Food Control 31 (2013) 1e4

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Food Control
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Short communication

Antifungal activity of orange (Citrus sinensis var. Valencia) peel essential oil
applied by direct addition or vapor contact
Maria José Velázquez-Nuñez a, Raúl Avila-Sosa b, Enrique Palou a, Aurelio López-Malo a, *
a
Departamento de Ingeniería Química, Alimentos y Ambiental, Universidad de las Américas Puebla, Cholula, Pue. 72810, Mexico
b
Facultad de Ciencias Químicas, Benemérita Universidad Autónoma de Puebla, 14 Sur y Av. San Claudio, Ciudad Universitaria, Puebla, Pue. 72420, Mexico

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

Article history: Vapor contact is an alternative when essential oils (EO’s) and microorganisms are placed separately in
Received 1 June 2012 some sealed environment. The aim of this study was to compare the antifungal efficacy of orange peel EO
Received in revised form at selected concentrations, applied either by vapor exposure or direct addition on the growth of
10 September 2012
Aspergillus flavus. Orange peel EO was obtained from fresh oranges (Citrus sinensis var. Valencia). EO
Accepted 18 September 2012
was obtained by vapor distillation, analyzed by means of GCeMS chromatography, and applied to potato-
dextrose agar inoculated with A. flavus, using the techniques of direct addition to the agar or by
Keywords:
generating EO vapors in airtight containers. Radial growth rate and lag phase were calculated using the
Orange peel EO
Vapor and direct contact assays
Gompertz equation. Main compounds identified in the orange peel EO were: limonene, b-myrcene, b-
A. flavus pinene, a-pinene, as well as citral Z and E; of which, limonene represented 96.62%. The minimum
inhibitory concentration for the growth of A. flavus by direct addition was 16,000 mg l
1, while for the
vapor contact was 8000 mg of EO l
1 of air. For both studied methods A. flavus growth decreased when
increasing EO concentration. Although the effect of orange peel EO direct addition was faster, orange peel
EO vapors were more effective, since lower concentrations were required to achieve the same antifungal
effect.
Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction separately in some sealed environment and therefore microbial

Essential oils (EO’s) and extracts obtained from many plants
have recently gained a great popularity and scientific interest.
Phenolic compounds present in essential oils have been recognized
as bioactive components with antimicrobial activity. Most plant
phenolic compounds are classified as Generally Recognized as Safe
(GRAS) substances, therefore they could be used to prevent growth
of many pathogenic and spoilage microorganisms in foods (Burt,
2004; Nedorostova, Kloucek, Kokoska, Stolcova, & Pulkrabek,
2009). However, EOs antimicrobial efficacy in foods is usually
achieved at higher concentrations, which many times entail
a sensory impact, caused by altering the natural taste and/or odor of
the food by exceeding the acceptable flavor and/or odor thresholds
(Nazer, Kobilinsky, Tholozana, & Dubois-Brissonneta, 2005). Hence,
for reducing EO’s sensory impact, one of the alternatives can be the
use of EO’s by vapor contact instead of its direct addition. Vapor
contact is an alternative when EO’s and microorganisms are placed
* Corresponding author. Tel.: þ52 222 229 2126; fax: þ52 222 229 2727.
E-mail address: aurelio.lopezm@udlap.mx (A. López-Malo).
inhibition is achieved from a distance without direct contact of the
antimicrobial agent with the food (Avila-Sosa et al., 2012). Few
studies have been published regarding inhibition of microorgan-
isms by the vapor-phase generated by EO’s (Inouye, Uchida,
Maruyama, Yamaguchi, & Abe, 2006; López, Sánchez, Battle, &
Nerin, 2007; Nielsen & Rios, 2000; Suppakul, Miltz, Sonneveld, &
Bigger, 2003), pointing out that EOs applied in vapor phase could
be effective against foodborne pathogens and spoilage microor-
ganisms at relatively lower concentrations than when applied in
liquid phase, thereby causing less effect on sensory attributes (Tyagi
& Malik, 2011).
Inouye (2003) reported that EO’s of cinnamon, thyme, and
lavender inhibited molds more effectively by vapor contact than by
direct contact in aqueous systems and explained that the dimin-
ished activity of the essential oil in aqueous media was due to the
presence of detergents and lipophilic materials. Arras and Usai
(2001) observed alterations in the morphology of Penicillium dig-
itatum caused by thyme EO applied by vapor contact, and Gómez-
Sánchez, Palou, and López-Malo (2011) established the antifungal
activity of Mexican oregano EO by vapor contact on Aspergillus
flavus.

0956-7135/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodcont.2012.09.029
2 M.J. Velázquez-Nuñez et al. / Food Control 31 (2013) 1e4

On the other hand the genus Citrus, which includes several Table 1
important fruits such as oranges, mandarins, limes, and lemons, is Main compounds identified in orange peel
essential oil.
the largest fruit crop in the world (100 million cubic tons per year)
and oranges account for 60% (Oreopoulou & Tzia, 2007). Farhat et al. Compound %
(2011) reports that orange peel accounts for approximately 45% of Limonene 96.62
the total bulk with significant amounts of it available as a by- b-Myrcene 1.72
b-Pinene 0.53
product after orange processing that create environmental prob-
a-Pinene 0.47
lems, particularly water pollution, due to the presence of bioma- Citral Z 0.31
terials such as EO, pectin, and sugars. Citrus spp. EO’s are present in Citral E 0.34
great quantities and it is known that can have an antimicrobial
effect against both bacteria and fungi (Chanthaphon, Chanthachum,
& Hongpattarakere, 2008; Jafari et al., 2011). Orange peel includes For the vapor contact assays, uncovered inoculated PDA plates
the epidermis covering the exocarp consisting of irregular paren- were placed on a perforated plastic sheet inside hermetically
chymatous cells, which are completely enclosing numerous glands closed plastic chambers (1.7-L capacity) with transparent lids,
or oil sacs (Lin, Sheu, Hsu, & Tsai, 2010). Nevertheless the antifungal leaving sufficient headspace (each airtight chamber utilized was
activity of orange peel EO has been scarcely studied, Caccioni, tested for leaks prior to experimenting with the essential oil). A
Guizzardi, Biondi, Renda, and Ruberto (1998), reported that vola- glass plate containing the essential oil (500, 1000, 2000, 4000,
tile compounds of orange and lemon peel are capable to inhibit 8000, or 16,000 mg of EO l
1 of air) was located under the
Penicillium spp., although it is claimed (using the agar dilution perforated plastic sheet (Gómez-Sánchez et al., 2011). Dishes were
technique) that are effective against the growth of some molds. incubated at 25  C. A growth control was prepared in parallel, to
The aim of this study was to compare the antifungal efficacy at ensure that viable organisms were present. For both studied
selected concentrations of orange peel EO applied either by vapor methods, radial growth was measured every 24 h during 20 d. The
exposure or direct addition on the growth of A. flavus. diameter of the growing mold colonies was daily measured in two
directions at right angles (in the case of the vapor contact assays,
2. Materials and methods through the transparent lid of the chamber in order to not disturb
the internal system equilibrium). Minimal inhibitory concentra-
2.1. Essential oil and chemical characterization tion (MIC) was defined as the lower concentration tested were no
fungal growth was detected. Every test was performed by
Orange peel was obtained from fresh oranges (Citrus sinensis var. triplicate.
Valencia) that were bought at a local market in Puebla, Mexico.
Oranges were washed and peeled, then peels were cut into 1 cm 2.3. Fungal growth modeling and statistical analysis
square pieces, EO was obtained by vapor distillation for 4 h with
a Clevenger-type apparatus. Growth data were fitted using the modified Gompertz model as
EO was analyzed with a GC Varian Star 3400cX coupled with an reported by Char, Guerrero, and Alzamora (2007):
MS Varian Saturn 2000 (Varian Inc., Walnut Creek, CA, USA) with

Dt n h e io
a splitless injector, equipped with a Zebron ZB-1 capillary column
ln ¼ Aexp
exp ymax $ ðl
tÞ þ 1 (1)
(30 m*0.32 I.D.*0.25 mm). Helium was used as the carrier gas, and Do A
the following conditions were set for the analysis: injector, 170  C,
detector 225  C; the initial oven temperature was 74  C held for where: Dt (mm) is the average colony diameter at time t (d), and Do
4 min, followed by a ramp-up of 3  C min
1 up to 95  C, and (mm) is the average colony diameter at initial time; A is the
a second ramp-up of 25  C min
1 up to 220  C, and held at the final maximum mold growth achieved during the stationary phase, ymax
temperature for 10 min, for a total run time of 30 min. The obtained is the maximum specific growth rate (d
1), l is the lag phase (d) and
spectra were compared with respective mass spectra of pure e ¼ exp (1).
compounds, and also with the mass profile of the same compounds
available from the US National Institute of Standard Technology
(NIST) library (Adams, 1995).

2.2. Direct addition and vapor contact assay

A. flavus (ATCC 18672) was obtained from Universidad de las

Américas Puebla Food Microbiology Laboratory and cultivated on
potato-dextrose agar plates (PDA, Merck, Mexico) for 5 days at
25  C, and then spores were harvested with 10 ml of 0.1% Tween 80
(Merck) solution sterilized through membrane (0.45-mm pore size)
filtration. The number of spores present in the suspension was
determined using a hemocytometer and an optical microscope
(Zeiss Primo Star, Göttingen, Germany), and expressed as number
of spores per milliliter. The spore suspension was adjusted with the
same solution to give a final spore concentration of 106 spores ml
1
and was utilized the same day (Avila-Sosa et al., 2012).
For the direct addition assays, EO was added to sterilized PDA
and mixed at 45  C to achieve final concentrations of 500, 1000,
Fig. 1. Effect of orange peel essential oil applied by direct addition at selected
2000, 4000, 8000, or 16,000 mg l
1; plates were allowed to solidify concentrations [0 (-), 500 (A), 1000 (:), 2000 (C), 4000 (,), 8000 (>) or 16,000
and then 10 ml of A. flavus spore suspension was inoculated in the (D) mg l
1] on Aspergillus flavus growth. Dt is the average colony diameter at time t and
center of each plate. Do is the average colony diameter at initial time.
 M.J. Velázquez-Nuñez et al. / Food Control 31 (2013) 1e4 3

For both studied methods, vapor exposure or direct addition of

Fig. 2. Effect of orange peel essential oil applied by vapor contact at selected
concentrations [0 (-), 500 (A), 1000 (:), 2000 (C), 4000 (,) and 8000 (>) mg of
EO l
1 of air] on Aspergillus flavus growth. Dt is the average colony diameter at time t
and Do is the average colony diameter at initial time.
Growth parameters were obtained by means of nonlinear
regression using the software Kaleidagraph V.3.51 (Synergy Soft-
ware, Reading, PA, USA). In order to compare both application
methods t-Student tests were performed, and Gompertz parame-
ters were compared with General Linear Model procedure in
Minitab 16 (Minitab Inc., State College, PA, USA) with 95% of
significance, different means were separated with Tukey’s test.
3. Results and discussion
The main compounds in the orange peel EO (Table 1) identified
by gas chromatography coupled with mass spectrometry were
limonene, b-myrcene, b-pinene, a-pinene, as well as citral Z and E;
of which, limonene represented 96.62%. Sawamura et al. (2004)
stated that citrus essential oils are a mixture of volatile
compounds that mainly consisted of monoterpene hydrocarbons
compounds that can be approximated into three fractions: terpene
hydrocarbons, oxygenated compounds and non-volatile
compounds. The monoterpene fraction can constitute from 50 to
more than 95% of the oil; however, it makes little contribution to
the flavor and fragrance of the oil (Chanthaphon et al., 2008). Tyagi
and Malik (2011) reported that monoterpenes concentrations in
EO’s are the main components with higher antimicrobial activity
in vapor phase than in direct contact and depends on their presence
in gaseous form facilitating their solubilization in cell membranes.
orange peel EO, A. flavus growth (Figs. 1 and 2) decreased when
increasing EO concentration. Although the effect of orange peel EO
direct addition was faster, orange peel EO vapors were more
effective, since lower concentrations were required to achieve the
same antifungal effect. MIC value for A. flavus growth by direct
addition was 16,000 mg l
1, while for the vapor contact was
8000 mg of EO l
1 of air. Sharma and Tripathi (2008) suggested that
citrus EO exhibits among other properties, antifungal activity by
reducing or totally inhibiting fungal growth in a doseeresponse
manner, Viuda-Martos, Ruiz-Navajas, Fernández-López, and
Pérez-Álvarez (2008) reported A. flavus inhibition by agar dilution
method with orange EO at 7900 mg l
1, while Inouye (2003)
inhibited Aspergillus fumigatus by vapor contact of yuzu (Citrus
junos) EO at 4000 mg of EO l
1 of air.
A. flavus growth curves were adequately adjusted by equation
(1) (R2 between 0.981 and 0.999). Modified Gompertz model
parameters (Table 2) demonstrated significant differences
(p < 0.05) between both studied methods. Maximum mold growth
(A) and maximum specific growth rate ðymax Þ show that increasing
the concentration of orange peel EO decreases these parameters. In
the direct addition technique, A and ymax parameters did not
exhibit significant differences (p > 0.05) as compared to control
parameters until 8000 mg l
1, but for vapor contact treatment,
these parameters were significantly (p < 0.05) lower than control
ones even at low concentrations. Lower values of A and ymax were
observed for the vapor contact than for direct addition assays.
Parameter A depends on the maximum growth diameter, which in
our case is the Petri dish diameter. For direct addition assays, the lag
phase parameter showed a fungistatic effect, since at 8000 mg l
1
its lambda value is nearly two times the value for the control. In
contrast with direct addition assays, lag phase values for vapor
contact assays decreased when EO concentration increased. Similar
effects were reported by Bluma, Landa, and Etcheverry (2009) for
A. flavus inhibition by vapor contact with boldus and poleo EO’s;
moreover, they determined that EO efficacy depends on the
complex interactions of tested environmental factors. Matan et al.
(2006) evaluated the effects of vapor phase EO concentration
(clove or cinnamon) and atmosphere composition in the number of
days required for visible mold growth, they observed that higher
concentrations of EO are required to delay fungal growth; and re-
ported a synergic inhibitory effect of the surrounding gas compo-
sition of EO volatile compounds. Carson, Me, and Riley (2002) and
Tyagi and Malik (2011) stated that EO causes different changes on
microbial cell membrane properties and functions by increasing
membrane fluidity and altering membrane permeability; while low
concentrations alter its permeability, high concentrations cause

Table 2
Modified Gompertz model parametersa (mean  standard deviation) for Aspergillus flavus growth curves subjected to selected concentrations of orange peel essential oil either
by direct addition or vapor contact.

EO concentrationc A ymax ð1=dayÞ l(day) R2

(mg/L)
Direct Vapor Direct Vapor Direct Vapor Direct Vapor
addition exposure addition exposure addition exposure addition exposure
0 2.28  0.03a,A 2.35  0.08a,A 0.35  0.02a,A 0.38  0.03a,A 2.65  0.17a,A 2.72  0.12a,A 0.999 0.999
500 2.30  0.01a,A 1.67  0.03b,B 0.41  0.07a,A 0.11  0.01b,B 2.16  0.51a,A 1.07  0.19b,B 0.991 0.998
1000 2.23  0.05a,A 1.70  0.03b,B 0.33  0.06a,A 0.13  0.01b,B 2.14  0.41a,A 0.23  0.29b,C 0.992 0.997
2000 2.08  0.06a,B 1.56  0.08b,C 0.35  0.05a,A 0.09  0.01b,C 2.61  0.19a,A 0.54  0.14b,C 0.985 0.992
4000 1.99  0.06a,B 0.98  0.02b,D 0.31  0.03a,A 0.08  0.01b,C 2.62  0.29a,A 0.26  0.03b,C 0.981 0.989
8000 1.80  0.05C _b 0.26  0.01B _b 4.41  0.21B >20D 0.987 0.991
16,000 _b _b _b _b >20C >20D 0.982 0.983

Means followed by a different superscript letter within a row for each parameter are significantly different (p < 0.05).
Means followed by a different superscript capital letter within a column for each parameter are significantly different (p < 0.05).
a
A: maximum mold growth in the stationary phase; ymax : maximum specific growth rate; l: lag phase.
b
No growth.
c
For direct addition assays (mg l
1); for vapor contact assays (mg of EO l
1 of air).
4 M.J. Velázquez-Nuñez et al. / Food Control 31 (2013) 1e4
severe damage, loss of homeostasis, and death. Nychas (1995) re-
ported that some EO’s components are able to denature the
enzymes responsible for spore germination, energy production and
synthesis of structural compounds or interfere with the amino acid
involved in germination.
Several researchers (Avila-Sosa et al., 2012; Bluma et al., 2009;
Caccioni et al., 1998; Gómez-Sánchez et al., 2011; Inouye, 2003;
Inouye et al., 2006; López et al., 2007; Nedorostova et al., 2009)
concur that a greater antifungal activity of EO is achieved in vapor
phase than in aqueous solution or agar contact. In our case although
direct addition of orange peel EO had a rapid effect on A. flavus
growth, exposure to orange peel EO vapors was more effective,
requiring lower concentrations of EO to inhibit mold growth.
Acknowledgments
We acknowledge financial support from the National Council for
Science and Technology of Mexico (CONACyT) for the project
“Combinación de Factores Físicos y Químicos para la Inactivación de
Microorganismos Relacionados con Alimentos”. Author Velázquez-
Núñez acknowledges financial support for her bachelor studies
from CONACyT.
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