 5 and 15 °C, respectively. Improper MAP detrimental effects were evident at the higher temperatures, reducing the shelf-life to about 6 days at 5 and to 3 days at 15 °C. These results are in line with shelf-life prediction in air reported by other authors (Koukounaras, Siomos, & Sfakiotakis, 2007) being 16 days at 0 °C, and 13 days at 5 °C; few days of difference may be due to the different raw material and to the storage conditions (open bags vs humidified air flow). The off-odor perception increased over time in all samples stored at 15 °C and in MAP stored leaves at 5 °C. Leaves stored at 15 °C reached the highest score for off-odors already at the third day of storage (Fig. 2b), almost reaching the limit of acceptability at 6 days. At these temperatures samples were scored high for off-odor even when stored in air, reaching the limit of marketability at 8 days. Results in Fig. 2b indicated a very slight perception of off-odor during the storage of rocket at 0 °C in both MAP and air storage with no differences over time, thus hindering the shelf-life calculation which was estimated to be higher than 12 days, for rocket stored in air at 0 °C and 5 °C, and in MAP at 0 °C. Also in this case the reduction of shelf-life may be strongly associated to the gas composition at 5 °C (about 10 days) and to the temperature during the storage at 15 °C as samples stored in air and MAP showed a shelf life respectively of 8 days and 7 days. A slight increase of ascorbic acid (AA) was observed in the first few days of storage, as also reported in a previous study from Cavaiuolo, Cocetta, Bulgari, Spinardi, and Ferrante (2015). Generally AA was well retained at 0 °C, without differences for storage in air and in MAP (fig. 2c). AA content showed a slight reduction in samples stored in air at 5 °C (from 70.5 ± 9.1 mg/100 g to 45.9 ± 6.0 mg/100 g). For the other conditions, a progressive decrease was observed, being more pronounced in and MAP at 15 °C, followed by air and by MAP at 5 °C

3.3. Volatile profile Volatiles determination was done on bags different from those used for sensorial and quality evaluation; data of gas composition showed 4

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pentyl isothiocyanate, one of the volatiles responsible for the typical odor of rocket, is reported in Fig. 4b. A sharp increase of its content was observed at the 6th storage day for samples in MAP at 15 °C (peak area of about 15 × 106) and a considerable increase at 10th storage day for samples in MAP at 5 °C (Fig. 4b). Significant differences were found comparing samples stored in MAP and in air at 5 °C; in fact degradation reactions seemed not to have occurred in air, as shown by the constant values over time (Fig. 4b). For both MAP at 5 °C and 15 °C gas composition reached anaerobic conditions and an increase up to 25 kPa of CO2 after 3 days of storage at 15 °C and after 6 days at 5 °C, respectively (Fig. 1). It can therefore be supposed that the established gas conditions enhanced the degradation rate of cell structures and that temperature played a key role in accelerating these reactions. A similar behavior was observed for 4-methylpenthyl isothiocyanate. In accordance with Luca et al. (2016), a production of DMS (Fig. 4c) and DMDS (Fig. 4d), known to be off-odors (Nielsen, Bergström, & Borch, 2008), strongly depending on the temperature, was observed. DMS is formed either from (+)-S-methyl-L-cysteine sulfoxide (Marks, Hilson, Leichtweis, & Stoewsand, 1992) and by the degradation of some volatiles derived from glucosinolates (Jin, Wang, Rosen, & Ho, 1999). Broccoli seedling stored in anaerobic conditions produced DMS and DMDS through the degradation of cysteine containing protein (Derbali, Makhlouf, & Vezina, 1998). DMS (Fig. 4c) and DMDS (Fig. 4d) contents in rocket stored at 15 °C started to increase from the second day of storage and showed a sharp peak increase at the 8th day., while in the increase at 5 °C was much lower. DMS accumulation was higher for MAP samples compared to samples stored in air (Fig. 4c); differently,DMDS production was higher in control samples than in MAP, starting from the sixth day of storage (Fig. 4d). It is likely that the presence of oxygen enhanced the oxidation of sulfurous amino acid yielding to dimethyl disulfide. Tetrahydrothiophene and thiophene are typical odor compounds of rocket (Jirovetz et al., 2002; Miyazawa et al., 2002) with alliaceous, sulfurous notes of cabbage-like. Thiophene amount slightly decreased during the storage time and was best preserved in MAP treatments at 0 °C and 5 °C; at 5 °C, MAP samples showed a higher content of thiophene than samples in air (Table 1), suggesting a slower compound degradation at a lower of temperature and in MA conditions. Tetrahydrothiophene increased at 15 °C (Table 1), starting from the 6th storage day (data not shown), whereas no statistical difference was revealed at 5 °C between samples stored in MAP and in air.

similar values if compared with those acquired in packaged rocket used for aroma evaluation as described in Fig. 1. Thirteen volatiles, all previously reported in the literature for rocket (Miyazawa, Maehara, & Kurose, 2002; Jirovetz, Smith, & Buchbauer, 2002; Spadafora et al., 2016) were identified in the rocket package headspace and reported in Table 1, grouped in four classes: sulfur compounds (including also isothiocyanates), furans, aldehydes and terpenes. Peak areas relevant to volatiles found at day 0, which depict the headspace fingerprint of fresh rocket at its maximum quality, are also reported, together with peak areas relevant to volatiles found after 8 days of MAP storage at 0, 5 and 15 °C, corresponding to the last storage day for 15 °C samples. Their changes allowed to appreciate the effect of temperature, and the consequent change in gas composition, on the headspace profile. Finally, Table 1 reported also volatiles found in air stored-samples at 5 °C to point out differences, with respect to 5 °C-MAP rocket, due only to the effect of gas composition. The terpenes D-limonene and α-pinene, which are secondary metabolites produced as a defense mechanism and known to give floral notes to vegetables and fruits, showed no statistical difference due to the temperature and differences depending on gas composition (Table 1). Furans significantly increased during MA storage at 15 °C with no differences between storage at 0 and 5 °C and no changes have been observed in relation to treatment in air and MA (Table 1) indicating a fast deterioration at high temperature. Börjesson, Stöllman, and Schnurer (1992) suggested that 3-methylfuran could be of fungal origin. It has also been suggested that 3-methylfuran is released during oxidation of isoprene (Gu, Rynard, Hendry, & Mill, 1985) or as a degradation product of catechol or phenols (Huber, Wunderlich, Schöler, & Williams, 2010), or that Vitamin C is an efficient precursor for furans (Fan, Huang, & Sokorai, 2008; Limacher, Kerler, CondePetit, & Blank, 2007; Mark, Pollien, Lindinger, Blank, & Mark, 2006; Owczarek-Fendor et al., 2011). Its concentration in the package headspace increased significantly during the 15 °C MAP storage; about hundred times compared with the initial value, likely due to an accelerated fungi growth caused by the high temperature or to a new synthesis starting from Vitamin C precursors. As regards the effect of gas composition, during the 5 °C MAP storage, at 10th storage day, a significant increase was observed, if compared to samples stored in air, and this may be explained by the higher degradation of Vitamin C in MAP, compared to air. 2-ethyl furan, and 2-pentyl furan, both known to be a result of lipid autoxidation (Nawar, 1999) showed very low and constant concentrations at 0 and 5 °C, whereas their content increased during the storage at 15 °C. No statistical difference was found between the control and 5 °C MAP samples. These results suggest that the temperature enhanced the metabolic pathway yielding to furans, and that the effect could not be attributed to anaerobic conditions reached at 15 °C after 3 days and at 5 °C after 6 days. As an example, the behavior of 2-ethyl furan is reported in Fig. 4a. Aldehydes, which are also derived by fatty acid breakdown (Buttery, Parker, Teranishi, Mon, & Ling, 1981), showed different behavior: (E,E)-2,4-heptadienal production was significantly higher in MA stored samples at 15 °C compared to the other temperatures and no effect was observed depending on gas composition (Table 1); nonanal slightly decreased during storage without clear differences among samples stored in MAP at different temperatures. On the other hand, control samples showed values significantly higher than MAP samples stored at 5 °C (Table 1). These results may suggest that the presence of oxygen enhanced the lipid oxidation pathway generating nonanal, whereas MAP storage inhibited its formation. Also for sulfur compounds, a higher amount was found at 15 °C compared to 0 and 5 °C, with the exception of thiophene (Table 1); the off-odour responsible volatile production was also susceptible to O2 availability. Their formation could be due to membrane degradation processes and the consequent advances in enzymatic reactions (Cavaiuolo & Ferrante, 2014). Particularly, in samples stored in MAP at 15 °C the increase of isothiocyanates was observed. The trend for n-

4. Conclusion Results of this work showed that suboptimal packaging condition may decrease the shelf-life of rocket leaves compared to storage in air, and that no additional benefit was obtained by optimal gas composition when bags were stored at 0 °C. The detrimental effect induced by anoxia and CO2 accumulation could be observed on the leaf appearance, but also on the production of off-odors and the complete oxidation of AA to DHAA,. Temperature strongly affected shelf-life of MA packaged rocket leaves enhancing the degradation rate, either in air than in MAP, but differences were more evident in MAP and, in air, between samples at 15 °C and at lower temperatures (0 and 5 °C). These results underlined that packaging design and control of the temperature during the whole product chain are crucial factors to preserve the aroma and quality of rocket leaves. The importance of adapting packaging conditions to the respiration rate of the incoming products clearly demonstrated; this can be achieved by adjusting the perforation level of the plastic material based on metabolic activity. As alternative, when the modification of the atmosphere is not decisive as for cut leaves (i.e. controlling browning of lettuce) and/or if monitoring respiraton is not possible, high permeable materials should be preferred.

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