Physical, Structural, Barrier, and Antifungal Characterization of Chitosan–Zein Edible Films with

14 Pages·2017·1.13 MB·English

International Journal o f
Molecular Sciences
Article
Physical, Structural, Barrier, and Antifungal
Characterization of Chitosan–Zein Edible Films
with Added Essential Oils
MonserratEscamilla-García1,GeorginaCalderón-Domínguez2,JorgeJ.Chanona-Pérez2,
AngélicaG.Mendoza-Madrigal3,ProsperoDiPierro4,BlancaE.García-Almendárez1,
AldoAmaro-Reyes1andCarlosRegalado-González1,* ID
1 DepartmentofFoodResearchandPostgraduateStudies,C.U.,AutonomousUniversityofQuerétaro,
CerrodelasCampanasS/N,LasCampanas,SantiagodeQuerétaro76010,Mexico;
[email protected](M.E.-G.);[email protected](B.E.G.-A.);[email protected](A.A.-R.)
2 DepartmentofBiochemicalEngineering,NationalPolytechnicInstitute,Av.WilfridoMassieuEsq.Cda.
MiguelStampaS/N,GustavoA.Madero,CiudaddeMéxico07738,Mexico;
[email protected](G.C.-D.);[email protected](J.J.C.-P.)
3 FacultyofNutrition,ResearchLaboratory,AutonomousUniversityoftheStateofMorelos,
VistaHermosaS/N,Cuernavaca62350,Mexico;[email protected]
4 DepartmentofChemicalSciences,UniversityofNaples“FedericoII”,ComplessoUniversitariodiMonte
Sant’Angelo,ViaCinthia,21,80126Napoli,Italy;[email protected]
* Correspondence:[email protected];Tel.:[phone]
Received:13October2017;Accepted:2November2017;Published:8November2017
Abstract: Edible films (EFs) have gained great interest due to their ability to keep foods safe,
maintaining their physical and organoleptic properties for a longer time. The aim of this work
was to develop EFs based on a chitosan–zein mixture with three different essential oils (EOs)
added: anise,orange,andcinnamon,andtocharacterizethemtoestablishtherelationshipbetween
their structural and physical properties. The addition of an EO into an EF significantly affected
(p < 0.05) the a* (redness/greenness) and b* (yellowness/blueness) values of the film surface.
TheEFspresentedarefractiveindexbetween1.35and1.55,andthusareclassifiedastransparent.
ThephysicalpropertiesofEFswithanaddedEOwereimproved,andfilmsthatincorporatedthe
anise EO showed significantly lower water vapor permeability (1.2 ± 0.1 g mm h−1 m−2 kPa−1)
andhighhardness(104.3 ± 3.22MPa). EFswithanaddedEOwereabletoinhibitthegrowthof
Penicillium sp. and Rhizopus sp. to a larger extent than without an EO. Films’ structural changes
weretheresultofchemicalinteractionsamongaminoacidsidechainsfromzein,glucosaminefrom
chitosan,andcinnamaldehyde,anethole,orlimonenefromtheEOsasdetectedbyaRamananalysis.
TheincorporationofanEOintheEFs’formulationcouldrepresentanalternativeuseascoatingsto
enhancetheshelflifeoffoodproducts.
Keywords: essentialoils;ediblefilms;ramanspectroscopy;chitosan;zein;Rhizopus;Penicllium
1. Introduction
Thegrowingdemandforhigh-qualityandsafefoodsisleadingtoincreasedsyntheticpackaging
materialsproduction. However,thereareenvironmentalconcernsabouttheirusedespitethebenefits
theyprovide,suchaspreservingandensuringfoodquality[1].Ediblefilms(EFs)maybeanalternative
toreducetheuseofsyntheticpolymers,whiletheadditionofessentialoils(EO)mayimprovephysical
andantimicrobialproperties[2]. EOshavebeenextensivelystudiedasnaturalproductstoprovide
benefits in food and human health. EOs or their components can be added directly to foods or
Int.J.Mol.Sci.2017,18,2370;doi:10.3390/ijms18112370 www.mdpi.com/journal/ijms
Int.J.Mol.Sci.2017,18,2370 2of14
incorporatedintocontainersmadeofnon-renewablematerials,orbiomaterials,suchaspolypropylene
orchitosan,respectively,tobereleasedduringtransportandstorage[3].
CinnamaldehydeisthemajoractivecomponentofthecinnamonEO(CN),andhasshownabroad
antimicrobial spectrum against bacteria, yeasts, and molds. Thus, CN could be used as a natural
foodpreservative,andmaybeaddedintoediblecoatingstoimprovetheshelflifeandsafetyoffood
products[4]. TherehavebeenreportsonthebactericidaleffectofCNagainstSalmonella,Campylobacter,
EscherichiacoliO157:H7,andListeriamonocytogenes,anditsantifungalactivityagainstAspergilliussp.,
Penicilliumsp.,andColletotrichumsp.[5,6].
ThereisincreasedinterestintheuseoftheaniseEO(AS)duetoitsantioxidantcapacityandantifungal
effectagainstPenicilliumsp. andAspergillussp.[7]. AnetholeisthemajoractivecomponentofAS[8],
whichhasbeenincorporatedintoediblecoatingsfordriedfishandfruitspreservation[9].Limoneneisthe
maincomponentoftheorangeEO(OR),andhasshowninsecticidal,antibacterial,andantifungalactivities.
IthasshownactivityagainstStreptococcussp.,Aspergillussp.,andPenicilliumsp.[10,11].Limonenehas
manyapplicationsinboththefoodandcosmeticindustries,butduetoitslipophiliccharacter,itexhibits
poorwaterabsorption,palatability,andoxidativedeterioration[12].
TheFoodandDrugAdministrationoftheU.S.listsalloftheessentialoilsusedinthisworkas
GenerallyRecognizedasSafe(GRAS).Watervaporpermeabilityandthemigrationofatmospheric
gasesaredependentonmanyinterrelatedfactors,includingthepolarityandstructuralfeaturesof
polymers’sidechains, hydrogenbondingcharacteristics, thedegreeofbranchingorcross-linking,
andthedegreeofcrystallinity[13]. Thespatialarrangementofatomsgivesarecognizableshapethatis
uniquetoeachmolecule,andX-raydiffractionhelpstoelucidatethepreferredmolecularstructureand
thepackingarrangement. Ultraviolet,infrared,andRamanspectroscopiesarehelpfulfordiagnosing
the presence of functional groups’ interactions and the presence of hydrogen bonds that provide
auniquespectralfingerprintthatenablesustoidentifyanddetectvariouscompounds[13,14].
Theobjectiveofthisworkwastoevaluate: (1)theeffectoftheadditionoftheEOscinnamon
(CN),anise(AS),andorange(OR)inEFspreparedfromachitosan–zeinblend(50%:50%);and(2)the
relationshipofthefilms’structuralmatrixwiththeirphysicalandmechanicalproperties. Theeffectof
CN,AS,andORincorporatedintoEFswasqualitativelytestedonfungalgrowthinhibition.
2. ResultsandDiscussion
2.1. Color,Transparency,andOpticalProperties
Theeffectoftheaddedessentialoilsonthetransparency,chromaticity,andrefractiveindexof
theediblefilmspreparedfromachitosan–zein50:50%blendarepresentedinTable1. Addingthe
EOs into the chitosan films significantly affected (p < 0.05) the a* (redness/greenness) and b*
(yellowness/blueness)valuesofthefilms’surface. WhenEFswereincorporatedwithORandCN,
thea*valuesshowed27%and6%morenegativevalues,indicatingarednesstendency,whereasthe
EFwithANaddedshowedthehighesta*valueamongthetreatments(greennesstendency). AllEFs
incorporatingEOsincreasedtheirb*values,indicatingayellownesstendency. Polysaccharidefilms
areusuallycolorless,essentialoilsshowaslightlyyellowappearance,andtheincorporationofEOs
intopolysaccharide-basedEFshavebeenreportedtoaffectcolorandtransparency[4,6].
AS showed the highest transparency value and the lowest refractive index of all of the EOs
incorporatedintoanEF.Nevertheless,allediblefilmspresentedarefractiveindexbetween1.35and
1.55,andbecausetheyfallintherange1.35–1.70theyareclassifiedastransparent[15]. Therefractive
index of EFs incorporating CN or OR showed similar values compared to the EFs without an EO.
However,therefractiveindexoftheEFincorporatingASwassignificantlydifferent(p<0.05)from
thevaluesoftheEFwithoutanEO,andtheEFwithCN.Nevertheless, evensmallchangesinthe
refractiveindexindicatethatEOadditionledtostructuralchanges[16]. Thevisualpropertiesofafilm
areanimportantfactorforconsumeracceptability.
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Table1.Colorandtransparencyofediblefilmszein–chitosan50:50%withessentialoils.
EdibleFilm %T a* b* η
Withoutoil 88.4±0.7a −1.34±0.02a 12.4±2.2a 1.45±0.02a
Anise 72.6±1.4b −1.10±0.01b 22.6±1.3b 1.35±0.02b
Orange 71.6±1.1b,c −1.70±0.02c 27.8±1.2c 1.55±0.01a,c
Cinnamon 69.3±1.1c −1.42±0.01d 28.8±1.3c 1.50±0.02c
Dataarethemean±standarddeviation. Superscriptlettersa–dareusednexttoreportedvalues,indicating
thatifthesameletterappearsinthesamecolumn,thevaluescomparedarenotsignificantlydifferent(p>0.05).
%T:%transparency;a*andb*:chromaticity;η:refractiveindex.
2.2. PhysicalProperties
EF thickness (e) and filmogenic suspension density (ρ) changed with EO addition, achieving
higherthicknessvaluesascomparedtothecontrolsample(Table2). Thiseffectcanbepartlyrelatedto
thefilmogenicsuspensiondensity,becauseahigherdensitywasdeterminedwithinthesesamples.
Inthisregard,Bonillaetal.[17]workingwithbasilreportedthatthehigherdensityofafilmogenic
suspensioncontaininganEOwasduetolargermolecularcontactbetweenthechitosan’sCHgroups
andtheoilcompounds,weakeningthepolymerchainaggregationforces,andproducingamoreopen
matrixleadingtoahigherfilmthickness.
Table2.Physicalpropertiesofediblefilm(EF)withdifferenttypesofessentialoils.
EdibleFilm e(µm) ρ(gcm−3) WVP(gmmh−1m−2kPa−1) Rq(nm) Ra(nm)
Withoutoil 20.02±1.45a 1.33±0.02a 2.92±0.16a 13.94±0.09a 12.63±0.06a
Anise 23.92±0.92b 1.72±0.01b 1.21±0.10b 11.63±0.14b 9.12±0.12b
Orange 21.43±0.51c 1.42±0.02a 1.62±0.02c 7.24±0.11c 5.64±0.16c
Cinnamon 22.54±0.32d 1.54±0.02c 1.53±0.20b,c 3.62±0.12d 2.84±0.09d
Dataarethemean±standarddeviation. Superscriptlettersa–dareusednexttoreportedvalues,indicating
thatifthesameletterappearsinthesamecolumn,thevaluescomparedarenotsignificantlydifferent(p>0.05).
e:EFthickness;ρ:densityofsolution;WVP:EFwatervaporpermeability;RaandRq:EFroughness.
The film prepared without an EO tended to show higher surface roughness than the others;
thiscouldbeduetotheformationofZagglomerates,resultingfromhydrogenordisulfidebonding
andhydrophobicinteractionspromotedbythepHatwhichthefilms’suspensionswereprepared.
ThiseffectwasnotedbyEscamillaetal.[18]whenworkingwithEFswithoutEOaddition,andwas
confirmedbyGuoetal.[19].
The additionof EOscould reduce suchinteractions, decreasing agglomerates’ formation and
promotingasmoothersurface. Theroughness(Ra,Rq)oftheEFdecreasedwiththeadditionofanEO,
inagreementwithAtarésetal.[20]. Theseauthorsworkedwithfilmsmadefromsoyproteinisolate
withaddedgingerandcinnamonEOsandreportedthatheatingenablestheintegrationoftheEOs
intotheproteinmatrix,resultinginsmoothersurfaces.
TheadditionofEOsimprovedthewatervaporbarrierpropertyofthechitosan-zein(CT-Z)EF,
achievingpermeabilityof1.2, 1.5, and1.6gmmh−1 m−2 kPa−1 forAS,OR,andCN,respectively
(Table 2). Aguirre et al. [21], working with whey protein isolate and the oregano EO, suggested
aprotein–EOinteractionthatimmobilizedtheproteinchain,producingamoreorderedandtightly
crosslinkedstructure,andconsequentlyalowerpermeability. Ontheotherhand,theinteractionofEO
componentssuchasethers,ketones,andaldehydeswiththeOHgroupsofpolymersincreasedthe
EFs’hydrophobicity,therebyimprovingthewatervaporbarrierproperty[22].
Another study produced sodium caseinate films incorporating the cinnamon or ginger EOs,
andthecinnamonEOwashomogenouslydistributedintheproteinmatrix,whereasthegingeroil
dropletsshowedagglomeration[20]. Theseauthorsconcludedthatstructuraldifferenceslinkedto
theoiltypeweretheresultofcomplexinteractionstakingplaceamonglipids,proteins,andsolvents.
Anotherstudyreportedthatwatervaporpermeability(WVP)dependsondifferentstructuralfactors,
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suchasthekindofmatrix,thecompositionandamountofoiladded,interactionswiththepolymers,
andthehydrophilic–hydrophobicbalanceinthematrix[23].
Nevertheless,onereportconcludedthatadecreaseintheWVPofchitosan(CT)-basedfilmswith
alemongrass,thyme,orcinnamonEOaddedwasduetoanincreaseinthetortuosityfactorofthe
watertransferwithinthefilmmatrix[24]. Bonillaetal.[17]reportedadecreaseintheWVPofCT
filmswithanincreasingconcentrationofthebasilEO,suggestingthatthewatermolecules’diffusivity
decreasedbecauseofthehydrophobicnatureoftheEOsthatpredominatedoverthecohesionforcesof
theCTmatrix.
MechanicalProperties
TheadditionofEOsdecreasedtheelasticmodulusandincreasedthehardnessoftheEFwithoutan
EO.AllEFsincorporatinganEOdidnotshowasignificantdifferenceintheelasticmodulus,whereas
theASEFexhibitedthehighesthardnessvalue(Table3).Theadditionoflemongrass,rosemarypepper,
andbasilEOsdecreasedtheelasticmodulusofacellulose-basedEF,suggestingthattheinteractionsof
EFpolymerswithanEOweresimilartothoseexertedbyplasticizers[25]. AnEFmadefromfishskin
gelatinwithaddedCN,basil,plai,andlemonEOsshowedadecreasedelasticmodulus,butalarger
elongationatbreakduetothereplacementofprotein–proteininteractionsbyEOadditioninthefilm
network[26]. ThisreportagreeswiththatofZinoviadou[26],whoworkedwithwheyproteinwiththe
oreganoEOadded. Inthisregard,thethree-dimensionalstructureofproteinsstabilizedbyhydrogen
anddisulfidebondsshouldbedisruptedtoobtainseparate,entangledmacromoleculestoachieve
plastic-likeproperties. TheEOandotherplasticizersareabletoreducetheinter-andintra-molecular
interactionsandincreasefilms’flexibilitydependingontheoilandproteincompatibility[20].
Table3.Mechanicalpropertiesofzein–chitosanfilmswithessentialoilsadded.
EdibleFilm Hardness(MPa) ElasticModulus(MPa)
Withoutoil 5.91±0.41a 66±6a
Anise 104.33±3.22b 22±3b
Orange 57.51±2.43c 20±2b
Cinnamon 25.54±1.85d 24±3b
Dataarethemean±standarddeviation.Superscriptlettersa–dareusednexttoreportedvalues,indicatingthatif
thesameletterappearsinthesamecolumn,thevaluescomparedarenotsignificantlydifferent(p>0.05).
2.3. X-rayDiffraction
ToconfirmstructuralchangesinanEFmatrix,X-raydiffractionandRamanspectroscopyanalyses
wereconducted. EFspreparedwithoutEOadditionshowedtwowell-definedpeaks,oneat7.5◦ and
theotherat20◦ (2θ);however,regardingEO-containingfilms,twopeaks,oneat2.7◦ andanotherat
about10◦ (2θ),canbenoted.
BasedonEquation(14),ASadditionpromotedhighercrystallinitythantheotherEOsaddedto
theEFs,whichwasprobablyassociatedwithhighercomponentsmiscibilityinthefilms’matrix[27].
Ontheotherhand,ORaddedtoanEFbarelydecreasedthecrystallinity(%C:21.0±0.4vs. 22.4±0.5)
(Figure1).AccordingtoSánchez-Gonzalezetal.[28],anEOaddedtoanEFbasedonCTonlyincreased
thecrystallinityupto50%,whereasglycerolincreasesresultedinalowerEFcrystallinityduetothe
highermobilityofthepolymerchains[27].
Thus,itissuggestedthatthepresenceofORleadstoadecreasedcrystallinityoftheprepared
films. ThisphenomenoncorrespondstonewlyformedinteractionsbetweenCTandORthatslightly
destroytheoriginalcrystallinestructure,whereasASandCNincorporationincreasedthecrystallinity,
suggesting that AS and CN reinforced CT films, leading to more dense crystalline domains in
comparisontopureCTandCT–OR;similarresultshavebeenreportedbyJahedetal.[29].
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Figure1. X-raydifractrogramsofEFbasedonamixtureofzein–chitosan(a)Withoutessentialoil
Figure 1. X-ray difractrograms of EF based on a mixture of zein–chitosan (a) Without essential oil (%
(%C=22.4±0.5);(b)Withthecinnamon(CN)essentialoil(%C=27±0.3);(c)Withtheanise(AS)
C = 22.4 ± 0.5); (b) With the cinnamon (CN) essential oil (% C = 27 ± 0.3); (c) With the anise (AS)
essentialoil(%C=32±0.2);(d)Withtheorange(OR)essentialoil(%C=21.0±0.4).
essential oil (% C = 32 ± 0.2); (d) With the orange (OR) essential oil (% C = 21.0 ± 0.4).
2.4. RamanSpectroscopy
2.4. Raman Spectroscopy
Raman spectroscopy showed that, for all EFs with an EO added, the signal at 1745 cm−1
Raman spectroscopy showed that, for all EFs with an EO added, the signal at 1745 cm−1
disappeared (Figure 2a), which, according to Gizem-Gezer et al. [30], is a characteristic Z signal
disappeared (Figure 2a), which, according to Gizem-Gezer et al. [30], is a characteristic Z signal that
that corresponds to the functional group O=S=O. This was attributed to interactions between the
corresponds to the functional group O=S=O. This was attributed to interactions between the protein
proteinandEOcomponents.
and EO components.
Thesignalat1666cm−1wasassociatedwiththepartialacetylationoftheNH groupofCT[31];
The signal at 1666 cm−1 was associated with the partial acetylation of the NH22 group of CT [31];
italsodisappearedinthepresenceofanEO.
it also disappeared in the presence of an EO.
InrelationtoCNediblefilms,cinnamaldehyde,whichiscomprisedofamono-substitutedbenzene
In relation to CN edible films, cinnamaldehyde, which is comprised of a mono-substituted
ring, an aldehyde functional group, and a conjugated double bond, reacted with CT, promoting
benzene ring, an aldehyde functional group, and a conjugated double bond, reacted with CT,
anucleophilicadditionreactiontypicalofaldehydes(Figure2b). WhenanalyzingtheEF’sstructural
promoting a nucleophilic addition reaction typical of aldehydes (Figure 2b). When analyzing the EF’s
components,thecarbonylgroup(C=O)providesareactivesitefornucleophilicaddition,withaSchiff
structural components, the carbonyl group (C=O) provides a reactive site for nucleophilic addition,
base(N=C)formation.
with a Schiff base (N=C) formation.
The presence of carbonyl and amino primary groups with a free electron pair allows for the
The presence of carbonyl and amino primary groups with a free electron pair allows for the
formationofaSchiffbaseduetotheelectron-deficientcarbonylcarbon. Thiswasconfirmedbythe
formation of a Schiff base due to the electron-deficient carbonyl carbon. This was confirmed by the
disappearanceofthecharacteristiccarbonyl(C=O)signalat1745.9cm−1whentheCNEOwasadded,
disappearance of the characteristic carbonyl (C=O) signal at 1745.9 cm−1 when the CN EO was added,
whichinvolvedasignaldecreaseat998–953cm−1 andat1081–1083.7cm−1, bothcharacteristicof
which involved a signal decrease at 998–953 cm−1 and at 1081–1083.7 cm−1, both characteristic of
C–O–C bonds. This finding agrees with those reported by Gao et al. [32], who studied reactions
C–O–C bonds. This finding agrees with those reported by Gao et al. [32], who studied reactions
betweenaldehydesandCT,nucleophilicadditionbeingthemosttypicalreaction.
between aldehydes and CT, nucleophilic addition being the most typical reaction.
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FFiigguurree 22.. SSppeeccttrrooggrraammss ooff EEFFss bbaasseedd oonn aa mmiixxttuurree ooff zzeeiinn––cchhiittoossaann ((11::11 ww//ww)).. ((aa)) WWiitthhoouutt eesssseennttiiaall ooiill;;
((bb)) WWiitthh cciinnnnaammoonn eesssseennttiiaall ooiill.. AArrrroowwss iinnddiiccaattee tthhee ddiissaappppeeaarraannccee ooff zzeeiinn ssiiggnnaallss..
The spectrogram of the edible film containing the OR EO (Figure 3a) did not show characteristic
ThespectrogramoftheediblefilmcontainingtheOREO(Figure3a)didnotshowcharacteristic
ssiiggnnaallss aatt 11008811,, 11444400,, aanndd 11774455..99 ccmm−−11 aassssoocciiaatteedd wwiitthh tthhee CC––OO––CC,, NN==NN,, aanndd OO==SS==OO bboonnddss,,
respectively. Limonene (an olefin) is the main active component of the OR EO, which in the presence
respectively. Limonene(anolefin)isthemainactivecomponentoftheOREO,whichinthepresence
of CT experiences olefin metathesis allowing for the synthesis of small and polymeric molecules by
ofCTexperiencesolefinmetathesisallowingforthesynthesisofsmallandpolymericmoleculesby
sscciissssiioonn aanndd tthhee rreeggeenneerraattiioonn ooff CC==CC mmoolleeccuulleess [[3333]],, wwhhiicchh wweerreed deetteecctteedda att1 1559977c cmm−−11,, wwhheerreeaass tthhee
signals listed above disappeared.
signalslistedabovedisappeared.
The EF with the AS EO added showed signal disappearance at 729.74 (C–S), 1110–1150 (C–O–
TheEFwiththeASEOaddedshowedsignaldisappearanceat729.74(C–S),1110–1150(C–O–C),
C), 1440 (N=N), and 1745 (C=O). These signal losses indicate the interaction of EF components
1440(N=N),and1745(C=O).ThesesignallossesindicatetheinteractionofEFcomponents(CT-Z)
(CT-Z) with the AS EO, causing bond disruption. The characteristic tyrosine signal disappeared upon
withtheASEO,causingbonddisruption. Thecharacteristictyrosinesignaldisappeareduponthe
athded iatidodnitoiofnth eofA tShEe OA(S8 2E1Oc m(8−211) ,cams−w1)e, lalsa swtheell saigs ntahlea tsi1g7n4a5l. 9actm 1−7415t.h9a tcmis−r1e ltahtaetd itso rtehleatZedfu tnoc ttihoen aZl
functional group O=S=O reacting with CT [30] (Figure 3b). This implies the disruption of the protein’s
groupO=S=OreactingwithCT[30](Figure3b). Thisimpliesthedisruptionoftheprotein’ssecondary
secondary structure (α-helix) [34] so that protein structural changes due to EO addition allow for a
structure (α-helix) [34] so that protein structural changes due to EO addition allow for a tyrosine
rtyearoctsiionne wreiathctiootnh ewrifithlm octhoemr pfoilnme nctosm. Tphoenernedtsu. cTehdes irgendaulcaetd1 s6i1g9n.a4l– 1a6t 5156.419c.m4–−1165in5.d4i ccamte−s1 cinhdemicaicteasl
chemical interactions due to C=N bonds losses. The addition of each EO resulted in a signal
interactionsduetoC=Nbondslosses. TheadditionofeachEOresultedinasignaldisappearanceat
7d2is9a.7p4pcemar−an1,cceh aatr a7c2t9e.r7is4t iccmo−f1,t hcehCar–aSctaelriipshtiact iocfg trhoeu pC–oSf caylsiptehiantei,c sgurgoguepst ionfg ctyhseterienaec,t isoungogfesetainchg otnhee
reaction of each one of the three EOs with the CT-Z EF.
ofthethreeEOswiththeCT-ZEF.
From the Raman spectra, the addition of any EO confirms the X-ray analysis, since an EF’s
From the Raman spectra, the addition of any EO confirms the X-ray analysis, since an EF’s
structure was modified through the formation of a Schiff base, metathesis of CT, or disappearance of
structurewasmodifiedthroughtheformationofaSchiffbase,metathesisofCT,ordisappearanceof
CS bonds.
CSbonds.
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Figure3. SpectrogramsofEFbasedonamixtureofzein–chitosan(50%:50%w/w). (a)Withorange
Figure 3. Spectrograms of EF based on a mixture of zein–chitosan (50%:50% w/w). (a) With orange
essentialoil;(b)Withaniseessentialoil.Arrowsindicatethedisappearanceofzeinsignals.
essential oil; (b) With anise essential oil. Arrows indicate the disappearance of zein signals.
22..55.. AAnnttiiffuunnggaall AAccttiivviittyy
AAllll EEFFss wwiitthh aann EEOO aaddddeedd sshhoowweedd iinnhhiibbiittoorryy eeffffeeccttss aaggaaiinnsstt RRhhiizzooppuuss sspp.. aanndd PPeenniicciilllliiuumm sspp..,,
wwhheerreeaass AASS aanndd CCNN sshhoowweedd aann iinnhhiibbiittoorryy eeffffeecctt oonn PPeenniicciilllliiuumm sspp..,, tthhoouugghh aa lloowweerr aannttiiffuunnggaall eeffffeecctt
wwaass oobbsseerrvveedd ffoorr RRhhiizzooppuuss sspp.. ((TTaabbllee 44)).. TThhee iinnhhiibbiittoorryy eeffffeecctt ooff tthhee AASS EEOO wwaass aattttrriibbuutteedd ttoo aanneetthhoollee,,
iittss mmaaiinn bbiiooaaccttiivvee ccoommppoouunndd [[88]],, wwhhiicchh iiss eeffffeeccttiivvee aaggaaiinnsstt mmyyccoottooxxiiggeenniicc ffuunnggii,, ssuucchh aass RRhhiizzooppuuss sspp..
aanndd PPeenniicciilllliiuumm sspp.. [[77]]. .AAmmonogn gthteh eEOEsO, sO,RO sRhoshwoewde tdhet hsemsamlleasltl eisnthiinbhitiobriyto erfyfeecftf oecnt eoanche aocfh thoef ttwheo
ttwesotedte sfutendgif.u Tnhgei. amThoeunatm oof uenactho fEOea cuhseEdO tou csoenddtuoctc tohned auncttiftuhnegaanl ttiefsutns gwalast e1s5ts.6 wpapsm1, 5w.6hipcphm is,
wbehloicwh itshbe edloawilyt hinegdeastiiloyni nlgimesitt ioonf 2li5m0 iptpomf2 5[305p].p Tmhu[3s5, ]h.iTghheurs ,dhoisgehse mrdaoys bees mmaoyreb eefmfecotrievee fafegcatiinvset
athgeasien sftutnhgeis weiftuhnoguit wexitcheoeudtinegx ctheeed pinergmthitetepde lremveitlt. eCdolnecveenl.trCatoionnces notfr a1t0i0o npspomf 1o0f 0anpeptmhoolef iannheitbhitoelde
iAnshpiebrigtieldlusA sfplaevrugisl luansdfl aAvuspsearngidlluAss ppearrgaislliutiscupsa,r awsihtiecruesa,sw ah ecroemaspalecteo minphliebtietiionnh iwbiatiso nacwhiaesvaecdh iuesviendg
ucosinncgencotrnactieonntrsa otifo 1n0s0o pfp1m00 apnpdm 20a0n dpp2m00, prepsmpe,crteisvpeelyc t[i3v6e]l.y [36].
TTaabbllee 44.. IInnhhiibbiittoorryy eeffffeecctt ooff EEFFss wwiitthh ddiiffffeerreenntt aaddddeedd eesssseennttiiaall ooiillss..
Average Diameter (cm)
AverageDiameter(cm)
Edible Film
EdibleFilm Penicillium sp. Rhizopus sp.
Penicilliumsp. Rhizopussp.
Without essential oil 0 0
AWnisiteh eosusteenstsiaeln otiial loil 2.2 ± 0.20 a 1.5 ±0 0.3 a
Aniseessentialoil 2.2±0.2a 1.5±0.3a
Cinnamon essential oil 1.9 ± 0.3 a 1.7 ± 0.3 a
Cinnamonessentialoil 1.9±0.3a 1.7±0.3a
Orange essential oil 0.7 ± 0.1 b 0.6 ± 0.2 b
Orangeessentialoil 0.7±0.1b 0.6±0.2b
Data are the mean ± standard deviation. Superscript letters a-b are used next to reported values,
Dataarethemean±standarddeviation.Superscriptlettersa-bareusednexttoreportedvalues,indicatingthatif
indicating that if the same letter appears in the same column, the values compared are not
thesameletterappearsinthesamecolumn,thevaluescomparedarenotsignificantlydifferent(p>0.05).
significantly different (p > 0.05).
Anetholetargetsafungi’smitochondrialdefensesystemagainstoxidativestress[37].Theantifungal
activiAtyneotfhtohlee EtaOrghetass ab efeunngoib’ss emrvietodchatonddifrfiearle ndtefsetangsees soyfstaemfu naggia’sinlsitf eocxyidclaet,ivine cslutrdeisnsg [3a7t]s. pTohree
gaenrtmifuinnagtaiol na,ctthiveitfyo romf athtieo nEOof hpaesn betereanti nogbssetrruvectdu raet sd,iafnfedremnyt csetaliguems oafn ad fsupnogrui’lsa ltiifoen cdyecvlee,l oinpcmluednitn[g3 8a]t.
Lspimoroen egneerimstihneataicotniv, etihneg refdoriemnattoifoOn Ro,fa npdeintectaruasteinsgc hasntrguecstuinreasm, iacnrodb iamlyceclellmiuemm barnande ’ssppororuplearttiioesn,
idnecvreealospinmgeintst f[l3u8i]d. iLtyi,maonndelneea disi nthget oacatlitveer eindgpreerdmieenatb oilfi tOyRa,n adndho imt ceaoussteass icshlaonssg.eIsn ina dad mitiiocrno,btihael cEeOll
membrane’s properties, increasing its fluidity, and leading to altered permeability and homeostasis
loss. In addition, the EO components of OR denature enzymes responsible for germination and
sporulation. A concentration of 500 ppm of OR produced a fungistatic effect on A. flavus [28].
Int.J.Mol.Sci.2017,18,2370 8of14
componentsofORdenatureenzymesresponsibleforgerminationandsporulation. Aconcentrationof
500ppmofORproducedafungistaticeffectonA.flavus[28].
CinnamaldehydeistheactivecompoundofCN,anditsmechanismofactioninvolvescellular
ultrastructuralchanges,includingorganellesdisappearanceandsolidificationandthedegenerationof
thecellwallandcytoplasm. Aninhibitoryconcentrationof0.5%(v/v)wasfoundagainstA.niger[38].
AlthoughEOs’mechanismofactionisnotwell-definedyet,theirinhibitoryeffecthasbeengenerally
associated with their hydrophobicity, which causes changes in the permeability, ion transport, and
solubilizationoflipidcomponentsofthecellmembrane.Morestudiesareneededtoevaluatethecombined
effectofthetestedEOstoascertainwhethertherearesynergisticeffectsagainstavarietyoffungi.
Microbialfoodspoilageisoneofthemainproblemsofthefoodindustry,andmorethan50%of
fruitsandfruitproducts,arespoiltbyfungi;inthebakingindustry,theselossesrepresentbetween1%
and3%. Inaddition,antifungalpackaginghasbeenproposedtoextendthesafetyandshelflifeof
ready-to-eatfoods[39].Thus,activeediblefilmsmayrepresentagoodalternativeforfoodpreservation
inthebakingandfruitindustry,andaresuitablefordirectfoodproductsconsumption.
3. MaterialsandMethods
3.1. Materials
Anise(Essencefleur,AceitesyEsenciasCat. No. 98019,CiudaddeMexico,Mexico),cinnamon
(Essencefleur, Cat. No. 91041) and orange (Essencefleur, Cat. No. 10002) EOs were used.
Chitosan(≥75%deacetylation,molecularweightof375kDa,Sigma-Aldrich,St. Louis,MO,USA),
Zein(Ingredion,SanJuandelRío,Qro.,Mexico),Penicilliumsp.,andRhizopussp. wereacquiredfrom
thetypeculturecollectionoftheEscuelaNacionaldeCienciasBiológicas(ENCB,IPN,Ciudadde
Mexico,Mexico).
3.2. FilmsPreparation
SuspensionsofCT(1%CTin1Naceticacid,w/v),andZ(1:10,Z:ethanol,w/v),werepreparedby
mixinginahotplate(Barnstead,Thermolyne,MN,USA)for15minat200rpmat90◦C;thesesolutions
weremixedina1:1ratio. ThreedifferentEFswereelaboratedincorporatinganEO(AS,CN,andOR)
to promote ingredients solubilization and films formation; the pH was adjusted to 9.4 with 0.5 M
NaOH[40]. AnAS,anOR,oraCNEOwasaddedtoreach250ppmandwasmixedfor10minat
roomtemperature(~24◦C).Glycerol(Sigma-Aldrich)wasaddedata3:1ratio(Z:glycerol,w/w)and
stirredfor10minforcompletehomogenization. Finally,10goftheCT-Z-EOmixtureswerepoured
intopetridishes(90×15mm)anddriedinaconvectionoven(Terlab,Puebla,Mexico)atconstant
relativehumidity(RH)(45%)andtemperature(40◦C)for24h,accordingtoAbugoch[41]. Allsamples
were separated from the dishes and stored at constant RH (59.7%) and temperature (20 ◦C) until
furtheranalysis.
3.3. PhysicalCharacterization
3.3.1. Color,Transparency,andOpticalProperties
The films’ thickness, absorption coefficient, and refractive index were determined according
to Murray and Dutcher [42] with modifications. Briefly, an ellipsometer (Uvisell LT M200 AGMS,
YvonHoriba,Longiumeau,France)ata250to800nmwavelengthwasused. Therefractiveindexand
thicknesswereevaluatedbythepolarizationchangesofincidentlightbeams(70◦C)onthesurface
of the film; these changes consider the ellipsometric angles of the equipment (∆ and Ψ), which in
turn are related by mathematical models (polymer and amino acids) included in the software of
the same company providing the values of the refractive index and thickness for the equipment.
Asecondmethodwasusedtoconfirmtheellipsometerresultsbyusingaprecisiondigitalmicrometer
(IP54,Newton,MA,USA),thatwasemployedtomeasurefivedifferentzonesofthefilmsatrandom
Int.J.Mol.Sci.2017,18,2370 9of14
locations. EFtransparencyandcolorweredeterminedbyusingacolorimeter(CR-400ChromaMeter,
KonicaMinolta,Tokyo,Japan)withaD65illuminationsourceata0◦viewingangle,whitebackground,
andbyassumingthatacompletelytransparentfilmwillgeneratethesameluminosityvalues(L*)as
thoseobtainedfromthewhitecalibrationplate(L*=100)andthatanydifferencewillbetheresult
ofamoreopaquematerial(L*<100);then,lightnessisconsideredasequaltotransparency. Inthis
experiment,thefilmwasplacedoverthecalibrationplateandmeasurementswereperformedatfive
differentpointsofthefilm(centerandouterparts)avoidingtheedges[18]. Priortotheformation
ofthefilms,samplesofeachofthesolutionsweretaken(CT-ZwithandwithoutanEO)andtheir
densitywasevaluatedasdescribedbyASTMD1217-12[43]. Inapycnometer(10mL)atconstant
weight,10mLofthefilmogenicsolutionwaspoured,theweightwasdetermined,andthedensitywas
obtainedbyEquation(1):
m
ρ = (1)
v
whereρisthesolutiondensity,visthevolume(10mL),andmisthemassof10mLofsolution.
3.3.2. AtomicForceMicroscopy(AFM)
AFMprovidesrelativeandabsoluteroughnessmeasurementsandallowsinvestigatorstoobtain
three-dimensional (3D) images showing the topographic characteristics of the sample. For this
evaluation,anatomicforcemicroscope(MultimodeV,Veeco,CA,USA)wasused,andthetapping
method was applied using silicon probes (RTESP Bruker cantilevers, Santa Barbara, CA, USA)
with a resonance frequency of 286–362 kHz and a spring constant of 20–80 N m−1. Samples of
0.5×0.5cmwereusedandthreeareasof5×5µmwerescannedataspeedof1Hzandresolutionof
256×256pixels. Roughnesswascalculated(Equations(2)and(3)),usingtheNanoScopeAnalysis
1.20program(Veeco)andapplyingaflatteningprocess(onegrade).
(cid:118)
(cid:117) (cid:16) (cid:17)
(cid:117)Σ Z2
(cid:116) i
Rq = (2)
N
1 ∑N
Ra = Z (3)
i
N
j=1
whereZ istheheightdeviationfromthemeanofheights,Z isthemaximumverticaldistancebetween
i j
thehighestandlowestdatapointsintheimagepriortotheplanefit,Rqisthestandarddeviationofthe
Z values,Raisthearithmeticaverageoftheabsolutevaluesofthesurfaceheightdeviationsmeasured
i
fromthemeanplane,andNisthenumberofpointsintheimage[44].
3.3.3. WaterVaporPermeability
WatervaporpermeabilitywasevaluatedusingthegravimetricmethodASTME96–80[45]with
modifications. Slightamountsofwatermayaccumulateonthesurfaceofhydrophilicediblefilms
usingthecupmethod,andtheresistanceofthestagnantairlayerbetweenthebottomsideofthefilm
andthesurfaceofthesaturatedsaltmaybesignificant,and,ifneglected,mayunderestimatethewater
vaportransmissionrate(WVTR)[18,46]. Permeabilitycupswithanopenmouthofknownarea(A)
wereused. Thefilmwassealedontopofapermeationcup,andplacedinadesiccatorwithasaturated
NaClsolutionatconstanttemperature(25◦C)andRH(75%). ThecupscontainedasaturatedKNO
3
solution(HR=95.6%),andwereweighedat1hintervalsuntilequilibriumwasreached. Theslopeof
theweightloss(W )versustime(t)plotwasobtainedbylinearregression,andthemeasuredWVTR
S
(WVTR )wascalculated(Equation(4)).
m
W
WVTR = S (4)
m
tA
Int.J.Mol.Sci.2017,18,2370 10of14
The corrected WVTR (WVTRc) was obtained following Gennadios et al. [47] using
Equations(5)–(9).
P −P
WVTR =WVTR × w1 w2 (5)
C m P −P
w3 w4
RH
P = P × 1 (6)
w1 0
100
RH
P = P × 2 (7)
w2 0
100
WVTR×R×T×hi
Pw3 = PT−(PT−Pw1) PT×D (8)
WVTR×R×T×h0
Pw4 = PT−(PT−Pw2) PT×D (9)
whereAisthecross-sectionalareaofthefilm,P isthepartialpressureinsidethepermeabilitycell,
w1
P isthepartialpressureinsidethedesiccator,P andP arethepressuresbelowandabovethe
w2 w3 w4
ediblefilm,respectively,Disthediffusivityofwatervaporthroughtheair(2.82m2/d),Risthegas
constant,Tisthetemperature(298K),andP istheatmosphericpressure(85kPainQuerétaro,Mexico).
T
The water vapor permeability (WVP) was calculated according to Gennadios et al. [47] and
Alvarado-Gonzálezetal.[46](Equation(10)).
L
WVP =WVTR (10)
CP −P
W1 W2
whereWVPisthewatervaporpermeability(gmmh−1m−2kPa−1),Listhefilmthickness(mm),andp
W1
andp arethewatervaporpartialpressures(kPa)overandunderthefilm’ssurface,respectively.
W2
3.3.4. MechanicalProperties
Themechanicalpropertiesevaluatedwereelasticmodulusandhardness. Thesepropertieswere
obtainedbyananoindenter(CSM,Peseux,Switzerland)measuringeightdifferentpartsofthefilms,
includingtheedgesandcentralparts. Theindentationconditionswere: amaximumloadof2.5mNat
loadingandunloadingratesof7.5mN/min,andapauseof35susingaBerkovichtipofknowncontact
area(Ac). Fromtheload-displacementcurve,themaximumload(P ),maximumpenetrationdepth
max
(h ),andstiffnessofthematerial(h )wereobtained. Usingtheseparameters,valuesofhardness
max S
(Equation(11))andelasticmoduluswereevaluated(Equations(12)and(13))[46].
P
H = max (11)
A×hc
√
S π
E = √ (12)
r
2 Ac
1−v2
E = (13)
m 1 − 1−v2
Er Ei
whereH(MPa)isthematerialhardness,E istheelasticmodulus(GPa),E isthereducedmodulus,
m r
S is the initial unloading stiffness in the load curve, hc is the difference between h and h , v is
max S
the Poisson’s ratio for polymeric samples estimated as 0.35, and E and v are the elastic modulus
i i
(1141GPa)oftheindenterandPoisson’sratio(0.07),respectively[47].