Food Control 35 (2014) 132e136

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Food Control
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The essential oils thymol and carvacrol applied in the packing lines
avoid lemon spoilage and maintain quality during storage
S. Castillo a, *, C.O. Pérez-Alfonso a, D. Martínez-Romero a, F. Guillén a,
M. Serrano b, D. Valero a
a
Dept. of Food Technology, University Miguel Hernández, Ctra. Beniel km. 3.2, 03312 Orihuela, Alicante, Spain
b
Dept. Applied Biology, University Miguel Hernández, Ctra. Beniel km. 3.2, 03312 Orihuela, Alicante, Spain

a r t i c l e i n f o a b s t r a c t

Article history: The objective of this paper was to study the effect of essential oils (EOs) thymol and carvacrol applied in
Received 6 March 2013 citrus packing lines in combination with commercial wax on microbial spoilage, lemon decay and quality
Received in revised form parameters during postharvest storage and compared with control (tap water), wax and wax þ imazalil
17 June 2013
as positive control. Results showed that the treatment with wax þ EOs was able to reduce yeast and
Accepted 25 June 2013
mould and total aerobe counts on lemon surface and decayed lemons after 8 weeks of storage, the ef-
ficacy being similar to that obtained from wax þ imazalil treatment. In addition, lemons coated with EOs
Keywords:
or imazalil showed similar quality attributes (weight loss, softening and colour) retention compared with
Antimicrobial
Fungal decay
control or waxed fruits.
Citrus limon L. Ó 2013 Elsevier Ltd. All rights reserved.
Firmness
Colour
Acidity

1. Introduction
Lemons are susceptible to a number of postharvest fungal dis-
eases, the most important being caused by Penicillium digitatum
and Penicillium italicum which are responsible for green and blue
mould, respectively (Valero & Serrano, 2010). The pathogens
penetrate the fruit through the wounds that occurred during har-
vesting and improper handling in the packing lines (Smilanick,
Mansour, Margosan, Gabler, & Goodwin, 2005) leading to signifi-
cant postharvest losses every year. To control these pre- and
postharvest fungal diseases imazalil has been widely used in the
last three decades. Imazalil is an N-substituted imidazole systemic
fungicide that inhibits 1,4 a-demethylation in the ergosterol
biosynthesis pathway in fungal cells, with an ability to inhibit
sporulation of P. digitatum and P. italicum (Siegel, Kerkenaar, &
Kaars-Sijpesteijn, 1977).
However, the alternate use of imazalil has led to occurrence of
resistant strains attributed to a 100-fold enhancement of the
constitutive expression of the CYP51 gene, which encodes the
ergosterol 1,4 a-demethylase enzyme (Hamamoto et al., 2000).
Residue loading can give an indication of the effectiveness of
* Corresponding author. Tel.: þ34 96674 9733; fax: þ34 96674 9677.
E-mail address: scastillo@umh.es (S. Castillo).
imazalil application, but control of green mould differ for the
method of application (Erasmus et al., 2011). The great postharvest
losses in the citrus industry by these resistant strains demonstrate
the need to find new active ingredients to be used as citrus post-
harvest fungicides. In this sense some chemicals as pyrimethanil,
fludioxonil and azoxystrobin have been recently registered
(Kanetis, Förster, & Adaskveg, 2007). On the other hand, significant
improvements of the imazalil effectiveness to control green mould
have been achieved when imazalil was applied at 50  C instead of
ambient temperature, which is the commercial procedure (Dore,
Molinu, Venditti, & D’Hallewin, 2009), although fungicide resi-
dues are enhanced in the lemon rind. Thus, new alternatives are
required with natural compounds exhibiting antifungal activity
without detrimental effect on human health, environmentally
friendly and less contaminant.
In this sense, essential oils (EOs) have been proven to be
inhibitory against a wide range of food spoiling microorganisms
alone (Tajkarimi, Ibrahim, & Cliver, 2010) or in combination with
modified atmosphere packaging (Serrano et al., 2008). Among
these EOs, thymol and carvacrol, the main active components of
thyme and oregano, respectively, have shown effectiveness on
reducing microbial counts in sweet cherry (Serrano, Martínez-
Romero, Castillo, Guillén, & Valero 2005) and table grape (Guillén
et al., 2007; Valero et al., 2006; Valverde et al., 2005), showing an
additional effect on maintaining the overall fruit quality during

0956-7135/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodcont.2013.06.052
 S. Castillo et al. / Food Control 35 (2014) 132e136
storage, including organoleptic, nutritive and functional properties.
Thyme oil controlled decay caused by Alternaria alternate in cherry
tomato, the effect being enhanced when the EO was applied as
vapour phase than by dipping (Feng, Chen, Zheng, & Liu, 2011). In
citrus fruits there are few studies about this issue, although
peppermint, spearmint, and thyme essential oils decreased green
and blue mould decay in ‘Valencia’ oranges (Du Plooy, Regnier, &
Combrinck, 2009; Fatemi, Jafarpour, Eghbaisaled, Rezapour, &
Borji, 2011). The essential oils are considered as Generally Recog-
nized as Safe (GRAS) food ingredients, and the World Health Or-
ganization (WHO) has stated that thymol and carvacrol residues in
food are without danger to the consumer as long as they do not
exceed 50 mg/kg (WHO, 2012), and thus they could be applied as
postharvest treatment for controlling fruit decay.
Recently, the antifungal activity of thymol and carvacrol against
P. digitatum and P. italicum has been reported in vitro tests and
inhibited decay in lemon artificially inoculated with P. digitatum in
a concentration-dependent manner (Pérez-Alfonso et al., 2012). A
further step would be application of these essential oils in the citrus
packing lines in combination with the wax used for commercial
purposes, which was the objective of this work and using imazalil
as positive control.
2. Material and methods
2.1. Experimental design
The experiment was carried out on commercial packing lines for
citrus fruits (Frutas Naturales, S.A.) located nearby the Postharvest
Laboratories. About 2500 lemons (Citrus limon L. cv. Fino) were
manually picked early in the morning and transferred to the
packinghouse. Fruits were selected based on colour and size uni-
formity and with absence of defects or injuries. Lemons were
passed over a series of soft bristled roller brushes to improve the
appearance by removal dirt and residues. Then, 4 lots of 600 fruits
were used for the following treatments: a) control (tap water), b)
commercial wax (WaterwaxÔ, Fomesa, Valencia, Spain), c)
wax þ imazalil at 2000 mL/L, and d) wax þ essential oils (EOs,
mixture of thymol and carvacrol, purchased from Sigma, Madrid
Spain, at 500 mL/L each). Treatments were applied by high pressure
spray solutions as lemons passes through the conveyor belt. Be-
tween treatments the packing line was washed with water to avoid
cross-contamination. Following treatments the fruits were dried
(force-air at room temperature), then numbered, individually
weighed, and packed in carton fibreboard box containing 40
lemons and transferred to the laboratory for storage at 10  C and RH
of 85e90% for 8 weeks. On bi-weekly basis, 3 boxes from each
treatment were taken out from chambers and decayed lemons
were counted for calculating percentage of decayed fruit. 10 healthy
fruits (absence of fungal growth) were taken at random from each
box for the following determinations: microbial counts (yeast and
mould and mesophilic aerobes), weight loss, colour, firmness, total
soluble solids (TSS) and total acidity (TA).
2.2. Weight loss, colour and firmness
Weight of individual fruits was recorded on the day of har-
vesting and after the different sampling dates. Cumulative weight
losses were expressed as percentage loss of original weight
(n ¼ 30). Superficial colour was determined by measuring L*, a* and
b* chromaticity co-ordinates of the CIE Lab scale and the total
colour difference (Konica Minolta, 2007) between day 0 and last
sampling date was calculated by using the formula:
DE ¼ O[(L0L)2 þ (a0a)2 þ (b0b)2], where L0, a0 and b0 are the
readings at day 0 and L, a, and b are the readings after 8 weeks of
133
storage (n ¼ 30). Firmness was determined on 30 lemons from each
treatment (10 for each of the 3 replicates) using a flat steel plate
probe coupled on a TX-XT2i Texture Analyzer (Stable Microsystems,
UK). A bevelled holder prevented bruising of the opposite side. For
each fruit, the diameter was measured and then a force that ach-
ieved a 3% deformation of the fruit diameter was applied. Results
were expressed as the ratio between the measured force and the
lemon diameter (N/mm).
2.3. Total soluble solids and total acidity
The juice from 10 lemons for each replicate was used to deter-
mine total soluble solids (TSS) and total acidity (TA). TSS were
determined with a digital refractometer Atago PR-101 (Atago Co.
Ltd., Japan) at 20  C and results were the mean  SE of 2 de-
terminations for each sub-sample and expressed as  Brix (g/100 g).
The pH of the juice was recorded and then TA was determined by
potentiometric titration with 0.1 N NaOH up to pH 8.1, using 1 mL of
diluted juice in 25 mL distilled H2O and results were the
means  SE of 2 determinations for each subsample and expressed
as g of citric acid equivalent per 100 g fresh weight.
2.4. Microbiological analysis and lemon decay
For each replicate and treatment 2 lemons were taken under
sterilized conditions (laminar fume cupboard and gloves). Each
lemon was washed in a bag containing 90 mL of sterile peptone
water with gentle shaking. Serial dilutions were carried out and
1 mL was added to plate count agar (PetrifilmÔ Aerobic and Yeast
and Mould Count Plates, Laboratories 3MÔ Santé, France). Samples
were loaded onto the plates and incubated during 3 days at 30  C
and 5 days at 25  C for mesophilic aerobes and yeast and mould,
respectively, and only counts of 30e300 colony forming units (CFU)
were considered. Results were expressed as CFU/mL (n ¼ 6). For
decay analysis, the number of decayed and faded lemons for each
replicate and treatment was counted, and then decay percentage
from the total fruits was calculated (n ¼ 3).
2.5. Statistical analysis
Data from analytical determinations were subjected to analysis
of variance (ANOVA). Sources of variation were treatments and
storage. Mean comparisons were performed using the high signif-
icant difference (HSD) Tukey’s test to examine if differences were
significant at P < 0.05. All analyses were performed with statistical
products and service solutions (SPSS) software package v. 11.0 for
Windows. Results from statistical analysis are shown in Table 1.
Table 1
ANOVA for dependent variables of lemon fruit affected by storage (time) and
treatment [control, wax, wax þ imazalil and wax þ EOs (essential oils)] and their
interactions for lemon fruit.a
Time Treatment Time  treatment
Yeast and Mould *** *** **
Mesophilic aerobic *** *** **
Decayed fruit ** ** *
Colour difference ** ** *
Weight loss ** ** *
Fruit firmness * * *
Total soluble solids * * *
Total acidity * NS NS
a
**, and* represent significance at the 0.01, and 0.05 levels, respectively, and NS
represents non-significance at P < 0.05.
134 S. Castillo et al. / Food Control 35 (2014) 132e136

3. Results and discussion

Control A a
B
Wa
Wax
3.1. Microbial counts and lemon decay Wax + Imazalil 12
80 Wax + EOs b
During postharvest storage of lemons, an increase in both yeast c c

Colour Difference (Units)

a 10
and mould and mesophilic aerobes was observed, the magnitude

Decayed Fruit (%)

being affected by treatments (Table 1, Fig. 1). The highest counts 60
were obtained for control and wax followed by wax þ imazalil and 8

wax-essential oils (EOs) treated lemons, although the application of

b
wax alone reduced yeast and mould counts and did not affect the 6
40
mesophilic aerobes. Interestingly, the application of wax þ EOs was
more effective than wax þ imazalil on reducing both yeast and
4
moulds and mesophilic aerobe counts along storage. This effect c
c
could be attributed to occurrence of imazalil-resistant spores which 20
appear when imazalil is applied at levels below MRL (Erasmus, 2

Lennox, Smilanick, Lesart, & Fourie, 2013). These authors postu-

late that adjusting the formulation, solution pH and exposure time
the control green mould without the risk of exceeding the imazalil
MRL could be achieved. This reduction in microbial spoilage was
also reflected in the percentage of decayed fruits (Table 1, Fig. 2),
which was y65, 40, 18 and 17% for control, wax, wax þ imazalil and
Treatment Treatment
Fig. 2. Percentage of decayed lemons (A) and colour difference (B) after 8 weeks of
storage in control and treated lemons with wax, wax þ imazalil and wax þ EOs
(essential oils). Data are the mean  SE. Bars with different letters show significant
differences (P < 0.05) among treatments for each sampling date.

Control A wax þ EOs treatments, respectively. These results confirm a pre-

35000 a
Wax vious report in which thymol, carvacrol or the mixture
Wax + Imazalil
thymol þ carvacrol were effective on reducing P. digitatum and
Yeast and Moulds (CFU/mL)

30000 Wax + EOs a

P. italicum growth in test tubes, potato dextrose agar (PDA) plates
b and PDA disks, the reduction being dependent on EO concentration
25000 a (Pérez-Alfonso et al., 2012). Similarly, carvacrol was effective on
inhibiting spore germination of Botrytis cinerea grown in PDA and
20000 b on artificially-inoculated table grapes (Martínez-Romero et al.,
b
2007), although the efficacy was higher in PDA than in grapes.
c
15000 When EOs were incorporated to modified atmosphere packages
(MAP), as an active packaging, higher reductions in microbial
counts were obtained compared with MAP alone, as has been
10000 c c
observed for cherries (Serrano et al., 2005; 2008) and grapes
d d (Valero et al., 2006; Valverde et al., 2005), with maintenance of fruit
d
5000 a aaa quality and shelf life for longer periods. Similarly, oregano and the
aaa a mixture of oregano þ thyme resulted similarly effective as chlorine
on decontamination of carrot and lettuce ready-to-eat (Gutierrez,
Control
a a
B Bourke, Longchamp, & Barry-Ryan, 2009).
1.4e+5 Wax From the point of view of decayed fruit similar results were
Wax + Imazalil obtained between imazalil and EOs application in combination of
Mesophilic Aerobes (CFU/mL)

1.2e+5
Wax + EOs wax. In ‘Valencia’ oranges, other EOs such as peppermint and
thyme controlled green mould (Fatemi et al., 2011), as well as
thyme and camphor on ‘Baladi’ oranges (Badawi, Sallam, Ibrahim, &
1.0e+5
Asran, 2011). In semi-industrial trials, the incorporation of essential
oils from Mentha spicata, Lippia scaberrima, and Cinnamomum
8.0e+4 a a zeylanicum into commercial citrus waxes reduced decay caused by
P. digitatum and P. italicum, the effective concentration of EOs in
6.0e+4 a wax being 10-fold lower than when EOs were applied by dipping in
a
b b ethanol solutions (Du Plooy et al., 2009; Kouassi, Bajji, & Jijakli,
4.0e+4 c b 2012). In addition, a synergistic effect has been found for essen-
b tial oils, since the antifungal effect of the combination of two EOs is
2.0e+4 c
higher than the sum of the individual effects (Tajkarimi et al., 2010).
a c c The mechanism by which EOs reduced fruit decay is not well
bb d d
c understood, although some authors have attributed it to their hy-
0 2 4 6 8 drophobicity, which enables them to partition in the lipids of the
cell membrane disturbing its integrity and the inorganic ions
Storage weeks at 10°C equilibrium (Lambert, Skandamis, Coote, & Nychas, 2001). Addi-
tionally, it has been postulated that the presence of the phenolic
Fig. 1. Counts for both yeast and moulds (A) and mesophilic aerobes (B) in control and
treated lemons with wax, wax þ imazalil and wax þ EOs (essential oils) during storage.
ring may be necessary for the antimicrobial activity of eugenol and
Data are the mean  SE. Bars with different letters show significant differences thymol (Ultee, Bennik, & Moezelaar, 2002). Moreover, the site(s)
(P < 0.05) among treatments for each sampling date. and number of hydroxyl groups on the phenol ring are thought to
 S. Castillo et al. / Food Control 35 (2014) 132e136 135

be related to their relative toxicity to microorganisms, with evi-

dence that increased hydroxylation results in increased toxicity Control a
Wax
(Cowan, 1999). On the contrary, imazalil inhibits 1,4 a-demethyla- 14
Wax + Imazalil
tion in the ergosterol biosynthesis in fungal cells with an ability to Wax + EOs
inhibit sporulation, although resistant strains of P. digitatum and 12 a
P. italicum have been reported (Hamamoto et al., 2000). Moreover, b
for controlling even sensitive strains, a double application of ima-

Weight Loss (%)

10 b
zalil has been proposed as necessary (as dip in the washing water b
and added into wax coating), but final residues exceeded frequently b
b
from the minimum residue limit (MRL), which has been established 8
as 5 mg/g, as has been observed in mandarins and oranges a c
(Njombolwana, Erasmus, & Fourie, 2013). From our results, it could 6 b b
be inferred that combination of EOs with imazalil would permit to b
decrease the imazalil concentration in the citrus industry, as has
4
been observed for the combination of EOs with mild heat or pulsed
electric fields on reducing the contamination of apple juice (Ait- a aa a
Ouazzou et al., 2012). 2

3.2. Lemon quality parameters

0 2 4 6 8
It is well known that during postharvest storage of fruits there
are some changes related to quality, such as colour, weight loss, Storage weeks at 10°C
firmness, total soluble solids and total acidity (Valero & Serrano,
Fig. 3. Percentage of weight loss in control and treated lemons with wax,
2010). Initial colour CIE Lab parameters were y75, 5 and 60, for wax þ imazalil and wax þ EOs (essential oils) during storage. Data are the mean  SE.
L*, a* and b*, respectively, without significant differences between Bars with different letters show significant differences (P < 0.05) among treatments for
control and treated fruits (data not shown). For a better under- each sampling date.
standing of colour changes along storage, colour difference (DE)
was calculated between day 0 and after 8 weeks, and results changes in the symplast/apoplast relation, and modification of the
showed that control lemons experienced higher colour changes cell wall structure and dynamics (Valero & Serrano, 2010). Although
than treated fruits, without significant differences between imazalil the mechanism involved in citrus softening is still unclear, the re-
or EOs treatment (Table 1, Fig. 2). These colour changes are due to sults reported herein could be attributed to higher turgor pressure
chlorophyll degradation and carotenoid synthesis in the flavedo in treated lemons, since lower weight losses and reduced softening
associated with the normal ripening process, since colour devel- are shown.
opment in citrus fruit involves temporary changes in chloroplast With respect to TSS and TA, the levels of TSS at harvest signifi-
ultrastructure and the metabolism of both chlorophylls and carot- cantly decreased during storage for all fruit without significant
enoids (Richardson & Cowan, 1995; Valero, Martínez-Romero, differences between control fruit and treated with wax or
Serrano, & Riquelme, 1998). wax þ imazalil, although the reduction was lower in those lemon
Weight loss significantly increased over storage in control fruits treated with wax þ EOs (Table 2). On the contrary, initial TA levels
reaching final values of 14.92  0.64% at the end of the experiment,
the weight loss being significantly reduced in those lemons coated
with wax, wax þ imazalil and wax þ EOs, ca. 9%, independently of 14 Control
Wax
the treatment (Table 1, Fig. 3). These results are in agreement with
aa aa Wax + Imazalil
the reported effect of wax for retarding moisture loss, since most of
12 Wax + EOs
the natural surface wax is removed during the cleaning step and the
Fruit Firmness (N/mm)

exogenous wax would replace the natural protection. Thus, the bb

natural openings get clogged by wax leading to a decrease in water 10 b
b bb c
vapour diffusion throughout the fruit surface (Mannheim & Soffer, bc
a b
1996), and improving visual characteristics by imparting a natural 8 a
looking-seen. This is the main reason by which natural or synthetic bb b
food grade waxes are used for coating in the citrus industry with a
the advantage of reducing weight loss and maintain quality 6 a
(Sánchez-González, Vargas, González-Martínez, Chiralt, & Chafer,
2011). 4
Firmness is one of the main fruit quality attributes determining
storability and usually during postharvest storage a softening
process occurs which is associated to the increase of fruit suscep- 2
tibility to fungal attack (Cantu et al., 2009). Control lemons showed
the greatest rates of softening with significant reduction of firm-
ness during storage (Table 1, Fig. 4), while the application of all 0 2 4 6 8
treatments led to a significant delay in softening. Thus, after 8
weeks, control fruits lost 56% of initial firmness while these losses Storage weeks at 10°C
were 42% in treated lemons. The metabolic events responsible for
Fig. 4. Fruit firmness in control and treated lemons with wax, wax þ imazalil and
textural changes leading to fruit softening during maturation and wax þ EOs (essential oils) during storage. Data are the mean  SE. Bars with different
ripening of citrus fruits involve loss in turgor pressure, degradation letters show significant differences (P < 0.05) among treatments for each sampling
and other physiological changes on the composition of membranes, date.
136 S. Castillo et al. / Food Control 35 (2014) 132e136

Table 2 mould and postharvest quality of Citrus sinensis cv. Valencia. African Journal of
Total soluble solids (TSS) and total acidity of lemon at day 0 and after 8 weeks of Biotechnology, 10, 14932e14936.
storage at 10  C.a Feng, W., Chen, J., Zheng, X., & Liu, K. (2011). Thyme oil to control Alternaria alter-
nata in vitro and in vivo as fumigant and contact treatments. Food Control, 22,
Treatment TSS (g/100 g) Total acidity (g/100 g) 78e81.
Guillén, F., Zapata, P. J., Martínez-Romero, D., Castillo, S., Serrano, M., & Valero, D.
Day 0 After storage Day 0 After storage
(2007). Improvement of the overall quality of table grapes stored under
Control 9.08  0.08Aa
8.07  0.07Ab
7.21  0.12Aa
7.98  0.16Ab modified atmosphere packaging in combination with natural antimicrobial
Wax 9.02  0.03Aa 8.17  0.03Ab 7.29  0.08Aa 7.71  0.15Ab compounds. Journal of Food Science, 72, S185eS190.
Imazalil 9.15  0.063Aa 8.23  0.09Ab 7.26  0.11Aa 7.62  0.05Ab Gutierrez, J., Bourke, P., Lonchamp, J., & Barry-Ryan, C. (2009). Impact of plat
essential oils on microbiological, organoleptic and quality markers of mini-
Essential oils 9.08  0.06Aa 8.40  0.08Bb 7.44  0.21Aa 7.73  0.21Aa
mally processed vegetables. Innovative Food Science & Emerging Technologies,
a 10, 195e202.
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(p < 0.05) along storage time. (2000). Tandem repeat of a transcriptional enhancer upstream of the sterol
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