Postharvest Biology and Technology 155 (2019) 130–139

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Postharvest Biology and Technology

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

Preharvest UV-C treatment improves the quality of spinach primary T

production and postharvest storage
Ascensión Martínez-Sáncheza,b, Pedro Lozano-Pastorc, Francisco Artés-Hernándeza,b,
⁎
Francisco Artésa,b, Encarna Aguayoa,b,
a
Food Quality and Health Group, Institute of Plant Biotechnology, Universidad Politécnica de Cartagena (UPCT), Campus Muralla del Mar, 30202, Cartagena, Spain
b
Postharvest and Refrigeration Group (UPCT). Paseo Alfonso XIII, 48, 30203, Cartagena, Spain
c
Department of Research and Development, Verdimed, 30730, San Javier, Spain

A R T I C LE I N FO A B S T R A C T

Keywords: Preharvest low UV-C doses applied during spinach growth in the field as a preharvest tool could improve the
Microbiological antioxidant status, increasing the antioxidant compounds content and improving the resistnce to fungal damage.
Total phenolic content This physical treatment could replace or decrease the use of chemical treatments to control fungal diseases,
Vitamin C which may leave unpleasant chemical residues in fruit and vegetables. In this study, two different UV-C (1.5 kJ
Antioxidant capacity
m−2 and 3 kJ m−2) treatments were applied at three different development stages (I, immature; II, baby; and III,
Quality
mature commercial stage) of the spinach in the field. Color, chlorophyll content, sensorial analysis, microbial
load (mesophilic, molds and yeast), vitamin C content, total phenolic content (TPC) and antioxidant capacity
evaluated by two different assays were evaluated after harvest and after storage for six days at 5 °C in darkness
conditions. The immature leaves were more susceptible to UV-C, and 1.5 kJ m−2 or higher dose could affect
plant development after several applications. In stage II a decrease in vitamin C content was observed after
applying 3 kJ m−2. However, the TPC increased with the UV-C treatment and the number of applications. On the
other hand, UV-C maintained the meshophilic and yeast counts during the spinach development in the field and
reduced the mesophilic count after storage. Additionally, both UV-C doses applied in the field also showed an
effect on the antioxidant status of spinach leaves during the storage. However, these UV-C treatments were not
enough to reduce the mold counts in spinach leaves growth in the field and needs to be applied in conjunction
with chemical treatments to prevent diseases.

1. Introduction
Spinach (Spinacia oleracea) is a leafy cool-season vegetable with a
broad cultivation area throughout the world, with a production of more
than 26,492,955 tonnes in 2016 (FAOSTAT, 2016). Spain is among the
top-twenty countries of spinach producers, in 14th position, with
3686 ha cultivated and a production of 67,300 tonnes. Green leafy ve-
getables are involved a part of a healthy diet, particularly, spinach is
rich in minerals such as potassium, folic acid, vitamins (A, B6, C and K),
α-lipoic acid and other bioactive compounds including polyphenols,
which have great health-promoting properties such as anti-cancer, anti-
obesity, insulin-sensitizing and anti-steatotic properties (Roberts and
Moreau, 2016).
However, fungal diseases such as Alternaria alternata, mildew
(Peronospora farinose), etc. can provoke detrimental spinach quality,
which is rejected by the food industry (Gilardi et al., 2018). These
symptoms sometimes appear during the plant development in the field,
although the damage can also appear during the postharvest period.
Therefore, during primary production it is necessary to treat the fresh
plants with chemical products to reduce or avoid pests, fungal attacks
and the development of damage in the product. However, the use of
pesticides could lead to residues in the vegetable crops (Keikotlhaile
and Spanoghe, 2011). Those chemical products are becoming in dis-
appoint with consumer preferences by their contaminating effects and
harmful effect on health (Gilden et al., 2010). The trend in agriculture is
to satisfacy society’s requirements, therefore the search for substitutes
to chemical treatments in crop vegetables is an important approach. As
an alternative to chemical products, UV-C is a physical and en-
vironmentally friendly treatment (Bintsis et al., 2000) which has been
applied in postharvest due to its antimicrobial effect (Artés et al., 2009).

⁎
Corresponding author at: Food Quality and Health Group, Institute of Plant Biotechnology, Universidad Politécnica de Cartagena (UPCT), Campus Muralla del
Mar, 30202, Cartagena, Spain.
E-mail address: encarna.aguayo@upct.es (E. Aguayo).

https://doi.org/10.1016/j.postharvbio.2019.05.021
Received 10 February 2019; Received in revised form 19 April 2019; Accepted 24 May 2019
0925-5214/ © 2019 Elsevier B.V. All rights reserved.
 A. Martínez-Sánchez, et al.

Table 1
Effect of UV-C treatments during the primary production of spinach samples, overall quality, color parameters (L*, a*, b* and AE) and in chlorophyll a (Chl a) and chlorophyll b (Chl b) content.
Days of growing Plant development stage/ UV-C applications UV-C treatments Overall quality L* a* b* AE Chl a (mg kg−1) Chl b (mg kg−1)

After harvest
CTRL 8.00Aa ± 0.00 44.97Aa ± 1.15 −16.87 ± 0.91 26.02 ± 1.66 344.95Bb ± 6.33 573.39Aa ± 43.06
15 I 1.5 kJ m−2 8.67Aa ± 0.29 44.93Aa ± 1.16 −17.60 ± 1.34 27.45 ± 2.35 2.25Ba ± 0.94 617.00Aa ± 56.71 229.69Aa ± 11.19
3 kJ m−2 8.23Aa ± 0.46 45.19Aa ± 1.07 −16.94 ± 0.99 27.17 ± 1.73 2.27Ba ± 1.20 215.69Bb ± 28.57 689.94Aa ± 35.90
CTRL 8.75Aa ± 0.35 43.29Ba ± 1.79 −16.95 ± 1.60 25.80 ± 2.84 393.66Bab ± 40.75 243.92ABa ± 214.55
21 II 1.5 kJ m−2 8.75Aa ± 0.35 43.27Ba ± 1.50 −16.72 ± 1.76 25.40 ± 3.35 3.79Aa ± 1.59 543.61Aa ± 4.13 224.61ABa ± 20.93
3 kJ m−2 8.5Aa ± 0.00 43.91Ba ± 1.72 −16.58 ± 2.01 25.84 ± 4.14 4.38Aa ± 1.54 354.32ABb ± 107.28 341.11ABa ± 199.47
CTRL 7.67Ba ± 0.76 43.84Ba ± 2.07 −16.62 ± 1.42 26.45 ± 2.63 571.78Aa ± 84.41 207.24Ba ± 16.50
27 III 1.5 kJ m−2 6.83Ba ± 0.29 43.78Ba ± 1.87 −16.77 ± 1.39 26.21 ± 2.87 3.46Aa ± 1.36 551.35Aa ± 54.13 251.53Ba ± 67.11
3 kJ m−2 6.50Ba ± 1.50 44.54Ba ± 2.99 −17.33 ± 2.00 28.12 ± 4.75 3.76Aa ± 2.20 562.83Aa ± 32.43 243.50Ba ± 1.60
Time (days of growing) *** *** ns ns * *** **
UV-C treatments ns ns ns ns ns *** ns

131
Time x UV-C treatments ns ns ns ns ns ** ns

After harvesting and storage of 6 days at 5 ºC

21 CTRL 8.88Aa ± 0.25 43.73 ± 2.26 −17.39 ± 1.17 27.21 ± 2.64 615.05Aa ± 38.29 250.48Aa ± 14.21
II 1.5 kJ m−2 6.38Ab ± 1.60 44.30 ± 2.33 −16.89 ± 1.59 26.52 ± 3.48 3.74 ± 2.74 563.40Ab ± 87.38 238.12Aa ± 40.02
3 kJ m−2 5.13Ab ± 2.17 46.39 ± 2.72 −17.04 ± 1.69 27.79 ± 3.91 5.13 ± 3.75 543.34Ab ± 33.19 216.60Aa ± 23.23
27 CTRL 7.00Aa ± 0.00 42.18 ± 1.96 −17.12 ± 1.48 26.42 ± 2.60 664.58Aa ± 27.67 268.06a ± 5.91
III 1.5 kJ m−2 5.50Ab ± 0.58 42.16 ± 2.94 −16.93 ± 1.31 26.33 ± 3.14 4.07 ± 2.42 505.58Ab ± 65.48 206.52Aa ± 44.74
3 kJ m−2 4.50Ab ± 0.71 44.35 ± 3.00 −17.11 ± 1.80 28.35 ± 4.23 4.96 ± 3.00 539.37Ab ± 138.66 275.60Aa ± 21.36
Time (days of growing) ns ns ns ns ns ns ns
UV treatments ** ns ns ns ns * ns
Time x UV-C treatments ns ns ns ns ns ns ns

Values are means of five replicates ± standard deviations (SD). Means followed by different letters in the same column for the same factor (capital letters for applications and lower-case letters for treatments) are
significantly different (P ≤ 0.05) according to Tukey’s HSD (Tukey’s honestly significant difference) test. Asterisks indicate significant differences at *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; NS: non‐significant. CTRL (0 kJ
m−2).
Postharvest Biology and Technology 155 (2019) 130–139
A. Martínez-Sánchez, et al.
Photo 1. Spinach samples in stage III grown in the field and treated with UV-C
(1.5 kJ m−2 (B) and 3 kJ m−2 (C)), with respect to the control (CTRL) treatment
(A).
In addition, UV-C can delay senescence and improve the phytochemical
content with healthy properties for consumers (Escalona et al., 2010).
The germicidal effect of UV-C treatment has been explained by the
damage in the nucleic acids of microorganisms (Artés-Hernández et al.,
2009). Moreover, UV-C treatment could promote the secondary meta-
bolism of determined healthy compounds while, at the same time, these
compounds confer antimicrobial properties to the plant (Cisneros-
Zevallos, 2003; Urban et al., 2016) and improve the antioxidant status
of fruit and vegetables (Jiang et al., 2010). Additionally, the
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Postharvest Biology and Technology 155 (2019) 130–139
accumulative UV-C dose in plants could help in the development of
different defense mechanisms, improving the antioxidant status (Jiang
et al., 2010) and resistence to pathogens (Obande et al., 2011; Jin et al.,
2017), and increasing the shelf life of the food (Liao et al., 2016).
Nevertheless, high UV-C doses could cause damage in the plants
through the development of yellow spots in the leaves due to the
chlorophyll losses and cellular death.
Recently, some research works have studied the effect of preharvest
UV-C treatment in fruit quality and resistence to diseases during crop
production, mainly in strawberry fruits (Jin et al., 2017; Xu et al.,
2018). The aim of this work was to study the effect of UV-C treatment,
applied in primary production at different doses, during three different
maturity stages of spinach plants. During the experiment, vegetable
quality was evaluated after the UV-C treatments applied in the field and
after six days of storage at 5 °C. Parameters such as sensorial quality,
color, chlorophyll content, antioxidant status (vitamin C, total phenolic
content, antioxidant capacity) and microbial quality were all evaluated.
2. Material and methods
2.1. Plant material, study design and UV-C treatment
Spinach seeds (cv. Maya) were sown and grown under commercial
conditions, but without the regular pesticide and fungicide applica-
tions, in the field located in’ Campo de Cartagena’ (Cartagena, Murcia,
Spain) from the end of November through to the middle of December
2016. Spinach samples were cultivated in three beds with a cultivated
area per replicate and treatment of 1.6 m x 9 m, which were randomly
distributed. Two different UV-C treatments, 1.5 kJ m−2 and 3 kJ m−2,
were applied in the spinach, at three different development stages im-
mature stage with only the first true leaf (stage I, at 15 days after
growing); baby commercial stage (stage II, at 21 days after growing);
and adult spinach commercial stage (stage III, at 27 days after
growing). Untreated spinach leaves were considered as the control
treatment (CTRL). Three replicates were randomly distributed in three
ridges.
The UV-C equipment was formed by eight UV-C lamps 1199.4 mm
in length and 28 mm in diameter (Philips TUV 36 W SLV/6, 15.0 W,
Glass Guard®, Thereford, UK) mounted on an adjustable wooden box
(2 m x 0.6 m x 0.75 m) and placed at a distance of 52.2 cm above the
ground. Before starting treatment, the equipment was switched on
30 min before the treatment and calibrated by a radiometer
(VLX254 nm, Marné-la ValléeCedex 2, France). Spinach leaves were
treated at day 15 after planting with 1.5 and 3 kJ m−2. After applying
the UV-C treatments, some spinach samples were harvested and carried
to the laboratory for analysis. Samples were stored in air for six days at
5 °C under darkness conditions at 95% relative humidity (RH). These
fresh samples were analysed for color, sensorial analysis and microbial
quality and frozen samples were used in chlorophyll, vitamin C, total
phenolic content (TPC) and antioxidant capacity analyses at days 0 and
after six days at 5 °C.
2.2. Sensorial analysis
The sensory evaluation was carried out using a triangular test by a
sensory panel of four members (aged 23–42 years). The panel evaluated
differences in crispiness, aroma, taste and color of spinach leaves scored
on an interval hedonic scale and overall quality was evaluated re-
garding the appearance features intensity as well as the acceptability of
spinach leaves (Martínez-Sánchez, et al., 2006) where the lower ex-
treme was scored as 1 (dislike extremely, no characteristic of the pro-
duct) and centre of the interval was represented as 5 (neither like nor
dislike, limit of acceptance from the consumers point of view) and the
upper extreme was scored as 9 (like extremely, very characteristic of
the product).
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Fig. 1. Effect of UV-C treatments (1.5 kJ m−2 and 3 kJ m−2), with respect to the control (CTRL) treatment, during the primary production of spinach samples, in the
Vitamin C (AA + DHAA) content, after harvest (A) and in the postharvest storage (after six days at 5 °C) (B). Values are the mean of three replicates ± SD. Different
letters for the same factor (capital letters for UV-C applications/plant development stage and lower-case letters for UV-C treatments) are significantly different
(P ≤ 0.05) according to Tukey’s HSD (Tukey’s honestly significant difference) test. NS: non‐significant.

2.3. Color analysis
The color of spinach leaves was determined at three equidistant
points using a colorimeter (Minolta CR-300 Series, Japan) and the re-
sults were expressed as CIE L*a*b* color space units. The results were
expressed as luminosity (CIE L*), L* (brightness), a* (plan red-green),
b* (plan yellow-blue) and color differences as ΔE = ½(⌊ (L0 − L*) 2
+ (a0 − a*)2 + (b0 − b*)2⌋ ) (Chisari et al., 2010).
2.4. Chlorophyll determination
The extraction of chlorophyll was performed as previously reported
by Martínez-Sánchez et al. (2006). The results were expressed in mg per
kg of fresh weight as the mean of five replicates per each UV-C dose and
development stage during preharvest and dose during postharvest.
2.5. Vitamin C analysis
Frozen samples (5 g) were homogenized in 15 ml of extraction
medium; standard solutions, column conditioning, mobile phase, flow
rate, wavelengths and derivatization procedures were previously re-
ported by Martínez-Sánchez et al. (2008). The results were expressed in
mg per kg of fresh weight as the mean of three replicates per each UV-C
dose and development stage during preharvest and dose during post-
harvest.
2.6. Total phenolic content (TPC)
Frozen samples (0.5 g) were homogenized by Ultra-Turrax (T-25,
Ika-Labortechnik, Staufen, Germany) with 15 ml of a mixture of MeOH/
water (8:2, v:v), containing 4 mM NaF and homogenized for 1 min. The
extracts were centrifuged at 12,500×g for 10 min at 4 °C and super-
natants were used to determine the TPC by the reduction of Folin-.
Ciocalteau reagent as previously described by Martínez-Hernández
et al. (2011). The results were expressed in mg gallic acid equivalents
(GAE) per 1 kg of fresh weight as the mean of three replicates per each
UV-C dose and development stage during preharvest and dose during
postharvest.
2.7. Antioxidant capacity
The antioxidant capacity was determined, by ABTS and FRAP as-
says, using extracts from vitamin C, without filtering through C18 Sep-
Pak cartridge (Waters, Milford, MA, USA) (Martínez Sánchez et al.,

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Fig. 2. Effect of UV-C treatments (1.5 kJ m−2 and 3 kJ m−2), with respect to the control (0 kJ m−2) treatment, during the primary production of spinach samples, in
the total phenolic content (TPC), after harvest (A) and after six days of storage at 5 °C (B). Values are the mean of three replicates ± SD. Different letters for the
same factor (capital letters for UV-C applications/plant development stage and lower-case letters for UV-C treatments) are significantly different (P ≤ 0.05) according
to Tukey’s HSD (Tukey’s honestly significant difference) test. NS: non‐significant.

2008), with some modifications. In the ABTS*+ assay, the spinach ex-
tract (11 μL) was mixed with 32 μM of water solution (200 μL) of the
free radical ABTS*+. In the FRAP assay, the spinach extract (6 μL) was
mixed with 198 μL of FRAP solution.
The two different reactions were mixed in a well plate and in-
cubated for 45 min at room temperature in darkness conditions. The
changes in absorbance were determined at 25 °C in a Tecan Infinite®
200 micro plate reader (Grödig, Austria) at 414 nm and 593 nm for the
ABTS*+ and FRAP assays, respectively. Trolox was used as the standard
and the antioxidant activity was expressed as mg trolox equivalent
antioxidant capacity (TEAC) per kg of fresh weight.
2.8. Microbiological analyses
The natural microflora concerned with mesophiles, molds and
yeasts were analysed in 15 g of spinach samples by standard enu-
meration methods according to Martínez-Sánchez et al. (2006). Mi-
crobial counts were reported as log colony forming units per gram of
product (log c.f.u. g−1 f.w.).
2.9. Statistical analysis
To compare the different doses, development stages and storage
times an ANOVA analysis was performed at a significance level of
P ≤ 0.05 using PASW Statistics 23 for Windows (SPSS Inc., Chicago, IL,
USA) and the Tukey’s HSD (Honestly Significant Difference) test was
applied when significant differences were observed.
3. Results
3.1. Sensorial quality
Overall quality was influenced by growing time, with the quality
being lower at stage III (Table 1). However, in the field, spinach in stage
II treated with 3 kJ m−2 showed a lower growth than the rest of sam-
ples and the leaves were bent, these symptoms were more marked in
development stage III, after two UV-C applications (Photo 1 ). Spinach
treated with 1.5 kJ m−2 showed the same symptoms in stage III, but on
a lower scale. In addition to this, spinach from the third stage showed a
loss of crispiness and flavor (data not shown) and a reduction in the
overall quality even in the CTRL samples. However, the overall quality
of spinach was above the threshold of commercial acceptability in all
treatments (Table 1). During the postharvest storage, the sensorial
quality decreased in the UV-C treated samples, while no differences
were found between the UV-C doses (Table 1).

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A. Martínez-Sánchez, et al.
3.2. Color
A slight decrease in L* was observed with the plant growth. In plant
stage I, the L* parameter was slightly higher than in stages II and III
(Table 1). An increase in the changes of color was also detected at
stages II and III compared to the first stage (Table 1). However, the UV-
C doses did not have a significant effect on any color parameters of
spinach leaves, independently of whether the UV-C treatments were
applied once or three times during spinach development. Throughout
the postharvest storage in darkness conditions, up to six days at 5 °C, a
lighter green color (minor L*) was only observed in the spinach samples
treated with UV-C (Table 1).
3.3. Chlorophyll
No significant differences were observed between the CTRL and UV-
C treatments in the total chlorophyll (chlorophyll a + chlorophyll b)
content (data not shown). Spinach samples from stage I showed the
highest total chlorophyll content, although with no significant differ-
ences with samples from stage III. The chlorophyll b content showed
significant differences between the development stages, with the lowest
chlorophyll content in the stage III with respect to stage I (Table 1).
However, the chlorophyll a content increased in stage III with respect to
that of stage I. In spite of this, the use of 1.5 kJ m−2 provided spinach
with the highest chlorophyll a content in development stages I and II
(Table 1).
During the postharvest storage for six days at 5 °C, the chlorophyll a
content showed significant differences between spinach samples, with
the lowest content being found in those treated with UV-C (Table 1).
3.4. Vitamin C content
In relation to the content of total vitamin C (AA + DHA) a decrease
was observed with the spinach development stage (Fig. 1A).
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Postharvest Biology and Technology 155 (2019) 130–139
Fig. 3. Effect of UV-C treatments (1.5 kJ m−2
and 3 kJ m−2), with respect to the control (0 kJ
m−2) treatment, during the primary produc-
tion of spinach samples, in the antioxidant
capacity evaluated by two different assays
(ABTS and FRAP), after harvest (A, C) and after
six days of storage at 5 °C (B, D). Values are the
mean of three replicates ± SD. Different let-
ters for the same factor (capital letters for UV-C
applications/plant development stage and
lower-case letters for UV-C treatments) are
significantly different (P ≤ 0.05) according to
Tukey’s HSD (Tukey’s honestly significant dif-
ference) test. NS: non‐significant.
Additionally, in the first spinach stage, a tendency to increase the vi-
tamin C content with the UV-C dose was observed, although no sig-
nificant differences were detected between the UV-C and CTRL samples.
However, in stage II (after two UV-C applications), the CTRL samples
showed a higher vitamin C content than those treated with 3 kJ m−2,
while no significant differences were observed between the CTRL and
1.5 kJ m−2 treated samples. Finally, in the third stage (after 3 UV-C
applications), the CTRL samples showed the highest vitamin C content.
At the end of the postharvest storage, the vitamin C content of
spinach leaves did not show significant differences between the UV-C
treated and CTRL samples (Fig. 1B). However, a similar trend to pri-
mary production was observed in relation to vitamin C content with the
development stage, with the lowest level being detected in stage III of
spinach development.
3.5. Total phenolic content (TPC)
In the first development stage no significant differences were ob-
served between the UV-C treatments and CTRL samples. However, in
the second development stage (after two applications of UV-C) spinach
treated with 3 kJ m−2 showed the highest TPC, and in the third de-
velopment stage (after three applications of UV-C) spinach treated with
UV-C (1.5 or 3 kJ m−2) showed the highest TPC (Fig. 2A). However,
contrary to what was observed in the vitamin C content, the TPC was
increasing with the development stages of spinach. Therefore, the third
stage showed the highest TPC content, independently of the UV-C
treatment applied. A similar trend was observed at the end of post-
harvest storage, the treatment with 3 kJ m-2 provided spinach with the
highest TPC, although without significant differences from those leaves
treated with 1.5 kJ m−2 in the third stage of development (Fig. 2B).
3.6. Antioxidant capacity
The antioxidant capacity of spinach samples of two different
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Fig. 4. Effect of UV-C treatments (T1, 1.5 kJ m−2 and T2, 3 kJ m−2), with respect to the control (0 kJ m−2) treatment, during the primary production of spinach
samples, in the microbial counting of mesophilic bacteria, molds and yeast, after harvest (A, C, E) and after six days of storage at 5 °C (B, D, F). Values are the mean of
three replicates ± SD. Different letters for the same factor (capital letters for UV-C applications/plant development stage and lower-case letters for UV-C treat-
ments) are significantly different (P ≤ 0.05) according to Tukey’s HSD (Tukey’s honestly significant difference) test. NS: non‐significant.

cultivars and treated with different UV-C treatments was evaluated by
two different assays: ABTS and FRAP assays.
3.6.1. ABTS assay
In the ABTS assay, in the first stage, no significant differences were
observed between the UV-C treated and CTRL samples (Fig. 3A).
However, in the second and third stages significant differences were
detected between the UV-C and CTRL samples; the antioxidant capacity
increased with the UV-C dose. During the postharvest storage, the UV-C
treatment of 1.5 kJ m−2 showed the highest antioxidant capacity in
development stages II and III, whitout significant differences with the
CTRL samples in stage II and with no significant differences with the
UV-C treatment of 3 kJ m−2 in stage III (Fig. 3B).
3.6.2. FRAP assay
The antioxidant capacity evaluated by FRAP assay showed, in the
CTRL samples, a tendency to decrease the antioxidant capacity with the
development of spinach samples, while the UV-C treatments showed
reduced effects of the development in the antioxidant capacity eval-
uated by FRAP assay (Fig. 3C). Thus, in the third development stage
spinach samples treated with UV-C showed the highest antioxidant
capacity. However, in the first and second stages, no significant dif-
ferences were observed between the UV-C treatments and CTRL sam-
ples. During the postharvest storage, in stage II the UV-C treatment of
3 kJ m−2 showed the lowest antioxidant capacity, while in stage III the
CTRL samples showed the lowest antioxidant capacity (Fig. 3D).

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A. Martínez-Sánchez, et al.
3.7. Microbiological counting
3.7.1. Effect of UV-C treatments in mesophilic bacteria
Spinach samples treated with UV-C maintained the mesophilic
counts in development stages II and III, whilst the CTRL samples
showed a significant bacterial growth more than 2 log c.f.u. g−1.
(Fig. 4A). During the postharvest storage, this same trend was found in
the third stage spinach, thus UV-C use during plant development pro-
vided an important microbial control (Fig. 4B).
3.7.2. Effect of UV-C treatments in yeast
In the first development stage the yeast count was below the de-
tection limit in all the samples (< 2 log c.f.u. g−1. The yeast count was
increased with the development stage of spinach plants, although the
UV-C treatments reduced yeast growth. The lowest counts were ob-
served in the 3 kJ m-2 UV-C treated samples (Fig. 4C). However, during
the postharvest storage, the yeast count was below the detection limit
in all the samples in the second stage, and no significant differences
were observed between the UV-C treatment and CTRL samples in the
third development stage (Fig. 4D).
3.7.3. Effect of UV-C treatments in molds
No fungal damage associated to Alternaria sp., mildew or Cercospora
sp. was detected in any of the spinach plant treatments. Spinach sam-
ples treated with UV-C showed minor mold counts, but no significicant
differences were found between the UV-C treatments and the CTRL
(Fig. 4E). This same trend was found during the postharvest storage
(Fig. 4F).
4. Discusion
The green color is an important quality attribute in spinach leaves
which is linked to their freshness, while degreening and yellowing is
associated to spinach senescence. A tendency to decrease L* with the
UV-C treatments was observed, however these changes were not sig-
nificant. With regard to this, Artés-Hernández et al. (2009) described an
L* decrease of around 10–15 % in spinach samples harvested and
treated with UV-C at 4.54, 7.94, and 11.35 kJ m−2. Leaf color has been
linked to the total chlorophyll content in the leaves. In our trials, the
UV-C treatments preserved or increased the total chlrophyll content in
spinach leaves, mainly when early spinach leaves were treated with UV-
C doses. The hormesis effect of UV-C could delay photosynthetic pig-
ment degradation and improve the photosynthetic capacity (Reyes Jara
et al., 2019). During the postharvest storage of rocket, Gutiérrez et al.
(2018) observed a minor decrease in the chlorophyll content in samples
treated with UV-C (10 and 20 kJ m-2) with respect to the CTRL. Similar
results have been described in broccoli treated with 10 kJ m-2 of UV-C
and stored at 20 °C for four days, due to a reduction in the enzymatic
acitivity liked to chlorophylls (Costa et al., 2006). Additionally, UV-C
can delay the increase of expression of a gene encoding a putative
chlorophyll b reductase (Reyes Jara et al., 2019). On the contrary,
previous studies have reported a reduction in the initial chlorophyll
content in broccoli treated with UV-C (around 23% for 4.5 kJ m-2 and
31% for 9 and 15 kJ m-2) (Martínez-Hernández et al., 2011) or a re-
duction in the chlorophyll content after the storage at 5 °C for 12 days
in rocket samples treated with UV-C (10, 20 and 30 kJ m-2) (Gutiérrez
et al., 2015). It is likely that the differences observed between the
previous studies and our trials could be due to the different UV-C doses
applied, whether the UV-C treatment was applied before or after har-
vest, and the different vegetables studied, and also when the effect was
evaluated. These changes in the chlorophyll content can be linked to
the color modifications observed during spinach development.
On the other hand, several studies described the activation of sec-
ondary metabolism to increase bioactive compounds. However, the
decrease in the vitamin C content with the UV-C treatment of 3 kJ m−2
observed in development stage II could be linked to the high oxidative
137
Postharvest Biology and Technology 155 (2019) 130–139
stress by the high UV-C treatment, which oxidates AA to DHA, which
could be oxidated, thus decreasing the vitamin C content (Xu et al.,
2018). These results could be linked to the different development pat-
tern of spinach leaves, since ascorbic acid is linked to plant growth. In
plants, ascorbic acid is one of the most important antioxidants to avoid
the increase in ROS content and it is involved in several physiological
processes controlling growth, development, and stress tolerance.
Therefore, plant growth and development can be affected by changes in
cellular ascorbic acid levels (Pastori et al., 2003). This decrease in the
vitamin C content can be correlated with the lower growth and the bent
leaves of spinach treated with 3 kJ m−2 observed in development stage
II or with both UV-C treatments in stage III (Photo 1). UV-C treatment
could have an accumulative effect in the vegetative cell, even though
the next treatment would be applied two weeks later. For that reason, it
is probable that lower doses applied with a higher frequency could have
a greater positive effect. In this sense, an increase in vitamin C content
due to the hormesis effect has been described in previous studies where
low UV-C doses (0.3 or 0.5 kJ m−2) were applied every 2 days in
strawberry plants (Severo et al., 2017). Additionally, after storage no
significant differences were observed in the vitamin C content of spi-
nach. In agreement with this, a reduction in ROS levels has been de-
scribed at 24 h of UV-C treatment (in strawberries harvested with an
accumulative dose of 6.6 kJ m−2), with similar ROS and AA levels in
the UV-C treated and control strawberries (Xu et al., 2018).
On the contrary, TPC increased with the UV-C dose applied and
number of applications. Topcu et al. (2015) described an increase in the
polyphenols content and antioxidant compounds in broccoli grown in
greenhouse under hydroponic system and treated with UV-C at 2.2, 8.8
and 16.4 kJ m−2 day-1. In addition, an increase in phenolic compounds
of around 14% was described in harvested broccoli treated with UV-C at
9 and 15 kJ m−2 (Martínez-Hernández et al., 2011). These results
concur in considering the UV-C radiation as an abiotic stress that can
improve the phytochemical compounds of vegetable and fruits
(Cisneros-Zevallos, 2003). In this sense, Nigro et al. (2000) reported an
increase in phenylalanine amonio lyase activity (PAL, key enzyme in
the synthesis of phenolic compounds) in strawberries treated with a low
UV-C dose (0.5 kJ m−2), while a higher UV-C dose (2.5 kJ m−2) showed
a lower effect. Furthermore, there is an increase in ROS content in
plants submitted to abiotic stresses (Xu et al., 2018). For this reason, it
is necessary to neutralize ROS molecules, and plants have two different
systems: the activation or production of antioxidant ROS enzymes and
the antioxidant compounds like phenolic compounds among others
(Gurunani et al., 2015). Therefore, the UV-C treatment applied in spi-
nach samples could be considered as an abiotic stress that increases the
TPC in spinach samples to maintain the balance between ROS com-
pounds and antioxidants. In our study, a greater antioxidant activity has
been observed under the UV-C treatments in the different assays eval-
uated: in ABTS after two UV-C applications and in FRAP after three UV-
C applications. The helpful UV-C properties to extend the shelf-life of
postharvest products are linked to the capacity to stimulate the anti-
oxidant defense system and reduce the oxidative damage (Urban et al.,
2016). Previous research works have described an antioxidant effect
linked to the phenolic compounds and antioxidant enzymes in straw-
berries treated with 2 kJ m−2 of UV-C (Jin et al., 2017) or in straw-
berries treated during preharvest with 0.94 kJ m-2 and 1.84 kJ m-2 and
applied several times (Xu et al., 2017). Additionally, an increase in the
TPC and in the antioxidant enzymes has been described in lettuce
treated in preharvest with 0.85 kJ m-2 of UV-C applied four times
(Vásques et al., 2017). Moreover, Martínez-Hernández et al. (2011)
observed a greater phenolic compounds content and antioxidant ca-
pacity with increasing the UV-C dose from 1.5 to 15 kJ m−2. However,
in our study the UV-C treatment of 3 kJ m-2 showed a decrease in the
antioxidant capacity after storage in both the ABTS and FRAP assays
evaluated. Similar results were described by Artés-Hernández et al.
(2009) in spinach leaves treated with UV-C (4.54, 7.94 and 11.35 kJ
m−2) and stored at 5 °C and 8 °C.
A. Martínez-Sánchez, et al.
Regarding the antimicrobial effect of UV-C, it has been previously
described in an extensive number of research works in postharvest
(Artés-Hernández et al., 2009; Martínez-Hernández et al., 2015). Baby
spinach leaves treated with high doses of UV-C (12 or 24 kJ m−2) had
reductions of between 1 or 1.5 log u.f.c. g-1 with respect to the control
samples and only these doses showed a reduction in mold counts
(Escalona et al., 2010). However, the susceptibility to UV-C light in
each tissue must be taken in account, additionally, early tissue can be
more susceptible to UV-C light than medium age or older tissues in the
same plant. This could be appreciated in spinach samples when early
plants were treated at the same doses as at the rest of the development
stages. In our study, the UV-C dose of 3 kJ m−2 showed a reduction in
yeast and mold counts, while the reduction in mesophilic bacteria was
observed after two applications of UV-C (1.5 or 3 kJ m-2). Some authors
have reported the positive effect of several UV-C applications in redu-
cing the microbial count in fruit and vegetables. Vàsquez et al. (2017)
described the positive effect of four applications of UV-C (0.85 kJ m−2
and 1.70 kJ m−2) in lettuce leaves inoculated with B. cinerea. Moreover,
de Oliveira et al. (2016), showed a reduction in mesophilic counts in
strawberries treated with UV-C (0.5 kJ m-2, every four days) with re-
spect to control samples. Additionally, a resistence in strawberries
treated with UV-C was observed against fungal pathogens such as gray
mold decay caused by Botrytis cinerea (Jin et al., 2017) or also against
Tetranychus urticae (D Short et al., 2018). The antimicrobial power of
UV-C treatment has been linked to the stimulation of antioxidant de-
fense systems (antioxidant secondary metabolites and antioxidant en-
zymes) (Urban et al., 2016). In addition to this defense system, the
production of phytoalexins could improve the antimicrobial effect in
the plant by the UV-C treatments (Charles et al., 2008). In our study, the
UV-C treatments helped to control the microbial counts, in particular
for mesophilic growth; however, the reductions observed were between
0.5 and 2 log u.f.c. g-1 depending on the microorganism evaluated and
the UV-C treatment applied.
5. Conclusions
A proper dose of UV-C light, used as a preharvest tool during pri-
mary production of spinach plant, can be an abiotic stress which sti-
mulates the plant defense mechanism, increases the healthy compounds
content for the consumer, and at the same time can reduce the micro-
bial counts without pesticide residues. In this study, UV-C treatment of
1.5 kJ m−2 was better than 3 kJ m-2, since improves the antioxidant
capacity, the TPC, reduce the microbial counts and spinach plants
showed sensitivity to the UV-C treatment of 3 kJ m-2. Although, UV-C
treatment of 1.5 kJ m-2 could affect the plant development after several
applications. It could be explaining because immature leaves are more
susceptible to UV-C and the UV-C treatments are cumulative in the
plant. These results can be useful in agriculture, although more studies
would be necessary in order to design the optimal dose and number of
applications to promote antioxidant compounds biosynthesis and con-
trol fungal damage during primary production and postharvest time.
Probably, an increase of UV applications at lower radiations would
improve the effect of UV-C as an antimicrobial preharvest treatment.
Acknowledgements
The authors are grateful to Verdimed S.A. for providing the spinach
field and samples. A. Martínez-Sánchez is the holder of a postdoctoral
grant (‘Juan de la Cierva’ FPDI-2013-17220) from the Spanish
Government (MINECO). We are also grateful to Josefa Guirao Martínez
for her technical assistance.
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