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Food Control 22 (2011) 1046e1053<br />
Contents lists available at ScienceDirect<br />
Food Control<br />
journal homepage: www.elsevier.com/locate/foodcont<br />
<strong>Antimicrobial</strong> <strong>activity</strong> <strong>of</strong> <strong>Pistacia</strong> <strong>lentiscus</strong> <strong>and</strong> <strong>Satureja</strong> <strong>montana</strong> essential oils<br />
against Listeria monocytogenes CECT 935 using laboratory media: Efficacy <strong>and</strong><br />
synergistic potential in minced beef<br />
Djamel Djenane b , Javier Yangüela a , Luis Montañés c , Mouloud Djerbal b,d , Pedro Roncalés a, *<br />
a Dpt. Producción Animal y Ciencia de los Alimentos, Universidad de Zaragoza, C/Miguel Servet, 177-50013 Zaragoza, Spain<br />
b Faculté des Sciences Biologiques et des Sciences Agronomiques, Dpt. Biochimie et Microbiologie, Université Mouloud Mammeri, Bastos, BP 17, 15000 Tizi-Ouzou, Algeria<br />
c Valero Analítica S.L., Laboratorio de Ensayos Físico Químicos y Microbiológicos, Autovía de Logroño Km 246, Polígono Europa 2, 50011 Zaragoza, Spain<br />
d Laboratoire Vétérinaire Régional, Draâ Ben Khedda, Tizi-Ouzou, Algeria<br />
article<br />
info<br />
abstract<br />
Article history:<br />
Received 21 September 2010<br />
Received in revised form<br />
9 December 2010<br />
Accepted 27 December 2010<br />
Keywords:<br />
Essential oils<br />
<strong>Pistacia</strong> <strong>lentiscus</strong><br />
<strong>Satureja</strong> <strong>montana</strong><br />
Growth inhibition<br />
Listeria monocytogenes<br />
Beef<br />
The aim <strong>of</strong> this study was to optimize the antimicrobial efficacy <strong>of</strong> plant essential oils (EOs) for control <strong>of</strong><br />
Listeria monocytogenes (L. monocytogenes) serovar 4b CECT 935 using laboratory media <strong>and</strong> minced beef<br />
stored at 5 1 C. Commercial EOs obtained from leave parts <strong>of</strong> Mediterranean <strong>Pistacia</strong> <strong>lentiscus</strong><br />
(P. <strong>lentiscus</strong>) <strong>and</strong> <strong>Satureja</strong> <strong>montana</strong> (S. <strong>montana</strong>) were analyzed by gas chromatographyemass spectrometry<br />
(GCeMS). The main components <strong>of</strong> EOs obtained were b-myrcene (15.18%) <strong>and</strong> carvacrol<br />
(29.19%), respectively for P. <strong>lentiscus</strong> <strong>and</strong> S. <strong>montana</strong>. The in vitro antimicrobial <strong>activity</strong> <strong>of</strong> both EOs was<br />
evaluated against L. monocytogenes using the agar diffusion technique, the minimum inhibitory<br />
concentrations (MIC) were also determined against the same microorganism using the broth microdilution<br />
method. According to the diameters <strong>of</strong> inhibition, S. <strong>montana</strong> EO had more antibacterial effects<br />
than that from P. <strong>lentiscus</strong>. MICs showed a range <strong>of</strong> 0.03 <strong>and</strong> 0.10% (vol/vol) respectively for S. <strong>montana</strong><br />
<strong>and</strong> P. <strong>lentiscus</strong>. S. <strong>montana</strong> <strong>and</strong> P. <strong>lentiscus</strong> EOs were added respectively in minced beef (tw<strong>of</strong>old MIC<br />
values) at 0.06 <strong>and</strong> 0.20%, experimentally inoculated with L. monocytogenes at a level <strong>of</strong> 3 10 5 CFU/g<br />
<strong>and</strong> stored at 5 1 C during one week. S. <strong>montana</strong> EO was the more effective (P < 0.05) against target<br />
bacteria. P. <strong>lentiscus</strong> EO also demonstrated antibacterial effect against the same bacterium. EO combinations<br />
were also investigated in minced beef <strong>and</strong> P. <strong>lentiscus</strong> combined with S. <strong>montana</strong> had synergistic<br />
effects. This work shows that the combined EOs might be more effective against L. monocytogenes when<br />
applied to minced beef at the ratio <strong>of</strong> 1/1 to 2/2 according to the MIC values. Sensory evaluation revealed<br />
that minced beef treated with EOs was acceptable by panelists at the levels used.<br />
Ó 2011 Elsevier Ltd. All rights reserved.<br />
1. Introduction<br />
Meat products are perishable <strong>and</strong> require protection from<br />
undesired spoilage <strong>and</strong> pathogenic bacteria during their preparation,<br />
storage, distribution <strong>and</strong> sale. Foodborne illness resulting from<br />
consumption <strong>of</strong> food contaminated with pathogenic bacteria is <strong>of</strong><br />
vital concern to public health.<br />
Listeria monocytogenes is widely distributed in nature <strong>and</strong> can be<br />
frequently found in a large number <strong>of</strong> food products, as well as in<br />
processing plants. Listeriosis is recognized as an important public<br />
health problem, affecting primarily pregnant women, newborns<br />
* Corresponding author. Tel.: þ34 976 761 582; fax: þ34 976 761 590.<br />
E-mail address: roncales@unizar.es (P. Roncalés).<br />
<strong>and</strong> adults with weakened immune systems. The majority <strong>of</strong><br />
human listeriosis infections are caused by the consumption <strong>of</strong><br />
contaminated food. It has been shown that L. monocytogenes is able<br />
to survive <strong>and</strong> grow to significant numbers on refrigerated meat<br />
products making post-process contamination a significant concern<br />
for ready-to-eat meat produce (Farber & Peterkin, 1991).<br />
Today, different strategies are applied in order to control pathogens<br />
in meats, <strong>and</strong> particular interest has been focused on the<br />
application <strong>of</strong> EOs as a safe <strong>and</strong> effective alternative to chemical<br />
preservatives; their application in controlling pathogens could<br />
reduce the risk <strong>of</strong> foodborne outbreaks <strong>and</strong> assure consumers safe<br />
meat products. In recent years the EOs <strong>and</strong> extracts <strong>of</strong> many plant<br />
species have become popular, <strong>and</strong> attempts to characterize their<br />
bioactive principles have gained momentum in many food-processing<br />
applications. The chemical composition <strong>and</strong> antimicrobial<br />
0956-7135/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved.<br />
doi:10.1016/j.foodcont.2010.12.015
D. Djenane et al. / Food Control 22 (2011) 1046e1053 1047<br />
properties <strong>of</strong> EOs extracted from diverse plant species have been<br />
demonstrated using a variety <strong>of</strong> experimental methods (Djenane<br />
et al., 2011; Özkan, Sagdic, Göktürk, Unal, & Albayrak, 2010).<br />
<strong>Pistacia</strong> <strong>lentiscus</strong> (Anacardiaceae), <strong>and</strong> <strong>Satureja</strong> <strong>montana</strong> (Lamiaceae)<br />
are distributed throughout the Mediterranean regions. The<br />
chemical composition <strong>of</strong> the EOs from leaves <strong>of</strong> both species <strong>of</strong><br />
diverse origins has been already reported ( Cavar, Maksimovic, Solic,<br />
Jerkovic-Mujkic, & Besta, 2008; Gardeli, Papageorgiou, Mallouchos,<br />
Theodosis, & Komaitis, 2008). EOs <strong>of</strong> both species are used as a food<br />
ingredient in the Mediterranean region as an aromatic <strong>and</strong><br />
flavoring agent. In Algeria, the leaves <strong>of</strong> <strong>Pistacia</strong> spp. <strong>and</strong> <strong>Satureja</strong><br />
spp. were used to purify water <strong>and</strong> increase the time <strong>of</strong> conservation<br />
<strong>of</strong> dry figs <strong>and</strong> sun-dried tomatoes; they are also used as<br />
natural preservatives for fish <strong>and</strong> meat products. The in vitro antimicrobial<br />
<strong>activity</strong> <strong>of</strong> P. <strong>lentiscus</strong> <strong>and</strong> S. <strong>montana</strong> EO has also been<br />
tested on bacteria <strong>and</strong> fungi ( Cavar et al., 2008; Iauk, Ragusa,<br />
Rapisadra, Franco, & Nicolosi, 1996). It has been generally used as<br />
traditional medicine for various diseases such as asthma, peptic<br />
ulcer, diarrheic, anti-inflammatory, antipyretic, antibacterial, <strong>and</strong><br />
antiviral medicines <strong>and</strong> insecticidal activities (Bakkali, Averbeck,<br />
Averbeck, & Idaomar, 2008).<br />
Most previous reports on the anti-listerial effect <strong>of</strong> EOs used<br />
only an in vitro approach (Oussalah, Caillet, Saucier, & Lacroix,<br />
2007). The review by Holley <strong>and</strong> Patel (2005) highlighted those<br />
reports in which a food matrix was used for studying the antimicrobial<br />
action <strong>of</strong> EOs. However, as far as we are aware, none <strong>of</strong> them<br />
focused on the effects on the sensory properties <strong>of</strong> food. It appears<br />
then to be necessary to determine the minimum concentration<br />
necessary to inhibit the growth <strong>of</strong> L. monocytogenes without<br />
affecting the sensory attributes <strong>of</strong> meat.<br />
The objective <strong>of</strong> this study was to determine the efficacy <strong>of</strong><br />
P. <strong>lentiscus</strong> <strong>and</strong> S. <strong>montana</strong> EOs <strong>and</strong> their combination in inhibiting<br />
L. monocytogenes growth both in vitro <strong>and</strong> on a model minced beef<br />
during refrigerated storage, affording a novel insight on their<br />
possible effect on meat sensory properties.<br />
2. Materials <strong>and</strong> methods<br />
2.1. Essential oils<br />
The pure essential oils used in this study were extracted from<br />
P. <strong>lentiscus</strong> <strong>and</strong> S. <strong>montana</strong> (density: 0.85 <strong>and</strong> 0.92 at 20 C,<br />
respectively). They were selected based on previously reported<br />
efficacy, <strong>and</strong> were obtained from Florame Aromathérapie (St Rémy<br />
de Provence, France). The EOs were certified by Ecocert SAS F32600<br />
(France) <strong>and</strong> that considered 100% pure <strong>and</strong> natural, obtained from<br />
Mediterranean biological culture. The isolated EOs were preserved<br />
in darkness in a sealed vial at 1 C until its analysis or its use in<br />
bioassays.<br />
2.2. Essential oils analysis<br />
2.2.1. Gas chromatography analysis<br />
Gas chromatography (GC) analyses <strong>of</strong> EOs obtained from dried<br />
material were performed using a Hewlett Packard 6890 gas chromatograph<br />
equipped with a flame ionization detector (FID) <strong>and</strong><br />
a Stabilwax (PEG) column (30 m 0.32 mm i.d., 1 mm film thickness)<br />
(Centre de Recherche en Analyses Physico-Chimiques-CRAPC,<br />
Algiers, Algeria).<br />
The operating conditions were as follows: injector <strong>and</strong> detector<br />
temperatures, 250 <strong>and</strong> 280 C, respectively; carrier gas, N 2 at a flow<br />
rate <strong>of</strong> 1 mL/min; oven temperature program, 3 min isothermal at<br />
50 C, raised at 2 C/min to 220 C, <strong>and</strong> finally held isothermal for<br />
15 min. The identities <strong>of</strong> the separated components on the polar<br />
column were determined by comparing their retention indices<br />
relative to aliphatic hydrocarbons injected under the above<br />
temperature program with literature values measured on columns<br />
with identical polarities.<br />
2.2.2. Gas chromatographyemass spectrometry analysis<br />
The gas chromatographyemass spectrometry (GCeMS) analysis<br />
was performed using a HewlettePackard 6890 series GC systems<br />
(Agilent Technologies) coupled to a quadrupole mass spectrometer<br />
(model HP 5973) equipped with an HP5 MS capillary column (5%<br />
phenyl methylsiloxane, 30 m 0.25 mm, 0.25 mm film thickness)<br />
(CRAPC, Algiers, Algeria). For GCeMS detection an electron ionization<br />
system with ionization energy <strong>of</strong> 70 eV was used over a scan<br />
range <strong>of</strong> 30e550 atomic mass units. Helium was the carrier gas, at<br />
a flow rate <strong>of</strong> 0.5 mL/min. Injector <strong>and</strong> detector MS transfer line<br />
temperatures were set at 250 <strong>and</strong> 280 C, respectively; the<br />
temperature <strong>of</strong> the ion source was 230 C. Column temperature was<br />
initially kept at 60 C for 8 min, then gradually increased to 280 C<br />
at 2 C/min, <strong>and</strong> finally held isothermal for 30 min. The volume <strong>of</strong><br />
injections was 0.2 mL <strong>of</strong> a hexaneeoil solution, injected in the<br />
splitless mode. The identity <strong>of</strong> the components was assigned by<br />
matching their spectral data with those detailed in the Wiley 7N,<br />
NIST 02, <strong>and</strong> NIST 98 libraries. The results were also confirmed by<br />
the comparison <strong>of</strong> their retention indices, relative to C7eC29<br />
n-alkanes assayed under GCeMS in the same conditions as the oils.<br />
Some structures were further confirmed by available authentic<br />
st<strong>and</strong>ards analyzed under the same conditions described above.<br />
The percentage composition <strong>of</strong> the oils was computed by the<br />
normalization method from the GC peak areas, calculated as the<br />
mean value <strong>of</strong> two injections from each essential oil (EO).<br />
2.3. <strong>Antimicrobial</strong> screening<br />
2.3.1. Bacterial strain <strong>and</strong> culture conditions<br />
The L. monocytogenes culture employed was provided by the<br />
Spanish Type Culture Collection (STCC). Strain used was L. monocytogenes<br />
serovar 4b CECT 935. Bacterial strain was cultured overnight<br />
at 37 C in Mueller Hinton agar (MHA, Oxoid, Basingstoke,<br />
UK). One milliliter <strong>of</strong> stock culture was st<strong>and</strong>ardized through two<br />
successive 24 h growth cycles at 37 1 C in 9 mL <strong>of</strong> BraineHeart<br />
Infusion Broth (BHIB, Oxoid, Basingstoke, UK). After 48 h, 100 mL <strong>of</strong><br />
the suspension were then inoculated in fresh BHIB <strong>and</strong> incubated at<br />
37 1 C for 12 h to obtain a working fresh culture containing about<br />
3 10 5 CFU/mL, determining by measuring transmittance at<br />
600 nm (Spectrophotometer: Spectronic 20 Bausch & Lomb).<br />
Bacterial strain was maintained frozen ( 80 C) in cryovials <strong>and</strong><br />
were subcultured every antibacterial test.<br />
2.3.2. Disc-diffusion method<br />
Screening <strong>of</strong> EOs for antibacterial <strong>activity</strong> was determined by<br />
the agar diffusion method as previously described (Hazzit,<br />
Baaliouamer, Veríssimo, Faleiro, & Miguel, 2009), which is normally<br />
used as a preliminary check efficient EOs. Petri plates were<br />
prepared by pouring 20 mL <strong>of</strong> MHA medium <strong>and</strong> allowed to<br />
solidify. Plates were dried for 30 min in a biological safety cabinet<br />
with vertical laminar flow <strong>and</strong> 0.1 mL <strong>of</strong> st<strong>and</strong>ardized inoculums<br />
suspension was poured <strong>and</strong> uniformly extend. The inoculums were<br />
allowed to dry for 5 min. To prepare the stock solution <strong>of</strong> the<br />
samples, the pure EOs were dissolved in 0.5% (v/v) dimethyl sulfoxide<br />
(DMSO) (Sigma Aldrich Ò -Química, S.A.). Then sterile filter<br />
paper disk (6 mm diameter, Filter LAB ANOIA, Barcelona, Spain) was<br />
impregnated with 05 mL EO, using a capillary micro-pipette<br />
(Finnpipette Ò , Thermo Fischer Scientific Inc.). The plates were left<br />
15 min at room temperature to allow the diffusion <strong>of</strong> the EO, <strong>and</strong><br />
then they were incubated at 37 C for 24 h. At the end <strong>of</strong> the period,<br />
the diameter <strong>of</strong> the clear zone around the disc was measured with
1048<br />
D. Djenane et al. / Food Control 22 (2011) 1046e1053<br />
a caliper (Wiha dialMax Ò ESD-Uhrmessschieber, CH) <strong>and</strong> expressed<br />
in millimeters (mm: disk diameter included) as its antimicrobial<br />
<strong>activity</strong>. The sensitivity to the different oils was classified by the<br />
diameter <strong>of</strong> the inhibition halos as follows: not sensitive (L) for<br />
diameter less than 8 mm; sensitive (D) for diameter 9e14 mm;<br />
very sensitive (DD) for diameter 15e19 mm <strong>and</strong> extremely<br />
sensitive (þþþ) for diameter larger than 20 mm (Ponce, Fritz, del<br />
Valle, & Roura, 2003). Negative controls were prepared using the<br />
same solvent employed to dissolve the samples. St<strong>and</strong>ard reference<br />
antibiotic, amoxicillin (4 mg/disc; Sigma Aldrich Ò -Química, S.A.),<br />
was used as positive control in order to control the sensitivity <strong>of</strong> the<br />
tested microorganism. Each assay in this experiment was replicated<br />
three times.<br />
2.3.3. Microdilution assays<br />
The minimal inhibitory concentration (MIC) values were also<br />
studied for the target bacterium which was determined as sensitive<br />
to the EOs in disc-diffusion assay, as described in Section 2.3.2. The<br />
inocula <strong>of</strong> L. monocytogenes were prepared from 12 h broth cultures<br />
<strong>and</strong> suspensions were adjusted to 0.5 McFarl<strong>and</strong> st<strong>and</strong>ard turbidity<br />
to give a final density <strong>of</strong> 3 10 5 CFU/mL. P. <strong>lentiscus</strong> <strong>and</strong> S. <strong>montana</strong><br />
EOs dissolved in 0.5% dimethyl sulfoxide (DMSO) were first diluted<br />
to the highest concentration (32 mL/mL) to be tested, <strong>and</strong> then serial<br />
tw<strong>of</strong>old dilutions were made in a concentration range from<br />
32 mL/mL to 0.3125 mL/mL in 10 mL sterile test tubes containing MH<br />
broth. MIC values <strong>of</strong> both EOs against L. monocytogenes were<br />
determined based on a microwell dilution method. The 96-well<br />
plates (Iwaki br<strong>and</strong>, Asahi Techno Glass, Japan) were prepared by<br />
dispensing into each well 95 mL <strong>of</strong> MH broth <strong>and</strong> 5 mL <strong>of</strong> the inoculum.<br />
A 100 mL aliquot from both EOs extracts initially prepared at<br />
the concentration <strong>of</strong> 32 mL/mL was added into the first wells. Then,<br />
100 mL from their serial dilutions were transferred into consecutive<br />
wells. The last well containing 195 mL <strong>of</strong> nutrient broth without<br />
compound <strong>and</strong> 5 mL <strong>of</strong> the inoculums on each strip was used as<br />
negative control. The final volume in each well was 200 mL. Lev<strong>of</strong>loxacin<br />
(SigmaeAldrich, Madrid, Spain) at the concentration range<br />
<strong>of</strong> 32e0.3125 mL/mL was prepared in MH broth <strong>and</strong> used as st<strong>and</strong>ard<br />
antibiotic for positive control. Contents <strong>of</strong> each well were<br />
mixed on a plate shaker at 300 rpm for 20 s <strong>and</strong> then incubated at<br />
appropriate temperatures for 24 h. After incubation at 37 C for<br />
18e24 h under agitation the wells were then examined for<br />
evidence <strong>of</strong> growth <strong>and</strong> MICs (mL/mL) values were determined as<br />
the lowest EO concentration that inhibited visible growth <strong>of</strong> the<br />
tested microorganism which was indicated by absence <strong>of</strong> turbidity.<br />
The negative control was set up with DMSO in amount corresponding<br />
to the highest quantity present in the test solution (0.5%).<br />
The tests were performed in duplicate <strong>and</strong> repeated twice.<br />
2.4. <strong>Antimicrobial</strong> <strong>activity</strong> in minced beef<br />
2.4.1. Preparation <strong>of</strong> meat<br />
The muscle Semimembranosus (initial pH 5.7e5.8) was excised<br />
from three beef carcasses 48 h post-slaughter from a local supplier<br />
(Boucherie Khatir, Draâ Ben Khedda, Algeria) <strong>and</strong> transported to the<br />
laboratory under refrigerated conditions within 30 min.<br />
2.4.2. Treatment <strong>of</strong> minced beef<br />
Prior to meat inoculation with L. monocytogenes <strong>and</strong> the addition<br />
<strong>of</strong> EOs <strong>and</strong>/or their combination, minced beef was also<br />
examined for any contamination by bacteria or the tested pathogen<br />
(results not shown).<br />
In order to evaluate the antimicrobial <strong>activity</strong> <strong>of</strong> both EOs in<br />
a meat system, a sufficient amount <strong>of</strong> fresh minced beef was<br />
prepared following good practices, <strong>and</strong> was tested using tw<strong>of</strong>old<br />
the MICs value found for both EOs. After the aseptic removal <strong>of</strong> the<br />
outer surface, seven (07) pieces <strong>of</strong> approximately 400 g <strong>of</strong> prepared<br />
meat were minced in a sterile grinder, <strong>and</strong> portions <strong>of</strong> 50 2 g were<br />
placed into polystyrene trays. A total <strong>of</strong> 56 meat samples were<br />
obtained. Two individual duplicate <strong>of</strong> each sample were performed<br />
in all cases. Minced beef samples (50 2 g) were placed in stomacher<br />
bags <strong>and</strong> inoculated with strain <strong>of</strong> L. monocytogenes at a level<br />
<strong>of</strong> 3 10 5 CFU/g. The inoculated samples were homogenized in<br />
a stomacher 400 (Stomacher Ò 400 Circulator. Seward. Worthing,<br />
UK) for 2 min at room temperature to ensure proper distribution <strong>of</strong><br />
the pathogen. Following homogenization, the individual EO <strong>and</strong><br />
their combination were added to the inoculated samples.<br />
Addition <strong>of</strong> S. <strong>montana</strong> <strong>and</strong> P. <strong>lentiscus</strong> EO were done at tw<strong>of</strong>old<br />
MIC values (0.06 <strong>and</strong> 0.2%, respectively). In addition, the combinations<br />
<strong>of</strong> EOs were also examined at followed concentrations: 1/1<br />
(0.10%/0.03%); 2/1 (0.20%/0.03%); 1/2 (0.10%/0.06%); 2/2 (0.20%/<br />
0.06%), respectively for P. <strong>lentiscus</strong> <strong>and</strong> S. <strong>montana</strong>.<br />
To attain uniform distribution <strong>of</strong> the added compounds, treated<br />
meat samples were further homogenized in stomacher, as previously<br />
described. All stomacher bags with samples from all treatments<br />
were wrapped <strong>and</strong> stored under aerobic conditions at<br />
5 1 C for 8 days, simulating a proper refrigeration storage.<br />
Microbial analyses <strong>of</strong> samples for populations <strong>of</strong> L. monocytogenes<br />
were carried out at 2 days intervals up to the 8th day <strong>of</strong> refrigerated<br />
storage.<br />
2.5. pH measurements<br />
The pH <strong>of</strong> meat samples was measured using a micro pH-meter<br />
model 2001 (Crison Instruments, Barcelona, Spain) after homogenizing<br />
3 g <strong>of</strong> sample in 27 mL distilled water for 10 s at 1300 rpm<br />
with an Ultra-Turrax T25 macevator (Janke & Kunkel, Staufen,<br />
Germany). Each value was the mean <strong>of</strong> three replicates.<br />
2.6. Sensory analysis<br />
For sensory analysis, the pieces (400 g) <strong>of</strong> meat prepared were<br />
minced, <strong>and</strong> portions <strong>of</strong> 50 2 g were obtained. A total <strong>of</strong> 56 meat<br />
samples were obtained. The samples were mixed with different<br />
concentrations <strong>of</strong> EOs at the same concentrations used for antibacterial<br />
screening. Eight samples were obtained for each treatment.<br />
The remaining 8 meat samples were served for controls. All<br />
samples were placed into polystyrene trays, <strong>and</strong> over-wrapped in<br />
polyethylene film (Sidlaw Packaging-Soplaril, Barcelona, Spain).<br />
Two individual duplicates <strong>of</strong> each sample were performed in all<br />
cases. Samples were evaluated for <strong>of</strong>f odor, discoloration <strong>and</strong> overall<br />
acceptability attributes by an eight-member trained panel. Panelists<br />
were selected among students <strong>and</strong> staff <strong>of</strong> the Department <strong>of</strong><br />
Veterinary Microbiology (Laboratoire Vétérinaire Régional de Draâ<br />
Ben Khedda, Algeria) <strong>and</strong> trained according to the method<br />
described by Djenane, Sánchez-Escalante, Beltrán, <strong>and</strong> Roncalés<br />
(2001). Though already skilled in this kind <strong>of</strong> evaluation, panelists<br />
received further training prior to analysis. Three open-discussion<br />
sessions were held to familiarize the individuals with the attributes<br />
<strong>and</strong> the scale to use. The attribute <strong>of</strong>f odor was evaluated using<br />
a 5-point scale, according to Sørheim, Kropf, Hunt, Karwoski, <strong>and</strong><br />
Warren (1996). Odor scores referred to the intensity <strong>of</strong> <strong>of</strong>f odors<br />
associated to meat spoilage: 1 ¼ none; 2 ¼ slight; 3 ¼ small;<br />
4 ¼ moderate; <strong>and</strong> 5 ¼ extreme. Discoloration scores referred to<br />
percentage <strong>of</strong> discolored surface: 1 ¼ none, 2 ¼ 0e10%, 3 ¼ 11e20%,<br />
4 ¼ 21e60%, <strong>and</strong> 5 ¼ 61e100%. For acceptability attribute, before<br />
evaluation, treated minced beef samples were wrapped in<br />
aluminum foil individually <strong>and</strong> cooked in a steam-cooker for<br />
20 min. Each sample was served warm in dishes coded with 3-digit<br />
r<strong>and</strong>om numbers <strong>and</strong> presented in individual booths to each<br />
panelist for evaluation. A 5-point hedonic scale was used to score
D. Djenane et al. / Food Control 22 (2011) 1046e1053 1049<br />
acceptability attribute, where 1 ¼ dislike extremely, 2 ¼ dislike,<br />
3 ¼ nor like or dislike, 4 ¼ like; 5 ¼ like extremely. Sensory evaluation<br />
was accomplished at 0 <strong>and</strong> 2 days intervals up to the end <strong>of</strong><br />
refrigerated storage at 5 1 C. Results were expressed as the<br />
predominant score given by panelists.<br />
2.7. Bacterial enumeration<br />
At each sampling time, samples (25 g) <strong>of</strong> minced beef in the<br />
stomacher bags were aseptically added with 225 mL <strong>of</strong> 0.1%<br />
peptone water. The content was macerated in the stomacher for<br />
2 min at room temperature. Resulting slurries were serially diluted<br />
(1:10) in 0.1% sterile peptone water. Sample dilutions (0.1 mL) <strong>of</strong><br />
minced beef were spread plated on appropriate media in duplicate.<br />
Populations <strong>of</strong> L. monocytogenes were determined on Agar Listeria<br />
according to Ottaviani <strong>and</strong> Agosti (ALOA, Biolife, Milan, Italy) at<br />
37 C for 24e48 h.<br />
2.8. Statistical analysis<br />
Variance analyses were used to test the significant difference<br />
among the results from the antibacterial assays <strong>and</strong> sensory analysis<br />
(SPSS 10.0 s<strong>of</strong>tware package, 1995). Means <strong>and</strong> st<strong>and</strong>ard errors<br />
(SE) <strong>of</strong> the samples were calculated. Three replicates were performed<br />
for each treatment. Differences between means were tested<br />
through LSD <strong>and</strong> values <strong>of</strong> P < 0.05 were considered significantly<br />
different.<br />
3. Results <strong>and</strong> discussion<br />
3.1. Chemical composition <strong>of</strong> the EOs<br />
Steam distillation is the most commonly used method for<br />
producing EOs on a commercial basis. Table 1 summarizes the<br />
results <strong>of</strong> chemical composition <strong>of</strong> both EOs. In order to simplify the<br />
analysis <strong>of</strong> the results, only compounds with more than 0.5%<br />
abundance were selected. Fifty-seven <strong>and</strong> 44 constituents, which<br />
represented 98.69% <strong>and</strong> 94.8% <strong>of</strong> the total EO <strong>of</strong> P. <strong>lentiscus</strong> <strong>and</strong><br />
S. <strong>montana</strong>, respectively, were identified. P. <strong>lentiscus</strong> EO was characterized<br />
by a high percentage <strong>of</strong> b-myrcene (15.18%) <strong>and</strong> 1.8-cineole<br />
(15.02%), followed by terpinen-4-ol (6.41%), a-pinene (5.54%) <strong>and</strong><br />
b-pinene (5.10%). S. <strong>montana</strong> EO was characterized by a high<br />
percentage <strong>of</strong> carvacrol (29.19%), thymol (15.41%) <strong>and</strong> p-cymene<br />
(11.77%), followed by g-terpinene (6.72%), b-caryophyllene (5.38%)<br />
<strong>and</strong> farnesol (4.10%). Similar findings have been reported by other<br />
authors (Barra, Coroneo, Dessi, Cabras, & Angioni, 2007; Cavar et al.,<br />
2008). However, the percentage <strong>of</strong> most <strong>of</strong> the individual constituents<br />
present in both EOs differed significantly (P < 0.05) from other<br />
findings.<br />
Seasonal variations in the distribution between the different<br />
compounds in the plant EOs could be related to changes<br />
throughout the plant’s vegetative cycle, along with environmental<br />
conditions prevailing in the Mediterranean regions. Environmental<br />
factors such as geography, temperature, day length, nutrients, etc.,<br />
were considered to play a key role in the chemical composition <strong>of</strong><br />
EOs (Gardeli et al., 2008; Hussain, Anwar, Nigam, Ashrafd, & Gilanif,<br />
2010). These factors influence the plant’s biosynthetic pathways<br />
<strong>and</strong> consequently the relative proportion <strong>of</strong> the main characteristic<br />
compounds. This leads to the existence <strong>of</strong> different chemotypes<br />
which distinguish EOs <strong>of</strong> different origins.<br />
3.2. <strong>Antimicrobial</strong> <strong>activity</strong> (disc assay)<br />
The in vitro antimicrobial activities <strong>of</strong> P. <strong>lentiscus</strong> <strong>and</strong> S. <strong>montana</strong><br />
EOs against L. monocytogenes were qualitatively <strong>and</strong> quantitatively<br />
Table 1<br />
Main constituents (%) <strong>of</strong> the EOs <strong>of</strong> <strong>Pistacia</strong> <strong>and</strong> <strong>Satureja</strong> species, as identified by<br />
GCeMS analysis.<br />
RT (min) a Compound b P. <strong>lentiscus</strong> S. <strong>montana</strong><br />
1 5.177 Tricyclene 0.64 e<br />
2 5.320 a-Thujene e 0.73<br />
3 5.527 a-Pinene 5.54 0.79<br />
4 5.932 Camphene 3.15 0.51<br />
5 6.844 b-Pinene 5.10 0.96<br />
6 7.287 Myrcene e 1.04<br />
7 7.335 b-Myrcene 15.18 e<br />
8 7.763 a-Phell<strong>and</strong>rene 3.83 e<br />
9 8.232 a-Terpinene 2.78 1.33<br />
10 8.577 p-Cymene 1.64 11.77<br />
11 8.742 Limonene e 0.64<br />
12 8.773 1.8-Cineole 15.02 e<br />
13 9.130 trans-b-Ocimene e 0.92<br />
14 9.544 ciseb-Ocimene 1.68 e<br />
25 9.887 Isoamyl butyrate 0.51 e<br />
16 10.034 g-Terpinene 4.10 6.72<br />
17 10.392 4-Thujanol trans e 1.05<br />
18 11.349 Terpinolene 2.21 e<br />
19 12.020 Linalool e 1.97<br />
20 14.725 Menthone e 0.59<br />
21 15.377 Borneol e 1.75<br />
22 16.063 Terpinen-4-ol 6.41 1.04<br />
23 16.854 a-Terpineol 2.97 e<br />
24 19.982 Thymol methyl ether e 0.95<br />
25 20.877 Farnesol e 4.10<br />
26 22.330 Bornyl acetate 1.88 e<br />
27 23.330 Carvacrol e 29.19<br />
28 23.854 Thymol e 15.41<br />
29 24.740 Myrtenyl acetate 0.73 e<br />
30 28.563 Geranyl acetate e 0.53<br />
31 30.215 Caryophyllene 4.03 5.38<br />
32 32.135 a-Caryophyllene 0.84 e<br />
33 33.802 Germacrene-D 0.87 e<br />
34 34.759 g-Elemene e 0.75<br />
35 35.040 a-Cadinene 0.56 e<br />
36 35.716 b-Bisabolene e 0.87<br />
37 36.428 d-Cadinene 1.80 0.23<br />
38 39.611 Caryophyllene oxyde e 0.98<br />
a Retention time.<br />
b Compounds present in trace amounts ( 0.05).<br />
All tests were performed in duplicate.<br />
a<br />
f: Inhibition zone in diameter around the discs impregnated with essential oils.<br />
The diameter (6 mm) <strong>of</strong> the disc is included.
1050<br />
D. Djenane et al. / Food Control 22 (2011) 1046e1053<br />
Table 3<br />
Minimal inhibitory concentrations values from P. <strong>lentiscus</strong> <strong>and</strong> S. <strong>montana</strong> EOs using<br />
broth microdilution method.<br />
MIC (%) a<br />
P. <strong>lentiscus</strong> S. <strong>montana</strong><br />
Listeria monocytogenes serovar 4b CECT 935 0.1 0.01 0.03 0.007<br />
All tests were performed in duplicate.<br />
a<br />
f: Minimal inhibitory concentrations values expressed by % (vol/vol).<br />
respectively. Elgayyar, Draughon, Golden, <strong>and</strong> Mount (2001)<br />
reported similar results regarding the effect <strong>of</strong> Rosmarinus <strong>of</strong>ficinalis<br />
(rosemary) on L. monocytogenes, showing inhibition zones<br />
ranging from 23 to 30 mm. This antimicrobial effects obtained<br />
with both EOs against L. monocytogenes, was comparable to that<br />
reported by other findings. Vagionas, Graikou, Ngassapa,<br />
Runyoro, <strong>and</strong> Chinou (2007) recorded close MICs (0.04e0.1%)<br />
when they tested the EO <strong>of</strong> <strong>Satureja</strong> spp. against various pathogens.<br />
A large number <strong>of</strong> studies have reported that the EOs <strong>of</strong><br />
<strong>Satureja</strong> species are among the most potent regarding their<br />
antimicrobial properties (Dikbas, Kotan, Dadasoglu, & Sahin,<br />
2008; Razzaghi-Abyaneh et al., 2008), which has been<br />
confirmed <strong>and</strong> extended in this study. The EOs <strong>of</strong> many species<br />
<strong>of</strong> <strong>Satureja</strong> are known to possess antibacterial <strong>and</strong> fungicidal<br />
properties (Özcan & Erkmen, 2001; Vagionas et al., 2007). The<br />
antibacterial <strong>activity</strong> assigned at both EOs could be attributed to<br />
their high content <strong>of</strong> compounds with known antimicrobial<br />
<strong>activity</strong>. Confirming previous reports, it was found that the<br />
strength <strong>and</strong> spectrum <strong>of</strong> <strong>activity</strong> varied between the investigated<br />
<strong>Satureja</strong> species <strong>and</strong> Gram type <strong>of</strong> target bacteria. Grampositive<br />
bacteria were generally more sensitive to the effects <strong>of</strong><br />
the EOs than Gram-negative bacteria. This general higher resistance<br />
among Gram-negative bacteria has been ascribed to the<br />
existence <strong>of</strong> their outer phospholipidic membrane, almost<br />
impermeable to lipophilic compounds (Nikaido & Vaara, 1985).<br />
The absence <strong>of</strong> this barrier in Gram-positive bacteria allows the<br />
direct contact <strong>of</strong> the essential oil’s hydrophobic constituents<br />
with the phospholipid bilayer <strong>of</strong> the cell membrane, where they<br />
bring about their effect, either causing an increase <strong>of</strong> ion<br />
permeability <strong>and</strong> leakage <strong>of</strong> vital intracellular constituents<br />
(Cowan, 1999).<br />
The antimicrobial activities <strong>of</strong> the EOs are difficult to correlate<br />
to a specific compound due to their complexity <strong>and</strong> variability.<br />
Nevertheless, some researchers reported that there is<br />
a relationship between the chemical composition <strong>of</strong> the most<br />
abundant components in the EO <strong>and</strong> the antimicrobial <strong>activity</strong>.<br />
For example, 1,8-cineole (abundant in Algerian P. <strong>lentiscus</strong> EO<br />
tested in this study) is well-known for its antimicrobial potential<br />
(Pattnaik, Subramanyam, Bapaji, & Kole, 1997). Lis-Balchin <strong>and</strong><br />
Deans (1997) showed that EOs containing large amounts <strong>of</strong><br />
1,8-cineole are better anti-listerial agents than EOs that do not<br />
contain 1,8-cineole. The antimicrobial effects <strong>of</strong> borneol were<br />
also reported elsewhere (Dorman & Deans, 2000). As a result <strong>of</strong><br />
these findings, the higher antimicrobial activities <strong>of</strong> P. <strong>lentiscus</strong><br />
<strong>and</strong> S. <strong>montana</strong> EOs could be attributed to their particular chemotypes<br />
characterized by their complexity. Moreover, many<br />
reports mentioned that carvacrol <strong>and</strong> thymol <strong>and</strong> their precursors<br />
(p-cymene <strong>and</strong> g-terpinene), are biologically <strong>and</strong> functionally<br />
closely associated (Ultee, Bennik, & Moezelaar, 2002). In that<br />
context, compared to the EO <strong>of</strong> P. <strong>lentiscus</strong>, p-cymene (1.64%)<br />
was more abundant in the EO <strong>of</strong> S. <strong>montana</strong> (11.77%). The MIC<br />
values indicated that the EO <strong>of</strong> S. <strong>montana</strong> was more efficient<br />
than that <strong>of</strong> P. <strong>lentiscus</strong>. However, the EOs that exhibited large<br />
inhibition zones for a given bacterium were confirmed as those<br />
with the lower MIC values.<br />
3.3. <strong>Antimicrobial</strong> <strong>activity</strong> in minced beef<br />
Figs. 1 <strong>and</strong> 2 summarize L. monocytogenes growth inhibition in<br />
minced meat by each plant EO <strong>and</strong> their combination effects,<br />
respectively. It appeared that both EOs remained more effective<br />
when they were applicated separately at tw<strong>of</strong>old MIC values<br />
(Fig. 1). The initially recorded population <strong>of</strong> 5.63 log CFU/g <strong>of</strong><br />
L. monocytogenes strain increased to approximately 8.39 log CFU/g<br />
by the end <strong>of</strong> storage in untreated samples. Indeed, a reduction <strong>of</strong><br />
1.4 <strong>and</strong> 4.25 log CFU/g was recorded in 4 days <strong>of</strong> storage, respectively<br />
by P. <strong>lentiscus</strong> <strong>and</strong> S. <strong>montana</strong>. Four days later (at day 8), the<br />
same effects were observed with reduced levels <strong>of</strong> L. monocytogenes<br />
during storage; a reduction <strong>of</strong> 2.14 <strong>and</strong> 5.54 log CFU/g, respectively<br />
for P. <strong>lentiscus</strong> <strong>and</strong> S. <strong>montana</strong>. The populations <strong>of</strong> L. monocytogenes,<br />
after an initial decrease to 4.1 log CFU/g on day 2, decreased with<br />
significant difference (P < 0.05) <strong>and</strong> reached 2.8 log CFU/g by the<br />
end <strong>of</strong> storage. When both EOs were applied individually, P. <strong>lentiscus</strong><br />
EO exhibited bacteriostatic <strong>activity</strong>. However, bactericidal<br />
<strong>activity</strong> was pronounced, especially in the case <strong>of</strong> S. <strong>montana</strong> EO.<br />
Addition <strong>of</strong> the combination <strong>of</strong> P. <strong>lentiscus</strong> EO at 0.1% plus S. <strong>montana</strong><br />
EO at 0.03% (Fig. 2) resulted in populations <strong>of</strong> L. monocytogenes<br />
significantly lower (P < 0.05) than in the samples treated with<br />
P. <strong>lentiscus</strong> EO alone, throughout storage at 5 1 C. The populations<br />
<strong>of</strong> L. monocytogenes were kept below 5 log CFU/g up to day<br />
4, <strong>and</strong> then decreased to reach 4.1 log CFU/g by the end <strong>of</strong> storage.<br />
On the other h<strong>and</strong>, the combination <strong>of</strong> P. <strong>lentiscus</strong> EO at 0.2% plus<br />
S. <strong>montana</strong> EO at 0.03% resulted in decreased populations <strong>of</strong> the<br />
pathogen strain without any significant difference (P > 0.05) than<br />
in the samples treated with P. <strong>lentiscus</strong> EO at 0.1% plus S. <strong>montana</strong><br />
EO at 0.03%, throughout storage, <strong>and</strong> reached 3.9 log CFU/g, by the<br />
end <strong>of</strong> storage. Combining P. <strong>lentiscus</strong> EO <strong>and</strong> S. <strong>montana</strong> at 0.1 <strong>and</strong><br />
0.06% respectively, resulted in populations <strong>of</strong> L. monocytogenes<br />
significantly lower (P < 0.05) than in the samples treated with<br />
P. <strong>lentiscus</strong> EO at 0.1e0.2% plus S. <strong>montana</strong> EO at 0.03%, throughout<br />
storage. Populations <strong>of</strong> L. monocytogenes were found below<br />
3 log CFU/g throughout storage. However, when a combination <strong>of</strong><br />
P. <strong>lentiscus</strong> EO at 0.2% plus S. <strong>montana</strong> EO at 0.06% was used, the<br />
populations <strong>of</strong> L. monocytogenes decreased to reach 1.2 log CFU/g by<br />
the end <strong>of</strong> storage, showing a high antibacterial effect (P > 0.05) to<br />
those <strong>of</strong> the combination use.<br />
The combination containing higher concentrations <strong>of</strong> S. <strong>montana</strong><br />
EO (0.06%) was more effective during the whole period <strong>of</strong><br />
Fig. 1. Inhibition <strong>of</strong> L. monocytogenes by P. <strong>lentiscus</strong> <strong>and</strong> S. <strong>montana</strong> EOs in minced beef<br />
stored at 5 1 C: (A) Control; (-) P. <strong>lentiscus</strong> (0.2%); (:) S. <strong>montana</strong> (0.06%).
D. Djenane et al. / Food Control 22 (2011) 1046e1053 1051<br />
Fig. 2. Inhibition <strong>of</strong> L. monocytogenes by combination <strong>of</strong> P. <strong>lentiscus</strong> <strong>and</strong> S. <strong>montana</strong> EOs<br />
at different concentrations in minced beef stored at 5 1 C: (A) Control; (&z.squf;)<br />
P. <strong>lentiscus</strong>/S. <strong>montana</strong> (1/1); (C) P. <strong>lentiscus</strong>/S. <strong>montana</strong> (2/1); (:) P. <strong>lentiscus</strong>/<br />
S. <strong>montana</strong> (1/2); (B) P. <strong>lentiscus</strong>/S. <strong>montana</strong> (2/2).<br />
storage. However, when both EOs were combined in minced beef, it<br />
appeared that all treatments containing 0.03% <strong>of</strong> S. <strong>montana</strong> EO,<br />
exerted bacteriostatic activities depending on the period <strong>of</strong> storage.<br />
For example, if the reduction rates were notably different in day 2<br />
(1.35e2.35 log CFU/g), they were closely similar in the rest <strong>of</strong><br />
storage. However, bactericidal <strong>activity</strong> was pronounced, especially<br />
in the case <strong>of</strong> S. <strong>montana</strong> EO applied at a concentration <strong>of</strong> 0.06%.<br />
The initial meat pH <strong>of</strong> 5.7e5.8 in the samples decreased to about<br />
5.6 after treatment with EOs (data not shown). The values <strong>of</strong> pH did<br />
not differ significantly (P > 0.05) within treatments throughout<br />
storage. The fact that initial meat pH decreased slowly in the<br />
presence <strong>of</strong> EOs <strong>and</strong> that there were no significant differences<br />
(P > 0.05) within treatments. The buffering capacity <strong>of</strong> meat may<br />
explain this phenomenon (Djenane et al., 2011). Further reviews<br />
<strong>of</strong> published literature revealed that other herbs are also as effective<br />
in inhibiting L. monocytogenes. Aureli, Costantini, <strong>and</strong> Zolea<br />
(1992) found that thyme EO at about 0.25% resulted in the reduction<br />
<strong>of</strong> initial populations <strong>of</strong> L. monocytogenes in minced pork by 2<br />
<strong>and</strong> 2.3 log CFU/g after 8 days <strong>of</strong> storage at 4 <strong>and</strong> 8 C, respectively.<br />
The means by which microorganisms are inhibited by EOs seems to<br />
involve different modes <strong>of</strong> action. The potent antimicrobial activities<br />
<strong>of</strong> S. <strong>montana</strong> observed in this study can be attributed to the<br />
presence <strong>of</strong> high concentrations <strong>of</strong> carvacrol, thymol <strong>and</strong> p-cymene,<br />
which have a well documented antibacterial <strong>and</strong> antifungic<br />
potential (Oussalah et al., 2007). Mourey <strong>and</strong> Canillac (2002) tested<br />
the bacteriostatic <strong>and</strong> bactericidal activities <strong>of</strong> six components <strong>of</strong><br />
P. <strong>lentiscus</strong> <strong>and</strong> S. <strong>montana</strong> EOs <strong>and</strong> found that a-pinene was the<br />
most active component with an average MIC <strong>of</strong> 0.019% against<br />
L. monocytogenes serovar 4b, though this concentration was directly<br />
bactericidal. Lis-Balchin <strong>and</strong> Deans (1997) showed that EOs containing<br />
large amounts <strong>of</strong> 1,8-cineole were better anti-listerial<br />
agents than EOs that do not contain 1,8-cineole (herein, abundant<br />
in P. <strong>lentiscus</strong> EO).<br />
The EOs <strong>of</strong> P. <strong>lentiscus</strong> chemotype b-myrcene, characterized by<br />
the presence <strong>of</strong> high concentrations <strong>of</strong> 1,8-cineole <strong>and</strong> terpinen-4-<br />
ol, compounds with well also documented antimicrobial <strong>activity</strong><br />
(Benhammou, Bekkara, & Panovska, 2008), inhibited growth <strong>of</strong><br />
L. monocytogenes <strong>and</strong> Staphylococcus aureus. Preliminary studies<br />
showed that the combination <strong>of</strong> EOs had a greater efficacy than the<br />
EOs separately against foodborne pathogens (Gutierrez, Barry-<br />
Ryan, & Bourke, 2008, 2009). Our findings suggest that the<br />
combined EOs <strong>of</strong> P. <strong>lentiscus</strong> <strong>and</strong> S. <strong>montana</strong> exhibited bacteriostatic<br />
<strong>activity</strong> at low concentrations (0.03%) <strong>of</strong> S. <strong>montana</strong> <strong>and</strong> bactericidal<br />
<strong>activity</strong> when S. <strong>montana</strong> was applied at higher concentrations<br />
(0.06%). Based on the chemical composition <strong>of</strong> the active EOs, it can<br />
be concluded that EO containing carvacrol <strong>and</strong> thymol compounds<br />
is the most potent (S. <strong>montana</strong>), followed by EO containing<br />
b-myrcene <strong>and</strong> 1,8-cineole (P. <strong>lentiscus</strong>). Generally, the major<br />
components are found to reflect quite well the antibacterial effects<br />
<strong>of</strong> the EOs from which they were isolated, the amplitude <strong>of</strong> their<br />
effects being just dependent on their concentration when they<br />
were tested alone or comprised in EOs. Thus, synergistic functions<br />
<strong>of</strong> the various molecules contained in an EO, in comparison to the<br />
action <strong>of</strong> one or two main components <strong>of</strong> the EOs, seems questionable.<br />
The effect <strong>of</strong> food ingredients <strong>and</strong> pH on the antimicrobial<br />
efficacy <strong>of</strong> EO was assessed by monitoring the lag phase <strong>and</strong> the<br />
maximum specific growth rate <strong>of</strong> L. monocytogenes grown in model<br />
media (Gutierrez et al., 2008). The pH <strong>of</strong> meat is an important factor<br />
affecting the <strong>activity</strong> <strong>of</strong> EOs. At low pH, the hydrophobicity <strong>of</strong> some<br />
EOs increases <strong>and</strong> while they may tend to partition in the lipid<br />
phase <strong>of</strong> the food, they can also dissolve more easily in the lipid<br />
phase <strong>of</strong> the bacterial membrane <strong>and</strong> have enhanced antimicrobial<br />
action (Juven, Kanner, Schved, & Weisslowicz, 1994). Synergism<br />
between EOs <strong>and</strong> other parameters in antimicrobial action must be<br />
therefore considered. Holley <strong>and</strong> Patel (2005) found that plant EOs<br />
were more effective in low than in high fat product. In agreement<br />
with these findings, Smith-Palmer, Stewart, <strong>and</strong> Fyfe (2001)<br />
showed that the active compounds present in various EOs had<br />
a stronger <strong>and</strong> a broader spectrum <strong>of</strong> antimicrobial <strong>activity</strong> against<br />
L. monocytogenes in low fat than in high fat product. Furthermore,<br />
since another important aspect for the optimized application <strong>of</strong> EOs<br />
in food is the evaluation <strong>of</strong> interaction with meat ingredients.<br />
3.4. Sensory analysis<br />
Sensory evaluation scores <strong>of</strong> minced beef meat samples treated<br />
with single EOs at tw<strong>of</strong>old MIC values <strong>and</strong> combined EOs at rations<br />
from 1/1 to 2/2 during refrigerated storage at 5 1 C, are shown in<br />
Tables 4e6. Results showed that the intensity <strong>of</strong> all attributes<br />
Table 4<br />
Effect <strong>of</strong> EOs on discoloration sensory scores (mean st<strong>and</strong>ard deviation) <strong>of</strong> minced beef stored at 5 1 C.<br />
Treatment<br />
Days <strong>of</strong> storage<br />
0 2 4 6 8<br />
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<br />
P. <strong>lentiscus</strong> 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<br />
S. <strong>montana</strong> 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<br />
P. <strong>lentiscus</strong>/S. <strong>montana</strong> (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<br />
P. <strong>lentiscus</strong>/S. <strong>montana</strong> (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<br />
P. <strong>lentiscus</strong>/S. <strong>montana</strong> (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<br />
P. <strong>lentiscus</strong>/S. <strong>montana</strong> (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<br />
Mean values in the same column <strong>and</strong> relating to discoloration are significantly different when accompanied by different letters (P < 0.05).<br />
*1 ¼ none, 2 ¼ 0e10%, 3 ¼ 11e20%, 4 ¼ 21e60%, <strong>and</strong> 5 ¼ 61e100%.
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D. Djenane et al. / Food Control 22 (2011) 1046e1053<br />
Table 5<br />
Effect <strong>of</strong> EOs on <strong>of</strong>f odor sensory scores (mean st<strong>and</strong>ard deviation) <strong>of</strong> minced beef stored at 5 1 C.<br />
Treatment<br />
Days <strong>of</strong> storage<br />
0 2 4 6 8<br />
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<br />
P. <strong>lentiscus</strong> 1.00 00 a 1.00 00 b 1.12 0.35 b 2.00 00 b 3.6 0.25 b<br />
S. <strong>montana</strong> 1.00 00 a 1.00 00 b 1.60 0.50 c 2.60 0.5 c 3.5 0.15 b<br />
P. <strong>lentiscus</strong>/S. <strong>montana</strong> (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<br />
P. <strong>lentiscus</strong>/S. <strong>montana</strong> (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<br />
P. <strong>lentiscus</strong>/S. <strong>montana</strong> (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<br />
P. <strong>lentiscus</strong>/S. <strong>montana</strong> (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<br />
Mean values in the same column <strong>and</strong> relating to <strong>of</strong>f odor are significantly different when accompanied by different letters (P < 0.05).<br />
*1 ¼ none; 2 ¼ slight; 3 ¼ small; 4 ¼ moderate; <strong>and</strong> 5 ¼ extreme.<br />
Table 6<br />
Effect <strong>of</strong> EOs on acceptability sensory scores (mean st<strong>and</strong>ard deviation) <strong>of</strong> minced beef stored at 5 1 C.<br />
Treatment<br />
Days <strong>of</strong> storage<br />
0 2 4 6 8<br />
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<br />
P. <strong>lentiscus</strong> 5.00 0.00 a 4.00 0.00 b 3.80 0.44 b 3.40 0.54 b 3.20 0.44<br />
S. <strong>montana</strong> 5.00 0.00 a 5.00 0.00 a 3.20 0.44 b 3.20 0.44 3.40 0.54<br />
P. <strong>lentiscus</strong>/S. <strong>montana</strong> (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<br />
P. <strong>lentiscus</strong>/S. <strong>montana</strong> (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<br />
P. <strong>lentiscus</strong>/S. <strong>montana</strong> (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<br />
P. <strong>lentiscus</strong>/S. <strong>montana</strong> (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<br />
Mean values in the same column <strong>and</strong> relating to acceptability are significantly different when accompanied by different letters (P < 0.05).<br />
*1 ¼ dislike extremely, 2 ¼ dislike, 3 ¼ nor like or dislike, 4 ¼ like; 5 ¼ like extremely.<br />
increased throughout storage in all samples, though not at the same<br />
rate. Control minced beef reached the highest value, corresponding<br />
to extreme <strong>of</strong>f odor <strong>and</strong> discoloration, at day 8 <strong>of</strong> storage. Untreated<br />
samples were assessed by the panelists with scores above (P < 0.05)<br />
the rejection limit (score 3), whereas samples treated with single or<br />
combined EOs were assessed by the panelists with scores below<br />
(P < 0.05) the rejection limit.<br />
The presence <strong>of</strong> EOs significantly (P < 0.05) extended fresh meat<br />
odor <strong>and</strong> color attributes. These results were in agreement with<br />
those reported by Sánchez-Escalante, Djenane, Torrescano, Beltrán,<br />
<strong>and</strong> Roncalés (2001) <strong>and</strong> Djenane, Sánchez-Escalante, Beltrán, <strong>and</strong><br />
Roncalés (2003), who showed that meat treated with natural plant<br />
extracts, either alone or in combination, maintained their fresh<br />
characteristics at higher levels than controls during refrigerated<br />
storage. It must be also emphasized that panelists did not perceive<br />
any extreme odor related to EOs in minced meat (results not<br />
shown). Consequently, the sensory properties <strong>of</strong> minced beef meat<br />
treated with EOs were acceptable by the panelists at the supplementation<br />
levels. Solomakos, Govarisa, Koidisb, <strong>and</strong> Botsoglou<br />
(2008) showed that the organoleptic properties <strong>of</strong> minced meat<br />
treated with EOs were acceptable at the supplementation levels <strong>of</strong><br />
0.3 <strong>and</strong> 0.6%, but unacceptable at the level <strong>of</strong> 0.9%. However,<br />
Ouattara, Sabato, <strong>and</strong> Lacroix (2001) reported that addition <strong>of</strong> EOs<br />
at 0.9% exerted no negative effects on the flavor <strong>and</strong> appearance <strong>of</strong><br />
cooked shrimps. Therefore, more work on the acceptability <strong>of</strong> these<br />
ingredients will be necessary. Table 6 shows also the acceptability<br />
scores <strong>of</strong> meat samples. The most preferred sample contained<br />
a combination <strong>of</strong> 2/2 <strong>of</strong> EO from P. <strong>lentiscus</strong> <strong>and</strong> S. <strong>montana</strong>,<br />
respectively. Furthermore, all other samples treated with single EO<br />
or with combined EOs were also acceptable by panelists. Nevertheless,<br />
untreated samples were unacceptable by the panelists after<br />
2 days <strong>of</strong> storage.<br />
The antioxidant <strong>activity</strong> may be ascribed to the same chemical<br />
components. Monoterpenes found in these EOs may act as radical<br />
scavenging agents (Tepe et al., 2004). These activities may be<br />
attributed to the presence <strong>of</strong> terpinolene, a- <strong>and</strong> g-terpinene,<br />
a-pinene, b-pinene, carvacrol, thymol, <strong>and</strong> p-cymene found in both<br />
EOs. The combined effects <strong>of</strong> both EOs for preservation <strong>of</strong> the meat<br />
at cold may play a key role. By using this method, a stable <strong>and</strong>, from<br />
a microbiological point <strong>of</strong> view, safe meat can be produced with<br />
low, if any, loss in sensory quality.<br />
4. Conclusion<br />
The results <strong>of</strong> the bioassays, together with the chemical pr<strong>of</strong>ile<br />
<strong>of</strong> the oils <strong>of</strong> P. <strong>lentiscus</strong> <strong>and</strong> S. <strong>montana</strong>, support the possibility <strong>of</strong><br />
using both EOs as potent natural preservatives to contribute in the<br />
reduction <strong>of</strong> experimentally inoculated L. monocytogenes in minced<br />
meat. These preservative effects were enhanced when combined<br />
EOs were used. Combining such results <strong>and</strong> those <strong>of</strong> sensory evaluation<br />
we could propose the use <strong>of</strong> these EOs, to reduce L. monocytogenes<br />
growth <strong>and</strong> extend the shelf-life <strong>of</strong> minced meat during<br />
refrigerated storage.<br />
Acknowledgment<br />
The authors are grateful to Ministerio de Asuntos Exteriores y<br />
Cooperación <strong>of</strong> Spain (AECID) for financial assistance to this work<br />
within the Programa de Cooperación Interuniversitaria e Investigación<br />
Científica PCI/MED Algeria e Spain (grants ALI A/019342/<br />
08 <strong>and</strong> A/023365/09).<br />
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