1. Antibacterial and antifungal LDPE films for
active packaging
Rotem Shemesha,b
, Maksym Krepkera
, Diana Goldmana
, Yael Danin-Polega
,
Yechezkel Kashia
, Nadav Nitzanc
, Anita Vaxmanb
and Ester Segala
*
Active antimicrobial packaging is a promising form of active packaging that can kill or inhibit microorganism growth
in order to maintain product quality and safety. One of the most common approaches is based on the release of vol-
atile antimicrobial agents from the packaging material such as essential oils. Due to their highly volatile nature, the
challenge is to preserve the essential oils during the high-temperature melt processing of the polymer, while main-
taining high antimicrobial activity for a desired shelf life. This study suggests a new approach in order to achieve this
goal. Antimicrobial active films are developed based on low-density polyethylene (LDPE), organo-modified montmo-
rillonite clays (MMT) and carvacrol (used as an essential oil model). In order to minimize carvacrol loss throughout
the polymer compounding, a pre-compounding step is developed in which clay/carvacrol hybrids are produced.
The hybrids exhibit a significant increase in the d-spacing of clay and enhanced thermal stability. The resulting
LDPE/(clay/carvacrol) films exhibit superior and prolonged antibacterial activity against Escherichia coli and Listeria
innocua, while polymer compounded with pure carvacrol loses the antibacterial properties within days. The films
also present an excellent antifungal activity against Alternaria alternata, used as a model plant pathogenic fungus.
Furthermore, infrared spectroscopy analysis of the LDPE/(clay/carvacrol) system displayed significantly higher carva-
crol content in the film as well as a slower out-diffusion of the carvacrol molecules in comparison to LDPE/carvacrol
films. Thus, these new films have a high potential for antimicrobial food packaging applications due to their long-
lasting and broad-spectrum antimicrobial efficacy. Copyright © 2014 John Wiley & Sons, Ltd.
Keywords: polyolefins; antimicrobial; essential oils; carvacrol; nanocomposites; clay
INTRODUCTION
A broad spectrum of microbial pathogens can contaminate hu-
man food and water supplies, and cause illness after they or their
toxins are consumed.[1]
An important challenge related to food
safety and shelf life extension is the development of antimicro-
bial packaging.[2]
These food-packaging systems act to reduce,
inhibit or retard the growth of microorganisms that may be pres-
ent in the packed food.[3,4]
The target microorganisms and the
food composition must be considered in antimicrobial packag-
ing. Antimicrobials have to be selected based on their spectrum
of activity, mode of action, chemical composition, as well as the
rate of growth and physiological state of the targeted microor-
ganisms.[3,5]
This approach can reduce the addition of larger
quantities of antimicrobial preservatives that are usually incorpo-
rated into the bulk of the food.[6]
There are several methods for the introduction of antimicro-
bial activity into polymeric materials, which include incorporat-
ing antimicrobial agents directly into the polymers, coating
antimicrobials onto polymer surfaces,[7,8]
immobilizing antimi-
crobials by chemical grafting[9,10]
or using polymers that exhibit
intrinsic antimicrobial properties (e.g. chitosan).[6]
Synthetic anti-
microbials, which are commonly integrated into polymers for
packaging, include organic or inorganic acids, metals, alcohols,
ammonium compounds or amines.[11,12]
Some of these mate-
rials, specifically silver-based additives, are already commercially
available and are applied for food packaging.[13,14]
However, the
increasing consumer health concern and growing demand
for healthy foods have stimulated the use of natural
biopreservatives, such as essential oils, e.g. carvacrol and
thymol.[15]
Essential oils are natural substances categorized as
GRAS (generally recognized as safe) by the Food and Drug
Administration (FDA), and most of them are derived from
plants.[16]
They have shown substantial antibacterial and antifun-
gal properties achieved both in direct contact and in vapor
phase.[17]
The possibility of achieving an antimicrobial action
by the release of the volatile compounds has increased the
interest of including them into the packaging. One major
drawback of essential oils molecules is their volatile nature;
therefore, the main applicative methodology for their incorpo-
ration into polymers is by coating technologies.[18,19]
Once the
essential oils are directly incorporated into the polymer matrix
by high-temperature melt processing, antimicrobial activity is
achieved.[16,18,20–33]
However, this activity is evaluated, in most
* Correspondence to: Ester Segal, Department of Biotechnology and Food
Engineering, Technion—Israel Institute of Technology, Haifa 32000, Israel.
E-mail: esegal@tx.technion.ac.il
a R. Shemesh, M. Krepker, D. Goldman, Y. Danin-Poleg, Y. Kashi, E. Segal
Department of Biotechnology and Food Engineering, Technion—Israel Insti-
tute of Technology, Haifa 32000, Israel
b R. Shemesh, A. Vaxman
Carmel Olefins Ltd., P.O. Box 1468, Haifa 31014, Israel
c N. Nitzan
D.S. Smith Plastics/StePac L.A., Tefen Industrial Park, Tefen, Western Galilee,
24959, Israel
Research article
Received: 10 October 2014, Accepted: 11 November 2014, Published online in Wiley Online Library: 1 December 2014
(wileyonlinelibrary.com) DOI: 10.1002/pat.3434
Polym. Adv. Technol. 2015, 26 110–116 Copyright © 2014 John Wiley & Sons, Ltd.
110

2. cases, immediately after film production,[29,33–35]
while the chal-
lenges associated with the controlled and prolonged activity of
these films are not addressed.
In recent years, polymer nanocomposites[36–41,2,42]
containing
exfoliated organoclay platelets have been extensively studied
for food packaging applications.[2,43]
In particular, these nano-
composites exhibit excellent barrier properties, which are as-
cribed to the clay layers, hindering the diffusing molecule
pathway.[2,44–46]
An emerging approach is to employ polymer
nanocomposites for potential antimicrobial applications.[47–49]
The prevailing methodology in most studies is to incorporate
organo-modified montmorillonite clays (MMT) as filler during
melt compounding.[26,31,50]
The rational for the incorporation of
the clay was to improve the barrier properties of the films in or-
der to protect the volatile and heat-sensitive essential oil
molecules.
Our novel approach is to use the layered clays as an “active”
carrier for antimicrobial essential oils, e.g. carvacrol. This is
achieved by a pre-compounding step in which clay/carvacrol hy-
brids are produced.[51]
Taking into consideration the unique lay-
ered structure of the clay platelets, our interest was to utilize this
character for active encapsulation of the carvacrol into the galler-
ies of the clay, as schematically illustrated in Scheme 1. In our
recent study[51]
we have investigated the effect of different com-
mercially available clays on their ability to retain carvacrol during
high-temperature melt processing. Herein, we use the most
promising clay/carvacrol combination to develop antimicrobial
polymer films with a broad spectrum of inhibitory activity
against Gram-negative (Escherichia coli) bacteria, Gram-positive
(Listeria innocua) bacteria and a common plant pathogenic fun-
gus, Alternaria alternata. Importantly, we investigate the films’
antimicrobial activity variation and out-diffusion kinetics of
carvacrol with storage time, which is crucial in a wide range of
applications to enhance food safety and shelf life.
EXPERIMENTAL
Preparation and characterization of clay/carvacrol hybrids
Carvacrol (98%, Sigma Aldrich Chemicals, Israel) is mixed with
natural montmorillonite clay modified with quaternary ammo-
nium salt (Dellite®72T, Laviosa Chimica Mineraria, Italy) by
ultrasonication (Vibra cell VCX 750, Sonics & Materials Inc., USA)
at a weight ratio of 2:1, respectively. Ultrasonication is performed
at room temperature for 20 min at an amplitude of 40% to form
uniform carvacrol–clay dispersions (Scheme 1).
X-ray diffraction (XRD)
The degree of carvacrol intercalation into the clay galleries is
evaluated by X-ray diffraction (XRD) using Bragg–Brentano
q-2q Philips PW3020 diffractometer, powered by a PW1710
generator. The diffraction data is collected by 2q step scanning
between 2θ of 1.2 and 10°, at 0.02° steps and a count time of
1 s/step. The experimental conditions are diffracted-beam graph-
ite (00.2) monochromator, Cu Ka radiation, 40 KV and 40 mA,
divergence and anti-scattering slits 1°, receiving slit 0.2 mm.
Thermogravimetric analysis (TGA)
Thermal measurements are performed using TGA Q5000 system
(TA instruments, USA) at a heating rate of 20°C/min under nitro-
gen atmosphere, starting at room temperature up to 600°C.
Preparation and characterization of LDPE/(clay/carvacrol)
films
Low-density polyethylene (LDPE), Ipethene 320 (Carmel Olefins
Ltd., Haifa, Israel) with a melt flow rate of 2 g/10 min is melt-
compounded with clay/carvacrol hybrids using a 16-mm twin-
screw extruder (Prism, England) L/D ratio of 25:1 with a screw
speed of 150 rpm and a feeding rate of 2 kg/h at 140°C. Table 1
specifies the composition of the different blends investigated
in the present study. Following the melt-compounding process,
120-μm-thick films are prepared by cast extrusion using 45-mm
screw diameter extruder (Dr. Collin, Germany) at 140°C.
Infrared spectroscopy
Carvacrol content in the LDPE-based films is characterized by
Fourier-transform infrared spectroscopy in transmission mode.
Spectra are recorded using a Thermo 6700 FTIR instrument and
OMNIC v8.0 software, and data analysis is performed using TQ
Scheme 1. A schematic illustration of organoclay galleries modified with carvacrol molecules as achieved by a pre-compounding step in which clay/
carvacrol hybrids are produced. This figure is available in colour online at wileyonlinelibrary.com/journal/pat
Table 1. The composition of the different blends investi-
gated in this work
Sample LDPE
(wt%)
Clay
(wt%)
Carvacrol
(wt%)
Neat LDPE 100 0 0
LDPE/clay 95 5 0
LDPE/carvacrol 90 0 10
LDPE/(clay/carvacrol) 85 5 10
ANTIBACTERIAL AND ANTIFUNGAL LDPE FILMS FOR ACTIVE PACKAGING
Polym. Adv. Technol. 2015, 26 110–116 Copyright © 2014 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/pat
111

3. analyst v8.0 software. Spectra are collected from at least three
different locations on the films. The peak area at 810 cmÀ1
,
which is attributed to out-of-plane deformation vibrations in
carvacrol molecule,[52]
is calculated and is used to quantify the
carvacrol concentration using a calibration curve.
Transmission electron microscopy (TEM)
The nanostructure of the resulting films is studied with a Philips
CM120 transmission electron microscope (TEM) operated at
120-kV accelerating voltage, using 400 mesh carbon-covered
Cu grid Pk/100 (SPI Supplies West Chester, USA). Images are re-
corded digitally by a Gatan MultiScan 791 CCD camera using
the Digital Micrograph 3.1 software. Ultra-thin cross sections of
approximately 100 nm thick are prepared at À100°C with a
Reichert E Ultracut microtome, using a glass knife.
Antimicrobial activity
The antibacterial activity of the different films against E. coli
(ATCC 8739) is evaluated utilizing two methods. Initially, the
Kirby–Bauer technique is used for potency screening.[53]
The
zone of bacterial inhibition around the circumference of a poly-
meric film disc is utilized to qualitatively assess whether or not
the polymeric matrix sample possesses antibacterial proper-
ties.[3,54,55]
Discs (of 12 mm in diameter) are cut out of sample
films and are placed onto the surface of a full concentration Luria
broth (LB) agar in 9 cm Petri plates that are inoculated with
0.1 ml of 108
colony forming units (CFU)/ml of bacterial culture.
The plates are incubated at 37°C for 18 h, and the antibacterial
activity is recorded by observing the presence or absence of an
inhibition zone around the studied sample. Films without carva-
crol are assayed as negative control. The inhibition zone tests are
performed in triplicates.
The second method is quantitative and is based on the
Japanese standard (JIS Z 2801: 2000 “Antimicrobial products—
test for antimicrobial activity and efficacy”). E. coli is cultured
overnight in Nutrient Broth media (NB, Sigma Aldrich Chemicals,
Israel) under continuous shaking (250 rpm) at 37°C. In the follow-
ing day, the overnight culture is diluted in fresh NB medium to
an optical density (OD) value of 0.1, which approximately corre-
sponds to 108
CFU/ml. The culture is incubated for an additional
1.5 h, allowing the cells to enter the logarithmic stage. As the
culture reaches an OD value of 0.6, it is diluted by 1:100 with
1% NB to obtain a bacterial stock solution at a concentration of
105
CFU/ml. Efficacy tests are carried out in 6-well plates. Each
film sample is placed in a well that is supplemented with 3 ml of
bacterial stock solution. The plates are incubated at 37°C (under
continuous agitation at 100 rpm) for 24h. Incubation is followed
by a 1:100 serial dilutions with 1% NB performed in 96-well plates.
The drop-plate technique is used to assess viable cell counts with
20-μl drops that are transferred onto 1% NB bacto-agar (Becton
Dickinson) in 9-cm Petri plates. The Petri plates are incubated at
37°C for 18–24 h; CFU are counted, and log reduction is calculated
in comparison to E. coli cultured in NB 1:100 medium (108
CFU/ml)
that is used as control. All measurements, including the growth
controls, are performed in triplicates.
L. innocua (ATCC 33090) is cultured overnight in brain heart in-
fusion media (BH, Difco, France) under continuous shaking
(250 rpm) at 37°C. The following day, the incubated culture is di-
luted in a fresh BH medium to an optical density (OD) of 0.1 and
is reincubated for 1.5 h to allow the cells to enter the logarithmic
stage until an OD value of 0.3 is reached, which approximately
corresponds to 108
CFU/ml. Then, the bacteria are diluted with
1% NB (1:100) to obtain a bacterial stock solution at a concentra-
tion of 105
CFU/ml. Film samples are placed in a 24-well plate
added with 1 ml of bacterial stock solution. The plates are incu-
bated at 37°C (under continuous agitation at 100 rpm) for 24 h
followed by serial dilutions with NB 1:100 in 96-well plates. The
drop plate technique is used for viable cell counts. Drops of
10 μl are placed onto NB bacto-agar (Becton Dickinson) in 9 cm
Petri plates to determine cell numbers. The plates are incubated
at 37°C for 18–24 h followed by CFU count and log reduction cal-
culation. L. innocua cultured in NB 1:100 medium (106–7
CFU/ml)
is used as control for comparisons. All tests are conducted in
triplicates.
The phytopathogenic and clinical fungus A. alternata originat-
ing from the surface of tomato fruits and cultured on 1% Corn
Meal Agar (Difco CMA: 10 g/l; Bacto Agar: 10 g/l in 1000 ml of
deionized water) is used in all tests. Efficacy tests are conducted fol-
lowing a modified ISO 16869: 2008 protocol (Plastics—Assessment
of the effectiveness of fungistatic compounds in plastic formula-
tions), utilizing 1% CMA instead of Nutrient Salt Agar. Sample film
discs (30 mm in diameter) are excised from the film with the aid
of a manual puncher. The sample discs are placed onto 1%
CMA in the center of 9-cm Petri plates. Fungal conidial suspen-
sion is prepared by harvesting conidia from 5-day-old cultures
with sterilized de-ionized water. The spore suspension is adjusted
with the aid of a hemocytometer and then transferred into soft
agar (pre-solidified 1% CMA cooled to 45°C), producing an
inoculum suspension at a final conidial concentration of
105
conidia/ml. The inoculum is poured over the disc and the base
agar layer, covering the sample with a thin layer of inoculum. The
Petri plates are left at room temperature for 1 h to allow the inoc-
ulum layer to solidify. Then the plates are sealed with Parafilm
and incubated at 28°C in the dark for 10 days. LDPE films amended
or not amended with 1% Folpet (Thiophthalamide; Folpan 50 WP
broad spectrum M group fungicide by Makhteshim–Agan, Israel)
are used as positive and negative controls, respectively. Following
incubation the antifungal activity of the tested film is quantified
with the aid of a stereomicroscope (Model SMZ 171, MRC Lab,
Israel) following fungal growth and sporulation. The activity is re-
corded using a non-parametric ordinal scale as follows: 4 = Very
High (VH) activity—no fungal growth; 3 = High (H) activity—slight
mycelial growth; 2 = Moderate (M) activity—mycelial growth pres-
ent with sporulation covering up to 10% of the tested sample;
1 = Low (L) activity—mycelial growth present with sporulation cov-
ering up to 30% of the tested sample and 0 = no activity (none)—
severe fungal sporulation. All tests are carried out in triplicates, and
the antifungal efficacy of the tested film sample is reported as the
median of the three replications.
RESULTS AND DISCUSSION
Clay/carvacrol hybrids
Organoclay and clay/carvacrol hybrid are characterized by X-ray
diffraction (XRD), as previously described,[56,57]
in order to evalu-
ate the extent of carvacrol intercalation into the clay galleries.
Figure 1 depicts the XRD patterns for the neat organoclay and
clay/carvacrol hybrid. The obtained 2θ and corresponding
d-spacing values are also summarized in Table 2 for clarity. The
diffraction pattern of the neat clay exhibits a peak at 2θ =3.5°,
corresponding to the mean interlayer spacing of the (001) plane.
R. SHEMESH ET AL.
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112

4. The broad peak at 7° is related to the (002) reflection
representing the half-length of the actual average distance
(25 Å) between two silicate layers. For the clay/carvacrol hybrid
a profound shift to lower 2θ values is observed for all order re-
flections, indicating expansion of the clay nanostructure. Indeed,
the interlayer spacing increases by 11 Å to a value of 36 Å for the
hybrid. These results are ascribed to the intercalation of the car-
vacrol molecules into the silicate galleries, where the clay serves
as a potential carrier for carvacrol molecules. Our previous
study[51]
demonstrated that organic modification of the clay
plays a vital role in intercalation the carvacrol molecules within
the silicate platelets. For neat clays (with no modification), no ex-
pansion of the clay galleries was observed.[51]
The thermal stability of the carvacrol within the clay/carvacrol
hybrids is studied by thermogravimetric analysis (TGA) and com-
pared to that of neat carvacrol (Fig. 2). The thermogram for the
neat carvacrol displays one distinct weight loss process, ascribed
for carvacrol evaporation, which is completed at ~165°C. For the
clay/carvacrol hybrid, carvacrol loss occurs at higher tempera-
tures ~200°C. Above 250°C the hybrid shows a moderate weight
loss, attributed to degradation of the organic modifier moieties
within the clay. Similar behavior is observed for the neat
organoclay. Using the thermograms we can calculate the inor-
ganic content within the hybrid to be ~22 wt%, corresponding
with the initial composition of the system. Thus, the TGA results
demonstrate that the clay platelets significantly enhance the
thermal stability of the volatile carvacrol molecules and may
function as “active” carriers during high-temperature polymer
melt compounding processes.
Characterization of LDPE/(clay/carvacrol) films
The clay/carvacrol hybrid is melt-compounded with LDPE at 140°C
using a twin-screw extruder. Following compounding, films are
produced by cast extrusion at 140°C. In order to study the effect
of the clay/carvacrol hybrids during the high-temperature melt
compounding and processing, the content of carvacrol within the
films (post processing) is studied by FTIR spectroscopy and TGA.
Table 3 summarizes carvacrol content in different films follow-
ing melt compounding and processing, as determined based
FTIR and TGA. The FTIR spectra of the carvacrol-containing films
depict a characteristic peak at 810 cmÀ1
, which is assigned to
out-of-plane deformation vibrations in carvacrol molecule (see
Fig. 3).[52]
The carvacrol content within the films is quantified
by measuring this peak area. The carvacrol content in
LDPE/carvacrol films is only 2.8 wt%, indicating that the majority
of carvacrol is lost during the high-temperature processing steps.
On the other hand, the LDPE/(clay/carvacrol) systems contain
significantly higher carvacrol content of ~7.9 wt%. TGA results
for these systems present a similar trend, see Table 3. These
results clearly show that the clay has a profound role in retaining
the highly volatile molecules within the polymer during process-
ing at elevated temperatures, in agreement with previous TGA
results for the clay/carvacrol hybrids (Fig. 2), which demon-
strated significant enhancement in the carvacrol thermal
stability, thus, supporting our hypothesis that the clay particles
act as “active” carriers, protecting the carvacrol during high-
temperature compounding processes.
Figure 3a depicts characteristic FTIR spectra of LDPE/carvacrol
films in comparison to neat LDPE. The peak at 810 cmÀ1
, which is
attributed to out-of-plane deformation vibrations in carvacrol
molecule,[52]
is only observed for the carvacrol-containing films.
Thus, carvacrol content within the films is quantified based on
this peak area by using a calibration curve. This allows us to sys-
tematically monitor the changes in carvacrol content with time
at room temperature (Fig. 3). As expected, in all films carvacrol
content is reduced with time, observed as a decrease of the
band (at 810 cmÀ1
) over time. This behavior is at assigned to
Figure 1. XRD patterns of the neat clay and the corresponding clay/car-
vacrol hybrid.
Table 2. 2θ and corresponding interlayer spacing, d(001)
and d(002), values for the neat clay and the clay/carvacrol
hybrid
Diffraction angle
2θ (°)
Interlayer
spacing,
d (Å)
Sample (001)
plane
(002)
plane
(001)
plane
(002)
plane
Neat clay 3.5 7 25 12
Clay/carvacrol
hybrid
2.5 5 36 18
Figure 2. TGA curves of neat clay, carvacrol and clay/carvacrol hybrid.
Table 3. Carvacrol content in different carvacrol-containing
films, determined by FTIR spectroscopy and TGA (Initial
carvacrol content, pre-processing, was 10 wt%)
Carvacrol content (wt%)
Film FTIR TGA
LDPE/carvacrol 2.8 ± 0.1 2.5 ± 0.1
LDPE/(clay/carvacrol) 7.9 ± 0.1 6.7 ± 0.1
ANTIBACTERIAL AND ANTIFUNGAL LDPE FILMS FOR ACTIVE PACKAGING
Polym. Adv. Technol. 2015, 26 110–116 Copyright © 2014 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/pat
113

5. the out-diffusion of the volatile carvacrol molecules from the
films. While in the LDPE/carvacrol films, carvacrol content is ob-
served to diminish within several days, in LDPE/(clay/carvacrol)
system (Fig. 3b), its release from the films can be monitored for
more than a month. The slower out-diffusion kinetics of carvacrol
from the LDPE/(clay/carvacrol) nanocomposite is attributed to
the clay carriers, which hinder the carvacrol release from the
clay/carvacrol hybrids. In addition, it is well established for
polymer/clay nanocomposites that the clay platelets act as im-
permeable barriers to the diffusing molecules, forcing them to
follow a significantly longer tortuous path, and accordingly
lengthening the diffusion path which results in a slower diffusion
kinetics.[2,40,58]
Indeed, TEM images of cryogenic cross-sectioned
LDPE/(clay/carvacrol) films (Fig. 4) clearly demonstrate fine dis-
persion of the clay platelets within the LDPE matrix; different in-
tercalation and exfoliation levels of the modified clays are
observed.
Antimicrobial studies are initially carried out using the Kirby–
Bauer inhibition zone method[53]
with E. coli (ATCC 8739) as
model Gram-Negative bacteria, for all freshly produced films.[4]
Typical results of these tests for LDPE/(clay/carvacrol) films are
depicted in Figure 5. A clear zone of bacterial growth inhibition
is observed around circumference of the LDPE/(clay/carvacrol)
film disc (Fig. 5b), while for the reference neat LDPE films no
zone of inhibition could be detected (Fig. 5a). As expected, all
films containing carvacrol exhibit a durable antibacterial efficacy,
with limited changes in the diameter of the inhibition zone,
while no clear zone for the control films without carvacrol.
In addition to the inhibition zone tests, which provide qualita-
tive assessment of the antibacterial properties of the films, quan-
titative antibacterial studies of different LDPE/(clay/carvacrol)
films are executed by incubating the films with E. coli suspen-
sions (108
CFU/ml, for 18–24 h, at 37°C), after which viable cell
counts and log reductions are calculated in comparison to con-
trol growth of E. coli. Figure 6 summarizes the results of these ex-
periments for LDPE/(clay/carvacrol) films (stored at 4°C) versus
storage time. All freshly produced films reduced E. coli counts
to undetectable levels, demonstrating the bacteriocidal efficacy
of carvacrol within the melt-compounded films. Nevertheless,
storage time has a profound effect on the antibacterial potency
of the films. LDPE/carvacrol films completely lose their efficacy
within the first month from production, while LDPE/(clay/carva-
crol) films preserve their efficacy up to a year. The films continue
to be active for approximately an additional 100 days, yet with a
2–3 logs reduction of E. coli.
The antibacterial activity of the films is also studied with
L. innocua (ATCC 33090), as a Gram-positive model bacteria,
which is a relevant indicator for the pathogenic Listeria
monocytogenes[59,60]
(Fig. 6). The LDPE/carvacrol films show high
Figure 3. FTIR spectra of (a) LDPE/carvacrol films as a function of storage
time and (b) LDPE/(clay/carvacrol) films as function of storage time. A neat
LDPE film spectrum is presented for reference. All films are stored at room
temperature, and FTIR measurements are carried out periodically in trans-
mission mode. The spectra present the peak at 810 cm
À1
, which is attrib-
uted to out-of-plane deformation vibrations in carvacrol molecule. This
figure is available in colour online at wileyonlinelibrary.com/journal/pat
Figure 4. TEM images (a–b) of cryogenic cross-sectioned LDPE/(clay/carvacrol) films at two different magnitude scales. This figure is available in colour
online at wileyonlinelibrary.com/journal/pat
R. SHEMESH ET AL.
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114

6. antibacterial activity against L. innocua. However, the activity
ceases after several days from production. In comparison, the
LDPE/(clay/carvacrol) films exhibit L. innocua reduction of 4 to
5 logs for a period of 13 months following production.
Antifungal tests are carried out with A. alternata as a model
fungus. A. alternata is a cosmopolitan mold, a phytopathogen,
one of the most common species in harvested fruits and vegeta-
bles and is the most important mycotoxin-producing species.
Due to its growth even at low temperature, A. alternata is also re-
sponsible for spoilage of these commodities during refrigerated
transport and storage.[61–63]
In addition, A. alternata is an aller-
gen,[64]
which is a part of the “sick houses” syndrome, and a clin-
ical fungus that may cause disease in immune-compromised
patients. Table 4 summarizes the results of the in vitro studies
conducted with A. alternata, indicating the ability of the
LDPE/(clay/carvacrol) system to sustain antifungal activity follow-
ing the high-temperature melting compounding and processing.
The LDPE/carvacrol films show only low antifungal activity, indic-
ative of mycelial growth present with sporulation covering up to
30% of the tested sample, in comparison to the very high activity
of the LDPE/(clay/carvacrol), which shows no evident growth.
Figure 7 shows images of different LDPE-based films incu-
bated with A. alternata at 28°C in the dark for 10 days. For neat
LDPE film, no effect on fungal growth is observed, while for the
LDPE/(clay/carvacrol) film a complete eradication of fungal
growth is observed. It should be noted that fungi is approxi-
mately 10 fold more resistant to the antimicrobial effect of es-
sential oils than bacteria. With this in mind, the outcome of the
study is a major step towards sustainable management of fungal
contamination utilizing active packaging.
CONCLUSIONS
The present study shows for the first time prolonged and
efficient bacteriocidal as well as fungicidal activity of melt-
compounded LDPE-containing carvacrol films. This is achieved
by using Dellite®72T clay as an active carrier for the highly vola-
tile carvacrol, i.e. efficient intercalation of carvacrol molecules
into the galleries of the organo-modified clay hinders their evap-
oration and degradation during the polymer melt compounding.
This evidenced by XRD studies of the resulting clay/carvacrol hy-
brids, showing a significant increase in the d-spacing of clay, and
enhanced thermal stability. The LDPE/(clay/carvacrol) system ex-
hibits significantly higher carvacrol content in the film as well as
a slower out-diffusion of the carvacrol molecules in comparison
to LDPE/carvacrol films. This is further manifested in the superior
and prolonged antibacterial activity against E. coli and L. innocua;
films containing clay/carvacrol hybrids preserve their efficacy for
a year, while carvacrol-containing films loss their activity within
the first weeks. Moreover, LDPE/(clay/carvacrol) films also exhibit
excellent antifungal activity against A. alternata, used as model
pathogenic fungus. Thus, these new films have a high potential
for antimicrobial food packaging applications due to their long-
lasting and broad-spectrum antimicrobial efficacy.
Acknowledgement
This study was supported by the Magnet Program of the Israeli
Ministry of Economy and the Israeli P^3 Consortium.
Figure 5. Images of the zone of inhibition for E. coli for: (a) control neat
LDPE film; (b) LDPE/(clay/carvacrol) film, depicting a clear inhibition zone
around the polymer film.
Figure 6. Antimicrobial activity against E. coli and Listeria innocua bacte-
ria of LDPE/carvacrol and LDPE/(clay/carvacrol) films as a function of stor-
age time. This figure is available in colour online at wileyonlinelibrary.
com/journal/pat
Table 4. Antifungal activity against A. alternata of neat
LDPE, LDPE/carvacrol and LDPE/(clay/carvacrol) films
Film Antifungal activity
Neat LDPE 0 = None
LDPE/carvacrol 1 = Low
LDPE/(clay/carvacrol) 4 = Very High
Figure 7. Images of (a) neat LDPE film, and (b) LDPE/(clay/carvacrol) film
incubated with A. alternata. The film margins are marked for clarity. This
figure is available in colour online at wileyonlinelibrary.com/journal/pat
ANTIBACTERIAL AND ANTIFUNGAL LDPE FILMS FOR ACTIVE PACKAGING
Polym. Adv. Technol. 2015, 26 110–116 Copyright © 2014 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/pat
115

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