Food Control 54 (2015) 188e199

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Food Control
journal homepage: www.elsevier.com/locate/foodcont

Antibacterial activity of Oregano, Rosmarinus and Thymus essential

oils against Staphylococcus aureus and Listeria monocytogenes in beef
meatballs
G. Pesavento a, 1, C. Calonico a, 1, A.R. Bilia b, 2, M. Barnabei a, 1, F. Calesini a, 1, R. Addona a, 1,
L. Mencarelli a, 1, L. Carmagnini a, 1, M.C. Di Martino c, 3, A. Lo Nostro a, *
a
Health Sciences Department, Applied Microbiology Laboratory, University of Florence, Viale Morgagni 48, 50132, Florence, Italy
b
Department of Chemistry Ugo Schiff, University of Florence, Via Ugo Schiff 6, 50019, Sesto Fiorentino, Florence, Italy
c
Experimental Medicine Department, Second University of Naples, Via Costantinopoli 16, 80138, Napoli, Italy

a r t i c l e i n f o a b s t r a c t

Article history: Antimicrobial activity of five essential oils (EOs) was investigated up to 72 h against foodborne pathogens
Received 7 October 2014 (Staphylococcus aureus, Listeria monocytogenes, Salmonella enteritidis, Campylobacter jejuni) through disk
Received in revised form diffusion and determination of Minimum Inhibitory Concentrations. The most active EOs were Thymus
26 January 2015
vulgaris and Origanum vulgare, followed by Cinnamomum zeylanicum, Rosmarinus officinalis, and Salvia
Accepted 28 January 2015
Available online 11 February 2015
officinalis. The antimicrobial activity of O. vulgare, Rosmarinus officinalis and T. vulgaris was investigated
against five enterotoxin producers of S. aureus and five L. monocytogenes strains, for different amounts of
time (up to 14 days), at 4
C, in meatballs. Concentrations of 2% and 1% restricted the growth of both the
Keywords:
Essential oils
pathogens but, as a result of panel tests, altered the meat flavor. The cooked meatballs containing 0.5% of
Food preservation EO were acceptable in terms of taste, and the oils were able to suppress concentrations of <102 CFU/g of
Minimum inhibitory concentration the pathogens, revealing the potential use of R. officinalis, T. vulgaris and O. vulgare as food preservatives
Thymus vulgaris at this concentration.
Origanum vulgare © 2015 Elsevier Ltd. All rights reserved.
Rosmarinus officinalis

1. Introduction is fundamental, and international health authorities have directed

One of the main public health problems, according to the World
Health Organization (2002), is food-related diseases, and in
particular, foodborne diseases that are the cause of numerous
complications and many deaths all over the world. In industrialized
countries, their spread is also favored by new lifestyles which have
led the entire population to increasingly resort to the catering in-
dustry, buying ready-to-eat, ready-to-cook and heat-and-eat foods.
To reduce the risks and incidence of foodborne diseases, prevention
* Corresponding author. Tel.: þ39 55 2751079; fax: þ39 55 2751093.
E-mail addresses: giovanna.pesavento@unifi.it (G. Pesavento), carmela.
calonico@unifi.it (C. Calonico), ar.bilia@unifi.it (A.R. Bilia), martina.barnabei@
libero.it (M. Barnabei), francescacalesini@gmail.com (F. Calesini), robadd84@
yahoo.it (R. Addona), lauramencarelli87@gmail.com (L. Mencarelli),
lauracarmagnini@hotmail.it (L. Carmagnini), mariachiaradimartino@gmail.com
(M.C. Di Martino), antonella.lonostro@unifi.it (A. Lo Nostro).
1 ables them to cross the bacterial membranes and act directly on
Tel.: þ39 55 2751079; fax: þ39 55 2751093.
2
Tel.: þ39 55 4573708.
3
Tel.: þ39 81 5665835.
their attention towards production and conservation of food (WHO,
2002). From the middle of the last century, in order to preserve
foods and give them a long shelf life, permitting international trade,
synthetic compounds have been used as additives in food pro-
duction. In recent years, people have expressed strong concerns
about the use of these substances as a result of diseases (Fleming-
Jones & Smith, 2003) associated with their consumption; this has
led to an increased interest in natural substances as food pre-
servatives (Goni et al., 2009; Lv, Liang, Yuan, & Li, 2011). Other
studies (Brenes & Roura, 2010; Burt, 2004) have shown that some
essential oils (EOs) have strong antioxidant and antimicrobial
properties; therefore, they could be used in food production as a
possible alternative to synthetic preservative additives, limiting the
growth of food pathogens and increasing the shelf life of some
foods.
The characteristic that most influences the antimicrobial activity
of these natural extracts is their high hydrophobicity, which en-
them, causing loss of ions and reduction of the membrane poten-
tial, loss of function of the proton pumps and ATP depletion (Di

http://dx.doi.org/10.1016/j.foodcont.2015.01.045
0956-7135/© 2015 Elsevier Ltd. All rights reserved.
 G. Pesavento et al. / Food Control 54 (2015) 188e199
Pasqua, Hoskins, Betts, & Mauriello, 2006), or damage to proteins,
lipids, and organelles present within the bacterial cell (Bakkali,
Averbeck, Averbeck, & Idaomar, 2008), causing cell death.
Many authors have performed studies in vitro on antibacterial
properties on several EOs, (Bakkali et al., 2008; Hyldgaard, Mygind,
& Meyer, 2012) finding minimal inhibitory concentrations (MIC100
and MIC90) values very low (<0.1%) against an initial concentration
higher than 105 CFU/mL of many pathogens, such as Staphylococcus
aureus, Listeria monocytogenes, Salmonella Enteritidis, Campylo-
bacter jejuni, Escherichia coli.
Although there is currently very little knowledge regarding the
effectiveness of the antibacterial activity of EOs in foods and their
mechanisms of interaction with food components (Burt, 2004;
Negi, 2012), some authors (Brenes & Roura, 2010; Gutierrez,
Barry-Ryan, & Bourke, 2008) have shown that, in food, higher
bactericidal concentrations of EO are required than in experimental
media, due to the interaction of some EO compounds with those of
food, such as meat fat (Hsieh, Mau, & Huang, 2001). These high EO
concentrations may lead to exceeding the threshold of acceptability
for the taste of food. However, lower concentrations may be used in
combination with traditional food preservation techniques, such as
refrigeration, modified atmosphere packaging or vacuum pack-
aging, with the aim of suppressing the multiplication of pathogens
and aerobic spoilage flora in perishable foods, such as ready-to-eat
foods, and ready-to-cook fish and meats, especially minced ones
(Chen & Brody, 2013; Nieri, Pesavento, Ducci, Calonico, & Lo Nostro,
2014; Skandamis & Nychas, 2001). In addition, in food in general,
the pathogen concentrations are much lower than in experimental
media, established often to values higher than 105 CFU/g (Liu &
Yang, 2012; Skandamis & Nychas, 2001): in food, Staphylococcus
aureus rarely exceeds 104 CFU/g (Sergelidis et al., 2012) and Listeria
monocytogenes 102 CFU/g (De Cesare, Mioni, & Manfreda, 2007;
Uyttendaele et al., 2009). Consequently, very low concentrations
of EOs might have potentially bactericidal or bacteriostatic effects
in food.
The purpose of this study is to evaluate the antimicrobial ac-
tivity of five essential oils (Origanum vulgare, Thymus vulgaris,
Rosmarinus officinalis, Cinnamomum zeylanicum, and Salvia offici-
nalis), classified as Substances Generally Recognized As Safe (GRAS)
by the FDA (2013), first, in vitro, against S. aureus, L. monocytogenes,
Salmonella enteritidis, and Campylobacter jejuni at optimal growth
conditions as culture broth, and then, in vivo, in a real food system
(only Origanum, Thymus and Rosmarinus) such as raw minced
meat experimentally contaminated with L. monocytogenes or S.
aureus and preserved at 4
C, for different amounts of time up to 14
days. Antibiotic susceptibility was determined for both the patho-
gens, as virulence factors, and a sensory evaluation of the cooked
meat samples was made to evaluate the flavor and organoleptic
properties.
The main antibacterial compounds of O. vulgare EO are carvacrol
and thymol which are present in concentrations close to 15% and
20% (vol/vol) respectively, depending on the chemotype (Burt,
2004; Russo, Galletti, Bocchini, & Carnacini, 1998).
Thymol and carvacrol are also the main antimicrobial constitu-
ents of the T. vulgaris EO. With concentrations ranging from 10% to
64%, and from 2% to 11% respectively, they represent the mono-
terpenes with the highest bactericidal power present in the
composition of many EOs, due to their phenolic nature (Yanishlieva,
Marinova, & Pokorny, 2006).
The antimicrobial properties of the Cinnamomum zeylanicum EO
are mainly due to the action of two compounds: cinnamaldehyde
(concentrations up to 80%), and eugenol (representing about 4%)
(Lens-Lisbonne, Cremieux, Maillard, & Balansard, 1987). At high
doses, cinnamaldehyde and eugenol can cause serious damage to
the bacterial wall leading to cell lysis (Yehouenou et al., 2012).
189
Rosmarinus officinalis EO contains 1.8-cineol (26e51%), camphor
(4.9e29%), a-pinene (7e11%), camphene (3.3e12, 8%) and borneol
(2.2e12%) (Zaouali, Bouzaine, & Boussaid, 2010). The mechanism of
action of these compounds has not yet been fully clarified, and only
in the case of 1.8-cineol was it possible to identify a specific activity
against the bacterial membrane (Burt, 2004).
The S. officinalis EO contains numerous active molecules; the
most abundant is a-thujone (1e36.9%), followed by a-pinene
(1.2e5.9%), camphene (0.5e5.9%), b - pinene (1.2e5.3%), 1,8-cineole
(6.7e20.5%), b-thujone (0.2e28.7%), camphor (3.2e12.3%), bornyl
(0.5e7.9%), b-caryophyllene (1.5e15.8%). Among them, sesqui-
terpens and b-thujone were shown to have the greater antibacterial
properties (Lamien-Meda et al., 2009).
This study was aimed at identifying “natural” substances that
could eventually replace synthetic additives in food (raw meat and
fish, ready-to-eat, ready-to-cook and heat-and-eat) preservation
(Faleiro et al., 2003; Hyldgaard et al., 2012) together with hurdle
technologies (Barbosa et al., 2009; Chen & Brody, 2013; Davies,
1995; Nieri et al., 2014), and in particular on raw meatballs that
could be prepared by food industries and preserved at 4
C until
home cooking.
2. Materials and methods
2.1. Essential oils
2.1.1. Preparation of dilutions of EOs
The EOs used in this study (Oregano, Cinnamomum, Rosmar-
inus, Salvia, and Thymus) were extracted by steam distillation
method, and purchased from the same retailer (Prodotti Phito-
cosmetici Dott. Vannucci di Vannucci Daniela e C. Sas). Dilutions of
the EO, for disk diffusion assay, were made in sterile glass using
distilled water; 0.5% Dimethyl Sulfoxide (DMSO, Carlo Erba
Reagenti) was added. The dilutions, in a final volume of 2 mL,
were: 25%, 50%, 75%, 100% (vol/vol). Dilutions used for MIC
determination were in Mueller Hinton Broth (MHB, Oxoid), con-
centrations were different for each EO and bacterial species; 0.5%
DMSO was added. The addition of DMSO, an aprotic organic sol-
vent belonging to the category of sulfoxides, had the purpose of
facilitating the solubilization of EOs in the culture media. All EOs
were stored at 4
C in darkness before use and utilized before the
expiration date. EO dilutions were prepared just before the
experiments.
Experiments “in vivo” were performed adding the suitable vol-
ume of EO to the meat without making any dilution and without
using DMSO which could be potentially toxic for eukaryotic cells.
2.2. “In vitro” experiments
2.2.1. Preparation of microbial suspensions and media
Two different strains of each microorganism were used; one was
an ATCC (American Type Culture Collection) strain, and the other
was previously isolated from a food product from the Health Sci-
ences Dept. (HSD) BioBank: S. aureus (ATCC 25923, HSD 3623), L.
monocytogenes (ATCC 7644, HSD 3509), S. Enteritidis (ATCC 13076,
HSD 3657) and C. jejuni (ATCC 33291, HSD 3486). All ATCC strains
and media were purchased from Oxoid. Before they were used, the
pathogens were cultured in Brain Heart Infusion Broth (BHI) for
24 h at 37 ± 1
C and then streaked on Tryptone Soy Agar (TSA), and
incubated at 37
C for 24 h. Tubes were prepared for each bacterial
strain in sterile deionized water, with a turbidity of 0.5 McFarland
or 1 McFarland, to perform disk diffusion assays and MIC deter-
mination, respectively, using a McFarland standard (bioMe
rieux).
Serial dilutions 101 to 105 of each bacterial suspension were
streaked on TSA Petri dishes in order to count the microorganisms
190 G. Pesavento et al. / Food Control 54 (2015) 188e199
and verify that the number of bacteria in the samples was appro-
priate for the performance of the tests.
Mueller Hinton Agar (MHA) and MHB, used respectively for
performing the disk diffusion assays and the determination of MIC,
were enriched with a suitable volume of DMSO, thus obtaining 0.5%
(vol/vol) solutions identified by the abbreviations of DMHA and
DMHB.
2.2.2. Agar disk diffusion assay
The preliminary determination of the inhibitory effect of EOs (O.
vulgare, T. vulgaris, R. officinalis, C. zeylanicum, and S. officinalis) on the
eight bacterial strains was determined by agar disk diffusion method
(Rota, Carramin  ana, Burillo, & Herrera, 2004). Bacterial suspensions,
of about 105 CFU/mL, were streaked on Petri dishes containing DMHA
obtaining confluent growth. Sterile filter paper disks (Oxoid) of 6 mm
diameter were soaked with 10 mL of each oil's dilution and placed on
the surface of the dishes. The antibiotic meropenem (10 mg, Oxoid)
and a solution of DMSO 0.5% (vol/vol) in sterile deionized water were
used respectively as positive and negative controls. The plates were
incubated at 37
C for 24 h aerobically. After incubation, the diameter
of the inhibition zones was measured in millimeters, including the
diameter of disks. The strain sensitivity to each EO dilution was
classified by the diameter of the inhibition zones as follows: Not
sensitive for total diameter smaller than 8 mm, Sensitive for total
diameter 9e14 mm, Very sensitive for total diameter 15e19 mm, and
Extremely sensitive for total diameter larger than 20 mm (Ponce, Fritz,
del Valle, & Roura, 2003). Each assay was performed in triplicate on
three separate experimental runs.
2.2.3. Determination of minimum inhibitory concentration (MIC100
and MIC90) and minimum bactericidal concentration (MBC)
MIC and MBC are generally considered as a measure of antimi-
crobial performance of EOs. Determination of MIC assay was per-
formed as described by Weerakkody, Caffin, Turner, and Dykes
(2010) with some modifications to the method. Aliquots of 100 mL
of each bacterial suspension prepared, with a turbidity of 1
McFarland, corresponding approximately to 107 CFU/mL, were
added to each tube containing the serial dilutions of the essential
oils (Cinnamomum, Oregano, Rosmarinus, Salvia, and Thymus). The
positive control was obtained by preparing a test tube containing
2 mL MHB and 100 mL of the bacterial suspension; the negative
control contained 2 mL of DMHB, 100 mL of the bacterial suspension,
and 10 mL of the antibiotic meropenem. As further controls, in order
to evaluate the sterility of the tested oil and the effect of DMSO on
bacterial cells, a solution of 2 mL of DMHB and 100 mL of each
essential oil and a solution of 2 mL of DMHB and 100 mL of the
bacterial suspension were prepared, respectively. All tubes were
tightly capped to avoid evaporation of EOs. At time “0”, from each
tube so arranged, 100 mL of the suspension was spread on TSA Petri
dishes, which were incubated at 37
C aerobically; after 24 h the
colonies were counted. The tubes were then incubated at 37
C
aerobically. The effect of EOs on bacteria was monitored at intervals
of 24 h up to 72 h by seeding 100 mL of the suspensions on TSA Petri
dishes, as previously described.
From the data obtained by all bacterial counts it was possible to
provide a description of the bactericidal and/or bacteriostatic ac-
tivity of each tested oil against each microorganism, in terms of
MIC100, MIC90 and MBC. The MIC100 value had been determined as
the lowest concentration of oil able to inhibit completely the
growth of microorganisms in tubes and on plates after incubation;
MIC90 was determined as the minimum concentration of essential
oil capable of inhibiting 90% of the bacterial growth; MBC was
defined as the minimum concentration of EO capable of killing all
bacteria. Each assay was performed in triplicate on three separate
experimental runs.
2.3. Determination of Origanum vulgare, Thymus vulgaris and
Rosmarinus officinalis EO composition
EO composition was determined only for the more effective EOs:
O. vulgare, Rosmarinus officinalis and T. vulgaris.
GC analyses were accomplished with an HP-5890 series II
instrument equipped with an HP-5 capillary column
(30 m  0.25 mm, 0.25 m film thickness), working with the
following temperature program: 60
C for 10 min, ramp of 5
C/
min to 220
C; injector and detector temperatures, 250
C; car-
rier gas, nitrogen (2 mL/min); detector, dual FID; split ratio, 1:30;
injection, 0.5 mL. The identification of the components was per-
formed, for both columns, by comparison of their retention times
with those of pure authentic samples and by means of their
linear retention indices (LRI) relative to the series of n-hydro-
carbons. Gas chromatographyeelectron impact mass spectrom-
etry (GCeEIMS) analyses were performed with a Varian CP 3800
gas chromatograph (Varian, Inc. Palo Alto, CA) equipped with a
DB-5 capillary column (Agilent Technologies HewlettePackard,
Waldbronn, Germany; 30 m  0.25 mm, coating thickness
0.25 mm) and a Varian Saturn 2000 ion trap mass detector.
Analytical conditions were as follows: injector and transfer line
temperature at 250 and 240
C, respectively; oven temperature
was programmed from 60 to 240
C at 3
C/min; carrier gas,
helium at 1 mL/min; splitless injector. Identification of the con-
stituents was based on comparison of the retention times with
those of the authentic samples, comparing their LRI relative to
the series of n-hydrocarbons, and on computer matching against
commercial [NIST 98 (U.S. National Institute of Standards and
Technology) and ADAMS (Adams, 1995)] and homemade library
mass spectra built from pure substances and components of
known samples and MS literature data (Adams, 1995; Davies,
1990; Jennings & Shibamota, 1980; Massada, 1976; Stenhager,
Abrahamsson, & Lafferty, 1974; Swigar & Silvestein, 1981).
Moreover, the molecular weights of all the identified substances
were confirmed by gas chromatographyechemical ionization
mass spectrometry (GCeCIMS), using methanol as chemical
ionization gas.
2.4. “In vivo” experiments
2.4.1. Preparation of bacterial strains
Five strains of L. monocytogenes and S. aureus isolated from food
samples and belonging to the HSD BioBank were used.
L. monocytogenes strains were n. HSD 2434, HSD 3261, HSD 3705,
HSD 3948, HSD 4210, respectively of serotypes 1/2b, 4b, 4b, 1/2a
and 1/2c; serotype determination was previously performed using
L. monocytogenes antiserum kit (Denka Seiken Co). S. aureus strains
were n. HSD 244, HSD 2866, HSD 2874, HSD 3320, HSD 4128
respectively producing Staphylococcal Enterotoxins (SE) E D, A,
A þ B, B, and C. SE determination was previously performed using
SET RPLA Kit (Oxoid).
In addition, ATCC strains (Oxoid) for S. aureus (25923) and
L. monocytogenes (7644) were tested.
All strains were maintained at 80 ± 1
C in BHI containing 10%
glycerol. The 12 strains were inoculated in 9 mL of BHI broth and
incubated at 35 ± 1
C for 24 h. The suspensions were used to obtain
inocula of 1 McFarland in sterile saline solution containing culture
in exponential growth phase of approximately 107 CFU/mL. To
obtain bacterial count, 100 mL of the serial diluted suspensions were
spread on TSA and incubated at 35 ± 1
C for 24 h, after which the
colonies were counted. The suspensions diluted were used to
experimentally inoculate the meat samples at the final desired
concentrations (about 104 CFU/g e Dilution (DIL) 1; 103 CFU/g e DIL
2; 102 CFU/g e DIL 3; 10 CFU/g e DIL 4).
 G. Pesavento et al. / Food Control 54 (2015) 188e199 191

2.4.2. Preparation of meat samples cooked meat (at 200
C for 20 min): one made of meat added
For the present study we purchased 3.3 Kg of raw beef minced only of the EOs at the three concentrations, and the other made
meat for each EO test on each microorganism. The meat was kept at in the same manner plus boiled and chopped potatoes (30%),
about 4
C until the start of the experiments. Each test was per- eggs (10%) and breadcrumbs (3%). The panelists had to fill in a
formed in duplicate. questionnaire in which 1 was the worst (unacceptable) and 5
12 samples of 25 g of raw minced meat were placed in plastic was the best (good). The samples that presented mean scores
bags. One of them was used at the initial time of the experiment lower than 3.5 were considered unacceptable; raw samples
(T0) to evaluate Total Viable Count (TVC), Staphylococcus and Lis- judged unacceptable were not used for the taste sensory evalu-
teria presence, following respectively ISO 4833 (2004), 6888-1 ation of cooked meat.
(2004), 11290-1 (2005). Five samples were used to obtain TVC at All experiments were performed in compliance with CFR
the experimental time intervals of 2, 4, 7, 11 and 14 days (respec- 46.101(b). All panelists were informed completely about the study
tively T1, T2, T3, T4, T5) after T0. The other 6 samples served for the and signed informed consent.
sensory evaluation of the raw meat at the 6 time intervals. These 12
samples were named White Samples (WSs).
18 samples of 25 g of minced meat were added with the EO in Table 1
the following concentrations: 0.5%, 1%, or 2% (v/w). These sam- Chemical composition (%) and principal chemical classes (%) of the tested essential
ples were used as Negative Controls (NCs) to calculate TVC in oils.
presence of EO at the experimental times (T0, T1, T2, T3, T4, T5). Constituents LRI Origanum Rosmarinus Thymus
Another 36 samples were prepared in the same way as the NCs: vulgare officinalis vulgaris
18 served for the sensory evaluation of the raw meat, and the tricyclene 928 0.2 tr
other 18 were used for sensory evaluation of color, odor and a-thujene 933 tr
flavor after cooking. a-pinene 941 1.7 11.5 4.3
96 samples of 25 g of minced meat were experimentally inoc- camphene 955 0.4 4.1 0.1
thuja-2,4(10)-diene 959 tr
ulated with the suspensions of the pathogen (Staphylococcus or
b-pinene 982 0.4 3.8 1.2
Listeria) at the following final concentrations of about 10, 102, 103 myrcene 993 1.3 1.3
and 104 CFU/g. 24 of them were analyzed, at each time interval, to a-phellandrene 1006 tr 0.2
evaluate the growth of the pathogen in absence of the EOs as d-3-carene 1013 tr
Positive Controls (PCs). To the other 72 samples, the appropriate 1,4-cineole 1018 0.1
a-terpinene 1020 0.8 0.4
volume of each EO was added to reach final concentrations of 0.5%, p-cymene 1027 11.6 1.9 47.9
1%, or 2% v/w. In these experimental samples the antimicrobial limonene 1032 1.1 1.8 0.2
activity of the three EO concentrations on the 4 pathogen con- 1,8-cineole 1034 0.6 43.9 0.2
centrations was evaluated. g-terpinene 1063 1.7 0.4
cis-sabinene hydrate 1070 tr
All plastic bags containing the samples were stored at 4
C until
terpinolene 1090 0.2 0.3
the microbiological analysis or sensory evaluation was performed 1-pentyl butyrate 1094 tr
(2, 4, 7, 11 and 14 days). linalool 1101 1.8 0.9 1.2
exo-fenchol 1118 tr tr
2.4.3. Initial microbiological analysis (T0) trans-pinocarveol 1141 tr
camphor 1145 tr 11.3
At T0 a WS was blended (Stomacher 400) for 60 s at room isoborneol 1158 0.2
temperature in 225 mL Buffered Peptone Water. Decimal di- trans-pinocamphone 1162 tr
lutions were carried out using the same diluent. TVC were ob- pinocarvone 1164 tr
tained on duplicate plates of Tryptone Glucose Yeast Agar borneol 1168 0.4 4.2
cis-pinocamphone 1175 tr
incubated at 32 ± 1
C for 48 h, S. aureus count was obtained on
4-terpineol 1178 0.2 0.8
Baird Parker agar incubated at 35 ± 1
C for 48 h and Listeria a-terpineol 1190 0.4 2.6 0.6
strains were isolated by using Palcam Agar, incubated for 24 h at verbenone 1206 0.2
37 ± 1
C. The suspected colonies of Listeria were characterized isobornyl acetate 1287 0.2 0.7
by Gram stain. Gram positive colonies were tested for hemolysis thymol 1292 1.6 43.1
carvacrol 1301 71.8 0.4
on Columbia Blood Agar and Bactident Catalase (Merk). Species a-cubebene 1352 tr
identification was made with API Listeria kit and Api Staph a-ylangene 1373 0.2
(bioMe
rieux). a-copaene 1377 tr 0.6
b-caryophyllene 1419 2.7 5.1 0.2
trans-a-bergamotene 1437 tr
2.4.4. Antimicrobial activity assay in food
a-guaiene 1440 0.2
Antimicrobial activity of Oregano, Rosmarinus, and Thymus was a-humulene 1455 0.2 0.5 tr
evaluated at the 6 time experimental intervals of preservation of g-muurolene 1478 0.6
the meat samples at 4
C, monitoring TVC count and each pathogen viridiflorene 1494 0.2
concentration, as described in Section 2.4.3, for each EO dilution. a-muurolene 1499 0.2
b-bisabolene 1509 0.2
trans-g-cadinene 1514 0.4
2.4.5. Sensory evaluation d-cadinene 1524 0.9
Sensory evaluation of all food samples, added or not of the caryophyllene oxide 1582 0.6 0.3 tr
EOs at the three concentrations (0.5%, 1%, 2% v/w), was assessed Monoterpene hydrocarbons 19.2 25.9 53.7
Oxygenated monoterpenes 77.2 64.6 45.6
by a panel consisting of 5 panelists selected from students and
Sesquiterpene hydrocarbons 2.9 9.1 0.2
staff of the laboratory. At the initial time of the experiment, and Oxygenated sesquiterpenes 0.6 0.3 tr
after 2, 4, 7, 11 and 14 days of preservation of meat samples at Phenylpropanoids e e e
4
C, the panelists had to judge the acceptance of color and odor Other derivatives tr e e
of the raw meat samples added of the EOs at the three con- Total identified 99.9 99.9 99.5

centrations, and the flavor, color and odor of two groups of tr ¼ traces (<0.1%); LRI ¼ Linear Retention Index.
192 G. Pesavento et al. / Food Control 54 (2015) 188e199

2.5. Statistical analysis

DMSO

0.5%

6
6
6
6
6
6
6
6
The growth data from plate counts were transformed to log10
values. The standard descriptive statistics of the contamination

Meropenem
(mean, standard deviations) and comparison test (Fisher's exact test)

10mg/disk

25. 7
31.3

33.3
32.3

22.7
were made using Stata/SE 8.0 (StataCorp, College Station, TX, USA).

29

33

23
2.6. Antibiotic susceptibility test

100%

33.5

31.5

32.3

31.7
30

27

33

28
Antibiotic susceptibility of all Staphylococcus and Listeria tested
strains was determined through the standard disk diffusion method

Thymus vulgaris
of KirbyeBauer (Bauer, Kirby, Sherris, & Turck, 1966; EUCAST, 2013a)

20.5
27.3
26.3

21.3
75%

22
24
26

29
on MHA and MHA added of 5% mechanically defribrinated horse
blood (Oxoid) and 20 mg/L b-NAD (SIGMA ALDRICH), respectively.

16.5

26.3
18.7
The Petri dishes were incubated at 37
C for 24 h. Reference strains

50%

18
17
19
11
12
were L. monocytogenes ATCC 19114 and S. aureus 25923.
Disks containing the following antibiotics (Oxoid) were spotted

11.5
11.5
13.5
10.3
10.7
25%

11

12
11
with a 3 cm interval on Petri dishes seeded with confluent growth
of S. aureus and L. monocytogenes: ampicillin 2 mg, cephalothin e

100%

15.7

22.7
9.67

10.3
30 mg, cefoxitin e 30 mg (only for Staphylococcus), clindamycin e

16
18

10
9
2 mg, chloramphenichol e 30 mg, erytromycin e 15 mg, gentamycin

Salvia officinalis
e 10 mg, kanamicin e 30 mg (only for Staphylococcus), linezolid e

10.3
11.3

15.3

7.33
8.33
7.33
75%

13

9
10 mg (only for Staphylococcus), meropenem e 10 mg (only for Lis-
teria), meticillin - 5 mg (only for Listeria), oxacillin e 1 mg, penicillin

7.33
8.67
G e 10 U.I., teicoplanin e 10 mg, tetracyclin e 30 mg, trimethoprim-

50%

12
9

8
7
7
6
sulfamethoxazole e 25 (1.25 þ 23.75) mg, vancomycin - 30 mg. Re-

Means of the inhibition diameters in mm from disk diffusion tests (n ¼ 3). Standard Deviations are not shown because they are all 1.
sults were interpreted following EUCAST breakpoint tables

25%
Essential oil concentration (Vol/Vol e mL/mL)

6
6
6
6
8
6
6
6
(EUCAST, 2013b) and, where not possible, according to CLSI (2012)
indications.

100%

19.7
27.3
20.3
20.7
16.3
20.3
25.3
24
3. Results and discussion
Origanum vulgare

22.7
16.3

15.7
15.3
24.7
75%

17

19

17
3.1. Essential oil composition

10.7

13.3
11.7
9.33

10.7
Constituents were identified by comparison of their retention
50%

18

15
13
times of both columns with those of pure authentic samples and by
means of their linear retention indices (LRI) relative to the series of
10.3
11.7
12.3
10.7
8.67
10.3
12.7
25%

10
n-hydrocarbons and MS data from a home-made library mass
spectra and literature (Table 1). Our results were comparable with
100%

28.7
13.3

20.3
18.3
19.7
28.3
those found in literature (Burt, 2004; Lamien-Meda et al., 2009;

HSD ¼ Strains from the Health Sciences Department (University of Florence) collection.
24

25
Cinnamomum zeylanicum

Lens-Lisbonne et al., 1987; Russo et al. 1998; Yanishlieva et al.,

2006; Yehouenou et al., 2012; Zaouali et al., 2010). Almost 100% of
21.3

24.7
19.3
18.3
19.3
75%

26
12

22

the volatiles of O. vulgare EO were identified, 77.2% being oxygen-

ated monoterpenes, principally represented by carvacrol repre-
senting 71.8% of the total essential oil, 19.2% of the constituents
20.3

23.7

17.3
14.3
17.7
50%

23

18
9

were represented by monoterpene hydrocarbons, principally p-

cymene, 2.9% were sesquiterpenes hydrocarbons, and 0.6%
13.7

11.7
25%

20

22

16
11
7
6

oxygenated sesquiterpenes.
Also in the case of R. officinalis EO, the identified volatiles were
99.9% and major constituents were represented by oxygenated
100%

16.3
19.7
23.3
26.7
9.67
14.7
17.7
11.3

monoterpenes (64.6%), the main volatile being 1,8-cineole (43.9%).

Rosmarinus officinalis

Monoterpene hydrocarbons were 25.9%, principally a-pinene.

10.3
9.33

12.7
8.67
8.67
13.3
9.33
75%

Sesquiterpene hydrocarbons were 9.1% and oxygenated sesquiter-

9

penes only 0.3%.

Total identified constituents of T. vulgaris EO were 99.5%. These
9.67

7.67

7.93
50%

8
6
6
6

volatiles were characterized by 53.7% of monoterpene hydrocar-

bons being 47.9% p-cymene and oxygenated monoterpenes 45.6%,
7.33
8.33
7.67
25%

principally thymol (43.1%). Only 0.2% of the volatiles were sesqui-

8
6
6
6
6

terpenes hydrocarbons.
S. enteriditis HSD
S. enteridis ATCC
L. monoc. ATCC

S. aureus ATCC

3.2. “In vitro” experiments

L. monoc. HSD

C. jejuni ATCC
S. aureus HSD

C. jejuni HSD

3.2.1. Agar disk diffusion assay

Table 2

Table 2 shows the antimicrobial inhibition zones, including the

diameter (6 mm) of the paper disk, of the five EOs, meropenem and
 G. Pesavento et al. / Food Control 54 (2015) 188e199 193

DMSO, against the eight tested microorganisms. Thymus was the
most effective EO: the inhibition diameters of 75% and 100% (v/v)
concentrations were larger than 20 mm on all the bacteria tested.
Furthermore, the diameters of all EO dilutions were larger than
9 mm (Ponce et al. 2003). Thymus showed the highest activity
against all tested microorganisms. Inhibition diameters measured
on meropenem were often larger than EO ones, with significant
differences (P < 0.05), while DMSO did not influence bacterial
growth.
3.2.2. Determination of MIC90, MIC100 and MBC
Through disk diffusion test, it was not possible to establish
which were the most inhibited bacteria between Gram-negative
and Gram-positive; thus we determined MIC100 and MIC90 as
Table 3 shows. Fig. 1 graphs show the changes in bacterial load over
time; among all tested foodborne pathogens, we chose to show
only the results of L. monocytogenes HSD and S. Enteritidis HSD since
they are pathogens of great concern in food safety and, in general,
foodborne bacteria are more resistant to EOs than ATCC strains. The
MIC values confirmed the results obtained by the agar disk
diffusion method. Salvia EO has been shown ineffective against all
tested microorganisms: bactericidal and bacteriostatic effects were
observed at concentrations too high to be used as additives in food
production (Fig. 1a and b).
Rosmarinus was more effective against Gram-negative bacteria
in lower concentrations than the Gram-positive ones. Listeria HSD
has proved to be much more resistant than the ATCC strain. After
1 h of contact with the oil, the microbial species with the lowest
MIC100 and MIC90 were respectively S. enteriditis ATCC and C. jejuni
ATCC. Both the graphs in Fig. 1c and d, and also the results in
Table 2, show that most values of MIC100 and MIC90 of Rosmarinus
remain constant from 24 h onwards, so we could deduce that
MIC100 corresponded to MBC.
The microorganism that was least inhibited by Cinnamomum
was Listeria. The two strains of Staphylococcus tested have shown a
similar trend except for concentration of 0.0625% (vol/vol) showing
higher EO sensitiveness of the ATCC strain than the HSD one. Even
the Salmonella HSD strain was a little more resistant than the ATCC
one. The HSD strain of Campylobacter was, instead, more sensitive
to C. zeylanicum EO than the ATCC one.

Table 3
MICs 100 and MICs 90 of essential oils against 4 microorganisms (ATCC and HSD). (Number of repetitions n ¼ 3).

MIC 100 (% Vol/Vol e mL/mL) MIC 90 (% Vol/Vol e mL/mL)

1h 24 h 48 h 72 h 1h 24 h 48 h 72 h

Salvia officinalis
L. monocytogenes ATCC 60 60 60 60 30 40 60 60
L. monocytogenes HSD 60 60 60 60 60 60 60 60
S. aureus ATCC 60 12.5 6.25 3.125 25 6.25 3.125 3.125
S. aureus HSD 60 12.5 6.25 3.125 25 3.125 3.125 3.125
S. enteritidis ATCC 1.5625 6.25 6.25 6.25 0.7812 1.5625 3.125 3.125
S. enteritidis HSD 25 6.25 6.25 6.25 1.5625 3.125 3.125 3.125
C. jejuni ATCC 6.25 6.25 6.25 6.25 0.7812 6.25 6.25 6.25
C. jejuni HSD 25 6.25 6.25 6.25 0.7812 6.25 6.25 6.25
Rosmarinus officinalis
L. monocytogenes ATCC 60 5 2 2 25 5 2 2
L. monocytogenes HSD 70 30 30 30 40 30 30 30
S. aureus ATCC 80 10 5 5 40 3 1 3
S. aureus HSD 85 12.5 2 2 30 1 2 2
S. enteritidis ATCC 2 0.5 0.5 0.5 1.25 0.5 0.5 0.5
S. enteritidis HSD 20 2 2 2 1 2 2 2
C. jejuni ATCC 5 1 1 1 1 1 1 1
C. jejuni HSD 60 1 1 1 1 1 1 1
Cinnamomum zeylanicum
L. monocytogenes ATCC 7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5
L. monocytogenes HSD 7.5 7.5 7.5 7.5 7.5 5 7.5 7.5
S. aureus ATCC 1 0.25 0.25 0.0625 0.0312 0.0312 0.0625 0.0625
S. aureus HSD 1 0.25 0.25 0.125 0.0312 0.0312 0.0625 0.125
S. enteritidis ATCC 0.25 0.0625 0.25 0.25 0.0312 0.0625 0.25 0.25
S. enteritidis HSD 0.25 0.25 0.25 0.25 0.125 0.25 0.0625 0.25
C. jejuni ATCC 0.25 0.25 0.25 0.25 0.125 0.25 0.25 0.25
C. jejuni HSD 0.25 0.25 0.25 0.125 0.0625 0.25 0.25 0.25
Thymus vulgaris
L. monocytogenes ATCC 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25
L. monocytogenes HSD 0.25 0.25 0.25 0.25 0.125 0.25 0.25 0.25
S. aureus ATCC 0.25 0.25 0.25 0.25 0.125 0.125 0.125 0.125
S. aureus HSD 0.25 0.25 0.25 0.25 0.125 0.25 0.25 0.25
S. enteritidis ATCC 0.125 0.125 0.125 0.125 0.0625 0.125 0.125 0.125
S. enteritidis HSD 0.125 0.125 0.125 0.125 0.0625 0.125 0.125 0.125
C. jejuni ATCC 0.125 0.125 0.125 0.125 0.0625 0.125 0.125 0.125
C. jejuni HSD 0.125 0.125 0.125 0.125 0.125 0.125 0.125 0.125
Origanum vulgare
L. monocytogenes ATCC 0.125 0.0625 0.0625 0.0625 0.0156 0.0156 0.0156 0.0156
L. monocytogenes HSD 0.125 0.125 0.0625 0.0625 0.0156 0.0156 0.0156 0.0156
S. aureus ATCC 0.5 0.5 0.25 0.25 0.125 0.125 0.0625 0.0625
S. aureus HSD 1 0.25 0.25 0.25 0.125 0.125 0.125 0.125
S. enteritidis ATCC 0.125 0.125 0.125 0.125 0.0312 0.0312 0.0312 0.0312
S. enteritidis HSD 0.125 0.25 0.25 0.25 0.0312 0.0312 0.0312 0.0312
C. jejuni ATCC 0.25 0.25 0.5 0.5 0.0156 0.0312 0.0625 0.0625
C. jejuni HSD 0.125 0.125 0.125 0.125 0.0312 0.0312 0.0312 0.0312

HSD ¼ Strains from the Health Sciences Department (University of Florence) collection.
194 G. Pesavento et al. / Food Control 54 (2015) 188e199

Fig. 1. Changes in bacterial load of L. monocytogenes (1a, 1c, 1e, 1g, 1i) and S. enteritidis (1b, 1d, 1f, 1h, 1j) at different concentrations of S. officinalis, R. officinalis, C. zeylanicum, T.
vulgaris and O. vulgare EO (n ¼ 3).
 G. Pesavento et al. / Food Control 54 (2015) 188e199 195

Oregano exerted its bactericidal activity at 0.125% (vol/vol) on
both strains of Staphylococcus and Listeria, after 48 h producing a
significant reduction (P < 0.05) in microbial counts that was greater
than 4 and 7 log respectively. The same concentration of Oregano
had different effects on the two Gram-negative bacteria: it showed
its bactericidal activity on Salmonella ATCC and on Campylobacter
HSD, but had bacteriostatic action on Salmonella HSD and
Campylobacter ATCC.
T. vulgaris EO at 0.125% (vol/vol) showed bactericidal activity on
both the Gram-negative after 24 h of incubation, while the per-
centage able to kill the Gram-positive was higher, than the Gram-
negative one, at 0.25% (vol/vol). T. vulgaris showed a strong anti-
microbial activity against the four pathogenic bacteria with a MIC
0.5% (vol/vol). Different MIC obtained by other authors (Burt,
2004; Oussalah, Caillet, Saucier, & Lacroix, 2007) are probably
due to different characteristics of the EOs used, thanks to the large
variability of the chemical composition of each essential oil
chemotype.
3.3. Susceptibility test
Due to the fact that, frequently, bacterial increased resistance
is accompanied by increased virulence thanks to plasmids that
carry both factors (Beceiro, Toma
s, & Bou, 2013), we decided to
analyze antibiotic resistance of Listeria and Staphylococcus
strains. Results of antibiotic resistance tests and characteristics of
the two pathogens are shown in Table 4. Although the tested
Listeria strains were virulent, being of the most frequently iso-
lated serotypes from cases of listeriosis, three of them were
susceptible to all antibiotics used in common therapy (Pesavento,
Ducci, Nieri, Comodo, & Lo Nostro, 2010), one strain was resistant
to vancomycin, and another to meropenem. The enterotoxin
producer Staphylococcus strains also showed sensitivity to anti-
biotics currently used in the treatment of staphylococcal in-
fections (Rayner & Munckhof, 2005), with the exception of the
producing SEA, which was resistant to erythromycin and
teicoplanin.
3.4. “In vivo” experiments
3.4.1. Microbiological analysis at T0
The microbiological analysis on the meat samples at T0 revealed
that the means of the TVC ranged from 103 to 7.4  104 CFU/g (mean
9.5  103 ± 3 CFU/g). None of the purchased meat samples were
contaminated by Staphylococcus and Listeria, confirming their low
Table 4
Antibiotic resistance and characteristics of L. monocytogenes and S. aureus strains.
prevalence on food (De Cesare et al., 2007; Pesavento, Ducci,
Comodo, & Lo Nostro, 2007, 2010; Sergelidis et al., 2012;
Uyttendaele et al., 2009).
3.4.2. EO activity against TVC
No significant differences (p > 0.05) were seen between the
growth of TVC in the absence and presence of the three concen-
trations of the EOs and between all analyzed samples. As an
example, the TVC growth curves of a sample, measured after
various days of preservation at 4
C in presence of 0.5% (v/w) of O.
vulgare EO, are shown in Fig. 2. The results indicated that after an
initial bacteriostasis, seen until the second day, TVC increased in
the presence of EOs (WSs) as in the absence of it (NCs). This is in
accordance with the results obtained by Skandamis and Nychas
(2001) and Barbosa et al. (2009). This phenomenon may be due
to the presence of Enterobacteriaceae and Pseudomonadaceae which
do not seem to be affected by the presence of EOs in aerobic storage.
In fact, from our meat samples we isolated some Enterobacteriaceae
such as: Escherichia coli, Proteus mirabilis, Proteus vulgaris, Cit-
robacter freundii, Enterobacter aerogenes and Enterobacter cloacae.
Other isolated species in decreasing order of magnitude were
Pseudomonas fluorescens, Acinetobacter haemolyticus, Alcaligenes
faecalis, Kocuria varians and Leuconostoc mesenteroides subsp.
cremoris.
3.4.3. Sensory analysis
Results of Panel test (Table 5) showed that raw minced meat
samples, preserved without EO, assumed unacceptable color after
the fourth day; instead it remained almost acceptable, up to the
end of the experiment, in samples added of EO as reported by
other authors (Ferna
ndez-Lo
pez, Zhi, Aleson-Carbonell, Pe

rez-
Alvarez, & Kuri, 2005). The odor of the samples, preserved at 4
C
without EO, was unacceptable after the fourth day, being typical of
putrid meat; instead, the odor of the meat samples (raw and
cooked) added of the three EOs, was perceptible only for the
concentration of 0.5% (v/w); at higher EO concentrations the
panelists could smell only the EO odor. The odor of Oregano was
too strong on all meat samples added with 2% EO, and samples
were judged as unacceptable.
The taste panel test gave the expected results: the cooked
meat without EOs was satisfactory for all the tested samples for
up to 4 days of preservation at 4
C. Only the meat samples
added with potatoes, eggs and breadcrumbs and containing 0.5%
of EO had scores greater than 3.5. Only EO concentrations lower
than or equal to 0.5 (v/w) could be organoleptically acceptable,
not exceeding of the flavor acceptability threshold observed by
the panelists, so they could be used as preservative food
additives.

Strains Antibiotic resistance

Listeria monocytogenes Serotype

HSD 2434 1/2b C, Cip, Ox, Tec, Van

HSD 3261 4b C, Cip, Da, Ox
HSD 3705 4b Te, Da, Ox
HSD 3948 1/2a C, Da, Met, Ox
HSD 4210 1/2c Mem, Ox
ATCC 7644 1/2c Ox
Staphylococcus aureus enterotoxins Antibiotic resistance
HSD 244 SED Amp, C, K, Kf, Ox, Pen
HSD 2866 SEA Amp, Ery, K, Pen, Tec, Tet
HSD 2874 SEA, SEB C, Pen
HSD 3320 SEB Amp, C, K, Pen
HSD 4128 SEC Amp, C, Pen
ATCC 25923 e e

Amp ¼ ampicillin, Kf ¼ cephalothin, Da ¼ clindamycin, C ¼ chloramphenicol, Fig. 2. Total Viable Count of the samples after various days of preservation at 4
C in
Ery ¼ erytromycin, K ¼ kanamicin, Mem ¼ meropenem, Ox ¼ oxacillin, presence of 0.5% of Oregano EO. Standard Deviations are not shown because they were
Pen ¼ penicillin G, Tec ¼ teicoplanin, Te ¼ tetracyclin, Van ¼ vancomycin. all <5% (n ¼ 2).
196 G. Pesavento et al. / Food Control 54 (2015) 188e199

3.4.4. Antimicrobial activity of EOs against L. monocytogenes and

0.3
0.1
0.1
0.1
Tastec S. aureus

0
0
0
0
0
0

0
0
1.4 ±
1.4 ±
1.4 ±
1.4 ±
1.4 ±
1.4 ±
1.4 ±
1.1 ±
1.5 ±
1.3 ±
1.2 ±
1.2 ±
To verify whether the EOs were responsible or not for the
n.p.
n.p.
n.p.
n.p.
n.p.
n.p.
decreased viability of tested pathogen populations in meat sam-
ples, we compared the experimental growth curves with the PC
Tasteb

1±0
1±0
1±0
1±0
1±0
1±0
1±0
1±0
1±0
1±0
1±0
1±0
n.p.
n.p.
n.p.
n.p.
n.p.
n.p.
ones that roughly followed the classical curve of microbial growth
With 2% EO

and were significantly different (p < 0.05) from the curves of all the
0.1

0.1
0.1
dilutions of both the pathogens (Fig. 1).
Odora

0
0

0
0
0
0
0
0
0
0
0
0
0
0
Pathogen count on contaminated raw meat after 2 h from T0
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
3.2
3.2
3.1

2.7
2.7
3.8
3.8
3.8
3.8
3.8
3.8
3.6
3.6
3.6
3.6
3.6
3.6
3

was lower than the expected one, probably thanks to the presence
of the EO which begins to act immediately after contact, damaging
Means (±Standard Deviation) of the results of the Panel Test performed on the meat samples added or not of the three concentrations of O. vulgare. R. officinalis and T. vulgaris EO (n ¼ 5).

0.11
0.23
0.1

0.3

0.2

0.6
0.1
microbial cells. Furthermore, Staphylococcus was more affected
a

0
0
0

0
0
0

0
0
0
0
Color

±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
than Listeria by all EOs, in accordance with other authors (Oussalah
5
5
5

5
5
5

5
5
5
5
4.9
4.8
3.8

4.87
4.67
3.8

4.4
3.9
et al. 2007) (Tables 6 and 7).
Rosmarinus was less effective than the other EOs against both
0.1
0.3
0.3
0.1

0.1
0.3

0.1

0.1

0.1
Listeria and Staphylococcus, although it was able to reduce bacterial
c

0
0
0
Taste

number according to Fern

pez et al. (2005) and Skandamis

2.4 ±
2.3 ±
2.4 ±
2.4 ±
2.3 ±

2.8 ±
2.3 ±
2.4 ±
2.8 ±
2.7 ±

2.4 ±
2.3 ±
2.4 ±
2.4 ±
2.4 ±
2.3 ±
andez-Lo
n.p.

n.p.

and Nychas (2001), thanks to the presence of 1.8 cineol (43.9%) and
a pinene (11.5%) (Table 1) (Burt, 2004; Lv et al., 2011). Both con-
0.1
0.1
0.1
0.1
0.1
0.1
b

0
0
0
0
0

0
0
0
0

centrations 0.5 and 1% (v/w) caused bacteriostasis on all the

Taste

1.8 ±
1.8 ±
1±
1±
1±

1.8 ±
1.8 ±
1.8 ±
1.8 ±

1.5 ±
1.5 ±
1.5 ±
1.3 ±
1.3 ±
1.3 ±
1.80
With 1% EO

n.p.

n.p.

pathogen concentrations (regression line slope coefficients of the

curves were all near zero), even in the more concentrated dilution
0.1
0.1
0.1
0.1
0.1

0.1
0.1
0.1
0.1
0.1

(DIL 1) of the pathogens. In particular, the 1% concentration showed

a

0
0
0
0
0
0
0
Odor

±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±

bactericidal activity against DIL 4 of Staphylococcus after 14 days of

4

4
4
4
4
4
4
4
4.7
4.7
4.7
4.5
4.3

4.9
4.7
4.7
4.3
4.3

preservation. Rosmarinus at 2% (v/w) caused a slight and gradual

reduction (time-dependent) of the microbial load of the three
0.3

0.4

0.1
0.1
0.3

0.1
0.1
a

0
0
0

0
0
0

0
0
0
0

higher Listeria concentrations and represented the MIC100 for DIL 4

Color

±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±

after 11 days of preservation at 4
C (Table 8). The regression line

5
5
5

5
5
5

5
5
5
5
4.8
4.6
3.3

4.9
4.5
3.2

4.9
3.5

slope coefficients of the time trend curves of each bacteria had

negative values, thus indicating a general decrease of the microbial
0.1
0.1
0.0

0.3
0.3
0.3
0.3
0.1

0.1
0.3
0.1
c

0
0

0
0
Taste

load; moreover, their absolute values had increasingly large values

3.5 ±
3.5 ±
3.5 ±
3.4 ±
3.4 ±

3.6 ±
3.6 ±
3.6 ±
3.6 ±
3.5 ±

3.6 ±
3.6 ±
3.5 ±
3.4 ±
3.3 ±
n.p.

n.p.

n.p.

going from DIL 1 to DIL 4, showing the increase of the EO action

with the decrease of the pathogen concentration. MBC90 and
With 0.5% O. vulgare EO

0.1

0.1
b

MBC100 determination was possible only for the lowest bacterial

0
0
0
0
0

0
0
0

0
0
0
0
0
Taste

2.8 ±
2.8 ±
2.8 ±
2.8 ±
2.8 ±

3.5 ±
3.4 ±
3.4 ±
3.4 ±
3.1 ±

3.2 ±
3.2 ±
3.2 ±
3±
3±

concentrations, respectively after 11 and 14 days of preservation for

n.p.

n.p.

n.p.

Listeria and Staphylococcus.

Thymus at a concentration of 0.5% (v/w) had a bacteriostatic
0.3
0.1

0.3
0.2
0.1
0.1
0.1
0.1
0.1
0.1
0.1
a

0
0
0
0

0
0
0
Odor

action for all the considered Listeria populations (DIL 1, DIL 2, DIL 3
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
5
5
5
5

5
5
5
4.4
4.1

4.8
4.3
4.1
4.3
4.3
4.3
4.3
3.9
3.7

and DIL 4) and the regression line slope coefficients of the curves
were all near zero. The 1% and 2% concentrations of the oil were,
0.1

0.3

0.1
0.2
0.2

0.1

0.3

instead, capable of determining a gradual reduction of the micro-

a

0
0
0

0
0
0

0
0
0

0
Color

±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±

bial load in meat. As seen for Rosmarinus 2% (v/w), the regression

5
5
5

5
5
5

5
5
5

3
4.9
4.4
3.2

4.9
4.3
3.2

4.9
4.2

line slope coefficients of the curves had negative values, thus

indicating a decrease of the microbial load with the prolongation of
0.1
c

0
0

0
0

0
0

the preservation at 4
C, and regression line slope coefficient ab-

Taste

Samples made of meat, potatoes, eggs and breadcrumbs (cooked).

5±
5±
4.7 ±

5±
5±

5±
5±
n.p.
n.p.
n.p.

n.p.
n.p.
n.p.
n.p.

n.p.
n.p.
n.p.
n.p.

solute values had increasingly large values going from DIL 1 to DIL 4
[respectively 0.14, 0.19, 0.20, 0.26 for 1% (v/w) and 0.36, 0.40, 0.46,
0.1

0.59 for 2% (v/w)], showing the increase of EO action with the

b

0
0

0
0

0
0
Taste

5±
5±
4.5 ±

5±
5±

5±
5±

decrease of the pathogen concentration and with the increase of EO

Without EOs

n.p.
n.p.
n.p.

n.p.
n.p.
n.p.
n.p.

n.p.
n.p.
n.p.
n.p.

concentration in meat (Tables 6 and 8). All the tested concentra-

tions of Thymus (0.5%, 1% and 2% v/w) were, instead, able to reduce
0.4
0.7
0.1

0.4
0.4

0.4
0.3
0.1
0.1
a

0
0
0

0
0
0
0

staphylococcal load in meat samples, during their preservation at

Odor

±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±

4
C, in a time dependent mode. Indeed, MBC90 and MBC100 values

4.3
3.1
1.5

4.5
3.1
1.6

4.5

1.5
5

1
1
5

1
1
5

1
1

Samples made only of meat (cooked).

(Tables 7 and 8) decreased with the prolongation of the sample

Results for raw and cooked meat.

preservation at 4
C. The chemical composition of this oil showed,

0.1
0.1
0.1
0.1

0.1
0.1

0.1

0.4

0.3
a

0
0

0
0
0

0
Color

in fact, high percentages of p-cimene (47.9%) and thymol (43.1%)

±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
5

1
5

1
5

1
4.7
3.7
2.1
1.1

4.7
3.5
2.4
1.1

4.8
3.7
2.8
1.2

(Table 1), two of the major antibacterial compounds (Burt, 2004;

Hyldgaard et al., 2012). In accordance with these results, the in-
Storage

crease of the slope coefficient absolute values of the regression

days

0
2
4
7
11
14
0
2
4
7
11
14
0
2
4
7
11
14

n.p. not performed.

lines from DIL 1 to DIL 4, for each tested EO concentration, dem-

onstrates the increase of the EO action with the decrease of the
R. officinalis

pathogen concentration.
T. vulgaris
O. vulgare
Essential

According to Skandamis and Nychas (2001), Oregano was found

Table 5

to affect bacterial multiplication both of Listeria and Staphylococcus.

oil

a
b
c

The major constituent of this EO was carvacrol (71.8%) (Table 1) and

 G. Pesavento et al. / Food Control 54 (2015) 188e199 197

Table 6
Means (±Standard Deviation) of the effect of preservation of minced meat samples at 4
C at various time ranges and at different EO concentrations on Listeria monocytogenes
growth (n ¼ 2).

Listeria Preservation Origanum vulgare Thymus vulgaris Rosmarinus officinalis

monocytogenes time at 4
C h
Essential oil concentrations
(h) or days (d)
0.5% 1% 2% 0.5% 1% 2% 0.5% 1% 2%

Log10 CFU/g Log10 CFU/g Log10 CFU/g Log10 CFU/g Log10 CFU/g Log10 CFU/g Log10 CFU/g Log10 CFU/g Log10 CFU/g

DIL1 2h 4.36 ± 0.01 4.15 ± 0.11 4.75 ± 0.07 4.38 ± 0.04 4.81 ± 0.07 4.75 ± 0.07 4.77 ± 0.01 4.83 ± 0.07 3.96 ± 0.03
2d 4.15 ± 0.26 3.97 ± 0.06 4.56 ± 0.11 4.72 ± 0.07 4.59 ± 0.03 4.56 ± 0.11 4.77 ± 0.001 4.75 ± 0 3.80 ± 0.03
4d 4.48 ± 0.10 3.83 ± 0.11 3.85 ± 0.11 4.60 ± 0.12 4.45 ± 0.19 3.85 ± 0.11 4.74 ± 0.01 4.78 ± 0 3.80 ± 0.03
7d 4.08 ± 0.09 3.76 ± 0.10 3.63 ± 0.09 4.69 ± 0.10 4.54 ± 0.22 3.63 ± 0.093 4.78 ± 0.01 4.76 ± 0.02 3.78 ± 0.06
11 d 4.04 ± 0.17 3.51 ± 0.13 3.37 ± 0.03 4.60 ± 0.13 4.15 ± 0.04 3.37 ± 0.03 4.75 ± 0.00 4.70 ± 0.02 3.41 ± 0
14 d 3.57 ± 0.06 3.28 ± 0.21 2.88 ± 0.07 4.18 ± 0.16 4.04 ± 0.28 2.88 ± 0.07 4.76 ± 0.03 4.70 ± 0.07 3.59 ± 0.05
DIL2 2h 3.62 ± 0.01 3.49 ± 0.11 3.76 ± 0.05 3.90 ± 0.07 3.89 ± 0.06 3.76 ± 0.05 3.58 ± 0.06 3.72 ± 0 2.79 ± 0.01
2d 3.19 ± 0.18 3.32 ± 0.02 3.54 ± 0.02 3.92 ± 0.05 3.71 ± 0.07 3.54 ± 0.02 3.65 ± 0.01 3.41 ± 0.07 2.92 ± 0.10
4d 3.36 ± 0.05 3.04 ± 0.04 3.26 ± 0.06 3.89 ± 0.05 3.41 ± 0.07 3.26 ± 0.06 3.68 ± 0.01 3.65 ± 0.07 2.77 ± 0.12
7d 3.34 ± 0.06 2.49 ± 0.14 2.85 ± 0.01 3.55 ± 0.02 3.51 ± 0 2.85 ± 0.01 3.72 ± 0.01 3.32 ± 0.10 2.66 ± 0.16
11 d 3.18 ± 0.22 2.28 ± 0.03 2.18 ± 0.21 3.83 ± 0.06 2.98 ± 0.07 2.18 ± 0.21 3.70 ± 0.01 3.40 ± 0.06 2.52 ± 0.02
14 d 3.06 ± 0.14 2.28 ± 0.10 1.78 ± 0.21 3.69 ± 0.11 2.92 ± 0.04 1.78 ± 0.21 3.67 ± 0.01 3.58 ± 0.10 2.20 ± 0.08
DIL3 2h 2.15 ± 0.09 2.51 ± 0.09 2.77 ± 0.07 2.85 ± 0.09 2.82 ± 0.04 2.77 ± 0.07 2.83 ± 0.08 2.73 ± 0.14 2.32 ± 0.03
2d 2.30 ± 0.19 2.04 ± 0.17 2.64 ± 0.11 2.99 ± 0.01 2.82 ± 0.71 2.64 ± 0.11 2.81 ± 0.03 2.56 ± 0.10 2.08 ± 0.10
4d 2.49 ± 0.06 2.04 ± 0.001 2.30 ± 0.06 2.96 ± 0.06 2.72 ± 0.37 2.30 ± 0.06 2.85 ± 0.04 2.65 ± 0.04 1.70 ± 0.12
7d 2.23 ± 0.11 1.78 ± 0.001 1.48 ± 0.55 2.88 ± 0.03 2.56 ± 0.07 1.48 ± 0.55 2.83 ± 0.07 2.64 ± 0.03 1.60 ± 0.34
11 d 2.00 ± 0.26 1.30 ± 0.34 1.00 ± 0.92 2.99 ± 0.06 2.04 ± 0.17 1.00 ± 0.92 2.82 ± 0.04 2.63 ± 0.07 1.48 ± 0.21
14 d 2.04 ± 0.17 1.00 ± 1.13 0 ± 0 2.68 ± 0 1.90 ± 0.16 0.00 ± 0 2.81 ± 0.07 2.62 ± 0 1.48 ± 0.21
DIL4 2h 1.60 ± 0.34 1.48 ± 0.21 1.60 ± 0.34 1.60 ± 0 1.95 ± 0.64 1.60 ± 0.34 1.70 ± 0.12 1.78 ± 0 1.30 ± 0.92
2d 1.30 ± 1.13 0 ± 0 0 ± 0 1.95 ± 0 1.60 ± 0.34 0 ± 0 1.95 ± 0.07 1.70 ± 0.12 1.30 ± 0.92
4d 1.78 ± 0.43 0 ± 0 0 ± 0 1.30 ± 0 1.30 ± 0.34 0 ± 0 1.95 ± 0.07 1.78 ± 0.21 1 ± 0.71
7d 1.30 ± 0.001 0 ± 0 0 ± 0 1.95 ± 0.07 1.00 ± 0 0 ± 0 1.95 ± 0.07 1.70 ± 1.20 1 ± 0.71
11 d 1.48 ± 0.21 0 ± 0 0 ± 0 1.90 ± 0.16 0.82 ± 0 0 ± 0 1.95 ± 0.07 1.78 ± 0 0 ± 0
14 d 0 ± 0 0 ± 0 0 ± 0 1.70 ± 0.43 0.52 ± 0.0 0 ± 0 1.30 ± 0.92 1.70 ± 0.12 0 ± 0

it is demonstrated as one of the most antibacterial EO components
(Hyldgaard et al., 2012). The lowest concentration of O. vulgare EO
(0.5% v/w) acted as bacteriostatic on DIL 1, DIL 2 and DIL 3 Listeria
populations; only after 11 days of preservation at 4
C, bacterial
concentration of DIL 4 (concentration higher than 40 CFU/g at T0)
reached 0 CFU/g (MBC). The 1% and 2% (v/w) concentration of O.
vulgare EO inhibited bacterial cells in time and bacterial concen-
tration dependent mode: Listeria concentration decreased contin-
uously reaching the lowest values at the end of the experiments (14
days of preservation at 4
C), and regression line slope coefficient

Table 7
Means (±Standard Deviation) of the effect of preservation of minced meat samples at 4
C at various time ranges and at different EO concentrations on Staphylococcus aureus
growth (n ¼ 2).

Staphylococcus Preservation Origanum vulgare Thymus vulgaris Rosmarinus officinalis

aureus time at 4
C h
Essential oil concentrations
(h) or days (d)
0.5% 1% 2% 0.5% 1% 2% 0.5% 1% 2%

Log10 CFU/g Log10 CFU/g Log10 CFU/g Log10 CFU/g Log10 CFU/g Log10 CFU/g Log10 CFU/g Log10 CFU/g Log10 CFU/g

DIL1 2h 3.45 ± 0.11 3.06 ± 0.02 3.77 ± 0.04 3.97 ± 0.03 3.79 ± 0.06 3.67 ± 0.01 4.13 ± 0.01 3.23 ± 0.19 3.45 ± 0.03
2d 3.41 ± 0.17 3.09 ± 0.02 2.84 ± 0.02 3.79 ± 0.02 3.69 ± 0.01 3.62 ± 0.02 4.51 ± 0 3.38 ± 0.01 3.51 ± 0.05
4d 3.21 ± 0.09 3.09 ± 0.02 2.43 ± 0.07 3.11 ± 0.05 2.93 ± 0 2.78 ± 0.01 4.59 ± 0.05 3.78 ± 0.08 3.96 ± 0.02
7d 2.82 ± 0.06 2.55 ± 0.09 1.88 ± 0.04 2.77 ± 0.01 2.69 ± 0.01 2.62 ± 0.01 4.55 ± 0.01 3.74 ± 0.02 3.48 ± 0.12
11 d 2.96 ± 0.01 2.28 ± 0.13 1.74 ± 0.06 2.56 ± 0.07 2.56 ± 0 2.28 ± 0.03 4.44 ± 0.01 3.62 ± 0.06 2.95 ± 0.14
14 d 2 ± 0.26 1.93 ± 0.11 1.48 ± 0 2.35 ± 0.07 2.16 ± 0.25 2.10 ± 0.02 4.14 ± 0 3.28 ± 0.10 2.60 ± 0.16
DIL2 2h 2.04 ± 0.05 2.12 ± 0.04 2.78 ± 0.01 3.33 ± 0.01 2.95 ± 0.06 2.88 ± 0 3.29 ± 0.07 2.20 ± 0 2.67 ± 0.01
2d 2.14 ± 0.09 2.21 ± 0.05 2.46 ± 0.02 2.91 ± 0.03 2.59 ± 0.06 2.48 ± 0.02 3.46 ± 0.01 2.15 ± 0 2.65 ± 0.10
4d 2 ± 0.12 2.06 ± 0.08 2.33 ± 0.04 2.03 ± 0.07 1.91 ± 0.02 1.95 ± 0.07 3.41 ± 0.03 2.74 ± 0.08 3.11 ± 0.05
7d 1.84 ± 0.09 1.60 ± 0 2.10 ± 0.02 1.78 ± 0 1.77 ± 0.06 1.72 ± 0.03 3.27 ± 0.26 3.06 ± 0.03 2.70 ± 0.29
11 d 1.9 ± 0 1.28 ± 0.03 1 ± 0 1.48 ± 0 1.36 ± 0 1.31 ± 0.01 3.27 ± 0.24 2.83 ± 0.07 1.78 ± 0
14 d 1.48 ± 0.21 0 ± 0 0 ± 0 1.51 ± 0.05 1.11 ± 0.14 0.90 ± 0 3.04 ± 0 2.77 ± 0.03 1.78 ± 0
DIL3 2h 1.79 ± 0.02 1.75 ± 0.01 0.95 ± 0.07 1.74 ± 0.11 1.56 ± 0.03 1.30 ± 0 2.28 ± 0.10 1.70 ± 0.12 1.78 ± 0.21
2d 1.18 ± 0.21 1.40 ± 0.12 0 ± 0 1.53 ± 0.02 1.30 ± 0.06 0.60 ± 0 2.28 ± 0.03 1.85 ± 0.28 1.78 ± 0.21
4d 0.74 ± 0.71 0 ± 0 0 ± 0 1.18 ± 0.04 1.04 ± 0.06 0 ± 0 2.63 ± 0.13 2.20 ± 0 1.95 ± 0.21
7d 0.74 ± 0.71 0 ± 0 0 ± 0 1 ± 0 0.85 ± 0.09 0 ± 0 2.49 ± 0.10 2.20 ± 0 1.60 ± 0
11 d 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0 2.48 ± 0.12 2.20 ± 0.08 1.30 ± 0
14 d 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0 2.08 ± 0 1.30 ± 0 1.02 ± 0.92
DIL4 2h 1.30 ± 0 0.60 ± 0 1 ± 0 1.00 ± 0 0.70 ± 0 0.30 ± 0 2.00 ± 0.26 1.30 ± 0 1.30 ± 0
2d 0.74 ± 0.71 0 ± 0 0 ± 0 0.78 ± 0 0 ± 0 0 ± 0 2.18 ± 0.04 1.60 ± 0 1.30 ± 0
4d 0 ± 0 0 ± 0 0 ± 0 0.18 ± 0.21 0 ± 0 0 ± 0 2.30 ± 0.06 1.95 ± 0.21 1.48 ± 0.21
7d 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0 1.95 ± 0.07 1.90 ± 0.16 1.30 ± 0
11 d 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0 1.85 ± 0.28 1.90 ± 0.16 1.02 ± 0.92
14 d 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0 1.48 ± 0.21 0 ± 0 0 ± 0
198 G. Pesavento et al. / Food Control 54 (2015) 188e199

Table 8
Minimal bactericidal concentrations of the O. vulgare, T. vulgaris and R. officinalis EOs against L. monocytogenes and S. aureus.

Pathogen Dilutions O. vulgare T. vulgaris R. officinalis

MBC 90 MBC 100 MBC 90 MBC 100 MBC 90 MBC 100

Listeria monocytogenes DIL1 2% (7d) n.d. 2% (7d) n.d. n.d. n.d.

DIL2 1% (7d) n.d. 2% (11d) n.d. n.d. n.d.
DIL3 1% (14d) 2%(14) 2% (7d) n.d. n.d. n.d.
2% (7d)
DIL4 0.5% (14d) 0.5% (14d) 1% (11d) 2% (2d) 2% (11d) 2% (11d)
1% (2d) 1% (2d)
Staphylococcus aureus DIL1 0.5% (14d) n.d. 0.5% (7d) n.d. n.d. n.d.
2% (4d)
DIL2 1% (14d) 1% (14d) 0.5% (4d) n.d. n.d. n.d.
2% (7d)
DIL3 0.5% (4d) 1% (4d) 0.5% (11d) 0.5% (11d) n.d. n.d.
2% (2d) 2% (4d) 2% (4d)
DIL4 0.5% (4d) 0.5% (4d) 0.5% (7d) 0.5% (7d) 1% (14d) 1% (14d)
1% (2d) 1% (2d) 1% (2d) 1% (2d)

absolute values had increasingly large values going from DIL 1 to
DIL 4 [respectively 0.17, 0.18, 0.29, 1.48 for 1% (v/w) and 0.37, 0.41,
0.56, 0.60 for 2% (v/w)], showing the increase of the EO action with
the decrease of the pathogen concentration and the increase of EO
concentration. Furthermore, only 1% and 2% concentrations (v/w)
of this EO were able to kill (MBC) all Listeria cells of DIL 4 (con-
taining more than 30 and 40 CFU/g respectively) at the second day
of preservation at 4
C, 2% (v/w) also being able to cause the
decrease of the Listeria load of DIL 3 (initial load of more than
590 CFU/g) till 0 CFU/g at the end of the experiment (MBC).
The three concentrations of Oregano (0.5%, 1% and 2% v/w)
reduced staphylococcal load in meat samples during their preser-
vation at 4
C in a time dependent mode, as did Thymus. Differently
from Thymus, Oregano had a dose dependent mode of inhibition,
2% (v/w) of the oil reducing the staphylococcal load more than the
1% (v/w), which had a greater bactericidal effect than 0.5% (v/w).
This is confirmed by the increase of the slope coefficient absolute
values of the regression lines from DIL 1 to DIL 4, in each EO con-
centration, and between the three EO concentrations and by MIC
values (Table 8).
4. Conclusions
We observed that only the 0.5% concentration of the three
tested EOs could be used as food additives, since it does not
substantially alter the flavor of food, being, however, able to act as
bacteriostatic and bactericidal against, respectively, high and low
(<102 CFU/g) pathogen concentrations. This suggests a possible
use of Rosmarinus, Thymus and Oregano as food preservatives at a
concentration of 0.5% (v/w) or lower, since L. monocytogenes and
S. aureus are usually found in raw meat at concentrations near
10 CFU/g or lower.
The tested essential oils were found to be active, as well as
against the ATCC strains, also against toxigenic strains of S. aureus
(SED, SEA, SEC, SEB and SEA þ SEB producers) and virulent strains
of L. monocytogenes (1/2a, 1/2b, 1/2c and 4b), showing the true
effectiveness of these compounds.
We are completely aware that sensitivity to a given antimicro-
bial compound may vary widely among different strains belonging
to the same bacterial species; even if EOs were more effective in
reducing microbial numbers during preservation at 4
C against
Staphylococcus than Listeria, we observed no significant differences
(p < 0.05) in susceptibility to EOs among all tested bacteria of the
same genus. Therefore, in our opinion, the data reported in this
paper are particularly encouraging, since they demonstrate that the
use of EOs might represent an alternative way to limit pathogen
and aerobic spoilage flora presence in perishable foods such as
meat, fish, ready-to-eat and ready-to-cook foods, when combined
with other preservative technologies, such as modified atmosphere
packaging. We think that the EOs we tested could be used by food
industries during the preparation of beef meatballs and other meat
ready-to-cook products, having a shelf life of at least 14 days at a
preservation temperature of 4
C.
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