Antibacterial activity of essential oils from plants of the genus origanum

Food Control 34 (2013) 539e546

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Food Control
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Short communication

Antibacterial activity of essential oils from plants of the genus
Origanum
Michalis K. Stefanakis a, Eleftherios Touloupakis a, Elias Anastasopoulos b,
Dimitrios Ghanotakis a, Haralambos E. Katerinopoulos a, *, Pavlos Makridis c
a
b
c

Department of Chemistry, University of Crete, Voutes, Heraklion 71003, Crete, Greece
Department of Plant Production, Plant Biotechnology Laboratory, Technological and Educational Institute of Larissa, 41110 Larissa, Greece
Institute of Aquaculture, Hellenic Centre for Marine Research, P.O. Box 2214, 71003 Heraklion, Crete, Greece

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 31 December 2012
Received in revised form
15 May 2013
Accepted 22 May 2013

In this study, three plant species, members of the family of Lamiaceae and the genus Origanum, namely,
Origanum vulgare subsp. hirtum, Origanum onites L., and Origanum marjorana L. were studied for their
chemical composition and antibacterial activity. Essential oils of these plants were received by means of
micro-steam distillation and their components were analyzed by gas chromatography and mass spectrometry (GCeEIMS). The major components identified in all three species are carvacrol and thymol. The
oils were assayed as potential food control antimicrobial agents. In vitro studies showed that the essential
oils showed strong antimicrobial activity against 5 bacterial and 1 yeast strains.
Ó 2013 Elsevier Ltd. All rights reserved.

Keywords:
Food control
Origanum
Essential oils
Antimicrobial activity

1. Introduction
Essential oils (EOs) are aromatic oily liquids obtained from
various plants generally localized in temperate to warm countries.
In nature as secondary metabolites, EOs play an important role in
the protection of the plants as antibacterials, antivirals, antifungals,
insecticides and also act against herbivores (Bakkali, Averbeck,
Averbeck, & Idaomar, 2008). An estimated 3000 essential oils are
known, of which about 300 are commercially available and
destined mostly for the flavor and fragrances market (Van de Braak
& Leijten, 1999, 116 pp.). EOs are very complex natural mixtures
containing hydrocarbons (mainly terpenoids) and oxygenated
compounds (alcohols, esters, ethers, aldehydes, ketones, lactones,
phenols and phenol ethers). Their composition may vary considerably between plant species and varieties, and, within the same
variety, from different geographical origin (Zygadlo & Juliani, 2003).
EOs are widely used in medicine, in perfumes, cosmetics and bath
products, as flavoring agents in food and drink, and in many other

manufacturing areas. EOs can constitute a powerful tool to reduce
the development and dissemination of antimicrobial resistance.
Nowadays essential oils are recognized as safe substances (ESO,

* Corresponding author. Tel.: ỵ30 2810 545026; fax: ỵ30 2810 545001.
E-mail address: (H.E. Katerinopoulos).
0956-7135/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved.
/>
GRAS e 182.20) by the Food and Drug Administration (2005) and
some contain compounds which can be used as antibacterial additives (Ait-Ouazzou et al., 2011; Cox et al., 2001; Muyima, Zulu,
Bhengu, & Popplewell, 2002; Nerio, Olivero-Verbel, & Stashenko,
2010).
They become increasingly popular as natural antimicrobial and
antioxidant agents that may be used in food preservation. Public
concern about the use of antibiotics in livestock feed has increased,
because of the emergence of antibiotic resistant bacteria and their
possible transmission from livestock to humans. In fact, in the European Union the use of synthetic antibiotics, health and growth
promoters as additives in livestock feed has been prohibited since
2006 by the European Parliament and Council Regulation (EC
No.1831/2003). In this context, one of the possible solutions is the
use of EOs such as those found in the genus Origanum and in its 3
species used for this study: Origanum vulgare subsp. hirtum, Origanum onites and Origanum marjorana.
Antimicrobials are used in the food industry for two main reasons: to control natural spoilage processes (food preservation), and
to prevent the growth of micro-organisms, including pathogenic
micro-organisms (food safety).
A large number of reports concerning the antioxidant and the
antimicrobial ability of essential oils have already been published
(Bagamboula, Uyttendaele, & Debevere, 2003; Bakkali, Averbeck,
Averbeck, Zhiri, & Idaomar, 2005; Botelho et al., 2007; Castilho,

540

M.K. Stefanakis et al. / Food Control 34 (2013) 539e546

Savluchinske-Feio, Weinhold, & Gouveia, 2012; Deba, Xuan, Yasuda,
& Tawata, 2008; Gỹlỗin, Elmastas¸, & Aboul-Enein, 2007; Kalemba &
Kunicka, 2003; Pauli, 2006; Pavela, 2009; Sacchetti et al., 2005;
Saidana et al., 2008; Si et al., 2006; Thuille, Fille, & Nagl, 2003;
Valero & Salmeron, 2003; Ye, Dai, & Hu, 2013). In these studies, EO
exhibited very good insecticide, bactericide and fungicide effects
(Kumar, Shukla, Singh, Prasad, & Dubey, 2008; Srivastava, 2008).
Burt (2004) gives an overview of the studies of the antibacterial
activity of essential oils in foods. As typical lipophiles, they cross
through the cell wall and cytoplasmic membrane, disrupt the
structure of their different layers of polysaccharides, fatty acids and
phospholipids and permeabilize them. In general, the cytotoxic
activity of essential oils is mostly due to the presence of phenols,
aldehydes and alcohols (Bruni et al., 2003; Sacchetti et al., 2005).
The aims of this study were a) to determine the chemical
composition of essential oils extracted from three species of the
genus Origanum, and b) evaluate their ability to inhibit in vitro
bacterial strains such as Vibrio, isolated from aquaculture facilities,
and the common microbial strains Escherichia coli and Saccharomyces cerevisiae.
2. Materials and methods
2.1. Materials
Samples of leaves, stems and/or seeds from three Origanum
species were collected from various regions of Greece, or made
available from commercial sources (Table 1).
The collection and/or identification of the plant material were

carried out by Assistant Professor Ilias Anastasopoulos, at the Plant
Biotechnology Laboratory of Larissa’s Technological Educational
Institute. Voucher specimens have been deposited at the Herbarium of the Institute. All non-commercial plants were grown from
seeds, except from samples 0 and 5 which were asexually propagated from cuttings of the same plant, and therefore genetically
identical. Plants were grown in a plastic greenhouse at the Technological Educational Institute, in Larissa, Greece, and received no
treatment apart from watering.
2.2. Extraction methods
After collection, the plant material was air dried in the dark at
room temperature (w25  C) for 10 days. The dried plant parts
underwent hydrodistillation for two hours on a Clevenger apparatus connected to a modified refrigerated essential oil receiver
(European Pharmacopoeia 5.0). Refrigeration was used to reduce
the byproducts of the thermal treatment. The essential oils were
then diluted with 2 mL of ether and filtered through anhydrous
sodium sulfate to remove water traces. The resulting essential oils
were stored at 4  C. The oil content was estimated in mL/100 g (dry
weight of the plant material).

2.3. Chemical analysis
2.3.1. GCeEIMS analysis
GCeEIMS analysis of the extracts was performed on a Shimadzu
GC-17A gas chromatograph coupled with a Shimadzu GCMS-QP
5050 mass-selective detector with the appropriate data system.
The GC was equipped with a Grob-type split-splitless injector. The
fused silica capillary column (Supelco, SBP-5 with 0.25 mm film
thickness, 30 m  0.25 mm i.d.) was directly coupled to the ion
source. Helium was used as a carrier gas with a back pressure of
0.8 Atm. The injector temperature was 250  C and the oven temperature program started at 50  C for 5 min and then increased at a
rate of 5  C/min up to 150  C, retained at this temperature for
10 min and increased again at a rate of 5  C/min up to 280  C, where
it remained for 20 min.

2.3.2. Identification
The scanning range was 30e700 m/z. The quantification of the
components was based on the total number of fragments (total ion
count) of the metabolites, as detected by the mass spectrometer.
The identification of the chemical components was carried out
based on the retention time of each component (Rt) compared with
those of commercially available compounds, by analysis of their
mass spectra, by the use of the NIST21, NIST107 and PMW_TOX2
mass spectra libraries (NIST, 2010) as well as by comparison with
literature data (Adams, 2007). Calculation of retention indices was
performed in accordance to the work of Van den Dool and Kratz
(1963), in comparison to the retention times of standard hydrocarbons (C9eC25). Also, when necessary, co-injection with standard
compounds was carried out.
2.4. Antimicrobial screening
The 6 microbial strains used as test organisms were as follows:
E. coli, S. cerevisiae, Listonella anguillarum (CECT 522), Vibrio
splendidus DMC-1 (kindly provided by Prof. T.H. Brikbeck, University of Glaskow), Vibrio alginolyticus, isolated from seabream
larvae (Sparus aurata) (kindly provided by Dr. P. Katharios, Institute of Aquaculture, Hellenic Center for Marine Research) and
Vibrio sp. isolated from enriched Artemia metanauplii homogenate
by Dr. P. Makridis at the Institute of Aquaculture, Hellenic Center
for Marine Research. L. anguillarum, V. splendidus, V. alginolyticus
and Vibrio sp. were grown on tryptic soy agar dishes. S. cerevisiae
were grown on YPD agar dishes and E. coli were grown on LB agar
dishes.
The agar disc diffusion method was employed for the determination of antimicrobial activity of the essential oil (NCCLS, 1997). For
this purpose, Watmann number 1 paper disks with 6 mm diameter
soaked with 2 mL of the essential oil were laid on top of the agar
culture medium plate previously inoculated (50 mLe108 CFU/mL)
with the different micro-organisms tested in this work. In addition

Table 1
Origanum samples used in the study.
Sample

Date

Species

0
1
2
3
4
5
6
7
8
9

Oct/2007
Aug/2006
Aug/2006
Aug/2006
Aug/2006
Oct/2007
Oct/2007
Aug/2006
Aug/2006
Oct/2007

O.
O.
O.
O.
O.
O.
O.
O.
O.
O.

vulgare subsp.
vulgare subsp.
vulgare subsp.
marjorana
vulgare subsp.
vulgare subsp.
vulgare subsp.
onites
onites
vulgare subsp.

hirtum
hirtum
hirtum
hirtum
hirtum
hirtum

hirtum

Place of collection or purchasing

Geographical coordinates (altitude)

Pilio, Greece
CC Botanicals Ltd (Warwick, UK)
Agrafa, Greece
Pieterpikzonen b.v. (Luinjeberd, NL)
CC Botanicals Ltd (Warwick, UK)
Pilio, Greece
Sisses, Greece
Naxos island, Greece
Naxos island, Greece
Agrafa, Greece

39 260 N 23 20 E (700 m)
e
39 80 N 21 380 E (700 m)
e
e
39 260 N 23 20 E (700 m)
35 220 N 24 280 E (500 m)
37 50 N 25 280 E (700 m)
37 50 N 25 280 E (700 m)
39 80 N 21 380 E (700 m)


M.K. Stefanakis et al. / Food Control 34 (2013) 539e546

541

Table 2
Results of qualitative and quantitative (%v/v) analysis of the Essential Oil Origanum (O. vulgare subsp. hirtum), in 7 different samples.
Season

Summer

Autumn

Components

Molecular
formula

Ret. time

R.I.b

R.I.c

Oil 1

Oil 2

Oil 4

Oil 5

Oil 6

Oil 9

Oil 0

a-Thujene
a-Pinenea

C10H16
C10H16
C10H16
C10H16
C8H16O
C8H16O
C10H16
C10H16
C10H16
C10H16
C10H14
C10H16
C10H18O
C10H14
C10H18O
C10H16
C10H18O
C10H18O
C10H18O
C10H18O
C10H14O
C10H18O

C10H16O
C10H16O
C11H16O
C10H14O
C11H16O
C10H14O
C10H14O
C12H16O2
C10H12O2
C15H24
C15H24
C15H24
C15H24
C15H24
C15H24
C15H24
C15H24
C15H24
C15H24
C15H24
C15H24
C15H24
C15H24
C15H22
C15H24O
C15H24O
C15H26O
C15H24O
C15H26O
C15H26O

C15H26O
C15H26O
C15H18
C15H26O

11.591
11.793
12.259
13.299
13.358
13.466
13.606
13.949
14.083
14.266
14.482
14.582
14.663
15.311
15.537
15.988
16.245
16.737
17.638
17.796
18.297
18.379
18.441
18.644
18.866

18.902
18.956
19.848
20.067
21.178
21.231
21.519
21.615
21.844
22.772
23.014
23.295
23.720
24.301
24.769
24.960
25.003
25.171
25.472
25.674
25.676
27.476
27.617
27.881
28.365
28.954
29.181
29.575
29.791
30.148

30.294

930
937
952
977
980
987
990
1002
1008
1017
1024
1028
1033
1059
1071
1090
1096
1127
1169
1177
1184
1190
1210
1218
1235
1244
1245
1290

1302
1352
1359
1376
1377
1388
1422
1435
1442
1454
1479
1485
1502
1508
1512
1526
1531
1531
1578
1583
1590
1622
1643
1648
1661
1672
1682
1685

930

939
954
979
979
987
990
1002
1011
1017
1024
1029
1031
1059
1070
1088
1096
1128
1169
1177
1182
1188
1210
1218
1235
1243
1244
1290
1299
1352
1359

1375
1376
1388
1419
1434
1441
1454
1479
1485
1500
1505
1512
1526
1531
1534
1578
1583
1590
1623
1643
1648
1662
1675
1682
1685

n.d.e
trd
n.d.
n.d.

0.43
0.28
0.13
n.d.
n.d.
n.d.
1.82
n.d.
n.d.
tr
0.05
n.d.
0.05
tr
0.09
0.67
0.07
0.17
n.d.
0.16
n.d.
n.d.
0.06
9.58
79.36
n.d.
0.08
n.d.
n.d.
n.d.

0.60
n.d.
n.d.
0.09
0.06
n.d.
n.d.
0.05
0.77
0.15
0.38
n.d.
0.23
3.12
tr
0.35
tr
0.28
0.05
n.d.
0.07
0.16

n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
0.07

n.d.
n.d.
n.d.
0.40
0.20
n.d.
tr
0.13
n.d.
0.05
n.d.
tr
0.31
n.d.
0.08
0.08
tr
n.d.
tr
tr
9.11
85.52
n.d.
0.07
tr
0.07
tr
0.89
n.d.
0.07

0.07
0.19
0.05
n.d.
0.05
0.63
0.17
0.52
n.d.
0.16
0.54
n.d.
0.05
tr
0.09
0.08
n.d.
n.d.
0.08

0.17
0.19
n.d.
0.11
0.28
0.10
1.18
0.33
0.12
0.67

3.33
0.28
tr
2.01
0.39
0.17
0.18
tr
0.21
2.09
tr
0.34
n.d.
0.13
2.30
0.29
n.d.
6.30
73.83
0.07
0.39
tr
n.d.
tr
1.09
n.d.
n.d.
0.14
0.20
0.05

n.d.
0.09
0.71
0.16
0.49
n.d.
0.09
0.92
n.d.
0.13
0.05
n.d.
0.08
n.d.
n.d.
0.16

0.08
0.19
0.05
n.d.
0.30
0.16
0.55
tr
tr
0.49
6.47
0.36
0.07

0.54
1.29
0.18
0.31
n.d.
0.80
1.76
0.14
0.50
0.15
n.d.
n.d.
0.05
0.09
67.43
9.10
n.d.
n.d.
n.d.
n.d.
n.d.
1.14
0.06
0.10
0.26
n.d.
n.d.
n.d.
n.d.
3.14

tr
0.20
n.d.
0.47
2.75
n.d.
0.52
n.d.
n.d.
n.d.
n.d.
n.d.
0.21

0.39
0.23
0.06
0.40
0.21
0.10
2.33
0.20
0.10
1.33
6.79
0.49
n.d.
3.46
0.36
0.26

0.25
n.d.
0.23
1.13
0.11
0.37
0.15
n.d.
n.d.
n.d.
0.05
56.26
8.87
n.d.
0.05
n.d.
n.d.
n.d.
6.83
n.d.
0.18
1.56
n.d.
n.d.
0.11
n.d.
1.43
0.11
n.d.
n.d.

0.34
3.41
n.d.
0.60
n.d.
n.d.
n.d.
1.12
n.d.
0.11

0.67
0.44
0.15
0.31
0.37
0.10
2.41
0.15
0.16
1.56
8.00
0.57
n.d.
1.63
0.62
0.46
0.30
0.21
0.47

1.60
0.19
0.79
n.d.
0.17
n.d.
n.d.
n.d.
55.30
10.45
n.d.
0.05
n.d.
n.d.
n.d.
3.98
0.12
n.d.
0.47
n.d.
n.d.
tr
n.d.
4.24
n.d.
n.d.
0.14
0.35
2.84
n.d.

0.31
n.d.
n.d.
n.d.
n.d.
n.d.
0.30

0.39
0.11
0.05
n.d.
0.19
0.10
0.53
n.d.
n.d.
n.d.
4.31
0.34
n.d.
2.41
1.70
0.08
0.27
1.17
0.34
0.63
n.d.
0.25

0.05
n.d.
n.d.
tr
n.d.
72.43
7.19
n.d.
n.d.
n.d.
n.d.
n.d.
1.45
tr
0.11
0.36
tr
n.d.
tr
n.d.
2.60
tr
n.d.
n.d.
0.30
1.62
0.33
0.33
n.d.
n.d.

n.d.
n.d.
n.d.
0.12

Total identified (%)

99.36

99.73

99.82

99.91

99.98

99.88

99.76

Monoterpene hydrocarbons
Oxygenated monoterpenes
Sesquiterpene hydrocarbons
Oxygenated sesquiterpenes
Essential oil content
(mL/100 g dry weight)

2.76
90.24

2.17
4.19
3.25 Ỉ 0.3

0.67
95.17
2.71
1.18
3.56 Ỉ 0.3

8.56
85.95
2.93
2.38
4.08 Ỉ 0.1

9.91
80.02
5.46
4.52
2.36 Ỉ 0.2

16.96
67.22
10.22
5.58
3.09 Ỉ 0.3

16.51
69.23

8.95
5.19
1.69 Ỉ 0.1

9.48
82.06
4.52
3.70
1.40 Ỉ 0.2

a

Camphene
b-Pinenea
1-Octen-3-ol
3-Octanone
Myrcenea
a-Phellandrene
d-3-Carene
a-Terpinenea
p-Cymenea
Limonenea
1,8-Cineolea
g-Terpinenea
cis-Sabinene hydrate
Terpinolene
Linaloola
trans-p-Menth-2-en-ol
Borneola
Terpinen-4-ol

p-Cymen-8-ol
a-Terpineola
cis-Dihydro carvone
trans-Dihydro carvone
Thymol methyl ether
Carvone
Carvacrol methyl ether
Thymola
Carvacrola
Thymyl acetate
Eugenol
a-Ylangene
a-Copaene
b-Bourbonene
b-Caryophyllene
trans-a-Bergamotene
Aromandendrene
a-Humulene
g-Muurolene
Germacrene-D
Leden
a-Muurolene
b-Bisabolene
g-Cadinene
d-Cadinene
cis-Calamenene
Spathulenol
Caryophyllene oxide
Globulol
Humulene epoxide

Cubenol
s-Cadinol
a-Cadinol
Eudesm-7(11)-en-4-ol
Cadalene
a-Bisabolol

a
b
c
d
e

Identification by comparison of retention times and co-injection with authentic compound.
R.I. (Retention Indices) from experimental using a SBP-5 column using a homologous series of n-alkanes (C9eC25).
R.I. (Retention Indices) from literature data and Adams (2007) for a DB-5 column.
tr: Traces of substances (<0.05%).
n.d.: Not detected.


542

M.K. Stefanakis et al. / Food Control 34 (2013) 539e546

to the tested essential oils, the same procedure was repeated with
thymol, carvacrol (98%, Sigma Aldrich), g-terpinene (Fluka, GC,
98.5%), dimethyl-sulfoxide (minimum 99.5%, GC, Sigma), and O/129
(2,4-diamino-6,7-diisopropylpteridine). Paper discs with no compound added were used as negative control. O/129, is a compound
which inhibits Vibrio bacteria in general, and was used as a positive
control. Ampicillin and Miconazole have been used as positive

controls for E. coli and S. cerevisiae, respectively. Four dishes were
used for each treatment. Petri dishes of L. anguillarum, V. splendidus,
V. alginolyticus and Vibrio sp. were incubated for one week in the

dark at room temperature (20e22  C). E. coli and S. cerevisiae cultures were incubated for 24 h at 35  C. The diameters of the inhibition zones were measured in millimeters.
2.5. Statistical analysis
Statistical calculations were carried out with the STATGRAPHICS
Plus 5.0. Results are expressed as the mean Ỉ S.E.M. Student’s t-test
was used for statistical analyses; P values > 0.05 were considered to
be significant. The Principal Coordinate Analysis (PCA) GenAlEx was

Table 3
Results of qualitative and quantitative (%v/v) analysis of the Essential Oil Origanum (O. onites), in 2 different samples.
Components

Molecular formula

Ret. time

R.I.b

R.I.c

Oil 7
Summer

Oil 8
Summer

a-Thujene

a-Pinenea

C10H16
C10H16
C10H16
C10H16
C10H16
C10H16
C10H16
C10H16
C10H14
C10H16
C10H18O
C10H14
C10H18O
C10H16
C10H18O
C10H18O
C10H18O
C10H14O
C10H18O
C10H16O
C10H16O
C11H16O
C12H22O2
C10H14O
C10H14O
C15H24
C15H24
C15H24

C15H24
C15H24
C15H24
C15H24
C15H24
C15H24
C15H24
C15H24
C15H24
C15H24O
C15H24O
C15H24O
C15H26O
C15H26O
C15H26O
C15H26O
C15H26O

11.591
11.793
12.259
13.299
13.606
13.949
14.083
14.266
14.482
14.582
14.663
15.311

15.537
15.988
16.245
17.638
17.796
18.297
18.379
18.441
18.644
18.956
19.375
19.848
20.067
21.615
21.844
22.772
23.014
23.295
23.720
24.301
24.769
24.960
25.171
25.472
25.674
27.476
27.617
28.365
28.954
28.965

29.181
29.526
30.294

930
937
952
981
990
1002
1008
1017
1026
1030
1033
1059
1071
1090
1098
1172
1177
1184
1190
1210
1218
1245
1272
1290
1302
1377

1388
1422
1435
1442
1454
1478
1485
1502
1512
1526
1531
1578
1583
1622
1643
1644
1648
1651
1685

930
939
954
979
990
1002
1011
1017
1024
1029

1031
1059
1070
1088
1098
1169
1177
1182
1188
1210
1218
1244
1275
1290
1299
1376
1388
1419
1434
1441
1454
1479
1485
1500
1512
1526
1531
1578
1583
1623

1643
1646
1648
1650
1685

0.11
0.18
0.07
0.07
0.59
tr
tr
0.79
5.63
0.55
0.14
0.89
0.25
0.06
0.19
0.38
1.50
n.d.
0.31
0.09
n.d.
n.d.
n.d.
34.62

46.65
tr
tr
2.35
n.d.
0.07
0.45
0.07
n.d.
tr
0.45
0.05
0.15
0.30
2.57
0.24
n.d.
n.d.
n.d.
n.d.
n.d.

n.d.e
trd
tr
n.d.
0.20
n.d.
n.d.
0.27

1.99
0.19
n.d.
0.52
tr
0.11
0.14
0.18
1.77
tr
0.19
0.05
tr
0.10
0.11
20.21
66.18
n.d.
n.d.
1.42
tr
0.17
0.21
0.07
tr
0.18
1.50
0.08
0.30
0.88

1.53
0.18
0.22
0.08
0.34
0.13
0.34

Total identified (%)

99.77

99.84

Monoterpene hydrocarbons
Oxygenated monoterpenes
Sesquiterpene hydrocarbons
Oxygenated sesquiterpenes
Essential oil content (mL/100 g dry weight)

8.98
84.13
3.55
3.11
0.72 Ỉ 0.1

3.28
88.93
3.93
3.7

1.97 Ỉ 0.1

Camphenea
b-Pinenea
Myrcenea
a-Phellandrene
d-3-Carene
a-Terpinenea
p-Cymenea
Limonenea
1,8-Cineolea
g-Terpinenea
cis-Sabinene hydrate
Terpinolene
Linaloola
Borneola
Terpinen-4-ol
p-Cymen-8-ol
a-Terpineola
cis-Dihydro carvone
trans-Dihydro carvone
Carvacrol methyl ether
Dihydro-Linalool acetate
Thymola
Carvacrola
a-Copaene
b-Bourbonene
b-Caryophyllene
trans-a-Bergamotene
Aromadendrene

a-Humulene
g-Muurolene
Germacrene-D
Leden
b-Bisabolene
g-Cadinene
d-Cadinene
Spathulenol
Caryophyllene oxide
Humulene epoxide
Cubenol
g-Eudesmol
s-Cadinol
b-Eudesmol
a-Bisabolol

a
b
c
d
e

Identification by comparison of retention times and co-injection with authentic compound.
R.I. (Retention Indices) from experimental using a SBP-5 column using a homologous series of n-alkanes (C9eC25).
R.I. (Retention Indices) from literature data and Adams (2007) for a DB-5 column.
tr: Traces of substances (<0.05%).
n.d.: Not detected.


M.K. Stefanakis et al. / Food Control 34 (2013) 539e546

543

Table 4
Results of qualitative and quantitative (%v/v) analysis of the Essential Oil of O. marjorana.
Components

Molecular formula

Ret. time

R.I.b

R.I.c

Oil 3
Summer

a-Thujene
a-Pinenea

C10H16
C10H16
C10H16
C10H16
C8H16O
C8H16O
C10H16
C10H16
C10H16

C10H14
C10H16
C10H18O
C10H14
C10H18O
C10H16
C10H18O
C10H18O
C10H18O
C10H18O
C10H16O
C10H16O
C10H14O
C11H16O
C10H14O
C10H14O
C15H24
C15H24
C15H24
C15H24
C15H24
C15H24
C15H24
C15H24
C15H24
C15H24
C15H24
C15H24O
C15H24O
C15H24O

C15H26O

11.591
11.793
12.259
13.299
13.358
13.466
13.606
13.949
14.266
14.482
14.582
14.663
15.311
15.537
15.988
16.245
17.638
17.796
18.379
18.441
18.644
18.902
18.956
19.848
20.067
21.519
21.615
21.844

22.772
23.720
24.301
24.769
25.003
25.171
25.472
25.676
27.476
27.617
28.365
29.575

930
937
952
977
980
987
990
1002
1017
1026
1030
1033
1059
1071
1090
1101
1172

1177
1190
1210
1218
1244
1245
1290
1302
1375
1377
1388
1422
1454
1478
1485
1508
1512
1526
1531
1578
1583
1622
1661

930
939
954
979
979
987

990
1002
1017
1024
1029
1031
1059
1070
1088
1101
1169
1177
1188
1210
1218
1243
1244
1290
1299
1375
1376
1388
1419
1454
1479
1485
1505
1512
1526
1531

1578
1583
1623
1665

0.11
0.18
0.06
trd
0.08
0.08
1.12
0.15
0.93
5.03
0.26
0.05
3.75
0.21
0.22
0.25
tr
2.25
0.15
0.40
0.25
0.40
0.09
11.03
63.24

0.11
0.05
0.20
3.56
0.49
0.48
0.12
0.24
0.85
0.43
0.99
0.25
1.24
0.19
0.08

Camphenea
b-Pinenea
1-Octen-3-ol
3-Octanone
Myrcenea
a-Phellandrene
a-Terpinenea
p-Cymenea
Limonenea
1,8-Cineolea
g-Terpinenea
cis-Sabinene hydrate
Terpinolene
Linaloola

Borneola
Terpinen-4-ol
a-Terpineola
cis-Dihydro carvone
trans-Dihydro carvone
Carvone
Carvacrol methyl ether
Thymola
Carvacrola
a-Ylangene
a-Copaene
b-Bourbonene
b-Caryophyllene
a-Humulene
g-Muurolene
Germacrene-D
a-Muurolene
b-Bisabolene
g-Cadinene
d-Cadinene
Spathulenol
Caryophyllene oxide
Humulene epoxide
a-Cadinol
Total identified (%)

99.64

Monoterpene hydrocarbons
Oxygenated monoterpenes

Sesquiterpene hydrocarbons
Oxygenated sesquiterpenes
Essential oil content (mL/100 g dry weight)

11.85
78.51
7.52
1.76
2.79 Ỉ 0.45

a
b
c
d

Identification by comparison of retention times and co-injection with authentic compound.
R.I. (Retention Indices) from experimental using a SBP-5 column using a homologous series of n-alkanes (C9eC25).
R.I. (Retention Indices) from literature data and Adams (2007) for a DB-5 column.
tr: Traces of substances (<0.05%).

applied in order to show initially the variability of the composition
of the essential oils in the 10 specimens deriving from the two
species of Origanum and the one from O. marjorana.
3. Results and discussion
3.1. Quantitative and qualitative analysis
Seven samples of O. vulgare subsp. hirtum were analyzed as
mentioned before. Their quantitative and qualitative analysis is
presented on Table 2. The analysis of the two samples of the species
O. onites is depicted on Table 3 and the components detected in the
essential oil of the species O. marjorana L. are shown on Table 4. The

essential oils of oregano and marjoram, in agreement with literature data (D’Antuono, 2000) contained, as major constituents,
carvacrol and/or thymol coupled with p-cymene and g-terpinene.
The content of the rest of the oil components was significantly

Fig. 1. PCA plot according to the chemical composition of the essential oil components
from data collected from 10 species of genus Origanum accessions via covariance
matrix with data standardization in respect to their correlation to the first principal
components (20.21 and 19.32%).


544

M.K. Stefanakis et al. / Food Control 34 (2013) 539e546

Table 5
Radius of inhibition area in mm (Average Ỉ Standard deviation).
Sample

L. anguillarum

V. splendidus

Oil 0
Oil 1
Oil 2
Oil 3
Oil 4
Oil 5
Oil 6
Oil 7

Oil 8
Oil 9
Thymol
Carvacrol
g-Terpinene
O/129

14.1 Ỉ
11.5 Ỉ
13.9 Ỉ
11.5 Ỉ
10.7 Ỉ
10.7 Ỉ
9.1 Ỉ
9.2 Ỉ
13.8 Æ
13.1 Æ
12.5 Æ
15.2 Æ
8.3 Æ
8.1 Æ

13.8
7.3
10.1
9.2
10.8
8.8
10.8
12.6

14.3
14.0
12.8
15.6
9.6
8.0

a

0.1
0.7
0.3
0.5
0.3
0.3
0.7
0.4
0.2
0.7
0.5
0.2
0.7
0.1

Æ 0.20
Æ 0.70
Æ 0.50
Æ 0.20
Æ 0.40
Ỉ 0.60

Ỉ 0.01
Ỉ 0.60
Ỉ 0.10
Ỉ 0.01
Ỉ 0.40
Ỉ 0.01
Ỉ 0.60
Ỉ 0.20

V. alginolyticus
9.8
8.8
11.5
7.8
7.9
8.6
8.6
8.6
13.6
9.2
12.0
14.7
5.2
6.5

Ỉ
Ỉ
Ỉ
Ỉ
Ỉ

Ỉ
Ỉ
Ỉ
Ỉ
Ỉ
Ỉ
Ỉ
Ỉ
Ỉ

0.20
0.01
0.50
0.20
0.70
0.40
0.60
0.20
0.60
0.40
0.01
0.30
0.10
0.50

Vibrio sp.
14.5
12.6
9.9
8.9

10.1
10.2
9.8
7.6
11.1
13.5
12.6
15.3
5.4
7.4

Ỉ
Ỉ
Ỉ
Ỉ
Ỉ
Ỉ
Ỉ
Ỉ
Ỉ
Ỉ
Ỉ
Ỉ
Ỉ
Ỉ

0.5
0.4
0.1
0.7

0.1
0.2
0.4
0.2
0.1
0.5
0.4
0.3
0.1
0.4

E. coli
16.7 Ỉ
11.9 Ỉ
12.8 Ỉ
13.4 Ỉ
13.7 Ỉ
15.7 Ỉ
11.2 Ỉ
9.4 Æ
11.0 Æ
11.3 Æ
n.d.a
13.8 Æ
n.d.
n.d.

S. cerevisiae
2.26
1.83

1.60
1.03
2.14
0.90
1.18
1.05
0.90
2.40
1.80

17.0
11.7
16.0
14.9
13.9
10.7
10.0
18.8
15.1
11.8
n.d.
18.8
n.d.
n.d.

Æ
Æ
Æ
Æ
Æ

Æ
Æ
Æ
Æ
Æ

2.9
2.9
1.3
1.4
2.7
1.0
1.8
1.9
2.7
2.9

Æ 2.8

n.d.: Not detected.

Fig. 2. The effect of essential oil against bacterial strains of the Vibrio sp. group in (A) oil 0 against bacterial strains V. alginolyticus, (B) oil 9 against bacterial strains V. anguillarum, (C)
thymol against bacterial strains V. alginolyticus, (D) oil 8 against bacterial strains of the Vibrio sp., (E) carvacol against bacterial strains V. alginolyticus, (F) g-terpinene against
bacterial strains V. anguillarums, (G) DMSO against bacterial strains of the Vibrio sp., (H) control against bacterial strains V. anguillarum, and (I) O/129 against bacterial strains of the
Vibrio sp.


M.K. Stefanakis et al. / Food Control 34 (2013) 539e546

lower than those of carvacrol and thymol, since the latter compounds are present in a total exceeding 90%, thus contributing to

the distinct differences in the scent of the plants. For a detailed
analysis of the data and their comparison to those available in the
literature, see “Supporting information” section.
3.2. Chemical classification of the species of genus Origanum
The Principal Coordinate Analysis (PCA) GenAlEx is one of the
oldest, widely used, and perhaps one the simplest classification
methods. It is a multivariate analysis which is linked to the numerical analysis. The deriving coordinates show the total percentage of variants of the data which is counted/estimated by the
matrix eigen values.
The figure below (Fig. 1) shows the classification of the specimens defined by the two initial coordinates of PCA, indicating a
39.16% of the total variants with the first coordinate rising to 20.21
and the second one to 19.32%.
According to the analysis results, the specimens are classified in
the first coordinate according to the harvesting period, with
autumn specimens being placed in the left part, and summer
specimens put in the right part of the first coordinate.
Accordingly, regarding the second coordinate (axon), the classification of the specimens is done with the taxonomic classification of the specimen. Specimens of the genus O. onites are placed in
the upper right part of the table whereas the specimen of the
O. marjorana is placed in the lower side of the second coordinate.
The specimens of O. vulgare subsp. hirtum are classified in the
middle and on either side of the coordinates with an inclination to
the second pole, mainly because the chemical composition of the
essential oils includes compounds that exist in the specimens of the
other two taxa. Taking into consideration that the amount of the
total variants of the two columns is the same, we concluded that
the chemical composition of the specimens varies according to the
harvesting period and to the taxonomic classification of each
specimen.
3.3. Antibacterial activity
The essential oils tested exhibited strong antibacterial activity
against all microbial strains used in this study as shown on Table 5.

Their activity became apparent in the Petri dishes with the emergence of a circular area of inhibition (Fig. 2). On the contrary, no
antibacterial activity was observed in the controls as well as in the
assays where only DMSO was used. Phenolic compounds such as
thymol and carvacrol showed higher antibacterial activity
compared to the essential oils, whereas the g-terpinene showed a
lower inhibitory effect compared with the bacterial strains. In this
study, carvacrol appeared to be the most active compound, presenting average values of 15.2 Ỉ 0.2 mm in all bacterial strains used.
Carvacrol increases the membrane fluidity and leading to a
decrease in the pH gradient across the membrane, a collapse of the
membrane potential, and the inhibition of ATP synthesis (Ultee,
Bennik, & Moezelaar, 2002; Ultee, Kets, & Smid, 1999). Ranking of
the oils according to the size of the inhibition zone showed that oil
0, oil 8, and oil 9 were the three samples that scored highest. It
should be noticed however, that although the phenolic content of
these oils is very high, the sum of the contents of the isomeric
phenols namely, carvacrol and phenol is not the decisive factor of
activity since some oils (e.g. oil 2) contain these phenols in higher
percentages. Activity therefore, should rest in the combined action
of a larger fraction of components.
The emphasis given to the Vibrio strains study is due to the fact
that in a previous study (Stefanakis, Anastasopoulos, Katerinopoulos,
& Makridis, 2013) the aforementioned EO were used in order to

545

eliminate the bacterial action in aquacultures, especially during the
rearing of marine fish larvae in which high mortality rates are often
observed especially during the first weeks after hatching. One of the
factors likely to affect the survival of the larvae is the bacterial load
associated with added live food, such as rotifers (Brachionus sp. and

Artemia sp.). Opportunistic bacteria include members of group of
Vibrio, which may prove highly virulent for the larvae. A prerequisite
for the successful rearing of larvae is the control of bacterial load in
live food and larval tanks (Conceiỗóo, Yỳfera, Makridis, Morais, &
Dinis, 2010). Several methods have been applied for this purpose
with variable success. The use of antibiotics should be avoided,
however, because of the risk of development of resistant strains as
well as the negative impact on the image of aquaculture industry by
the consumers. An alternative method proposed by our group was
that of the use of natural products from plant essential oils and
extracts, which would be more accepted by the public.
4. Conclusion
In conclusion, this study suggests that essential oils of oregano
and marjoram have a strong and broad spectrum antibacterial effect
against bacterial strains of E. coli, S. cerevisiae, L. anguillarum as well
as those of the Vibrio sp. group. This antibacterial activity depends on
the type of herb as well as its composition; however, the content in
phenolic components is not the only factor contributing to the oils’
activity. All oils exhibited stronger antibacterial behavior than O/129
against the bacterial strains at the concentrations tested. Moreover,
among the oil components, pure carvacrol, when tested, showed the
greatest antibacterial activity against all bacterial strains.
Acknowledgment
This study is implemented through the Operational Program
“Education and Lifelong Learning” and is co-financed by the European Union (European Social Fund) and Greek national funds.
Appendix A. Supporting information
Supporting information related to this article can be found online at />References
Adams, R. P. (2007). Identification of essential oil components by gas chromatography/
mass spectrometry (4th ed.). Carol Stream IL: Allured Publishing Corporation.
Ait-Ouazzou, A., Cherrat, L., Espina, L., Lorán, S., Rota, C., & Pagán, R. (2011). The

antimicrobial activity of hydrophobic essential oil constituents acting alone or
in combined processes of food preservation. Innovative Food Science & Emerging
Technologies, 12, 320e329.
Bagamboula, C. F., Uyttendaele, M., & Debevere, J. (2003). Antimicrobial effect of
spices and herbs on Shigella sonnei and Shigella flexneri. Journal Food Protection,
66, 668e673.
Bakkali, F., Averbeck, S., Averbeck, D., & Idaomar, M. (2008). Biological effects of
essential oils e a review. Food and Chemical Toxicology, 46, 446e475.
Bakkali, F., Averbeck, S., Averbeck, D., Zhiri, A., & Idaomar, M. (2005). Cytotoxicity
and gene induction by some essential oils in the yeast Saccharomyces cerevisiae.
Mutation Research/Genetic Toxicology and Environmental Mutagenesis, 585, 1e13.
Botelho, M. A., Nogueira, N. A., Bastos, G. M., Fonseca, S. G., Lemos, T. L., Matos, F. J.,
et al. (2007). Antimicrobial activity of the essential oil from Lippia sidoides,
carvacrol and thymol against oral pathogens. Brazilian Journal of Medical and
Biological Research, 40, 349e356.
Bruni, R., Medici, A., Andreotti, E., Fanti, C., Muzzoli, M., Dehesa, M., et al. (2003).
Chemical composition and biological activities of Ishpingo essential oil, a
traditional Ecuadorian spice from Ocotea quixos (Lam.) Kosterm. (Lauraceae)
flower calices. Food Chemistry, 85, 415e421.
Burt, S. (2004). Essential oils: their antibacterial properties and potential applications
in foods e a review. International Journal of Food Microbiology, 94, 223e253.
Castilho, P. C., Savluchinske-Feio, S., Weinhold, T. S., & Gouveia, S. C. (2012). Evaluation of the antimicrobial and antioxidant activities of essential oils, extracts
and their main components from oregano from Madeira Island, Portugal. Food
Control, 23, 552e558.


546

M.K. Stefanakis et al. / Food Control 34 (2013) 539e546

Conceiỗóo, L. E. C., Yúfera, M., Makridis, P., Morais, S., & Dinis, M. T. (2010). Live feeds
for early stages of fish rearing. Aquaculture Research, 41, 613e640.
Cox, S., Mann, C., Markham, J., Gustafson, J., Warmington, J., & Wyllie, S. (2001).
Determining the antimicrobial actions of tea tree oil. Molecules, 6, 87e91.
D’Antuono, L. F., Galletti, G. C., & Bocchini, P. (2000). Variability of essential oil
content and composition of Origanum vulgare L. populations from a North
Mediterranean area (Liguria Region, Northern Italy). Annals of Botany, 86, 471e
478.
Deba, F., Xuan, T. D., Yasuda, M., & Tawata, S. (2008). Chemical composition and
antioxidant, antibacterial and antifungal activities of the essential oils from
Bidens pilosa Linn. var. Radiata. Food Control, 19, 346e352.
European Parliament and Council Regulation (EC) No. 1831/2003. opa.
eu/food/food/animalnutrition/feedadditives/legislen.print.htm.
European Pharmacopoeia Commission. (2004). European pharmacopoeia (5th ed.).
Strasbourg Cedex, France: Council of Europe.
Food and Drug Administration. (2005). GRAS notifications.
(retrieved 30.06.10).
_ Elmastas¸, M., & Aboul-Enein, H. Y. (2007). Determination of antioxidant
Gỹlỗin, I.,
and radical scavenging activity of Basil (Ocimum basilicum L. Family Lamiaceae)
assayed by different methodologies. Phytotherapy Research, 21, 354e361.
Kalemba, D., & Kunicka, A. (2003). Antibacterial and antifungal properties of
essential oils. Current Medicinal Chemistry, 10, 813e829.
Kumar, A., Shukla, R., Singh, P., Prasad, C. S., & Dubey, N. K. (2008). Assessment of
Thymus vulgaris L. essential oil as a safe botanical preservative against post
harvest fungal infestation of food commodities. Innovative Food Science &
Emerging Technologies, 9, 575e580.
Muyima, N. Y. O., Zulu, G., Bhengu, T., & Popplewell, D. (2002). The potential
application of some novel essential oils as natural cosmetic preservatives in an
aqueous cream formulation. Flavour and Fragrance Journal, 17, 258e266.

National Committee for Clinical Laboratory Standards. (1997) (6th ed.).Performance
standards for antimicrobial disc susceptibility tests: Approved standard, NCCLS
document no. M2-A6, Vol. 17, no. 1. Villanova, Pennsylvania.
Nerio, L. S., Olivero-Verbel, J., & Stashenko, E. (2010). Repellent activity of essential
oils: a review. Bioresource Technology, 101, 372e378.
National Institute of Standards and Technology (NIST). (2010). t.
gov/chemistry/name-ser.html Accessed 15.05.13.
Pauli, A. (2006). Anticandidal low molecular compounds from higher plants with
special reference to compounds from essential oils. Medicinal Research Reviews,
26, 223e268.
Pavela, R. (2009). Larvicidal effects of some Euro-Asiatic plants against Culex quinquefasciatus Say larvae (Diptera: Culicidae). Parasitology Research, 105, 887e892.

Sacchetti, G., Maietti, S., Muzzoli, M., Scaglianti, M., Manfredini, S., Radice, M., et al.
(2005). Comparative evaluation of 11 essential oils of different origin as functional antioxidants, antiradicals and antimicrobials in foods. Food Chemistry, 91,
621e632.
Saidana, D., Mahjoub, M. A., Boussaada, O., Chriaa, J., Chéraif, I., Daami, M., et al.
(2008). Chemical composition and antimicrobial activity of volatile compounds
of Tamarix boveana (Tamaricaceae). Microbiological Research, 163, 445e455.
Si, W., Gong, J., Tsao, R., Zhou, T., Yu, H., Poppe, C., et al. (2006). Antimicrobial activity of essential oils and structurally related synthetic food additives towards
selected pathogenic and beneficial gut bacteria. Journal of Applied Microbiology,
100, 296e305.
Srivastava, B., Singh, P., Shukla, R., & Dubey, N. K. (2008). A novel combination of the
essential oils of Cinnamomum camphora and Alpinia galanga in checking aflatoxin B-1 production by a toxigenic strain of Aspergillus flavus. World Journal of
Microbiology and Biotechnology, 24, 693e697.
Stefanakis, M. K., Anastasopoulos, E., Katerinopoulos, H. E., & Makridis, P. (2013).
Use of essential oils extracted from three Origanum species for disinfection of
cultured rotifers (Brachionus plicatilis). Aquaculture Research. />10.1111/are.12137 (in press).
Thuille, N., Fille, M., & Nagl, M. (2003). Bactericidal activity of herbal extracts. International Journal of Hygiene and Environmental Health, 206, 217e221.
Ultee, A., Bennik, M. H., & Moezelaar, R. (2002). The phenolic hydroxyl group of
carvacrol is essential for action against the food-borne pathogen Bacillus cereus.

Applied and Environmental Microbiology, 68, 1561e1568.
Ultee, A., Kets, E. P., & Smid, E. J. (1999). Mechanisms of action of carvacrol on the
food-borne pathogen Bacillus cereus. Applied and Environmental Microbiology,
65(10), 4606e4610.
Valero, M., & Salmeron, M. C. (2003). Antibacterial activity of 11 essential oils
against Bacillus cereus in tyndallized carrot broth. International Journal of Food
Microbiology, 85, 73e81.
Van de Braak, S., & Leijten, G. (1999). Essential oils and oleoresins: A survey in the
Netherlands and other major markets in the European Union. Rotterdam: CBI,
Centre for the Promotion of Imports from Developing Countries.
Van Den Dool, H., & Dec. Kratz, P. (1963). A generalization of the retention index
system including linear temperature programmed gas-liquid partition chromatography. Journal of Chromatography A, 11, 463e471.
Ye, C.-L., Dai, D.-H., & Hu, W.-L. (2013). Antimicrobial and antioxidant activities of
the essential oil from onion (Allium cepa L.). Food Control, 30, 48e53.
Zygadlo, J. A., & Juliani, H. R. (2003). Recent progress in medicinal plants. In
D. K. Majundar, J. N. Govil, V. K. Singh, M. S. Shailaja, & S. V. Gangal (Eds.), Phytochemistry and Pharmacology II, Vol. VIII (pp. 273e281). Texas: Studium Press LLC.