ate

elhrazilof S

Antimicrobial activity of cinnamon and clove essential oils is widely known; however their application to

renewable source, inexpensive and widely available (Souza,Ditchield, & Tadini, 2010). Beyond this, it has good lm-formingproperties.

Cassava starch hasbeen extensively used to producelms and theresults indicated that these carbohydrates are promisingmaterials in

sky, 2006; Mller,012; Souza et al.,Vercelheze et al.,arini, 2008). Filmsodorless, tasteless,al., 2010).was described thewith sugars and

. Results reportedbecause they gaveoduce composites

lms with better mechanical and barrier properties. Now, authorsare trying to incorporate antimicrobial agents in the formulation ofcassava starch lms since carrying natural additives could beconsidered as a new tendency of functional food packaging in thenear future. Active packaging provides microbial safety for con-sumers, reducing, inhibiting or retarding the growth of microor-ganisms, and then, could extend the shelf life of the packaged food.

Based on results presented by Kechichian et al. (2010), cinna-mon essential oil and clove essential oil were chosen to continue

* Corresponding author. Department of Chemical Engineering, Escola Politcnica,University of So Paulo, SP, Brazil. Tel.: 55 11 30912258; fax: 55 11 30912255.

Contents lists availab

LWT - Food Science

w.e

LWT - Food Science and Technology 54 (2013) 346e352E-mail address: catadini@usp.br (C.C. Tadini).current tendencies include the development of packagingmaterialsthat interact with the product. One of the several possibilities,which are being extensively studied, is the incorporation of activesubstances within the package material, as lms based on cassavastarch (Kechichian, Ditchield, Veiga-Santos, & Tadini, 2010).

Although many types of new polymers are being industriallyproduced (PLA, PHA, PCL, PEA and others), polymers from agri-cultural sources are the most studied by researchers, especiallypolysaccharides. Among the lms made from polysaccharides,those obtained from starch are themost important because it is oneof the most commonly used agricultural raw materials, since it is a

2010; Mali, Grossmann, Garca, Martino, & ZaritYamashita, & Laurindo, 2008; Pelissari et al., 22012; Veiga-Santos, Ditchield, & Tadini, 2011;2012; Veiga-Santos, Suzuki, Nery, Cereda, & Scampdeveloped from starch are described as isotropic,colorless, non-toxic and biodegradable (Souza et

In a previous study (Souza et al., 2012), itdevelopment of cassava starch lms plasticizedglycerol and reinforced with clay nanoparticleswere important for the continuity of the researchinformation about optimal formulation to prIn order to keep food quality and freshness, it is necessary toselect correct materials and packaging technologies. In this way,

Gerschenson, 2007; Fam, Flores, Gerschenson, & Goyanes, 2006;Kaisangsri, Kerdchoechuen, & Laohakunjit, 2012; Kechichian et al.,Accepted 20 June 2013

Keywords:Cassava starchAntimicrobial activityMicrostructureMechanical propertiesBarrier properties

1. Introduction0023-6438/$ e see front matter 2013 Elsevier Ltd.http://dx.doi.org/10.1016/j.lwt.2013.06.017evaluated on antimicrobial activity, mechanical and barrier properties of lms and the results werecompared with those of control lms (without antimicrobial agent). ANOVA (P < 0.05) showed that theessential oil content inuenced signicantly the properties of the lms. The release of antimicrobialagent and the microstructure of cassava lms incorporated with cinnamon essential oil were alsostudied. Furthermore, all lms, containing different amounts of essential oil, showed effective antimi-crobial activity against P. commune and E. amstelodami, fungi commonly found in bread products.

2013 Elsevier Ltd. All rights reserved.

this regard (Bertuzzi, Castro Vidaurre, Armanda, & Gottifredi, 2007;Chen & Lai, 2008; Chillo et al., 2008; Fam, Goyanes, &Received in revised form20 May 2013 cassava starch lms in order to develop an active packaging. The effect of cinnamon essential oil wereReceived 3 March 2013 polymeric materials is already limited. Based on results of their minimum inhibitory concentrationagainst Penicillium commune and Eurotium amstelodami, cinnamon was chosen to be incorporated intoCassava starch composite lms incorporoil: Antimicrobial activity, microstructurproperties

A.C. Souza a, G.E.O. Goto a, J.A. Mainardi a, A.C.V. CoaDepartment of Chemical Engineering, Escola Politcnica, University of So Paulo, SP, BbDepartment of Metallurgical and Materials Engineering, Escola Politcnica, UniversitycNAPAN e Food and Nutrition Research Center, University of So Paulo, SP, Brazil

a r t i c l e i n f o

Article history:

a b s t r a c t

journal homepage: wwAll rights reserved.ed with cinnamon essential, mechanical and barrier

o b, C.C. Tadini a,c,*

o Paulo, SP, Brazil

le at ScienceDirect

and Technology

lsevier .com/locate/ lwt

ce atheir research, which was developed by the same research group ofthe present work. Other authors also demonstrated the antimi-crobial efcacy of these agents in literature (Goi et al., 2009; Kim,Park, & Park, 2004; Nielsen & Rios 2000; Oussalah, Caillet, Saucier,& Lacroix, 2006; Oussalah, Caillet, Saucier, & Lacroix, 2007). Cin-namon and clove has been used as spices for thousands of years.The main constituents of their oils are cinnamaldehyde andeugenol, respectively, two well known agents due to their antimi-crobial activities.

Oussalah et al. (2006) reported that cinnamon essential oilshowed a strong antimicrobial activity against Pseudomonas putidastrain isolated from meat. Kim et al. (2004) suggested that theantimicrobial activity of cinnamaldehyde is bactericidal againstEscherichia coli O157:H7. Scanning electron microscopic observa-tions revealed that the bacterial cells treated with cinnamaldehydesuffered severe damages in their surface structure. Nielsen & Rios(2000) tested the effect of essential oils against the most impor-tant spoilage fungi of bread and demonstrated that cinnamonessential oil had high activity. Results obtained by Oussalah et al.(2007) showed that one of the most active essential oil againstfour pathogenic bacteria was the cinnamon. Moreover, Goi et al.(2009) tested a combination of cinnamon and clove essential oilsagainst awide range of bacteria in the vapor phase as a preservativemethod to prevent microorganism proliferation.

In the present work, the minimum inhibitory concentration(MIC) of two essential oils, cinnamon (Cinnamomum cassia) andclove (Eugenia caryophyllata), were established. In a second step,cinnamon essential oil was incorporated into cassava starch lmselaborated by casting. The main goal was to develop active com-posite lms, and to verify the inuence of cinnamon essential oiladdition on microstructure, mechanical (tensile strength andpercent elongation at break) and barrier (water vapor permeabilityand oxygen permeability coefcient) properties of produced lms.Also, the antimicrobial activity against fungi commonly found inbread was tested by two different techniques: disk diffusionmethod and release mass experiments by UVevis spectroscopy.

2. Materials and methods

2.1. Materials

Native cassava starch, kindly supplied by Cargill Agrcola, Brazil(amylose: 19.7 g/100 g; moisture: 12.5 g/100 g) was used as thelm-forming component to provide a continuous matrix of lms.Glycerol (Synth, Brazil) and natural -Na montmorillonite clay(commercial product Argel T, used as received, without purica-tion, Bentonit Unio, Brazil) were used as plasticizer and rein-forcement ller, respectively. Cinnamon essential oil (Ferquima,Brazil) with 82.5 g/100 g of cinnamaldehyde and clove essential oil(Ferquima, Brazil) with 75.0 g/100 g of eugenol were used asantimicrobial agents. Sucrose ester of fatty acids was used asemulsier, specic for oil/water emulsion, in order to incorporatethe cinnamon essential oil into the lms (commercial name: SP70,Sisterna, Brazil). Distilled water and ethanol (Synth, Brazil) wereused as solvents of the lmogenic solutions. Penicillium communeand Eurotium amstelodami were obtained in a lyophilized form(Andr Tosello Foundation, Brazil). All growth experiments werecarried out on amedium for fungi preparedwith Czapek Dox (Difco,USA) and agar (Synth, Brazil).

2.2. Film preparation

The lms were produced by casting technique, using themethodology and optimum contents of cassava starch, glycerol and

A.C. Souza et al. / LWT - Food Scienclay nanoparticles proposed by Souza et al. (2012). The lmogenicsolutionwas prepared according to the following procedure: rstly,0.1 g of clay nanoparticles were suspended in 25 g of distilled waterfor 1 h, under stirring (500 rpm), and, after rest for 24 h, they wereblended with a suspension of 5.0 g of starch and 70 g of distilledwater. After that, cinnamon essential oil (0.40 g, 0.60 g or 0.80 g)was mixed with emulsier (0.010 g, 0.015 g or 0.020 g), corre-spondent to 0.025 for emulsier content/essential oil contentproportion; and glycerol (0.75 g, 1.13 g or 1.50 g) at (38 2) C,correspondent to 1.88 for glycerol content/essential oil contentproportion, using a magnetic stirrer (200 rpm). Both mixturesprepared were then homogenized and heated in a domestic mi-crowave oven (Panasonic, model Family Plus, Brazil) until starchgelatinization, which occurs at (69 2) C. After cooling, the l-mogenic solution was diluted with 14.25 g of ethanol, and, for eachformulation, a specic content of lmogenic solution was pouredonto rectangular plates (97.5 cm2 of area) of polytetrauoro-ethylene (Teon) to obtain a constant thickness of (100 10) mm,followed by drying at (35 2) C for approximately (18e24) h, in aconventional chamber dryer with forced air circulation (Nova tica,series N480, Brazil).

The quantities of glycerol, emulsier and cinnamon essential oilwere dened according preliminary tests and based on previouswork (Souza et al., 2012), taking into account the maximum levelsof cinnamon essential oil which could be incorporated into thematrix without oil phase separation during lm drying. Compositelm without cinnamon essential oil and emulsier was also pro-duced and considered as control.

After drying, all lms were placed in a controlled relative hu-midity of 75% and at ambient temperature of (23 2) C and storedprior to testing.

2.3. Antimicrobial activity e disk diffusion method

Using the pour plate method, inoculums (1 mL) of P. communeand E. amstelodami were spread on the surface of Petri dishesprepared with the selected medium for fungi. The inoculums wereadjusted to each microorganism to yield a cell concentration of108 CFU/mL.

Theminimum inhibitory concentration (MIC) was dened as thelowest essential oil concentration resulting in the lack of visiblemicroorganism growth using the disk diffusion method.

Circular disk samples of lter paper (25 mm of diameter) wereabsorbed with alcoholic solutions of each essential oil at differentconcentrations: (0.0, 0.5,1.0, 2.0, 4.0, 8.0,16.0 and 32.0) g/100 g, andplaced on the solidied medium surface. Petri dishes were thenincubated at (25 2) C for 5 days and the inhibitory zone wasdetermined measuring its diameter.

In order to evaluate the efciency of each cinnamon essentialoil contents incorporated into composite lms, circular disk sam-ples of these lms were placed, instead of lter paper, on the so-lidied medium surface. Petri dishes were then incubated at(25 2) C for 5 days. Antimicrobial agent efciency was evaluatedby the formation of an inhibition zone around the disk samples,which was characterized by surrounding clear areas. To a betterpresentation of the results, the diameter of inhibition zone of eachPetri dish was measured and, knowing the total area of the Petridish, inhibition results were converted to percentage of inhibitionareas. All tests were performed in triplicate, seven days after thelm elaboration.

2.4. Antimicrobial agent release

The amount of antimicrobial agent incorporated in cassavastarch lms was quantied by UVevis spectroscopy (Spectropho-

nd Technology 54 (2013) 346e352 347tometer JASCO, model 550, Japan) measuring at 289 nm,

sensor determined the amount of oxygen transmitted through thelm into the carrier gas. The oxygen transmission rate was deter-mined for all formulations in duplicate. The permeance (PO2) of thelms was calculated according to Equation (2):

PO2 OTRp

(2)

wherein:PO2 is the permeance of the lms [cm3m2 d1 Pa1]; OTR is the

oxygen transmission rate [cm3 m2 d1]; and p is the partialpressure of oxygen, which is the mol fraction of oxygen multipliedby the total pressure (nominally, 1 atm) in the test gas side of thediffusion cell. The partial pressure of O2 on the carrier gas side isconsidered to be zero.

The oxygen permeability coefcient (P0O2) was calculated asfollows:

PO2 PO2 t (3)

wherein:P0O is the oxygen permeability coefcient [cm3 m1 d1 Pa1];

ce acorresponding to the maximum absorption wavelength of cinna-mon essential oil, and using a pre-determined calibration curve.The release experiments were carried out at room temperaturewith the lms immersed in distilled water (150 mL) for 2 h. Afterthe rst 2 h, tested samples were once more immersed in distilledwater and a new spectroscopy quanticationwas done at 289 nm inorder to ensure that this time was enough to guarantee full releaseof antimicrobial agent from the lms (in this case, the contentquantied should be zero). Assays were performed in duplicate.

2.5. Film thickness

The thickness (t) [mm] was measured using a at parallel sur-face micrometer (MITUTOYO Sul Americana Ltda., model 103-137,Brazil, precision 0.002 mm), at ve random positions of the lms.

2.6. Scanning Electron Microscopy (SEM)

Small strips (5 mm 5 mm) of cassava starch lms weremounted on aluminum stubs, coated with a thin layer of gold andobserved on a Scanning Electron Microscope (Philips, model XL-30FEG), at an accelerate voltage of 5 kV.

2.7. Mechanical properties

Tensile strength (TS) [MPa] and percent elongation at break (E)[%] were evaluated by a tensile test performed on a textureanalyzer (TA.XT2i e Stable Micro Systems, UK) with a load cell of5 kg, using the A/TGT self-tightening roller grips xture, accordingto ASTM D882-09 (2009). Twenty strips (130 mm 25 mm) werecut from each formulation of preconditioned lms and each onewas mounted between the grips of the equipment for testing.Initial grip separation and test speed were set to 50 mm and0.8 mm s1, respectively. Tensile strength (nominal) was calculateddividing the maximum load by the original minimum cross-sectional area of the specimen (related to minimum thickness).Percent elongation at break (nominal) was calculated by dividingthe extension at the moment of rupture of the specimen by itsinitial gage length and multiplying by 100. All formulations wereevaluated in triplicate.

2.8. Water vapor permeability

Water vapor transmission (WVT) was determined by a gravi-metric method based on ASTM E96/E96M-05 (2005), using theDesiccant Method. This property was reported as water vaporpermeability (WVP), which is the rate of water vapor transmission(WVT) through a unit area of at material of unit thickness inducedby unit vapor pressure difference between two surfaces, underspecied humidity condition of 75%. Each lm sample was sealedwith parafn over a circular opening of 44 cm2 at the permeationcell (PVA/4, REGMED, Brazil) that was stored, at ambient temper-ature, in a desiccator. To maintain 75% of relative humidity (RH)gradient across the lm, a constant mass of silica gel was placedinside the cell and a sodium chloride saturated solution (75% RH)was used in the desiccator. Two cells without silica gel were pre-pared and submitted to the same conditions to account for weightchanges occurring in the lm, since it is a hydrophilic material. TheRH inside the cell was always lower than the outside, and watervapor transport was determined from the weight gain of thepermeation cell. After steady state conditions were reached (about2 h), tenweight measurements were made over 48 h Fig. 1 shows atypical curve indicating that the weight gain from the straight line

A.C. Souza et al. / LWT - Food Scien348was 3.15 102 g h1. WVP was calculated according Equation (1):WVP wq

24 tA Dp

(1)

wherein:WVP is thewater vapor permeability [gmmm2 d1 kPa1];w is

the weight gain (from the straight line) [g]; q is the time duringwhichw occurred [h]; t is the average lm thickness [mm]; A is thetest area (cell top area) [m2] and Dp is the vapor pressure difference[kPa]. All formulations were evaluated in triplicate.

2.9. Oxygen permeability coefcient

Oxygen transmission rate (OTR) of the lms was measured at23 C and 75% RH on a 50 cm2 circular lms using an oxygenpermeation system (OXTRAN 2/21, MOCON, USA), in accordancewith ASTM F1927-07 (2007). A starch based lm was sealed be-tween two chambers (each one with two channels), the lower onesuppliedwith O2 at a controlled ow rate (20mLmin1) to keep thepressure constant in that compartment, and the other one waspurged by a stream of nitrogen carrier gas (98% of nitrogen and 2%of hydrogen), at controlled ow rate (10 mL min1). A coulometric

Fig. 1. Typical curve of weight gain (w) of the samples as a function of time (t) incomparison with weight gain of the samples in the cell without silica gel.

nd Technology 54 (2013) 346e3522and t is the average thickness of the specimen [mm].

2.10. Statistical analysis

Analysis of variance (ANOVA) was applied on the results usingthe statistical program Statgraphics Centurion program v.15.2.06(StatPoint, Inc., Warrenton, USA) and the Tukey test was used toevaluate average differences (at a 95% of condence interval).

3. Results and discussion

The study was conducted in two steps: rstly, antimicrobialactivities of cinnamon and clove essential oils were evaluated, us-ing the disk diffusion method, against P. commune and E. amstelo-dami, fungi commonly found in bread products (Saranraj & Geetha,2012). It was possible to quantify the minimum amount of each

inhibition began with 0.5 g/100 g of cinnamon essential oil solu-tion (diameter: 4 mm) and with 4.0 g/100 g of clove essential oilsolution (diameter: 6 mm) and was completed (100% of inhibition)with 2.0 g/100 g and 16 g/100 g, respectively. For E. amstelodami,inhibition was completed with only 0.5 g/100 g of cinnamonessential oil solution and began with 4.0 g/100 g of clove essentialoil solution (diameter: 14 mm) and was completed with 16 g/100 g.

With these results, it can be concluded that cinnamon essentialoil was more effective against the fungi selected for this work, sinceit presented a better inhibition with lower concentration. In thisway, cinnamon essential oil was chosen to be incorporated incomposite lms based on cassava starch.

different contents of cinnamon essential oil against each studied

A.C. Souza et al. / LWT - Food Science and Technology 54 (2013) 346e352 349essential oil necessary to be incorporate in cassava starch lms inorder to develop lms with antimicrobial properties.

In the second step, cinnamon and clove essential oils wereincorporated in cassava starch lms. In preliminary assays, it wasnoted that the amount of clove essential oil necessary to providelms with effective antimicrobial activity against fungi tested wastoo high and, therefore, it became infeasible to obtain lms withsuitable visual and handling properties. Thus, it was decided toproduce the active lms with only cinnamon essential oil, since thisagent presented more promising results in the rst step.

Despite initial results of microbiological inhibition were quitesatisfactory, indicating an almost complete inhibition of fungi,materials produced showed a compromised surface because lmsbecame more and more brittle with the increase of essential oilcontent in the formulation. To overcome this hurdle, it wasnecessary to vary the plasticizer content in accordance with theincrease of essential oil content in the formulation.

Since it is known that it is impossible to make homogeneoussuspensions of oil in water (that was used as the solvent of thelmogenic solution), an emulsier in the formulation of cassavastarch lms was added in order to avoid a phase separation.Therefore, active cassava starch lms with emulsier and plasti-cizer in contents varying according to the amount of cinnamonessential oil were developed and characterized.

After preliminary results and based on previous work (Souzaet al., 2012), proportions of 1.88 for glycerol content/essential oilcontent; and 0.025 for emulsier content/essential oil content, werechosen to provide lms with good visual and tactile characteristics.

3.1. Determination of minimum inhibitory concentration (MIC) ofcinnamon and clove essential oils

Different results of inhibition were obtained for each essentialoil and for each microorganism studied (Table 1). For P. commune,

Table 1Inhibition areas [%] for each essential oil evaluated against P. commune andE. amstelodami.

Essential oilsolution [g/100 g]

Inhibition area [%]

P. Commune E. amstelodami

Cinnamonessential oil

Cloveessential oil

Cinnamonessential oil

Cloveessential oil

0.0 0.0 0.0 0.0 0.00.5 10.4 0.0 100.0a 0.01.0 32.1 0.0 100.0 0.02.0 100.0a 0.0 100.0 0.04.0 100.0 11.9 100.0 18.78.0 100.0 40.1 100.0 62.316.0 100.0 100.0a 100.0 100.0a

32.0 100.0 100.0 100.0 100.0a MIC: minimum inhibitory concentration.Table 2Inhibition areas [%] against P. commune and E. amstelodami of active cassava starchlms produced with different contents of glycerol, emulsier and cinnamonessential oil.

Formulation Glycerol[g/100 g]

Emulsier[g/100 g]

Cinnamonessential oil[g/100 g]

Inhibition area [%]

P. commune E. amstelodami

Control 0.75 0 0 0.00 0.00a 0.00 0.00aA 0.75 0.010 0.40 1.93 0.74b 14.83 3.79bB 1.13 0.015 0.60 10.77 5.31c 47.02 4.69cC 1.50 0.020 0.80 25.94 5.72d 91.06 15.48dTukey

HSD 5%10.25 21.50microorganism are shown in Table 2. ANOVA indicated that therewere signicant differences among antimicrobial activity of lmswith different cinnamon essential oil contents (P < 0.05). As pre-dictable, no inhibition zone against the microorganisms wasobserved for lm disks without incorporation of essential oil(control lms). Comparing themicroorganisms, it can be concludedthat E. amstelodami is more sensitive for cinnamon essential oilbecause its inhibition was greater, reaching approximately 91% ofinhibition with the highest concentration used. Fig. 2 shows theinhibition of P. commune caused by active lms produced withthree different contents of cinnamon essential oil. As expected, abetter inhibition was observed with higher content of cinnamonessential oil (Fig. 3).

Even at minimum concentration applied into the lm formula-tion, cinnamon essential oil showed inhibition against both mi-croorganisms, which was considered an important result since thathigher concentrations could imply a sensorial impact, altering thenatural taste of the food packaged by exceeding the acceptableavor thresholds.

A great number of studies on the antimicrobial characteristics oflms made from starch have been carried out earlier. Nevertheless,no information has been presented about the effect of cinnamonessential oil on P. commune and E. amstelodami, which plays animportant role in the spoilage of bread products.

3.3. Release of cinnamon essential oil

Cinnamon essential oil (CEO) release proles from cassavastarch lms, for a monitoring period of 2 h, are shown in Fig. 2.3.2. Antimicrobial activity of active cassava starch lmsincorporated with cinnamon essential oil

Inhibition areas yielded by cassava starch lm disks with*Means in the same column with the same letter are not signicantly different(P > 0.05).

Fig. 4. Cassava starch lm incorporated with 0.4 g of cinnamon essential oil/100 g of

A.C. Souza et al. / LWT - Food Science and Technology 54 (2013) 346e352350Released amounts of CEO varied from (0.88 0.10) mg CEO/g lmto (1.19 0.02) mg CEO/g lm for lms incorporated with differentcontents of antimicrobial agent. It can also be observed that in 2 hall added CEO was released, since the results for the new quanti-cation done after 2 h were zero. Moreover, it should be pointedout that cassava starch lm samples did not dissolve in the water

Fig. 2. Petri dishes with circular disks of lms incorporated with three differentcontents of cinnamon essential oil (CEO), showing the inhibitory zone against Peni-cillium commune in comparison with Petri dish without the active lms.after 2 h, but their volumewere increased, demonstrating that lmswere susceptible to water uptake.

The pronounced initial increase of mass released content sug-gests that it is necessary to incorporate the antimicrobial agent intomatrix by another technique, like as supercritical solvent impreg-nation, if a slower release is desired.

Fig. 3. Mass released of cinnamon esse3.4. Appearance and microstructure of cassava starch lms

Homogeneous, thin and exible cassava starch lms were ob-tained. They could be easily removed from the Teon plates afterdrying. Visually, all lms were colorless and slightly opaque (Fig. 4).

Fig. 5 shows SEM micrographs of the surface of active cassavastarch lms with remarkable differences. A continuous matrix wasobserved for active lms elaborated with emulsier (Fig. 5a).Smooth, uniform and regular surface was observed in all samples.On the other hand, the absence of the emulsier caused a discon-tinuous structure, with lipid droplets embedded in the polymernetwork (Fig. 5b).

3.5. Mechanical and barrier properties of cassava starch lms

Data of tensile strength, elongation at break, water vapor

lmogenic solution (from Eduardo de Oliveira, with permission).permeability and oxygen permeability coefcient obtained from

ntial oil from cassava starch lms.

Compared to most commonly used synthetic polymers, TS and Ewere rather low, but sufcient for use in many food applications. Inprevious work, Souza et al. (2012) tested lms based on cassavastarch reinforced with 1.0 g/100 g of clay, at the same conditions ofthis work, and found that the increase of glycerol content from(0.75e1.25) g/100 g, decreased the TS from (3.96 0.60 to2.07 0.33) MPa and increased E from (123.61 19.57 to200.24 33.50) %. Considering these previous results, the increaseof the glycerol content in cassava starch lms elaborated in thispresent work can also contributed with the decrease of TS.

A.C. Souza et al. / LWT - Food Science and Technology 54 (2013) 346e352 351cassava starch lms produced with cinnamon essential oil asantimicrobial agent are shown in Table 3. All data were analyzed byANOVA and the results indicated there were signicant differencesamong lms properties with different cinnamon essential oil con-tents (P < 0.05).

Tensile strength (TS) and elongation at break (E) of lms withcinnamon essential oil incorporated varied from (2.32 0.40 to1.05 0.16) MPa and from (264.03 35.06 to 191.27 22.62) %,respectively, therefore an increase of cinnamon essential oil, glyc-erol and emulsier contents lowered the TS and the E of the lms,indicating a loss of macromolecular mobility. From presented data,it was realized that control lms (without essential oil) presentedhigher TS (3.96 0.60) MPa and lower E (123.61 19.57) %

Fig. 5. Micrographs of surface of cassava starch lms: active lm elaborated (a) withemulsier and (b) without emulsier.

Table 3Tensile strength (TS), elongation at break (E), water vapor permeability (WVP) and oxygencontents of glycerol, emulsier and cinnamon essential oil.

Formulation TS [MPa] E [%]

Control 3.75 0.70d 128.81 18.67aA 2.32 0.40c 256.13 48.57cB 1.36 0.18b 264.03 35.06cC 1.05 0.16a 191.27 22.62bTukey HSD 5% 0.12 14.02

*Means in the same column with the same letter are not signicantly different (P > 0.05When comparing lms prepared according formulation A withthe control ones, it can be observed that the presence of emulsierplus cinnamon essential oil also decreased signicantly the TS from(3.75 0.70 to 2.32 0.40) MPa and increased the E from(128.8118.67 to 256.13 48.57) %. Analyzing these values, we canconclude that the introduction of essential oil reduced the inter-molecular interaction between polymeric chains, resulting in ma-terials with lower tensile strength.

Concerning barrier properties, a rise of contents of glycerol,emulsier and cinnamon essential oil caused an increase in bothpermeabilities. Water vapor permeability (WVP) and oxygen perme-ability coefcient (P0O2) of lms with cinnamon essential oil incor-poratedvaried from(9.781.40 to 14.79 2.76)gmmm2 d1 kPa1and from (27.50 0.60 to 143.47 8.30) 109 cm3 m1 d1 Pa1 ,respectively.

In previous work, Souza et al. (2012) tested lms based oncassava starch reinforced with 1.0 g/100 g of clay, at the sameconditions of this work, and found that the increase of glycerolcontent from (0.75e1.25) g/100 g also decreased barrier properties:WVP from (3.81 0.58 to 5.38 0.80) g mm m2 d1 kPa1 andP0O2 from (22.51 0.79 to 94.00 3.90) 109 cm3 m1 d1 Pa1).Considering these results, the increase of the glycerol content incassava starch lms elaborated in this work can also contributedwith the decrease of the studied barrier properties.

Glycerol is a relatively small hydrophilic molecule, which can beentrapped between adjacent polymeric chains, decreasing inter-molecular attractions and increasing molecular mobility, facili-tating migration of water vapor molecules (Rodrguez, Oss, Ziani,& Mat, 2006). However, in this work, when comparing lmselaborated according formulation A with the control ones, bothwith the same glycerol content, it can be veried that the WVPincreased signicantly from (3.81 0.58 to 9.78 1.40)g mm m2 d1 kPa1.

It is known that the addition of a lipidic component in theformulation could act as a barrier in the lms. Therefore, it is ex-pected that cinnamon essential oil was not the responsible agentfor the elevation of permeabilities values.

Based on previous observations, the emulsier, as a hydrophilicagent, probably it is the most component responsible for the WVPincreasing. However, it should be pointed that its presence in thelm elaboration process is necessary, because it promotes theincorporation of the antimicrobial agent into the aqueous solution,resulting in a homogeneous polymer matrix.

permeability coefcient (P0O2) of active cassava starch lms producedwith different

WVP[g mm m2 d1 kPa1]

P0O2 109 [cm3 m1 d1 Pa1]

3.61 0.69a 21.50 0.89a9.78 1.40b 27.50 0.60a

10.11 1.63b 67.44 0.81b14.79 2.76c 143.47 8.30c3.52 19.95).

4. Conclusions

Cinnamon and clove essential oils proved to be effective againsttwo microorganisms commonly found in bread. Minimum inhibi-tory concentration of them, which provided 100% of inhibitionagainst P. commune and E. amstelodami, were established, respec-tively, with 2.0 g/100 g of cinnamon essential oil and 16.0 g/100 g ofclove essential oil.

for lms incorporated with different contents of antimicrobial

cinnamon essential oil contents have a statistically signicant effect

Chen, C.-H., & Lai, L.-S. (2008). Mechanical and water vapor properties of tapiocastarch/decolorized hsian-tsao leaf gum lms in the presence of plasticizer. FoodHydrocolloids, 22, 1584e1595.

Chillo, S., Flores, S., Mastromatteo, M., Conte, A., Gerschenson, L., & Del Nobile, M. A.(2008). Inuence of glycerol and chitosan on tapioca starch-based edible lmproperties. Journal of Food Engineering, 88, 159e168.

Fam, L., Flores, S. K., Gerschenson, L., & Goyanes, S. (2006). Physicalcharacterization of cassava starch biolms with special reference to dy-namic mechanical properties at low temperatures. Carbohydrate Polymers,66, 8e15.

Fam, L., Goyanes, S., & Gerschenson, L. (2007). Inuence of storage time at room

A.C. Souza et al. / LWT - Food Science and Technology 54 (2013) 346e352352on TS, E, WVP and P0O2. Although the results established that cas-sava starch lms can be considered as a potential active alternativepackaging material, further research is necessary to improve theirmechanical and barrier properties since adequate mechanicalproperties are generally required for a packaging lm to withstandexternal stress and maintain its integrity as well as barrier prop-erties during applications as food packaging.

Acknowledgements

This research was supported by FAPESP (The State of So PauloResearch Foundation) and CAPES (Brazilian Committee for Post-graduate Courses in Higher Education).

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Cassava starch composite films incorporated with cinnamon essential oil: Antimicrobial activity, microstructure, mechanical ...1 Introduction2 Materials and methods2.1 Materials2.2 Film preparation2.3 Antimicrobial activity disk diffusion method2.4 Antimicrobial agent release2.5 Film thickness2.6 Scanning Electron Microscopy (SEM)2.7 Mechanical properties2.8 Water vapor permeability2.9 Oxygen permeability coefficient2.10 Statistical analysis

3 Results and discussion3.1 Determination of minimum inhibitory concentration (MIC) of cinnamon and clove essential oils3.2 Antimicrobial activity of active cassava starch films incorporated with cinnamon essential oil3.3 Release of cinnamon essential oil3.4 Appearance and microstructure of cassava starch films3.5 Mechanical and barrier properties of cassava starch films

4 ConclusionsAcknowledgementsReferences