Chemical composition, antimicrobial, and antioxidant activities of orange essential oil and its concentrated oils

Abstract

In this study, the chemical composition, antimicrobial, and antioxidant activities of orange essential oil (AN), its folded orange oils (5×, 10×, 20×) and d-limonene (LN) were investigated. The results observed in the chemical composition showed a decrease in the major component LN, in contrast to other minor components, which increased their concentration, such as decanal, linalool, and α-terpineol. The antimicrobial activity was determined for foodborne pathogens using the disk diffusion method followed by the minimum inhibitory concentration and minimum bactericidal concentration. Results showed that folded orange oils (5×, 10×, 20×) had better antimicrobial activity than AN and LN. The antioxidant activity was carried out by 2,2ʹ-azinobis-3-ethylbenzthiazoline-6-sulphonate and 1,1-diphenyl-2-picrylhydrazyl methods; folded orange oil 20× presented significantly better results (p ≤ 0.05) than other oils studied. Using folded oils from AN could be a natural alternative in food processing as ingredients with antimicrobial and antioxidant effect.

Full text

FOOD SCIENCE AND TECHNOLOGY
Chemical composition, antimicrobial, and antioxidant activities of orange
essential oil and its concentrated oils
C. Torres-Alvarez
a
, A. Núñez González
a
, J. Rodríguez
b
, S. Castillo
a
, C. Leos-Rivas
a
and J. G. Báez-
González
a
a
Facultad de Ciencias Biológicas, UANL, San Nicolás de los Garza, México;
b
Centro de Calidad Ambiental, Tecnológico de Monterrey, Monterrey,
México
ABSTRACT
In this study, the chemical composition, antimicrobial, and antioxidant activities of orange essential oil
(AN), its folded orange oils (5×, 10×, 20×) and d-limonene (LN) were investigated. The results observed
in the chemical composition showed a decrease in the major component LN, in contrast to other
minor components, which increased their concentration, such as decanal, linalool, and α-terpineol.
The antimicrobial activity was determined for foodborne pathogens using the disk diffusion method
followed by the minimum inhibitory concentration and minimum bactericidal concentration. Results
showed that folded orange oils (5×, 10×, 20×) had better antimicrobial activity than AN and LN. The
antioxidant activity was carried out by 2,2ʹ-azinobis-3-ethylbenzthiazoline-6-sulphonate and 1,1-diphe-
nyl-2-picrylhydrazyl methods; folded orange oil 20× presented significantly better results (p≤0.05)
than other oils studied. Using folded oils from AN could be a natural alternative in food processing as
ingredients with antimicrobial and antioxidant effect.
Perfil químico, actividad antimicrobiana y antioxidante del aceite esencial de
naranja y sus aceites concentrados
RESUMEN
En este estudio, se investigó la composición química, actividad antimicrobiana y antioxidante del
aceite esencial de naranja (AN), sus concentrados (5×, 10×, 20×) y d-limoneno (LN). Los resultados
en la composición química mostraron una disminución del componente mayoritario d-limoneno,
contrario a otros componentes minoritarios que aumentaron su concentración como es el decanal,
linalol y α-terpinol. La actividad antimicrobiana fue determinada para bacterias de importancia en
alimentos por el método de difusión en disco seguido de la concentración mínima inhibitoria (CMI)
y concentración mínima bactericida (CMB). Los resultados mostraron que los aceites concentrados
(5×, 10×, 20×) presentaron significativamente mayor actividad antimicrobiana que el AN y LN. La
actividad antioxidante se realizó por los métodos de ABTS y DPPH obteniendo que la fracción 20×,
presentó actividad antioxidante significativamente mayor (p≤0,05) que los demás aceites estudia-
dos. Los aceites concentrados del aceite esencial de naranja podrían ser una alternativa en la
aplicación en alimentos como ingredientes con efecto antimicrobiano y antioxidante
ARTICLE HISTORY
Received 19 April 2016
Accepted 28 July 2016
KEYWORDS
Fold oil; orange essential oil;
composition; antimicrobial;
antioxidant
PALABRAS CLAVE
Aceite concentrado; aceite
esencial de naranja;
composición; antimicro-
biano; antioxidante
Introduction
Essential oils (EOs) from aromatic and medicinal extracts
obtained from a wide variety of plants have recently become
popular and raised scientific interest as potential natural agents
for food preservation because of their biological activity
(Velázquez-Nuñez, Avila-Sosa, Palou, & López-Malo, 2013).
Citrus EOs have been a part of the human diet for hundreds
of years, and they belong to the group of substances generally
recognized as safe (GRAS) by U.S. Food and Drug
Administration (FDA) (Nannapaneni et al., 2009). It has been
suggested that the use of such compounds as additives in
foods may act as preservatives, preventing the growth of
pathogens and spoiling microorganisms of the product con-
cerned (Fisher & Phillips, 2006; Velázquez-Nuñez et al., 2013).
In recent years, consumers have become aware of the
health problems (oxidative-stress-related disease) caused by
synthetic chemical ingredients used in processed foods.
Therefore, a natural alternative to the chemical preservation
of foods is the use of essential oils. Industrially, citrus EOs
have been widely applied in a variety of products, including
foods, beverages, cosmetics, and medicines because of their
flavor, fragrance, and certain properties (Liu, Chen, Liu, Zhou,
& Wang, 2012; Settanni et al., 2012). Citrus EOs are complex
mixtures containing volatile (85–99%) and nonvolatile (1–
15%) compounds mainly from monoterpenes, sesquiterpene
hydrocarbons, and their oxygenated derivatives, including
aldehydes, ketones, acids, alcohols, and esters. D-Limonene
(LN), (1-methyl-4-(1-methylethenyl) cyclohexane) is one of
the main component of citrus EOs (25–98%), and its content
varies significantly between species and/or varieties of the
same (Fisher & Phillips, 2008; Jing et al., 2014). The nonvola-
tile residue may contain hydrocarbons, sterols, fatty acids,
waxes, carotenoids, coumarins, psoralens, and flavonoids;
this latter group of compounds is useful for differentiating
CONTACT J. G. Báez-González juan.baezgn@uanl.edu.mx Facultad de Ciencias Biológicas, UANL, Av. Universidad s/n, Cd. Universitaria CP, 66450 San
Nicolás de los Garza, Nuevo Leon, México
© 2016 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.
CYTA –JOURNAL OF FOOD, 2017
VOL. 15, NO. 1, 129–135
http://dx.doi.org/10.1080/19476337.2016.1220021

between species (Jing et al., 2014; Tranchida, Bonaccorsi,
Dugo, Mondello, & Dugo, 2012).
The chemical composition of the citrus EOs is influenced
by several factors, such as the genetic differences between
varieties and species, which are the main determinants of the
composition and content of essential oils. Environmental fac-
tors, such as soil type, cropping practices, stages of maturity,
and types of weather can contribute to quantitative variations
in the content of essential oils; in addition, they could affect
the biological activity of the oils (Jing et al., 2014). Citrus EOs
contain a high amount of terpene hydrocarbons; however,
these compounds do not contribute much to the flavor and
fragrance of the oil. In addition, they are unstable when
exposed to heat or light and the solubility of the whole oil
in alcohol decreases. Because of this, in order to stabilize the
final product, these kinds of compounds should be removed.
Furthermore, the oxygenated fraction provides much of the
intense characteristic flavor of citrus EOs and consists mainly
of alcohols, aldehydes, ketones, and esters (Lopes, Raga,
Stuart, & Oliveira, 2003).
Industrially, orange essential oil (AN) is produced from
orange peel through the cold-pressed method. Different pro-
cedures can be applied for the reduction of undesirable com-
pounds, such as terpene hydrocarbons (including LN), which
conventionally may be reduced by vacuum distillation
(O’Bryan, Crandall, Chalova, & Ricke, 2008), solvent extraction,
and adsorption chromatography. During vacuum distillation,
it is difficult to indicate general limits for obtaining defined
physicochemical properties of the concentrated fractions
because these depend on the degree of concentration, oper-
ating conditions, applied technology, relative proportions of
the oxygenated components of origin, and economic factors
(Lopes et al., 2003). Commercially, the main folding degrees
available are 2–5-fold for lime and mandarin oils, 2–10-fold for
lemon and grapefruit oils, and 2–20-fold for orange oil. Folded
oils are less prone to oxidation, they have a high solubility in
water, and they have high organoleptic qualities (López-
Muñoz & Balderas-López, 2014).
Moreover, the application of antioxidants plays an impor-
tant role in inhibiting oxidative reactions in various products;
in addition, these could prevent diseases related to the
oxidative stress in the human body (Liu et al., 2012).
Application of synthetic antioxidants, such as butylated
hydroxyanisole (BHA) and butylated hydroxytoluene (BHT),
has resulted in the appearance of significant side effects,
which explains the growing interest in search for natural
antioxidants (Olmedo, Nepote, & Grosso, 2014).
Another important issue among the food, cosmetics, and
pharmaceutical industries is microbial contamination. The
continuous use of synthetic preservatives can cause the
development of resistance in some microorganisms, as well
as residual toxicity. To solve these problems, natural and safe
antimicrobial additives have been suggested. Recently, nat-
ural products, such as essential oils or compounds obtained
from plants have gained scientific interest and acceptance
by these industries, since they have shown little or no harm
to human health (Char, Cisternas, Pérez, & Guerrero, 2016;
Liu et al., 2012). Biological activities of citrus EOs as antifun-
gal, antioxidant, and antimicrobial among others, have been
studied (Espina et al., 2011; Jing et al., 2014; Liu et al., 2012;
Singh et al., 2010). However, there are few reports on the
antioxidant and antimicrobial activity of the individual com-
ponents and/or folded oil obtained from the AN.
The aim of this research was to compare and evaluate the
chemical composition of AN, its folded oils (5×, 10×, 20×)
and LN (as main component) as well as evaluating their
antimicrobial and antioxidant activity.
Materials and methods
AN and folded oil
AN was provided by Frutech International (NL, Mexico),
industrially obtained through cold-pressed method; subse-
quently, the folded oils were prepared in a vacuum-distilled
column with structured packing equivalent to 20 theoretical
plates. The equipment was operated in a batch mode. The
conditions of the parameters used during vacuum distilla-
tion were adjusted based on the initial composition of AN in
order to obtain a final product with a desired composition
according to the specification of each folded oil with the
following parameters: pressure range 5–20 mbar and the
reflux at temperatures between 80°C and 100°C. Table 1
shows some physicochemical characteristics of the AN and
folded orange oils (5×, 10×, 20×). LN 97% and Trolox were
purchased from Sigma–Aldrich (Sigma Chemical Co., St.
Louis, MO, USA). The reagents used were analytical grade.
Gas chromatography–mass spectrometry (GC–MS)
analysis
The analysis for AN and its folded oils (5×, 10×, 20×) was
performed according to Liu et al. (2012), with some modifi-
cations. These oils were analyzed by GC–MS (7890B, Agilent
Technologies, Santa Clara, CA, USA). GC was conducted on
HP-5 MS (30 m × 0.25 mm × 0.25 µm) capillary column. The
GC conditions were as follows: injection temperature 250°C;
the oven temperature was controlled at 70°C for 1 min with
the heating rate from 10°C/min to 200°C for 2 min, and
finally from 10°C/min to 300°C for 5 min. Helium gas was
used as carrier gas at a constant flow rate of 1 mL/min,
sample size at 1 μL. Parameters for MS analysis 5977A were
with EI ion source, electron energy 70 eV, the temperature of
quadrupoles 150°C, temperature of interface 230°C, m/z =
30–400 amu. Identification of compounds was carried out by
comparing their mass spectra with those of Wiley 7 n.L
library, considering a quality match >85%. In addition, essen-
tial oils’analyses were performed in one more capillary
column (DB-1 MS, 60 m × 0.25 mm × 0.25 µm) for a more
accurate identification. Results were confirmed with the
injection of some standards of essential oils components
(LN, linalool, α-terpineol, decanal, and valencene) (Sigma–
Aldrich) (data not shown).
Table 1. Chemical properties of the oils tested.
Tabla 1. Propiedades químicas de los aceites probados.
Code Commercial name
Specific
gravity (20°C)
Refractive
index (20°C)
Solubility
in water
AN Orange essential oil 0.846 1.477 Negligible
LN D-Limonene 0.840 1.472 Negligible
5× Fivefold concentrated
orange oil
0.884 1.482 Negligible
10× Tenfold concentrated
orange oil
0.920 1.494 Negligible
20× Twentyfold
concentrated
orange oil
0.945 1.497 Negligible
130 C. TORRES-ALVAREZ ET AL.

Bacterial strains and growth conditions
The following strains used in the study were acquired from
American Type Culture Collection (ATCC), Salmonella typhi
(ATCC 19430), Bacillius cereus (ATCC13061), Staphylococcus
aureus (ATCC 6538), and Listeria monocytogenes (ATCC
7644), and kindly provided by the Laboratory of Sanitary
Microbiology,FCB,UANL.Thestrainswerestoredat−80°C
in brain heart infusion with 20% (v/v) glycerol (Difco
Laboratories, Sparks, MD, USA). Fresh cultures were
obtained in Mueller Hinton broth or agar (MHB and MHA,
Difco Laboratories). L.monocytogenes was cultivated on
trypticase soy broth (TSB). An aliquot (50 μL) was taken
from a frozen culture and was added to a tube containing
5 mL of MHB or TSB depending of each strain and were
incubated at 37°C for 18 h (30°C for L. monocytogenes);
thereafter an aliquot (10 μL) of each culture was added to
the tubes with MHB or TSB.
Antimicrobial activity assay (disk diffusion assay)
Preliminary antimicrobial activity of AN, LN, and folded
orange oils (5×, 10×, 20×) was tested against four food-
borne pathogens, using disk diffusion technique
(Klančnik, Piskernik, Jeršek, & Možina, 2010). An aliquot
(100 μL) of bacterial suspension (adjusted to 0.5
McFarland ≈10
8
) was distributed homogeneously on
MHA plates. Five disks of 6 mm in diameter were sepa-
rately impregnated with 10 μL of each oil and placed on
the agar surface. The plates were incubated at 37°C for
24 h. Antimicrobial activity was defined as absence of
bacterial growth in the area surrounding the disk. The
diameter of the inhibition zones was measured with a
caliper. A test for L.monocytogenes was conducted the
same way, but TSA was used instead of MHA, and it was
incubated at 30°C. Duplicate analyses were performed at
least three times. Disks impregnated with sterile distilled
water and/or DMSO served as negative controls and disks
with an antibiotic (gentamicin) as positive controls.
Determination of minimum inhibitory concentration
(MIC) and minimum bactericidal concentration (MBC)
After determining preliminary antimicrobial activity, the MIC
and MBC were obtained for the four pathogens. The dilution
method according to Castillo, Heredia, Contreras, and García
(2011), with minor modifications was carried out. Briefly, an
aliquot (50 μL) of fresh cultures adjusted to McFarland stan-
dard (0.5 ≈1×10
8
) of each strain were added separately in
tubes containing 5 mL of MHB with various concentrations
of AN, LN, 5×, 10×, 20× (0.25, 0.5, 1.0, 1.5, 2, 2.5 mg/mL final
concentration in each tube for every oil). Cultures were
incubated at 37°C for 24 h. After that, bacterial survival was
determined by plate counting. MIC was defined as the low-
est concentration of oil that decreased growth about 90% of
the bacterial population after 24 h of incubation, while the
MBC was defined as the lowest concentration that comple-
tely inhibited growth on the MHA plate after 24 h of incuba-
tion (Klančnik et al., 2010). L.monocytogenes was tested the
same way under the conditions referred above. Triplicate
analyses of MIC and MBC were conducted at least three
times.
Antioxidant activity assay
The antioxidant activity of the oils was measured using the
2,2ʹ-azinobis-3-ethylbenzthiazoline-6-sulphonate (ABTS) radi-
cal and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical. Trolox
(Sigma–Aldrich) a water-soluble analogue of vitamin E, show-
ing a potent antioxidant activity, was used as a standard
reference (positive control). ABTS radical scavenging assay
was determined following the method of Re et al. (1999),
with some modifications, the blue–green ABTS radical cation
chromophore (ABTS·
+
) was prepared by reacting ABTS stock
solution (7 mmol) with potassium persulphate (2.45 mmol)
and allowing the mixture to stand at room temperature in the
dark during 16 h. The solution was diluted with ethanol to
obtain an absorbance of 0.700 ± 0.02 at 734 nm in a Genesys
5 (spectronic) spectrophotometer. The DPPH radical scaven-
ging assay was conducted according to Brand-Williams,
Cuvelier, and Berset (1995), with some modifications. DPPH
solution was prepared with 3.9 mg of radical and 100 mL of
ethanol, the solution was measured to 517 nm in a spectro-
photometer to obtain an absorbance of 1.000 ± 0.05. Aliquots
of 10–300 μL of solution of each oil with ethanol were mixed
with 2.7 mL of ABTS·
+
solution. The mixture was allowed to
react at room temperature for 7 min in dark conditions. The
absorbance was recorded by spectrophotometer at 734 nm.
For DPPH assay, different concentrations (200–600 μL) of each
oil were added separately to 2.25-mL DPPH solution. The
mixture was shaken and left at room temperature for
240 min in the dark. Thereafter, the absorbance was mea-
sured at 517 nm in spectrophotometer.
The antioxidant activity was calculated as a percentage of
inhibition according to the following equation:
%Inhibition ¼Ab As=Ab½100
fg
;
where Ab represents the absorbance of the control (without
test oil), and As represents the absorbance of the test oil. A
calibration curve was determined for Trolox standard for
each radical (ABTS and DPPH) at a concentration range of
10–160 μmol. The curve equations were y= 0.4447x+
0.3721, R
2
= 0.99 for ABTS and y= 0.7371x+ 0.7321,
R
2
= 0.99 for DPPH. Triplicate analyses of each oil were
made. The antioxidant activity values were expressed as
μmol Trolox equivalent (TE)/mL citrus oil (CO). In addition,
the antioxidant activity expressed as half-maximal inhibitory
concentration IC
50
(mg/mL) was defined as the amount of
antioxidant necessary to decrease the initial ABTS and DPPH
concentration by 50% (Liu et al., 2012).
Statistical analysis
All experiments were performed in duplicate or triplicate at
least three times. Statistical analyses were performed using
SPSS software (IBM version 22, SPSS Inc, Chicago, IL). Data
were analyzed by analyses of variance test (one-way ANOVA)
and a post hoc test of Tukey’s multiple range. Differences
between means were considered significant at pvalues ≤0.05.
Results and discussion
Chemical composition of the citrus EOs
The analysis of the main components of AN, 5×, 10×, 20×
showed differences in the composition and percentage of
CYTA –JOURNAL OF FOOD 131

the compounds observed (Table 2). These results agree with
results of previous studies (Espina et al., 2011; Ruiz & Flotats,
2014; Singh et al., 2010) in which differences in the chroma-
tographic profile of AN were found depending on the type
of extraction, retaining its major components, such as the
LN. This finding is consistent with our results, which showed
LN as majority compound. Lopes et al. (2003) performed a
study with different parameters during vacuum distillation
and they found quantitative variations in the chromato-
graphic profiles. Our results showed the same tendency
indicating that these variations might be influenced by the
type of extraction used. It is well known that vacuum dis-
tillation process produces folded oils from orange EO,
through a selective decrease of LN (main constituent) and
other terpene hydrocarbons; therefore, the concentration of
oxygenated compounds, primarily aldehydes and alcohols
are increased (Lopes et al., 2003;O’Bryan et al., 2008).
Terpene hydrocarbons (including LN) must be removed in
order to obtain a stable final product because they do not
contribute much to the flavor and fragrance of the oil; also,
they are unstable when exposed to heat or light and their oil
solubility is decreased as well (Beneti et al., 2011; Liu et al.,
2012; Lopes et al., 2003). This study analyzed the chemical
composition of AN and its folded oils 5×, 10×, and 20× to
estimate the differences between the constituents, since the
folded orange oils, provide greater flavor intensity by
increasing the concentration of oxygenated compounds. In
addition, they are less susceptible to oxidation and off-flavor
development, showing better stability with greater industrial
interest (Beneti et al., 2011; Lopes et al., 2003). Considering
the information mentioned above, it is preponderant to
evaluate the antimicrobial and antioxidant activity of the
folded orange oil. Analysis by GC–MS gave the chemical
composition and relative abundance of the major compo-
nents that comprise AN, 5×, 10×, 20×. The components are
grouped into several classes: hydrocarbon monoterpenes,
oxygenated monoterpenes, hydrocarbon sesquiterpenes,
oxygenated sesquiterpenes, and others (Table 2). These
results showed that there are qualitative similarities between
components present in oils although some of them showed
quantitative differences when tested. For AN, 5×, 10×, and
20×, were identified 16, 20, 18, and 26 compounds, respec-
tively, representing the 99.57%, 98.49%, 98.25%, and 90.05%
of the total detected constituents, respectively. There was a
decrease in the mixture of hydrocarbon monoterpenes
(including LN) in all fractions compared with AN (97.37%),
keeping the 20× fraction the minor amount (23.24%) fol-
lowed by 5× and 10× with 72.51% and 74.88%, respectively.
For oxygenated monoterpenes, an increase in the concen-
tration of folded orange oils with respect to AN was
observed, being these 1.78% (AN), 17.20% (5×), 15.45%
(10×), and 43.07% (20×), coinciding with Lopes et al. (2003)
who reported an inversely proportional diminution on
hydrocarbon compounds, such as LN, myrcene, among
others, respect to the folded oils, while for oxygenated
compounds as decanal and linalool, an inversely propor-
tional increase with respect to the folded oils was reported.
It is important to note the variation about the quantity of
oxygenated monoterpenes between the oils because some
compounds from this group are recognized with biological
and antimicrobial activity (Espina et al., 2011). According to
some reports (Espina et al., 2011; Njoroge, Koaze, Karanja, &
Sawamura, 2005; Singh et al., 2010), LN is found at around
90%, recognized as the major component of AN, coinciding
with our results, in which AN presented 91.12% of LN; how-
ever in the folded oils, a decrease of this compound was
observed, resulting in 72.08% and 73.77% of LN in 5× and
10×, respectively, while in the 20×, a lower amount was
observed (23.24%). Nevertheless, LN was still the leading
component in all oils analyzed. Other compounds detected
over 1%, as decanal, an aldehyde that provides fragrance
(Liu et al., 2012) increased significantly in 5×, 10× and 20×
with 3.60%, 4.78%, and 18.46%, respectively, while in AN it
was detected only 0.44%. In addition, compounds such as
linalool, recognized for its importance in fragrance and bio-
logical activity (Fisher & Phillips, 2006) presented an incre-
ment in 5× (3.33%), 10× (3.99%), and 20× (5.53%), with
respect to AN (0.65%). The same tendency was observed
for hydrocarbon sesquiterpenes, in which an increase was
detected for 5× (7.94%), 10× (6.83%), and 20× (17.82%)
compared to AN (0.17%), being the 20× the major concen-
tration of these compounds. Finally, oxygenated sesquiter-
penes were detected only in 20× (2.67%), while in AN, 5×
and 10× were not.
Table 2. Chemical composition of AN, 5×, 10×, 20×.
Tabla 2. Composición química de AN, 5×, 10×, 20×.
No. Compound
Percentage (%)
a
AN 5× 10× 20×
1α-Pinene 1.11
2 Sabinene 0.41
3 Myrcene 4.11 0.43 1.11
4α-Phellandrene 0.42
5 Octanal 0.15
6D-Limonene 91.12 72.08 73.77 23.24
7β-Ocimene 0.11
8 Terpinolene 0.10
9 Octanol 0.10
10 Linalool 0.65 3.33 3.99 5.53
11 trans-p-Mentha-2,8-dienol 0.80 0.46 0.98
12 cis-p-Mentha-2,8-dienol 1.00
13 cis-Limonene oxide 1.94
14 trans-Limonene oxide 1.01
15 Citronellal 0.09 0.52 0.69 1.04
16 α-Terpineol 0.60 1.37 2.16 4.60
17 Decanal 0.44 3.60 4.78 18.46
18 trans-Carveol 1.07 1.59
19 Citronellol 0.61
20 cis-Carveol 0.76
21 Neral 0.67 0.79 1.70
22 Carvone 1.25 0.68 2.01
23 Geranial 1.12 1.33 3.48
24 Perilla aldehyde 0.52 0.57 1.31
25 Geraniol acetate 0.45
26 Dodecanal 0.84 1.09 2.80
27 α-Cubebene 0.71 0.98 2.32
28 β-Cubebene 0.05 0.82 2.22
29 Caryophyllene 0.64 1.35
30 Germacrene D 0.05 0.71 0.94 0.91
31 Valencene 4.89 2.14 6.35
32 γ-Muurolene 0.65 1.46
33 δ-Cadinene 0.06 0.98 1.31 3.21
34 Elemol 0.96
35 Caryophyllene oxide 0.77
36 Nootkatone 0.94
Hydrocarbon monoterpenes 97.37 72.51 74.88 23.24
Oxygenated monoterpenes 1.78 17.20 15.45 43.07
Hydrocarbon sesquiterpenes 0.17 7.94 6.83 17.82
Oxygenated sesquiterpenes ND ND ND 2.67
Others 0.25 0.84 1.09 3.25
Total identified components 99.57 98.49 98.25 90.05
a
Given in the relative peak area percent, mean of triplicate determination. ND:
not detected.
a
Dada en el porcentaje de área relativa del pico, con una media de
determinación por triplicado. ND = No detectado.
132 C. TORRES-ALVAREZ ET AL.

Antimicrobial activity by the disk diffusion assay and
determination of MIC and MBC
Antimicrobial activity was performed for AN, LN, 5×, 10×,
and 20× by disk diffusion method against four foodborne
pathogens. The antimicrobial activity of the oils studied is
summarized in Table 3. The oils tested showed inhibition
zone in ranges of 12–28 mm. B. cereus and S. aureus showed
greater inhibition at 10× and 20×, L. monocytogenes showed
inhibition with 5× and 20×, while for S. typhi, the greatest
inhibition was in 5× and 10×, and it was comparable with
positive control (gentamicin). When comparing the antimi-
crobial activity of AN and LN, with 5×, 10×, and 20× there
were significant differences (p≤0.05) in inhibition; the major
inhibition zones were for the folded orange oils in most
cases. These results are similar to those previously reported
by Espina et al. (2011), who found that lemon and orange
EOs showed lower inhibition against foodborne pathogen
than other essential oils, which could be attributed to the
portion of oxygenated monoterpenes.
The MIC and MBC were determined for the four food-
borne pathogens for the oils tested, the values were com-
pared finding differences between AN and LN with 5×, 10×,
and 20× (Table 4). The MIC varied among folded orange oils
ranged from ˂0.25 to 1 mg/mL while the MBC from 0.25 to
2.5 mg/mL. Major values of MIC and MBC for AN and LN
were evidenced. The MIC for these two oils ranged from 1.5
to 2.5 mg/mL and MBC from 2 to >2.5 mg/mL, depending on
the strain tested. MIC and MBC values were lowest in the
entire folded orange oils fractions being the 20× with the
higher antimicrobial potential. However, the values varied
very little between the folded orange oils for the same
microorganism, but on comparing the MIC and MBC values
with AN and LN, significant differences were observed. LN
showed the lowest antimicrobial activity with MIC values of
0.5–2 mg/mL and MBC values >2.5 mg/mL. Even AN pre-
sented lower values of MIC and MBC than LN, no significant
differences were evidenced in most cases. These results
agree with Frassinetti, Caltavuturo, Cini, DellaCroce, and
Maserti (2011) and Nannapaneni et al. (2009), who reported
a low or null antimicrobial activity of LN compared with
citrus essential oils against foodborne pathogens.
When MIC/MBC were determined, slight differences in sus-
ceptibility with respect to the results obtained in disk diffusion
assay were noted; the most sensitive bacteria was L. monocyto-
genes with MIC/MBC (<0.25/0.5 mg/mL), followed by B. cereus
(0.5/1.5 mg/mL), S. aureus (0.5/2 mg/mL) while the more resis-
tant one was S. typhi (1.0/2.5 mg/mL). This could be attributed to
the disk diffusion method, which is limited due to changes in the
diffusivity of the active compounds in agar; for this reason, the
size of the inhibition zone did not necessarily predict the MIC/
MBC determined in liquid medium (Nannapaneni et al., 2009;
O’Bryan et al., 2008). On the other hand, MIC/MBC values varied
between bacteria, confirming the observations of Kim, Marshall,
and Wei (1995); Rivera, Bocanegra-García, and Monge (2010)
who reported that essential oils could vary in their antimicrobial
potential among different microorganisms. This will depend on
several factors including the structure of bacterial cell wall and
variability of compounds present in the oil, which sometimes
couldexertasynergismbetweenthem(O’Bryan et al., 2008). In
previous reports, it has been demonstrated that compounds
present in low concentrations have a crucial role in antimicrobial
activity due to a synergic effect that potentiates biological activ-
ity. In this study, the folded orange oil 20× presented the best
antimicrobial activity followed by 10× and 5×; all of them had
major activity than AN and LN. This could be attributed to the
increment of oxygenated monoterpenes considering that this
portion was lower for AN (1.78%) than the fractions 5× (17.21%),
10× (15.45%), and 20× (43.07%). In addition, other minority
compounds, which have been reported to possess antimicrobial
activity (linalool, decanal, geranial, among others), could be
implicated in synergism or biological activity (Jing et al., 2014;
Nannapaneni et al., 2009;Singhetal.,2010). These findings were
consistent with those found in this study, in which these com-
ponents are present, and are actually increased in the concen-
trated oils.
There are some studies about fractions from cold-pressed
orange oil, terpeneless orange oil, terpenes from orange
juice essence, or fivefold orange oil (Nannapaneni et al.,
2009;O’Bryan et al., 2008), and also of isolated compounds
Table 3. Zones of inhibition of AN, LN, 5×, 10×, 20× against foodborne
pathogens by a disk diffusion assay.
Tabla 3. Zonas de inhibición del AN, LN, 5×, 10×, 20× contra patógenos
transmitidos por alimentos mediante ensayo de difusión en disco.
Zone of inhibition (mm)
Oil B. cereus S. aureus L. monocytogenes S. typhi
AN 13 ± 1
d
13 ± 1
d
12 ± 0.7
d
16 ± 0.7
c
LN 15.4 ± 1.1
d
17 ± 1
c
14 ± 0.7
d
17 ± 0.7
c
5× 23.4 ± 2.0
c
17.4 ± 0.5
c
24.4 ± 1.3
b,c
28 ± 1.4
a
10× 28 ± 1.2
b
20.4 ± 2.4
b
22.6 ± 1.8
c
27.2 ± 1.6
a
20× 27.8 ± 1.3
b
18.6 ± 0.8
b,c
26.2 ± 1.1
b
24.8 ± 1.1
b
Gentamicin 45.2 ± 0.8
a
37 ± 1.4
a
35.2 ± 0.8
a
28 ± 0.7
a
Zone of inhibition are average values of three replicates ± the standard
deviation of the mean.
a–d
Mean values with different letter in the same
column are significantly different (p≤0.05).
Zona de inhibición son valores promedio de tres repeticiones ± desviación
estándar de la media.
a–d
Los promedios en una columna con diferente letra
difieren significativamente (p≤0,05).
Table 4. Minimal inhibitory concentration (MIC) and minimal bactericide concentration (MBC) (mg/mL) of AN, LN, 5×, 10×, 20×.
Tabla 4. Concentración mínima inibitoria (CMI) y concentracion minima bactericida (CMB) (mg/mL) de AN, LN, 5×, 10×, 20×.
Microorganisms
B.cereus S. aureus L. monocytogenes S. typhi
Oil MIC MBC MIC MBC MIC MBC MIC MBC
AN 1.5 ± 0.3
a,b
˃2
a
1 ± 0.3
b
˃2
a
0.5 ± 0.2
a
2 ± 0.3
b
2 ± 0.5
a
˃2.5
a
LN ˃2
a
˃2
a
2 ± 0.2
a
˃2
a
0.5 ± 0.2
a
˃2
a
2.5 ± 0.3
a
˃2.5
a
5× 1 ± 0.3
b,c
1.5 ± 0.3
b
0.5 ± 0.2
b
1.5 ± 0.3
b
0.25 ± 0.1
a
0.5 ± 0.2
c
1 ± 0.3
b
2.5 ± 0.3
a,b
10× 1 ± 0.3
b,c
1.5 ± 0.3
b
0.5 ± 0.3
b
2 ± 0.3
a,b
0.25 ± 0.1
a
0.5 ± 0.2
c
1 ± 0.3
b
2.5 ± 0.3
a,b
20× 0.5 ± 0.3
c
1 ± 0.3
b
0.5 ± 0.2
b
1.5 ± 0.3
b
˂0.25
b
0.25 ± 0.1
c
1 ± 0.6
b
2 ± 0.3
b
MIC and MBC are average values of three replicates ± the standard deviation of the mean.
a–c
Mean values with different letter in the same column are
significantly different (p≤0.05).
CMI y CMB son valores promedio de tres repeticiones ± desviación estándar de la media.
a–c
Los promedios en una columna con diferente letra difieren
significativamente (p≤0,05).
CYTA –JOURNAL OF FOOD 133

from orange EO (Liu et al., 2012), a different variety of
orange (Settanni et al., 2012; Singh et al., 2010) and a differ-
ent variety of citrus EOs (Espina et al., 2011; Frassinetti et al.,
2011; Settanni et al., 2012). However, there are no studies
about folded oils (5×, 10× and 20×) from AN obtained by
vacuum distillation, as studied in this work.
Antioxidant activity
The antioxidant activity values obtained for AN, LN, and folded
orange oils were evaluated by the methods of ABTS and DPPH
(Table 5).Thesewereexpressedas[μmol Trolox equivalent (TE)/
mL citrus oil (CO)]. The values obtained by ABTS method were
23.25–156.25 μmol TE/mL CO and DPPH method showed values
between 3.01 and 21.24 μmol TE/mL CO. The folded orange oil
20× showed the highest concentration equivalent Trolox with
156.25 μmol TE/mL CO in ABTS and 21.24 μmol TE/mL CO with
DPPH, followed by 10× and 5× with values 141.53 and 86.02
μmol TE/mL CO, respectively, for ABTS, and 16.96 and 7.69 μmol
TE/mL CO, respectively, for DPPH. In the case of AN, it showed
values of 23.25 and 3.01 μmol TE/mL CO in ABTS and DPPH,
respectively. Finally, in LN, activity for any of the two methods
was not detected. The IC
50
values are shown in Table 6,where
the folded orange oil 20× showed higher antioxidant activity
with values of 3.80 and 10.25 mg/mL to inhibit 50% of ABTS and
DPPH radicals, respectively, followed by 10× and 5× with values
6.49 and 16.27 mg/mL, respectively, for ABTS while 15.50 and
37.25 mg/mL, respectively, for DPPH. The AN showed values of
68.40 and 70.17 mg/mL for ABTS and DPPH, respectively. The LN
did not present antioxidant activity for inhibiting the radicals
studied. The results showed significant differences in antioxidant
activity between 20×, 10×, and 5× with respect to AN and LN,
highlighting the best antioxidant activity for 20× compared to
the others oils studied. The folded orange oils (5×, 10× and 20×)
are mainly used in the food and cosmetics pharmaceutical
industries for their flavor and fragrance; however, few studies
have determined their antimicrobial and antioxidant activities.
According to the results obtained in this work, the folded orange
oils showed better antioxidant activity than AN. This could be
attributed to the potentiation of minority compounds and
reduction of LN, which in normal conditions remain at 90%.
In some studies, it has been proposed that the antioxi-
dant activity of citrus essential oils could be related to the
presence of LN and other monoterpenes as γ–terpinene and
terpinolene (Choi, Song, Ukeda, & Sawamura, 2000;
Frassinetti et al., 2011). However, in this study the best
results were obtained by the folded orange oils (5×, 10×
and 20×), which according to the chromatographic profile,
the amount of LN decreased while other compounds such as
decanal and linalool increased. These results agree with Choi
et al. (2000), who found that although LN was the majority
compound in ANs, it would not play the main role in deter-
mining antioxidant activity.
Some investigations have studied the antioxidant activity of
individual compounds. According to Liu et al. (2012), the anti-
oxidant activity of AN was major compared to individual com-
pounds, such as linalool, decanal, octanal, and valencene; this
evidence could confirm, based on the results obtained in this
study, that antioxidant activity is not due to a single compound
as LN, but a mixture of compounds, that alter their amount
when the orange EO is processed by vacuum distillation. Some
of them were found in lesser proportion while others increased
their concentration; this effect could potentiate biological or
synergistic action among compounds that derive in higher
antimicrobial and antioxidant activities. In that case, the folded
orange oil could provide different biological characteristics of
those found in the AN.
A major concern in this context is the toxicity of the com-
pounds. A variety of EO components have been registered by
the European Commission for being used as flavorings in food-
stuffs. Therefore, presently no risk to human health, including
the AN and LN (LN), which are GRAS. Meticulous studies of
toxicology support the safety of the substance for its intended
use (Gavarićet al., 2015). Studies in rats revealed the oral LD
50
for AN and LN and is established at >5 g/kg, also for the
concentrated oils used in this study. According to the health
hazard definition, the AN and its folded oils are classified as
non-toxic (Tisserand & Young, 2014).
In addition, further research could address these results’
focus on strategic applications of these compounds, consid-
ering their efficiency in antimicrobial and antioxidant activ-
ities (Choi et al., 2000).
Conclusions
The folded orange oils showed the best potential as anti-
microbials, as well as in their antioxidant activity. The food-
borne pathogens were affected at significant low
concentrations than the AN and LN when treated with the
concentrated oils, attributing these differences to their che-
mical composition. The results of this research may provide
the knowledge of the antimicrobial and antioxidant poten-
tial of folded orange oils obtained from AN, and these could
be used for control of foodborne pathogens as an alterna-
tive to replace the additives used today. Finally, the study of
the enormous range of biological activities of essential oils
and their prospective industrial applications in order to
increase the food safety is suitable; at the same time, it
may provide alternatives for the development of new, safer
products that are accepted by consumers who prefer natural
ingredients rather than synthetic ingredients.
Table 5. Antioxidant activity of AN, LN, 5×, 10×, 20×.
Tabla 5. Actividad antioxidante de AN, LN, 5×, 10×, 20×.
Method
μmol TE/mL CO
AN LN 5× 10× 20×
ABTS 23.25 ± 0.84
d
ND 86.02 ± 1.12
c
141.53 ± 0.16
b
156.25 ± 0.62
a
DPPH 3.01 ± 0.20
d
ND 7.69 ± 0.35
c
16.96 ± 0.81
b
21.24 ± 0.32
a
Mean values of three replicates ± the standard deviation of the mean.
a–d
Mean values with different letter in the same row are significantly
different (p≤0.05). ND = Not detected.
Valores promedio de tres repeticiones ± desviación estándar de la media.
a–d
Los promedios en la misma fila con diferente letra difieren significativa-
mente (p≤0,05). ND: No detectado.
Table 6. Inhibition concentration (IC
50
) of AN, LN, 5×, 10×, 20×.
Tabla 6. Concentración Inhibitoria (CI
50
) de AN, LN, 5×, 10×, 20×.
Method
IC
50
(mg/mL)
AN LN 5× 10× 20×
ABTS 68.40 ± 0.39
a
ND 16.27 ± 0.32
b
6.49 ± 0.10
c
3.80 ± 0.06
d
DPPH 70.17 ± 5.15
a
ND 37.25 ± 2.85
b
15.50 ± 1.20
c
10.25 ± 0.50
c
Mean values of three replicates ± the standard deviation of the mean.
a–d
Mean values with different letter in the same row are significantly
different (p≤0.05). ND = Not detected.
Valores promedio de tres repeticiones ± desviación estándar de la media.
a–d
Los promedios en la misma fila con diferente letra difieren significativa-
mente (p≤0,05). ND: No detectado.
134 C. TORRES-ALVAREZ ET AL.

Acknowledgements
We would like to thank Frutech International (N.L., Mexico) for providing
the commercial EOs samples and Juan José Ledezma for his support and
the technical information provided.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This study was supported by Consejo Nacional de Ciencia y Tecnología
de México (CONACyT) [No. 269983/275113].
ORCID
C. Torres-Alvarez http://orcid.org/0000-0001-6664-2063
A. Núñez González http://orcid.org/0000-0002-7415-0736
J. Rodríguez http://orcid.org/0000-0001-8924-2644
S. Castillo http://orcid.org/0000-0001-8354-3200
C. Leos-Rivas http://orcid.org/0000-0002-3626-7263
J. G. Báez-González http://orcid.org/0000-0003-0509-4678
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