1. No category

Use of rosemary, oregano, and a commercial blend of essential oils in broiler chickens:
In vitro antimicrobial activities and effects on growth performance
N. Mathlouthi, T. Bouzaienne, I. Oueslati, F. Recoquillay, M. Hamdi, M. Urdaci and R.
Bergaoui
J ANIM SCI 2012, 90:813-823.
doi: 10.2527/jas.2010-3646 originally published online November 7, 2011
The online version of this article, along with updated information and services, is located on
the World Wide Web at:
http://www.journalofanimalscience.org/content/90/3/813
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Use of rosemary, oregano, and a commercial blend of essential oils
in broiler chickens: In vitro antimicrobial activities
and effects on growth performance1
N. Mathlouthi,*†2 T. Bouzaienne,‡ I. Oueslati,† F. Recoquillay,§ M. Hamdi,‡
M. Urdaci,# and R. Bergaoui†
*Department of Animal Production, Ecole Supérieure d’Agriculture du Kef, University of Jendouba,
7119 Le Kef, Tunisia; †Research Laboratory “Economie Agroalimentaire,” Institut National Agronomique
de Tunisie, University of Carthage, 1082 Tunis, Tunisia; ‡Research Laboratory “Ecologie et Technologie
Microbienne,” Institut National des Sciences Appliquées et de Technologie, University of Carthage, 1080 Tunis,
Tunisia; §Department of Research and Development, Phytosynthèse, 63203 Riom Cedex, France;
and #UPR-000263 JE 1, Ecole Nationale des Ingénieurs en Techniques Agricoles de Bordeaux,
University of Bordeaux, 33175 Gradignan, France
ABSTRACT: The present study was conducted to
characterize the in vitro antimicrobial activities of 3
essential oils [oregano, rosemary, and a commercial
blend of essential oils (BEO)] against pathogenic and
nonpathogenic bacteria and to evaluate their effects on
broiler chicken performances. The chemical composition of the essential oils was determined using the gas
chromatography interfaced with a mass spectroscopy.
The disc diffusion method, the minimum inhibitory
concentration (MIC), and the minimum bactericidal
concentration (MBC) were applied for the determination of antimicrobial activities of essential oils. In vivo
study, a total of seven hundred fifty 1-d-old male broiler
chickens were assigned to 6 dietary treatment groups:
basal diet (control; CON), CON + 44 mg of avilamycin/kg (A), CON + 100 mg of rosemary essential oil/
kg (ROS), CON + 100 mg of oregano essential oil/kg
(OR), CON + 50 mg of rosemary and 50 mg of oregano
essential oils/kg (RO), and CON + 1,000 mg of BEO/
kg (essential oil mixture, EOM). The essential oils isolated from rosemary and oregano were characterized
by their greater content of 1,8-cineole (49.99%) and
carvacrol (69.55%), respectively. The BEO was mainly
represented by the aldehyde (cinnamaldehyde) and the
monoterpene (1,8-cineole) chemical groups. The results
of the disc diffusion method indicated that the rosemary essential oil had antibacterial activity (P ≤ 0.05)
against only 3 pathogenic bacteria, Escherichia coli (8
mm), Salmonella indiana (11 mm), and Listeria innocua
(9 mm). The essential oil of oregano had antimicrobial
activities (P ≤ 0.05) on the same bacteria as rosemary
but also on Staphylococcus aureus (22 mm) and Bacillus
subtilis (12 mm). Oregano essential oil had greater (P
≤ 0.05) antimicrobial activities against pathogenic bacteria than rosemary essential oil but they had no synergism between them. The BEO showed an increased
antimicrobial activity (P ≤ 0.05) against all studied
bacteria (pathogenic and nonpathogenic bacteria) except for Lactobacillus rhamnosus. The supplementation
of the basal diet with avilamycin or essential oils improved (P ≤ 0.05) broiler chicken BW, BW gain, and
G:F compared with the CON diet. There were no differences in growth performances among birds fed A,
ROS, OR, RO, or EOM diets. In general, essential oils
contained in rosemary, oregano, and BEO can substitute for growth promoter antibiotics. Although the 3
essential oils had different antimicrobial activities, they
exhibited the same efficiency in broiler chickens.
Key words: antimicrobial activity, broiler chicken, essential oil, growth performance, oregano, rosemary
©2012 American Society of Animal Science. All rights reserved.
J. Anim. Sci. 2012. 90:813–823
http://dx.doi.org/10.2527/jas.2010-3646
1
The authors thank the Ministry of High Education, Research
and Technology of Tunisia (Tunisia and Egypt Joint Project; Tunis, Tunisia) and Phytosynthèse (Riom, France) for their financial
support of this experiment. The authors are grateful to M. Larbier
(INRA, Nouzilly, France) and T. Sassi (Cristal, Sousse, Tunisia) for
their critical reading of the manuscript.
2
Corresponding author: [email protected]
Received October 29, 2010.
Accepted October 27, 2011.
813
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814
Mathlouthi et al.
INTRODUCTION
Components of essential oils have shown biological
properties such as antioxidant and antimicrobial activities (Jang et al., 2007; Windisch et al., 2008). In vitro
antimicrobial activity of essential oils from the Labiatae
family such as oregano and rosemary has been reported
(Sivropoulou et al., 1996; Burt and Reinders, 2003).
Helander et al. (1998) and Faleiro et al. (1997) showed
antimicrobial properties against only pathogenic bacteria. Oussalah et al. (2006) and Celiktas et al. (2007)
reported that oregano and rosemary essential oils were
active against pathogenic bacteria such as Escherichia
coli and Staphylococcus aureus. Lee and Ahn (1998) observed that the Cinnamomum cassia essential oil exhibited growth-inhibiting activity against nonpathogenic
bacteria. However, to our knowledge, there are few
data, in vitro, on the antimicrobial activities of oregano
and rosemary essential oils against nonpathogenic bacteria.
The effect of rosemary and oregano essential oils on
broiler growth performances has not been well documented (Botsoglou et al., 2002, 2004; Hernández et al.,
2004). Several studies indicated that the use of essential
oils improved broiler feed conversion ratio (Hertrampf,
2001; Windisch et al., 2008). However, most data available in literature are obtained from commercial products containing blends of different essential oils. Thus,
it is difficult to evaluate the efficiency of each essential oil in terms of its botanical origin and active compounds. In addition, we have no information about the
relationship between in vitro antimicrobial potential
and efficiency of essential oils in broiler chickens. Perhaps essential oils, which inhibit pathogenic and nonpathogenic bacteria, are more efficient in broiler chickens. The objective of the present study was to evaluate
the antimicrobial activities of 3 essential oils (oregano,
rosemary, and a blend of essential oils) against pathogenic and nonpathogenic bacteria and their effects on
broiler chicken performances.
MATERIALS AND METHODS
The animal experiment was conducted in accordance
with the principles and specific guidelines presented in
decree number 87-848 of October 19, 1987, from the
French Rural Code on experiments conducted with animals.
Resources of Essential Oils
Three essential oils were used in the present study:
rosemary (Rosmarinus officinalis), oregano (Origanum
vulgare), and a blend of essential oils. Rosemary and
oregano plants were collected (Hadjeb Elayoun, Tunisia), dried, and subjected to water distillation according to the method of Zaouali et al. (2010). The 3 essential oils preparations were stored at 4°C in dark glass
bottles until assays. The essential oils of rosemary and
oregano were provided by a commercial company (Carthago, Sousse, Tunisia), and the blend of essential oils
(BEO) was provided by another commercial company
(Phytosynthèse, Riom, France).
Composition of the Essential Oils
The composition of the essential oils were determined
using gas chromatography (GC; Agilent 6890N, Agilent Technologies, Paris, France) interfaced with mass
spectroscopy (MS; Agilent 5973N, Agilent Technologies). The capillary column used was the HP5-MS 5%
phenyl methyl siloxan (length: 30 m; internal diameter:
0.25 mm; film thickness: 0.25 µm) and an automatic
passer (Agilent 7683B; Agilent Technologies). Helium
was the carrier gas at a flow rate of 1 mL/min. The
column temperature was initially adjusted at 5°C (during 1 min) then increased progressively at a rate of
2°C/min to reach 300°C within 130 min. The samples
were diluted in ethanol (1/10) then 1 µL was injected
into GC-MS (Bampidis et al., 2005). The components
were identified by comparing their relative retention
times and mass spectra with the standard data (NIST
05, Mass Spectra Library, National Institute of Standards and Technology, Gaithersburg, MD). The GCMS analyses were conducted at Institut National de
la Recherche et d’Analyses Physico-chimiques (SidiThabet, Tunisia).
In Vitro Study
Microbial Strains. Essential oils were individually tested against a panel of microorganisms including
lactic acid bacteria and undesirable bacteria. Microorganisms were provided from the culture collection
of the Laboratory of Microbial Technology and Ecology at Institut National des Sciences Appliquées et de
Technologie (Tunis, Tunisia) and UPR- 000263 JE 1
at Ecole Nationale des Ingénieurs en Techniques Agricoles (Bordeaux, France). The lactic acid bacteria were
Lactobacillus plantarum (T25), Lactobacillus rhamnosus (TB1), Lactobacillus reuteri (J1), Enterococcus
faecium (T10), Enterococcus fecalis (T8), and Lactobacillus salivarius (Ecole Nationale des Ingénieurs en
Techniques Agricoles). The undesirable bacteria were
E. coli 011 (CIP), Salmonella indiana (CIP), Listeria
innocua 8011T (CIP), Bacillus subtilis 168 (CIP), and
S. aureus 20256 (CIP). Trypticase soy broth (TSB)
and de Man-Rogosa-Sharpe (MRS) broth (Pronadisa,
Madrid, Spain) were used, respectively, for growing and
diluting undesirable and lactic acid bacterial cultures.
Undesirable strains were cultured overnight at 37°C in
TSB (Pronadisa), and lactic acid bacteria were cultured
overnight at 37°C in MRS (Pronadisa).
Disk Diffusion Method. Antibacterial activities
of the 2 essential oils and the BEO were assessed using the paper disk agar diffusion method according to
Sacchetti et al. (2005) and Rasooli et al. (2006). Each
tested microorganism was set up 16 h before the as-
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815
In vitro and in vivo essential oil effects
says to reach the log phase of growth (optic density at
a wavelength of 600 nm = 0.4 to 0.5). Molten agar (5
mL, 40 to 45°C) containing 0.1 mL of microorganism
suspension (107 cfu/mL) was spread over the surface
of agar plates containing appropriate medium of each
microorganism and left to solidify. Absorbent disks
(Whatman disk No. 3 of 6-mm diameter) were placed
in the inoculated Petri dishes than impregnated with
10 µL of different essential oils. The blend of rosemary
and oregano was used to study the effect of the one-half
of the amount of each compound (vol/vol) from each
essential oil on growth bacteria. Before incubation, all
the Petri dishes were kept in a refrigerator (4°C) for 2
h to stop the bacteria from multiplication. Then they
were incubated at 37°C for 24 h and the diameters of
the inhibition zones, including the 6-mm disk, were
measured (mm). Ethanol and chloramphenicol (Biomérieux, Craponne, France) were used as negative and
positive control standards, respectively, to determine
the sensitivity of each bacterial species tested. All the
tests were performed in triplicate.
Minimum Inhibitory Concentration
and Minimum Bactericidal
Concentration Determination
The minimum inhibitory concentration (MIC) values were determined for undesirable bacterial strains,
which were sensitive to the essential oils in the disk diffusion assay. The inocula of the bacterial strains were
prepared from 16-h broth cultures. The molten agar (5
mL) containing 100 µL of bacterial suspension (107 cfu/
mL) was poured into sterile Petri plates containing the
appropriate medium of each microorganism and left to
solidify. Serial dilutions (1/2, 1/4, 1/8, and 1/10) of
each essential oil were made with ethanol and spread
(10 µL) over the sterile paper disks (Whatman disk No.
3 of 6 mm diameter). Petri dishes were incubated at
37°C for 24 h. Ethanol served as the negative control.
The MIC was calculated as the least concentration of
essential oil showing complete inhibition of the tested
microorganism.
The minimum bactericidal concentration (MBC)
was determined as the least concentration of essential
oils showing complete absence of bacterial growth. In
this assay, 50 µL from each dilution of essential oil
showing growth inhibition zone in disc diffusion method was added to 5 mL of TSB tubes containing 105 cfu/
mL. The tubes were then incubated at 37°C for 24 h on
an incubator shaker to disperse the oil throughout the
broth in the tubes. From the tubes showing no bacterial
growth, 0.1 mL of the cells was spread on trypticase soy
agar plates in triplicate.
In Vivo Study
The experiment was performed at Centre de Formation Professionnelle Agricole dans le secteur de
l’Aviculture (Sidi-Thabet, Tunisia). Seven hundred and
fifty 1-d-old male broiler chickens (Arbor Acres, Société Tunisienne d’Aviculture, Borj-Cedria, Tunisia) were
weighed, placed in 30 floor pens (i.e., 25 birds per pen;
size: 2.4 × 1.3 m), and randomly assigned to 6 dietary
treatment groups (5 replicates per treatment). Used litter, top-dressed with 7 cm of wood shavings, was utilized as bedding. The room temperature was gradually
decreased from 32°C at d 1 to 24°C at d 22. The light
was continuous during the first 3 d; then the lighting
regimen was 23 h per day. One basal diet based on corn
and soybean meal was formulated (Table 1) according
to the nutritional requirements for chickens (Larbier
and Leclercq, 1992) and calculated using a software
(version 2.0, PORFAL, ITP-INRA, Paris, France). The
basal diet was fed in mash form and contained no antibiotics or other growth factors (control; CON). The
basal diet was supplemented with antibiotic (Avilamycin Maxus, Elanco Animal Health, Madrid, Spain) as
antibiotic growth promoters (A) at the rate of 44 mg/
kg of diet. To have the same amount of plant essential
oils in the diet, the essential oil preparations were added
to the basal diet at 100 mg/kg for rosemary (ROS) and
oregano (OR), 50 mg of rosemary + 50 mg of oregano/
kg (RO), or 1,000 mg/kg of BEO (essential oil mixture,
EOM). The 6 diets were manufactured at a commercial company (Aliments Composés du Nord, Soliman,
Tunisia). The diets were given to broiler chickens from
1 to 42 d of age. Feed and water were supplied ad libitum throughout the entire experiment. Fasted birds
were weighed individually at 1, 21, and 42 d of age to
determine BW. Feed intake was recorded by pen during
the entire experiment.
Statistical Analysis
Data (antimicrobial activities and growth performances) were statistically analyzed for treatment effect
by the ANOVA procedures of Statview software (SAS
Inst. Inc., Cary, NC). The experimental unit was the
floor pen, and the mean differences were determined
using Fisher’s test of LSD. The level of statistical significance was set at P ≤ 0.05.
RESULTS
Chemical Composition
The essential oils isolated from rosemary and oregano were characterized by their increased content of
1,8-cineole (49.99%) and carvacrol (69.55%), respectively (Table 2). The monoterpenes, which represented approximately 75% of the mixture, were the major
chemical class of the rosemary essential oil. They were
dominated by 1,8-cineole, the 5 other main components
in this chemical class being α-pinene, β-pinene, camphene, cymol, and β-myrcene. Ketones were the second
major class of compounds in rosemary essential oil with
camphor (11.88%) being the only compound detected.
In the group of alcohols, borneol and p-menth-1-en-8-ol
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816
Mathlouthi et al.
Table 1. Formulation and calculated composition of
the broiler chicken diet (as-fed basis)
Item
Ingredient, g/kg
Corn
Soybean meal
Calcium carbonate
Dicalcium phosphate
dl-Methionine
NaCl
Mineral and vitamin mixtures1
Nutrient composition2
ME, MJ/kg
CP, g/kg
Lys, g/kg
Met, g/kg
Met + Cys, g/kg
Trp, g/kg
Ca, g/kg
Available P, g/kg
Content
 
628
332
12.7
18.8
1.5
2.5
4.5
 
12.17
205
11.5
4.8
8.4
2.4
10.8
4.2
1
Mineral and vitamin mixtures supplied per kilogram of diet: Cu, 8.7
mg as copper sulfate pentahydrate; I, 1.2 mg as potassium iodate; Se,
0.2 mg as sodium selenite; Zn, 84 mg as zinc oxide; Fe, 44 mg as iron
carbonate; Mn, 106 mg as manganese sulfate; vitamin A, 10,000 IU;
cholecalciferol, 1,500 IU; vitamin E, 15 mg; butylated hydroxytoluene,
125 mg; menadione, 5 mg; thiamine, 0.5 mg; riboflavin, 4 mg; calcium
pantothenate, 8 mg; niacin, 25 mg; pyridoxine, 1 mg; vitamin B12,
0.008 mg; folic acid, 1 mg; biotin, 0.2 mg; and choline chloride, 750 mg.
2
Calculated using PORFAL software version 2.0 (ITP-INRA, Paris,
France).
are the most abundant. Caryophyllene is the main component in the sesquiterpenes group, which represented
more than 4% of the whole essential oil of rosemary. Finally, there are no phenols in the rosemary essential oil.
In the essential oil of oregano, the phenols (73.64%)
represented the main chemical class. They were dominated by carvacrol (69.55%). The monoterpenes,
which represented more than 18% of the whole oregano essential oils, were mainly represented by cymol,
δ-terpinene, limonene, and α-pinene. Caryophyllene and
α-bisabolene were the only 2 components of the sesquiterpenes group, which represented more than 4% of the
whole essential oil of oregano. Alcohols were the minor
chemical group in the oregano essential oil, and they
were only represented by linalool and 1-terpinen-4-ol.
The BEO were mainly represented by the aldehydes
(cinnamaldehyde) and the monoterpenes (1,8-cineole)
chemical groups.
Antimicrobial Activity of Essential Oils
The results of the present study showed that the essential oils had varying degrees of growth inhibition
against the microorganisms tested. The disc diameters
of the inhibition zones, the MIC and the MBC values of the rosemary, oregano, and BEO are shown in
Tables 3, 4, and 5. The results of the disc diffusion
method (Table 3) indicated that the rosemary essential
oil had antibacterial activity against only 3 pathogenic
bacteria, including E. coli, S .indiana, and L. innocua
among the 5 tested bacteria. The E. coli, S. indiana, L.
innocua, S. aureus, and B. subtilis were more sensitive
to the oregano essential oil than the rosemary essential
oil. The combination of the rosemary and oregano essential oils (vol/vol) did not exhibit greater antibacterial activity than the separate use of the rosemary
or oregano essential oils. Thus, there was no synergy
between compounds of rosemary and oregano essential
oils except for B. subtilis. The essential oils of rosemary
and oregano used alone or in combination were not efficient against L. plantarum, L. rhamnosus, L. reuteri, L.
salivarius, E. faecium, and E. fecalis. The BEO showed
an antimicrobial activity against all studied bacteria
Table 2. Composition of essential oils of rosemary and
oregano, % peak area
Compound1
Monoterpene
α-Pinene
Camphene
β-Pinene
β-Myrcene
α-Phellandrene
α-Terpinene
Cymol
1,8-Cineole
δ-Terpinene
Carene
Limonene
Total
Ketone
Camphor
Alcohol
Linalool
Borneol
1-Terpinen-4-ol
p-Menth-1-en-8-ol
Total
Phenol
Thymol
Carvacrol
Total
Sesquiterpene
Copaene
Caryophyllene
α-Humulene
Naphthalene
α-Bisabolene
Total
Other
Bicyclo(3.1.0)hexane
1-Octen-3-ol
Bicyclo(4.1.0)hept-3-ene
Bicyclo(2.2.1)heptan-2-ol
1.4.7-Cycloundecatriene
1.5-Heptadiene
Total
Total
KI2
Rosemary
 
930
941
960
974
1,011
1,018
1,026
1,147
1,192
1,239
1,449
 
 
1,421
 
1,251
1,555
1,562
1,567
 
 
2,080
2,125
 
 
1,585
1,600
1,855
1,904
2,192
 
 
860
968
985
1,572
2,162
2,188
 
 
 
9.95
3.66
6.53
1.08
ND
0.40
1.60
49.99
0.75
0.40
ND
74.36
 
11.88
 
0.83
3.45
0.95
2.23
7.46
 
ND
ND
0
 
0.22
3.89
0.43
0.26
ND
4.80
 
0.33
ND
0.26
0.91
ND
ND
1.50
100
Oregano
 
1.37
ND3
ND
0.62
0.10
ND
10.57
ND
3.05
0.89
1.64
18.24
 
ND
 
1.08
ND
1.15
ND
2.23
 
4.09
69.55
73.64
 
ND
3.97
ND
ND
0.13
4.10
 
0.22
0.11
ND
ND
0.22
0.21
0.76
98.97
1
Identification by gas chromatography coupled to mass spectroscopy
(GC-MS): National Institute of Standards and Technology (Gaithersburg, MD).
2
KI: Kovats index on HP5-MS capillary column (Agilent Technologies, Paris, France). It converts retention times into system-independent constants.
3
ND: compounds are not detected.
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In vitro and in vivo essential oil effects
Table 3. Antibacterial activities by the disc diffusion method of essential oils against selected bacterial strains1
Inhibition zone, mm
Rosemary2
Bacterial strain
Escherichia coli
Salmonella indiana
Listeria innocua
Staphylococcus aureus
Bacillus subtilis
Lactobacillus plantarum
Lactobacillus rhamnosus
Lactobacillus reuteri
Lactobacillus salivarius
Enterococcus faecium
Enterococcus fecalis
d
8 ± 0.6
11 ± 1.0d
9 ± 0.6d
NA
NA
NA
NA
NA
NA
NA
NA
Oregano2
27
23
21
22
12
Rosemary + oregano,
vol/vol
a
± 1.0
± 0.6a
± 0.6a
± 2.0b
± 1.0b
NA
NA
NA
NA
NA
NA
17
21
16
17
16
b
± 1.0
± 0.6b
± 0.6c
± 1.0c
± 0.6a
NA
NA
NA
NA
NA
NA
BEO3
15
21
17
21
11
13
14
14
13
15
CON−4
c
± 0.6
± 1.0b
± 1.0c
± 1.0b
± 0.6b
± 1.0b
NA
± 0.6b
± 0.6a
± 1.0a
± 0.6a
CON+5
6
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
17
17
18
25
16
28
33
32
10
8
8
±
±
±
±
±
±
±
±
±
±
±
P-value
b
1.0
0.6c
0.6b
0.6a
0.6a
0.6a
0.6
1.0a
0.6b
0.6b
0.6b
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
—
<0.01
<0.01
<0.01
<0.01
a–d
Mean values within a row having different superscripts are significantly different by the least significant difference test (P < 0.05).
Values are means ± SD of 3 values per test.
2
Carthago, Sousse, Tunisia.
3
BEO: commercial blend of essential oils (Phytosynthèse, Riom, France).
4
CON−: negative control, ethanol.
5
CON+: positive control, antibiotic (Chloramphenicol, Biomérieux, France).
6
NA: no antimicrobial activity.
1
(pathogenic and lactic acid bacteria), except for L.
rhamnosus.
The MIC value of rosemary essential oil against E.
coli was 4.40 mg/mL (Table 4). The L. innocua and
S. indiana were inhibited only when the essential oil
of rosemary was used at the concentration of 8.8 mg/
mL. It is observed also that the essential oil of oregano
exhibited a decreased MIC value (0.9 mg/mL) against
E. coli, L. innocua, and S. indiana compared with the
rosemary essential oil. The greatest MIC value (2.25
mg/mL) for oregano essential oil was obtained in the
presence of B. subtilis. The combination of the essential
oils of rosemary and oregano exhibited the same MIC
values (1.11 mg/mL) for the 5 pathogenic bacteria but
greater than those obtained for the oregano essential
oil when used alone. Among pathogenic bacteria, S.
indiana and S. aureus were more sensitive (MIC: 1.15
mg/mL) to the BEO compared with E. coli, B. subtilis,
and L. innocua (MIC: 2.3 mg/mL). The 5 pathogenic
bacteria showed increased susceptibility to the oregano
essential oil. Moreover, S. aureus and B. subtilis were
resistant only to the rosemary essential oil.
In many instances, the MBC values (Table 5) of the
essential oils studied were equivalent to the MIC values
(bactericidal effect). The essential oil of rosemary inhibited only the E. coli completely, and the MBC value
detected was equivalent to the MIC value (4.4 mg/mL).
Our results also showed that E. coli, S. indiana, L. innocua, and S. aureus were inhibited by the oregano essential oil at the concentration of 1.12 mg/mL, whereas
B. subtilis was inhibited at the concentration of 2.25
mg/mL. The MBC values obtained in the case of the
combination of essential oils of rosemary and oregano
ranged from 1.11 to 4.45 mg/mL for S. indiana, L. innocua, and S. aureus, and they were greater than those
observed in the presence of the oregano essential oil.
The BEO exhibited a bactericidal effect against the 5
pathogenic bacteria, and the MBC values were greater
Table 4. Antimicrobial activity expressed as the minimum inhibitory concentration
(MIC) of essential oils against selected bacterial strains1
Essential oil (MIC, mg/mL)
Bacterial strain
Escherichia coli
Salmonella indiana
Listeria innocua
Staphylococcus aureus
Bacillus subtilis
Rosemary2
Oregano2
Rosemary + oregano,
vol/vol
BEO3
4.4
8.8
8.8
NA4
NA
0.9
0.9
0.9
0.9
2.25
1.11
1.11
1.11
1.11
1.11
2.3
1.15
2.3
1.15
2.3
1
MIC was considered the least concentration of each essential oil showing a clear zone of inhibition.
Carthago, Sousse, Tunisia.
3
BEO: commercial blend of essential oils (Phytosynthèse, Riom, France).
4
NA: no antimicrobial activity.
2
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Mathlouthi et al.
Table 5. Antimicrobial activity expressed as minimal bactericidal concentration
(MBC) of essential oils against selected bacterial strains1
Essential oil (MBC, mg/mL)
Bacterial strain
Escherichia coli
Salmonella indiana
Listeria innocua
Staphylococcus aureus
Bacillus subtilis
Rosemary2
Oregano2
Rosemary + oregano,
vol/vol
BEO3
4.4
NA4
NA
NA
NA
1.12
1.12
1.12
1.12
2.25
1.11
4.45
2.22
2.22
1.11
2.3
1.15
4.6
1.15
2.3
1
The MBC was considered as the greatest dilution (least concentration) at which no growth occurred on the
plates.
2
Carthago, Sousse, Tunisia.
3
BEO: commercial blend of essential oils (Phytosynthèse, Riom, France).
4
NA: no antimicrobial activity.
than those obtained in the presence of oregano essential
oil. The MBC values ranged from 1.15 to 2.3 mg/mL
for S. indiana, S. aureus, E. coli, and B. subtilis; however, L. innocua needed a concentration of 4.6 mg/mL
of BEO essential oil to be destroyed completely.
Effect of Essential Oils on Broiler
Performance
The effects of essential oils on BW of broiler chickens are presented in Table 6. Mortality was low (<5%)
and independent of treatments. From 1 to 21 d of age,
broiler chickens fed the CON diet grew less (P ≤ 0.05)
than those fed the A, ROS, OR, RO, or EOM diets.
Moreover, during the same trial period (1 to 21 d of
age), there was no synergy between the essential oils of
rosemary and oregano. Nevertheless, the broiler chickens receiving the ROS diet had better BW gain (P ≤
0.05) than the groups fed A or OR diets from 1 to 21
d of age. In addition, from 22 to 42 d of age, broiler
chickens fed the A diet grew faster (P ≤ 0.05) than
those fed the CON diet. As observed during the first
3 wk of the trial, the decreased BW and BW gain obtained in the CON group from 22 to 42 d of age was
completely alleviated (P ≤ 0.05) by rosemary, oregano,
and BEO addition. The last 3 wk of the experiment,
no differences in BW or BW gain were observed when
animals were fed the A, ROS, OR, RO, or EOM diets. Throughout the entire experimental period, broiler
chickens fed the CON diet had the least BW and BW
gain (P ≤ 0.05). The supplementation of the basal diet
with antibiotics or essential oils improved (P ≤ 0.05)
broiler chicken BW and BW gain compared with the
CON diet during the whole trial period. There were no
differences in BW at 42 d of age or BW gain from 1 to
42 d of age when broiler chickens were fed A, ROS, OR,
RO, or EOM diets.
Furthermore, broiler chickens receiving the A diet ate
less feed (P ≤ 0.05) compared with the other groups
fed CON, ROS, OR, RO, or EOM diets during the
entire trial period. The greatest feed intake (P ≤ 0.05)
was recorded in the RO group (3,892 g). The addition
of oregano (3,771 g) and BEO (3,803 g) essential oils
decreased (P ≤ 0.05) the feed intake of broiler chickens
compared with the CON group (3,833 g). However, the
ROS and CON groups had similar feed intake.
During the first 3 wk of the experimental period (1 to
21 d), there were no differences in feed efficiency among
broiler chickens fed the CON diet and the ROS, OR,
RO, or EOM diet. Moreover, the results of the present
study showed that A, ROS, OR, RO, or EOM diets
increased (P ≤ 0.05) feed efficiency compared with the
CON diet from 22 to 42 d of age. In addition, throughout the entire experimental period (1 to 42 d of age),
we observed the same feed efficiency as during the second growing period (22 to 42 d of age). In fact, the addition of antibiotic (group A) or essential oils (groups
ROS, OR, RO and EOM) in the basal diet improved (P
≤ 0.05) the feed efficiency of broiler chickens compared
with the CON group. Besides, there were no differences
in feed efficiency when birds fed A, ROS, OR, RO, or
EOM diets.
DISCUSSION
Chemical Composition
The present study showed that the essential oil isolated from rosemary was characterized by its increased
content of 1,8-cineole (49.99%). This result is close to
work describing different essential oils of rosemary, in
which the major component was 1,8-cineole and constituted 50.7% of the essential oil (Celiktas et al., 2007).
Pintore et al. (2002) also reported that the major constituent of rosemary essential oils, collected from Morocco, Tunisia, Turkey, Greece, Yugoslavia, Italy, and
France, was 1,8-cineole, which accounted for more than
40% of the essential oil. Moreover, Daferera et al. (2000)
showed that the chemical composition of rosemary essential oil was also characterized by the predominant
presence of 1,8-cineole, which accounted for 88.9% of
the total essential oil. The rosemary essential oil studied in this study contained also camphor, α-pinene,
β-pinene, caryophyllene, camphene, and borneol as mi-
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47 ± 5
597 ± 58c
2,066 ± 204b
 
551 ± 58c
1,473 ± 230b
2,025 ± 215b
 
807 ± 15d
3,028 ± 115b
3,833 ± 126b
 
0.701 ± 0.079
0.476 ± 0.032b
0.526 ± 0.018b
Control2
 
47 ± 5
615 ± 56b
2,140 ± 226a
 
566 ± 53b
1,540 ± 228ab
2,093 ± 226a
 
802 ± 11e
2,907 ± 45e
3,709 ± 55e
 
0.689 ± 0.061
0.521 ± 0.017a
0.558 ± 0.012a
Antibiotic3
 
48 ± 4
633 ± 65a
2,167 ± 243a
 
585 ± 65a
1,521 ± 240ab
2,119 ± 243a
 
819 ± 11a
3,008 ± 47bc
3,827 ± 54b
 
0.698 ± 0.080
0.502 ± 0.036a
0.549 ± 0.017a
Rosemary4
 
48 ± 5
619 ± 52ab
2,144 ± 232a
 
569 ± 53b
1,525 ± 236ab
2,096 ± 232a
 
803 ± 10e
2,968 ± 53d
3,771 ± 46d
 
0.681 ± 0.081
0.513 ± 0.032a
0.548 ± 0.012a
Oregano5
1
Mean values within a row having different superscripts are different (P < 0.05).
Values are means ± SD of 5 floor pens per dietary group.
2
Without antibiotic or other growth factors.
3
Included antibiotic (Avilamycin Maxus; Elanco Animal Health, Madrid, Spain) at the rate of 44 mg/kg.
4
Rosemary (Carthago, Sousse, Tunisia) at the rate of 100 mg/kg.
5
Oregano (Carthago, Sousse, Tunisia) at the rate of 100 mg/kg.
6
Rosemary (50 mg/kg) and oregano (50 mg/kg).
7
EOM: control + commercial blend of essential oils (Phytosynthèse, Riom, France) at the rate of 1,000 mg/kg.
a–e
BW
d 0, g
d 21, g
d 42, g
BW gain
d 0 to 21, g
d 21 to 42, g
d 0 to 42, g
Feed intake
d 0 to 21, g
d 21 to 42, g
d 0 to 42, g
G:F
d 0 to 21
d 21 to 42
d 0 to 42
Variable
Table 6. Effect of the essential oils on BW, BW gain, feed intake, and G:F in broiler chickens1
 
48 ± 5
627 ± 58ab
2,174 ± 257a
 
580 ± 58ab
1,565 ± 263a
2,133 ± 265a
 
816 ± 16b
3,075 ± 103a
3,892 ± 110a
 
0.690 ± 0.085
0.503 ± 0.088a
0.545 ± 0.012a
Rosemary + oregano6
 
48 ± 5
627 ± 53ab
2,143 ± 224a
 
579 ± 52ab
1,512 ± 238ab
2,095 ± 223a
 
812 ± 16c
2,995 ± 110c
3,803 ± 122c
 
0.694 ± 0.056
0.503 ± 0.029a
0.549 ± 0.013a
EOM7
 
0.68
<0.01
0.01
 
<0.01
0.04
0.02
 
<0.01
<0.01
<0.01
 
0.48
<0.01
<0.01
P-value
In vitro and in vivo essential oil effects
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819
820
Mathlouthi et al.
nor components, whereas no verbenone was detected.
Celiktas et al. (2007) showed that the essential oils
of rosemary collected from Izmir in Turkey contained
not only camphor (10.2%), α-pinene (11.5%), β-pinene
(1.1%), caryophyllene (0.6%), camphene (2.6%), and
borneol (5.3%), but also verbenone (11.8%).
The oregano essential oil was the second product
studied in the present study; it contained carvacrol
(69.55%) as the major constituent of the total oil. This
carvacrol content is greater than those reported by Oussalah et al. (2006), who studied 3 oregano varieties: the
first (Origanum heracleoticum) from France, rich in carvacrol (54%); the second (Origanum compactum) from
Morocco, rich both in carvacrol (22%) and γ-terpinene
(23%); and the third (Origanum majorana) from Egypt,
rich in terpinene-4-ol (26%) and γ-terpinene (12%).
The reasons for this variability, in the chemical composition of the rosemary and oregano essential oils, can be
due to the different geographical sources, the climate,
the harvesting seasons, the genotype, the drying procedure, the distilled part of the plant (e.g., seeds, leaf,
root, or bark), and the technique for processing (e.g.,
cold extraction, steam distillation, or extraction with
nonaqueous solvents). All of these factors influence the
relative concentration of each constituent in the essential oils (McGimpsey and Douglas, 1994; Venskutonis,
1996). Finally it seems that essential oils of rosemary
and oregano analyzed in this work could be classified
as 1,8-cineole type and carvacrol type, respectively. The
BEO contained mainly aldehydes (cinnamaldehyde)
and monoterpenes (1,8-cineole) as chemical groups.
The presence of these components and others gives antimicrobial agent properties to rosemary, oregano, and
BEO.
Antimicrobial Activity of Essential Oils
Our results showed that the essential oils of rosemary
and oregano had antimicrobial activity against only
the pathogenic bacteria, whereas the BEO was efficient
against all the 11 studied bacteria (pathogenic and lactic acid bacteria). In fact, the essential oil of rosemary
inhibited the growth of 3 pathogenic bacteria (E. coli,
S. indiana, and L. innocua), but it destroyed only E.
coli. Similar findings were obtained by Celiktas et al.
(2007), who reported that E. coli was sensitive to the
essential oil of rosemary. The antimicrobial activities of
the rosemary essential oil can be attributed, in part, to
the presence of increased concentrations of 1,8-cineole
(Knobloch et al., 1989). Nevertheless, Lopes-Lutz et al.
(2008) demonstrated that potent antimicrobial activities of the rosemary essential oils were not only correlated to the presence of 1,8-cineole. Camphor and borneol
were previously reported to have potent antimicrobial
activities in vitro against pathogenic bacteria (Kordali
et al., 2005). Moreover, other minor constituents have
also been reported for their antimicrobial activity such
as α-pinene, β-pinene, limonene, α-terpinene, caryophyllene, and camphene (Sökmen et al., 2004). Recently,
Randrianarivelo et al. (2009) reported that E. coli was
more sensitive to pure linalool than pure 1,8-cineole.
Moreover, Celiktas et al. (2007) observed that rosemary
essential oil containing verbenone (45.2%) exhibited the
greatest antimicrobial activities compared with other
rosemary essential oils characterized by a lack or low
content of verbenone. Therefore, the weak antimicrobial activities of rosemary essential oils against pathogenic bacteria tested in the present study were likely
due to the lack of verbenone. In addition, we observed
in our study that there were no antimicrobial activities of the rosemary essential oil against nonpathogenic
bacteria (4 strains of Lactobacillus and 2 strains of Enterococcus). Our results were not consistent with those
reported by Celiktas et al. (2007), who reported that
essential oils of rosemary, collected from Canakkale in
Turkey, which contained a greater proportion of verbenone (45.2%), had a great antibacterial activity against
E. fecalis. Therefore, the antibacterial activity potential
of rosemary essential oil against pathogenic and nonpathogenic bacteria was related to the rate of verbenone, camphor, and linalool rather than to the increased
content of 1,8-cineole.
In the present study, the oregano essential oil showed
interesting antimicrobial effects against the 5 studied
pathogenic bacteria. These results are similar to those
obtained by Oussalah et al. (2006) who studied 3 species of oregano (Origanum heracleoticum, Origanum
compactum, and Origanum majorana). Their studies
showed that O. heracleoticum and O. compactum had
the greatest antimicrobial activities against 4 pathogenic bacteria (E. coli, Listeria monocytogenes, Salmonella typhimurium, and S. aureus) because they had
a greater concentration of carvacrol. Thus, the antimicrobial activities of the oregano essential oil can be
attributed to the presence of increased concentration
of carvacrol (Dorman and Deans, 2000; Chorianopoulos et al., 2004). Thymol had also antimicrobial effects
on the number of coliforms as reported by Michiels et
al. (2009). In fact, carvacrol and thymol are isomeric
chemicals with a phenolic functional moiety, and they
had comparable antimicrobial properties (Michiels et
al., 2009). Like rosemary, the nonpathogenic bacteria
were not sensitive to the oregano essential oil in the
present study. This is in disagreement with the results
obtained by Michiels et al. (2009), who demonstrated
that carvacrol had antimicrobial effects against lactobacilli. This could be explained by differences in the
sensitivity of lactobacilli bacteria species and strain
tested in our study and that of Michiels et al. (2009).
Second, we used a crude oregano essential oil that contained many components in the present experiment, but
Michiels et al. (2009) studied the antimicrobial effects
of pure carvacrol. It has been reported in the literature
that essential oil samples, studied as complex mixtures,
may exhibit antimicrobial activities which differ from
those of their major component studied alone (Delaquis
et al., 2002). In fact, major or trace compounds contained in an essential oil might increase or decrease its
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In vitro and in vivo essential oil effects
antimicrobial activity because it should be taken into
consideration the possible synergistic and antagonistic
effect of compounds in the oil (Lopes-Lutz et al., 2008).
Third, the methods used to evaluate the antimicrobial
activities of essential oil were different in those studies. In our work, we used the disc diffusion method,
but Michiels et al. (2009) used an in vitro incubation
model, simulating the fermentation in the animal gastrointestinal tract.
However, we observed in the present study that the
antimicrobial activities of oregano essential oils were
greater than those of rosemary essential oil. In addition, there were no synergistic effects between essential
oils of rosemary and oregano, except for B. subtilis.
Ultee et al. (2002) showed that p-cymene probably acts
synergistically with carvacrol, and Savelev et al. (2003)
found a minor synergy in 1,8-cineole/α-pinene and an
antagonistic effect in 1,8-cineole/camphor combinations. However, to our knowledge, there are no data
in the literature about the combination of rosemary
and oregano essential oil compounds. Also, the present
study indicates that the combination of oregano and
rosemary essential oils (vol/vol) exhibited antibacterial activities only against studied pathogenic bacteria.
This strongly indicates selective antimicrobial effects
of rosemary and oregano essential oils used alone or in
combination on pathogenic bacteria vs. nonpathogenic
bacteria. Unfortunately, there is a little information on
the effect of rosemary and oregano essential oils in the
same study on both pathogenic and nonpathogenic bacteria growth to establish clear and consistent results
about their selective properties. On the basis of their
antimicrobial effects on gram-positive vs. gram-negative bacteria, the oregano and rosemary essential oils
can be considered as nonselective because they inhibited E. coli and S. indiana, which are gram-negative,
and L. innocua and S. aureus, which are gram-positive.
This is in agreement with the results obtained by Burt
(2004) and Michiels et al. (2009).
The BEO essential oil evaluated in the present study
exhibited potent antimicrobial activities against all
studied bacteria (i.e., pathogenic or nonpathogenic and
gram-positive or gram-negative bacteria). Therefore, it
can be considered as nonselective for both criteria. This
might be attributed to the presence of aldehydes (cinnamaldehyde) in the BEO, which is in line with the
results of Lee and Ahn (1998), Oussalah et al. (2006),
and Michiels et al. (2009). In the current study, it seems
that the ranking of antimicrobial activities against
studied bacteria was cinnamaldehyde > carvacrol >
1,8-cineole. This is consistent with the results of Michiels et al. (2009), who found the following order: cinnamaldehyde > carvacrol > thymol > eugenol, but they
did not test 1,8-cineole in their study. Nevertheless, the
results of Friedman et al. (2002) showed the following
rank: carvacrol > cinnamaldehyde > thymol > eugenol.
Globally, the antimicrobial mode of action of the
BEO and the 2 essential oils tested in the present study
is considered to arise mainly from their hydrophobic
821
potential to introduce into the bacterial cell membrane,
disintegrate membrane structures, and cause ion leakage (Windisch et al., 2008). Moreover, it is likely that
essential oil components can penetrate into the interior
of the cell and interact with intracellular sites critical
for bacterial activities (Cristani et al., 2007). More precisely, they are able to inhibit glucosyltransferase enzyme activity, which is responsible in bacteria adhesion
to its sites (Tsai et al., 2007). In summary, the essential
oil preparations (rosemary, oregano, and BEO) tested
in the present study had different chemical composition
and different antimicrobial activities. Thus, we expect
that they have different effects on broiler chicken performances.
Effect of Essential Oils on Broiler
Performances
The results of the present study show that the suppression of antibiotic growth promoter markedly depressed growth performance of broiler chickens. Emborg et al. (2001) obtained similar results when broiler
chickens were fed a diet without antibiotic growth promoter. The exact mode of action of the antibiotic
growth promoter is not well established, and different
mechanisms have been elaborated. Most are based on a
reduction in bacterial numbers with a reduction in the
production of growth-depressing microbial metabolites
and in the competition for nutrients with the host (Anderson et al., 1999). Moreover, the selection of healthier
microbial groups could constitute another mechanism
of action (Castillo et al., 2006). The addition of rosemary, oregano, and BEO to a corn-based diet in the
present study improved broiler chicken performance,
but there were no interactions between rosemary and
oregano essential oils as reported by Basmacioglu et
al. (2004). This beneficial effect of essential oils has
previously been reported in numerous studies (Hertrampf, 2001; Alçiçek et al., 2003). The improvements
of broiler chicken growth performance could be partly
explained by the increase in the apparent digestibility
of dietary protein and the prececal digestive capacity
in general, which increase the intestinal availability of
nutrients for absorption and consequently lead animals
to grow faster (Windisch et al., 2008). Another mode
of action of growth-promoting feed essential oils arises
from stabilizing the ecosystem of the gastrointestinal
microbiota (Windisch et al., 2008) by decreasing microbial activity and controlling potential pathogenic
microorganisms in the gastrointestinal tract of animals
(Castillo et al., 2006). In fact, animals receiving feed
supplemented with essential oils had more stabilized
intestinal health and are less exposed to microbial toxins and other undesired microbial metabolites, such as
ammonia and biogenic amines (Jamroz et al., 2003;
Windisch et al., 2008). Moreover, decreasing microbial
activity led to reduced production of VFA, which contributed to the stabilization of the intestinal pH and,
therefore, ensured an optimum activity of digestive en-
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822
Mathlouthi et al.
zymes (Jamroz et al., 2003). In addition, Jamroz et al.
(2006) demonstrated that essential oil feed additives
improved the absorptive capacity of the intestinal mucosa by increasing intestinal villi size and crypt depth.
The depression of intraepithelial lymphocyte number
in the jejunum of animals treated with essential oils
proved that there is a relationship between the immune
system and essential oils (Nofrarías et al., 2006).
All these findings might explain the beneficial effect
of essential oils; however, some studies indicate that
these feed additives are not efficient in broiler chickens (Lee et al., 2003; Botsoglou et al., 2004). In fact,
Basmacioglu et al. (2004) and Botsoglou et al. (2005)
showed that the use of rosemary and oregano essential oils alone or in combination did not improve laying
hen performance or broiler chicken feed conversion. The
large variation in the effects of essential oils on broiler
chicken performance is due to intrinsic and extrinsic
factors such as physiological status of animals, rearing
environment, infections, diet composition, the content
of active substances in essential oil sample, and the different experimental approaches used by the authors to
test the suitability of these substances as growth-promoting feed additives for broiler chickens (Giannenas et
al., 2003; Windisch et al., 2008).
Although the 3 essential oil preparations tested in
the present study had different antimicrobial activities,
they exhibited the same efficiency in broiler chickens.
Thus, there was no relationship between in vitro essential oil antimicrobial potential and their efficiency in
broiler chickens. To our knowledge, this finding has not
previously been demonstrated. One possible hypothesis
to explain this result is the decreased doses used in the
in vivo trial compared with those used in the in vitro
experiment. This may indicate that the antimicrobial
potential of the essential oils should not be the same
in in vivo and in vitro studies. Moreover, the essential
oil antimicrobial activity tests were performed on pure
bacteria and not on a mixture of microorganisms as
found in the gastrointestinal tract of broiler chickens.
In these circumstances, it seems that the effects of essential oils would be related to changes in the ecological structure and metabolic activity of the microbial
community rather than to a reduction in the number
of some bacteria groups (Castillo et al., 2006). It is
likely that rosemary, oregano, and BEO used in smaller
amounts had similar and beneficial effects on the ecosystem of broiler chicken gastrointestinal microbiota. A
second plausible hypothesis is that the beneficial effects
of the essential oils should not only arise from their
antimicrobial properties, but also from their interference with digestive and absorptive processes and the
immune system (Windisch et al., 2008).
In summary, the suppression of antibiotic growth promoters from feed decreased zootechnical performance
of broiler chickens. The addition of rosemary, oregano,
and BEO counteracted these effects. These 3 essential oil preparations had the same efficiency in broiler
chickens despite their different antimicrobial activities.
Thus, the selection of the essential oils as effective feed
additives on the basis of their in vitro antimicrobial
activities is not sufficient and should be completed by
in vivo experiments. More specific studies are required
to clarify how essential oils affect the gastrointestinal
microbiota of broiler chickens for better use under field
conditions.
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