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Antibacterial and Antioxidant Activity of

Essential Oils from Citrus spp
Article in Journal of Essential Oil Research January 2011
DOI: 10.1080/10412905.2011.9700427

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Citrus

Antibacterial and Antioxidant Activity of Essential

Oils from Citrus spp.
S. Frassinetti,* L. Caltavuturo, M. Cini and C. M. Della Croce
National Research Council, Institute of Biology and Agricultural Biotechnology (IBBA), Pisa Unit, Research Area of Pisa,
Via Moruzzi 1, 56124, Pisa, Italy

B. E. Maserti
National Research Council, Institute of Biophysics (IBF), Pisa Unit, Research Area of Pisa,
Via Moruzzi 1, 56124, Pisa, Italy
Abstract
The antibacterial and antioxidant activities of essential oils from Bitter orange, Sweet orange, Lemon and Mandarin
were investigated. The antimicrobial capability of these oils was determined against ten strains of Gram-negative and
Gram-positive bacteria, including some phytopathogenic strains. The antibacterial activity of the oils was expressed
as minimum inhibitory concentrations (MICs). All oils showed good antibacterial activity against both Gram-negative
and Gram-positive bacteria. The MICs for selected oils ranged 15250 g/mL. The lowest MICs were 15 g/mL and
20 g/mL against Xanthomonas citri strains, respectively. The antioxidant and antiradical scavenging properties of the
selected oils were tested by means of 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay. All examined oils exhibited a free
radical scavenging activity, ranging 2070% of DPPH inhibition. Lemon oil showed the most antioxidant capacity,
with DPPH inhibition rate of 70%.
Key Word Index
Citrus aurantium, Citrus sinensis, Citrus limon, Citrus reticulata, Rutaceae, essential oil composition, antibacterial activity, antioxidant activity.

Introduction
Essential oils have been shown to exhibit antibacterial,
antifungal and antioxidant properties (1, 2). The concern over
the use of essential oils as antimicrobial agents has increased,
due to an emergent microbial resistance towards conventional
synthetic antimicrobial preservatives. Essential oils are widely
used in medicine (2-4), in pharmaceutical and cosmetic industries (5), and in the food industry, where they are used both
as flavoring additives and as antioxidants for preservation of
stored food crops (6, 7) instead of synthetic chemicals charged
to be cytotoxic (8).
Among essential oils, those from Citrus plants are particularly interesting, because they could be used in food both as
antioxidants (9, 10) and as flavoring compounds. Moreover,
Rossi (11) reported the antimicrobial activity of C. sinensis and
C. reticulata against both Gram-negative and Gram-positive
bacteria. Citrus oils can have antifungal activity, even if their
complexity makes it difficult to correlate their action to a specific
component. The effects of the volatile component of the Citrus
oils and peel extracts against Penicillium have been reported
(12, 13); the fungitoxicity of C. sinensis oils against Aspergillus
niger has also been described (14). However, there are very

few detailed reports on both antimicrobial and antioxidant

properties of Citrus oils.
The aim of the present research was to study both antibacterial and antioxidant activities of four Citrus (Bitter and
Sweet orange, Lemon and Mandarin) oils, and to test their
possible use as preservatives and antioxidant additives in the
food industry.

Experimental
Essential oils: The essential oilsBitter orange, Sweet
orange, Lemon and Mandarinused in this work are commercially available from Aboca (Perugia, Italy) The chemical
analysis of the oils had been performed by Aboca using gas
chromathographic analyses (GC Trace 2000 Termoquest
Instrument , according to GC-FID ISO 7609: 1985 method).
Aboca kindly provided the authors both the products and the
main constituent chemical composition. The plant species and
the organs used for oil extraction (by cold-pressing method)
are listed in Table I.
Microorganisms: Citrus oils were assayed against Gramnegative and Gram-positive bacteria (Table II). Three bacterial
strains of phytopathogen Xanthomonas campestris pv. citri

*Address for correspondence: frassinetti@ibba.cnr.it

Rec: July 2008

Rev: Dec 2008

1041-2905/11/0001-027$14.00/0 2011 Allured Business Media

Vol. 23, January/February 2011

Acc: March 2009

Journal of Essential Oil Research/27

Frassinetti et al.

Table I. Sources of Citrus oils used in this study and main components
Plant species

Common name

Product tested

Part of plant

Citrus aurantium L.
Orange
EO
Fruit

Citrus sinensis (L.) Osbeck
Sweet Orange
EO
Fruit

Citrus limon (L.) N.L. Burm.
Lemon
EO
Fruit

Citrus reticulata Blanco
Mandarin
EO
Fruit

Table II. Bacterial strains used in this study

Bacteria
Gram-negative
Xanthomonas campestris pv citri
Xanthomonas campestris pv citri
Xanthomonas campestris pv citri
Escherichia coli
Escherichia coli
Salmonella choleraesuis
Pseudomonas aeruginosa

Gram-positive
Staphylococcus aureus
Enterococcus faecalis
Enterobacter aerogenes

strain
NCPPB 3236
NCPPB 3562
NCPPB 3832
ATCC 10536
ATCC 25922
ATCC 14028
ATCC 27853

ATCC 25923
ATCC 19433
ATCC 13048

NCPPB: National Collection of Plant Pathogenic Bacteria, Central Science

Laboratory, York, U.K.

ATCC: American Type Culture Collection, Rockville, MD

from the NCCPB (National Collection of Plant Pathogenic

Bacteria, Central Science Laboratory, York, UK) and seven
strains of bacteria from the ATCC (American Type Culture
Collection, Rockville, MD) were used. The bacterial strains
and their ATCC numbers are listed in Table II. Subcultures
were obtained by growing bacteria for 24 h in Oxoid Nutrient
Broth (NB) at 37C.
Antimicrobial Activity Assay: The Broth microdilution assay method (15,16) was used for the determination of
antimicrobial activity of the oils, with some modifications, as
follows.
Bacterial cultures grown over 16 h were diluted with sterile
physiological saline solution with reference to the McFarland
standard (bioMrieux, Mary lEtoile, France) to achieve inoculums of approximately 105 cell/mL; these cultures (100 L)
were inoculated in 2 mL of Mueller Hinton Broth medium
(MHB; Oxoid, Basingstoke, UK). Then, 100 L of different
dilutions of the oils in DMSO (Sigma) were added to triplicate
test tubes, to give final concentration ranging 10500 g/mL.
The cultures were incubated at 37C for 24 h. Control samples
and blank samples were incubated under the same conditions.
The final concentration of DMSO did not exceed 2% v/v and
did not affect the bacterial growth. The bacterial growth in
the presence of oil samples (at different concentrations) was
compared visually with the growth of control cultures.
Determination of minimum inhibitory concentration
(MIC): The optical density (750 nm) of the bacterial cultures,
performed as described above, was successively determined by
28/Journal of Essential Oil Research

Main constituent
d-limonene 93% myrcene 1.85%
d-limonene 95% myrcene 1.88%
d-limonene 70%

g-terpinene 9.5%

d-limonene 67%

g-terpinene 17%

a Perkin-Elmer spectrophotometer. The backgrounds for each

sample and the growth of control cultures were also measured.
All assays were repeated three times. From these results, the
antibacterial activity was expressed as MIC (17). MIC was
defined as the lowest EO concentration giving a reduction of
> 90% in the observed absorbance (16).
Antioxidant Activity Assay/DPPH radical scavenging assay: The antioxidant activity was measured using the
1,1-diphenyl-2-picryl-hydrazyl (DPPH) radical (Sigma-Aldrich)
reduction assay, according to the method of Brand-Williams
(18). The free radical-scavenging activity of each oil was determined according to the method of Tepe (19), with some
modification. Aliquots 0.1 mL of the oil solutions in methanol
80% (ranging 501000 g/mL) were mixed with 1.9 mL of
DPPH solution (0.2 mM in methanol 80%). The mixture was
shaken and left at room temperature for 30 min in the dark.
The absorbance of the solution was measured at 517 nm in
a Perkin-Elmer spectrophotometer. The radical scavenging
activities, expressed as percentage inhibition of DPPH, were
obtained from the following equation:
Scavenging effect (%) = {[A0 (A Ab)] /A0 ] }
where A0 was the control (Absorbance 517 of DPPH solution), Ab was the blank (Absorbance 517 of essential oil solutions),
and A was the sample absorbance (Absorbance 517 of the oils
with DPPH). Trolox (Sigma-Aldrich), a water-soluble analogue
of vitamin E, showing a potent antioxidant activity, was used
as a standard reference. A Trolox standard calibration curve
for DPPH radical was measured at the concentration range
of 10300 m. The curve equation was: y = 12.6 + 0.17x r =
0.96.
Each oil sample was tested in triplicate. Data were expressed
as a mean of three independent experiments standard deviation, and analyzed by the students t test. Differences are
considered significant when p = 0.05.

Results and Discussion

Four different, commercially available Citrus oils were
tested for their antimicrobial activities against ten strains of
Gram-negative and Gram-positive bacteria, including some
phytopathogenic strains (Table II).
The antimicrobial activities have been determined by
measuring the minimum inhibitory concentration (MIC)
(Table III). All of the Citrus oils tested showed a great potential against the examined species. The MIC for selected
Vol. 23, January/February 2011

Citrus

Figure I. Antibacterial activity of the Citrus essential oils against the Gram-negative Escherichia coli ATCC 10536. Growth
on MHB medium at 37C for 24 h. Data were expressed as means sd of three different experiments.

Figure II. Antibacterial activity of the Citrus essential oils against the Gram-positive Staphylococcus aureus ATCC 25293.
Growth on MHB medium at 37C for 24 h. Data were expressed as means sd of three different experiments.

oils ranged 15230 g/mL. In particular, C. limon showed the

lowest MIC values of 15 g/mL and 20 g/mL, respectively,
against Xanthomonas citri strains. This antimicrobial activity
might be attributed to the presence of limonene and gamma
Vol. 23, January/February 2011

terpinene (Table I), which have been reported to exert their

toxic effects through the disruption of bacterial or fungal
membrane integrity and the inhibition of respiration and ion
transport processes (20). Among the examined bacteria, both
Journal of Essential Oil Research/29

Frassinetti et al.

Table III. Minimum inhibitory concentrations (MICs) of Citrus oils against tested bacteria

Citrus aurantium
Bitter Orange

Citrus sinensis
Sweet Orange

Citrus limon
Lemon

Citrus reticulata
Mandarin

Gram-negative
Xanthomonas campestris pv citri

NCPPB 3236
20
20
15
Xanthomonas campestris pv citri

NCPPB 3562
20
20
15
Xanthomonas campestris pv citri

NCPPB 3832
25
25
20
Escherichia coli

ATCC 10536
35
35
40
Escherichia coli

ATCC 25922
25
25
30
Salmonella cholerasuis

ATCC 14028
50
50
50
Pseudomonas aeruginosa

ATCC 27853
75
70
70

Gram-positive
Staphylococcus aureus

ATCC 25923
200
200
200
Enterococcus faecalis

ATCC 29212
150
150
150
Enterobacter aerogenes

ATCC 13048
100
100
130

20
20
25
40
35
55
70

230
150
120

Figure III. Free radical-scavenging activity of Citrus essential oils evaluated by the DPPH assay, and comparison with that
of the reference (Trolox 100 g/mL). Data were expressed as means sd of three different experiments.

Gram-positive and Gram-negative bacteria were sensitive to the

Citrus oils, but the Gram-positive strains were more resistant,
showing the highest MIC values. For the better elucidation of
the antibacterial activity of Citrus oils, the bacterial growth of
representative Gram-negative (Escherichia coli ATCC 10536),
and Gram-positive strain (Staphylococcus aureus ATCC 25293)
are shown in Figures I and II.
The Gram-negative strains showed a very high sensitivity
to the Citrus oils; in particular Pseudomonas aeruginosa was
inhibited at 7075 g/mL (MIC), while E. coli strains were
most susceptible, showing MIC values ranging 2545 g/mL
(Table III). The Gram-positive strains Enterococcus faecalis,
Enterobacter aerogenes and S. aureus were inhibited at oils
concentrations ranging 100250 g/mL (MIC).
Although Gram-negative organisms are generally reported
to be more resistant to active antimicrobial compounds (21),
30/Journal of Essential Oil Research

other studies have found Gram-positive bacteria less sensitive

to essential oils than Gram-negative strains (22, 23). The results
obtained in the present study are in agreement with the latter
literature data. Moreover, other authors suggested that the simple
relation involving cell structure and microbial sensitivity to essential oils is not yet well established, and possible antagonistic
or synergistic effects among the various active constituents of
the oils should be taken into consideration (24).
The use of natural antioxidants are of great interest in
the food industry, since their possible use as natural additives
emerged from a growing tendency to replace synthetic antioxidants with natural ones, because most antioxidants currently
employed, such as BHA (butylated hydroxyanisole) and BHT
(butylated hydroxytoluene) can be cytotoxic and increase the
development of cancerous cells (8, 10).
Hence in this work the antioxidant properties of Citrus oils
Vol. 23, January/February 2011

Citrus

were also tested. Free radical generation is directly related to

oxidation in foods and biological systems; therefore, the determination of free radical scavenging in food is important (25).
Among the numerous of methods that can be used, TEAC,
DPPH, and PCL are functional for determining the activity
of both hydrophilic and lipophilic species, thus ensuring a
better comparison of the results and proposing a wider range
of possible applications. Taking this into account, the antioxidant activity was determined by the DPPH test, because this
method is simple, reproducible and is widely used in the food
industry (26).
All the tested Citrus oils showed a good antioxidant activity
depending on the concentration. Lemon oil showed the best
antioxidant capacity, with an inhibition rate of 70%, comparable to that of Trolox, a potent synthetic antioxidant and also
comparable with those found in Oregano oil by Kulisic et al.
(27). The present works results are also in agreement with
those obtained by Wei and Shibamoto, demonstrating the
scavenging abilities ranging 3990% at a level of 200 g/mL
of various essential oils (28).
The significant antioxidant activity showed by all the tested
Citrus oils might be correlated to the presence of monoterpenes,
particularly g-terpinene and limonene, which are the most
abundant compounds in the oils examined (Table I) and have
been reported to posses good antioxidant activity (28, 29).
In conclusion, our results provide a further confirmation
that the oils of Citrus spp. may be used as a potential natural
antioxidant and antimicrobial agents in the food industry.
Acknowledgements:

This study was part of the project Programma Internazionale

Pic Interreg Iii- A Italia-Francia Isole (2000-2006): Il Citrus come
sistema modello per larea mediterranea: studio varietale per resistenza
a stress biotici e abiotici. Le Citrus comme modle de systme pour
la zone mditerrranenne: tude varitale de rsistance aux Stress
biotiques et abiotiques.
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