Journal of Food Engineering 124 (2014) 110

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Journal of Food Engineering

journal homepage: www.elsevier.com/locate/jfoodeng

Antimicrobial effects of different combined non-thermal treatments

against Listeria monocytogenes in broccoli orets
Renato Severino a,b, Khanh Dang Vu b, Francesco Dons c, Stphane Salmieri b, Giovanna Ferrari a,c,
Monique Lacroix b,
a
ProdAl Scarl, Competence Center on Agro-Food Productions, via Ponte don Melillo, 84084 Fisciano, SA, Italy
b
Research Laboratories in Sciences Applied to Food, Canadian Irradiation Center, INRS-Institut Armand-Frappier, Institute of Nutraceutical and Functional Foods, 531, Boulevard
des Prairies, Laval, Qubec H7V 1B7, Canada
c
Department of Industrial Engineering, University of Salerno, via Ponte don Melillo, 84084 Fisciano, SA, Italy

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

Article history: The antilisterial effect of three non-thermal treatments in combination with a bioactive coating formu-
Received 22 April 2013 lation on broccoli orets inoculated with Listeria monocytogenes was evaluated. The nanoemulsions of
Received in revised form 6 August 2013 carvacrol, bergamot, lemon and mandarin essential oils (EO) were incorporated in native and modied
Accepted 20 September 2013
chitosan coating formulations and the antilisterial effect in inoculated samples was evaluated. The mod-
Available online 30 September 2013
ied chitosan based coating containing mandarin EO was the best treatment which caused a load reduc-
tion of 1.46 log CFU/g after 6 days of storage. The antilisterial effects of this coating formulation in
Keywords:
combination with ozonated water, UV-C and c-ray treatments on inoculated samples were evaluated
Listeria monocytogenes
Essential oil
during 13 days storage at 4 C. The combined coating and ozonated water showed very high antilisterial
Gamma irradiation ozonated water effects at days 1 and 3; however, their antilisterial activity was reduced after day 5. The combined coating
UV-C and UV-C did not show any additive effect against L. monocytogenes as compared to coating alone. The
Coating best antilisterial activity was obtained in the combined coating and c-rays. This combined treatment
caused an increase in relative radiation sensitivity of L. monocytogenes by 1.33-fold. Further, this com-
bined treatment ensured microbial safety during storage with a reduction of L. monocytogenes by
2.5 log CFU/g after 13 days.
2013 Elsevier Ltd. All rights reserved.

1. Introduction
According to the Center for Disease Control and Prevention
(CDC), the annual incidence of serious foodborne illnesses is very
high: CDC estimates that each year in the United States 48 million
people get sick, 128,000 are hospitalized, and 3000 die due to food-
borne diseases. Therefore, ensuring microbiological safety of food
products, while maintaining their nutritional and organoleptic
properties, is still a priority nowadays.
Vegetables can be easily contaminated: cutting or slicing oper-
ations increase tissue damage, causing the release of intracellular
contents (Gonzlez-Aguilar et al., 2009) that can support and in-
crease the activity of pathogenic and saprophytic microorganisms.
A frequent problem is contamination with Listeria monocytogenes
(Beauchat, 1996), Gram-positive, rod-shaped bacteria which are
widely distributed in the environment and are pathogenic to hu-
mans and animals (Hein et al., 2000). Heat treatments can ensure
microbial inactivation, but frequently they unacceptably affect
the nutritional and organoleptic properties of food. Consumers de-
Corresponding author. Tel.: +1 450 687 5010x4489; fax: +1 450 686 5501.
E-mail address: Monique.Lacroix@iaf.inrs.ca (M. Lacroix).
mand for high-quality foods that are microbiologically safe and
stable has awakened a growing interest in non-thermal preserva-
tion techniques (Espina et al., 2012). Particularly the combination
of non-thermal methods with antimicrobial treatments can en-
hance the lethal effects of non-thermal processing, reduce the
severity or the exposure time of non-thermal treatment needed
to obtain a given level of microbial inactivation, preserve food
physico-chemical properties without affecting the nutritional va-
lue (Raso and Barbosa-Cnovas, 2003).
In this context, the use of active edible coatings in combination
with non-thermal treatments may represent a viable approach for
achieving microbial stability and preserving quality. Due to their
characteristics, in fact, edible coatings have been traditionally used
to improve food appearance and maintain microbiology quality
(Khwaldia et al., 2004). Chitosan is a polycationic polymer obtained
by partial deacetylation of chitin, the most abundant natural carbo-
hydrate after cellulose. Chitosan is non-toxic, biodegradable and
biocompatible and is also easily modied by physical or chemical
methods (Le Tien et al., 2003). Several studies reported the antimi-
crobial activity of chitosan and its derivatives in coating formula-
tions (Kanatt et al., 2013; Bordenave et al., 2010). The
mechanism of action of chitosan against bacteria has not been

0260-8774/$ - see front matter 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.jfoodeng.2013.09.026
2 R. Severino et al. / Journal of Food Engineering 124 (2014) 110
completely explained yet, but several hypotheses have been postu-
lated: most likely, the antimicrobial activity can be attributed to a
change in cell permeability due to interactions between the amine
groups of chitosan and the electronegative charges on the cell sur-
face. This interaction leads to the leakage of intracellular electro-
lytes and proteinaceous constituents (Papineau et al., 1991).
In addition to native chitosan, several derivatives have been
studied for their antimicrobial activity, for example N-carboxybu-
tyl chitosan (Muzzarelli et al., 1990), quaternary N-alkylchitosan
(Jia, 2001). A new formulation was also elaborated, based on chito-
san acylation with fatty acids derivatives, with the aim of enhanc-
ing the hydrophobic properties of the polymer (Han et al., 2008).
Essential oils have also gained interests as natural antimicrobial
agents for food preservation against foodborne pathogens and
spoilage bacteria (Caillet et al., 2006a,b). The antimicrobial activity
of several essential oils and essential oil components has been suc-
cessfully proved on different microorganism species (Di Pasqua
et al., 2007; Gill and Holley, 2006). However, being essential oil
constituents characterized by low solubility in water, their ef-
ciency is promoted when encapsulated in appropriate delivery sys-
tems that can enhance their dispersibility in the aqueous part of
foods, where microorganisms grow and proliferate (Weiss et al.,
2009).
Recently, the use of nanoscale delivery systems for essential oils
was shown to offer the potential of improving not only the phys-
ico-chemical stability of encapsulated bioactive compounds in
foods, but also their bioactivity through the activation of passive
mechanisms of cell absorption, owing to their subcellular size,
therefore enabling the reduction of the dose of essential oils re-
quired to ensure antimicrobial activity in foods, minimizing the
impact on aroma, avor and taste (Dons et al., 2011, 2012).
In parallel, physical non-thermal treatments have known an
increasing development, with the aim of ensuring microbial safety
without undesirable sensorial and nutritional changes, such as col-
or degradation, softening of tissues, and vitamin losses on foods.
Recently, new processing technologies have been developed, based
on ozone treatments, UV-C irradiation and gamma ray irradiation,
which are capable of prolonging product shelf life without the
unfavorable effects of severe heating (Alexandre et al., 2011).
Owing to its powerful oxidizing effect, ozone is one of the most
potent disinfectant agents (Gzel-Seydim et al., 2004). Ozone is
appropriate for several applications in food industry, such as food
surface hygiene and preservation, equipment and food plant sani-
tation and reuse of waste water. In several studies ozone has been
found to be effective against a wide spectrum of microorganisms,
including viruses, gram-negative and gram-positive bacteria,
spores and fungi (Manousaridis et al., 2005).
Several studies highlighted that c-irradiation is an excellent
process for reducing or eliminating foodborne pathogens (Caillet
et al., 2006b; Turgis et al., 2009) by breaking DNA chemical bonds,
or altering the membrane permeability and other cellular functions
(Lopez-Gonzales et al., 1999). It has also been shown that the com-
bined use of antimicrobial compound incorporated or not in edible
coating and c-irradiation can increase the radiosensitivity of bacte-
ria, resulting in lower radiation doses required for lethality (Caillet
et al., 2006a,b; Ouattara et al., 2001; Vu et al., 2012).
Another postharvest physical treatment developed in recent
years that can be used to ensure fruits and vegetables safety is
the UV-C irradiation (Alexandre et al., 2012). Its germicidal activity
against several pathogens, due to the formation of pyrimidine di-
mers in DNA strands, crosslinks of aromatic amino acids leading
to membrane depolarization and abnormal ionic ow, has been re-
ported in several studies (Sommer et al., 1996; Wright et al., 2000).
It has also been demonstrated that UV-C illumination can delay
postharvest fruit senescence and especially control decay in differ-
ent fruit and vegetable species (Erkan et al., 2001). For example,
UV-C exposition decreases the activity of enzymes involved in to-
mato cell wall degradation and delays the fruit softening (Barka
et al., 2000).
The aim of this study is the evaluation of antimicrobial effect of
combined non-thermal treatments against L. monocytogenes on
broccoli orets. In particular, the combined non-thermal treat-
ments will be based on the use on an antimicrobial edible coating,
based on native and modied chitosan incorporating nanoemul-
sions of different essential oil, in combination with a physical
non-thermal treatment, such as ozone treatment, UV-C exposition
and c-ray irradiation. This approach is innovative both in the
development of novel antimicrobial edible coatings, and in the
study of the possible synergistic effect of its use in combination
with a physical non-thermal treatment.
2. Materials and methods
2.1. Bacterial preparation
The ve L. monocytogenes strains (Health Canada, Health Prod-
ucts and Food Branch, Ottawa, Canada) used in this study were iso-
lated from foodborne outbreak (Table 1).
Before each experiment, stock cultures of 5 strains of L. mono-
cytogenes were propagated separately through two consecutive
24 h growth cycles in tryptic soy broth (TSB, Difco) at 37 C. Subse-
quently, 2 ml from each culture broth were combined together.
The combined broth of mixed strains was centrifuged at 4000 g
for 20 min at 4 C and washed twice in sterile saline solution
(0.85% w/v) to eliminate residual components of media culture.
The obtained biomass was then suspended in sterile saline water,
to obtain a working culture containing approximately 109 CFU/ml.
2.2. Characterization of the essential oils
The analysis of the essential oils was carried out in a GCMS
Finnigan-Focus (Thermo-Fisher Scientic, UK), as previously de-
scribed (Dons et al., 2012). Briey, an RTX-5 SIL MS capillary col-
umn (30 m long, 0.25 mm i.d. and 0.25 lm lm thickness) was
used with a cross-linked stationary phase of polyethylene glycol
(Restek). The chromatographic conditions were as follows: He as
the carrier gas; the injector in split-mode with a split ow of
20 ml/min and a temperature of 250 C; the temperature of the
ion source was 250 C; the temperature of the transfer line was
260 C. The compounds were separated using a temperature pro-
gram with an initial oven temperature of 80 C for 10 min and a
temperature gradient of 25 C/min to a nal temperature of
250 C, which was maintained for 10 min. 3 ll of the sample was
injected, using the split technique. The ionization was produced
by electronic impact at 70 eV. The eluted compounds were identi-
ed using the retention times and by comparing their mass spectra
with a spectral library of known standard compounds. The identi-
cation was carried out in full scan mode between 40 and 400
amu.
Table 1
Source and serotypes of L. monocytogenes strains used in the study.
Strain Serotype Source
HPB2558 b Beef hot dogs
HPB2812 a Homemade salami
HPB1043 a Turkey frank factory isolate
HPB2569 a Cooked cured sliced turkey
HPB2371 b Raw turkey
 R. Severino et al. / Journal of Food Engineering 124 (2014) 110
2.3. Nanoemulsion of essential oils preparation
Carvacrol (P98% FCC SigmaAldrich, Germany), bergamot
essential oil, mandarin essential oil and lemon essential oil (a kind
gift of the Stazione Sperimentale per le Industrie delle Essenze e
dei Derivati dagli Agrumi, Italy) are the four antimicrobial agents
used in nanoemulsions preparation. In the emulsion fabrication,
a mixture of sunower oil (Sagra, Italy), glycerol monooleate (Sig-
maAldrich, Germany) and the essential oil were dispersed in
bidistilled water containing Tween 20 (SigmaAldrich, Germany),
using an Ultra Turrax T25 (IKA Labortechnik, Germany) at
24,000 rpm for 5 min, to form a primary emulsion. Subsequently,
the primary emulsions were subjected to a high pressure homoge-
nization treatment in a Nano DeBEE Electric Bench-top Laboratory
homogenizer (BEE International, USA) for ten times at 350 MPa, to
reach a nanometric size.
A photon correlation spectrometer (HPPS, Malvern Instruments,
Malvern, UK) was used for the particle size measurement of the
nanoemulsion droplets. The droplet size distribution was charac-
terized in terms of the mean droplet size (z-diameter) and polydis-
persity index (PDI) by measuring the backscattered (173) light
through samples diluted 1:100 with bidistilled water to avoid mul-
tiple scattering effects within polystyrene cuvettes. Measurements
were carried out at 25 C. Each measurement was replicated twice,
with the means and the standard deviations being calculated. The
composition, the z-diameter and PDI of the different nanoemul-
sions tested are reported in Table 2.
2.4. Preparation of bioactive coating
In this study, both native chitosan (NC, Kitomer, Mw
1600 kDa, 83% deacetylation, Marinard Biotech, Canada) and mod-
ied chitosan (MC) were used as coating matrix for incorporation
of antimicrobial emulsions. Modied chitosan (3% N-palmitoyl
chitosan) was prepared by N-acylation of NC using palmitoyl chlo-
ride based on the method developed in our lab (Le Tien et al.,
2003). The functionalization of the MC was characterized by FTIR
structural analysis. The MC exhibited the changes in band intensi-
ties that were correlated to a chemical modication in the presence
of palmitoyl chains linked to the polymers by acylation (data not
shown) (Le Tien et al., 2003; Han et al., 2008).
NC and MC were dissolved in 1% (v/v) acetic acid solution and
were stirred for 24 h to ensure total solubility. The nal concentra-
tion of NC and MC, separately prepared in the coating suspensions,
was 1% (w/v). Essential oil (EO) nanoemulsions were added into
coating suspensions and mixed vigorously using an Ultra Turrax
T25 (IKA Labortechnik, Germany) at 19,000 rpm for 5 min. The nal
concentration of EO in the coating formulations was 0.05% (w/v).
2.5. Sample preparation
Broccoli orets were purchased from a local supermarket (IGA,
Laval, Quebec, Canada). Florets were packaged in 0.5-mil metalized
polyester-2-mil ethylene vinyl acetate copolymer bags (205
 355 mm; Winpak Division Ltd, Montreal, Canada). The packaged
orets were sterilized by gamma-irradiation at the Canadian
Table 2
Composition of the four different nanoemulsions of essential oils.
Sample Formulation
Carvacrol Carvacrol 1%, Sunower oil 3%, Tween 20 0.75%, Glycerol monooleate 0.75%, Water 94.5%
Bergamot oil Bergamot oil 2%, Sunower oil 2%, Tween 20 0.75%, Glycerol monooleate 0.75%, Water 94.5%
Lemon oil Lemon oil 2%, Sunower oil 2%, Tween 20 0.75%, Glycerol monooleate 0.75%, Water 94.5%
Mandarin oil Mandarin oil 2%, Sunower oil 2%, Tween 20 0.75%, Glycerol monooleate 0.75%, Water 94.5%
3
Irradiation Center using a UC-15 A (SS canister) underwater cali-
brator (Nordion Inc., Kanata, Ontario, Canada) equipped with a
60
Co source. A radiation dose of 10 kGy was delivered at a dose rate
of 16.74 kGy/h to sterilize the broccoli orets. The packages were
then stored at 4 C.
2.6. Coating application
The coating was applied on broccoli samples (2022 g) using al-
ways the same procedure: the samples were sprayed with the se-
lected coating formulation for 10 s using a compressed air-assisted
sprayer, set at the pressure of 20 psi (1.4 bar). The coated samples
were allowed to dry for 1 h on sterile aluminum sheets placed in a
biological safety cabinet.
2.7. Preliminary experiment
A preliminary experiment on the antimicrobial effects of differ-
ent coating formulations, reported in Table 3, against L. monocytog-
enes inoculated in broccoli orets was conducted to identify the
best coating formulation. Sterilized broccoli samples were coated
with different formulations. The coated samples were then placed
into sterile bags and inoculated, by adding 500 ll of diluted work-
ing culture of L. monocytogenes (107 CFU/ml), to reach a nal pop-
ulation of 105 CFU/g. The samples were stored at 4 C and microbial
analysis was conducted at days 0, 2 and 6.
2.8. Antimicrobial effects of different combined treatments against L.
monocytogenes inoculated in broccoli orets
2.8.1. Combined treatment using ozonated water and antimicrobial
coating
Broccoli samples were inoculated with 500 ll of diluted work-
ing culture of L. monocytogenes (108 CFU/ml) to reach a nal con-
centration of 106 CFU/g. Inoculated samples were immersed in
500 ml ozonated water (7 0.5 ppm), under constant stirring for
2.5 min. Ozone, produced from puried oxygen (PSA Oxygen Gen-
erator, AIRSEP Corporation, NY) by an A2Z-S 16GLAB Corona Dis-
charge ozone generator (A2Z Ozone Systems INC, NY), was
bubbled in distilled water using an inlet dispenser and ozone con-
centration was measured in real time using a dissolved ozone
monitor (Q-45H, Ozone Solutions, INC). The washing time and
ozone concentration used based on the results of preliminary test
in which different ozone concentrations (07 ppm) and washing
periods (0.55 min) were conducted. Samples were allowed to
dry on sterile aluminum sheets for 30 min in a biological safety
cabinet and then sprayed with the coating formulation MC + M
(which resulted the optimal one from the preliminary experi-
ments) and then placed in sterile bags and stored at 4 C. Microbial
analysis was conducted after 24 h of treatment.
2.8.2. Combined treatment using antimicrobial coating and UV-C
radiation
UV-C irradiation experiments were conducted either before or
after applying the antimicrobial coating. In the rst case, broccoli
samples were coated with the optimal formulation (MC  M),
z-Diameter (nm) PDI
133.4 5.8 0.21 0.01
161.5 7.2 0.19 0.07
163.7 6.3 0.21 0.05
176.4 14.5 0.22 0.02
4 R. Severino et al. / Journal of Food Engineering 124 (2014) 110

Table 3 (log CFU/g) were plotted against radiation doses, and the reciprocal
List of the coating formulations tested on broccoli orets inoculated with L. of the slope of the trendline was extracted from the plot. Moreover,
monocytogenes.
the relative radiation sensitivity was also determined using the fol-
Sample Active compounds in 1.0% acetic acid solution lowing equation:
NC 1.0% Native chitosan
Dcontrol
10
NC + C 1.0% Native chitosan SR 1
5% Carvacrol nanoemulsion Dcoating
10
(Carvacrol content of 0.05%)
NC + B 1.0% Native chitosan where SR is the relative radiation sensitivity, Dcontrol
10 is the radiation
2.5% Bergamot oil nanoemulsion D10 value of the control sample and Dcoating
10 is the radiation D10 value
(Bergamot oil content of 0.05%) of sample treated in the presence of antimicrobial coating.
NC + L 1.0% Native chitosan
2.5% Lemon oil nanoemulsion
(Lemon oil content of 0.05%) 2.9. Antimicrobial effects of different combined treatments against L.
NC + M 1.0% Native chitosan
monocytogenes during storage of broccoli orets
2.5% Mandarin oil nanoemulsion
(Mandarin oil content of 0.05%) 2.9.1. Ozonated water and coating combined treatment
MC 1.0% Modied chitosan Fresh sterile broccoli samples were inoculated by adding 500 ll
MC + C 1.0% Modied chitosan
of diluted working culture of L. monocytogenes (105 CFU/ml) to
5.0% Carvacrol nanoemulsion reach a nal concentration of 103 CFU/g. Inoculated samples were
(Carvacrol content of 0.05%) immersed in 500 ml ozonated water (7 0.5 ppm), under constant
MC + B 1% Modied chitosan stirring for 2.5 min. Ozone was produced with the same system ex-
2.5% Bergamot oil nanoemulsion plained above. Samples were allowed to dry on sterile aluminum
(Bergamot oil content of 0.05%) sheets for 30 min in a biological safety cabinet and then were
MC + L 1.0% Modied chitosan coated sprayed with the selected formulation (MC + M). They were
2.5% Lemon oil nanoemulsion then placed in sterile bags and stored at 4 C.
(Lemon oil content of 0.05%)
MC + M 1% Modied chitosan
2.5% Mandarin oil nanoemulsion 2.9.2. UV-C and coating combined treatment
(Mandarin oil content of 0.05%) Fresh sterile broccoli samples were rst coated with the se-
lected formulation (MC + M) and then inoculated by adding
500 ll of diluted working culture of L. monocytogenes (105 CFU/
and then inoculated with 500 ll of diluted working culture of L. ml) to reach a nal concentration of 103 CFU/g. Subsequently, the
monocytogenes (106 CFU/ml) to reach a nal concentration of 106 samples were placed in sterile petri dishes and exposed to doses
CFU/g. Subsequently, the samples were placed in sterile petri of 8 kJ/m2 of UV-C radiation for each side. The samples were pack-
dishes and exposed to different UV-C (255 nm) doses correspond- aged in sterile bags and stored at 4 C.
ing to 2, 4, 8 and 10 kJ/m2 for each side. The UV-C irradiation cham-
ber BS-04 (Dr. Grobel UV-Elektronik GmbH, Germany), equipped 2.9.3. Gamma irradiation and coating combined treatment
with an irradiation controller UVMAT (Dr. Grobel UV-Elektronik Fresh sterile broccoli samples were rst coated with the se-
GmbH, Germany) was used for UV-C treatments. The interior irra- lected formulation (MC + M) and then inoculated by adding
diation chamber has a footprint of 60  60 cm and a height of 500 ll ofdiluted working culture of L. monocytogenes (105 CFU/
28 cm. Treated samples were then placed in sterile bags and stored ml) to reach a nal concentration of 103 CFU/g. The samples were
at 4 C. Microbial analysis was conducted after 24 h of treatment. irradiated with doses of 0.25 kGy. Samples were packaged in sterile
In the second case, broccoli samples were inoculated with a - bags and stored at 4 C.
nal L. monocytogenes concentration of 106 CFU/g and were treated For all the different combined treatments, microbial analysis
with different UV-C doses (255 nm) corresponding to 2, 4, 8, 10 kJ/ was conducted at days 1, 3, 5, 7, 9, 11, and 13 during storage.
m2 for each side, using the same UV-C system above. Subsequently,
samples were coated with the formulation MC + M and placed on
2.10. Microbial analysis
sterile bags and stored at 4 C. Microbial analysis was conducted
after 24 h of treatment.
Samples were homogenized for 2 min at 230 rpm in 80 ml pep-
tone water (0.1% w/v) using a Lab-blender 400 Stomacher (Labora-
tory Equipment, London, UK). From the homogenate serial decimal
2.8.3. Combined treatment using antimicrobial coating and gamma
dilutions were prepared and plated on petri dishes containing Pal-
irradiation
cam Agar (Alpha Biosciences, INC., USA), which contained acryla-
Broccoli samples were rst coated with the selected formula-
vine (5 mg/L), polymixin B (10 mg/L) and ceftazidime (8 mg/L). The
tion (MC + M) and then were inoculated with 500 ll of diluted
petri plates were incubated at 37 C for 48 h. After incubation, the
working culture of L. monocytogenes (106 CFU/ml) to reach a nal
colonies producing black precipitates were considered as L. mono-
concentration of 106 CFU/g. Subsequently, the samples were irradi-
cytogenes and were enumerated.
ated at the Canadian Irradiation Center using a UC-15 A (SS canis-
ter) underwater calibrator (Nordion Inc., Kanata, Ontario, Canada)
equipped with a 60Co source at room temperature. The radiation 2.11. Statistical analysis
treatments were conducted at different doses from 0 to 2.24 kGy.
Microbial analysis of samples was conducted after 24 h of All experiments were conducted in duplicates. For each repli-
treatments. cate, two samples were analyzed (n = 4). The data were analyzed
For the calculation of D10 value (irradiated dose required to using STATISTICA (StatSoft, Tulsa, OK, USA), and the means com-
eliminate one log CFU of population), the kinetics of bacterial parison among treatments was based on Tukeys HSD (Honestly
destruction was evaluated by linear regression. Bacterial counts Signicantly Difference) tests (p 6 0.05).
 R. Severino et al. / Journal of Food Engineering 124 (2014) 110 5

3. Results and discussion for samples treated by NC + C, NC + B, NC + L and NC + M, respec-

tively. After 6 days of storage, a microbial load of 4.00 log CFU/g
3.1. Preliminary experiments was observed in samples coated with MC alone. There were no sig-
nicant difference (p > 0.05) in microbial count among the treat-
The antimicrobial efcacy against L. monocytogenes of native ments of MC + C, MC + B, MC + L, however, these treatments were
chitosan and modied chitosan based coatings, containing four dif- signicant difference in microbial count as compared to the control
ferent nanoemulsion of essential oils (EO) and EO extracts, are without any treatment. Among all the treatments, MC + M caused
shown in Table 4 for up to 6 days storage at 4 C. The concentration highest reduction (1.46 log CFU/g or 96.5% bacterial population
of L. monocytogenes in untreated samples was 4.29 log CFU/g at the reduction) as compared to the control and therefore, this treatment
day 0 of storage; after 6 days of storage at 4 C it was, for uncoated was chosen for use in combination with the physical non-thermal
samples, equal to 4.91 log CFU/g. For samples coated with native treatments.
chitosan alone (NC), a microbial load equal to 3.83, 3.83 and 4.34 The antimicrobial activity of EOs against pathogenic bacteria
log CFU/g was observed at days 0, 2 and 6, respectively. The addi- such as L. monocytogenes, Salmonella typhimurium, Escherichia coli
tion of nanoemulsion of carvacrol, mandarin, bergamot and lemon has already been established (Cosentino et al., 1999). The major ac-
EO to the NC-based coating formulation (NC + C, NC + M, NC + B, tive components of EOs can be classied into phenols, terpenes,
NC + L) did not give a signicantly reduction of the initial L. mono- and aldehydes. Several works reported that all three classes of
cytogenes load at day 0 in comparison to NC. Samples coated with components principally act against the cell cytoplasmic membrane
modied chitosan alone (MC) exhibited a microbial load of 3.68 log (Ceylan and Fung, 2004; Sikkema et al., 1995; Ultee et al., 2000),
CFU/g at day 0, which is not signicantly different from the sam- especially because of their hydrophobic nature, which can affect
ples coatedwith NC containing the four different EO nanoemul- the unsaturated fatty acid on the bacterial membrane and thus al-
sions. There were no signicant difference (p > 0.05) in microbial ter its structure (Burt and Reinders, 2003). Phenolic compounds act
count among the treatments of MC + C, MC + B, MC + L, and as protonophore, a carrier of protons across the lipid bilayers, caus-
MC + M, however, these treatments were signicant difference ing the dissipation of the proton motive force (Ultee et al., 2002).
(p 6 0.05) in microbial count as compared to the control at day 0. The site of action of terpenes, such as limonene, is also the cyto-
After 2 days of storage samples coatedwith only MC and NC (Ta- plasmic membrane; they cause the loss of membrane integrity
ble 4) had a microbial load equal to, respectively, 3.92 log CFU/g and dissipation of the proton-motive force (Sikkema et al., 1994).
and 3.83 log CFU/g. The addition of nanoemulsion of essential oils The hypothesized mechanism of action of aldehyde is also based
to NC did not signicantly improved the antimicrobial effect after on the dissipation of the proton motive force, but differently from
2 days of storage, with a microbial load for samples treated with phenols, in this case the antimicrobial action is related to the leak-
NC + C, NC + B, NC + L and NC + M of 3.80, 3.95, 3.69, and 3.70 log age of small ions rather than large molecules (Gill and Holley,
CFU/g, respectively. On the other side, the samples treated with 2004). The antimicrobial activity of the NC and its derivatives have
MC + B, MC + L and MC + M exhibited a signicant reduction of also been established previously (Entsar et al., 2003). The most
the microbial load after 2 days of storage as compared to the sam- acceptable mechanistic model is based on the interaction between
ples treated with MC alone, causing a reduction of 0.97, 0.95 and positively charged chitosan molecules and negatively charged
1.08 log CFU/g in comparison to the control, respectively. microbial cell membranes. In this model the interaction is medi-
After 6 days of storage samples treated with NC showed a ated by the electrostatic forces between the protonated NH 3
microbial load of 4.34 log CFU/g, with a reduction of 0.57 log groups and the negative residues, presumably by competing with
CFU/g as compared to the control. The addition of the four different Ca2+ for electronegative sites on the membrane surface (Oussalah
EO nanoemulsions to NC did not cause a signicant change et al., 2006; Tsai and Su, 1999).
(p > 0.05) in the antimicrobial activity as compared to NC alone,
with a microbial load of 4.31 log, 4.44, 4.30, and 4.24 log CFU/g 3.2. Antimicrobial effects of different combined treatments against L.
monocytogenes inoculated in broccoli orets
Table 4
Effect of coating application on population of L. monocytogenes on broccoli orets 3.2.1. Antimicrobial effects of combined treatment using ozonated
during storage at 4 C*. water and antimicrobial coating
Treatment L. monocytogenes population (log CFU/g) The antimicrobial effect of combined treatment using ozonated
water and coating application against L. monocytogenes in broccoli
Day 0 Day 2 Day 6
orets was evaluated, with the results being reported in Fig. 1. Pre-
Control 4:29  0:08Aa 4:47  0:14Ba 4:91  0:16Ca liminary experiments showed that ozonated water washing alone
Native chitosan 3:83  0:13Ab 3:83  0:18Ab;c 4:34  0:08Bb caused a statistically signicant reduction (p 6 0.05) of microbial
NC + C 3:70  0:06Ab;c 3:80  0:14Ab;c;d 4:31  0:07Bb load only at an ozone concentration of 7 ppm and treatment times
NC + B 3:50  0:24Ac;d 3:95  0:21Bb 4:44  0:16Cb of 2.5 min and 5 min, which resulted in a reduction of 1.02 and
NC + L 3:41  0:25Ac;d 3:69  0:20Ab;c;d;e 4:30  0:23Cb;c 1.15 log, respectively, as compared to the control (no washing).
NC + M 3:64  0:22Ab;c 3:70  0:18Ab;c;d 4:24  0:06Bb;c;d The antimicrobial effects of these washing treatments with ozonat-
Modied chitosan 3:68  0:19Ab;c 3:92  0:17AB
b 4:00  0:11Bc;d;e ed water was also higher than that for samples washed with sterile
MC + C 3:50  0:18Ac;d 3:91  0:08Bb 3:99  0:12Bd;e water, which caused a reduction from 0.63 to 0.86 log CFU/g after
MC + B 3:46  0:22Ac;d 3:50  0:11Ad;e 3:93  0:19Be 2.5 min and 5 min, respectively (data not shown). Therefore, the
MC + L 3:29  0:08Ad 3:52  0:22Ac;d;e 3:86  0:16Be
washing of broccoli samples using ozonated water at 7 ppm for
2.5 min was selected for the further experiments in combination
MC + M 3:60  0:14Ab;c;d 3:39  0:10Be 3:45  0:15AB
f
with antimicrobial coating. A shorter exposure time to ozonated
NC, native chitosan; MC, modied chitosan; C, carvacrol nanoemulsion; B, bergamot water is in fact desirable, in order to preserve the organoleptic
essential oil nanoemulsion; L, lemon essential oil nanoemulsion; and M, mandarin properties of broccoli samples.
essential oil nanoemulsion.
*
Values are means standard deviations. Means with different lowercase letters
The application of the MC + M coating on samples washed with
within the same column are signicantly different (p 6 0.05), while means with 7 ppm ozonated water for 2.5 min allowed to increase signicantly
different uppercase letters within each treatment lot are signicantly different the antimicrobial activity against L. monocytogenes in broccoli o-
(p 6 0.05). rets. Samples treated only with the coating formulation of
6 R. Severino et al. / Journal of Food Engineering 124 (2014) 110
7
a
6 UV-C followed by coating
b
c
5 b
d 6
Log CFU/g
4
3
2
0 0
control coating 7 ppm 2.5 min 7 ppm 2.5 min +
coating
Fig. 1. Effect of combined treatment of ozonated water (7 ppm for 2.5 min) and
coating formulation on the population of L. monocytogenes on broccoli orets.
Means with different letters are signicantly different.
MC + M did not exhibit a signicantly microbial load reduction
compared to untreated samples, while samples treated with ozo-
nated water (7 ppm) for 2.5 min showed a microbial reduction of
1.13 log CFU/g. The combined treatment of washing with ozonated
water followed by the addition of the coating formulation of
MC + M caused a microbial load reduction of 1.51 log CFU/g, show-
ing an additive effect between the two different antimicrobial
treatments.
Several studies have been conducted on the use of ozonated
water for washing vegetables to eliminate L. monocytogenes. For
example, Rodgers et al. (2004) were able to reduce L. monocytoge-
nes level in green leaf lettuce to undetectable levels, while Yuk
et al. (2006) had no signicantly reduction of L. monocytogenes
population inoculated on lettuce. The effectiveness of ozone treat-
ment is in fact inuenced by several factors, such as the ozonation
delivery methods, the variety and structure of analyzed vegetables,
and also the conditions of temperature and inoculum level. For
example, in case of samples that contain high level of organic mat-
ters, the antimicrobial effect of ozonated water might be reduced
as explained by the fact that ozone may react with organic matter
released by food samples before acting on the bacteria present at
the food surfaces (Ketteringham et al., 2006).
3.2.2. Antimicrobial effects of combined treatment using UV-C and
antimicrobial coating
The germicidal effect of UV-C radiation on L. monocytogenes
inoculated in broccoli orets was evaluated alone and in combina-
tion with a coating treatment with MC + M at different exposition
doses, with the results being reported in Fig. 2. The exposition of
inoculated samples to UV-C doses of 2 kJ/m2, 4 kJ/m2, 8 kJ/m2 and
10 kJ/m2 did not show any signicant reduction of the microbial
load. UV-C radiation is mainly a surface treatment (Jagger, 1965),
and therefore the physical location of the microorganisms in the
product and the different composition and structure of the product
may play an important role in the antimicrobial efciency of the
UV-C radiation in reducing microbial loads. For example, Yaun
et al. (2004)found that UV-C was more effective at reducing bacte-
rial populations on the surface of apples than on tomatoes and
lettuce.
The used antimicrobial coating formulation (MC + M) was
tested on broccoli samples either before or after the UV-C treat-
ment, in order to identify the better application ensuring L. mono-
cytogenes reduction on broccoli samples. The exposition of
UV-C
8 Coating followed by UV-C
b
b b
c c cd
cd e de e
Log CFU/g
4
2
2 kJ/m 2 4 kJ/m 2 8 kJ/m 2 10 kJ/m2
Fig. 2. Effect of UV-C treatment at different doses (2 kJ/m2, 4 kJ/m2, 8 kJ/m2, and
10 kJ/m2) and UV-C treatment (8 kJ/m2) in combination with coating treatment on
L. monocytogenes population on broccoli orets. Coating treatment is tested before
and after UV-C treatment. Means with different letters are signicantly different.
inoculated samples to UV-C doses of 2 or 4 kJ/m2 after the coating
treatment caused a microbial load reduction of 0.8 log CFU/g, with-
out any signicant difference between the two different doses. An
improvement of the antimicrobial effectiveness was obtained
when inoculated samples were exposed to UV-C doses of 8 or
10 kJ/m2 after the coating treatment, with a resulting signicant
(p 6 0.05) microbial reduction of 1.10 log CFU/g.
The exposition of broccoli orets inoculated with L. monocytog-
enes to UV-C doses of 2 or 4 kJ/m2 before the coating treatment
caused a microbial reduction of 0.85 log CFU/g, while the exposi-
tion to UV-C doses of 8 or 10 kJ/m2 before the coating treatment
caused a signicant (p 6 0.05) microbial load reduction of 1 log
CFU/g. The exposition of broccoli orets to UV-C doses not exceed-
ing 10 kJ/m2 was reported to delay yellowing and chlorophyll deg-
radation, to reduce chlorophyll peroxidase and chlorophyllase
activity, as well as to maintain the antioxidant capacity during
storage (Costa et al., 2006), while UV-C radiations are in general
not characterized by a strong antimicrobial activity against L. mon-
ocytogenes in broccoli orets. The use of a MC + M coating formula-
tion caused the antimicrobial activity of UV-C treatment against L.
monocytogenes to increase, although from obtained data there are
no signicantly differences between samples treated with coating
formulation before or after the UV-C exposition. This suggests that
the UV-C exposition on samples treated with the coating formula-
tion does not affect in any way the structure and antimicrobial
activity of the coating formulation itself.
3.2.3. Antimicrobial effects of combined treatment using c irradiation
and antimicrobial coating
Fig. 3 shows the radiosensitization of L. monocytogenes in broc-
coli orets treated with the coating formulation MC + M. The pres-
ence of the bioactive coating increased the radiosensitivity of L.
monocytogenes, reported as microbial inactivation as a function of
c-ray dose for the uncoated and coated samples. A radiation D10-
value of 0.32 kGy was observed for control samples. It was ob-
served that the addition of antimicrobial compound increased
the radiosensitization of analyzed bacteria: the D10-value of L.
monocytogenes in the samples coated with MC + M was 0.24 kGy.
This caused an increase in the relative radiation sensitivity SR by
1.33-fold, considered as the ratio between the radiation D10-value
of the control and the radiation D10-value of samples in presence of
antimicrobial coating.
 R. Severino et al. / Journal of Food Engineering 124 (2014) 110 7

in fact psychotropic bacteria, able to grow between 0.4 C and

Irradiation
Coating plus Irradiation
50 C (Farber and Peterkin, 1991), and therefore able to grow and
0 proliferate in foods at refrigeration temperatures (Knabel et al.,
1990).
During 13 days storage at 4 C L. monocytogenes load on control
-2 samples increased from 3.13 log CFU/g to 5.33 log CFU/g. Samples
Log N/N0

treated with MC + M exhibited a signicant 1 log CFU/g reduction

D10 =0.32
-4
D10=0.24
-6
-8
0.0 0.5 1.0 1.5 2.0 2.5
Irradiation doses (kGy)
Fig. 3. Radiosensitization of L. monocytogenes in broccoli orets after coating
treatment. The D10-value is indicated for each curve. The relative radiation
sensitivity SR, considered as the ratio between the radiation D10-value of the
control and the radiation D10-value of samples in presence of antimicrobial coating,
increased by 1.33-fold.
These results indicate that the tested coating increased the
radiosensitivity of L. monocytogenes in broccoli orets. In different
study about gamma irradiation treatment in combination with
other non-thermal treatments, the authors proposed that survived
microorganisms after irradiation treatment will probably be more
sensitive than untreated cells to adverse environmental conditions
(Lacroix et al., 2000; Mahrour et al., 2003). The mechanism of ac-
tion of microbial inhibition/elimination by irradiation is expressed
by the breakdown of chemical bonds in the DNA molecules, alter-
ation of microbial membrane permeability, and alteration of some
cellular functions (Lopez-Gonzales et al., 1999; Murano, 1995).
These changes may promote the interaction between antimicrobial
molecules and cell membranes.
3.3. Antimicrobial effects of different combined treatments against L.
monocytogenes during storage of broccoli orets
The antimicrobial effect of the MC + M coating in combination
with different non-thermal treatments against L. monocytogenes
on broccoli orets was also evaluated during 13 days of refriger-
ated storage at 4 C. Results are reported in Table 5. The coating
formulation was tested in combination with ozonated water wash-
ing with a continuous ow of 7 ppm for 2.5 min, done before the
coating treatment, in combination with UV-C treatment using
doses of 8 kJ/m2 for each side of the sample, and in combination
with c-ray treatment using dose of 0.28 kGy, to evaluate the anti-
microbial activity during refrigerated storage. L. monocytogenes are
(p 6 0.05) of the microbial load on day 1; after 13 days their micro-
bial load was 4.22 log CFU/g, with a microbial load reduction, as
compared to control samples, of 1.11 log CFU/g. Despite the ob-
served increase of the microbial load on broccoli orets treated
with the coating formulation during storage time, the antimicro-
bial activity of MC + M coating was able to maintain a microbial
load lower of at least 1 log CFU/g than control during storage time.
In a study of Le Tien et al. (2003) about the use of acylated chitosan
3.0 as hydrophobic matrix for controlled drug release, it was found
that palmitoylated chitosan, due to its hydrophobic stabilization,
resulted the best matrix, compared to other modied chitosan
matrices, in controlled release of active compounds. This feature
can contribute to the prolonged antimicrobial activity against L.
monocytogenes showed by the coating formulation of palmitoylat-
ed chitosan and nanoemulsion of mandarin EO during the 13 days
storage.
The combination of coating and ozone treatments showed the
highest microbial reduction on day 1 of storage, with a signicant
(p 6 0.05) microbial load reduction of 1.67 log CFU/g. Despite
ozone being recognized as an effective sanitizer, with approxi-
mately 50% greater oxidation capacity than chlorine (Kim et al.,
1998), there are conicting studies about its antimicrobial efcacy.
The antimicrobial efcacy of ozone is inuenced in fact by several
factors, such as the organic matter, the initial microbial load, the
temperature of storage and the ozone delivery system. In the pres-
ent study a system based on bubbling ozone in water with strong
stirring was developed, according to the results obtained by Kim
et al. (1998), who showed that bubbling ozone gas in water with
rapid stirring provides the best ozone delivery system for the inac-
tivation of microorganisms on vegetables. Farooq et al. (1977)
found that the presence of ozone bubbles was more effective in
inactivating microorganisms than dissolved ozone alone. Their
observation was explained by a conceptual model using the lm
theory. According to this theory, a liquid lm is formed at the
gasliquid interface, and ozone is more concentrated in the liquid
lm than in the bulk liquid. This implies that inactivation of micro-
organisms would be greater when they are in contact with ozone
bubbles or when present within liquid lm than with bulk liquid.
Therefore, smaller bubbles provide larger surface area and greater
inactivation.
The combination of ozone treatment followed by coating appli-
cation showed a strong antimicrobial activity limited to the early
days of storage, while from day 5, the residual antimicrobial activ-
ity decreased, and a signicant increase in the microbial load was

Table 5
Effect of coating formulation in combination with non-thermal treatments on population of L. monocytogenes on broccoli orets during storage at 4 C*.

Treatment L. monocytogenes population (log CFU/g)

Day 1 Day 3 Day 5 Day 7 Day 9 Day 11 Day 13
Control 3:13  0:12Aa 3:25  0:13Aa 3:70  0:15Ba 4:51  0:12Ca 5:04  0:17D
a 5:28  0:09Ea 5:33  0:18Ea
Coating 2:10  0:08Ab 2:21  0:11Ab 2:34  0:22Ac 2:98  0:16Bc 3:34  0:23Cc 3:96  0:15D
c 4:22  0:13D
c
Ozone + coating 1:46  0:15Ac 1:73  0:20Ac 2:79  0:15Bb 3:23  0:21Cb 4:13  0:16D
b 4:39  0:18D
b 4:76  0:20Eb
UV-C + coating 1:90  0:10Ab 2:03  0:14Ab 2:11  0:13Ad 2:65  0:17Bd 2:92  0:20Cd 3:32  0:19D
d 3:97  0:21Ec
c ray + coating 2:01  0:11Ab 2:21  0:14ABC
b 2:15  0:10AB
cd 2:25  0:09BC
e 2:40  0:17CD
e 2:49  0:12D
e 2:74  0:19Ed
*
Values are means standard deviations. Means with different lowercase letters within the same column are signicantly different (p 6 0.05), while means with different
uppercase letters within each treatment lot are signicantly different (p 6 0.05).
8 R. Severino et al. / Journal of Food Engineering 124 (2014) 110

observed. After 13 days of storage, the observed reduction in com-
parison to the control was of only 0.57 log CFU/g, with an increase
of the microbial load on 13 days storage of 3 log CFU/g.
Samples treated with the coating formulation and UV-C doses of
8 kJ/m2 for each side showed a signicant (p 6 0.05) microbial load
reduction of 1.23 log CFU/g which was not signicantly different
from the samples treated with coating alone after 1 day of storage.
A signicant reduction of the antimicrobial activity was observed
in the rst 7 days of storage at 4 C, while in the second 7 days
the population of L. monocytogenes increased 1.5 log CFU/g, proba-
bly due the formation of slight damages on the surface of the veg-
etables as a consequence of the high doses of irradiation, making
nutrients more accessible for bacterial growth. Ultraviolet radia-
tions cause crosslinks of aromatic amino acids at their carboncar-
bon double bonds which lead to membrane depolarization and
abnormal ionic ow, photochemical oxidation and pyrimidine di-
mer formation in DNA strands (Miller et al., 1999). The DNA muta-
tions block the transcription and replication of the cell,
compromising cellular functions.
The application of the coating formulation can improve the
antimicrobial efcacy of the UV-C treatment; however, being UV-
C irradiation a surface treatment, its antimicrobial effect is strongly
inuenced by the structure and topography of the treated samples.
The residual antimicrobial activity observed after the combined
treatment of UV-C radiations and coating application was shown
not to be able to ensure microbial safety against L. monocytogenes
during 13 days storage at 4 C.
It can be observed that samples treated with coating formula-
tion, with the combined treatment of ozone washing followed by
coating formulation and with the combined treatment of UV-C
light and coating formulation showed a relatively similar increase
of L. monocytogenes population during 13 days storage, with an in-
crease of approximately 2 log CFU/g of the initial microbial
population.
The combined treatment of gamma irradiation and active coat-
ing based on modied chitosan and nanoemulsion of mandarin EO
caused a signicant (p 6 0.05) microbial reduction of about 1 log
CFU/g on day 1 of storage, as expected from the calculated D10-va-
lue. This combined treatment showed a strong residual antimicro-
bial activity during storage, with an increase of L. monocytogenes
population during 13 days of storage of only 0.73 log CFU/g. After
13 days of storage, the microbial load on broccoli orets treated
with coating formulation and gamma irradiation was 2.74 log
CFU/g which was lower of 2.5 log CFU/g than for control.
Microorganisms that are able to survive after c-ray treatment
are probably more sensitive to adverse environmental conditions,
such as changes in temperature, pH, nutrients, or the presence of
other antimicrobial substances, like essential oils, as suggested in
some previous works (Lacroix et al., 2000). This hypothesis is sup-
ported by the data obtained by Mahrour et al. (2003), who treated
poultry with a combined treatment of irradiation and marination
in rosemary, thyme, and lemon juice, obtaining a signicant in-
crease in bacterial radiosensitization. According to Chiasson et al.
(2004), the increased radiosensitivity is caused by the antimicro-
bial properties of EOs, that are predominantly associated with their
main components. Chemical analysis of mandarin EO, shown in Ta-
ble 6, has indicated that the major compounds of this EO are lim-
onene (70.6%) and terpinene (18.6%). The site of action of terpenes
is the cytoplasmic membrane, causing a loss of membrane integ-
rity, the inhibition of respiratory enzymes and dissipation of the
proton-motive force (Sikkema et al., 1994). This mechanism is en-
hanced by the encapsulation in nanoemulsion, that improves their

Table 6
GCMS analysis of bergamot, lemon and mandarin essential oils: retention time, measured peak area and relative concentration of the compounds constituting the essential oils.

Component Bergamot oil Lemon oil Mandarin oil

Retention time Area Area percent Retention time Area Area percent Retention time Area Area percent
a-thujene 6.00 7.7E+05 0.36 6.07 1.4E+06 0.51 6.03 1.1E+07 0.78
a-pinene 6.28 3.0E+06 1.39 6.35 6.1E+06 2.28 6.32 3.0E+07 2.12
camphene 6.87 8.0E+04 0.04 6.95 1.8E+05 0.07 6.90 2.5E+05 0.02
sabinene 7.92 2.4E+06 1.14 8.02 5.3E+06 1.99 7.96 3.4E+06 0.24
b-pinene 8.11 1.4E+07 6.55 8.22 3.3E+07 12.29 8.15 2.2E+07 1.56
myrcene 8.75 1.9E+06 0.90 8.86 4.2E+06 1.56 8.82 2.4E+07 1.71
3-octanol 9.26 2.9E+04 0.01 9.39 3.2E+04 0.00
d-3-carene 9.67 2.2E+05 0.08 9.57 2.8E+06 0.20
a-terpinene 10.33 3.1E+05 0.14 10.45 3.7E+05 0.14 10.40 4.9E+06 0.35
p-cymene 10.86 2.1E+05 0.10 10.99 1.5E+06 0.57 11.01 4.1E+06 0.29
limonene 11.18 7.2E+07 33.57 11.37 1.8E+08 67.55 11.63 1.0E+09 70.64
(z)-b-ocymene 11.69 6.7E+04 0.03 11.82 1.8E+05 0.07 11.87 1.1E+05 0.01
(e)-b-ocymene 12.34 6.2E+05 0.29 12.46 2.6E+05 0.10 12.45 3.0E+05 0.02
c-terpinene 13.00 1.4E+07 6.49 13.12 2.2E+07 8.18 13.30 2.6E+08 18.58
terpinolene 14.88 9.1E+05 0.34 15.26 4.7E+04 0.00
linalool 15.67 3.9E+07 18.24 15.67 5.5E+05 0.21 15.62 1.6E+06 0.12
nonanal 15.85 1.0E+05 0.05 15.94 6.5E+05 0.24
a-terpineol 20.74 1.6E+05 0.07 20.80 3.0E+05 0.11 20.74 2.3E+06 0.16
decanal 21.52 7.9E+04 0.04 21.61 5.5E+04 0.02 21.53 1.3E+06 0.09
nerol 22.74 2.6E+05 0.12 22.81 7.3E+04 0.03 22.79 3.0E+05 0.02
neral 23.39 3.9E+05 0.18 23.43 1.7E+06 0.63 23.58 8.2E+04 0.01
linalyl acetate 24.16 5.9E+07 27.34 24.19 4.1E+04 0.02 24.47 7.9E+03 0.00
geranial 24.89 5.7E+05 0.27 24.93 2.8E+06 1.03 25.03 8.6E+05 0.06
anethole 26.66 3.0E+04 0.01 26.19 9.7E+05 0.07
eugenol 29.11 5.8E+05 0.27
neryl acetate 29.15 7.0E+05 0.26 29.14 5.8E+04 0.00
isosafrole 29.48 1.8E+05 0.01
geranyl acetate 29.90 2.5E+05 0.12 29.94 4.5E+05 0.17 29.93 5.9E+04 0.00
methyl-eugenol 31.04 4.0E+04 0.02 31.09 5.0E+04 0.02
methyl n-methyl anthranilate 31.20 5.3E+05 0.25 31.25 4.1E+05 0.16 31.21 1.6E+06 0.12
b-caryophyllene 31.81 4.2E+05 0.20 31.85 6.5E+05 0.24 32.48 1.9E+05 0.01
valencene 34.02 6.6E+04 0.03 34.06 8.6E+04 0.03 33.97 6.1E+05 0.04
a-sinensal 34.39 6.0E+05 0.28 34.23 7.6E+04 0.03 34.38 2.8E+06 0.20
b-bisabolene 34.62 1.6E+04 0.01 34.43 8.8E+05 0.33 34.92 1.2E+05 0.01
 R. Severino et al. / Journal of Food Engineering 124 (2014) 110
bioactivity by increasing dispersion in aqueous phase and activat-
ing passive mechanisms of cell adsorption (Dons et al., 2012;
Weiss et al., 2009). In addition, the interaction with palmitoylated
chitosan is likely to signicantly contribute to the observed in-
crease of antimicrobial activity, that due to its hydrophobic charac-
ter, allows to improve hydrophobic interactions with bacterial
cytoplasmic membrane.
4. Conclusions
In the present study the antimicrobial activity of native and
modied chitosan in combination with nanoemulsion of essential
oils and extract from essential oils against L. monocytogenes inocu-
lated on broccoli orets was evaluated. Modied chitosan-based
coating containing 0.05% (w/v) mandarin essential oil, encapsulated
in nanoemulsion, was found as the most efcient coating formula-
tion among those tested, causing a microbial load reduction by 1.46
log CFU/g after 6 days of storage. This coating formulation was also
tested in combination with several non-thermal treatments such as
ozonated water, UV-C and gamma irradiation. The combination of
ozonated water and coating formulation showed an additive anti-
microbial effect in the rst days of treatments, but was character-
ized by a low residual antimicrobial activity during 13 days
storage at 4 C. The combination of coating formulation and UV-C
treatment on samples inoculated with L. monocytogenes was able
to increase the antimicrobial activity of UV-C radiation, with a
microbial load reduction, after 13 days of storage, of 1.3 log CFU/
g. The combination of coating formulation and gamma irradiation
was able to increase the radiosensitivity of L. monocytogenes in
broccoli orets, showing a synergistic effect between the two treat-
ments. This permits to obtain a good microbial load reduction by
using low c-ray doses and low essential oil concentrations. This
combined treatment was able to control the growth of L. monocyt-
ogenes during storage in which there was less than one log increase
in bacterial concentration after 13 days of storage at 4 C, as com-
pared to the microbial load after 1 day from the treatment. This
combined treatment could be used as a potential approach to con-
trol effective the growth of L. monocytogenes in other food products.
Acknowledgements
This work was conducted in the frame of the NanoBioSafe
project for the use of nanoencapsulated bioactive molecules in
combination with treatments with emerging technologies for the
food safety. The research was supported by the Italian Ministry
for Foreign Affair and ProdAl and by the Natural Sciences and Engi-
neering Research Council of Canada (NSERC) Discovery Program
and by the International Atomic Energy Agency (IAEA) RCM Project
F22063-CR-1. Renato Severino thanks the Project CARINA (Sicu-
rezza, sostenibilit e competitivit nelle produzioni agroalimentari
campane) POR Campania FSE 20072013, for his stay at INRS. The
authors acknowledge the Stazione Sperimentale per le Industrie
delle Essenze e dei Derivati dagli Agrumi Italy, for the composi-
tional determination of essential oils. Nordion Int. is acknowledged
for irradiation treatment of samples.
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