Postharvest Biology and Technology 114 (2016) 4553

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Postharvest Biology and Technology

journal homepage: www.elsevier.com/locate/postharvbio

Modied atmosphere generated during storage under light conditions

is the main factor responsible for the quality changes of baby spinach
Yolanda Garridoa , Juan A. Tudelaa , Jos A. Hernndezb , Mara I. Gila,*
a
Research Group on Quality, Safety and Bioactivity of Plant Foods, Food Science and Technology Department, CEBAS-CSIC, P.O. Box 164, E-30100 Espinardo,
Spain
b
Department of Plant Breeding, CEBAS-CSIC, P.O. Box 164, E-30100 Espinardo, Spain

A R T I C L E I N F O A B S T R A C T

Article history: Minimally processed products are generally exposed to low temperature and uncontrolled light
Received 11 August 2015 conditions during the supply chain. The positive effect of low temperature on quality of baby spinach is
Received in revised form 23 November 2015 widely reported, but there is little information available about the light effect. The objective of this study
Accepted 1 December 2015
was to obtain insight about the cause-effect of light exposure of baby spinach on leaf quality and
Available online xxx
senescence parameters. Minimally processed baby spinach was stored in passive Modied Atmosphere
Packaging (MAP) and in Controlled Atmosphere (CA) under different light conditions. In passive MAP,
Keywords:
three very different headspace gas compositions within the bags due to photosynthesis and respiration
Fresh-cut produce
Baby leaves
reactions were generated that strongly affected the quality characteristics. To isolate the light effect from
Physiology the atmosphere composition inuence, and thus understand the mechanisms causing the quality
Quality changes, baby spinach was stored under two CA of 0.5 kPa O2 + 10 kPa CO2 (low O2 + high CO2 levels) and
air combined with 2 light conditions (continuous light and darkness). The changes observed under the
different light conditions were mainly caused by the differences in gas composition. Under light, MAP
with high O2 partial pressure (pO2) and low CO2 partial pressure (pCO2) was detrimental because of the
growth of Pseudomonas spp. and the progress in tissue senescence due to oxidative stress, increasing cell
damage, lipid peroxidation and chlorophyll degradation. Under darkness, MAP with low pO2 and high
pCO2 was also detrimental because of the intense off-odour developments, the increase in pH and
electrolyte leakage and the reduction in chlorophyll uorescence. Our results showed that the modied
atmosphere generated with exposure to the different light conditions affects the quality of baby spinach
mainly because of the high pO2 under light and high pCO2 under darkness.
2015 Elsevier B.V. All rights reserved.

1. Introduction the recommended storage temperatures (Evans et al., 2007; Kou

Baby spinach constitutes an important source of bres and
bioactive compounds such as vitamin C, vitamin A and poly-
phenols in the diet (Gil et al., 1999; Pandrangi and LaBorde, 2004;
Xie et al., 2013). As a minimally processed product, baby spinach is
characterized by a limited shelf life due to product quality
deterioration and off-odours development (Wang, 2003; Tudela
et al., 2013). Shelf life of minimally processed baby spinach
packaged in perforated bags is about 8 days at 8
C (Kou et al., 2014)
and under 710 kPa O2 and 510 kPa CO2 moderate benet is
offered to spinach by delaying yellowing (Suslow and Cantwell,
1998). To ensure the optimal product quality it is vital to maintain
* Corresponding author. Fax: +34 968 396 213.
E-mail address: migil@cebas.csic.es (M.I. Gil).
et al., 2015).
Minimally processed products, including baby spinach, are
usually displayed under uncontrolled light condition through the
supply chain (Glowacz et al., 2014). The inuence of light on quality
and shelf life of minimally processed vegetables is controversial
since both positive and negative effects have been observed
depending on the product (Lester et al., 2010; Costa et al., 2013;
Gergoff Grozeff et al., 2013; Braidot et al., 2014; Glowacz et al.,
2014). In general, light conditions in minimally processed
vegetables affect some quality parameters during storage,
promoting a decrease of leaf water content (Lester et al., 2010;
Zhan et al., 2013; Glowacz et al., 2014; Xiao et al., 2014), and visual
quality (Martnez-Snchez et al., 2011; Xiao et al., 2014), preserving
the cholorophyll content (Costa et al., 2013; Gergoff Grozeff et al.,
2013; Zhan et al., 2013), promoting microbial internalization
(Kroupitski et al., 2009) and increasing the content of bioactive
compounds (Noichinda et al., 2007; Lester et al., 2010; Zhan et al.,

http://dx.doi.org/10.1016/j.postharvbio.2015.12.001
0925-5214/ 2015 Elsevier B.V. All rights reserved.
46 Y. Garrido et al. / Postharvest Biology and Technology 114 (2016) 4553
2012; Gergoff Grozeff et al., 2013; Braidot et al., 2014). Moreover,
exposure of the bags to different light conditions affects the
headspace gas composition because under light leaf tissues
continue the photosynthetic activity that depletes CO2 and
releases O2 (Olarte et al., 2009; Martnez-Snchez et al., 2011;
Glowacz et al., 2014).
The effects of modied atmosphere packaging (MAP) on the
quality maintenance of baby spinach have been extensively
reviewed (Babic and Watada, 1996; Ko et al., 1996; McGill et al.,
1996; Tudela et al., 2013). The major problem associated with
minimally processed baby spinach is the strong off-odour
development when stored under low O2 and high CO2 partial
pressures (Tudela et al., 2013). No information is available on
whether the quality changes observed when the product is stored
under light are a direct consequence of the light exposure or the
indirect consequence of the headspace gas composition generated
in the bags. Hence, the objective of this study was to evaluate the
effect of different light conditions and atmosphere composition on
the quality of minimally processed baby spinach during storage.
The quality parameters evaluated were related to leaf quality
(sensory evaluation, water content, leaf colour and microbiological
quality) and leaf senescence (cell morphology, lipid peroxidation,
electrolyte leakage, chlorophyll uorescence, chlorophyll content,
and pH changes).
2. Materials and methods
2.1. Plant material and growing conditions
Baby spinach (Spinacia oleracea L.) was cultivated under
commercial conditions in elds near Pulp (Almera, Spain) by
Primaor S.L. Two different spinach varieties were evaluated
because of the short period of time that each variety covers in the
winter season. Sowing was performed directly on elevated beds
using a plant density of 700 plants m2. Plant material (20 kg) was
mechanically harvested on January 16th 2014 (trial 1) and March
7th 2014 (trial 2). The growing cycle was 57 days and 52 days for
trial 1 and trial 2, respectively.
2.2. Processing, packaging and storage conditions
After harvest, baby spinach leaves were transported (150 km)
under refrigerated conditions in polystyrene boxes to the CEBAS-
CSIC laboratory (Murcia, Spain). Then, baby leaves were kept for
24 h at 4
C and 70% relative humidity (RH) in darkness. After that,
baby leaves were processed at 4
C as described by Garrido et al.
(2015). Samples were washed in a cold (4
C) chlorine solution
(NaOCl 100 mg L1) adjusted to pH 6.5 with citric acid, drained and
then rinsed with tap water. The excess of water was removed by
spinning in an automatic salad spinner (K-50, Kronen GmbH, Kehl
am Rhein, Germany)
The study of the light exposure was carried out in two different
trials: Modied Atmosphere Packaging (MAP) (trial 1) and
Controlled Atmospheres (CA) (trial 2). In the rst trial, the
objective was to evaluate the effect of different light conditions
on the visual quality of baby spinach during storage in MAP.
Samples of 100 g were mechanically packed in a vertical packaging
machine (Etna 280-X model, Ulma, Oati, Spain) using a 35 mm
polypropylene (PP) lm (Amcor Flexibles, Bristol, UK) with O2
permeance of 2.629 E12 mol m2 s1 Pa1, CO2 permeance of
9.84 E12 mol m2 s1 Pa1 and H2O permeance of 5.408 E6 mol
m2 d1 at 7
C and 97% RH. Package size was 230 mm  310 mm. A
passive modied atmosphere was created by the respiration rate of
the product and the lm permeability characteristics as bags were
sealed under ambient air conditions. Bags were randomly
separated into three groups and stored in shelves in a cold room
for 12 days at 7
C under 3 light conditions: continuous light,
photoperiod 12 h and continuous darkness. Shelves with the bags
subjected to darkness condition were covered with black
polyethylene plastic lm, avoiding light exposure. In the second
trial, the objective was to investigate if the responsible factor for
the quality changes was the light or the modied atmosphere
generated. For this, samples of 300 g were stored in 5 L glass jars
connected to a ow-through board system providing different CAs
at a constant ow rate. A total of 32 jars were randomly separated
into two batches of 16 jars connected to air (21 kPa O2 + 0.04 kPa
CO2) and 16 jars to CA (0.5 kPa O2 + 10 kPa CO2). These atmospheres
were humidied by bubbling through a previous connected ask
containing 100 mL of water with a constant ow rate of 40 and
10 mL/min for air and CA, respectively. Under each atmosphere
composition, half of the jars were stored in continuous light and
half of them in darkness. Jars subjected to darkness were covered
with black polyethylene plastic lm to avoid light exposure. The
jars were placed in a cold room and stored for 10 days at 7
C. In
both trials, samples under light conditions were exposed to
conditions similar to those of retail sale with a total radiation of
5 1 mmol m2 s1 obtained from uorescent lights (Philips Ibrica
S.A., Madrid, Spain) of 58 W each. The storage conditions were
named as: Light + Air, Light + CA, Darkness + Air and Darkness + CA.
2.3. Headspace gas composition and respiration rate
Headspace gas composition within the bags and the atmo-
sphere composition within the jars were monitored daily with an
O2 analyser with a ceramic oxide-zirconia electrochemical
detection cell (CG-1000, Ametek, Thermox Instruments Co.,
Pittsburgh, PA, USA) and an infrared CO2 detector (Via 510, Horiba
Instruments Co., Irvine, CA, USA). Four replicates were used for
each storage condition and sampling date. Partial pressures of O2
(pO2) and CO2 (pCO2) were expressed in kPa. Respiration rate was
measured in the same bags used in trial 1 by means of the
permeable system as described by Luna et al. (2013) with some
modications. Changes in O2 in the bag headspace from the initial
20.9% were monitored as a function of time and tted to a curve to
determine a respiration rate function as proposed by Lee et al.
(1996). Respiration rate was calculated when the headspace O2
concentration within the bags stored at 7
C reached a level of
17 kPa.
2.4. Leaf quality parameters: sensory evaluation, water content, leaf
colour and microbiological quality
Sensory quality was evaluated by a ve member trained panel
(Tudela et al., 2013). About 250 g of baby spinach from 4 individual
replicates were mixed together in the same tray for each storage
condition and sampling date. Sensory quality was evaluated at 0,
10, and 12 days in MAP and at 0, 7, and 10 days in CA. Visual quality
was evaluated using a 19 hedonic scale where 9 = excellent,
5 = limit of marketability and 1 = inedible. Off-odour development
was evaluated in a 5 point scale where 5 = severe, 3 = moderate and
1 = none.
Leaf water content (WC) was analysed as described by Viacava
et al. (2010) with some modications. Fifteen samples from
3 individual replicates (5 samples per replicate) were measured
per storage condition and sampling date. For WC determination,
leaves were weighed to obtain fresh mass (FM). Samples were
dried for 48 h in a forced air oven at 65
C until constant weight to
determine the dried mass (DM). Values of FM and DM were used to
calculate WC according to the equation (Viacava et al., 2010):
FM  DM
WC%  100
FM
 Y. Garrido et al. / Postharvest Biology and Technology 114 (2016) 4553
Leaf colour was measured in the same samples using a compact
tristimulus colorimeter (Minolta CR-300, Ramsey, NJ) with an
8 mm diameter viewing (Garrido et al., 2015) and L*, a*, b*
parameters were measured. Leaf colour was evaluated as Hue
angle [tan1 (b*/a*)] and Chroma index [(a*2 + b*2)1/2]. Leaf water
content and colour were evaluated at 0 and 10 days in MAP and 0, 7,
and 10 days in CA.
For the microbiological analysis, samples of 30 g were analysed
as previously described by Medina et al. (2012). Samples were
homogenized for 2 cycles of 60 s in 0.1% sterile buffered peptone
water (BPW) (AES Laboratoire, Combourg, France) (1:10 dilution).
Pseudomonas spp. were counted in Pseudomonas agar (Oxoid, Ltd.,
Basingstoke, Hampshire, England) at 30
C for 2448 h. Three
samples from 3 individual replicates were analysed for each
storage condition at 0, 7 and 10 days in CA. Microbiological counts
were expressed as log CFU g1.
2.5. Leaf senescence parameters: leaf anatomy and cytology, lipid
peroxidation, electrolyte leakage, chlorophyll uorescence, chlorophyll
content and pH
Tissue samples of approximately 3  5 mm were excised from
the centre of the leaf lamina (away from the midrib). Samples were
xed, dehydrated, embedded in Spurr resin, sectioned and stained
with 0.5% toluidine blue in borate buffer (Tudela et al., 2013).
Microphotographs of semi-thin sections were captured with a
Leica DMR light microscope (Leica, Wetzlar, Germany) using the
20 objective. Images were processed using the software ImageJ
(NIH Image, National Institute of Health, USA). Cell morphology
was evaluated to identify symptoms of cell damage as described by
Garrido et al. (2015). Cells with symptoms such as a visible gap
between the cell wall and the plasma membrane, cells with
cytoplasmic retraction and cell corpse (cells with rupture of
plasma membrane and unstructured cytoplasm) were considered
as damaged cells. Samples of 15 leaves from 3 individual replicates
(5 leaves per replicate) were evaluated per storage condition at
0 and 10 days in CA. At least 150 cells per leaf were measured and
the level of damaged cells was expressed as percentage of total
damaged cells per leaf.
The extent of lipid peroxidation was determined as described by
Hernndez et al. (2004) via the thiobarbituric acid (TBA) reaction
which measures the malondialdehyde (MDA) content of the tissue.
Fresh sample (0.5 g) was homogenized with 0.1% trichloroacetic
Light
25 25
O2
A B
CO2
20 20
15 15
kPa
kPa
10 10
5 5
0 0
0 1 2 3 4 5 6 7 8 9 10 11 12
Days of storage
Fig. 1. Headspace partial pressure of O2 and CO2 of minimally processed baby spinach stored for 12 d at 7 C under different light conditions: light (A), photoperiod (B) and
darkness (C). Each symbol is the mean  standard deviation of 4 bags.
47
acid (TCA) and centrifuged (12,000 g 10 min.). The supernatant
(0.5 mL) was mixed with 0.5% TBA diluted with 20% of TCA and
incubated at 95
C for 30 min. and the reaction was stopped by
placing the reaction tubes in an ice-water bath. Then, the
absorbance of supernatant at 532 nm was corrected for non-
specic absorbance by subtracting the absorbance at 600 nm. Four
samples from 2 individual replicates (2 samples per replicate) were
measured per storage condition at 0 and 10 days in CA. The results
were expressed in nmol MDA per g of fresh weight (FW).
Electrolyte leakage was measured as described by Fan and
Sokorai (2005) with slight modications. Six samples from
3 individual replicates (2 samples per replicate) were measured
for each storage condition at 0, 7 and 10 days in CA. Twelve leaves
were randomly taken per sample and a piece of 1 cm2 per leaf was
cut and immersed in 15 mL of deionized water. The electrical
conductivity of the bathing solution was measured after 120 min of
incubation at room temperature using a conductivity meter
(Model CM35, Crison, Barcelona, Spain). After that, samples were
exposed to 95
C for 20 min and the electrical conductivity of the
bathing solution measured (total tissue electrolytes). The results
were expressed as percent of the conductivity of total tissue
electrolytes.
Fluorescence of chlorophyll was measured with a chlorophyll
uorometer (IMAGIM-PAM M-series, Heinz Walz, Effeltrich,
Germany) as described by Faize et al. (2011) with some
modications. After dark-incubation of leaves for 30 min, the
minimum and the maximal uorescence yields were monitored.
Kinetic analyses were carried out with actinic light (81 mmol
quanta m2 s1 PAR) and repeated pulses of saturating light at
2700 mmol quanta m2 s1 PAR for 0.8 s at intervals of 20 s. The
maximum quantum yield of photosystem II (PSII) (Fv/Fm), the
effective PSII quantum yield (Y(II)), the quantum yield of
nonregulated energy dissipation Y(NO), and the quantum yield
of regulated energy dissipation Y(NPQ) were analysed. Two
reference areas per leaf were established. Twenty leaves from
4 individual replicates (5 leaves per replicate) were analysed for
each storage condition at 0 and 10 days in MAP and at 0, 7 and
10 days in CA.
The content of chlorophylls in spinach leaves was extracted in
80% acetone at room temperature and quantied by measuring the
absorbance at 646 and 663 nm for chlorophyll b and chlorophyll a
according to Lichtenthaler and Wellburn (1983). Results were
expressed in mg per g FW. Four samples from 2 individual
Photoperiod Darkness
25
O2 O2 C
CO2 CO2
20
15
kPa
10
5
0
0 1 2 3 4 5 6 7 8 9 10 11 12 0 1 2 3 4 5 6 7 8 9 10 11 12
Days of storage Days of storage


48 Y. Garrido et al. / Postharvest Biology and Technology 114 (2016) 4553

replicates (2 samples per replicate) were measured for each
storage condition at 0 and 10 days in CA.
To measure the pH, 8 g of fresh tissue were homogenized in
125 mL of distilled water. The pH of homogenate was measured
with a pH-meter Metrohm (model 785 DMP Tritino, Herisau,
Switzerland). Four samples from 2 individual replicates (2 samples
per replicate) were measured for each storage condition at 0 and
10 days in both MAP and CA.
2.6. Statistical analysis
All data were analysed using PASW Statistics 20 for Windows
(SPSS Inc., Chicago, IL, USA). The different trials were evaluated
separately. Prior to all statistical analyses, the test for normality
(Saphiro-Wilk) was performed (P > 0.05). For data showing normal
distribution, Anova test was carried out and when signicant
differences were observed, the Tukeys HSD (Honestly Signicant
Difference) test was applied. KruskalWallis test was carried out
when data did not show normal distribution and when signicant
differences were observed, pairwise comparison with Kruskal
Wallis test was applied. The differences were considered to be
statistically signicant if the probability value was less than 5%
(P < 0.05) and 1% (P < 0.01). Anova test was carried out to evaluate
3.2. Sensory quality, water content and leaf colour of baby spinach
stored in MAP at different light conditions
Signicant differences in the sensory attributes were observed
among light conditions. Baby spinach stored under darkness was
scored with the highest visual quality after 12 days of storage
(Table 1). However, off-odours were more perceptible in darkness,
reaching high intensity at the end of storage (Table 1). Light
conditions signicantly affected leaf water content and leaves
stored under light showed the lowest water content while under
darkness showed the highest (89.2, 89.7 and 90.2% for light,
photoperiod and darkness, respectively). Leaf colour measured as
Chroma did not show signicant differences among light
conditions (data not shown).
Light
A
Photoperiod
1.0
Darkness
0.8

Y(II)
0.6 a
the effect of storage condition on the respiration rate, leaf water
content, leaf colour, damaged cells, lipid peroxidation, electrolyte
leakage, chlorophyll content and pH. KruskalWallis test was 0.4
carried out to evaluate the effect of storage condition on sensory b b
evaluation, microbiological analysis and chlorophyll uorescence. 0.2

3. Results
a B
3.1. Headspace gas composition and respiration rate of baby spinach 1.0
stored in MAP at different light conditions
b
0.8
The MAP under light achieved levels of 17 kPa O2 + 2 kPa CO2
Y(NO)

after 2 days, meaning a slight modication of the initial headspace 0.6

gas composition (Fig. 1A). When bags were stored in photoperiod,
CO2 and O2 reached similar levels of 7 kPa O2 + 7 kPa CO2 at the
0.4 c
steady state (Fig. 1B). The highest modication of the headspace
gas composition was observed under darkness, with levels of
0.5 kPa O2 + 10 kPa CO2 at the steady state (Fig. 1C). Respiration rate 0.2
of baby spinach was inuenced by light condition with mean
values of O2 consumption of 195, 262 and 532 nmol kg1 s1 for
continuous light, photoperiod and continuous darkness, respec- C
tively. Therefore, in continuous darkness baby spinach showed the 1.0
highest level of respiration rate with signicant differences
compared with continuous light and photoperiod. 0.8
Y(NPQ)

0.6
Table 1
Changes in visual quality and off-odour of minimally processed baby spinach stored 0.4 a a
for 12 d at 7
C under light, photoperiod and darkness.

Storage time Storage condition Visual quality Off-odour 0.2

0d 7.4  0.3 1.0  0.0 b
10 d Light 6.7  0.2 1.4  0.4b
Photoperiod 7.0  0.3 1.6  0.4b 0 10
Darkness 7.0  0.0ns 2.5  0.4a
Days of storage
12 d Light 5.8  0.3b 1.6  0.2ab
Fig. 2. Changes in the effective PSII quantum yield (Y(II)) (A), the quantum yield of
Photoperiod 6.1  0.5b 1.1  0.1b
nonregulated energy dissipation Y(NO) (B), and the quantum yield of regulated
Darkness 6.9  0.3a 3.8  0.2a
energy dissipation Y(NPQ) (C) of minimally processed baby spinach stored for
Values are the mean of 5 replicates  standard deviation. For each day of storage, 10 days at 7
C under different light conditions: light, photoperiod and darkness.
values with different letters in the same column are signicantly different (P < 0.05) Baby spinach was evaluated at 0 days (white bar) and after 10 days of storage. Bars
according to KruskalWallis test. Visual quality was scored as 9 = excellent, 5 = limit are the mean  standard deviation of 40 replicates. For the same storage date, bars
of marketability and 1 = inedible. Off-odour was scored as 5 = severe, 3 = moderate with different letters are signicantly different (P < 0.05) according to Kruskal
and 1 = none. Wallis test.
 Y. Garrido et al. / Postharvest Biology and Technology 114 (2016) 4553 49

3.3. Chlorophyll uorescence and pH of baby spinach stored in MAP at independently of light conditions (Fig. 4A). After 10 days, the lipid
different light conditions peroxidation extension in CA was maintained close to the initial
value (Fig. 4B). However, when baby spinach was stored in air, a
Chlorophyll uorescence was signicantly affected by the higher level of lipid peroxidation was observed without the
different light conditions during storage (Fig. 2). Baby spinach inuence of the light conditions (Fig. 4B). Electrolyte leakage was
stored under light showed the highest level of the effective PSII higher in CA than in air after 7 days, and for each atmosphere
quantum yield (Y(II)) compared to photoperiod and darkness composition it was higher in darkness (Fig. 4C). After 10 days,
(Fig. 2A). On the contrary, darkness showed the highest level of the electrolyte leakage showed the same trend and darkness + CA
quantum yield of non-regulated energy dissipation (Y(NO)) but the showed the highest value compared with the rest of conditions
lowest quantum yield of regulated energy dissipation Y(NPQ) (Fig. 4C).
(Fig. 2B, C). After 10 days, baby spinach stored under light showed
the maximum quantum yield of PS II (Fv/Fm) with mean values of 3.5. Chlorophyll uorescence, chlorophyll content and pH of baby
0.8, 0.5 and 0.4 for light, photoperiod and darkness, respectively. spinach stored in CA at different light conditions
During storage, the pH was also affected by the different light
conditions. After 10 days, baby spinach under darkness showed the Chlorophyll uorescence, chlorophyll content and pH were
highest level of pH with mean values of 6.94, 6.99 and 7.32 for light, signicantly affected by the atmosphere composition but not by
photoperiod and darkness, respectively. the light conditions (Table 2). The images of baby spinach obtained
using imaging-PAM uorometer are shown in Fig. 5. Changes in
3.4. Leaf cytology, lipid peroxidation and electrolyte leakage of baby photosynthetic activities could be readily discerned from false-
spinach stored in CA at different light conditions colour images. The highest negative effects occurred in response to
CA without the inuence of light whereas in air the photosynthetic
Light conditions (light and darkness) and atmosphere compo- activities were maintained similarly to that of day 0 in both light
sition (air and CA) were separately evaluated and the statistical conditions (Fig. 5). During storage, the maximum quantum yield of
signicance of the individual factors analysed (Table 2). The level of PS II (Fv/Fm) showed a similar response to the effective PSII
damaged cells and lipid peroxidation (MDA content) were quantum yield (Y(II)) (Table 3). In CA, Fv/Fm and Y(II) decreased at
signicantly affected by the atmosphere composition but not by both light conditions while in air similar values to those of day
the light exposure. Electrolyte leakage was signicantly affected by 0 were observed. In addition, Y(NO) and Y(NPQ) were higher in CA
the light conditions and atmosphere composition after 10 days and after 7 days and the relation Y(NO)/Y(NPQ) was 3 times higher after
only by the atmosphere composition after 7 days (Table 2). 10 days than after 7 days (Table 3).
Symptoms of cell damage were clearly shown in the microphoto- After 10 days, chlorophyll a content decreased in air compared
graphs captured by the light microscope (Fig. 3). Damaged cells to the initial value, while the content was similar to the initial value
differed from the undamaged cells (Fig. 3A) by the presence of a in CA independently of the light conditions (Table 3). Chlorophyll b
cytoplasmic retraction (Fig. 3B), a visible gap between the cell wall content did not show signicant differences with the storage
and the plasma membrane (Fig. 3C) and the rupture of the plasma conditions (Table 2).
membrane (cell corpse) (Fig. 3D). The level of damaged cells The pH of baby spinach stored for 10 days showed mean values
measured as percent of total cells was higher in air than in CA, of 6.4, 7.4, 6.5 and 7.5 in light + air, light + CA, darkness + air, and
darkness + CA, respectively. So, at both light conditions, pH was
higher in CA than in air.
Table 2
Statistical signicance by the Anova test and KruskalWallis test of leaf quality and
3.6. Sensory evaluation, water content, leaf colour and microbiological
leaf senesce parameters of baby spinach stored for 10 d at 7
C under different
storage conditions: light + air, light + CA (0.5 kPa O2 + 10 kPa CO2), darkness + air, analysis of baby spinach stored in CA at different light conditions
darkness + CA (0.5 kPa O2 + 10 kPa CO2).
Visual quality, water content and leaf colour were not affected
Statistical analysis 7 days 10 days
by any storage conditions, while off-odour development and
Light Atmosphere Light Atmosphere Pseudomonas counts were signicantly affected by the atmosphere
Anova test composition but not by the light exposure (Table 2). Visual quality
Water content (%) ns ns ns ns remained above the limit of marketability with a scored value of
Leaf colour ns ns ns ns
Damaged cells (%) na na ns ***
6.7 after 10 days (Table 4). During storage, off-odour development
Malondialdehyde (nmol g1 FW) na na ns ** was notably more perceptible under CA without differences
Electrolyte leakage (%) ns *** * * between light and darkness conditions (Table 4). After 7 days,
Chlorophyll a content (mg g1 FW) na na ns * microbiological analysis showed that Pseudomonas counts was
Chlorophyll b content (mg g1 FW) na na ns ns
1 log higher in air than in CA. However, after 10 days, Pseudomonas
pH na na ns ***
counts reached a mean value of 6.7 log CFU g1 with no signicant
KruskalWallis test differences among storage conditions (Table 4).
Visual quality ns ns ns ns
Off-odour development ns * ns ** 4. Discussion
Pseudomonas counts (log CFU g1) ns ** ns ns
Fv/Fm ns *** ns ***
Y(II) ns *** ns *** Headspace gas composition was affected during storage by the
Y(NO) ns *** ns *** light conditions. Continuous darkness showed the highest
Y(NPQ) ns *** ns *** modication, while continuous light showed the lowest and
Fv/Fm, maximum quantum yield of photosystem II (PSII); Y(II), effective PSII photoperiod an intermediate modication. Consumption of O2
quantum yield, Y(NO), quantum yield of nonregulated energy dissipation, Y(NPQ), measured as respiration rate of samples exposed to darkness was
quantum yield of regulated energy dissipation. higher than in photoperiod or light conditions. These results agree
na, not analysed, ns, not signicant.
*Signicant at P  0.05.
with those described in processed lettuce stored under low
*Signicant at P  0.01. temperature and similar radiation conditions of 6 mmol m2 s1
*** Signicant at P  0.001. obtained from uorescent lights (Martnez-Snchez et al., 2011).
50 Y. Garrido et al. / Postharvest Biology and Technology 114 (2016) 4553

Fig. 3. Images captured by optical microscope of cells of baby spinach stored for 10 days at 7
C under different storage condition: light + air, light + CA (0.5 kPa O2 + 10 kPa
CO2), darkness + air, darkness + CA (0.5 kPa O2 + 10 kPa CO2). Images of intact cells (A), damaged cell with a cytoplasmic retraction (B), damaged cell with a visible gap between
the cell wall and the plasma membrane (C) and cell corpse (D). dc (damaged cell); cc (cell corpse); ic (intact cell); c (cytoplasm); cw (cell wall); pm (plasma membrane). Bars:
20 mm.

Moreover, higher radiation levels of at least 30 mmol m2 s1 have
been also reported to cause lower modication of headspace gas
composition in leafy greens when compared to darkness (Glowacz
et al., 2014; Xiao et al., 2014). These authors attributed the least
modication of headspace gas composition during light exposure
to the consumption of O2 compensated by its production because
of photosynthesis (Martnez-Snchez et al., 2011; Glowacz et al.,
2014; Xiao et al., 2014). The sensory attributes, leaf water content,
chlorophyll uorescence and pH of baby spinach were differently
affected by the different atmospheres generated under the
different light conditions. Controlled atmospheres allowed isola-
tion of the light effect from the atmosphere composition effect, and
thus insight into the mechanisms causing the quality changes.
Controlled atmospheres of 0.5 kPa O2 + 10 kPa CO2 retarded leaf

Fig. 4. Changes in the level of damaged cells (A), lipid peroxidation (measured as malondialdehyde content) (B) and electrolyte leakage (C) of minimally processed baby
spinach stored for 10 days at 7
C under different storage conditions: light + air, light + CA (0.5 kPa O2 + 10 kPa CO2), darkness + air, darkness + CA (0.5 kPa O2 + 10 kPa CO2). Baby
spinach was evaluated at 0 days (white bar) and during storage. Bars are the mean  standard deviation of 15, 4 and 6 replicates for damaged cells, lipid peroxidation and
electrolyte leakage, respectively.
 Y. Garrido et al. / Postharvest Biology and Technology 114 (2016) 4553 51

Fig. 5. Changes in chlorophyll uorescence parameters of minimally processed baby spinach stored for 10 days at 7
C under different storage conditions: light + air, light + CA
(0.5 kPa O2 + 10 kPa CO2), darkness + air, darkness + CA (0.5 kPa O2 + 10 kPa CO2). Images of maximum quantum yield of photosystem II (PSII) (Fv/Fm), effective PSII quantum
yield (Y(II)), quantum yield of nonregulated energy dissipation Y(NO), and quantum yield of regulated energy dissipation Y(NPQ) captured with an Imaging-PAM M-series
system. The false-colour code depicted between the images ranged from 0 (black) to 1 (purple).

senescence as observed by the lower levels of damaged cells, lipid
peroxidation contents and chlorophyll degradation, without the
inuence of light. Generally, low partial pressures of O2 and CO2
decrease respiration and the rate of product deterioration (Kader
et al., 1989). Histological studies carried out by Tudela et al. (2013)
showed that baby spinach stored with low pO2 and high pCO2
showed less symptoms of cell damage compared to leaves stored
with moderate pO2 and high pCO2.
During storage, the effect of light exposure on chlorophyll
content of leafy greens is controversial. While some studies
reported an increase in the chlorophyll content with light exposure
(Noichinda et al., 2007; Zhan et al., 2012; Costa et al., 2013; Gergoff
Grozeff et al., 2013), others reported the opposite (Glowacz et al.,
2014). The results of this study showed that baby spinach under
low pO2 and high pCO2 preserved better the chlorophyll content
than in air, without the inuence of light. Moreover, several studies
have demonstrated that chlorophyll content of leafy greens is
maintained under high pCO2 (Herner, 1988; Philosoph-Hadas et al.,
1993; Fernndez-Len et al., 2013), while delaying other charac-
teristic changes of leaf senescence such as lipid peroxidation,
protein degradation and decay (Philosoph-Hadas et al., 1993; Lers
et al., 1998; Kenigsbuch et al., 2014).
In spinach, increases in pH have been reported when leaves are
stored in high pCO2 (Burgheimer et al., 1967). The increase in pH
has been described to be due to the accumulation of alkaline
compounds and not to the loss of organic acids (Burg, 2004). In

Table 3
Changes in maximum quantum yield of photosystem II (PSII) (Fv/Fm), effective PSII quantum yield (Y(II)), quantum yield of nonregulated energy dissipation Y(NO), quantum
yield of regulated energy dissipation Y(NPQ) and chlorophyll a content of minimally processed baby spinach stored for 10 days at 7
C under different storage condition:
light + air, light + CA (0.5 kPa O2 + 10 kPa CO2), darkness + air, darkness + CA (0.5 kPa O2 + 10 kPa CO2).

Storage time Storage condition Fv/Fm Y(II) Y(NO) Y(NPQ) Chlorophyll a content (mg g1 FW)
0d 0.76  0.02 0.51  0.06 0.29  0.03 0.20  0.02 0.86  0.03

7 d Light + Air 0.75  0.03a 0.50  0.06a 0.32  0.03b 0.18  0.04b na
Light + CA 0.61  0.08b 0.14  0.12b 0.66  0.19a 0.21  0.11a na
Darkness + Air 0.75  0.02a 0.51  0.05a 0.32  0.03b 0.16  0.03b na
Darkness+ CA 0.63  0.07b 0.10  0.06b 0.64  0.10a 0.26  0.06a na

10 d Light + Air 0.71  0.05a 0.45  0.06a 0.34  0.05b 0.21  0.03a 0.75 0.08ab
Light + CA 0.51  0.12b 0.08  0.07b 0.85 0.15a 0.09  0.08c 0.86  0.06a
Darkness + Air 0.73  0.03a 0.47  0.06a 0.33  0.03b 0.20  0.04a 0.74  0.08b
Darkness + CA 0.51  0.11b 0.06  0.05b 0.79  0.12a 0.15 0.10b 0.83  0.06ab

Values are the mean of 40, 40, 40, 40 and 4 replicates  standard deviation for Fv/Fm, Y(II), Y(NO), Y(NPQ) and chlorophyll a content, respectively. For each day of storage,
values with different letters in the same column are signicantly different (P < 0.05) according to KruskalWallis test and Tukeys HSD (Honestly Signicant Difference) test
for chlorophyll uorescence parameters and chlorophyll content, respectively. na, not analysed.
52 Y. Garrido et al. / Postharvest Biology and Technology 114 (2016) 4553
Table 4
Changes in visual quality, off-odour and Pseudomonas counts of minimally processed baby spinach stored for 10 d at 7
C under different storage conditions: light + air,
light + CA (0.5 kPa O2 + 10 kPa CO2), darkness + air, darkness + CA (0.5 kPa O2 + 10 kPa CO2).
Storage time Storage condition
0d
7d Light + Air
Light + CA
Darkness+ Air
Darkness+ CA
10 d Light + Air
Light + CA
Darkness+ Air
Darkness+ CA
Values are the mean of 5, 5 and 3 replicates  standard deviation for visual quality, off-odour and Pseudomonas counts, respectively. For each day of storage, values with
different letters in the same column are signicantly different with P < 0.05 (sensory attributes) and P < 0.01 (Pseudomonas counts) according to KruskalWallis test. Visual
quality was scored as 9 = excellent, 5 = limit of marketability and 1 = inedible. Level of off-odour was scored as 5 = severe, 3 = moderate and 1 = none.
previous studies, baby spinach exposed to CO2 accumulates
ammonia (Cantwell et al., 2010; Tudela et al., 2013). Ammonia
can be toxic to plant cells (Toivonen, 1997) and therefore, it has
been reported as a good indicator of CO2 injury in spinach
(Rodriguez and Cantwell, 2002). The results of this study showed
that independently of the light conditions, pH was higher in CA
because of the high pCO2. The pH of baby spinach was also higher
when the pCO2 increased in the MAP headspace, showing the
highest pCO2 under darkness. The results showed that electrolyte
leakage was mainly affected by the atmosphere composition, with
higher level in CA, in agreement with the increase in pH. An
increase in electrolyte leakage has been traditionally attributed to a
disruption of cell membranes. However, in spinach it has been
reported that electrolyte leakage could increase due to ammonium
accumulation even if there is no disruption of cell membranes
during senescence (Rolny et al., 2011). The results related to the
electrolyte leakage would explain the damage caused by the
ammonia content and the results related to the lipid peroxidation
and cell morphology would support oxidative damage of cell
membrane.
The measure of chlorophyll a uorescence in leafy greens
provides the information related to the efciency of photosynthe-
sis and the energy dissipation by a non-destructive technique
(Maxwell and Johnson, 2000). This technique was used to
monitoring senescence during storage in different products such
as lettuce, spinach and lambs lettuce (Schoeld et al., 2005;
Ferrante and Maggiore, 2007; Baldasarre et al., 2011) as well as
decay in lettuce (Simko et al., 2015). The potential efciency and
the achieved efciency of PSII were reected by Fv/Fm and Y(II),
and both represent the proportion of the light absorbed by
chlorophyll associated with PSII used for photochemistry (Maxwell
and Johnson, 2000). In mature spinach leaves stored in darkness
Gergoff Grozeff et al. (2013) reported that a previous treatment
with low intensity light pulses (30 mmol m2 s1) delayed the
progress of Fv/Fm declination during storage. The results in our
study showed that the efciency of photosynthesis was better
preserved in air independently of the light conditions, showing the
highest levels of Fv/Fm and Y(II). In addition, high level of Y(NO)
was observed under high pCO2 in both CA and MAP. The parameter
Y(NO) reects the fraction of energy that is dissipated passively as
heat and uorescence. The large values of Y(NO) reect a
suboptimal capacity of photoprotective reactions (Klughammer
and Schreiber, 2008; Brling et al., 2010). Moreover, lower
photosynthetic efciency corresponded with the conditions that
led to higher pH and electrolyte leakage, and it could be related
with the stressful or toxic effect of CO2 to the cells.
Darkness preserved a high water content and visual quality
compared to light and photoperiod. In minimally processed
Visual quality Off-odour Pseudomonas spp. (log CFU g1)
8.1  0.5 1.0  0.0 2.0  0.0
6.3  0.3 1.0  0.0b 6.7 0 .8a
6.9  0.5 2.0  0.4a 5.6  0.4b
6.8  0.3 1.1  0.1b 6.4  0.8ab
6.8 0.2ns 1.6  0.3ab 5.6  0.4b
6.1  0.1 1.1  0.3b 6.5  0.5
6.1  0.1 1.6  0.3ab 6.6  0.5
6.1  0.1 1.1  0.3b 7.0  1.0
6.2  0.2ns 1.7  0.2a 6.9  0.2ns
products, light exposure during storage generally affects leaf
hydration because of the increase in transpiration rate due to a
higher stomatal conductance that accelerates the loss of water
from the tissue (Noichinda et al., 2007; Lester et al., 2010; Zhan
et al., 2013; Glowacz et al., 2014; Xiao et al., 2014). In the present
study, visual quality and water content in CA were not affected by
the light conditions. The atmosphere compositions were constant-
ly humidied in order to prevent the dehydration effect caused by
the ow through system, decreasing water loss due to light.
Low pO2 and high pCO2 led to an inhibition of the growth of
Pseudomonas spp. These results were similar to those observed in
baby spinach stored under low pO2 and high pCO2 (Tudela et al.,
2013). This means that the growth of Pseudomonas spp. was not
responsible for the off-odours in baby spinach because the
development of off-odours increased under low pO2 and high
pCO2 with lower counts. There is a need to study the mechanisms
that promote off-odours in baby spinach and discuss strategies to
control them because they are still the major problem (Tudela
et al., 2013).
The absence of a direct effect of light in some parameters
evaluated in this study may be due to low light intensity used
(5 mmol m2 s1) compared to others which were at least 20 mmol
m2 s1. However, the results showed that a light exposure, even of
low intensity, can affect the water content and respiration activity
of baby spinach and led to changes in the quality of the product.
The inuence of these conditions should be taken into account
during distribution and retail sale.
5. Conclusions
Modied atmosphere (MA) is the main cause of quality changes
when baby spinach is exposed to different light conditions during
storage. Benecial and stressful effects of light exposure and
modied atmosphere generated depended on the parameter
measured. Under light, a less MA is generated with higher pO2
and lower pCO2. These storage conditions are detrimental as the
tissue senescence progresses with all characteristics of oxidative
stress (damaged cells, lipid peroxidation and chlorophyll degrada-
tion) and bacterial growth. Under darkness, a more MA is
generated with a lower pO2 and higher pCO2. These storage
conditions are also detrimental because of the intense off-odour
development, the increase in pH and electrolyte leakage and the
reduced Fv/Fm and Y(II) values. The only direct effect observed due
to light exposure is the water loss and the consequence on visual
quality, although under CA it was prevented by the humidied
atmosphere. Moderate levels of O2 and CO2 that were achieved in
this study in photoperiod can be recommended to delay
senescence, CO2 damage and oxidative stress.
 Y. Garrido et al. / Postharvest Biology and Technology 114 (2016) 4553
Acknowledgements
Authors thank the MINECO (projects AGL2010-20070 and
AGL2013-48529-R) for nancial support. Yolanda Garrido benet-
ted from a grant (BES-2011-044616). Authors are also grateful to
Primaor S.L. for supplying plant material, and Silvia Andjar,
Emilia Gonzlez, Nathalie Hernndez and Macarena Moreno for
the assistance with processing and evaluations.
References
Babic, I., Watada, A.E., 1996. Microbial populations of fresh-cut spinach leaves
affected by controlled atmospheres. Postharvest Biol. Technol. 9, 187193.
Baldasarre, V., Cabassi, G., Ferrante, A., 2011. Use of chlorophyll a uorescence for
evaluating the quality of leafy vegetables. Aust. J. Crop Sci. 5, 735741.
Braidot, E., Petrussa, E., Peresson, C., Patui, S., Bertolini, A., Tubaro, F., Whlby, U., et
al., 2014. Low-intensity light cycles improve the quality of lambs lettuce
(Valerianella olitoria [L.] Pollich) during storage at low temperature. Postharvest
Biol. Technol. 90, 1523.
Burg, S.P., 2004. Oxygen, carbon dioxide, ammonia and cyanide. In: Burg, S.P. (Ed.),
Postharvest Physiology and Hypobaric Storage of Fresh Produce. CABI
Publishing, UK, pp. 81131 (Chapter 4).
Burgheimer, F., McGill, J.N., Nelson, A.I., Steinberg, M.P., 1967. Chemical changes in
spinach stored in air and controlled atmosphere. Food Technol. 21, 109111.
Brling, K., Hunsche, M., Noga, G., 2010. Quantum yield of non-regulated energy
dissipation in PSII (Y (NO)) for early detection of leaf rust (Puccinia triticina)
infection in susceptible and resistant wheat (Triticum aestivum L.) cultivars.
Precis. Agric. 11, 703716.
Cantwell, M., Hong, G., Nie, X., 2010. Using tissue ammonia and fermentative
volatile concentrations as indicators of benecial and stressful modied
atmospheres for leafy and oral vegetables. Acta Hortic. 876, 165172.
Costa, L., Millan Montano, Y., Carrin, C., Rolny, N., Guiamet, J.J., 2013. Application of
low intensity light pulses to delay postharvest senescence of Ocimum basilicum
leaves. Postharvest Biol. Technol. 86, 181191.
Evans, J.A., Scarcelli, S., Swain, M.V.L., 2007. Temperature and energy performance of
refrigerated retail display and commercial catering cabinets under test
conditions. Int. J. Refrig. 30, 398408.
Faize, M., Burgos, L., Faize, L., Piqueras, A., Nicolas, E., Barba-Espin, G., Clemente-
Moreno, M.J., Alcobendas, R., Artlip, T., Hernandez, J.A., 2011. Involvement of
cytosolic ascorbate peroxidase and Cu/Zn-superoxide dismutase for improved
tolerance against drought stress. J. Exp. Bot. 62, 25992613.
Fan, X., Sokorai, K.J.B., 2005. Assessment of radiation sensitivity of fresh-cut
vegetables using electrolyte leakage measurement. Postharvest Biol. Technol.
36, 191197.
Fernndez-Len, M.F., Fernndez-Len, A.M., Lozano, M., Ayuso, M.C., Gonzlez-
Gmez, D., 2013. Different postharvest strategies to preserve broccoli quality
during storage and shelf life: controlled atmosphere and 1-MCP. Food Chem.
138, 567573.
Ferrante, A., Maggiore, T., 2007. Chlorophyll a uorescence measurements to
evaluate storage time and temperature of Valeriana leafy vegetables.
Postharvest Biol. Technol. 45, 7380.
Garrido, Y., Tudela, J.A., Gil, M.I., 2015. Comparison of industrial precooling systems
for minimally processed baby spinach. Postharvest Biol. Technol. 102, 18.
Gil, M.I., Ferreres, F., Toms-Barbern, F.A., 1999. Effect of postharvest storage and
processing on the antioxidant constituents (avonoids and vitamin C) of fresh-
cut spinach. J. Agric. Food Chem. 47, 22132217.
Glowacz, M., Mogren, L.M., Reade, J.P., Cobb, A.H., Monaghan, J.M., 2014. High- but
not low-intensity light leads to oxidative stress and quality loss of cold-stored
baby leaf spinach. J. Sci. Food Agric. 95, 18211829.
Gergoff Grozeff, G.E., Chaves, A.R., Bartoli, C.G., 2013. Low irradiance pulses improve
postharvest quality of spinach leaves (Spinacia oleraceae L. cv Bison).
Postharvest Biol. Technol. 77, 3542.
Hernndez, J.A., Rubio, M., Olmos, E., Ros-Barcel, A., Martnez-Gmez, P., 2004.
Oxidative stress induced by long-term Plum Pox virus infection in peach (Prunus
persica L. cv. GF305). Physiol. Plant. 122, 486495.
Herner, R.C., 1988. High CO2 effects on plant organs. In: Weichmann, J. (Ed.),
Postharvest Physiology of Vegetables. Marcel Dekker, New York, pp. 239253.
Kader, A.A., Zagory, D., Kerbel, E.L., 1989. Modied atmosphere packaging of fruits
and vegetables. Crit. Rev Food Sci. 28, 130.
Kenigsbuch, D., Ovadia, A., Shahar-Ivanova, Y., Chalupowicz, D., Maurer, D., 2014.
Rock-Ad a new wild rocket (Diplotaxis tenuifolia) mutant with late owering
and delayed postharvest senescence. Sci. Hortic. 174, 1723.
Klughammer, C., Schreiber, U., 2008. Complementary PS II quantum yields
calculated from simple uorescence parameters measured by PAM uorometry
and the saturation pulse method. PAM Appl. Notes 1, 2735.
Ko, N.P., Watada, A.E., Schlimme, D.V., Bouwkamp, J.C., 1996. Storage of spinach
under low oxygen atmosphere above the extinction point. J. Food Sci. 61,
398401.
Kroupitski, Y., Golberg, D., Belausov, E., Pinto, R., Swartzberg, D., Granot, D., Sela, S.,
2009. Internalization of Salmonella enterica in leaves is induced by light and
involves chemotaxis and penetration through open stomata. Appl. Environ.
Microbiol. 75, 60766086.
53
Kou, L., Luo, Y., Park, E., Turner, E.R., Barczak, A., Jurick, W.M., 2014. Temperature
abuse timing affects the rate of quality deterioration of commercially packaged
ready-to-eat baby spinach. Part I: sensory analysis and selected quality
attributes. Postharvest Biol. Technol. 91, 96103.
Kou, L., Luo, Y., Ingram, D.T., Yan, S., Jurick, W.M., 2015. Open-refrigerated retail
display case temperature prole and its impact on product quality and
microbiota of stored baby spinach. Food Control 47, 686692.
Lee, D., Song, Y., Yam, K.L., 1996. Application of an enzyme kinetics based respiration
model to permeable system experiment of fresh produce. J. Food Eng. 27,
297310.
Lers, A., Jiang, W., Lomaniec, E., Aharoni, N., 1998. Gibberellic acid and CO2 additive
effect in retarding postharvest senescence of parsley. J. Food Sci. 63, 6668.
Lester, G.E., Makus, D.J., Hodges, D.M., 2010. Relationship between fresh-packaged
spinach leaves exposed to continuous light or dark and bioactive contents:
effects of cultivar, leaf size, and storage duration. J. Agric. Food Chem. 58,
29802987.
Lichtenthaler, H.K., Wellburn, A.R., 1983. Determinations of total carotenoids and
chlorophylls a and b of leaf extracts in different solvents. Biochemical Soc. Trans.
11, 591592.
Luna, M.C., Tudela, J.A., Martnez-Snchez, A., Allende, A., Gil, M.I., 2013. Optimizing
water management to control respiration rate and reduce browning and
microbial load of fresh-cut romaine lettuce. Postharvest Biol. Technol. 80, 917.
Martnez-Snchez, A., Tudela, J.A., Luna, C., Allende, A., Gil, M.I., 2011. Low oxygen
levels and light exposure affect quality of fresh-cut Romaine lettuce.
Postharvest Biol. Technol. 59, 3442.
Maxwell, K., Johnson, G.N., 2000. Chlorophyll uorescencea practical guide. J. Exp.
Bot. 51, 659668.
McGill, J.N., Nelson, A.I., Steinberg, M.P., 1996. Effects of modied atmospheres on
ascorbic acid and other quality characteristics of spinach. J. Food Sci. 31, 510.
Medina, M.S., Tudela, J.A., Marn, A., Allende, A., Gil, M.I., 2012. Short postharvest
storage under low relative humidity improves quality and shelf life of minimally
processed baby spinach (Spinacia oleracea L.). Postharvest Biol. Technol. 67, 19.
Noichinda, S., Bodhipadma, K., Mahamontri, C., Narongruk, T., Ketsa, S., 2007. Light
during storage prevents loss of ascorbic acid, and increases glucose and fructose
levels in Chinese kale (Brassica oleracea var. alboglabra). Postharvest Biol.
Technol. 44, 312315.
Olarte, C., Sanz, S., Echavarri, J.F., Ayala, F., 2009. Effect of plastic permeability and
exposure to light during storage on the quality of minimally processed broccoli
and cauliower. LWTFood Sci. Technol. 42, 402411.
Pandrangi, S., LaBorde, L.F., 2004. Retention of folate, carotenoids, and other quality
characteristics in commercially packaged fresh spinach. J. Food Sci. 69,
C702C707.
Philosoph-Hadas, S., Jacob, D., Meir, S., Aharoni, N., 1993. Acta Horticulturae 343,
117122.
Rodriguez, S.C., Cantwell, M., 2002. Efecto de atmsferas controladas en la conser-
vacin de espinacas mnimamente procesadas. 3er Cong. Iberoamericano de
Tecnologa Postcosecha y Agroexportaciones, Santiago de Chile, December.
Rolny, N., Costa, L., Carrin, C., Guiamet, J.J., 2011. Is the electrolyte leakage assay an
unequivocal test of membrane deterioration during leaf senescence? Plant
Physiol. Biochem. 49, 12201227.
Schoeld, R.A., DeEll, J.R., Murr, D.P., Jenni, S., 2005. Determining the storage
potential of iceberg lettuce with chlorophyll uorescence. Postharvest Biol.
Technol. 38, 4356.
Simko, I., Jimenez-Berni, J.A., Furbank, R.T., 2015. Detection of decay in fresh-cut
lettuce using hyperspectral imaging and chlorophyll uorescence imaging.
Postharvest Biol. Technol. 106, 4452.
Suslow, T.V., Cantwell, M. 1998. Spinach: Recommendations for Maintaining
Postharvest Quality. Perishables Handling #94, http://postharvest.ucdavis.edu/
pfvegetable/Spinach/ (accessed July, 2015).
Toivonen, P.M.A., 1997. Non-ethylene, non-respiratory volatiles in harvested fruits
and vegetables: their occurrence, biological activity and control. Postharvest
Biol. Technol. 12, 109125.
Tudela, J.A., Marn, A., Garrido, Y., Cantwell, M., Medina-Martnez, M.S., Gil, M.I.,
2013. Off-odour development in modied atmosphere packaged baby spinach is
an unresolved problem. Postharvest Biol. Technol. 75, 7585.
Viacava, G.E., Roura, S.I., Agero, M.V., 2010. Improvement of water status
methodology for leafy vegetables reduces consumption of time, skilled labor,
and laboratory resources. Food Anal. Methods 4, 307312.
Wang, C.Y., 2003. Leafy, oral and succulent vegetables. In: Bartz, J.A., Brecht, J.K.
(Eds.), Postharvest Physiology and Pathology of Vegetables. Marcel Dekker, Inc.,
New York, pp. 599623.
Xiao, Z., Lester, G.E., Luo, Y., Xie, Z.K., Yu, L.L., Wang, Q., 2014. Effect of light exposure
on sensorial quality, concentrations of bioactive compounds and antioxidant
capacity of radish microgreens during low temperature storage. Food Chem. 151,
472479.
Xie, J., Zhu, J., Lin, Y., 2013. Effect of vacuum pre-cooling on quality changes of
spinach during cold storage. Adv. Mater. Res. 641642, 886889.
Zhan, L., Hu, J., Li, Y., Pang, L., 2012. Combination of light exposure and low
temperature in preserving quality and extending shelf-life of fresh-cut broccoli
(Brassica oleracea L.). Postharvest Biol. Technol. 72, 7681.
Zhan, L., Hu, J., Ai, Z., Pang, L., Li, Y., Zhu, M., 2013. Light exposure during storage
preserving soluble sugar and L-ascorbic acid content of minimally processed
romaine lettuce (Lactuca sativa L.var. longifolia). Food Chem. 136, 273278.