Journal of Food Engineering 109 (2012) 513–519

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Journal of Food Engineering

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Characterization and antimicrobial activity studies of polypropylene filmswith carvacrol and thymol for active packaging

Marina Ramos ⇑, Alfonso Jiménez, Mercedes Peltzer, María C. GarrigósAnalytical Chemistry, Nutrition and Food Sciences Department, University of Alicante, P.O. Box 99, 03080 Alicante, Spain

a r t i c l e i n f o

Article history:Received 29 May 2011Received in revised form 24 October 2011Accepted 30 October 2011Available online 7 November 2011

Keywords:PolypropyleneActive packagingCarvacrolThymolCharacterizationAntimicrobial film

0260-8774/$ - see front matter � 2011 Elsevier Ltd. Adoi:10.1016/j.jfoodeng.2011.10.031

⇑ Corresponding author. Tel.: +34 965903400x1187E-mail address: marina.ramos@ua.es (M. Ramos).

a b s t r a c t

Antimicrobial active films based on polypropylene (PP) were prepared by incorporating thymol and car-vacrol at three different concentrations: 4, 6 and 8 wt.% of both additives as well as an equimolar mixtureof them. A complete thermal, structural, mechanical and functional characterization of all formulationswas carried out. SEM micrographs showed certain porosity for films with high additives concentrations.A decrease in elastic modulus was obtained for the active formulations compared with neat PP. The pres-ence of additives did not affect the thermal stability of PP samples, but decreased PP crystallinity and oxy-gen barrier properties. The presence of thymol and carvacrol also increased stabilization against thermo-oxidative degradation, with higher oxidation induction parameters. Finally, thymol showed higher inhi-bition against bacterial strain present in food compared with carvacrol, leading to higher antimicrobialactivity. The obtained results proved the permanence of certain amounts of the studied additives inthe polymer matrix after processing making them able to be used as active additives in PP formulations.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction

Studies within the area of food active packaging are experienc-ing a great development due to the consumer’s demand and mar-ket trends. Food active packaging systems are based on materialsin which some additives with antimicrobial and/or antioxidantproperties are added into the polymer matrix with the aim ofextending foodstuff shelf-life and improving consumer’s safety(Vermeiren et al., 1999; Álvarez, 2000; Appendini and Hotchkiss,2002; Del Nobile et al., 2009).

Antimicrobial packaging is increasing the attention from foodand packaging industries due to the increasing consumer demandsfor minimally processed and preservative-free products (Lópezet al., 2007a). Food-packaging films allow a controlled release ofthe additives into the food in prolonged times (including storageand distribution operations) and limit possible undesirable flavorscaused by the direct addition of these additives into food (Suppakulet al., 2003, 2006; Ho Lee et al., 2004; López et al., 2007a; Peltzeret al., 2009).

The demand for the use of natural additives has produced in re-cent years a clear increase in the number of studies based on nat-ural extracts such as essential oils, which are categorized asGenerally Recognised as Safe (GRAS) by US Food and Drug Adminis-tration (Persico et al., 2009), and they could be considered poten-tial alternatives to synthetic additives, such as butylated

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hydroxytoluene (BHT) (Valentao et al., 2002). Essential oils ex-tracted from plants or spices are rich sources of biological activecompounds, such as terpenoids and phenolic acids (Bakkali et al.,2008), and it has been long recognized that some of them showantimicrobial properties (Burt, 2004; López et al., 2007b). In partic-ular, carvacrol and thymol are present as major compounds inthyme and oregano essential oils (Al-Bandak and Oreopoulou,2007). These two compounds are phenolic monoterpenes and iso-mers that exhibit a significant in vitro antibacterial activity (Didryet al., 1994). Carvacrol shows antifungal, insecticidal, antitoxigenicand antiparasitic activities (Veldhuizen et al., 2006). On the otherhand, thymol has received considerable attention as an antimicro-bial agent showing very high antifungal activity, being also anexcellent food antioxidant (Youdim and Deans, 2000; Sanchez-Gar-cia et al., 2008). The antimicrobial activity of both compounds hasbeen already studied and reported against several bacterial strains(Halliwell et al., 1995).

Active compounds have been usually added to packaging mate-rials by the incorporation of their precursor essential oils (Salafran-ca et al., 2009). Polyolefin-based films are usually used for thedevelopment of active packaging systems combining the polymergood general properties (mechanical, barrier, optical and thermal)and the antimicrobial or antioxidant efficiency given by the addi-tives (Suppakul et al., 2006; López et al., 2007a; Peltzer et al.,2007, 2009). Nevertheless, there is a current trend in the use of di-rect antimicrobial additives derived from essential oils, which areperceived by consumers as low health risk compounds. Therefore,there is an increasing interest in the evaluation and possible

514 M. Ramos et al. / Journal of Food Engineering 109 (2012) 513–519

application of these compounds for minimizing the superficial con-tamination of foods such as meats, fruits and vegetables, decreas-ing the microbial growth rate of microorganisms responsible forfood degradation (Appendini and Hotchkiss, 2002; Lundbäcket al., 2006; Sanchez-Garcia et al., 2008; Persico et al., 2009; Peltzeret al., 2010; Suppakul et al., 2011a). In particular, the use of carva-crol and thymol might represent an advantage due to a possiblesynergistic effect produced by the addition of both additives intothe polymer matrix (Didry et al., 1994). This effect has been suc-cessfully studied against several microorganisms for food applica-tions (Lambert et al., 2001; Guarda et al., 2011). The use of highinitial concentrations for these volatile additives has been previ-ously reported, since some loss during processing is expectable(Sanchez-Garcia et al., 2008; Persico et al., 2009; Del Nobileet al., 2009; Mascheroni et al., 2011; Tunç and Duma, 2011).

This study is focused on the development of films based on PPwith carvacrol and thymol, in order to develop an antimicrobial ac-tive packaging system. For this purpose, melt blending followed bycompression molding was used in order to obtain films at labora-tory scale. The efficiency of both compounds will be compared atdifferent concentrations. A full characterization was carried outby the determination of thermal, structural, mechanical and func-tional properties. Finally, the antimicrobial activity of films wasalso evaluated against two typical food born bacteria: Staphylococ-cus aureus (gram-positive) and Escherichia coli (gram-negative).

2. Experimental

2.1. Materials

The polymer used in this work was polypropylene PP ECOLENHZ10K (Hellenic Petroleum, Greece), kindly supplied in pellets byAshland Chemical Hispania (Barcelona, Spain). Melt flow index(MFI) was 3.2 g 10 min�1 determined according to ASTM D1238standard (230 �C, 2.16 kg), and density 0.9 g cm�3. Carvacrol(98%) and thymol (99.5%) were purchased from Sigma–Aldrich(Madrid, Spain). The chemical structures of carvacrol and thymolare shown in Fig. 1.

2.2. Sample preparation

The different formulations were obtained by melt blending in aHaake Polylab QC mixer (ThermoFischer Scientific, Walham, USA)at 190 �C for 6 min at rotation speed of 50 rpm. Both additives wereintroduced in the mixer once the polymer was already in the meltstate, in order to avoid unnecessary losses and to ensure the pres-ence of some amount of them in the final materials. The 50 cm3

mixing chamber was filled with 50 g total mass. Nine active formu-lations were obtained: PP containing 4, 6 and 8 wt.% of thymol

Fig. 1. Chemical structures of the two additives used in this study.

(PPT4, PPT6 and PPT8) or carvacrol (PPC4, PPC6 and PPC8); andPP with the combination of an equimolar mixture of both additivesat 4, 6 and 8 wt.% (PPTC4, PPTC6 and PPTC8) to study a possibleadditive effect of both compounds. An additional sample withoutany active compound was also prepared and used as control (PP0).

The active antimicrobial films were obtained by compression-moulding at 190 �C in a hot press (Carver Inc., Model 3850, USA).The material was kept between the plates at atmospheric pressurefor 5 min until melting and then it was successively pressed under2 MPa for 1 min, 3.5 MPa for 1 min and finally 5 MPa for 5 min, inorder to liberate the trapped air bubbles. The average thickness ofthe films was around 200 lm measured with a Digimatic Microm-eter Series 293 MDC-Lite (Mitutoyo, Japan) at five random posi-tions around the film. The final appearance of the films wascompletely transparent and homogenous.

2.3. Material characterization

The active films were characterized by using different tech-niques in order to study their thermal, mechanical and oxygen bar-rier properties as well as their antimicrobial activity.

2.3.1. Scanning electronic microscopy (SEM)The films surfaces and cross sections were analyzed by using a

JEOL model JSM-840 (Jeol USA Inc., Peabody, MA, USA) microscopeoperated at 12 kV. Samples were coated with gold layer prior toanalysis in order to increase their electrical conductivity. Imageswere registered at 300� and 500� of magnification in order tostudy their homogeneity.

2.3.2. Mechanical propertiesTensile tests were carried out by using a 3340 Series Single Col-

umn System Instron Instrument, LR30K model (Fareham Hants,UK) equipped with a 2 kN load cell. Tests were performed in rect-angular probes (dimensions: 100 � 10 mm2), an initial grip separa-tion of 60 mm and crosshead speed of 25 mm min�1. Averagetensile strength, elongation at yield and elastic modulus were cal-culated from the resulting stress–strain curves according to ASTMD882-09 Standard procedure (ASTM D882-09. 2009). Results werethe average of five measurements (±standard deviation).

2.3.3. Thermogravimetric analysis (TGA)TGA tests were performed in a TGA/SDTA 851 Mettler Toledo

thermal analyzer (Schwarzenbach, Switzerland). Approximately5 mg samples were weighed in alumina pans (70 lL) and wereheated from 30 �C to 700 �C at a heating rate of 10 �C min�1 underinert nitrogen atmosphere (flow rate 50 mL min�1).

2.3.4. Differential scanning calorimetry (DSC)2.3.4.1. Determination of thermal parameters in inert atmo-sphere. DSC tests were conducted by using a TA DSC Q-2000instrument (New Castle, DE, USA) under inert nitrogen atmo-sphere. 3 mg of films were introduced in aluminum pans (40 lL)and were submitted to the following thermal program: heatingfrom 0 �C to 180 �C at 10 �C min�1 (3 min hold), cooling at10 �C min�1 to 0 �C (3 min hold) and heating to 180 �C at10 �C min�1. The percentage of crystallinity (v %) for each materialwas calculated according to the following equation,

v ð%Þ ¼ DHm=WDH�m� �

� 100 ð1Þ

where DHm (J g�1) is the latent heat of fusion of the sample, W is thePP weight fraction in the sample, and DH�m is the theoretical latentheat of fusion for 100% crystalline PP, 138 J g�1 (Joseph et al., 2003).

M. Ramos et al. / Journal of Food Engineering 109 (2012) 513–519 515

2.3.4.2. Evaluation of antioxidant induction parameters. The antioxi-dant performance of carvacrol and thymol was studied by DSC bydetermining the oxidation induction parameters, i.e. oxidation on-set temperature, OOT (�C) and oxidation induction time, OIT (min)(Pospíšil et al., 2003; Archodoulaki et al., 2006). OOT can be definedas the minimum temperature where oxidation takes place in pureoxygen atmosphere. The OIT value is defined as the time to the on-set of an exothermic oxidation peak in oxygen atmosphere and itwas determined as the difference between the time at the intersec-tion between the base line and the tangent of the exothermic oxi-dation peak and the time for the gas switching in two differentatmospheres: pure oxygen and air. All tests were performed intriplicate for each formulation.

OOT (�C) is a relative measurement of the degree of thermo-oxidative stability of the material evaluated at a given heating rateand oxidative environment (Peltzer and Jiménez, 2009). Sampleswere heated up at 10 �C min�1 under a pure oxygen atmosphere(50 mL min�1) from 30 �C to the observation of the exothermic oxi-dation peak. OOT was calculated as the temperature for the inter-section between the base line and the slope of the exothermic peakin each case.

On the other hand, the OIT test was carried out by heating sam-ples at 100 �C min�1 under nitrogen (flow rate 50 mL min�1) to theset temperature (200 �C). After 5 min, the atmosphere wasswitched to pure oxygen or air (50 mL min�1). The heat flow wasthen recorded in isothermal conditions up to the detection of theexothermic peak indicating the beginning of the oxidationreaction.

2.3.5. Oxygen transmission rate (OTR)OTR is defined as the quantity of oxygen passing through a

determined area of the parallel surface of a plastic film per timeunit. An oxygen permeation analyzer (8500 model Systech, Metro-tec S.A., Spain) was used. Tests were carried out by introducingpure oxygen into the upper half of the diffusion chamber whilenitrogen was injected into the lower half, where an oxygen sensorwas located. Films were cut into 14 cm diameter circles for eachformulation and they were clamped in the diffusion chamber at25 �C before testing.

2.4. Antimicrobial activity of the films

The evaluation of the antimicrobial activity of the polymer con-taining carvacrol, thymol and the equimolar mixture of both addi-tives was carried out by using two test microorganisms: E. coli(gram-negative, ATCC (American Type Culture Collection) 25922)and S. aureus (gram-positive, ATCC 6538P). The PP0 sample wasalso tested as control. Overnight cultures of E. coli and S. aureuswere grown in Tryptic Soy Broth at 35 �C for 24 h. The strains selec-tion represented typical spoilage organism groups commonlyoccurring in various kinds of food products.

Antimicrobial activity tests were carried out by using the agardisk diffusion method. Disks cut from films were placed on Petridishes containing Mueller–Hinton agar (MHA) supplied by INSU-LAB S.L., (Valencia, Spain), previously spread with 0.1 mL of eachinoculum. The bacterial cultures concentration in the inoculumwas 106 CFU mL�1, corresponding to the concentration that couldbe found in contaminated food, and standardized in the McFarlandscale 0.5, as it has been indicated by other authors (Suppakul et al.,2011b). The Petri dishes were then incubated at 37 �C for 24 h. Theantimicrobial activity of each material was evaluated by observingthe growth inhibition zone and measuring the diameter (mm) by aruler. The bacterial growth under the film disks (area of contactwith the agar surface) was also observed in Petri dishes. Tests werecarried out in duplicate for each formulation.

3. Results and discussion

3.1. Scanning electron microscopy (SEM)

Fig. 2 shows SEM images obtained for PP0 and samples with8 wt.% additives. Homogeneous surface morphologies were ob-served for all samples with no apparent effect of the addition ofthe different compounds to PP. However, certain porosity on thesurface of the materials with additives at each concentration(4 wt.%, 6 wt.% and 8 wt.%) was observed. This behavior could bedue to the presence of a certain amount of carvacrol and thymolin the materials surface and the eventual partial evaporation fromthe polymer matrix during processing leading to a potential loss ofsome additives amount in the final material. This fact will be fur-ther evaluated by using other techniques.

3.2. Mechanical properties

Tensile tests were performed in order to study the effect of thy-mol and carvacrol on polymer mechanical properties, by the eval-uation of different parameters, such as elastic modulus, elongationat yield and tensile strength, in all materials (Table 1). The additionof carvacrol and thymol to PP resulted in a slight modification oftensile properties. A significant decrease in elastic modulus wasobserved for the materials with additives when compared withPP0, being this effect more pronounced for PPC8 and PPT8 films.A certain increase in elongation at yield for these samples was alsoobserved. This behavior could be explained by some plasticizing ef-fect caused by the addition of both additives to the polymer matrixresulting in the increase in ductile properties, which would also re-sult in changes in the materials crystallinity. This behavior hasbeen also reported for LDPE-based samples with carvacrol (Persicoet al., 2009).

3.3. Thermogravimetric analysis (TGA)

The effect of carvacrol and thymol on the thermal stability of PPfilms was studied by TGA under nitrogen atmosphere. Fig. 3 showsthe TGA curves obtained for PP0 and PPC samples. As it is well re-ported for PP thermal degradation, a single degradation step wasobserved for PP0 sample (Navarro et al., 2003). However, sampleswith carvacrol showed a first degradation step at low temperatures(about 115 �C) and a second step corresponding to the thermaldegradation of the polymer matrix. The TGA patterns of other for-mulations were quite similar in all cases. In this way, the first deg-radation step observed in active films was associated to thedegradation of carvacrol and/or thymol. Therefore, it was possibleto determine the remaining amount of additive in the polymer ma-trix after processing. The remaining concentrations were approxi-mately 1 wt.%, 2 wt.% and 3.5 wt.% for formulations with initialantioxidants amount 4 wt.%, 6 wt.% and 8 wt.%, respectively. Inconclusion, TGA results gave an indirect confirmation of the pres-ence of both compounds in the polymer matrix after processingand consequently their ability to act as active agents in these mate-rials as it has been reported by other authors (Persico et al., 2009).

Table 2 summarizes the initial degradation temperature Ti,determined at 5% of weight loss, and maximum rate temperature(Tmax) for the main degradation step, ascribed to thermal degrada-tion of PP. As it can be seen, no appreciable differences were ob-served for Ti and Tmax values in all samples. These results showedthat the addition of carvacrol and thymol to the polymer matrixdoes not significantly affect its thermal degradation profile in inertnitrogen atmosphere. However, it is expectable that a certainamount of carvacrol and thymol would be lost during processing,because the materials are submitted to temperatures above the

Fig. 2. SEM micrographs (300�) of the edge surfaces for PP0 and samples with 8 wt.% of the studied additives.

Table 1Mechanical properties of samples according to ASTM D882-09 (n = 5, mean ± SD).

Sample Elastic modulus(MPa)

Tensile strength(MPa)

Elongation at yield(%)

PP0 851 ± 37 30 ± 1 19 ± 1PPC4 601 ± 25 27 ± 2 23 ± 2PPC6 597 ± 42 27 ± 1 23 ± 1PPC8 543 ± 44 27 ± 3 24 ± 2PPT4 593 ± 25 28 ± 2 23 ± 2PPT6 680 ± 96 28 ± 1 24 ± 1PPT8 585 ± 40 28 ± 2 25 ± 1PPTC4 681 ± 30 28 ± 2 23 ± 2PPTC6 646 ± 34 28 ± 1 22 ± 2PPTC8 677 ± 53 27 ± 3 22 ± 1

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volatilization point of these additives. Therefore, the processingparameters, in particular temperature and time should be opti-mized to avoid excessive evaporation and therefore the loss ofthese additives incorporated to PP (Dobkowski, 2006).

Fig. 3. TGA curves obtained for PP0 and form

3.4. Differential scanning calorimetry (DSC)

3.4.1. Determination of thermal parameters in inert atmosphereThermal properties of samples were also studied by DSC analy-

sis where four parameters were determined: crystallization tem-perature, Tc (�C); melting temperature, Tm (�C); crystallizationenthalpy, DHc (J g�1); and melting enthalpy, DHm (J g�1). These re-sults are summarized in Table 2. As it can be seen, melting andcrystallization temperatures and crystallization enthalpy did notshow important differences for all materials. Nevertheless, itshould be highlighted that the melting enthalpy of PP0 samplewas clearly higher than those obtained for the active materials.This observation could indicate a higher crystallinity of PP withno additives. In this sense, crystallinity, v (%), of samples was cal-culated according to Eq. (1). This parameter is also shown in Ta-ble 2, where a higher value for v (%) was determined for PP0sample. Thus, it could be concluded that the crystallinity of thematerial decreases significantly with the addition of thymol andcarvacrol, confirming the observed changes in the mechanical

ulations with carvacrol under nitrogen.

Table 2TGA and DSC parameters obtained for all samples.

Sample Ti (�C) Tmax (�C) Tc (�C) Tm (�C) DHc (J g�1) DHm (J g�1) v (%)

PP0 411 461 119 161 95.2 99.1 72

PPC4 417 462 119 161 91.4 49.6 37PPC6 414 461 117 160 93.6 52.2 40PPC8 407 462 118 161 89.2 48.1 38

PPT4 417 462 119 161 92.2 52.0 39PPT6 406 461 117 160 86.5 47.3 36PPT8 408 462 115 159 88.9 49.5 39

PPTC4 412 462 118 160 93.9 53.5 40PPTC6 398 461 117 160 89.6 49.0 38PPTC8 404 463 114 162 83.0 52.6 41

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properties where a decrease in the elastic modulus was noticed.This decrease in crystallinity could be due to the interactions be-tween the polymer matrix and additive molecules in the PP macro-molecular network. A similar effect was reported for PP with theaddition of some antioxidants (Alin and Hakkarainen, 2010).

3.4.2. Evaluation of antioxidant induction parameters (OIT and OOT)The evaluation of the antioxidant performance of carvacrol and

thymol as PP antioxidants is important since they are supposed notonly to play the role as active additives for food, but also to protectthe polymer to oxidative degradation during processing and fur-ther use. The determination of OOT and OIT parameters is consid-ered a reliable, simple and fast method for the evaluation ofantioxidants efficiency (Pomerantsev and Rodionova, 2005). Bothparameters correspond to relative measurements of the stabilityagainst oxidation of materials.

Table 3 shows OOT results. The onset degradation temperaturesfor the active materials were higher at least in 25 �C than the ob-tained value for PP0 sample. Therefore, it is remarkable that therewas some antioxidant effect in the active films. In particular, theformulations with thymol showed higher values than their coun-terparts with carvacrol. Similar results were previously reportedfor PP with a-tocopherol and hydroxytyrosol, two other naturalantioxidants (Peltzer and Jiménez, 2009).

According to ASTM D3895-07 Standard, the results obtained inOIT studies are dependent on the type of atmosphere used for theanalysis (ASTM D3895-07. 2007). For this reason, the evaluation ofOIT was carried out in two different atmospheres at 200 �C. Air wasselected since this environment would represent a similarsituation to the real conditions during materials processing or shelflife, while the use of pure oxygen would represent the most aggres-sive conditions for oxidative degradation. Table 3 shows the resultsobtained for OIT in both atmospheres.

Table 3Oxidation induction parameters, oxygen transmission rate and inhibition zone against S. a

Sample OOT (�C)a OIT (min), oxygena OIT (min), aira

PP0 195 ± 1 0.9 ± 0.3 1.3 ± 0.4PPC4 219 ± 3 5.9 ± 0.5 13.1 ± 1.6PPC6 225 ± 1 8.2 ± 0.2 17.8 ± 2.0PPC8 224 ± 1 8.5 ± 1.0 20.7 ± 2.8PPT4 234 ± 2 11.4 ± 1.8 28.9 ± 2.2PPT6 233 ± 3 13.2 ± 2.8 33.7 ± 1.2PPT8 235 ± 1 15.4 ± 1.7 38.8 ± 0.6PPTC4 223 ± 2 8.4 ± 1.2 24.1 ± 1.6PPTC6 226 ± 2 8.7 ± 1.1 26.4 ± 2.3PPTC8 230 ± 3 10.7 ± 1.2 35.5 ± 1.7

nd: No inhibition zone detected.a Mean ± SD (n = 3).b e: Thickness, mm.c Mean ± SD (n = 2).

For pure oxygen atmosphere, as in the case of OOT, it was pos-sible to confirm the higher efficiency of thymol as an antioxidant insuch aggressive conditions when compared to carvacrol. Thisbehavior was also reported by other authors who demonstratedthat the antioxidant efficiency of thymol was higher than that ofcarvacrol in sunflower oil samples (Yanishlieva et al., 1999). Inthe case of air atmosphere, as expected, OIT values were higherthan those obtained in pure oxygen atmosphere, since the experi-ment under air is less aggressive to materials (Riga et al., 1998).

In all cases, the increase in OOT and OIT values for PP with addi-tives showed certain antioxidant effect after processing. These re-sults are an additional confirmation that certain amounts ofthymol and carvacrol are still remaining in all formulations afterprocessing and they would be able to be released from the materialto foodstuff as active additives. Finally, an increase in both param-eters was also observed when the additive concentration in-creased. So, it can be concluded that the best results forantioxidant performance in these formulations would be obtainedin those samples with higher amount of additives.

3.5. Oxygen transmission rate (OTR)

Barrier properties to oxygen were studied by determination ofoxygen transmission rate per film thickness (e), OTR � e (Table 3).As it can be seen, PP0 showed lower OTR � e values than all activefilms, obtaining the maximum values for formulations with8 wt.% of carvacrol or thymol. This increase in oxygen transmissionfor the active films could be due to the modification of the polymermatrix structure in the presence of the additives, consequentlyreducing the resistance of films to oxygen diffusion through them(Sothornvit and Krochta, 2000). This behavior would be due to tworelated causes. On one hand, the increase in free volume in thepolymer structure caused by the chemical interaction between

ureus obtained for all formulations.

OTR � e (cm3 mm m�2 day)b S. aureus inhibition zone diameter (cm)c

82.2 nd104.7 nd146.9 nd159.6 2.75 ± 0.07114.3 nd142.5 nd155.5 3.70 ± 0.14112.2 nd126.7 nd158.0 3.25 ± 0.07

Fig. 4. Antimicrobial activity of PP films with 8 wt.% active additives. (a) S. aureus; (b) E. coli.

518 M. Ramos et al. / Journal of Food Engineering 109 (2012) 513–519

polymer chains and additive molecules and, on the other hand, thedecrease in the material crystallinity already observed by DSC andthe presence of certain porosity in the films surface observed bySEM (Amstrong, 2002).

3.6. Antimicrobial properties of films

Fig. 4 shows the results of the antimicrobial tests performedagainst S. aureus and E. coli of active films with 8 wt.% of additivesby the agar disk diffusion method. This method is very simple andit is based on the measurement of the clear zone caused by growthinhibition produced by a film disk containing the antimicrobialagent when putting in direct contact with a bacterial culture (Singhet al., 2003; Mendoza, 2009; Weerakkody et al., 2010). In thissense, when the PP films with the active agents are placed on topof the culture media, it is expected that both additives will diffusefrom the polymer matrix into the agar in a radial manner, produc-ing a clear zone of growth inhibition around the active film.

As it can be seen in Fig. 4a, films containing 8 wt.% of thymolwere the most effective against S. aureus showing the largest inhi-bition zone. This behavior was also observed for the rest of sampleswith 8 wt.% of additives, but with a lower inhibition zone (Table 3).These results demonstrate the antimicrobial action of both addi-tives at high concentrations (8 wt.%). The study of the combinedactivity by carvacrol and thymol in the same film (PPTC8) showedthat some additive effect between them took place considering theresults obtained for samples with 4 wt.% of each compound (PPC4and PPT4) separately, where an insufficient inhibition with nogrowth under the film was shown, as it was reported by Guardaet al. (2011). Finally, samples with additives at initial concentra-tions 6 wt.% were also not enough to achieve an adequate inhibi-tion at these conditions.

On the other hand, lower inhibition was observed when usingthese materials for E. coli (Fig. 4b). There was just some effect onlyunder the film, but the inhibition hale was not observed for thesebacteria. However, it is possible to attribute certain antibacterialproperties of these films against E. coli, since this inhibition is cat-egorized as ‘‘sufficient’’ as it is described in SNV 195920-1992Standard and it was reported by Pollini et al. (Pollini et al., 2009).The low antibacterial activity against E. coli could be due to thehigher resistance of gram-negative microorganisms to these com-pounds making necessary either the use of higher concentrationsof carvacrol and thymol in these films or the use of active agents

in vapor phase which has been reported to be more efficientagainst E. coli (Becerril et al., 2007).

Other studies have described the antimicrobial effect of thymoland carvacrol against E. coli (Xu et al., 2008), attributing this effectto its ability to permeate and depolarize the cytoplasmic membrane.These authors observed areas with coagulated material in the outerwall of cells caused by the precipitation of some proteins. However,other authors reported higher effectiveness of oregano essential oilin gram-positive bacteria (Lopez et al., 2005). Therefore it is con-cluded that experimental conditions for such tests are importantto get high or low resistance of specific microorganisms againstthese compounds (López et al., 2007a; Weerakkody et al., 2010).

As a general conclusion, the addition of carvacrol and thymol toPP films demonstrated some antimicrobial activity in bacterialstrains potentially present in food.

4. Conclusions

Carvacrol and thymol have demonstrated their potential to beused as active additives in PP films for food packaging applicationswith the double effect of their controlled antimicrobial release tofoodstuff and their possibility to substitute the common syntheticantioxidants used in PP formulations. Characterization of activefilms was carried out by using different analytical techniques in or-der to evaluate the effect of these additives in the polymer matrixand their material stabilization performance during processing.The addition of carvacrol and thymol did not significantly affectthe thermal behavior of PP, but these additives modified the filmsoxygen barrier and mechanical properties. PP films with carvacroland thymol showed a significant increase in OOT and OIT values;which means that the polymer is well stabilized and a certainamount of these compounds remains in the polymer matrix afterprocessing at relatively high temperatures. These additives couldbe furthermore released playing their role as active additives.Therefore, it could be concluded that the addition of antimicrobialadditives as carvacrol and thymol at 8 wt.% to PP shows some po-tential to improve product quality and safety aspects in food pack-aging applications. Further work is currently on-going to evaluateother important issues, such as organoleptic behavior or kineticsfor their release from the polymer matrix, to ensure their abilityto be used in food packaging applications. A final up-scale of thisstudy to include industrial processing (i.e. extrusion techniques)would be also necessary to obtain a commercial product basedon these formulations.

M. Ramos et al. / Journal of Food Engineering 109 (2012) 513–519 519

Acknowledgments

Authors would like to thank the ‘‘Instituto Alicantino de CulturaJuan Gil Albert’’ for the financial support and Ashland ChemicalHispania for kindly supplying ECOLEN HZ10K PP (HellenicPetroleum).

References

ASTM D3895-07, 2007. Standard test method for oxidative-induction time ofpolyolefins by differential scanning calorimetry. Annual Book of ASTMStandards. Amer. Soc. for Testing and Materials, Philadelphia, PA.

ASTM D882-09, 2009. Standard test method for tensile properties of thin plasticsheeting. 468 Annual Book of ASTM Standards. Amer. Soc. for Testing andMaterials, Philadelphia, PA.

Al-Bandak, G., Oreopoulou, V., 2007. Antioxidant properties and composition ofMajorana syriaca extracts. European Journal of Lipid Science and Technology109 (3), 247–255.

Alin, J., Hakkarainen, M., 2010. Type of polypropylene material significantlyinfluences the migration of antioxidants from polymer packaging to foodsimulants during microwave heating. Journal of Applied Polymer Science 118(2), 1084–1093.

Álvarez, M.F., 2000. Revisión: Envasado activo de los alimentos/review: active foodpackaging. Food Science and Technology International 6 (2), 97–108.

Amstrong, R.B., 2002. Effects of polymer structure on gas barrier of ethylene vinylalcohol (EVOH) and considerations for package development. Tappi PlaceConference 285, 311.

Appendini, P., Hotchkiss, J.H., 2002. Review of antimicrobial food packaging.Innovative Food Science and Emerging Technologies 3 (2), 113–126.

Archodoulaki, V.M., Lüftl, S., Seidler, S., 2006. Oxidation induction time studies onthe thermal degradation behaviour of polyoxymethylene. Polymer Testing 25(1), 83–90.

Bakkali, F., Averbeck, S., Averbeck, D., Idaomar, M., 2008. Biological effects ofessential oils–a review. Food and Chemical Toxicology 46 (2), 446–475.

Becerril, R., Gómez-Lus, R., Goñi, P., López, P., Nerín, C., 2007. Combination ofanalytical and microbiological techniques to study the antimicrobial activity ofa new active food packaging containing cinnamon or oregano against E. coli andS. aureus. Analytical and Bioanalytical Chemistry 388 (5), 1003–1011.

Burt, S., 2004. Essential oils: their antibacterial properties and potential applicationsin foods–a review. International Journal of Food Microbiology 94 (3), 223–253.

Del Nobile, M.A., Conte, A., Buonocore, G.G., Incoronato, A.L., Massaro, A., Panza, O.,2009. Active packaging by extrusion processing of recyclable and biodegradablepolymers. Journal of Food Engineering 93 (1), 1–6.

Didry, N., Dubreuil, L., Pinkas, M., 1994. Activity of thymol, carvacrol,cinnamaldehyde and eugenol on oral bacteria. Pharmaceutica Acta Helvetiae69 (1), 25–28.

Dobkowski, Z., 2006. Thermal analysis techniques for characterization of polymermaterials. Polymer Degradation and Stability 91 (3), 488–493.

Guarda, A., Rubilar, J.F., Miltz, J., Galotto, M.J., 2011. The antimicrobial activity ofmicroencapsulated thymol and carvacrol. International Journal of FoodMicrobiology 146 (2), 144–150.

Halliwell, B., Aeschbach, R., Löliger, J., Aruoma, O.I., 1995. The characterization ofantioxidants. Food and Chemical Toxicology 33 (7), 601–617.

Ho Lee, C., Soon An, D., Cheol Lee, S., Jin Park, H., Sun Lee, D., 2004. A coating for useas an antimicrobial and antioxidative packaging material incorporating nisinand [alpha]-tocopherol. Journal of Food Engineering 62 (4), 323–329.

Joseph, P.V., Joseph, K., Thomas, S., Pillai, C.K.S., Prasad, V.S., Groeninckx, G.,Sarkissova, M., 2003. The thermal and crystallisation studies of short sisal fibrereinforced polypropylene composites. Composites Part A: Applied Science andManufacturing 34 (3), 253–266.

Lambert, R.J.W., Skandamis, P.N., Coote, P.J., Nychas, G.-J.E., 2001. A study of theminimum inhibitory concentration and mode of action of oregano essential oil,thymol and carvacrol. Journal of Applied Microbiology 91 (3), 453–462.

Lopez, P., Sanchez, C., Batlle, R., Nerin, C., 2005. Solid and vapor-phase antimicrobialactivities of six essential oils: susceptibility of selected foodborne bacterial andfungal strains. Journal of Agricultural and Food Chemistry 53 (17), 6939–6946.

López, P., Sánchez, C., Batlle, R., Nerín, C., 2007a. Development of flexibleantimicrobial films using essential oils as active agents. Journal ofAgricultural and Food Chemistry 55 (21), 8814–8824.

López, P., Sánchez, C., Batlle, R., Nerín, C., 2007b. Vapor-phase activities ofcinnamon, thyme, and oregano essential oils and key constituents againstfoodborne microorganisms. Journal of Agricultural and Food Chemistry 55 (11),4348–4356.

Lundbäck, M., Hedenqvist, M.S., Mattozzi, A., Gedde, U.W., 2006. Migration ofphenolic antioxidants from linear and branched polyethylene. PolymerDegradation and Stability 91 (7), 1571–1580.

Mascheroni, E., Guillard, V., Gastaldi, E., Gontard, N., Chalier, P., 2011. Anti-microbialeffectiveness of relative humidity-controlled carvacrol release from wheatgluten/montmorillonite coated papers. Food Control 22 (10), 1582–1591.

Mendoza, P.G., 2009. Desarrollo de películas activas para el envasado de alimentos.Tesis. Instituto Politécnico Nacional, Escuela Nacional de Ciencias Biológicas.

Navarro, R., Torre, L., Kenny, J.M., Jiménez, A., 2003. Thermal degradation of recycledpolypropylene toughened with elastomers. Polymer Degradation and Stability82 (2), 279–290.

Peltzer, M., Jiménez, A., 2009. Determination of oxidation parameters by DSC forpolypropylene stabilized with hydroxytyrosol (3,4-dihydroxy-phenylethanol).Journal of Thermal Analysis and Calorimetry 96 (1), 243–248.

Peltzer, M., Navarro, R., López, J., Jiménez, A., 2010. Evaluation of the meltstabilization performance of hydroxytyrosol (3, 4-dihydroxy-phenylethanol)in polypropylene. Polymer Degradation and Stability 95 (9), 1636–1641.

Peltzer, M., Wagner, J., Jiménez, A., 2007. Thermal characterization of UHMWPEstabilized with natural antioxidants. Journal of Thermal Analysis andCalorimetry 87 (2), 493–497.

Peltzer, M., Wagner, J., Jiménez, A., 2009. Migration study of carvacrol as a naturalantioxidant in high-density polyethylene for active packaging. Food Additivesand Contaminants: Part A: Chemistry, Analysis, Control Exposure and RiskAssessment 26 (6), 938–946.

Persico, P., Ambrogi, V., Carfagna, C., Cerruti, P., Ferrocino, I., Mauriello, G., 2009.Nanocomposite polymer films containing carvacrol for antimicrobial activepackaging. Polymer Engineering and Science 49 (7), 1447–1455.

Pollini, M., Russo, M., Licciulli, A., Sannino, A., Maffezzoli, A., 2009. Characterizationof antibacterial silver coated yarns. Journal of Materials Science. Materials inMedicine 20 (11), 2361–2366.

Pomerantsev, A.L., Rodionova, O.Y., 2005. Hard and soft methods for prediction ofantioxidants’ activity based on the DSC measurements. Chemometrics andIntelligent Laboratory Systems 79 (1–2), 73–83.

Pospíšil, J., Horák, Z., Pilar, J., Billingham, N.C., Zweifel, H., Nešpurek, S., 2003.Influence of testing conditions on the performance and durability of polymerstabilisers in thermal oxidation. Polymer Degradation and Stability 82 (2), 145–162.

Riga, A., Collins, R., Mlachak, G., 1998. Oxidative behavior of polymers bythermogravimetric analysis, differential thermal analysis and pressuredifferential scanning calorimetry. Thermochimica Acta 324, 135–149.

Salafranca, J., Pezo, D., Nerín, C., 2009. Assessment of specific migration to aqueoussimulants of a new active food packaging containing essential oils by means ofan automatic multiple dynamic hollow fibre liquid phase microextractionsystem. Journal of Chromatography A 1216 (18), 3731–3739.

Sanchez-Garcia, M.D., Ocio, M.J., Gimenez, E., Lagaron, J.M., 2008. Novelpolycaprolactone nanocomposites containing thymol of interest inantimicrobial film and coating applications. Journal of Plastic Film andSheeting 24 (3–4), 239–251.

Singh, A., Singh, R.K., Bhunia, A.K., Singh, N., 2003. Efficacy of plant essential oils asantimicrobial agents against Listeria monocytogenes in hotdogs. Lebensmittel-Wissenschaft und-Technologie 36 (8), 787–794.

Sothornvit, R., Krochta, J.M., 2000. Oxygen permeability and mechanical propertiesof films from hydrolyzed whey protein. Journal of Agricultural and FoodChemistry 48 (9), 3913–3916.

Suppakul, P., Miltz, J., Sonneveld, K., Bigger, S.W., 2003. Active packagingtechnologies with an emphasis on antimicrobial packaging and itsapplications. Journal of Food Science 68 (2), 408–420.

Suppakul, P., Miltz, J., Sonneveld, K., Bigger, S.W., 2006. Characterization ofantimicrobial films containing basil extracts. Packaging Technology andScience 19 (5), 259–268.

Suppakul, P., Sonneveld, K., Bigger, S.W., Miltz, J., 2011a. Diffusion of linalool andmethylchavicol from polyethylene-based antimicrobial packaging films. LWT -Food Science and Technology 44 (9), 1888–1893.

Suppakul, P., Sonneveld, K., Bigger, S.W., Miltz, J., 2011b. Loss of AM additives fromantimicrobial films during storage. Journal of Food Engineering 105 (2), 270–276.

Tunç, S., Duman, O., 2011. Preparation of active antimicrobial methyl cellulose/carvacrol/montmorillonite nanocomposite films and investigation of carvacrolrelease. LWT - Food Science and Technology 44 (2), 465–472.

Valentao, P., Fernandes, E., Carvalho, F., Andrade, P.B., Seabra, R.M., Bastos, M.L.,2002. Antioxidative properties of cardoon (Cynara cardunculus L.) infusionagainst superoxide radical, hydroxyl radical, and hypochlorous acid. Journal ofAgricultural and Food Chemistry 50 (17), 4989–4993.

Veldhuizen, E.J.A., Tjeerdsma-van Bokhoven, J.L.M., Zweijtzer, C., Burt, S.A.,Haagsman, H.P., 2006. Structural requirements for the antimicrobial activityof carvacrol. Journal of Agricultural and Food Chemistry 54 (5), 1874–1879.

Vermeiren, L., Devlieghere, F., van Beest, M., De Kruijf, N., Debevere, J., 1999.Developments in the active packaging of foods. Trends in Food Science andTechnology 10 (3), 77–86.

Weerakkody, N.S., Caffin, N., Turner, M.S., Dykes, G.A., 2010. In vitro antimicrobialactivity of less-utilized spice and herb extracts against selected food-bornebacteria. Food Control 21 (10), 1408–1414.

Xu, J., Zhou, F., Ji, B.-P., Pei, R.-S., Xu, N., 2008. The antibacterial mechanism ofcarvacrol and thymol against Escherichia coli. Letters in Applied Microbiology 47(3), 174–179.

Yanishlieva, N.V., Marinova, E.M., Gordon, M.H., Raneva, V.G., 1999. Antioxidantactivity and mechanism of action of thymol and carvacrol in two lipid systems.Food Chemistry 64 (1), 59–66.

Youdim, K.A., Deans, S.G., 2000. Effect of thyme oil and thymol dietarysupplementation on the antioxidant status and fatty acid composition of theageing rat brain. British Journal of Nutrition 83 (01), 87–93.