Antimicrobial activity of Pistacia lentiscus and Satureja montana ...

Food Control 22 (2011) 1046e1053
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Antimicrobial activity of Pistacia lentiscus and Satureja montana essential oils
against Listeria monocytogenes CECT 935 using laboratory media: Efficacy and
synergistic potential in minced beef
Djamel Djenane b , Javier Yangüela a , Luis Montañés c , Mouloud Djerbal b,d , Pedro Roncalés a, *
a Dpt. Producción Animal y Ciencia de los Alimentos, Universidad de Zaragoza, C/Miguel Servet, 177-50013 Zaragoza, Spain
b Faculté des Sciences Biologiques et des Sciences Agronomiques, Dpt. Biochimie et Microbiologie, Université Mouloud Mammeri, Bastos, BP 17, 15000 Tizi-Ouzou, Algeria
c Valero Analítica S.L., Laboratorio de Ensayos Físico Químicos y Microbiológicos, Autovía de Logroño Km 246, Polígono Europa 2, 50011 Zaragoza, Spain
d Laboratoire Vétérinaire Régional, Draâ Ben Khedda, Tizi-Ouzou, Algeria
article
info
abstract
Article history:
Received 21 September 2010
Received in revised form
9 December 2010
Accepted 27 December 2010
Keywords:
Essential oils
Pistacia lentiscus
Satureja montana
Growth inhibition
Listeria monocytogenes
Beef
The aim of this study was to optimize the antimicrobial efficacy of plant essential oils (EOs) for control of
Listeria monocytogenes (L. monocytogenes) serovar 4b CECT 935 using laboratory media and minced beef
stored at 5 1 C. Commercial EOs obtained from leave parts of Mediterranean Pistacia lentiscus
(P. lentiscus) and Satureja montana (S. montana) were analyzed by gas chromatographyemass spectrometry
(GCeMS). The main components of EOs obtained were b-myrcene (15.18%) and carvacrol
(29.19%), respectively for P. lentiscus and S. montana. The in vitro antimicrobial activity of both EOs was
evaluated against L. monocytogenes using the agar diffusion technique, the minimum inhibitory
concentrations (MIC) were also determined against the same microorganism using the broth microdilution
method. According to the diameters of inhibition, S. montana EO had more antibacterial effects
than that from P. lentiscus. MICs showed a range of 0.03 and 0.10% (vol/vol) respectively for S. montana
and P. lentiscus. S. montana and P. lentiscus EOs were added respectively in minced beef (twofold MIC
values) at 0.06 and 0.20%, experimentally inoculated with L. monocytogenes at a level of 3 10 5 CFU/g
and stored at 5 1 C during one week. S. montana EO was the more effective (P < 0.05) against target
bacteria. P. lentiscus EO also demonstrated antibacterial effect against the same bacterium. EO combinations
were also investigated in minced beef and P. lentiscus combined with S. montana had synergistic
effects. This work shows that the combined EOs might be more effective against L. monocytogenes when
applied to minced beef at the ratio of 1/1 to 2/2 according to the MIC values. Sensory evaluation revealed
that minced beef treated with EOs was acceptable by panelists at the levels used.
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
Meat products are perishable and require protection from
undesired spoilage and pathogenic bacteria during their preparation,
storage, distribution and sale. Foodborne illness resulting from
consumption of food contaminated with pathogenic bacteria is of
vital concern to public health.
Listeria monocytogenes is widely distributed in nature and can be
frequently found in a large number of food products, as well as in
processing plants. Listeriosis is recognized as an important public
health problem, affecting primarily pregnant women, newborns
* Corresponding author. Tel.: þ34 976 761 582; fax: þ34 976 761 590.
E-mail address: roncales@unizar.es (P. Roncalés).
and adults with weakened immune systems. The majority of
human listeriosis infections are caused by the consumption of
contaminated food. It has been shown that L. monocytogenes is able
to survive and grow to significant numbers on refrigerated meat
products making post-process contamination a significant concern
for ready-to-eat meat produce (Farber & Peterkin, 1991).
Today, different strategies are applied in order to control pathogens
in meats, and particular interest has been focused on the
application of EOs as a safe and effective alternative to chemical
preservatives; their application in controlling pathogens could
reduce the risk of foodborne outbreaks and assure consumers safe
meat products. In recent years the EOs and extracts of many plant
species have become popular, and attempts to characterize their
bioactive principles have gained momentum in many food-processing
applications. The chemical composition and antimicrobial
0956-7135/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodcont.2010.12.015

D. Djenane et al. / Food Control 22 (2011) 1046e1053 1047
properties of EOs extracted from diverse plant species have been
demonstrated using a variety of experimental methods (Djenane
et al., 2011; Özkan, Sagdic, Göktürk, Unal, & Albayrak, 2010).
Pistacia lentiscus (Anacardiaceae), and Satureja montana (Lamiaceae)
are distributed throughout the Mediterranean regions. The
chemical composition of the EOs from leaves of both species of
diverse origins has been already reported ( Cavar, Maksimovic, Solic,
Jerkovic-Mujkic, & Besta, 2008; Gardeli, Papageorgiou, Mallouchos,
Theodosis, & Komaitis, 2008). EOs of both species are used as a food
ingredient in the Mediterranean region as an aromatic and
flavoring agent. In Algeria, the leaves of Pistacia spp. and Satureja
spp. were used to purify water and increase the time of conservation
of dry figs and sun-dried tomatoes; they are also used as
natural preservatives for fish and meat products. The in vitro antimicrobial
activity of P. lentiscus and S. montana EO has also been
tested on bacteria and fungi ( Cavar et al., 2008; Iauk, Ragusa,
Rapisadra, Franco, & Nicolosi, 1996). It has been generally used as
traditional medicine for various diseases such as asthma, peptic
ulcer, diarrheic, anti-inflammatory, antipyretic, antibacterial, and
antiviral medicines and insecticidal activities (Bakkali, Averbeck,
Averbeck, & Idaomar, 2008).
Most previous reports on the anti-listerial effect of EOs used
only an in vitro approach (Oussalah, Caillet, Saucier, & Lacroix,
2007). The review by Holley and Patel (2005) highlighted those
reports in which a food matrix was used for studying the antimicrobial
action of EOs. However, as far as we are aware, none of them
focused on the effects on the sensory properties of food. It appears
then to be necessary to determine the minimum concentration
necessary to inhibit the growth of L. monocytogenes without
affecting the sensory attributes of meat.
The objective of this study was to determine the efficacy of
P. lentiscus and S. montana EOs and their combination in inhibiting
L. monocytogenes growth both in vitro and on a model minced beef
during refrigerated storage, affording a novel insight on their
possible effect on meat sensory properties.
2. Materials and methods
2.1. Essential oils
The pure essential oils used in this study were extracted from
P. lentiscus and S. montana (density: 0.85 and 0.92 at 20 C,
respectively). They were selected based on previously reported
efficacy, and were obtained from Florame Aromathérapie (St Rémy
de Provence, France). The EOs were certified by Ecocert SAS F32600
(France) and that considered 100% pure and natural, obtained from
Mediterranean biological culture. The isolated EOs were preserved
in darkness in a sealed vial at 1 C until its analysis or its use in
bioassays.
2.2. Essential oils analysis
2.2.1. Gas chromatography analysis
Gas chromatography (GC) analyses of EOs obtained from dried
material were performed using a Hewlett Packard 6890 gas chromatograph
equipped with a flame ionization detector (FID) and
a Stabilwax (PEG) column (30 m 0.32 mm i.d., 1 mm film thickness)
(Centre de Recherche en Analyses Physico-Chimiques-CRAPC,
Algiers, Algeria).
The operating conditions were as follows: injector and detector
temperatures, 250 and 280 C, respectively; carrier gas, N 2 at a flow
rate of 1 mL/min; oven temperature program, 3 min isothermal at
50 C, raised at 2 C/min to 220 C, and finally held isothermal for
15 min. The identities of the separated components on the polar
column were determined by comparing their retention indices
relative to aliphatic hydrocarbons injected under the above
temperature program with literature values measured on columns
with identical polarities.
2.2.2. Gas chromatographyemass spectrometry analysis
The gas chromatographyemass spectrometry (GCeMS) analysis
was performed using a HewlettePackard 6890 series GC systems
(Agilent Technologies) coupled to a quadrupole mass spectrometer
(model HP 5973) equipped with an HP5 MS capillary column (5%
phenyl methylsiloxane, 30 m 0.25 mm, 0.25 mm film thickness)
(CRAPC, Algiers, Algeria). For GCeMS detection an electron ionization
system with ionization energy of 70 eV was used over a scan
range of 30e550 atomic mass units. Helium was the carrier gas, at
a flow rate of 0.5 mL/min. Injector and detector MS transfer line
temperatures were set at 250 and 280 C, respectively; the
temperature of the ion source was 230 C. Column temperature was
initially kept at 60 C for 8 min, then gradually increased to 280 C
at 2 C/min, and finally held isothermal for 30 min. The volume of
injections was 0.2 mL of a hexaneeoil solution, injected in the
splitless mode. The identity of the components was assigned by
matching their spectral data with those detailed in the Wiley 7N,
NIST 02, and NIST 98 libraries. The results were also confirmed by
the comparison of their retention indices, relative to C7eC29
n-alkanes assayed under GCeMS in the same conditions as the oils.
Some structures were further confirmed by available authentic
standards analyzed under the same conditions described above.
The percentage composition of the oils was computed by the
normalization method from the GC peak areas, calculated as the
mean value of two injections from each essential oil (EO).
2.3. Antimicrobial screening
2.3.1. Bacterial strain and culture conditions
The L. monocytogenes culture employed was provided by the
Spanish Type Culture Collection (STCC). Strain used was L. monocytogenes
serovar 4b CECT 935. Bacterial strain was cultured overnight
at 37 C in Mueller Hinton agar (MHA, Oxoid, Basingstoke,
UK). One milliliter of stock culture was standardized through two
successive 24 h growth cycles at 37 1 C in 9 mL of BraineHeart
Infusion Broth (BHIB, Oxoid, Basingstoke, UK). After 48 h, 100 mL of
the suspension were then inoculated in fresh BHIB and incubated at
37 1 C for 12 h to obtain a working fresh culture containing about
3 10 5 CFU/mL, determining by measuring transmittance at
600 nm (Spectrophotometer: Spectronic 20 Bausch & Lomb).
Bacterial strain was maintained frozen ( 80 C) in cryovials and
were subcultured every antibacterial test.
2.3.2. Disc-diffusion method
Screening of EOs for antibacterial activity was determined by
the agar diffusion method as previously described (Hazzit,
Baaliouamer, Veríssimo, Faleiro, & Miguel, 2009), which is normally
used as a preliminary check efficient EOs. Petri plates were
prepared by pouring 20 mL of MHA medium and allowed to
solidify. Plates were dried for 30 min in a biological safety cabinet
with vertical laminar flow and 0.1 mL of standardized inoculums
suspension was poured and uniformly extend. The inoculums were
allowed to dry for 5 min. To prepare the stock solution of the
samples, the pure EOs were dissolved in 0.5% (v/v) dimethyl sulfoxide
(DMSO) (Sigma Aldrich Ò -Química, S.A.). Then sterile filter
paper disk (6 mm diameter, Filter LAB ANOIA, Barcelona, Spain) was
impregnated with 05 mL EO, using a capillary micro-pipette
(Finnpipette Ò , Thermo Fischer Scientific Inc.). The plates were left
15 min at room temperature to allow the diffusion of the EO, and
then they were incubated at 37 C for 24 h. At the end of the period,
the diameter of the clear zone around the disc was measured with

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D. Djenane et al. / Food Control 22 (2011) 1046e1053
a caliper (Wiha dialMax Ò ESD-Uhrmessschieber, CH) and expressed
in millimeters (mm: disk diameter included) as its antimicrobial
activity. The sensitivity to the different oils was classified by the
diameter of the inhibition halos as follows: not sensitive (L) for
diameter less than 8 mm; sensitive (D) for diameter 9e14 mm;
very sensitive (DD) for diameter 15e19 mm and extremely
sensitive (þþþ) for diameter larger than 20 mm (Ponce, Fritz, del
Valle, & Roura, 2003). Negative controls were prepared using the
same solvent employed to dissolve the samples. Standard reference
antibiotic, amoxicillin (4 mg/disc; Sigma Aldrich Ò -Química, S.A.),
was used as positive control in order to control the sensitivity of the
tested microorganism. Each assay in this experiment was replicated
three times.
2.3.3. Microdilution assays
The minimal inhibitory concentration (MIC) values were also
studied for the target bacterium which was determined as sensitive
to the EOs in disc-diffusion assay, as described in Section 2.3.2. The
inocula of L. monocytogenes were prepared from 12 h broth cultures
and suspensions were adjusted to 0.5 McFarland standard turbidity
to give a final density of 3 10 5 CFU/mL. P. lentiscus and S. montana
EOs dissolved in 0.5% dimethyl sulfoxide (DMSO) were first diluted
to the highest concentration (32 mL/mL) to be tested, and then serial
twofold dilutions were made in a concentration range from
32 mL/mL to 0.3125 mL/mL in 10 mL sterile test tubes containing MH
broth. MIC values of both EOs against L. monocytogenes were
determined based on a microwell dilution method. The 96-well
plates (Iwaki brand, Asahi Techno Glass, Japan) were prepared by
dispensing into each well 95 mL of MH broth and 5 mL of the inoculum.
A 100 mL aliquot from both EOs extracts initially prepared at
the concentration of 32 mL/mL was added into the first wells. Then,
100 mL from their serial dilutions were transferred into consecutive
wells. The last well containing 195 mL of nutrient broth without
compound and 5 mL of the inoculums on each strip was used as
negative control. The final volume in each well was 200 mL. Levofloxacin
(SigmaeAldrich, Madrid, Spain) at the concentration range
of 32e0.3125 mL/mL was prepared in MH broth and used as standard
antibiotic for positive control. Contents of each well were
mixed on a plate shaker at 300 rpm for 20 s and then incubated at
appropriate temperatures for 24 h. After incubation at 37 C for
18e24 h under agitation the wells were then examined for
evidence of growth and MICs (mL/mL) values were determined as
the lowest EO concentration that inhibited visible growth of the
tested microorganism which was indicated by absence of turbidity.
The negative control was set up with DMSO in amount corresponding
to the highest quantity present in the test solution (0.5%).
The tests were performed in duplicate and repeated twice.
2.4. Antimicrobial activity in minced beef
2.4.1. Preparation of meat
The muscle Semimembranosus (initial pH 5.7e5.8) was excised
from three beef carcasses 48 h post-slaughter from a local supplier
(Boucherie Khatir, Draâ Ben Khedda, Algeria) and transported to the
laboratory under refrigerated conditions within 30 min.
2.4.2. Treatment of minced beef
Prior to meat inoculation with L. monocytogenes and the addition
of EOs and/or their combination, minced beef was also
examined for any contamination by bacteria or the tested pathogen
(results not shown).
In order to evaluate the antimicrobial activity of both EOs in
a meat system, a sufficient amount of fresh minced beef was
prepared following good practices, and was tested using twofold
the MICs value found for both EOs. After the aseptic removal of the
outer surface, seven (07) pieces of approximately 400 g of prepared
meat were minced in a sterile grinder, and portions of 50 2 g were
placed into polystyrene trays. A total of 56 meat samples were
obtained. Two individual duplicate of each sample were performed
in all cases. Minced beef samples (50 2 g) were placed in stomacher
bags and inoculated with strain of L. monocytogenes at a level
of 3 10 5 CFU/g. The inoculated samples were homogenized in
a stomacher 400 (Stomacher Ò 400 Circulator. Seward. Worthing,
UK) for 2 min at room temperature to ensure proper distribution of
the pathogen. Following homogenization, the individual EO and
their combination were added to the inoculated samples.
Addition of S. montana and P. lentiscus EO were done at twofold
MIC values (0.06 and 0.2%, respectively). In addition, the combinations
of EOs were also examined at followed concentrations: 1/1
(0.10%/0.03%); 2/1 (0.20%/0.03%); 1/2 (0.10%/0.06%); 2/2 (0.20%/
0.06%), respectively for P. lentiscus and S. montana.
To attain uniform distribution of the added compounds, treated
meat samples were further homogenized in stomacher, as previously
described. All stomacher bags with samples from all treatments
were wrapped and stored under aerobic conditions at
5 1 C for 8 days, simulating a proper refrigeration storage.
Microbial analyses of samples for populations of L. monocytogenes
were carried out at 2 days intervals up to the 8th day of refrigerated
storage.
2.5. pH measurements
The pH of meat samples was measured using a micro pH-meter
model 2001 (Crison Instruments, Barcelona, Spain) after homogenizing
3 g of sample in 27 mL distilled water for 10 s at 1300 rpm
with an Ultra-Turrax T25 macevator (Janke & Kunkel, Staufen,
Germany). Each value was the mean of three replicates.
2.6. Sensory analysis
For sensory analysis, the pieces (400 g) of meat prepared were
minced, and portions of 50 2 g were obtained. A total of 56 meat
samples were obtained. The samples were mixed with different
concentrations of EOs at the same concentrations used for antibacterial
screening. Eight samples were obtained for each treatment.
The remaining 8 meat samples were served for controls. All
samples were placed into polystyrene trays, and over-wrapped in
polyethylene film (Sidlaw Packaging-Soplaril, Barcelona, Spain).
Two individual duplicates of each sample were performed in all
cases. Samples were evaluated for off odor, discoloration and overall
acceptability attributes by an eight-member trained panel. Panelists
were selected among students and staff of the Department of
Veterinary Microbiology (Laboratoire Vétérinaire Régional de Draâ
Ben Khedda, Algeria) and trained according to the method
described by Djenane, Sánchez-Escalante, Beltrán, and Roncalés
(2001). Though already skilled in this kind of evaluation, panelists
received further training prior to analysis. Three open-discussion
sessions were held to familiarize the individuals with the attributes
and the scale to use. The attribute off odor was evaluated using
a 5-point scale, according to Sørheim, Kropf, Hunt, Karwoski, and
Warren (1996). Odor scores referred to the intensity of off odors
associated to meat spoilage: 1 ¼ none; 2 ¼ slight; 3 ¼ small;
4 ¼ moderate; and 5 ¼ extreme. Discoloration scores referred to
percentage of discolored surface: 1 ¼ none, 2 ¼ 0e10%, 3 ¼ 11e20%,
4 ¼ 21e60%, and 5 ¼ 61e100%. For acceptability attribute, before
evaluation, treated minced beef samples were wrapped in
aluminum foil individually and cooked in a steam-cooker for
20 min. Each sample was served warm in dishes coded with 3-digit
random numbers and presented in individual booths to each
panelist for evaluation. A 5-point hedonic scale was used to score

D. Djenane et al. / Food Control 22 (2011) 1046e1053 1049
acceptability attribute, where 1 ¼ dislike extremely, 2 ¼ dislike,
3 ¼ nor like or dislike, 4 ¼ like; 5 ¼ like extremely. Sensory evaluation
was accomplished at 0 and 2 days intervals up to the end of
refrigerated storage at 5 1 C. Results were expressed as the
predominant score given by panelists.
2.7. Bacterial enumeration
At each sampling time, samples (25 g) of minced beef in the
stomacher bags were aseptically added with 225 mL of 0.1%
peptone water. The content was macerated in the stomacher for
2 min at room temperature. Resulting slurries were serially diluted
(1:10) in 0.1% sterile peptone water. Sample dilutions (0.1 mL) of
minced beef were spread plated on appropriate media in duplicate.
Populations of L. monocytogenes were determined on Agar Listeria
according to Ottaviani and Agosti (ALOA, Biolife, Milan, Italy) at
37 C for 24e48 h.
2.8. Statistical analysis
Variance analyses were used to test the significant difference
among the results from the antibacterial assays and sensory analysis
(SPSS 10.0 software package, 1995). Means and standard errors
(SE) of the samples were calculated. Three replicates were performed
for each treatment. Differences between means were tested
through LSD and values of P < 0.05 were considered significantly
different.
3. Results and discussion
3.1. Chemical composition of the EOs
Steam distillation is the most commonly used method for
producing EOs on a commercial basis. Table 1 summarizes the
results of chemical composition of both EOs. In order to simplify the
analysis of the results, only compounds with more than 0.5%
abundance were selected. Fifty-seven and 44 constituents, which
represented 98.69% and 94.8% of the total EO of P. lentiscus and
S. montana, respectively, were identified. P. lentiscus EO was characterized
by a high percentage of b-myrcene (15.18%) and 1.8-cineole
(15.02%), followed by terpinen-4-ol (6.41%), a-pinene (5.54%) and
b-pinene (5.10%). S. montana EO was characterized by a high
percentage of carvacrol (29.19%), thymol (15.41%) and p-cymene
(11.77%), followed by g-terpinene (6.72%), b-caryophyllene (5.38%)
and farnesol (4.10%). Similar findings have been reported by other
authors (Barra, Coroneo, Dessi, Cabras, & Angioni, 2007; Cavar et al.,
2008). However, the percentage of most of the individual constituents
present in both EOs differed significantly (P < 0.05) from other
findings.
Seasonal variations in the distribution between the different
compounds in the plant EOs could be related to changes
throughout the plant’s vegetative cycle, along with environmental
conditions prevailing in the Mediterranean regions. Environmental
factors such as geography, temperature, day length, nutrients, etc.,
were considered to play a key role in the chemical composition of
EOs (Gardeli et al., 2008; Hussain, Anwar, Nigam, Ashrafd, & Gilanif,
2010). These factors influence the plant’s biosynthetic pathways
and consequently the relative proportion of the main characteristic
compounds. This leads to the existence of different chemotypes
which distinguish EOs of different origins.
3.2. Antimicrobial activity (disc assay)
The in vitro antimicrobial activities of P. lentiscus and S. montana
EOs against L. monocytogenes were qualitatively and quantitatively
Table 1
Main constituents (%) of the EOs of Pistacia and Satureja species, as identified by
GCeMS analysis.
RT (min) a Compound b P. lentiscus S. montana
1 5.177 Tricyclene 0.64 e
2 5.320 a-Thujene e 0.73
3 5.527 a-Pinene 5.54 0.79
4 5.932 Camphene 3.15 0.51
5 6.844 b-Pinene 5.10 0.96
6 7.287 Myrcene e 1.04
7 7.335 b-Myrcene 15.18 e
8 7.763 a-Phellandrene 3.83 e
9 8.232 a-Terpinene 2.78 1.33
10 8.577 p-Cymene 1.64 11.77
11 8.742 Limonene e 0.64
12 8.773 1.8-Cineole 15.02 e
13 9.130 trans-b-Ocimene e 0.92
14 9.544 ciseb-Ocimene 1.68 e
25 9.887 Isoamyl butyrate 0.51 e
16 10.034 g-Terpinene 4.10 6.72
17 10.392 4-Thujanol trans e 1.05
18 11.349 Terpinolene 2.21 e
19 12.020 Linalool e 1.97
20 14.725 Menthone e 0.59
21 15.377 Borneol e 1.75
22 16.063 Terpinen-4-ol 6.41 1.04
23 16.854 a-Terpineol 2.97 e
24 19.982 Thymol methyl ether e 0.95
25 20.877 Farnesol e 4.10
26 22.330 Bornyl acetate 1.88 e
27 23.330 Carvacrol e 29.19
28 23.854 Thymol e 15.41
29 24.740 Myrtenyl acetate 0.73 e
30 28.563 Geranyl acetate e 0.53
31 30.215 Caryophyllene 4.03 5.38
32 32.135 a-Caryophyllene 0.84 e
33 33.802 Germacrene-D 0.87 e
34 34.759 g-Elemene e 0.75
35 35.040 a-Cadinene 0.56 e
36 35.716 b-Bisabolene e 0.87
37 36.428 d-Cadinene 1.80 0.23
38 39.611 Caryophyllene oxyde e 0.98
a Retention time.
b Compounds present in trace amounts ( 0.05).
All tests were performed in duplicate.
a
f: Inhibition zone in diameter around the discs impregnated with essential oils.
The diameter (6 mm) of the disc is included.

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D. Djenane et al. / Food Control 22 (2011) 1046e1053
Table 3
Minimal inhibitory concentrations values from P. lentiscus and S. montana EOs using
broth microdilution method.
MIC (%) a
P. lentiscus S. montana
Listeria monocytogenes serovar 4b CECT 935 0.1 0.01 0.03 0.007
All tests were performed in duplicate.
a
f: Minimal inhibitory concentrations values expressed by % (vol/vol).
respectively. Elgayyar, Draughon, Golden, and Mount (2001)
reported similar results regarding the effect of Rosmarinus officinalis
(rosemary) on L. monocytogenes, showing inhibition zones
ranging from 23 to 30 mm. This antimicrobial effects obtained
with both EOs against L. monocytogenes, was comparable to that
reported by other findings. Vagionas, Graikou, Ngassapa,
Runyoro, and Chinou (2007) recorded close MICs (0.04e0.1%)
when they tested the EO of Satureja spp. against various pathogens.
A large number of studies have reported that the EOs of
Satureja species are among the most potent regarding their
antimicrobial properties (Dikbas, Kotan, Dadasoglu, & Sahin,
2008; Razzaghi-Abyaneh et al., 2008), which has been
confirmed and extended in this study. The EOs of many species
of Satureja are known to possess antibacterial and fungicidal
properties (Özcan & Erkmen, 2001; Vagionas et al., 2007). The
antibacterial activity assigned at both EOs could be attributed to
their high content of compounds with known antimicrobial
activity. Confirming previous reports, it was found that the
strength and spectrum of activity varied between the investigated
Satureja species and Gram type of target bacteria. Grampositive
bacteria were generally more sensitive to the effects of
the EOs than Gram-negative bacteria. This general higher resistance
among Gram-negative bacteria has been ascribed to the
existence of their outer phospholipidic membrane, almost
impermeable to lipophilic compounds (Nikaido & Vaara, 1985).
The absence of this barrier in Gram-positive bacteria allows the
direct contact of the essential oil’s hydrophobic constituents
with the phospholipid bilayer of the cell membrane, where they
bring about their effect, either causing an increase of ion
permeability and leakage of vital intracellular constituents
(Cowan, 1999).
The antimicrobial activities of the EOs are difficult to correlate
to a specific compound due to their complexity and variability.
Nevertheless, some researchers reported that there is
a relationship between the chemical composition of the most
abundant components in the EO and the antimicrobial activity.
For example, 1,8-cineole (abundant in Algerian P. lentiscus EO
tested in this study) is well-known for its antimicrobial potential
(Pattnaik, Subramanyam, Bapaji, & Kole, 1997). Lis-Balchin and
Deans (1997) showed that EOs containing large amounts of
1,8-cineole are better anti-listerial agents than EOs that do not
contain 1,8-cineole. The antimicrobial effects of borneol were
also reported elsewhere (Dorman & Deans, 2000). As a result of
these findings, the higher antimicrobial activities of P. lentiscus
and S. montana EOs could be attributed to their particular chemotypes
characterized by their complexity. Moreover, many
reports mentioned that carvacrol and thymol and their precursors
(p-cymene and g-terpinene), are biologically and functionally
closely associated (Ultee, Bennik, & Moezelaar, 2002). In that
context, compared to the EO of P. lentiscus, p-cymene (1.64%)
was more abundant in the EO of S. montana (11.77%). The MIC
values indicated that the EO of S. montana was more efficient
than that of P. lentiscus. However, the EOs that exhibited large
inhibition zones for a given bacterium were confirmed as those
with the lower MIC values.
3.3. Antimicrobial activity in minced beef
Figs. 1 and 2 summarize L. monocytogenes growth inhibition in
minced meat by each plant EO and their combination effects,
respectively. It appeared that both EOs remained more effective
when they were applicated separately at twofold MIC values
(Fig. 1). The initially recorded population of 5.63 log CFU/g of
L. monocytogenes strain increased to approximately 8.39 log CFU/g
by the end of storage in untreated samples. Indeed, a reduction of
1.4 and 4.25 log CFU/g was recorded in 4 days of storage, respectively
by P. lentiscus and S. montana. Four days later (at day 8), the
same effects were observed with reduced levels of L. monocytogenes
during storage; a reduction of 2.14 and 5.54 log CFU/g, respectively
for P. lentiscus and S. montana. The populations of L. monocytogenes,
after an initial decrease to 4.1 log CFU/g on day 2, decreased with
significant difference (P < 0.05) and reached 2.8 log CFU/g by the
end of storage. When both EOs were applied individually, P. lentiscus
EO exhibited bacteriostatic activity. However, bactericidal
activity was pronounced, especially in the case of S. montana EO.
Addition of the combination of P. lentiscus EO at 0.1% plus S. montana
EO at 0.03% (Fig. 2) resulted in populations of L. monocytogenes
significantly lower (P < 0.05) than in the samples treated with
P. lentiscus EO alone, throughout storage at 5 1 C. The populations
of L. monocytogenes were kept below 5 log CFU/g up to day
4, and then decreased to reach 4.1 log CFU/g by the end of storage.
On the other hand, the combination of P. lentiscus EO at 0.2% plus
S. montana EO at 0.03% resulted in decreased populations of the
pathogen strain without any significant difference (P > 0.05) than
in the samples treated with P. lentiscus EO at 0.1% plus S. montana
EO at 0.03%, throughout storage, and reached 3.9 log CFU/g, by the
end of storage. Combining P. lentiscus EO and S. montana at 0.1 and
0.06% respectively, resulted in populations of L. monocytogenes
significantly lower (P < 0.05) than in the samples treated with
P. lentiscus EO at 0.1e0.2% plus S. montana EO at 0.03%, throughout
storage. Populations of L. monocytogenes were found below
3 log CFU/g throughout storage. However, when a combination of
P. lentiscus EO at 0.2% plus S. montana EO at 0.06% was used, the
populations of L. monocytogenes decreased to reach 1.2 log CFU/g by
the end of storage, showing a high antibacterial effect (P > 0.05) to
those of the combination use.
The combination containing higher concentrations of S. montana
EO (0.06%) was more effective during the whole period of
Fig. 1. Inhibition of L. monocytogenes by P. lentiscus and S. montana EOs in minced beef
stored at 5 1 C: (A) Control; (-) P. lentiscus (0.2%); (:) S. montana (0.06%).

D. Djenane et al. / Food Control 22 (2011) 1046e1053 1051
Fig. 2. Inhibition of L. monocytogenes by combination of P. lentiscus and S. montana EOs
at different concentrations in minced beef stored at 5 1 C: (A) Control; (&z.squf;)
P. lentiscus/S. montana (1/1); (C) P. lentiscus/S. montana (2/1); (:) P. lentiscus/
S. montana (1/2); (B) P. lentiscus/S. montana (2/2).
storage. However, when both EOs were combined in minced beef, it
appeared that all treatments containing 0.03% of S. montana EO,
exerted bacteriostatic activities depending on the period of storage.
For example, if the reduction rates were notably different in day 2
(1.35e2.35 log CFU/g), they were closely similar in the rest of
storage. However, bactericidal activity was pronounced, especially
in the case of S. montana EO applied at a concentration of 0.06%.
The initial meat pH of 5.7e5.8 in the samples decreased to about
5.6 after treatment with EOs (data not shown). The values of pH did
not differ significantly (P > 0.05) within treatments throughout
storage. The fact that initial meat pH decreased slowly in the
presence of EOs and that there were no significant differences
(P > 0.05) within treatments. The buffering capacity of meat may
explain this phenomenon (Djenane et al., 2011). Further reviews
of published literature revealed that other herbs are also as effective
in inhibiting L. monocytogenes. Aureli, Costantini, and Zolea
(1992) found that thyme EO at about 0.25% resulted in the reduction
of initial populations of L. monocytogenes in minced pork by 2
and 2.3 log CFU/g after 8 days of storage at 4 and 8 C, respectively.
The means by which microorganisms are inhibited by EOs seems to
involve different modes of action. The potent antimicrobial activities
of S. montana observed in this study can be attributed to the
presence of high concentrations of carvacrol, thymol and p-cymene,
which have a well documented antibacterial and antifungic
potential (Oussalah et al., 2007). Mourey and Canillac (2002) tested
the bacteriostatic and bactericidal activities of six components of
P. lentiscus and S. montana EOs and found that a-pinene was the
most active component with an average MIC of 0.019% against
L. monocytogenes serovar 4b, though this concentration was directly
bactericidal. Lis-Balchin and Deans (1997) showed that EOs containing
large amounts of 1,8-cineole were better anti-listerial
agents than EOs that do not contain 1,8-cineole (herein, abundant
in P. lentiscus EO).
The EOs of P. lentiscus chemotype b-myrcene, characterized by
the presence of high concentrations of 1,8-cineole and terpinen-4-
ol, compounds with well also documented antimicrobial activity
(Benhammou, Bekkara, & Panovska, 2008), inhibited growth of
L. monocytogenes and Staphylococcus aureus. Preliminary studies
showed that the combination of EOs had a greater efficacy than the
EOs separately against foodborne pathogens (Gutierrez, Barry-
Ryan, & Bourke, 2008, 2009). Our findings suggest that the
combined EOs of P. lentiscus and S. montana exhibited bacteriostatic
activity at low concentrations (0.03%) of S. montana and bactericidal
activity when S. montana was applied at higher concentrations
(0.06%). Based on the chemical composition of the active EOs, it can
be concluded that EO containing carvacrol and thymol compounds
is the most potent (S. montana), followed by EO containing
b-myrcene and 1,8-cineole (P. lentiscus). Generally, the major
components are found to reflect quite well the antibacterial effects
of the EOs from which they were isolated, the amplitude of their
effects being just dependent on their concentration when they
were tested alone or comprised in EOs. Thus, synergistic functions
of the various molecules contained in an EO, in comparison to the
action of one or two main components of the EOs, seems questionable.
The effect of food ingredients and pH on the antimicrobial
efficacy of EO was assessed by monitoring the lag phase and the
maximum specific growth rate of L. monocytogenes grown in model
media (Gutierrez et al., 2008). The pH of meat is an important factor
affecting the activity of EOs. At low pH, the hydrophobicity of some
EOs increases and while they may tend to partition in the lipid
phase of the food, they can also dissolve more easily in the lipid
phase of the bacterial membrane and have enhanced antimicrobial
action (Juven, Kanner, Schved, & Weisslowicz, 1994). Synergism
between EOs and other parameters in antimicrobial action must be
therefore considered. Holley and Patel (2005) found that plant EOs
were more effective in low than in high fat product. In agreement
with these findings, Smith-Palmer, Stewart, and Fyfe (2001)
showed that the active compounds present in various EOs had
a stronger and a broader spectrum of antimicrobial activity against
L. monocytogenes in low fat than in high fat product. Furthermore,
since another important aspect for the optimized application of EOs
in food is the evaluation of interaction with meat ingredients.
3.4. Sensory analysis
Sensory evaluation scores of minced beef meat samples treated
with single EOs at twofold MIC values and combined EOs at rations
from 1/1 to 2/2 during refrigerated storage at 5 1 C, are shown in
Tables 4e6. Results showed that the intensity of all attributes
Table 4
Effect of EOs on discoloration sensory scores (mean standard deviation) of minced beef stored at 5 1 C.
Treatment
Days of storage
0 2 4 6 8
Discoloration* Control 1.00 0.00 a 1.00 0.0 a 3.50 0.75 a 4.75 0.46 a 5.00 0.00 a
P. lentiscus 1.00 0.00 a 1.00 0.0 a 1.12 0.35 b 2.00 0.00 b 2.60 0.89 a
S. montana 1.00 0.00 a 1.00 0.0 a 1.60 0.50 c 2.60 0.50 c 2.60 0.54 a
P. lentiscus/S. montana (1/1) 1.00 0.00 a 1.00 0.0 a 1.25 0.46 bc 2.00 0.53 b 2.20 0.44 a
P. lentiscus/S. montana (1/2) 1.00 0.00 a 1.00 0.0 a 1.25 0.46 bc 2.60 0.50 c 2.80 0.44 a
P. lentiscus/S. montana (2/1) 1.00 0.00 a 1.00 0.0 a 1.40 0.50 c 2.12 0.35 b 2.60 0.54 a
P. lentiscus/S. montana (2/2) 1.00 0.00 a 1.00 0.0 a 1.40 0.50 c 1.75 0.46 b 2.20 0.44 a
Mean values in the same column and relating to discoloration are significantly different when accompanied by different letters (P < 0.05).
*1 ¼ none, 2 ¼ 0e10%, 3 ¼ 11e20%, 4 ¼ 21e60%, and 5 ¼ 61e100%.

1052
D. Djenane et al. / Food Control 22 (2011) 1046e1053
Table 5
Effect of EOs on off odor sensory scores (mean standard deviation) of minced beef stored at 5 1 C.
Treatment
Days of storage
0 2 4 6 8
Off odor* Control 1.00 00 a 2.25 0.46 a 3.50 0.75 a 4.75 0.46 a 5.00 00 a
P. lentiscus 1.00 00 a 1.00 00 b 1.12 0.35 b 2.00 00 b 3.6 0.25 b
S. montana 1.00 00 a 1.00 00 b 1.60 0.50 c 2.60 0.5 c 3.5 0.15 b
P. lentiscus/S. montana (1/1) 1.00 00 a 1.00 00 b 1.25 0.46 bc 2.00 0.53 b 3.2 0.45 b
P. lentiscus/S. montana (1/2) 1.00 00 a 1.12 0.35 b 1.25 0.46 bc 2.60 0.5 c 3.7 0.2 b
P. lentiscus/S. montana (2/1) 1.00 00 a 1.00 00 b 1.40 0.50 c 2.12 0.35 b 3.5 0.35 b
P. lentiscus/S. montana (2/2) 1.00 00 a 1.00 00 b 1.40 0.50 c 1.75 0.46 d 2.2 0.2 c
Mean values in the same column and relating to off odor are significantly different when accompanied by different letters (P < 0.05).
*1 ¼ none; 2 ¼ slight; 3 ¼ small; 4 ¼ moderate; and 5 ¼ extreme.
Table 6
Effect of EOs on acceptability sensory scores (mean standard deviation) of minced beef stored at 5 1 C.
Treatment
Days of storage
0 2 4 6 8
Acceptability* Control 5.00 0.00 a 4.75 0.46 a 2.50 0.75 a 2.25 0.46 a 1.20 0.44
P. lentiscus 5.00 0.00 a 4.00 0.00 b 3.80 0.44 b 3.40 0.54 b 3.20 0.44
S. montana 5.00 0.00 a 5.00 0.00 a 3.20 0.44 b 3.20 0.44 3.40 0.54
P. lentiscus/S. montana (1/1) 5.00 0.00 a 5.00 0.00 a 4.25 0.46 c 4.00 0.00 a 3.20 0.44
P. lentiscus/S. montana (1/2) 5.00 0.00 a 5.00 0.00 a 4.20 0.44 c 4.00 0.00 a 3.60 0.54 a
P. lentiscus/S. montana (2/1) 5.00 0.00 a 5.00 0.00 a 4.25 0.44 c 3.20 0.33 a 3.00 0.00 a
P. lentiscus/S. montana (2/2) 5.00 0.00 a 5.00 0.00 a 5.00 0.00 c 4.25 0.46 a 4.00 0.00 a
Mean values in the same column and relating to acceptability are significantly different when accompanied by different letters (P < 0.05).
*1 ¼ dislike extremely, 2 ¼ dislike, 3 ¼ nor like or dislike, 4 ¼ like; 5 ¼ like extremely.
increased throughout storage in all samples, though not at the same
rate. Control minced beef reached the highest value, corresponding
to extreme off odor and discoloration, at day 8 of storage. Untreated
samples were assessed by the panelists with scores above (P < 0.05)
the rejection limit (score 3), whereas samples treated with single or
combined EOs were assessed by the panelists with scores below
(P < 0.05) the rejection limit.
The presence of EOs significantly (P < 0.05) extended fresh meat
odor and color attributes. These results were in agreement with
those reported by Sánchez-Escalante, Djenane, Torrescano, Beltrán,
and Roncalés (2001) and Djenane, Sánchez-Escalante, Beltrán, and
Roncalés (2003), who showed that meat treated with natural plant
extracts, either alone or in combination, maintained their fresh
characteristics at higher levels than controls during refrigerated
storage. It must be also emphasized that panelists did not perceive
any extreme odor related to EOs in minced meat (results not
shown). Consequently, the sensory properties of minced beef meat
treated with EOs were acceptable by the panelists at the supplementation
levels. Solomakos, Govarisa, Koidisb, and Botsoglou
(2008) showed that the organoleptic properties of minced meat
treated with EOs were acceptable at the supplementation levels of
0.3 and 0.6%, but unacceptable at the level of 0.9%. However,
Ouattara, Sabato, and Lacroix (2001) reported that addition of EOs
at 0.9% exerted no negative effects on the flavor and appearance of
cooked shrimps. Therefore, more work on the acceptability of these
ingredients will be necessary. Table 6 shows also the acceptability
scores of meat samples. The most preferred sample contained
a combination of 2/2 of EO from P. lentiscus and S. montana,
respectively. Furthermore, all other samples treated with single EO
or with combined EOs were also acceptable by panelists. Nevertheless,
untreated samples were unacceptable by the panelists after
2 days of storage.
The antioxidant activity may be ascribed to the same chemical
components. Monoterpenes found in these EOs may act as radical
scavenging agents (Tepe et al., 2004). These activities may be
attributed to the presence of terpinolene, a- and g-terpinene,
a-pinene, b-pinene, carvacrol, thymol, and p-cymene found in both
EOs. The combined effects of both EOs for preservation of the meat
at cold may play a key role. By using this method, a stable and, from
a microbiological point of view, safe meat can be produced with
low, if any, loss in sensory quality.
4. Conclusion
The results of the bioassays, together with the chemical profile
of the oils of P. lentiscus and S. montana, support the possibility of
using both EOs as potent natural preservatives to contribute in the
reduction of experimentally inoculated L. monocytogenes in minced
meat. These preservative effects were enhanced when combined
EOs were used. Combining such results and those of sensory evaluation
we could propose the use of these EOs, to reduce L. monocytogenes
growth and extend the shelf-life of minced meat during
refrigerated storage.
Acknowledgment
The authors are grateful to Ministerio de Asuntos Exteriores y
Cooperación of Spain (AECID) for financial assistance to this work
within the Programa de Cooperación Interuniversitaria e Investigación
Científica PCI/MED Algeria e Spain (grants ALI A/019342/
08 and A/023365/09).
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