Food Control 26 (2012) 326e330
Contents lists available at SciVerse ScienceDirect
Food Control
journal homepage: www.elsevier.com/locate/foodcont
Short communication
Inhibition of foodborne pathogen bacteria by essential oils extracted from citrus
fruits cultivated in Sicily
Luca Settanni a, Eristanna Palazzolo b, Valeria Guarrasi a, c, Aurora Aleo d, Caterina Mammina d,
Giancarlo Moschetti a, Maria Antonietta Germanà a, *
a
DEMETRA Department, University of Palermo, Viale delle Scienze 4, 90128 Palermo, Italy
SAGA Department, University of Palermo, Viale delle Scienze 13, 90128 Palermo, Italy
Institute of Biophysics at Palermo, Italian National Research Council, Via U. La Malfa 153, 90146 Palermo, Italy
d
Department of Sciences for Health Promotion “G. D’Alessandro”, Section of Hygiene, University of Palermo, Palermo, Italy
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 6 December 2011
Received in revised form
17 January 2012
Accepted 27 January 2012
The antagonistic activity of the essential oils (EOs) extracted by hydrodistillation from the fruit peel of
several citrus genotypes (pummelo, grapefruit, orange, kumquat, mandarin and lemon) was evaluated
against foodborne pathogen bacteria (43 strains of Listeria monocytogenes, 35 strains of Staphylococcus
aureus and 14 strains of Salmonella enterica). Five commercial EOs were used for comparison. Most of the
EOs were more effective against the Gram-positive bacteria rather than Salmonella. EOs of lemon genotypes 14 and 15 showed the best results in terms of number of strains inhibited and width of the inhibition
zone. The most susceptible strain of each species (L. monocytogenes 133, St. aureus 473 and Salmonella
Newport 50404) was used as indicator for the evaluation of the minimum inhibition concentration (MIC) of
the EOs 14 and 15. The last two EOs, EO 16 (showing the weakest inhibition power among lemon EOs), and
the commercial EO L (lemon) were analysed for their chemical composition. A total of 30 major compounds
were identified by gas chromatography/mass spectrometry (GC/MS) and the oxygenated monoterpenes
were suggested to be implicated in the process of bacterial inhibition by citrus EOs.
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Antibacterial activity
Citrus fruits
Essential oils
Foodborne pathogens
1. Introduction
Essential oils (EOs) and extracts of various plants, herbs, spices
and fruits show antimicrobial potential (Nychas, Tassou, &
Skandamis, 2003; Oussalah, Caillet, Saucier, & Lacroix, 2006). The
composition of EOs include a complex mixture of several
compounds. When the inhibitory activities are directed towards
foodborne pathogens, EOs may be employed in the food preservation, since they are referred to as GRAS (Generally Recognised As
Safe) (Viuda-Martos, Ruiz-Navajas, Fernández-López, & PérezÁlvarez, 2008). In the last years, consumers have become aware of
the health concerns of the food ingredients determining an
increase in the request of foods processed without the addition of
synthetic chemical preservatives. Thus, the use of EOs may provide
a “natural” alternative to the chemical preservation of foods.
Citrus EOs have been industrially applied in many products,
including foods and beverages, cosmetics and medicines (Uysal,
Sozmen, Aktas, Oksal, & Odabas Kose, 2011) and the activities
against some of the most important foodborne pathogens have
* Corresponding author. Tel.: þ39 091 23896094; fax: þ39 091 6515531.
E-mail address: mariaantonietta.germana@unipa.it (M.A. Germanà).
0956-7135/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodcont.2012.01.050
been proved (Fisher & Phillips, 2006; Kim, Marshall, & Wei, 1995).
Since citrus EOs are mainly located in the fruit peel, their extraction
is economically sustainable, because the fruit peel constitutes
a waste for the fruit juice industry (Tirado, Stashenko, Combariza, &
Martinez, 1995).
The objectives of this study were: to evaluate the inhibitory
potential of EOs extracted from the fruit peel of several citrus
varieties cultivated in Sicily against the most common foodborne
pathogens; and to determine the chemical composition of the most
effective EOs.
2. Materials and methods
2.1. Citrus samples and EO extraction
Citrus fruit samples used in this study (Table 1) were collected in
April 2011. Samples 1, 2, 3, 4, 5, 6, 9, 10 and 11 were provided by the
Azienda “Sperimentale Palazzelli” C.R.A.eCentro di ricerca per
l’agrumicoltura e le colture mediterranee Contrada Palazzelli
Scordia (CT, Italy), while samples 7, 8, 12, 13, 14, 15, 16, 17 and 18
were collected in the orchard “Parco d’Orleans”eAgricultural
Faculty of Palermo (Italy). Commercial mandarin, lemon,
327
L. Settanni et al. / Food Control 26 (2012) 326e330
grapefruit, blood orange and blond orange EOs were kindly
provided by Eurofood s.r.l. of Capo d’Orlando (ME, Italy).
Citrus fruit peels were subjected to hydrodistillation using
a Clevenger-type apparatus (Comandè, Palermo, Italy) and the EOs
were collected for 3 h in hexane.
Table 1
List of citrus peel EOs used in the antimicrobial assays.
EO
Species
Variety
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
P
A
AR
L
M
Pummelo (Citrus maxima (Burm.) Merrill)
Grapefruit (Citrus paradisi Macf.)
Orange (Citrus sinensis L. Osbeck)
Orange (Citrus sinensis L. Osbeck)
Kumquat (Fortunella margarita)
Orange (Citrus sinensis L. Osbeck)
Orange (Citrus sinensis L. Osbeck)
Orange (Citrus sinensis L. Osbeck)
Orange (Citrus sinensis L. Osbeck)
Orange (Citrus sinensis L. Osbeck)
Orange (Citrus sinensis L. Osbeck)
Mandarin (Citrus reticulata Blanco)
Lemon (Citrus limon L. Burm.)
Lemon (Citrus limon L. Burm.)
Lemon (Citrus limon L. Burm.)
Lemon (Citrus limon L. Burm.)
Lemon (Citrus limon L. Burm.)
Orange (Citrus sinensis L. Osbeck)
Grapefruit (Citrus paradisi Macf.)
Orange (Citrus sinensis L. Osbeck)
Orange (Citrus sinensis L. Osbeck)
Lemon (Citrus limon L. Burm.)
Mandarin (Citrus reticulata Blanco)
Reinking pummelo
Pink
Tarocco Meli
Lane Late Nucellar C2611
Ovale
Valencia
Frost Valencia
Lane Late VCR
Tarocco S. Alfio
Frost Washington Navel
Washington Navel 3033
Mandarino Tardivo di Ciaculli
Femminello apireno
Femminello Santa Teresa
Monachello
Femminello Continella
Femminello vecchio clone
Valencia
Grapefruits (commercial)
Blond oranges (commercial)
Blood oranges (commercial)
Lemons (commercial)
Mandarins (commercial)
2.2. Bacterial strains
The strains used in this study (Tables 2e4), associated with
foodborne diseases, belonged to the culture collection of the
Section of Hygiene, Department of Sciences for Health Promotion
“G. D’Alessandro” (Palermo, Italy). All strains were subcultured in
Brain Heart Infusion (BHI) agar (Oxoid, Milan, Italy) and incubated
overnight at 37 C.
2.3. Screening of the antibacterial activity
EOs were tested against a concentration of approximately
107 CFU mL 1 of each bacterial strain applying the paper disc
diffusion method reported by Militello et al. (2011). Sterile water
and streptomycin (10%, w/v) were used as negative and positive
control, respectively. Petri dishes were incubated at 37 C for 24 h
Table 2
Inhibitory activitya of citrus EOs against Listeria monocytogenes.
Strain
129b
130b
131b
132b
133b
134b
135b
136b
137b
138b
139b
140b
179c
180d
182d
184e
185f
186g
187h
188i
1BOl
2BOm
3BOm
4BOm
5BOm
6BOm
7BOm
8BOm
11BOn
12BOm
13BOo
14BOo
15BOo
16BOo
17BOo
18BOo
19BOo
20BOo
21BOo
22BOp
23BOp
24BOp
Citrus samples
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
P
A
AR
M
L
e
e
e
e
e
e
e
e
e
þ
e
e
e
e
e
þ
e
e
e
þ
e
e
e
e
e
e
e
e
e
þ
e
e
e
e
e
e
e
e
e
e
e
þ
þ
e
e
e
e
þ
þ
e
þ
þ
e
e
e
þ
þ
þ
þ
e
e
e
þ
e
e
e
þþ
þ
þ
e
e
e
þ
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
þ
þ
e
e
e
þ
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
þ
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
þ
þ
e
þ
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
þ
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
þ
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
þ
e
e
þ
e
e
þ
þ
þ
þ
þ
þ
þ
þ
þ
e
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þþ
þþ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þþ
þ
þ
þ
þþ
þþ
þþ
þ
þ
þ
þþ
þþ
þþ
þþ
þþ
þþ
þþ
þþ
þ
þþ
þþ
þ
þþ
þþ
þ
þ
þþ
þþ
þ
þþ
þþ
þþ
þ
þ
þ
þ
þþ
þþ
þ
þþ
þ
þþ
þþ
þ
þ
þþ
þþ
þþ
þþ
þ
þ
þ
þ
þþ
þ
þþ
þþ
þþ
þþ
þþ
þþ
þþ
þ
þþ
þþ
þ
þ
þ
þ
þ
þþ
þþ
þþ
þ
þ
þ
þþ
þ
þ
þ
þþ
e
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þþ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
þ
e
e
e
e
e
e
e
e
þ
e
e
e
e
þ
e
e
þ
þ
e
þ
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
þ
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
þ
þ
þ
þ
e
e
e
e
þ
þ
e
e
e
þ
þ
þ
þ
þ
þ
þ
þþþ
þ
þ
þ
þ
þ
þ
Source of isolation: b, human stools; c, salmon; d, Ricotta cheese; e, rice salad; f, beef; g, mozzarella salad; h, roasted chicken; i, green salad; l, chopped meat; m, salami; n, meat
factory; o, Gorgonzola cheese; p, Taleggio cheese.
a
e, no inhibition; , inhibition (<9 mm diameter); þ clear inhibition (9e12 mm diameter); þþ, strong inhibition (13e15 mm diameter); þþþ, huge inhibition (>18 mm diameter).
328
L. Settanni et al. / Food Control 26 (2012) 326e330
Table 3
Inhibitory activitya of citrus EOs against Staphylococcus aureus.
Strain
C1/5634-MSSAb
C4/6561.1-MSSAc
C6/5145-MSSAb
C38/249.1-MSSAb
C45/12425-MSSAb
195-MRSAd
TUM-MRSd
9-MRSd
14-MRSAd
189-MRSAd
1313-MRSAd
1369-MRSAd
581-MRSAd
340-MRSAd
4ADI MRSAd
7ADI MSSAd
14LU MRSAd
16 MSSAd
19 MRSAd
20ADI MRSAd
21ADI MRSAd
62 MRSAd
68 MRSAd
106 MRSAd
109 MRSAd
156 MRSAd
168 MRSAd
206 MSSAd
473 MRSAd
493 MRSAd
637 MRSAd
734 MSSAd
735 MSSAd
750 MSSAd
E36GI MRSAd
Citrus samples
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
P
A
AR
M
L
þ
þ
þ
þ
þ
þ
þ
e
þ
þ
þ
þ
e
e
e
e
þ
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
þ
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
þ
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þþ
þþ
þþ
þþ
þþ
þ
þþ
þ
þ
þ
þþ
þþ
þþþ
þþ
þþ
e
þþ
þþ
þþ
þþ
þþ
þþ
þþ
þþ
þþ
þþ
þþ
þ
þþþ
þþ
þþ
þþþ
þ
þþ
þþ
þþ
þþ
þ
þþ
þþ
þ
e
þ
þ
þ
þþ
þþ
þþ
þþ
e
þþ
þþ
þ
þþ
þþ
þþ
þþ
þþ
þþ
þþ
þþ
þ
þþþ
þþ
þþ
þþþ
þ
þþ
þþ
þ
e
e
e
e
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þþ
þþ
þ
þ
þ
þ
þþ
þ
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
þ
þ
e
þ
þ
þþ
þ
e
e
þ
þ
þþ
þ
þ
Source of isolation: b, cheese; c, raw milk; d, human stools.
a
e, no inhibition; , inhibition (<9 mm diameter); þ clear inhibition (9e12 mm diameter); þþ, strong inhibition (13e15 mm diameter); þþþ, huge inhibition (>18 mm
diameter).
and the inhibitory activity was evaluated as positive if a definite
clear area was detected around the paper disc. Each test was performed in duplicate and the experiments were repeated twice.
of EO antibacterial performances (Burt, 2004). MIC is defined as the
lowest concentration of an active compound inhibiting visible
growth of test organisms (Karapinar & Aktug, 1987).
The EOs were serially diluted (dilution factor ¼ 2) in acetone
(SigmaeAldrich, Milan, Italy). Each test tube, containing 990 mL of
broth medium and 10 mL of EO dilution, was inoculated with
approximately 106 CFU mL 1 of a given sensitive strain. Acetone
alone was used as negative control.
2.4. Determination of the minimum inhibitory concentration
The antibacterial activity was measured as minimum inhibitory
concentration (MIC), which represents the most common expression
Table 4
Inhibitory activitya of citrus EOs against Salmonella enterica.
Strain
S.
S.
S.
S.
S.
S.
S.
S.
S.
S.
S.
S.
S.
S.
Abony 50398b
Agona 50360b
Blockley 50314b
Bredeney 50374b
Derby 50399b
Enteritidis 50339c
Hadar 50272d
Infantis 50270b
Muenchen 50393b
Napoli 50376b
Newport50404b
Panama 50347b
Saintpaul 50415b
Thompson 50280e
Citrus samples
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
P
A
AR
M
L
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
þ
þ
e
þ
þ
e
þ
þ
e
þ
e
þþ
þ
þþ
þþ
þ
þþ
þþ
þ
þþ
þþ
þþ
þþ
þþ
e
þþ
þ
þþ
þþ
þþ
þþ
þþ
þ
þþ
þþ
þþ
þþ
þþ
e
e
e
e
e
þ
e
e
e
þ
þ
e
e
e
þ
þþ
þ
þþ
þþ
þ
þ
þþ
þþ
þþ
þþ
þ
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
Source of isolation: b, human stools; c, food preparation; d, egg; e, chicken-pork meat.
a
e, no inhibition; , inhibition (<9 mm diameter); þ clear inhibition (9e12 mm diameter); þþ, strong inhibition (13e15 mm diameter).
329
L. Settanni et al. / Food Control 26 (2012) 326e330
2.5. Determination of EO composition
Samples were analysed by gas chromatography/mass spectrometry (GC/MS) (EI) on a GCMS-QP2010 (Shimadzu, Milan, Italy)
using a fused silica capillary column, SLB-5MS (5% diphenyl:95%
methylsiloxane) : 30 m 0.25 i.d. 0.25 mm film thickness
(Supelco, Milan, Italy); temperature program 50e280 C at
3 C min 1; carrier gas He at a constant linear rate: 30 cm s 1
(30.6 kPa); 1.0 mL of solution (1:10 v/v, essential oil/hexane) injected
on a splitesplitless injector; injector temperature 250 C; injection
mode split (split ratio 100:1). MS scan conditions: source temperature 200 C, interface temperature 250 C, EI energy 70 eV; mass
scan range 40e400 amu.
Data were handled through the use of GCMS-Solution software
and the peak identification was carried out with NIST21,107,147
Library according to a similarity larger than 90%.
3. Results and discussion
3.1. Inhibition of bacterial growth
The antibacterial activity of the EOs analysed is shown in
Tables 2e4. The active extracts were mainly effective against the
Gram-positive bacteria (Tables 2 and 3), while only three lemon
EOs (13, 14 and 17) clearly inhibited the growth of Salmonella
(Table 4). This finding is not surprising, since also other studies
showed that Gram-positive bacteria were more susceptible to EOs
of different origin, including citrus fruits, than Gram-negative
bacteria (Al-Reza, Rahman, Lee, & Kang, 2010; Burt, 2004;
Calsamiglia, Busquet, Cardozo, Castillejos, & Ferret, 2007;
Davidson & Naidu, 2000). The presence of the outer membrane
provides a strong impermeable barrier for the Gram-negative
bacteria (Nikaido, 1994).
The commercial EOs were characterised by a lower inhibitory
power than Sicilian EOs, although commercial lemon and mandarin
EOs showed some antagonistic properties vs Listeria and staphylococci. Salmonella growth was not inhibited by any commercial EO.
In general, the strains belonging to the Gram-positive species
showed a different level of susceptibility to the treatments.
Undoubtedly, the most interesting results were displayed by EOs 14
and 15 which inhibited all 42 Listeria monocytogenes and almost all
35 St. aureus strains tested (34 strains by EO 14 and 33 by EO 15). For
the majority of the susceptible strains, the diameter of the halo was
in the range 13e15 mm (scored as strong inhibition). Except for S.
Thompson 50280, which was quite resistant to the EOs, all other
Salmonella strains were strongly inhibited by EOs 14, 15 and 17.
L. monocytogenes 133, St. aureus 473 and S. Newport 50404 were
found highly sensitive to the EOs 14 and 15 and, for this reason, they
were chosen for the calculation of the MIC (Table 5). In particular, S.
Newport 50404 showed the most interesting result, because it was
characterised by the highest susceptibility, despite it is a Gramnegative bacterium. Previous studies showed that the inhibition
of some Gram-negative (Escherichia coli and Campylobacter jejuni)
strains was comparable with that of Gram-positive bacteria (Fisher
& Phillips, 2006). Fisher and Phillips (2008) suggested that the
antibacterial effects of citrus EOs are not uniform among bacteria
and could depend on the compounds and the species/isolate under
study.
MIC data of EOs 14 and 15 were of paramount importance, since
these two EOs were effective at concentrations extremely low. The
first aspect to be considered for the application of EOs in foods is
represented by their impact on the organoleptic properties of the
final product, because the main constituents of EOs are responsible
for their fragrance. Hence, naturally derived preservatives can alter
the taste of foods or exceed acceptable flavour thresholds (Hsieh,
Table 5
MIC of Sicilian lemon EOs 14 and 15 against the most sensitive pathogenic strains.
MIC (mL mL
Strains
L. monocytogenes 133
St. aureus 473
S. Newport 50404
1
)
EO 14
EO 15
0.156
0.039
0.019
0.156
0.078
0.019
Mau, & Huang, 2001; Nazer, Kobilinsky, Tholozana, & DuboisBrissonneta, 2005). Moreover, once the antibacterial activity is
evaluated in vitro, a major problem to the in situ efficacy of a given
EO derives from its interaction with the food components and/or
other additives. For this reason, Burt (2004) suggested that a higher
concentration of EOs could be needed to achieve the same effect in
food as in vitro. Thus, the high inhibitory activity of EOs at low
concentrations determines a lower amount of EOs to be added to
the food matrices, limiting the unwanted changes of the sensory
features.
3.2. Chemical characterization of EOs
The EOs 14 and 15, which showed the highest inhibitory activities, EO 16, that within the lemon EO group displayed the lowest
inhibitory efficacy, and the commercial EO L were chemically analysed by GC and GCeMS. The qualitative and quantitative
Table 6
Chemical composition of commercial and Sicilian lemon EOs.
Chemical compounds
Monoterpene hydrocarbons
a-Thujene
a-Pinene
Camphene
Sabinene
b-Pinene
b-Myrcene
a-Terpinene
(þ)-4-Carene
o-Cymene
D-Limonene
p-Cymene
a-Terpinolene
Oxygenated monoterpenes
1-Octanol
Linalol
Nonanal
(þ)-Citronellal
Nonanol
4-Terpineol
a-Terpineol
cis-Geraniol
b-Citronellale
b-Citral
Nerol
a-Citral
Neryl acetate
Geranyl acetate
Sesquiterpene hydrocarbons
Caryophyllene
a-Bergamotene
b-Bisabolene
Others
Tetratetracontane
LRIa
%b
EO L
EO 14
EO 15
EO 16
1419
1493
1502
77.75
0.58
2.34
0.07
1.95
14.12
2.10
t
0.29
0.07
55.32
0.25
0.56
6.18
t
0.14
0.21
0.17
t
0.06
0.20
0.03
t
1.21
0.03
2.18
1.02
0.96
1.96
0.34
0.64
0.98
68.74
0.32
1.49
0.05
1.71
11.77
1.57
0.20
t
1.28
49.65
0.18
0.52
19.21
0.22
0.76
0.24
0.13
0.16
1.13
1.61
2.12
0.45
3.18
2.90
4.29
1.24
0.78
1.27
0.16
0.43
0.68
67.61
0.36
1.64
0.05
1.89
13.19
1.51
t
0.32
1.62
46.31
0.14
0.58
19.35
0.31
0.66
0.26
0.11
0.27
1.43
2.17
2.86
0.35
2.46
3.94
3.32
0.71
0.50
1.47
0.18
0.50
0.79
78.23
0.34
1.50
0.03
1.53
10.29
1.97
0.06
0.18
2.59
60.90
0.21
0.47
7.69
0.19
0.55
0.17
0.05
0.07
0.73
0.98
0.90
0.18
0.67
1.09
0.89
0.68
0.54
1.54
0.13
0.55
0.86
1689
0.02
t
0.36
t
930
939
954
976
981
990
1014
1020
1024
1029
1026
1088
1090
1098
1015
1145
1155
1177
1188
1255
1314
1337
1347
1358
1365
1383
t, trace (<0.02%).
a
Linear retention index on SLB-5MS column.
b
Percentage of each compound on the total oil (computed from the total GC peak
area).
330
L. Settanni et al. / Food Control 26 (2012) 326e330
composition of the EOs is reported in Table 6. Chemical compounds
were classified by phytochemical groups and listed in order of their
elution. A total of 82 chemicals were identified, most of which (52)
were detected in trace (percentage lower than 0.10). The total
recovery was: 85.91% for EO L, 89.22% for EO 14, 88.79% for EO 15
and 87.46% for EO 16. All EOs were dominated by the monoterpene
hydrocarbon fraction that reached almost the 78% for the EOs L and
16 and almost 68% for EOs 14 and 15. On the contrary, the
oxygenated monoterpene fraction of EOs 14 and 15 (more than 19%)
was about three times that of EOs L and 16. Sesquiterpene hydrocarbons did not exceed 2%, while among the other compounds,
tetratetracontane was detected at a percentage higher than 0.10
only in EO 15.
The efficacy of EOs 14 and 15 may be imputable to the oxygenated
monoterpenes, in particular to 4-terpineol, a-terpineol, cis-geraniol,
b-citral, nerol and a-citral. Our results confirmed previous observations that this class of chemicals, especially phenolic substances,
exhibits strong antimicrobial activity than hydrocarbon monoterpenes (Knobloch, Weigand, Weis, Schwarm, & Vigenschow, 1986;
Sokovi
c & van Griensven, 2006; Sokovic, Tzakou, Pitarokili, &
Couladis, 2002). Hydrocarbon monoterpenes are characterised by
a low water solubility which limits their diffusion through the
medium and their inactivity is closely related to their limited
hydrogen bound capacity (Griffin, Markham, & Leach, 2000).
Acknowledgements
Authors wish to thank Eurofood s.r.l. of Capo d’Orlando (ME,
Italy) for providing the commercial EO samples and Dr. G. Russo, Dr.
Reforgiato Recupero and S. Recupero (Centro di ricerca per l’agrumicoltura e le colture mediterranee) for providing some citrus
samples.
References
Al-Reza, S. M., Rahman, A., Lee, J., & Kang, S. C. (2010). Potential roles of essential oil
and organic extracts of Zizyphus jujuba in inhibiting food-borne pathogens. Food
Chemistry, 119, 981e986.
Burt, S. (2004). Essential oils: their antibacterial properties and potential application in foods e a review. International Journal of Food Microbiology, 94, 223e253.
Calsamiglia, S., Busquet, M., Cardozo, P. W., Castillejos, L., & Ferret, A. (2007). Invited
review: essential oils as modifiers of rumen microbial fermentation. Journal of
Dairy Science, 90, 2580e2595.
Davidson, P. M., & Naidu, A. S. (2000). Phyto-phenols. In A. S. Naidu (Ed.), Natural
food antimicrobial systems (pp. 265e293). Boca Raton, FL: CRC Press.
Fisher, K., & Phillips, C. A. (2006). The effect of lemon, orange, and bergamot
essential oils and their components on the survival of Campylobacter jejuni,
Escherichia coli O157, Listeria monocytogenes, Bacillus cereus and Staphylococcus
aureus in vitro and in food systems. Journal of Applied Microbiology, 101,
1232e1240.
Fisher, K., & Phillips, C. A. (2008). Potential antimicrobial uses of essential oils in
foods: is citrus the answer? Trends in Food Science and Technology, 19, 156e164.
Griffin, G. S., Markham, L. J., & Leach, N. D. (2000). An agar dilution method for the
determination of the minimum inhibitory concentration of essential oils.
Journal of Essential Oil Research, 12, 249e255.
Hsieh, P.-C., Mau, J.-L., & Huang, S.-H. (2001). Antimicrobial effect of various
combinations of plant extracts. Food Microbiology, 18, 35e43.
Karapinar, M., & Aktug, S. E. (1987). Inhibition of foodborne pathogens by thymol,
eugenol, menthol and anethole. International Journal of Food Microbiology, 4,
161e166.
Kim, J., Marshall, M. R., & Wei, C.-I. (1995). Antibacterial activity of some essential oil
components against five foodborne pathogens. Journal of Agricultural and Food
Chemistry, 46, 1739e1745.
Knobloch, K., Weigand, H., Weis, N., Schwarm, H. M., & Vigenschow, H. (1986).
Action of terpenoids on energy metabolism. In E. J. Brunke (Ed.), Progress in
essential oil research (pp. 429e445). Berlin: Walter de Gruyter.
Militello, M., Settanni, L., Aleo, A., Mammina, C., Moschetti, G., Giammanco, G. M.,
et al. (2011). Chemical composition and antibacterial potential of Artemisia
arborescens L. essential oil. Current Microbiology, 62, 1274e1281.
Nazer, A. I., Kobilinsky, A., Tholozana, J.-L., & Dubois-Brissonneta, F. (2005).
Combinations of food antimicrobials at low levels to inhibit the growth of
Salmonella sv. Typhimurium: a synergistic effect? Food Microbiology, 22,
391e398.
Nikaido, H. (1994). Prevention of drug access to bacterial targets: permeability
barriers and active efflux. Science, 264, 382e388.
Nychas, G. J. E., Tassou, C. C., & Skandamis, P. (2003). Antimicrobials from herbs and
spices. In S. M. Roller (Ed.), Natural antimicrobials for the minimal processing of
foods (pp. 176e200). New York: Woodhead Publishers, CRC Press.
Oussalah, M., Caillet, S., Saucier, L., & Lacroix, M. (2006). Inhibitory effects of
selected plant essential oils on the growth of four pathogenic bacteria: E. coli
O157:H7, Salmonella Typhimurium, Staphylococcus aureus and Listeria
monocytogenes. Food Control, 18, 414e420.
Sokovi
c, M., Tzakou, O., Pitarokili, D., & Couladis, M. (2002). Antifungal activities of
selected aromatic plants growing wild in Greece. Nahrung, 46, 317e320.
Sokovi
c, M., & van Griensven, L. J. L. D. (2006). Antimicrobial activity of essential oils
and their components against the three major pathogens of the cultivated
button mushroom, Agaricus bisporus. European Journal of Plant Pathology, 116,
211e224.
Tirado, C. B., Stashenko, E. E., Combariza, M. Y., & Martinez, J. R. (1995). Comparative
study of Colombian citrus oils by high-resolution gas chromatography and gas
chromatography-mass spectrometry. Journal of Chromatography A, 697,
501e513.
Uysal, B., Sozmen, F., Aktas, O., Oksal, B. S., & Odabas Kose, E. (2011). Essential oil
composition and antibacterial activity of the grapefruit (Citrus Paradisi L.) peel
essential oils obtained by solvent-free microwave extraction: comparison with
hydrodistillation. International Journal of Food Science and Technology, 46,
1455e1461.
Viuda-Martos, M., Ruiz-Navajas, Y., Fernández-López, J., & Pérez- Álvarez, J. (2008).
Antifungal activity of lemon (Citrus lemon L.), mandarin (Citrus reticulata L.),
grapefruit (Citrus paradisi L.) and orange (Citrus sinensis L.) essential oils. Food
Control, 19, 1130e1138.
Aurora Aleo