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Postharvest Biology and Technology 75 (2013) 75–85

Contents lists available at SciVerse ScienceDirect

Postharvest Biology and Technology

jou rna l h omepa g e: www.elsev ier .com/ locate /postharvbio

ff-odour development in modified atmosphere packaged baby spinach is annresolved problem

uan A. Tudelaa, Alicia Marína, Yolanda Garridoa, Marita Cantwellb,aría S. Medina-Martíneza, María I. Gila,∗

Research Group on Quality, Safety and Bioactivity of Plant Foods, Food Science and Technology Department, CEBAS-CSIC, P.O. Box 164, E-30100 Espinardo, Murcia, SpainDepartment of Plant Science, University of California, One Shields Avenue, Mann Laboratory, Davis, CA 95616-8631, USA

r t i c l e i n f o

rticle history:eceived 22 June 2012ccepted 19 August 2012

eywords:pinacia oleracea L.aby leafinimal processingAPualityicrobiology

a b s t r a c t

A major problem associated with minimally processed baby spinach (Spinacia oleracea L.) is strong off-odours when stored under modified atmosphere packaging (MAP) with low O2 and high CO2. Although theinfluence of O2 and CO2 levels on the quality and shelf-life of baby spinach has been extensively studied,results have been inconsistent and the benefits and disadvantages are not well understood. In this study,the effects of 3 different MAP conditions with low O2 with CO2 (stabilizing near 1% O2 + 11% CO2), lowO2 alone (stabilizing near 1% O2, CO2 scrubber) and moderate O2 with CO2 (stabilizing near 10% O2 + 9%CO2) were studied during storage at 7 ◦C for 12 days. Different parameters related to physiology, tissuestructure, microbial population and metabolite production were evaluated. Samples exposed to low O2

with CO2 had the lowest quality at the end of storage due to high development of off-odours, while off-odours of spinach in low O2 alone were intermediate but higher than in moderate O2 with CO2. Increasing

CO2 concentration significantly increased tissue damage with ammonia release and decreased proteincontent. Decreasing O2 concentration significantly reduced the development of aerobic psychrophilicbacteria and Pseudomonas. Senescence occurred more rapidly in baby spinach held in moderate O2 withCO2. Baby spinach quality remained acceptable during 7 days of storage at 7 ◦C, independent of MAPconditions tested. Appropriate MAP for baby spinach must be associated with maintenance of qualityand extension of shelf-life.

© 2012 Elsevier B.V. All rights reserved.

. Introduction

Minimally processed baby spinach is a very perishable leafyegetable with a shelf-life of only 7 days when stored at 7 ◦C.ajor problems associated with baby spinach are the develop-ent of strong off-odours, decay associated with breakage, and

issue softening (Medina et al., 2012). Off-odour development inodified atmosphere packaging (MAP) of baby spinach is still an

nresolved problem. MAP allows accumulation of significant lev-ls of volatiles in the restricted headspace volume and can lead tohanges in volatile generation (Kader et al., 1989). Accumulation ofon-respiratory volatiles can occur in MAP and some of them mayave negative effects (Toivonen, 1997). For example, ethanol andcetaldehyde can be accumulated if MAP conditions induce anaer-

bic metabolism (Ke et al., 1993). Some volatiles can be produceds a consequence of tissue starvation, as carbohydrate reserves areonsumed in postharvest storage (King et al., 1990). However, in

∗ Corresponding author. Tel.: +34 968 396 315; fax: +34 968 396 213.E-mail address: migil@cebas.csic.es (M.I. Gil).

925-5214/$ – see front matter © 2012 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.postharvbio.2012.08.006

the case of spinach and as a consequence of protein catabolism,ammonia is produced (Cantwell et al., 2010). Ammonia accumula-tion is the cause for dark-induced deterioration of leaves (Chibnall,1939).

The effects of controlled atmospheres (CA) or MAP on the qualitymaintenance of fresh-cut spinach have been extensively reviewed.In general, gas compositions inside MA packages are low in oxy-gen (O2) and high in carbon dioxide (CO2), depending primarilyon temperature, product fill weight and respiration rate, pack-age film O2 and CO2 transmission rates and the total respiringsurface area (Cameron et al., 1995). In well-designed equilibriummodified atmosphere packaging (EMAP), physiological processesare slowed down, resulting in a better preservation of plant tis-sue structure, which in turn can reduce microbiological processes(Jacxsens et al., 2003; Ragaert et al., 2007). Generally, low O2 orhigh CO2 concentrations decrease respiration rates and the rate ofproduct deterioration (Kader, 1986). It has been reported that baby

spinach requires high O2 in the package to maintain quality becauseof the high respiration rate (Allende et al., 2004). However, otherauthors have reported the benefits of low O2 atmospheres (0.8–3%)when spinach leaves are stored at higher temperatures (Izumi et al.,

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997). Low O2 atmospheres have been shown to be beneficial ineducing respiration, weight loss and antioxidant loss such as ascor-ic acid and flavonoids (Ko et al., 1996; Gil et al., 1999). Low O2tmospheres (0.8%) reduced the number of microorganisms, par-icularly the main spoilage agent Pseudomonas, but the control of

icrobial development on fresh-cut spinach is possible if the tem-erature does not exceed 5 ◦C (Babic and Watada, 1996). However,he beneficial effects of CA can be reversed by too low O2 or too highO2 concentrations. Ko et al. (1996) reported that O2 concentra-ion must be kept above 0.8% to prevent quality loss due to anoxia.n addition, increasing CO2 concentrations up to 13% caused theevelopment of off-odours and spinach was not acceptable after

week at 7 ◦C (McGill et al., 1996). It has been demonstrated thatO2 could decrease the pH of growth media (Daniels et al., 1985;ixon and Kell, 1989) but the bacteriostatic effect of CO2 is notell known. Allende et al. (2004) also reported that packages pre-ared with barrier films with an initial O2 level at 21% accumulatedO2 during storage and exhibited a significant reduction in aer-bic mesophilic bacteria growth compared to the perforated filmackages (control). However, this treatment also developed strongff-odours and loss of tissue integrity. Adding super atmospheric O2o the packages was beneficial in maintaining quality of fresh-cutaby spinach as it alleviated tissue injury in addition to reducingicrobial growth (Allende et al., 2004).Regulating the amount of O2 and CO2 in the environment of the

issue can control respiration rate of spinach and also off-odours.ncreasing the concentration of CO2 tends to reduce respiration rateBurgheimer et al., 1967). Decreasing the O2 concentration mayugment this reduction. However, if O2 is reduced below a criti-al level (<0.4%), undesirable anaerobic metabolism of spinach cane induced. Therefore, the control of the concentration of these twoases combined with refrigeration is critical for the maintenance ofroduct quality, avoiding the development of off-odours.

The objective of this study was the evaluation of the ben-ficial or detrimental effects of O2 and CO2 levels as possibletress factors for the development of off-odours during storagef baby spinach. Quality parameters such as sensory evalu-tion, colour and texture as well as parameters related toissue deterioration such as electrolyte leakage, tissue anatomynd cell microstructure, protein content, ammonia, pH, chloro-hyll content, carotenoid content, individual and total sugars,s well as microbiological quality were evaluated for up to2 d at 7 ◦C. Physiological and technological issues are dis-ussed.

. Materials and methods

.1. Plant material and sample preparation

Spinach (Spinacia oleracea L.) was cultivated under commer-ial conditions in Pulpí (Almería, Spain) by Primaflor S.L. Sowingas performed directly on elevated beds using a plant density of

00 plants m−2. Different quantities of plant material were har-ested several times to adjust MAP conditions, protocols, sampleariability, and number of replicates. The results presented corre-ponded to the same experiment in which 10 kg of plant materialere harvested mechanically on 13th October 2011. After harvest,

aby spinach leaves were transported (150 km) under refriger-ted conditions in polystyrene boxes to the CEBAS-CSIC laboratoryMurcia, Spain). On arrival, leaves with defects such as bruising oriscolouration were removed by hand. Then, baby leaves were kept

4 h at 4 ◦C and 70% relative humidity (RH) in darkness. A samplef 20 leaves was taken to measure leaf characteristics, which cor-esponded to length (8.5 ± 1.3 cm), maximum width (5.2 ± 0.5 cm)nd stem length (2.7 ± 1.0 cm).

nd Technology 75 (2013) 75–85

2.2. Processing, packaging and storage conditions

Baby leaves were processed in an isolated clean minimal pro-cessing room at 4 ◦C. Samples were washed for 30 s in a cold (4 ◦C)100 mg L−1 chlorine solution (NaOCl) adjusted to pH 6.5 with cit-ric acid, drained for 30 s and then rinsed with tap water for 30 s.Excess water was removed by spinning for 1 min at 440 rpm inan automatic salad spinner (K-50, Kronen GmbH, Kehl am Rhein,Germany). Three passive modified atmosphere packaging (MAP)conditions were created adjusting the film permeability character-istics, sample weight, package size and the optimal CO2 scrubber.Samples of 100 g were manually packed in a package size of230 mm × 310 mm. Packages were sealed under air conditions.MAP of low O2 with CO2 was obtained using a 35 �m polypro-pylene (PP) film (Amcor Flexibles, Bristol, UK), with permeabilityof 474 mL O2 m−2 d−1 atm−1 and 1507 mL CO2 m−2 d−1 atm−1 at7 ◦C and 97% RH (Moyls, 2004). MAP of low O2 without CO2 wasobtained with the same film but adding 20 g of Ca(OH)2 as CO2scrubber (Kader, 2002). MAP of moderate O2 with CO2 was obtainedusing the same film mentioned above but with 1 macro-perforation(approx. 2 mm diameter), increasing the permeability to 5410 mLO2 m−2 d−1 atm−1 and 5637 mL CO2 m−2 d−1 atm−1 at 7 ◦C and 97%RH. Packages were stored in darkness for 12 d at 7 ◦C.

Gas composition, sensory evaluation, colour, chlorophyll andcarotenoid content, texture, electrolyte leakage, protein content,ammonia content, pH, sugar content and fermentative metaboliteswere evaluated after processing (0 d) and during storage (3, 7, 10,12 d), whereas leaf anatomy and cell microstructure were evaluatedafter 0 and 12 d, and microbiological quality after 12 d.

2.3. Headspace analysis

Gas composition (O2 and CO2 kPa) of individual packages wasmeasured during storage using a gas chromatograph equipped witha thermal conductivity detector (TCD) (Shimadzu CG-14B, Kyoto,Japan). Samples of 0.25 mL of headspace gas were taken through aseptum (a patch of silicone sealant applied to the film) using a 1 mLplastic tuberculin syringe with 25-gauge needle. At each samplingday, 3 packages from each MAP condition were measured.

2.4. Sensory evaluation, colour, chlorophyll content, carotenoidcontent and texture

Baby spinach from 3 packages were combined and examinedby a 5 member trained panel at the beginning of the experimentand during the storage period. Panel members were requested toassess spinach off-odours (just after opening the bag and again5 min later) using a 5 point scale where 5 = severe, 3 = moderateand 1 = none. Visual quality was evaluated considering freshness,appearance, colour uniformity, and brightness following a 9 pointrating scale where 9 = excellent, 7 = good, 5 = fair (limit of consumeracceptability), 3 = poor and 1 = extremely poor (Medina et al., 2012).

Leaf colour was determined on photosynthetic tissue using acompact tristimulus colorimeter (Minolta CR-300, Ramsey, NJ, USA)with an 8 mm diameter-viewing aperture. Twenty leaves per MAPcondition were measured at 0 d and after 3, 7, 10 and 12 d ofstorage. Results were expressed as L*, a*, b* and chroma index[(a*2 + b*2)1/2]. Extraction and quantification of chlorophylls andcarotenoids were done as described by Wellburn (1994). Samplesof 0.3 g of tissue were mixed with 10 mL of pure methanol andwere incubated in darkness for 24 h at room temperature. Afterthis period, absorbance at 470, 653 and 666 nm was measured for

carotenoids, chlorophyll b and chlorophyll a, respectively.

Texture was measured by a compression-shear test with aKramer shear cell consisting of a five blade probe on a univer-sal testing machine (model TA.TX.plus texture analyser, Stable

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icro Systems, Godalming, UK) equipped with a 294.2 N loadell. Twenty five leaves without stem were placed in the Kramerhear cell perpendicularly oriented to the blades. The test waserformed to 10 mm distance using a test speed of 1.67 mm s−1.orce–deformation relationships were analysed and results werexpressed as maximum force.

.5. Electrolyte leakage, protein content, ammonia and pH

Electrolyte leakage was measured as described by Fan andokorai (2005) with slight modifications. Leaves randomly takenere cut in 4 pieces, and 2 of them were used to obtain 5 g of tissue

hat were immersed in 125 mL of deionized water. The electricalonductivity of the bathing solution was measured at 1 min, 60 minnd after autoclaving (121 ◦C for 20 min) at room temperature using

conductivity meter (Model CM35, Crison, Barcelona, Spain). Foureplicates were evaluated per MAP condition.

Soluble protein was determined by Bradford (1976) usingovine serum albumin as standard. Twenty grams of spinachere homogenized for 1 min in 10 mL of 50 mM Tris–Acetic

cid–EDTA (ethylendiamintetracetic acid) buffer (pH 6.0), contain-ng 2 mM cysteine, 1 mM phenylmethylsulphonylfluoride (PMSF),olyvinylpolypyrrolidone 2% (w/v) (PVPP) and 0.2% (w/v) Triton-100. The mixture was filtered and centrifuged at 10,600 × g for0 min at 4 ◦C. Then, 12 �L of supernatant was mixed with 988 �Lf Bradford’s reagent. The mixture was incubated at room temper-ture for 20 min and the absorbance measured at 595 nm.

For the determination of ammonia content, 5 g of frozen tissueere homogenized with an Ultra Turrax (Ika, Staufen, Germany) in

0 mL of water on an ice bath and centrifuged in a centrifuge (model408R, Eppendorf AG, Hamburg, Germany) at 10,600 × g for 10 mint 4 ◦C. Then, 0.1 mL of supernatant was mixed with 1 mL of phe-ol nitroprusside reagent and 1 mL of alkaline hypochlorite. Theixture was incubated at 30 ◦C for 15 min and the absorbance waseasured at 625 nm at room temperature (Weatherburn, 1967).

he calibration curve was prepared using ammonium sulphate.esults were expressed as mg 100 g−1 f.w. For measuring pH, 2 g of

resh tissue were homogenized in 30 mL of distilled water. The pHf homogenate was measured with a pH-meter Metrohm (model85 DMP Tritino, Herisau, Switzerland).

.6. Fermentative metabolites, glucose, fructose, sucrose and totalugar content

Analysis of fermentative metabolites was performed byead-space technique following the methodology described byelayo-Zaldívar et al. (2007). Samples were injected into a Perkinlmer AutoSystem gas chromatograph (Waltham, MA) equippedith a flame ionization detector (FID) and separated on a 60/80arbopack glass column (1.8 m × 2 mm) containing 5% CarbowaxSupelco, Bellefonte, PA).

For the analysis of individual and total sugar content, 5 g ofresh baby spinach were homogenized in 10 mL of water (Pelayo-aldívar et al., 2007). The homogenate was centrifuged at 10,600 × gor 10 min at 4 ◦C. The supernatant was flushed through an acti-ated Sep-Pak C-18 cartridge (Waters, Milford, MA) and thenltered through a 0.45 �m membrane filter. Samples of 20 �Lere analysed using a HPLC system (VWR-Hitachi) equipped with

pump (model L-6200), a refractive index detector (model L-490) and an autosampler (model AS2000A). Separations of sugarsere achieved on a Supelcogel C-610H column (300 mm × 7.8 mm;

upelco, Bellefonte, PA). Sugars were quantified by comparisonsith authentic markers from Sigma–Aldrich (St. Louis, MO). Resultsere expressed in mg per 100 g−1 f.w. Three replicates per MAP

ondition and storage period were analysed.

nd Technology 75 (2013) 75–85 77

2.7. Leaf anatomy and cell microstructure

The study of leaf anatomy and cell microstructure was carriedout in two of the three MAP conditions, low O2 with CO2 and moder-ate O2 with CO2, on days 0 and 12. Tissue samples of approximately3 mm × 5 mm were excised from the centre of the leaf lamina nearthe midrib and parallel to it. Samples were fixed for 2.5 h at 4 ◦C ina 0.1 M sodium phosphate-buffered (pH 7.2) mixture of 2.5% glu-taraldehyde and 4% paraformaldehyde (Morales et al., 2001). Tissuewas post-fixed with 1% osmium tetraoxide for 2 h. The sampleswere then dehydrated in a graded alcohol series and embeddedin Spurr resin (Spurr, 1969). Blocks were sectioned by an ultra-cutmicrosystem (Leica Mikrosysteme, Hernalser Hauptstraße, Vienna,Austria). Semi-thin sections (0.5 �m) were stained with 0.5% tolui-dine blue in borate buffer. Microphotographs of semi-thin sectionswere captured with a Leica DMR light microscope (Leica, Wet-zlar, Germany) using the 10× objective. Images were processedusing the software Leica QWin Pro V3 (Leica, Wetzlar, Germany).A minimum of 25 images from 15 leaves were studied. In order todefine the area occupied by epidermis, parenchyma and intercellu-lar spaces, a rectangular reference area from the cut leaf section wasdefined as follows: the longer side corresponding to the complete‘leaf thickness’ and the shorter side of 350 �m long (Supplementaryinformation). Leaf anatomy was carried out by the analysis of 305and 1134 epidermal and parenchymal cells, respectively. Withinthis reference area, the leaf surface cells were included, but cellsbisected by or touching the long side were excluded, as well asvascular bundles. In the remaining space, the areas occupied by epi-dermis, parenchyma and intercellular spaces were measured andexpressed as % of total area.

2.8. Microbiological analysis

Leaf samples, including blade and stem, of 30 g were analysed formicrobiological evaluation as previously described (López-Gálvezet al., 2010). Samples were homogenized for 2 cycles of 60 s in 0.1%sterile buffered peptone water (BPW) (AES Laboratoire, Combourg,France) (1:10 dilution). Psychrophilic bacteria were enumeratedusing plate count agar (PCA) (Scharlau Chemie S.A., Barcelona,Spain) incubated at 7 ◦C for 7 d. Pseudomonas spp. were counted inPseudomonas agar (Oxoid, Ltd., Basingstoke, Hampshire, England)at 30 ◦C for 24–48 h. Total coliforms were isolated in Chromocultagar (Merck, Darmstadt, Germany) at 37 ◦C for 24 h. Colonies ofListeria spp. were enumerated on plates of Listeria selective agar(Oxford formulation, Oxoid, Basingstoke, Hampshire, UK) at 37 ◦Cfor 48 h. Three replicates were analysed in duplicate and microbi-ological counts were expressed as log CFU g−1 of tissue.

2.9. Statistical analysis

Analysis of variance (ANOVA) with a significant level of P < 0.05was performed to compare the influence of MAP conditions duringstorage. When significant differences were observed, the Tukey’sHSD (Honestly Significant Difference) test was applied using PASWStatistics 18 for Windows (SPSS Inc., Chicago, IL, USA). Least signif-icant differences (LSD) at a significant level of P < 0.05 are shown infigures.

3. Results

3.1. Headspace analysis

Changes in the headspace gas composition of baby spinach atdifferent MAP conditions are shown in Fig. 1. Reductions in O2 downto 1.1% were observed during storage of baby spinach in packagesof non-perforated film with and without CO2 scrubber (Fig. 1A). In

78 J.A. Tudela et al. / Postharvest Biology and Technology 75 (2013) 75–85

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2 (

%)

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%)

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25

Low O2 + CO2

Low O2

Mode rate O2+ CO2

A B

F ach sO fferen

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ig. 1. Changes in headspace gas composition [% O2 (A) and % CO2 (B)] of baby spin2 with CO2. Each symbol represents a mean of three packages. Least significant di

on-perforated packages, the stabilizing MAP was near 1% O2 + 11%O2 because of the high CO2/O2 ratio ( ̌ = 3.2) (Fig. 1B). However, inerforated packages, CO2 and O2 reached similar levels because ofhe similar CO2/O2 ratio ( ̌ = 1). As expected, CO2 was not detectedn packages of baby spinach with the CO2 scrubber.

.2. Sensory evaluation, colour, chlorophyll and carotenoidontent and texture

Significant differences in the sensory attributes were observedmong MAP conditions. Off-odours increased during storage, beingotably more perceptible under low O2 with CO2 (Fig. 2A). After0 d, baby spinach exposed to low O2 with CO2 was rejectedecause of the severe off-odours. Five minutes after opening the

ags and placing spinach on a tray, severe off-odours persisted andere similar to those detected just after opening the bags (Fig. 2B).

he off-odours were not so noticeable when baby spinach wastored under low O2 alone. When baby spinach was stored under

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ig. 2. Changes in off-odours of baby spinach stored at 7 ◦C in MAP of low O2 with CO2, loB). Slashed line indicates the limit of acceptance. Values are the mean of 6 replicates. Lea

tored at 7 ◦C in three MAP conditions: low O2 with CO2, low O2 alone or moderatece (LSD) at a significant level of P < 0.05.

moderate O2 with CO2, development of off-odours was minor. Atthe end of storage, off-odours exceeded the limit of acceptabilityand caused the rejection of samples exposed to low O2 with CO2.When the overall visual quality was evaluated, it was observed thatduring storage the scores decreased and caused the rejection ofbaby spinach after 12 d without significant differences among MAPconditions (Fig. 3).

Statistical analysis of colour parameters (L*, a*, b* and chroma)showed significant differences during storage with slight differ-ences among MAP conditions. In general, the behaviour of samplesstored under moderate O2 with CO2 differed from the other twoMAP conditions (low O2 with and without CO2). At the end ofstorage, the colour of baby spinach stored under moderate O2with CO2 showed significantly lower a*, higher b* and higher

chroma than samples stored in low O2 (with and without CO2)(Fig. 4B–D).

The content of chlorophyll decreased when baby spinach wasstored in MAP with CO2 independently of the O2 level while in

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w O2 alone or moderate O2 with CO2 just after opening the bags (A) and 5 min laterst significant difference (LSD) at a significant level of P < 0.05.

J.A. Tudela et al. / Postharvest Biology a

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Fig. 3. Changes in visual quality of baby spinach stored at 7 ◦C in MAP of low Owaa

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Fa

2

ith CO2, low O2 alone or moderate O2 with CO2. Slashed line indicates the limit ofcceptance. Values are the mean of 6 replicates. Least significant difference (LSD) at

significant level of P < 0.05.

pinach stored in low O2 alone there was no significant changeuring storage (Fig. 5A). The content of carotenoids decreased dur-

ng storage with no significant differences among MAP conditionsFig. 5B).

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b*

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L*

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ig. 4. Changes in colour as L* (A), a* (B), b* (C) and chroma index (D) of baby spinach storre the mean of 25 replicates. Least significant difference (LSD) at a significant level of P <

nd Technology 75 (2013) 75–85 79

Maximum force to shear leaves did not vary significantly duringstorage or in relation to MAP conditions (data not shown).

3.3. Electrolyte leakage, protein content, ammonia and pH

Significant differences in electrolyte leakage were observed dur-ing storage (Fig. 6A). These differences were more pronounced atthe end of the storage period where the electrolyte leakage sig-nificantly increased in samples exposed to low O2 without CO2.The content of proteins decreased noticeably from 3 to 7 d of stor-age, particularly in MAP with CO2, and then was maintained untilthe end of storage (Fig. 6B). We observed that extensive proteincatabolism occurred after 3 d of storage and as a consequence anaccumulation of ammonia in the tissues was detected. Changes inammonia content and pH showed a very similar trend. Ammoniacontent increased during storage with the highest concentrationin baby spinach stored in MAP with CO2 and the lowest in leavesstored in low O2 (Fig. 7A). Tissue pH increased progressively from6.6 to 7.2 in MAP with CO2 but increased only slightly during storagein low O2-stored samples (Fig. 7B).

3.4. Glucose, fructose, sucrose, total sugar content and

fermentative metabolites

Glucose, fructose, sucrose and total sugar content were alsodetermined to evaluate if they were substrates associated with low

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ed at 7 ◦C in MAP of low O2 with CO2, low O2 alone or moderate O2 with CO2. Values 0.05.

80 J.A. Tudela et al. / Postharvest Biology and Technology 75 (2013) 75–85

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a of P < 0

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ig. 5. Changes in the content of chlorophyll (A) and carotenoid (B) of baby spinachre the mean of 4 replicates. Least significant difference (LSD) at a significant level

2 and high CO2 storage conditions. The total sugar content as theum of the content of individual sugars decreased during storageith no differences among MAP conditions (Fig. 8A). Glucose con-

ent of baby spinach was well maintained during storage with noifferences among MAP conditions (Fig. 8B). However, the contentf fructose and sucrose decreased greatly during storage. Fructoseeclined by 80% between 3 and 7 d and remained unchanged dur-

ng the rest of storage (Fig. 8C). Sucrose also declined by 80% at d 3nd gradually declined to the limit of detection after 10 d (Fig. 8D).

Ethanol and acetaldehyde were analysed as fermentativeetabolites induced under low O2 with and without CO2. How-

ver, accumulation of these metabolites was not observed in babypinach stored under any of the MAP conditions tested.

.5. Leaf anatomy and cell microstructure

Difference in leaf thickness was observed between storagetmospheres (Table 1). Leaf thickness increased in low O2 whereas

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ig. 6. Changes in electrolyte leakage (A) and protein content (B) of baby spinach storedre the mean of 3 replicates. Least significant difference (LSD) at a significant level of P < 0

d at 7 C in MAP of low O2 with CO2, low O2 alone or moderate O2 with CO2. Values.05.

it did not change in moderate O2. Changes in epidermal andparenchymal cell areas were examined. When the epidermal cellarea was measured, no differences were observed during storageand between MAP conditions (Table 1). However, the parenchy-mal cell area increased when leaves were stored in MAP with thelargest increase observed in low O2 (Table 1). The area occupied bythe epidermis, parenchyma, and intercellular spaces differed sig-nificantly between MAP conditions (Table 1). The area occupiedby parenchyma increased while the intercellular spaces decreasedwhen samples were stored under moderate O2 with CO2.

The study of the ultrastructural appearance of cells was carriedout by optical microscopy. At d 0, just after processing, althoughsome cells showed some damage, most of them showed a continu-ous cytoplasm close to the cellular margin because of the pressure

of vacuoles, which invaded most of the cellular volume (Fig. 9A).After 12 d, leaves that were stored in low O2 showed a discon-tinuous cytoplasm along the cell margin, (Fig. 9B). Tonoplast wasseparated from the cell wall, meaning that the vacuole did not

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at 7 ◦C in MAP of low O2 with CO2, low O2 alone or moderate O2 with CO2. Values.05.

J.A. Tudela et al. / Postharvest Biology and Technology 75 (2013) 75–85 81

A B

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10

20

30

40

50

60

70

80

Days of storage

121086420

pH

6.4

6.6

6.8

7.0

7.2

7.4

LSD = 0.0 2LSD = 4

Low O2+ CO2

Low O2

Mod erate O2+ CO2

Fig. 7. Changes in ammonia (A) and pH (B) of baby spinach stored at 7 ◦C in MAP of low O2 with CO2, low O2 alone or moderate O2 with CO2. Values are the mean of 3replicates. Least significant difference (LSD) at a significant level of P < 0.05.

Days of storage

0 2 4 6 8 10 12

Su

cro

se

co

nte

nt

(mg

10

0 g

-1 f

w)

0

20

40

60

80

Days of storage

0 2 4 6 8 10 12

Fru

cto

se

co

nte

nt

(mg

10

0 g

-1 f

w)

0

20

40

60

80

Glu

co

se

co

nte

nt

(mg

10

0 g

-1 f

w)

0

100

200

300

400

To

tal su

ga

r co

nte

nt

(mg

10

0 g

-1 f

w)

0

100

200

300

400

5=DSL21=DSL

41=DSL2=DSL

Low O2+ CO2

Low O2

Mode rate O2+ CO2

A B

C D

Fig. 8. Changes in total sugar content (A), glucose (B), fructose (C) and sucrose (D) of baby spinach stored at 7 ◦C in MAP of low O2 with CO2, low O2 alone or moderate O2

with CO2. Values are the mean of 3 replicates. Least significant difference (LSD) at a significant level of P < 0.05.

8 logy a

oaCta

Ft

2 J.A. Tudela et al. / Postharvest Bio

ccupy the whole cellular space. Leaves that were stored in moder-

te O2 showed more evident symptoms of deterioration (Fig. 9C).ytoplasm showed more fracture and vacuoles were more con-racted as well as there was an increased appearance of vesicless cell degradation products.

ig. 9. Transverse section (a) and details of cells (b) of baby spinach at d 0 (A) and after 1he upper epidermis (ue), parenchyma (p), intercellular space (i), lower epidermis (le), va

nd Technology 75 (2013) 75–85

3.6. Microbiological analysis

The influence of MAP on the microbial populations of babyspinach was significant. After 12 d, microbial population of babyspinach stored in MAP of low O2 with CO2 was significantly lower

2 d at 7 ◦C in MAP of low O2 with CO2 (B) or moderate O2 with CO2 (C). Details ofcuole (v), cytoplasm (c), (t) tonoplast and (ve) vesicles.

J.A. Tudela et al. / Postharvest Biology and Technology 75 (2013) 75–85 83

Table 1Leaf thickness, area of epidermal cell and parenchyma cell, and percentage of area occupied by epidermis, parenchyma and intercellular spaces of baby spinach at day 0 andafter 12 days of storage at 7 ◦C in MAP of low O2 with CO2 and moderate O2 with CO2.

Days of storage MAP condition Leaf thickness(�m)

Area of epidermalcell (�m2)

Area of parenchymacell (�m2)

Area occupied byepidermis (%)

Area occupied byparenchyma (%)

Area occupied byintercellular spaces (%)

0 608b 554 1032b 5a 35ab 60b12 Low O2 + CO2 670a 547 1202a 4b 33b 63a

Moderate O2 + CO2 606b 521 ns 1135a 5ab 36a 59b

Values are the mean of at least 60, 305, 1134 and 25 replicates for leaf thickness, area of epidermal cell and parenchyma cell and area occupied by epidermis, parenchymaand intercellular spaces, respectively. ns, not significant.

C

Packaging condition

Low O2 + CO

2Low O

2Moderate O

2 + CO

2

Tota

l colif

orm

s (

log C

FU

g-1

)

0

1

2

3

4

5

6

7

8

9 D

Packaging condition

Low O2 + CO

2Low O

2Moderate O

2 + CO

2

List

eria

spp.

(lo

g C

FU

g-1

)

0

1

2

3

4

5

6

7

8

9

A

Psych

roph

iles (

log C

FU

g-1

)

0

1

2

3

4

5

6

7

8

9

10

B

Pse

udom

onas

spp

.(lo

g C

FU

g-1

)

0

1

2

3

4

5

6

7

8

9

10

LSD = 0.22 LSD = 0.23

LSD = 0.15

LSD = 0.37

ab

c

ns

c

ab

ns

Fig. 10. Psychrophilic bacteria counts (A), Pseudomonas (B), coliforms (C) and Listeria spp. (D) (log CFU g−1) of baby spinach stored at 7 ◦C for 12 days in MAP of low O2 withCO , low O alone or moderate O with CO . Bars are the mean of 3 replicates plus standard deviation. Bars with different letters are significantly different (P < 0.05) accordingt

(oOtlottlpcCOt

2 2 2 2

o HSD (Honestly Significant Difference) test. Ns, not significant.

approx. 0.4–0.70 log) than that in MAP of low O2 alone and MAPf moderate O2 with CO2 (Fig. 10A). Baby spinach exposed to low2 alone showed slightly higher microbial counts consistent with

he anticipated antimicrobial effect of CO2. Low O2 resulted inower microbial counts when compared with moderate O2. MAPf moderate O2 with CO2 was shown to be the gas composi-ion that most allowed psychrophilic growth because it was closeo the optimum O2 for the growth of aerobic bacteria. The CO2evel reduced the bacteria growth on baby spinach. Pseudomonasopulations showed similar behaviour to those observed for psy-

hrophilic bacteria. Modified atmosphere packaging of low O2 withO2 reduced Pseudomonas significantly compared with moderate2 with CO2 (Fig. 10B). However, populations of coliforms and Lis-

eria spp. were not greatly affected by the type of atmosphere. After

12 d, these groups of microorganisms increased similarly regardlessof the atmosphere composition (Fig. 10C and D).

4. Discussion

MAP significantly affected off-odours of baby spinach duringstorage. MAP of low O2 with CO2 exhibited the lowest overallquality at the end of storage primarily due to the developmentof off-odours, followed by MAP of low O2 alone, which were theconditions in which baby spinach showed loss of freshness. The

fermentative volatiles, acetaldehyde and ethanol, routinely used asindicators of stressful MA for a broad range of vegetables, were notresponsible for the off-odours in baby spinach. We observed thatbaby spinach exposed to CO2 increasingly accumulatedammonia

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dooacalwoateb(oaaaMisittcb

b2rs

4 J.A. Tudela et al. / Postharvest Bio

p to 7 d, but then only slight increases were observed to thend of storage. The ammonia production of baby spinach storedithout CO2 was very small. Ammonia content was previously

eported as a good indicator of CO2 injury in spinach (Rodriguez andantwell, 2002). Spinach stored under a range of CO2-containingtmospheres was reported to show important increases in ammo-ia concentrations (Cantwell et al., 2010). However, in our study,mmonia was not the only compound associated with the develop-ent of off-odours as those leaves stored in MAP without CO2 also

eveloped off-odours despite the low ammonia content. Increasesn ammonia have been associated with senescence symptoms

hen spinach was stored in air or in CA with elevated CO2 levelsMartínez-Damian and Cantwell, 1999). In our study, we observedhat ammonia accumulation was not the only cause of leaf senes-ence as baby spinach with the lowest content of ammonia showedhe highest tissue damage and leaf senescence. Ammonia can beoxic to plant cells (Toivonen, 1997), although in our study, babypinach exposed to moderate O2 with CO2 showed clearer symp-oms of cell damage, in agreement with the accumulation of toxic

olecules such as free radicals causing the early phase of senes-ence (Wagstaff et al., 2007). The senescence effect in baby spinachould be attributed more to oxidative metabolism as differencesetween MAPs were related to O2 levels.

Although no significant differences in colour between MAPsere observed, samples stored under moderate O2 with CO2

howed slight variations in all colour parameters, indicating a lessark and more yellow colouration. One of the symptoms of senes-ence in harvested leafy vegetables is loss of greenness with theegradation of chlorophyll (Chl). Quantitative changes in Chl andegradation products of Chl and the hydrolysing enzymes haveeen monitored in spinach (Yamauchi and Watada, 1991). Webserved degradation of Chl in spinach leaves during storage buthe less Chl degradation appeared to be related to the absence ofO2.

Several authors suggested that the ammonia accumulation wasue to protein catabolism (Levitt, 1972; Schwerdtfeger, 1978). Inur study, the progressive decrease in protein content up to 7 df storage was well correlated with the progressive increase inmmonia. However, after 7 d, protein catabolism remained almostonstant to the end of the storage for all MAP conditions whereasmmonia accumulation progressed, except in samples stored inow O2 with CO2. Losses of specific volatiles can be enhanced by

ater loss (Toivonen, 1997). We have found that the developmentf off-odours in baby spinach was independent from water loss. Theccumulation of off-odours was similar in baby spinach exposedo different RH with different evapotranspiration rates (Medinat al., 2012). No difference in the conversion from protein to solu-le nitrogen between CA and air storage of spinach was reportedBurgheimer et al., 1967). Toivonen (1997) suggested that the modef action for ammonia injury may be through effects on tissue pHnd thus plant tissues high in organic acids are less susceptible tommonia injury (Levitt, 1972). In our study, pH of spinach macer-tes increased 0.6 units during storage in baby spinach stored inAP with CO2. Burgheimer et al. (1967) also reported the increase

n pH to over 7 in CA storage (4% O2 + 9.2% CO2) of spinach while airtorage caused only a slight rise in pH. The increase in pH observedndicated that baby spinach was injured by CO2 and was a responseo ammonia. In previous work, we also observed that an increase inhe pH of fresh-cut spinach stored in MAP corresponded with thehange in the equilibrium between ascorbic acid and dehydroascor-ic acid towards dehydroascorbic acid (Gil et al., 1999).

The respiration rate of baby spinach has been reported to

e higher than other fresh leafy vegetables, ranged from 4.6 to3.0 �L CO2 g−1 h−1 (Cantwell et al., 1998). Izumi et al. (1997)eported that when spinach leaves where held in low O2 atmo-pheres, CO2 production was not reduced but little to no off-odours

nd Technology 75 (2013) 75–85

were detected. The accumulation of volatiles was not a conse-quence of carbohydrates consumed by respiration during storageas no variation in individual and total sugars were observed inbaby spinach held in different MAP conditions. The effect low O2and high CO2 at low temperatures on microorganisms of fresh-cut spinach leaves has been studied. Izumi et al. (1997) reportedthat low O2 atmospheres did not affect the total number of aerobicmesophilic bacteria, lactic acid bacteria, or development of decay.Baby spinach leaves harboured high numbers of psychrophilic aer-obic bacteria, identified mainly as a pectinolytic species (Babic andWatada, 1996). These authors reported that low O2 atmospherescould be used to control spoilage microorganisms on cut spinachleaves at least for 7 d. We observed that low O2 with or without CO2reduced the number of psychrophiles and Pseudomonas microor-ganisms in baby spinach leaves compared to moderate O2 withCO2. Several authors reported that enriched CO2 atmospheres hada significant inhibitory effect on the growth of aerobic microor-ganisms on broccoli (Berrang et al., 1990), chicory leaves (Carlinand Nguyen-the, 1994) and fresh-cut spinach (Babic and Watada,1996). Wells (1974) reported a decrease in growth rates of P. fluo-rescens by concentrations of CO2 in excess of 10%. Ibe and Grogan(1983) demonstrated that O2 concentrations of 4% or less combinedwith CO2 concentrations of 10% or more reduced the growth of P.fluorescens by 50% compared to air. The inhibitory effect of low O2with CO2 was not due to an acidification of the microbial environ-ment, but probably to decreased oxygen availability in agreementwith Babic and Watada (1996) who reported that low O2, ratherthan high CO2 seemed to be the limiting factor for the growth ofaerobic microorganisms on spinach leaves at 5 ◦C but not at 10 ◦C.We found that baby spinach stored to moderate O2 with CO2 hadhigh microbiological counts probably because of the high degreeof nutrient release by the senescence tissue. Moreover, after 12 d,leaves that were stored in moderate O2 with CO2 showed a discon-tinuous cytoplasm along the cell margin, which indicated increasedmembrane permeability and the characteristic process of necroticdeath in vegetative cells (Van Doorn et al., 2011).

5. Conclusions

Off-odours from baby spinach were greatly increased by thepackaging headspace composition in terms of low O2 combinedwith CO2. Beneficial and stressful effects of MAP conditionsdepended on the parameter measured. Low O2 with CO2 reducedshelf-life because of the development of strong off-odours. MAPwith CO2 was directly correlated with ammonia accumulation andpH increase but the levels reached were not the cause of plantsenescence. Although moderate O2 with CO2 controlled the devel-opment of off-odours, this storage condition reduced shelf-life byaccelerating senescence and cell death. Low O2 preserved the phys-iological integrity of the tissue, which was associated with lowerpopulations of microorganisms, while moderate O2 was associatedwith high populations. For commercial handling of baby spinach,shelf-life may not be prolonged over 7 d at 7 ◦C unless other tech-nologies such as micro-perforated films with high CO2 permeabilityor removal of off-odours are available. For commercial packagingdesign, perforated polymeric packages can reduce off-odour butthe shelf-life is reduced, whereas non-perforated packages can pro-long shelf-life but develop off-odours. There is a need to discussstrategies for off-odours control in the case of spinach becauseit is an unresolved problem. Toivonen (1997) suggested severaladsorbents to remove ammonia from the headspace atmosphereof packages. He also noted there were few reports concerned with

the use of desiccants to remove components associated with off-flavours, improving shelf-life and quality. Appropriate integratedpre- and postharvest handling will help to improve strategies foroff-odours control of baby spinach. A comprehensive approach is

logy a

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A

tud0fFtSh

A

i2

R

A

B

B

B

B

C

C

C

C

C

D

D

F

G

I

J.A. Tudela et al. / Postharvest Bio

equired, including selection of varieties, improvements in agro-omical practices and postharvest handling and storage to reducehe development of off-odours and to provide baby spinach ofxcellent quality with adequate shelf-life.

cknowledgements

The authors are grateful to MICINN (project AGL2010-20070),he European Community’s Seventh Framework Programme (FP7)nder grant agreement no 244994 (project VEG-i-TRADE) and Fun-ación Seneca de la Región de Murcia (grupo de excelencia GERM6, 04486) for financial support. We are thankful to Primaflor S.L.or providing the plant material. We thank Francisco Salar, Lauraernández, Nathalie Hernández and Macarena Moreno for assis-ance with processing and evaluations. Thanks are also due to Faraáez from the Image Analysis Service of University of Murcia forer help.

ppendix A. Supplementary data

Supplementary data associated with this article can be found,n the online version, at http://dx.doi.org/10.1016/j.postharvbio.012.08.006.

eferences

llende, A., Luo, Y., McEvoy, J.L., Artés, F., Wang, C.Y., 2004. Microbial and qualitychanges in minimally processed baby spinach leaves stored under super atmo-spheric oxygen and modified atmosphere conditions. Postharvest Biology andTechnology 33, 51–59.

abic, I., Watada, A.E., 1996. Microbial populations of fresh-cut spinach leavesaffected by controlled atmospheres. Postharvest Biology and Technology 9,187–193.

errang, M.E., Bracket, R.E., Beuchat, L.R., 1990. Microbial, color and textural qualitiesof fresh asparagus, broccoli, and cauliflower stored under controlled atmo-sphere. Journal of Food Protection 53, 391–395.

radford, M., 1976. A rapid and sensitive method for the quantitation of microgramquantities of protein utilizing the principle of protein-dye binding. AnalyticalBiochemistry 72, 248–254.

urgheimer, F., McGill, J.N., Nelson, A.I., Steinberg, M.P., 1967. Chemical changes inspinach stored in air and controlled atmosphere. Food Technology 21, 109–111.

antwell, M., Rovelo, J., Nie, X., Rubatzky, V., 1998. Specialty salad greens: posthar-vest physiology and shelf-life. Acta Horticulturae 467, 371–377.

antwell, M., Hong, G., Nie, X., 2010. Using tissue ammonia and fermentative volatileconcentrations as indicators of beneficial and stressful modified atmospheres forleafy and floral vegetables. Acta Horticulturae 876, 165–172.

ameron, A.C., Talasila, P.C., Joles, D.W., 1995. Predicting permeability needs formodified atmosphere packaging of lightly processed fruits and vegetables.HortScience 30, 25–34.

arlin, F., Nguyen-the, C., 1994. The microbiology of minimally processed freshfruits and vegetables. Critical Reviews in Food Science and Nutrition 34,371–401.

hibnall, A.C., 1939. Protein metabolism in the plant. In: The Role of Proteins inthe Respiration of Detached Leaves. Yale University Press, New Haven, CT, pp.211–243 (Chapter X).

aniels, J.A., Krishnamurthi, R., Rizvi, S.S.H., 1985. A review of effects of carbondioxide on microbial growth and food quality. Journal of Food Protection 48,532–537.

ixon, N.M., Kell, D.B., 1989. The inhibition by CO2 of the growth and metabolismof microorganisms. Journal of Applied Bacteriology 67, 109–136.

an, X., Sokorai, K.J.B., 2005. Assessment of radiation sensitivity of fresh-cutvegetables using electrolyte leakage measurement. Postharvest Biology andTechnology 36, 191–197.

il, M.I., Ferreres, F., Tomás-Barberán, F., 1999. Effect of postharvest storageand processing on the antioxidant constituents (flavonoids and vitamin C)

of fresh-cut spinach. Journal of Agricultural and Food Chemistry 47, 2213–2217.

be, S.N., Grogan, G., 1983. Effect of controlled oxygen and carbon dioxide atmo-spheres on bacterial growth rate and soft rot of tomato fruits caused byPseudomonas marginalis. Plant Disease 67, 1005–1008.

nd Technology 75 (2013) 75–85 85

Izumi, H., Nonaka, T., Muraoka, T., 1997. Physiology and quality of fresh-cut spinachstored in low O2 controlled atmospheres at various temperatures. In: Gorny,J.R. (Ed.), Proceedings of the 7th International Controlled Atmosphere ResearchConference, vol. 5. Davis, University of California, pp. 130–132.

Jacxsens, L., Devlieghere, F., Ragaert, P., Vanneste, E., Debevere, J., 2003. Relationbetween microbiological quality, metabolite production and sensorial qualityof equilibrium modified atmosphere packaged fresh-cut produce. InternationalJournal of Food Microbiology 83, 263–280.

Kader, A.A., 1986. Biochemical and physiological basis for effects of controlled andmodified atmospheres on fruits and vegetables. Food Technology 40, 99–100,and 102–104.

Kader, A.A., 2002. Modified atmospheres during transport and storage. In: Kader,A.A. (Ed.), Postharvest Technology of Horticultural Crops, Publ. 3311, Universityof California, pp. 135–144.

Kader, A.A., Zagory, D., Kerbel, E.L., 1989. Modified atmosphere packaging of fruitsand vegetables. Critical Reviews in Food Science and Nutrition 28, 1–30.

Ke, D., Mateos, M., Kader, A.A.,1993. Regulation of fermentative metabolism in fruitsand vegetables by controlled atmospheres. In: Proc. 6th Int. CA Research Conf.,NRAES-71, vol. 1. Cornell University, Ithaca, NY, USA, pp. 63–77.

King, G.A., Woollard, D.C., Irving, D.E., Borst, W.N., 1990. Physiological changes inasparagus spear tips after harvest. Physiologia Plantarum 80, 393–400.

Ko, N.P., Watada, A.E., Schlimme, D.V., Bouwkamp, J.C., 1996. Storage of spinachunder low oxygen atmosphere above the extinction point. Journal of Food Sci-ence 61, 398–400.

Levitt, J., 1972. Responses of plants to environmental stresses. In: MiscellaneousStresses. Academic Press, New York, pp. 531–543 (Chapter 20).

López-Gálvez, F., Allende, A., Truchado, P., Martínez-Sánchez, A., Tudela, J.A., Selma,M.V., Gil, M.I., 2010. Suitability of aqueous chlorine dioxide vs sodium hypochlo-rite as an effective sanitizer preserving quality of fresh-cut lettuce whileavoiding by-product formation. Postharvest Biology and Technology 55, 53–60.

Martínez-Damian, M.T., Cantwell, M., 1999. Quality changes of spinach stored incontrolled and modified atmospheres. HortScience 34, 503.

McGill, J.N., Nelson, A.I., Steinberg, M.P., 1996. Effects of modified atmospheres onascorbic acid and other quality characteristics of spinach. Journal of Food Science31, 510.

Medina, M.S., Tudela, J.A., Marín, A., Allende, A., Gil, M.I., 2012. Short postharveststorage under low relative humidity improves quality and shelf life of minimalprocessed baby spinach. Postharvest Biology and Technology 67, 1–9.

Morales, M.A., Olmos, E., Torrecillas, A., Sánchez-Blanco, M.J., Alarcón, J.J., 2001. Dif-ferences in water relations, leaf ion accumulation and excretion rates betweencultivated and wild species of Limonium sp. grown in conditions of saline stress.Flora 196, 345–352.

Moyls, A.L., 2004. Whole bag method for determining oxygen transmission rate.Transactions of the ASAE 47, 159–164.

Pelayo-Zaldívar, C., Ben Abda, J., Ebeler, S.E., Kader, A.A., 2007. Quality and chemicalchanges associated with flavor of ‘Camarosa’ strawberries in response to a CO2-enriched atmosphere. HortScience 42, 299–303.

Ragaert, P., Devlieghere, F., Debevere, J., 2007. Role of microbiological and physio-logical spoilage mechanisms during storage of minimally processed vegetables.Postharvest Biology and Technology 44, 185–194.

Rodriguez, S.C., Cantwell, M., 2002. Efecto de atmosferas controladas en la conser-vación de espinacas mínimamente procesadas. In: 3er Cong. Iberoamericano deTecnología Postcosecha y Agroexportaciones, Santiago de Chile, December.

Schwerdtfeger, E., 1978. Proteolytic processes in short-time storage of some leafyvegetable. Qualitas Plantarum: Plant Foods for Human Nutrition 28, 147–163.

Spurr, A.R., 1969. A low-viscosity epoxy resin embedding medium for electronmicroscopy. Journal of Ultrastructure Research 26, 31–43.

Toivonen, P.M.A., 1997. Non-ethylene, non-respiratory volatiles in harvested fruitsand vegetables: their occurrence, biological activity and control. PostharvestBiology and Technology 12, 109–125.

Van Doorn, W.G., Beers, E.P., Dangl, J.L., Franklin-Tong, V.E., Gallois, P., Hara-Nishimura, I., Jones, A.M., Kawai-Yamada, M., Lam, E., Mundy, J., Mur, L.A.J.,Petersen, M., Smertenko, A., Taliansky, M., Van Breusegem, F., Wolpert, T.,Woltering, E., Zhivotovsky, B., Bozhkov, P.V., 2011. Morphological classificationof plant cell deaths. Cell Death Differentiation 18, 1241–1246.

Wagstaff, C., Clarkson, G.J.J., Rothwell, S.D., Page, A., Taylor, G., Dixon, M.S., 2007.Characterisation of cell death in bagged baby salad leaves. Postharvest Biologyand Technology 46, 150–159.

Weatherburn, M.W., 1967. Phenol–hypochlorite reaction for determination ofammonia. Analytical Chemistry 39, 971–974.

Wellburn, A.R., 1994. The spectral determination of chlorophylls a and b, as well astotal carotenoids, using various solvents with spectrophotometers of differentresolution. Journal of Plant Physiology 144, 307–313.

Wells, J.M., 1974. Growth of Erwinia carotovora, E. atroseptica and Pseudomonas fluo-rescens in low oxygen and high carbon dioxide. Phytopathology 64, 1012–1015.

Yamauchi, N., Watada, A.E., 1991. Regulated chlorophyll degradation in spinachleaves during storage. Journal of the American Society for Horticultural Science116, 58–62.