shemesh - pat - 2014

Antibacterial and antifungal LDPE films foractive packagingRotem 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 growthin 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, thechallenge 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 thisgoal. 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 throughoutthe 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 resultingLDPE/(clay/carvacrol) films exhibit superior and prolonged antibacterial activity against Escherichia coli and Listeriainnocua, while polymer compounded with pure carvacrol loses the antibacterial properties within days. The filmsalso 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/carvacrolfilms. 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 theirtoxins are consumed.[1] An important challenge related to foodsafety 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 thefood composition must be considered in antimicrobial packag-ing. Antimicrobials have to be selected based on their spectrumof activity, mode of action, chemical composition, as well as therate of growth and physiological state of the targeted microor-ganisms.[3,5] This approach can reduce the addition of largerquantities 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, coatingantimicrobials onto polymer surfaces,[7,8] immobilizing antimi-crobials by chemical grafting[9,10] or using polymers that exhibitintrinsic antimicrobial properties (e.g. chitosan).[6] Synthetic anti-microbials, which are commonly integrated into polymers forpackaging, include organic or inorganic acids, metals, alcohols,ammonium compounds or amines.[11,12] Some of these mate-rials, specifically silver-based additives, are already commerciallyavailable and are applied for food packaging.[13,14] However, theincreasing consumer health concern and growing demandfor healthy foods have stimulated the use of natural

biopreservatives, such as essential oils, e.g. carvacrol andthymol.[15] Essential oils are natural substances categorized asGRAS (generally recognized as safe) by the Food and DrugAdministration (FDA), and most of them are derived fromplants.[16] They have shown substantial antibacterial and antifun-gal properties achieved both in direct contact and in vaporphase.[17] The possibility of achieving an antimicrobial actionby the release of the volatile compounds has increased theinterest of including them into the packaging. One majordrawback 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 theessential oils are directly incorporated into the polymer matrixby high-temperature melt processing, antimicrobial activity isachieved.[16,18,20–33] However, this activity is evaluated, in most

* Correspondence to: Ester Segal, Department of Biotechnology and FoodEngineering, Technion—Israel Institute of Technology, Haifa 32000, Israel.E-mail: [email protected]

a R. Shemesh, M. Krepker, D. Goldman, Y. Danin-Poleg, Y. Kashi, E. SegalDepartment of Biotechnology and Food Engineering, Technion—Israel Insti-tute of Technology, Haifa 32000, Israel

b R. Shemesh, A. VaxmanCarmel Olefins Ltd., P.O. Box 1468, Haifa 31014, Israel

c N. NitzanD.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

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cases, immediately after film production,[29,33–35] while the chal-lenges associated with the controlled and prolonged activity ofthese films are not addressed.In recent years, polymer nanocomposites[36–41,2,42] containing

exfoliated organoclay platelets have been extensively studiedfor 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 moleculepathway.[2,44–46] An emerging approach is to employ polymernanocomposites for potential antimicrobial applications.[47–49]

The prevailing methodology in most studies is to incorporateorgano-modified montmorillonite clays (MMT) as filler duringmelt compounding.[26,31,50] The rational for the incorporation ofthe clay was to improve the barrier properties of the films in or-der to protect the volatile and heat-sensitive essential oilmolecules.Our novel approach is to use the layered clays as an “active”

carrier for antimicrobial essential oils, e.g. carvacrol. This isachieved 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 thischaracter for active encapsulation of the carvacrol into the galler-ies of the clay, as schematically illustrated in Scheme 1. In ourrecent study[51] we have investigated the effect of different com-mercially available clays on their ability to retain carvacrol duringhigh-temperature melt processing. Herein, we use the mostpromising clay/carvacrol combination to develop antimicrobialpolymer films with a broad spectrum of inhibitory activityagainst 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 ofcarvacrol with storage time, which is crucial in a wide range ofapplications to enhance food safety and shelf life.

EXPERIMENTAL

Preparation and characterization of clay/carvacrol hybrids

Carvacrol (98%, Sigma Aldrich Chemicals, Israel) is mixed withnatural montmorillonite clay modified with quaternary ammo-nium salt (Dellite®72T, Laviosa Chimica Mineraria, Italy) byultrasonication (Vibra cell VCX 750, Sonics & Materials Inc., USA)at a weight ratio of 2:1, respectively. Ultrasonication is performedat room temperature for 20min at an amplitude of 40% to formuniform carvacrol–clay dispersions (Scheme 1).

X-ray diffraction (XRD)

The degree of carvacrol intercalation into the clay galleries isevaluated by X-ray diffraction (XRD) using Bragg–Brentanoq-2q Philips PW3020 diffractometer, powered by a PW1710generator. The diffraction data is collected by 2q step scanningbetween 2θ of 1.2 and 10°, at 0.02° steps and a count time of1 s/step. The experimental conditions are diffracted-beam graph-ite (00.2) monochromator, Cu Ka radiation, 40 KV and 40mA,divergence and anti-scattering slits 1°, receiving slit 0.2mm.

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 OlefinsLtd., Haifa, Israel) with a melt flow rate of 2 g/10min is melt-compounded with clay/carvacrol hybrids using a 16-mm twin-screw extruder (Prism, England) L/D ratio of 25:1 with a screwspeed of 150 rpm and a feeding rate of 2 kg/h at 140°C. Table 1specifies the composition of the different blends investigatedin the present study. Following the melt-compounding process,120-μm-thick films are prepared by cast extrusion using 45-mmscrew diameter extruder (Dr. Collin, Germany) at 140°C.

Infrared spectroscopy

Carvacrol content in the LDPE-based films is characterized byFourier-transform infrared spectroscopy in transmission mode.Spectra are recorded using a Thermo 6700 FTIR instrument andOMNIC 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 0LDPE/clay 95 5 0LDPE/carvacrol 90 0 10LDPE/(clay/carvacrol) 85 5 10

ANTIBACTERIAL AND ANTIFUNGAL LDPE FILMS FOR ACTIVE PACKAGING

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analyst v8.0 software. Spectra are collected from at least threedifferent locations on the films. The peak area at 810 cm�1,which is attributed to out-of-plane deformation vibrations incarvacrol molecule,[52] is calculated and is used to quantify thecarvacrol concentration using a calibration curve.

Transmission electron microscopy (TEM)

The nanostructure of the resulting films is studied with a PhilipsCM120 transmission electron microscope (TEM) operated at120-kV accelerating voltage, using 400mesh carbon-coveredCu grid Pk/100 (SPI Supplies West Chester, USA). Images are re-corded digitally by a Gatan MultiScan 791 CCD camera usingthe Digital Micrograph 3.1 software. Ultra-thin cross sections ofapproximately 100 nm thick are prepared at �100°C with aReichert 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, theKirby–Bauer technique is used for potency screening.[53] Thezone of bacterial inhibition around the circumference of a poly-meric film disc is utilized to qualitatively assess whether or notthe polymeric matrix sample possesses antibacterial proper-ties.[3,54,55] Discs (of 12mm in diameter) are cut out of samplefilms and are placed onto the surface of a full concentration Luriabroth (LB) agar in 9 cm Petri plates that are inoculated with0.1ml of 108 colony forming units (CFU)/ml of bacterial culture.The plates are incubated at 37°C for 18 h, and the antibacterialactivity is recorded by observing the presence or absence of aninhibition zone around the studied sample. Films without carva-crol are assayed as negative control. The inhibition zone tests areperformed in triplicates.

The second method is quantitative and is based on theJapanese standard (JIS Z 2801: 2000 “Antimicrobial products—test for antimicrobial activity and efficacy”). E. coli is culturedovernight 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 toan optical density (OD) value of 0.1, which approximately corre-sponds to 108 CFU/ml. The culture is incubated for an additional1.5 h, allowing the cells to enter the logarithmic stage. As theculture reaches an ODvalue of 0.6, it is diluted by 1:100 with1% NB to obtain a bacterial stock solution at a concentration of105 CFU/ml. Efficacy tests are carried out in 6-well plates. Eachfilm sample is placed in a well that is supplemented with 3ml ofbacterial stock solution. The plates are incubated at 37°C (undercontinuous agitation at 100 rpm) for 24h. Incubation is followedby a 1:100 serial dilutions with 1% NB performed in 96-well plates.The drop-plate technique is used to assess viable cell counts with20-μl drops that are transferred onto 1% NB bacto-agar (BectonDickinson) in 9-cm Petri plates. The Petri plates are incubated at37°C for 18–24h; CFU are counted, and log reduction is calculatedin comparison to E. coli cultured in NB 1:100 medium (108 CFU/ml)that is used as control. All measurements, including the growthcontrols, 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 andis reincubated for 1.5 h to allow the cells to enter the logarithmic

stage until an ODvalue of 0.3 is reached, which approximatelycorresponds to 108 CFU/ml. Then, the bacteria are diluted with1% 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 plateadded with 1ml of bacterial stock solution. The plates are incu-bated at 37°C (under continuous agitation at 100 rpm) for 24 hfollowed by serial dilutions with NB 1:100 in 96-well plates. Thedrop plate technique is used for viable cell counts. Drops of10μl are placed onto NB bacto-agar (Becton Dickinson) in 9 cmPetri plates to determine cell numbers. The plates are incubatedat 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 intriplicates.The phytopathogenic and clinical fungus A. alternata originat-

ing from the surface of tomato fruits and cultured on 1% CornMeal Agar (Difco CMA: 10 g/l; Bacto Agar: 10 g/l in 1000ml ofdeionizedwater) is used in all tests. Efficacy tests are conducted fol-lowing a modified ISO 16869: 2008 protocol (Plastics—Assessmentof the effectiveness of fungistatic compounds in plastic formula-tions), utilizing 1% CMA instead of Nutrient Salt Agar. Sample filmdiscs (30mm in diameter) are excised from the film with the aidof 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 cultureswith sterilized de-ionized water. The spore suspension is adjustedwith the aid of a hemocytometer and then transferred into softagar (pre-solidified 1% CMA cooled to 45°C), producing aninoculum suspension at a final conidial concentration of105 conidia/ml. The inoculum is poured over the disc and the baseagar layer, covering the sample with a thin layer of inoculum. ThePetri plates are left at room temperature for 1 h to allow the inoc-ulum layer to solidify. Then the plates are sealed with Parafilmand incubated at 28°C in the dark for 10 days. LDPE films amendedor not amended with 1% Folpet (Thiophthalamide; Folpan 50 WPbroad spectrum M group fungicide by Makhteshim–Agan, Israel)are used as positive and negative controls, respectively. Followingincubation the antifungal activity of the tested film is quantifiedwith 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=VeryHigh (VH) activity—no fungal growth; 3 =High (H) activity—slightmycelial 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, andthe antifungal efficacy of the tested film sample is reported as themedian of the three replications.

RESULTS AND DISCUSSION

Clay/carvacrol hybrids

Organoclay and clay/carvacrol hybrid are characterized by X-raydiffraction (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 andclay/carvacrol hybrid. The obtained 2θ and correspondingd-spacing values are also summarized in Table 2 for clarity. Thediffraction pattern of the neat clay exhibits a peak at 2θ =3.5°,corresponding to the mean interlayer spacing of the (001) plane.

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The broad peak at 7° is related to the (002) reflectionrepresenting the half-length of the actual average distance(25 Å) between two silicate layers. For the clay/carvacrol hybrida 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 thehybrid. These results are ascribed to the intercalation of the car-vacrol molecules into the silicate galleries, where the clay servesas a potential carrier for carvacrol molecules. Our previousstudy[51] demonstrated that organic modification of the clayplays a vital role in intercalation the carvacrol molecules withinthe 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/carvacrolhybrids is studied by thermogravimetric analysis (TGA) and com-pared to that of neat carvacrol (Fig. 2). The thermogram for theneat carvacrol displays one distinct weight loss process, ascribed

for carvacrol evaporation, which is completed at ~165°C. For theclay/carvacrol hybrid, carvacrol loss occurs at higher tempera-tures ~200°C. Above 250°C the hybrid shows a moderate weightloss, attributed to degradation of the organic modifier moietieswithin the clay. Similar behavior is observed for the neatorganoclay. Using the thermograms we can calculate the inor-ganic content within the hybrid to be ~22wt%, correspondingwith the initial composition of the system. Thus, the TGA resultsdemonstrate that the clay platelets significantly enhance thethermal stability of the volatile carvacrol molecules and mayfunction as “active” carriers during high-temperature polymermelt compounding processes.

Characterization of LDPE/(clay/carvacrol) films

The clay/carvacrol hybrid is melt-compounded with LDPE at 140°Cusing a twin-screw extruder. Following compounding, films areproduced by cast extrusion at 140°C. In order to study the effectof the clay/carvacrol hybrids during the high-temperature meltcompounding and processing, the content of carvacrol within thefilms (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 basedFTIR and TGA. The FTIR spectra of the carvacrol-containing filmsdepict a characteristic peak at 810 cm�1, which is assigned toout-of-plane deformation vibrations in carvacrol molecule (seeFig. 3).[52] The carvacrol content within the films is quantifiedby measuring this peak area. The carvacrol content inLDPE/carvacrol films is only 2.8wt%, indicating that the majorityof carvacrol is lost during the high-temperature processing steps.On the other hand, the LDPE/(clay/carvacrol) systems containsignificantly higher carvacrol content of ~7.9wt%. TGA resultsfor these systems present a similar trend, see Table 3. Theseresults clearly show that the clay has a profound role in retainingthe highly volatile molecules within the polymer during process-ing at elevated temperatures, in agreement with previous TGAresults for the clay/carvacrol hybrids (Fig. 2), which demon-strated significant enhancement in the carvacrol thermalstability, thus, supporting our hypothesis that the clay particlesact as “active” carriers, protecting the carvacrol during high-temperature compounding processes.

Figure 3a depicts characteristic FTIR spectra of LDPE/carvacrolfilms in comparison to neat LDPE. The peak at 810 cm�1, which isattributed to out-of-plane deformation vibrations in carvacrolmolecule,[52] is only observed for the carvacrol-containing films.Thus, carvacrol content within the films is quantified based onthis peak area by using a calibration curve. This allows us to sys-tematically monitor the changes in carvacrol content with timeat room temperature (Fig. 3). As expected, in all films carvacrolcontent is reduced with time, observed as a decrease of theband (at 810 cm�1) 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/carvacrolhybrid

Diffraction angle2θ (°)

Interlayerspacing,d (Å)

Sample (001)plane

(002)plane

(001)plane

(002)plane

Neat clay 3.5 7 25 12Clay/carvacrolhybrid

2.5 5 36 18

Figure 2. TGA curves of neat clay, carvacrol and clay/carvacrol hybrid.

Table 3. Carvacrol content in different carvacrol-containingfilms, determined by FTIR spectroscopy and TGA (Initialcarvacrol content, pre-processing, was 10 wt%)

Carvacrol content (wt%)

Film FTIR TGA

LDPE/carvacrol 2.8 ± 0.1 2.5 ± 0.1LDPE/(clay/carvacrol) 7.9 ± 0.1 6.7 ± 0.1



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the out-diffusion of the volatile carvacrol molecules from thefilms. 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 formore than a month. The slower out-diffusion kinetics of carvacrol

from the LDPE/(clay/carvacrol) nanocomposite is attributed tothe clay carriers, which hinder the carvacrol release from theclay/carvacrol hybrids. In addition, it is well established forpolymer/clay nanocomposites that the clay platelets act as im-permeable barriers to the diffusing molecules, forcing them tofollow a significantly longer tortuous path, and accordinglylengthening the diffusion path which results in a slower diffusionkinetics.[2,40,58] Indeed, TEM images of cryogenic cross-sectionedLDPE/(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 areobserved.Antimicrobial studies are initially carried out using the Kirby–

Bauer inhibition zone method[53] with E. coli (ATCC 8739) asmodel Gram-Negative bacteria, for all freshly produced films.[4]

Typical results of these tests for LDPE/(clay/carvacrol) films aredepicted in Figure 5. A clear zone of bacterial growth inhibitionis observed around circumference of the LDPE/(clay/carvacrol)film disc (Fig. 5b), while for the reference neat LDPE films nozone of inhibition could be detected (Fig. 5a). As expected, allfilms 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 cellcounts 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) versusstorage time. All freshly produced films reduced E. coli countsto undetectable levels, demonstrating the bacteriocidal efficacyof carvacrol within the melt-compounded films. Nevertheless,storage time has a profound effect on the antibacterial potencyof the films. LDPE/carvacrol films completely lose their efficacywithin the first month from production, while LDPE/(clay/carva-crol) films preserve their efficacy up to a year. The films continueto be active for approximately an additional 100 days, yet with a2–3 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 Listeriamonocytogenes[59,60] (Fig. 6). The LDPE/carvacrol films show high

Figure 3. FTIR spectra of (a) LDPE/carvacrol films as a function of storagetime and (b) LDPE/(clay/carvacrol) films as function of storage time. A neatLDPE film spectrum is presented for reference. All films are stored at roomtemperature, and FTIR measurements are carried out periodically in trans-mission mode. The spectra present the peak at 810 cm�1, which is attrib-uted to out-of-plane deformation vibrations in carvacrol molecule. Thisfigure 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 colouronline at wileyonlinelibrary.com/journal/pat

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antibacterial activity against L. innocua. However, the activityceases after several days from production. In comparison, theLDPE/(clay/carvacrol) films exhibit L. innocua reduction of 4 to5 logs for a period of 13months 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 refrigeratedtransport 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-compromisedpatients. Table 4 summarizes the results of the in vitro studiesconducted 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 to30% of the tested sample, in comparison to the very high activityof 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 neatLDPE film, no effect on fungal growth is observed, while for theLDPE/(clay/carvacrol) film a complete eradication of fungalgrowth 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 thestudy is a major step towards sustainable management of fungalcontamination utilizing active packaging.

CONCLUSIONS

The present study shows for the first time prolonged andefficient bacteriocidal as well as fungicidal activity of melt-compounded LDPE-containing carvacrol films. This is achievedby using Dellite®72T clay as an active carrier for the highly vola-tile carvacrol, i.e. efficient intercalation of carvacrol moleculesinto 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, andenhanced thermal stability. The LDPE/(clay/carvacrol) system ex-hibits significantly higher carvacrol content in the film as well asa slower out-diffusion of the carvacrol molecules in comparisonto LDPE/carvacrol films. This is further manifested in the superiorand prolonged antibacterial activity against E. coli and L. innocua;films containing clay/carvacrol hybrids preserve their efficacy fora year, while carvacrol-containing films loss their activity withinthe first weeks. Moreover, LDPE/(clay/carvacrol) films also exhibitexcellent antifungal activity against A. alternata, used as modelpathogenic fungus. Thus, these new films have a high potentialfor 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 IsraeliMinistry of Economy and the Israeli P^3 Consortium.

Figure 5. Images of the zone of inhibition for E. coli for: (a) control neatLDPE film; (b) LDPE/(clay/carvacrol) film, depicting a clear inhibition zonearound 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 neatLDPE, LDPE/carvacrol and LDPE/(clay/carvacrol) films

Film Antifungal activity

Neat LDPE 0=NoneLDPE/carvacrol 1 = LowLDPE/(clay/carvacrol) 4 = Very High

Figure 7. Images of (a) neat LDPE film, and (b) LDPE/(clay/carvacrol) filmincubated with A. alternata. The film margins are marked for clarity. Thisfigure is available in colour online at wileyonlinelibrary.com/journal/pat



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