materials Article The Antifungal Activity of Functionalized Chitin Nanocrystals in Poly (Lactid Acid) Films AsierM.Salaberria1,ReneH.Diaz1,MaríaA.Andrés1,SusanaC.M.Fernandes2,*and JalelLabidi1,* 1 BiorefineryProcessesResearchGroup,DepartmentofChemicalandEnvironmentalEngineering, FacultyofEngineering,UniversityoftheBasqueCountry(UPV/EHU),Pza.Europa1, 20018Donostia-SanSebastian,Spain;[email protected](A.M.S.); [email protected](R.H.D.),[email protected](M.A.A.) 2 CNRS/UniversitédePauetdesPaysdel’Adour,InstitutdesSciencesAnalytiquesetdePhysico-Chimie pourl’EnvironnementetlesMateriaux,UMR5254,2Av.PdtAngot,64053Pau,France * Correspondence:[email protected](S.C.M.F.);[email protected](J.L.); Tel.:+33-540-175-015(S.C.M.F.);+34-943-017-178(J.L.) AcademicEditor:Jun-ichiAnzai Received:4April2017;Accepted:12May2017;Published:18May2017 Abstract: As, in the market, poly (lactic acid) (PLA) is the most used polymer as an alternative toconventionalplastics,andasfunctionalizedchitinnanocrystals(CHNC)canprovidestructural and bioactive properties, their combination sounds promising in the preparation of functional nanocompositefilmsforsustainablepackaging. Chitinnanocrystalsweresuccessfullymodifiedvia acylationusinganhydrideaceticanddodecanoylchlorideacidtoimprovetheircompatibilitywith thematrix,PLA.Thenanocompositefilmswerepreparedbyextrusion/compressionapproachusing differentconcentrationsofbothsetsoffunctionalizedCHNC.Thisinvestigationbringsforwardthat bothsetsofmodifiedCHNCactasfunctionalagents,i.e.,theyslightlyimprovedthehydrophobic characterofthePLAnanocompositefilms,and,veryimportantly,theyalsoenhancedtheirantifungal activity. Nonetheless,thenanocompositefilmspreparedwiththeCHNCmodifiedwithdodecanoyl chlorideacidpresentedthebestproperties. Keywords:poly(lacticacid);chitinnanocrystals;nanocomposites;barrierproperties;fungalinhibition 1. Introduction Among the available biodegradable polymers in the market, poly(lactic acid) (PLA) is the most used as a sustainable alternative to conventional plastics [1–4]. Lactic acid can be produced through microbial fermentation of agricultural by-products, namely corn, sugar beets, wheat and potato starch [1,4,5]; and PLA, which is an aliphatic thermoplastic polyester, can be obtained by polymerizationoflacticacidmonomers(hydroxylcarboxylicacids). Becauseofitsproperties,namely goodmechanicalstrengthandstiffness,UVstabilityandgloss,PLAisoftencomparabletopolystyrene andpolypropylene. Thus,increasingattentioninthedesignanddevelopmentPLA-basedmaterials forfoodpackaging,biomedicaldevicesandautomotiveindustryhasbeenobserved[6–8]. Similar to petrochemical derivatives, several strategies have been developed to improve the structural and/or functional properties of the polymer-based materials from renewable resources, includingtheincorporationoffillers.Naturalfillers,suchasnanofibersandnanocrystalsfromcellulose andchitin,andnanocrystalsfromstarcharegainingconsiderableinterestbecauseoftheirabundance, chemical structure, low toxicity and biodegradability [9–16]. Moreover, chitin nanofillers provide antifungalorantibacterialproperties[17–19]. Materials2017,10,546;doi:10.3390/ma10050546 www.mdpi.com/journal/materials Materials2017,10,546 2of16 Chitin is a crystalline high molecular weight linear polysaccharide consisting of β-(1 → 4)-2-acetamido-2-deoxy-D-glucopyranose repeating units, which is found in the crustacean shells, insectcuticlesandinthecellwallsoffungi,yeastandgreenalgae. Itoccursasahighly-organized micro-andnano-fibrilstructureincreasingfromthenanometertothemillimeterscale[20].Thesefibrils formhighlycrystallineregionsanddisordered(amorphous)regionsthatcanbeturnedinnanocrystals viatop-downmethodssuchasacidhydrolysesbydissolvingtheamorphousregions[18,21–24]. Nonetheless,theuseofnaturalnanofillers,includingnanochitin,oncompositematerialspresents some drawbacks, in particular, their high polar character is responsible for a very low interfacial compatibilitywithcommonthermoplasticmatrices,includingPLA.Consequently,differentapproaches havebeenemployedtoovercomethisissue[25,26]. Amongthem,thesurfaceacylationofthenatural nanofillersbytheintroductionofhydrophobicfunctionalgroupsunderheterogeneousconditions seemstobeanefficientwaytosolvethecompatibilityissue[9,27–32]. Thusfar,fewstudiesregardingtheuseofchitinnanofillersasreinforcingagentsinPLAmatrices have been described in literature [25,26,33–36]. Following our interest in the preparation of chitin nanofillers-basedmaterials[18,20–23,37,38],thepresentstudygoesastepfurtherthantheusechitin nanofillers as reinforcing agents. The aim of this work was to improve the compatibility of chitin nanocrystalswithPLAmatrixandevaluatetheirbehaviorasfunctionalagentsforpotentialuseas activefoodpackaging. 2. MaterialsandMethods 2.1. Materials Cervimunidajohnilobster(knownasyellowlobster)wastesintheformofpowderwerekindly suppliedbyAntarticSeafoodS.A.(Coquimbo,Chile). Poly(lacticacid)(PLA,L-poly-lactidecontent is ≥99%w/w)withMwof180,000gmol−1 intheformofpelletswaskindlyprovidedbyFuterro S.A.(Celles,Belgium). Dodecanoylchlorideacid(98.0%)waspurchasedfromAldrich. Hydrochloric acid(HCl,ACSreagent,37.0%),sulfuricacid(H SO ,ACSreagent,≥95%),aceticanhydride(Ac O, 2 4 2 ACSreagent,≥98.0%),dimethylformamide(DMF,anhydrous,99.8%),acetone(analyticalstandard, ≥99%),ethanol(analyticalstandard,70%)andpyridine(ACSreagent,99%)werepurchasedbyPanreac (Barcelona,Spain). Allreagentswereusedasreceivedwithexceptionofpyridinethatwaspurifiedby distillationoversodiumhydroxide. 2.2. ChitinExtraction α-Chitin(CH)wasextractedfromyellowlobsterwastesaccordingourpreviousworks[18,21–23]. Threemainstepswereappliedinthefollowingsequence: deproteinization—removalofproteinsina solutionof2MNaOHatroomtemperaturefor24h;demineralization—removalofminerals(CaCO ) 3 inasolutionof2MHClatroomtemperaturefor3h; anddecolorization—extractionofpigments andlipidsusingacetonefollowedbyethanolunderrefluxfor6h. Afterwards,α-chitinwasfiltered andwashedwithdeionizedwatertwice. Theresultingα-chitinwasdriedat60.0±0.5◦Cinanoven overnight. ThedegreeofN-acetylationwasfoundtobe96%by13C-NMR[39]. 2.3. α-ChitinNanocrystalsExtraction Chitin nanocrystals (CHNC) were isolated from the obtained α-chitin based on our previous work[18,21–23]adaptedfromthemethoddevelopedbyPailletetal.[24]. Tohydrolyzetheamorphous regions of chitin, α-chitin powder was dispersed in a solution of 3 M HCl at 100 ± 2 ◦C and left for 90 min under vigorous stirring and refluxing. After acid hydrolysis, the obtained suspension wasdispersedindeionizedwaterandwashedbycentrifugation. Thisprocedurewasrepeatedthree times. Thesuspensionwasthendialyzedchangingdeionizedwaterevery12huntilpH6. Thenthe suspensionwassubjectedtoultrasonictreatmentfor10min(Vibracell75043fromBioblockScientific) todisintegratechitinnanocrystalaggregatesandobtainahomogeneoussuspension. Finally,chitin Materials2017,10,546 3of16 nanocrystalswerefilteredtogetafinalsuspensionof4wt%andstoredat4◦Cbeforeused. CHNC presentedarod-likemorphologywithaveragewideof60nmandlengthof300nm. Theirdegreeof acetylationwasestimatedtobe92%by13C-NMRspectroscopy[39]andcrystallinityindextobe89% usingthemethodofFocheretal.[40]. Materials 2017, 10, 546 3 of 17 2.4. SurfaceFunctionalizationofα-ChitinNanocrystals 2.4. Surface Functionalization of α-Chitin Nanocrystals 2.4.1. AcylationwithAceticAnhydride 2.4.1. Acylation with Acetic Anhydride TheacylationofCHNCusingaceticanhydrideandsulfuricacidascatalystwasadaptedfromthe The acylation of CHNC using acetic anhydride and sulfuric acid as catalyst was adapted from methodusedbyFrisonietal.[41](Figure1a). the method used by Frisoni et al. [41] (Figure 1a). (a) (b) Figure 1. Scheme representing the heterogeneous chemical functionalization of chitin nanocrystals Figure1. Schemerepresentingtheheterogeneouschemicalfunctionalizationofchitinnanocrystals using: anhydride acetic (a); and dodecanoyl chloride acid (b). using:anhydrideacetic(a);anddodecanoylchlorideacid(b). Never dried CHNC (6 wt % suspension in water) were submitted to solvent exchange to acetone Nvieav eethradnroile ads CinHteNrmCed(6iawte tso%lvseunst,p weinthsi tohnreien dwispateerrsi)own/efirletrastuiobnm sietqteudentcoess ofolvr eeancthe sxoclhveanntg (e50t omaLc)e, tone viaetahnadn othleans iinn taecremtice daniahtyedsroidlvee (n50t, mwLit)h wtihthre tewdoi sdpisepresriosino/n/fifillttrraattiioonn sseeqquueenncceess fofor r1e5 amchins. olvent(50mL), Afterwards, CHNC were dispersed in 15 mL of acetic anhydride containing 3.6 wt % sulfuric andtheninaceticanhydride(50mL)withtwodispersion/filtrationsequencesfor15min. acid (0.2 mL) of the dry chitin weight. The reaction mixture was stirred at 30 °C for 7 h. At the end of Afterwards,CHNCweredispersedin15mLofaceticanhydridecontaining3.6wt%sulfuric acid(t0h.e2 rmeaLc)tioonf,t hthee darcyylcahteidti nchwitieni gnhatn.oTcrhyestraelas c(tCio2CnHmNiCxt)u wreerwe failstesrteirdr eadnda tse3q0u◦eCntifaollry7 wha.sAhetdt hweitehn dof acetone and ethanol. To remove residual trace of acetic anhydride and other impurities, the C2CHNC thereaction,theacylatedchitinnanocrystals(C CHNC)werefilteredandsequentiallywashedwith were Soxhlet extracted with ethanol overnight a2nd finally dried at 60 °C for 24 h. acetoneandethanol. Toremoveresidualtraceofaceticanhydrideandotherimpurities,theC CHNC 2 were2S.o4.x2h. lAectyelxattiroanc twedithw Ditohdeecthanaonyoll Cohvleorrnidieg hAtcaidn (dFafitntya lAlycidd)r iedat60◦Cfor24h. The acylation of CHNC using dodecanoyl chloride acid was carried out according the method 2.4.2. AcylationwithDodecanoylChlorideAcid(FattyAcid) described by Freire et al. [42] (Figure 1b). Dodecanoyl chloride acid (1 eq. relative to the total OH Tghroeuapcsy olfa cthioitnino) wfCasH pNlacCedu isni nag 25d0o mdLe craonunody-lbcohttloomri dfleasakc. iDdMwFa (s10c0a mrrLie),d poyruidtiancec aonrdd iCnHgNthCe (2m ge) thod descrwibeerde tbhyenF ardedireede atnadl .m[a4i2n]ta(iFniegdu urend1ebr) s.tiDrroindge acta 1n1o5y °lCc fholro 6r hid. Aefatecrid es(t1ereifqic.artieolna,t tihvee mtoixtthueret wotaasl OH groupfislteorfecdh aitnidn )wwaashsepdl awciethd DinMaF2, 5a0cemtoLner oanudn de-thbaonttool.m Tofl aresmk.oDveM aFny( 1r0es0idmuLal) ,trpaycer idofi nfaettayn daciCdH, NC (2g)waceyrleattehde nchaitdind endanaoncdrymstaalisn (tCai1n2CeHdNunCd) ewresrtei rSroinxghlaett e1x1t5ra◦cCtedfo wr6ithh .eAthfatneorle osvteerrinfiicgahtti oannd,t fhineamllyix ture dried at 60 °C in an oven for 24 h. wasfilteredandwashedwithDMF,acetoneandethanol. Toremoveanyresidualtraceoffattyacid, acylatedchitinnanocrystals(C CHNC)wereSoxhletextractedwithethanolovernightandfinally 2.5. Preparation of PLA/C2CHN1C2 and PLA/C12CHNC Nanocomposite driedat60◦Cinanovenfor24h. PLA/C2CHNC and PLA/C12CHNC nanocomposites were prepared by compression molding approach as following: Materials2017,10,546 4of16 2.5. PreparationofPLA/C CHNCandPLA/C CHNCNanocomposite 2 12 PLA/C CHNCandPLA/C CHNCnanocompositeswerepreparedbycompressionmolding 2 12 approachasfollowing: (1) PLApelletsweredissolvedinchloroform(10mLg−1)intoa250mLglassbeaker. (2) Differentamounts(i.e.,0.5,1.0and2.0wt%)ofeachC CHNCandC CHNCsetswereaddedin 2 12 thedifferentglassbeaker. (3) Themixtureswerestirredfor5minandthenleftat30◦Cfor1daytoevaporatethesolvent. (4) After solvent evaporation, the PLA/C CHNC and PLA/C CHNC mixtures were extruded 2 12 usingaHAAKEMiniLabmicrocompounderat180±2◦Cand120rpm. (5) Subsequently,thenanocompositesweremoldedbycompressionusingaMetrotecpressat190◦C and50barfor1min,followedbyfastcooled. (6) Finally,thesampleswerekeptat50%relativehumidity(RH)and25±2◦Cfor72hbeforebeingtested. SamplesidentificationislistedinTable1. Table1.IdentificationofthePLA/C CHNCandPLA/C CHNCsamples. 2 12 SampleIdentification Acylation CHNC(wt%) PLA – – PLA/C20.5 Aceticanhydride 0.5 PLA/C21 Aceticanhydride 1 PLA/C22 Aceticanhydride 2 PLA/C120.5 Dodecanoylchlorideacid 0.5 PLA/C121 Dodecanoylchlorideacid 1 PLA/C122 Dodecanoylchlorideacid 2 2.6. PhysicochemicalandMorphologicalCharacterization Attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) spectra were recorded on a Nicolet Nexus 670 equipped with a KRS-5 crystal of refractive index 2.4 and using anincidenceangleof45◦. Thespectraweretakeninatransmittancemodeinthewavenumberrange of 750–4000 cm−1, with resolution of 4 cm−1 and after 128 scan accumulations. The spectra were normalizedusingthesoftwareOmnic. X-raydiffractionpatternswerecollectedusingaPhilipsX’pertProautomaticdiffractometerusing Cu-Kαradiationoperatingat40kVand40mAovertheangularrangeof5to40◦ 2θ(stepsize=0.04 andtimeperstep=353s)atroomtemperature. Thecrystallinityindex(C.I.)ofchitinnanocrystal sampleswascalculatedasfollows(Equation(1))[40,43]: C.I.(%) = [(I −I /I ]×100 (1) 110 am 110 whereI isthemaximumintensity(arbitraryunits)ofthe110crystallographicplaneandI isthe 110 am amorphousportiondiffraction,whichusuallyisfoundabout2θ=12.5–13.5◦. Atomic force microscopy (AFM) images were performed in a Dimension 3100 NanoScope IV (Veeco,SantaBarbara,CA,USA).Theimageswerescannedintappingmodeunderambientconditions usingsiliconnitridecantilevershavingatipnominalradiusof10nmatafrequencyof1Hz. Chitin nanocrystalsampleswerefirstwelldilutedinwaterandsonicatedfor5minuntilcolloidalsuspension (Vibracell75043fromBioblockScientific). Thenadropofthesuspensionwascastedontoamicapiece, whichislatesubjectedtoaspincoatingequipment(SpecialtycoatingsystemsINC,SpinCoatermodel P6700series)at2000rpmfor2min. Thedimensionsofnanocrystalswerecalculatedbymeasuring 100differentnanocrystals. ThelighttransmittancespectraofthematerialsweremeasuredusingaShimadzu(Kyoto,Japan) UV-3600UV-VIS-NIRspectrophotometer. Spectrawererecordedatroomtemperatureinstepsof1nm, intherange400–700nm. Materials2017,10,546 5of16 ContactanglemeasurementswereperformedatroomtemperatureusingDataphysicsOCA20 ContactAngleSystem(Filderstadt,Germany). Threedifferentliquids,withdifferentdispersiveand polarsurfacetensions(water,diiodomethaneandethyleneglycol),wereusedtodeterminethesurface energyofthesamplesfollowingtheOwensandWendtapproach[44]. Toperformthemeasurements, 50 ± 5 mg of each functionalized CHNC were placed into a cylindrical holder and pressed using ahydraulicpresstoobtainuniformdiscs(13mmindiameterand0.4mminthickness). Thetestwas doneintriplicate. 2.7. BarrierProperties Watervaportransmissiontestsofthematerials(thicknessrangefrom0.06to0.10±0.01mm) wereperformedat30.0±0.2◦Conagravimetriccellinwhichasmallamountofliquidwater(2mL) wassealedbyamembrane. Thecellwasputonananalyticalbalance(±10−5g),andtheweightloss ofthecell,onlyduetothepermeationofthewatervaporthroughthemembrane,wasfollowedby acomputerconnectedtothebalance. Wateractivityinsidethecellwas1,whereasthedownstream averagehumiditywas33±2%asrecordedwithathermohygrometer. Changesintheweightofthe cellwererecordedasafunctionoftime. Thewatervaportransmissionrate(WVTR)wasdetermined fromthefollowingEquation(2): (cid:18) g.mm (cid:19) m.(cid:96) WVTR = (2) m2.day A(1−a) whereWVTRisthewatervaporflowpassingthroughthefilmsperunittime;“(cid:96)”isthethicknessofthe films;“A”istheareaofthefilms(2.54cm2)exposedtothewatervaporflow;and“a”istherelativehumidity inthethermostaticsystem.Foreachfilm,WVPmeasurementswereperformedatleastthreetimes. 2.8. AntifungalActivityagainstAspergillusNiger In order to evaluate the anti-fungal activity of the composite films against a common rot of food and indoor environments, the mold fungus Aspergillus niger (CBS 554.65) was cultured and incubatedaerobicallyfor72hat25◦CinPetridisheswithsolidsubstrateofMalteExtractAgar(MEA, Sharlab).Aftergrowing,analiquotofsporeswasdilutedinRingersolution(14mL)andwasaseptically inoculatedontheMEAsurfacesbyspraymethod(with40µLofdissolutionat1.21×106SporesmL−1). Finally, 1 cm × 1 cm shaped samples (with an average thickness of 0.08 mm) of each nanocompositefilmsandPLAfilms(thelatterwasusedascontrol)wereintroducedintotheMEA platestoassessthefungalinhibition. Eachsamplewaspreparedintriplicate. After7daysofincubationat25±1.5◦C,thefilmsweregentlyextractedfromtheagarandwashed with Ringer solution (1 mL) to obtain a spore solution. Then samples were vortexed and stained withLPCBtaking20µLofeachsampletocountsporesconcentrationwithanautomatedcellcounter (Cellometer® MiniNexcelomBioscienceLLC,Lawrence,MA,USA).Thefungalgrowthinhibition (FGI)wascalculatedasconcentrationofspores(conidia)permilliliter,accordingtotheEquation(3): C −T g g FGI (%)= ×100 (3) C g whereC istheaverageconcentrationinthecontrolsamplesset(PLAfilmsandfungaldish)andT is g g theaverageconcentrationinthetreatedset(filmtested),bothexpressedasconcentrationofspores (conidia)permilliliter[18]. 2.9. ThermalStabilityandMechanicalProperties Thermogravimetricanalysis(TGA)assayswerecarriedoutinaTGA/SDTA851MettlerToledo instrument. Allsamples(~8mg)wereheatedataconstantrateof10◦Cmin−1from25to900◦Cunder anitrogenatmosphereof20mLmin−1. Materials2017,10,546 6of16 TensiletestswereconductedunderambientconditionsusingaMaterialTestingSystems(MTS Insight10)deviceusingaloadcellof250Nandadeformationrateof3mmmin−1. Tenreplicates ofeachsample(rectangular-shapespecimensof6×1cm2 andgaugelengthof2cm)wereusedto determinetheaveragevaluesofYoung’smodulus,tensilestrengthandelongationatbreakusingMTS TestWorks4software(Version4.09A,MTSSystemCorporation,EdenPrairie,MN,USA). 3. ResultsandDiscussion 3.1. GeneralCharacterizationofChitinDerivatives: C CHNCandC CHNC Materials 2017, 10, 546 2 12 6 of 17 The preparation of PLA-based nanocomposite films with chitin nanocrystals as reinforcing 3. Results and Discussion nanofillerrequireschemicalmodificationofthenanofillers’surface. Twosetsoffunctionalizedchitin nanocry3s.1t.a Glsen(eCra2lC CHhaNracCterainzadtioCn 1o2f CCHhitNinC D,eFriivgatuivrees:1 C)2CwHeNreCp anredp Ca12rCeHdNuCs ingheterogeneousconditionsto increasetheThhye dprroeppahroabtioicn soufr fPaLcAe-cbhasaerda cntaenr,oaconmdptohsuitse ifmilmpsr owvietht hcehidtinis pneanrsoacbryislittayls inast oretihnefoPrcLinAg matrix. Fignuarneof2ilalerd riesqpuliaryess cAheTmRi-cFalT mIRodsifpiceacttiorun mof tohfe CnaHnoNfiCllerasn’ sduriftascde.e Trwivoa steitvse osf, fui.nec.,tiConaCliHzeNd cChi(tmin odified 2 withanhnayndorcirdyestaalcse (tCic2)CaHnNdCC andC CH1N2CCHN(mC,o Fdiigfiuered 1w) witherefa pttryepaacriedd) .usTinhge hsuetcecroegssenoefotuhse comnodditiiofincsa ttoio nswas 12 clearlycinocnrefiarsme tehde ,hmydaroinplhyobbiac sseudrfaocne cthhaeraecmtere, ragnedn tcheuso ifmapnroevwe tbhea ndidspaetrsaarboiluitny din1to7 t3h5e cPmLA− m1,aatrsisxi.g nedto Figure 2a displays ATR-FTIR spectrum of CHNC and its derivatives, i.e., C2CHNC (modified thecarbonylestergroupstretchingmode(C=O,Figure2a)displayintheC CHNCandC CHNC with anhydride acetic) and C12CHNC (modified with fatty acid). The success of2 the modification1s2 spectra[41,45–47].ThispeakissmallerinthecaseofthenanocompositefilmspreparedwithC CHNC, was clearly confirmed, mainly based on the emergence of a new band at around 1735 cm−1, assigne1d2 possiblytod thuee ctaorbaonsyml easltleerr gerxoutepn sttroeftcthhinegs murofdaec (eCs=uOb, sFtigituurtei o2an). dAispnlainy cinre tahsee Ci2nCHthNeCi nantedn Cs1i2tCyHoNfCth eC–H bandinstpheectrraan [g4e1,4258–6407–].2 9T0h0isc mpe−ak1 aisr issminagllefrro imn tthhee acalispe hoaft itchea cnidancohcaoimnpwosaitsea flislomos bpsreerpvaereddi nwtihthe caseof C CHNCC12CsHamNCp,l epo[4ss8i,b4ly9 ]d.uIne taol la sspmeacltlerar etxhteencth oafr tahcet esurirsfaticce asbubsostritputtiioonn. bAann idnscroefasαe- icnh tihtien ini.tee.n,saitty1 o5f5 5cm−1 12 ascribedthteo CN–HH bbaenndd inin tghe( aramnigde e28II6)0,–a2t90106 1cm9−a1 nadris1in6g5 5frocmm −th1e caolirprheastpico ancdidin cghation cwaarsb aolnsoy olbssterrevtcedh iinng bands (amideIth)ea ncadsea otf3 C2102C0HanNdC 3s4am00pclem [4−81,4o9]f. NIn– aHll sapnedctrOa –thHe cshtraeratcchteirnisgticb aabnsdo,rpretisopne bcatnivdesl oyf [α5-0c,h5i1ti]n ai.ree., present. at 1555 cm−1 ascribed to NH bending (amide II), at 1619 and 1655 cm−1 corresponding to carbonyl Nonethsetlreestcsh,iangd beacnrdesa (saemiindei In) taennds aitt y32o00f atnhde 3b4r0o0 acmd−b1 oafn Nd–Hat aanbdo Ou–tH3 s4t0re0tcchming− b1a,nind, preasrpteicctuivlealry [f5o0r,5C1]2 CHNC sample,awre apsreosebnste. rNvoendetihnedleiscsa, ati ndegcrtehaeses iunb instteitnusittiyo onf othfeh byrdoardo xbyanldg raot aubposutb 3y40a0c ecmty−l1, ginr opuaprtsic.uTlahri sfodr ecrease andnoCd2iCsaHpNpCe asarmanpclee, wofasO o–bHsersvterde tinchdiicnagtinbga tnhde ssuobnstitthuetiofnu nofc htiyodnroaxliyzl egdrosuapms bpyl eacsestyulg ggroeusptss.t Thhaits: (1)the decrease and no disappearance of O–H stretching bands on the functionalized samples suggests that: modificationhastakenplaceonthesurfaceofthechitinnanocrystalsremainingintactinsidehydroxyl (1) the modification has taken place on the surface of the chitin nanocrystals remaining intact inside group;and(2)anincompletesubstitutionofallofthesurfaceOHgroups[47,52]. hydroxyl group; and (2) an incomplete substitution of all of the surface OH groups [47,52]. Figure 2. (a) ATR-FTIR spectra; and (b) X-ray diffraction of CHNC and acylated chitin nanocrystals Figure2.(a)ATR-FTIRspectra;and(b)X-raydiffractionofCHNCandacylatedchitinnanocrystals (C2CHNC and C12CHNC). (C CHNCandC CHNC). 2 12 The crystal structure of CHNC and its derivatives was analyzed by X-ray diffraction (Figure 2b). The four typical crystalline reflections of α-chitin at 9.5°, 19.5°, 21.0° and 23.5° indexed as 020, 110, 120 and 101 crystallographic planes, with a basal spacing of 9.31, 4.55, 4.25 and 3.80 Å, respectively, were observed [43,53,54]. The stronger reflections were clearly observed at 9.5° and 21.0° and the lower reflections at higher 2θ values. CHNC presented a crystallinity index of 89%. The crystalline Materials2017,10,546 7of16 ThecrystalstructureofCHNCanditsderivativeswasanalyzedbyX-raydiffraction(Figure2b). Thefourtypicalcrystallinereflectionsofα-chitinat9.5◦,19.5◦,21.0◦ and23.5◦ indexedas020,110,120 and101crystallographicplanes,withabasalspacingof9.31,4.55,4.25and3.80Å,respectively,were observed[43,53,54]. Thestrongerreflectionswereclearlyobservedat9.5◦ and21.0◦ andthelower reflectionsathigher2θvalues. CHNCpresentedacrystallinityindexof89%. Thecrystallinestructure oftheMfautenriaclst i2o01n7a, 1li0z, 5e4d6 c hitinnanocrystalsremainedthesameasCHNCandtheC.I.werefo7u onf d17 tobe 90%and82%forC CHNCandC CHNC,respectively. ThedecreaseofthecrystallinityofC CHNC 2 12 12 structure of the functionalized chitin nanocrystals remained the same as CHNC and the C.I. were ismainlyduetothedestructionofthecrystallineregionsofthenanocrystals,whichledtoareduction found to be 90% and 82% for C2CHNC and C12CHNC, respectively. The decrease of the crystallinity ofnanoparticlelength(confirmedbyAFM,seebelow). Thefactthat,modifiedchitinnanocrystals of C12CHNC is mainly due to the destruction of the crystalline regions of the nanocrystals, which led showteod at rheedusacmtioen doiff fnraancotipoanrtpicalett leernngtthh a(cnounfnirmmoeddi fibye dAcFhMit,i sne,es bueglgowes)t. tThhaet ftahcet tmhaotd, mifiocdaitfiioend tcohoitkinp lace atthensaunorfcarycestaolfs tshheowneadn othcer ysasmtael sdaifnfrdacotinont hpeatmteronr ethaacnc uenssmiboldeifaiemd ochrpitihno, ususgdgoesmt tahiants t.he modification Ttohoiks pdlaatcae caot trhroe bsuorrfaatcee wofi tthhet hnaensoiczreysstoaflsC aHndN oCn athned mfuornec atciocensasliibzlee damchoritpihnonuas ndoocmryaisntsa.l sthatwere roughly eTshtiism daattead coursroinbgoraAteF Mwitihm thaeg isnizges( oFfi gCuHrNeC3) aannd dfuwncetiroenafoliuzendd chtoitibne naanpopcrroysxtiamlsa tthealty w3e0r0e , 290 and 1ro8u5gnhmly eisntimleantegdth us(iLn)g fAoFrMC iHmNagCin,gC (FCigHurNe C3) aanndd wCereC foHuNndC t,o rbees papepctriovxeimlya.teAlyc 3tu00a,l 2ly9,0 aafntde r the 2 12 heter1o8g5e nnmeo inu lsensugtrhf a(Lce) fmor oCdHifiNcCa,t Cio2CnH,tNhCe eanndsu Ci1n2CgHCNCC, rHesNpeCctisvheolyw. Aedctuaanlliym, apftoerr ttahne thedteercorgeeanseeooufs their 12 lengtshu,rjfuasceti fimeoddibfiycatthioenp, tahret ieanlsdueintagc Ch1m2CeHnNtoCf sthhoewmedo danifi imedpofirbtarnilts dferocrmeasteh eofc trhyesitra llelningeths, tjruustcitfuierde [55]. by the partial detachment of the modified fibrils from the crystalline structure [55]. No significant Nosignificantchangeswereobservedinthewidth(Ø)ofthesamplesafterthereactions. Thetypical changes were observed in the width (Ø) of the samples after the reactions. The typical rod-like rod-likemorphologyofthesamplesremainedafterthechemicalmodification. morphology of the samples remained after the chemical modification. Figure 3. AFM height images of CHNC, C2CHNC and C12CHNC (L, length; and Ø, width). Figure3.AFMheightimagesofCHNC,C CHNCandC CHNC(L,length;andØ,width). 2 12 The results have demonstrated that the modification was essentially limited to the nanocrystals’ Tsuhrefarcees ourl ttso hthaev meodreem acocnessstirbaltee admthorapthtohuesm choitdinifi dcoamtioainnsw, naost easfsfeecntitniagl lsyiglnimificitaendtlyto ththe ecrnyastnaollcinrey stals’ surfarceegoiorntso otfh tehem inonreera lcacyeesrssi ibnl ethaem noanrpohcroyustsaclsh. itindomains,notaffectingsignificantlythecrystalline regionsofTthhee tihnenremralla ybeerhsaivniotrh eonf atnhoec rCyHstNalCs. before and after the reactions was evaluated by thermogravimetric analysis (Figure 4 and Table 2). Derivative TGA (dTGA) tracing of CHNC showed The thermal behavior of the CHNC before and after the reactions was evaluated by one weight-loss-step with a maximum degradation step at 387 °C associated with the degradation of thermogravimetricanalysis(Figure4andTable2). DerivativeTGA(dTGA)tracingofCHNCshowed onewcheiitginh tm-loolsesc-uslteesp. Twheit hthaermmaalx dimeguramdadtieognr apdroaftilieo nofs Cte2pCHatN3C8 7sh◦oCweadss tooc biaet eledssw stiathbleth, esindceeg trhaedya tion started to decompose at lower temperatures. Derivative TGA curves of C2CHNC displayed two of chitin molecules. The thermal degradation profile of C CHNC showed to be less stable, since degradation steps: the first is a wide degradation that starte2d at around 180 until 310 °C; and the theyssteacrotnedd sttoepd oecccoumrsp aots 3e6a4t °lCo.w Tehrestee mthpeermraatlu dreecso.mDperoisvitaiotinv ebeThGavAiocrus rdveerisvoedf Cfr2oCmH cNhiCtind disipacleatyaeted two degraadndat icohnitisnt e[4p5s]:. Tthheis fisrlisgthits daecwreiadsee dine gthraerdmaatilo sntatbhilaittys otaf rCteHdNaCt aisr ocoumndmo1n8l0y ufonutinld3 1in0 s◦uCrf;aacen-d the seconadcesttyelpatoedcc cuhristiant. 3T6h4e ◦dCer.iTvahteivsee tThGeArm caulrvdee coof mC1p2CosHitNioCn dbiesphlaavyieodr stwdoer sievpeadrafrteo mdecghraitdiantidoina csetetaptse and chitinat[ 4352]2. °TCh aisndsl 3ig9h0 t°Cd eacsrseoacsiaeteind ttho ethrme daelgsrtaadbailtiitoyn ooff CesHteNrifCiedis cchoimtinm froanctliyonfo aunndd uinnmsoudriffaiecde- cahciettiny lated chitinfr.aTcthioend, erreisvpaetcitviveeTlyG. AIntceurersvteinogflyC, a CdHecNreCased iosfp lwayateedr tcwonotesnetp ianr atthee dCe1g2CraHdNatCio snamstpepless awta3s2 2◦C 12 observed, suggesting that the modification of CHNC made them more hydrophobic [56,57]. Materials2017,10,546 8of16 and390◦Cassociatedtothedegradationofesterifiedchitinfractionandunmodifiedchitinfraction, respectively. Interestingly, a decrease of water content in the C CHNC samples was observed, 12 suggestingthatthemodificationofCHNCmadethemmorehydrophobic[56,57]. Materials 2017, 10, 546 8 of 17 FFiigguurree 44.. TTGGAA ((aa));; aanndd ddTTGGAA ((bb)) pprroofifilleess ooff CCHHNNCC,, CC2CCHHNNCC aanndd CC12CCHHNNCC.. 2 12 TTaabbllee 22.. DDeeggrraaddaattiioonn tteemmppeerraattuurreess ooff CCHHNNCC,, CC2CCHHNNCC,, CC12CCHHNNCC aanndd PPLLAA--bbaasseedd nnaannooccoommppoossiitteess.. 2 12 Sample Temp. Main Degradation (°C) Temp. Second Degradation (°C) Sample Temp.MainDegradation(◦C) Temp.SecondDegradation(◦C) CHNC 387.1 C2CCHHNNCC 364.33 87.1 180–310 C CHNC 364.3 180–310 C12CC2HCNHCN C 390.23 90.2 3223.282 .8 12 PLA 364.3 PLA 364.3 PLPAL/AC/2C0.250 .5 362.33 62.3 PLPAL/AC/2C1 21 364.43 64.4 PLA/C 2 363.5 PLA/C22 2 363.5 PLA/C 0.5 363.3 12 PLAPL/CA1/20C.5 1 363.33 60.2 12 PLPAL/AC/1C211 22 360.23 58.0 PLA/C122 358.0 Figure5displaystheprofileobtainedforthedynamicwatercontactanglemeasurementsplaced Figure 5 displays the profile obtained for the dynamic water contact angle measurements placed onthesurfaceoftheCHNCpellets,beforeandafterthesurfacechemicalmodificationswithacetic on the surface of the CHNC pellets, before and after the surface chemical modifications with acetic anhydrideanddodecanoylchlorideacid. WhereastheunmodifiedCHNCshowedthetypicalhigh anhydride and dodecanoyl chloride acid. Whereas the unmodified CHNC showed the typical high wateraffinityassociatedwithafastdecreaseintheinitiallylowcontactangle(55◦)[58,59],modified water affinity associated with a fast decrease in the initially low contact angle (55°) [58,59], modified C CHNCandC CHNCsamplesshowedahigher(68.5◦and95.6◦,respectively)andconstantcontact 2 12 C2CHNC and C12CHNC samples showed a higher (68.5° and 95.6°, respectively ) and constant contact angle [47]. The drop contact angle remained constant during the time (Figure 5) as a result of the angle [47]. The drop contact angle remained constant during the time (Figure 5) as a result of the hydroxylgroups’replacementbyhydrophobicchains(acetylgroupsanddodecanoylchains)onthe hydroxyl groups’ replacement by hydrophobic chains (acetyl groups and dodecanoyl chains) on the surfaceofacylatedchitinnanocrystals. Asexpected,C CHNCdisplayedthelargervalueofcontact 12 saunrgflaece(9 o5f. 6a◦c)ydlauteedt ochitistinlo nnagnaolcipryhsattailcs.c hAasi nex.pTehcetesdu,r Cfa1c2CeHenNeCrg dyicspomlaypeodn ethnets la(prgoelar rvγalu,e doifs pcoenrstiavcet sp aγnglaen (d95t.o6t°a)l dγu)e otof Cits lCoHngN aClipsahmatpicle cshwaienr.e Tdheet esrumrfianceed eannedrgayr ecolimstpedoninenTtasb (lpeo3l.arT γhesp,d deicsrpeearsseivine tγhsed sd s 12 apnodla rtoctoaml γpso) noef nCt1(2γCH)NvCal usaemsfprolems w16e.r3e( CdeHteNrmC)intoed0 .a7nmdJ amre− l2is(tCed CinH TNaCbl)ec o3n. fiTrhme eddectrheeacshe eimn itchael sp 12 polar component (γsp) values from 16.3 (CHNC) to 0.7 mJ m−2 (C12CHNC) confirmed the chemical modificationontheCHNCsurface. modification on the CHNC surface. Materials2017,10,546 9of16 Materials 2017, 10, 546 9 of 17 Materials 2017, 10, 546 9 of 17 FigurFeig5u.rDe y5.n Damyniacmwica tweartcero ncotanctatcat nagnlgeleo fofC CHHNNCC aanndd ffuunnccttiioonnaalilziezde dC2CCHCNHCN aCnda nCd12CCHNCCH aNndC and 2 12 image of the water drop on the surface of the respective pellet. imageofthewaterdroponthesurfaceoftherespectivepellet. Figure 5. Dynamic water contact angle of CHNC and functionalized C2CHNC and C12CHNC and 3.2. Characterization of PLA-Based Nanocomposites (PLA/C2CHNC and PLA/C12CHNC) 3.2. Charaimctaegriez oaft itohne wofatPerL dAr-oBpa osne dthNe saunrofaccoem opf othseit reess(pPecLtAive/C peClleHt. NCandPLA/C CHNC) 2 12 3.2.1. Optical Properties of the Nanocomposites 3.2.1.3.O2.p CthicaaralcPterroizpaetirotnie osf oPfLAth-BeaNseadn Noacnoomcopmopsoistietess (PLA/C2CHNC and PLA/C12CHNC) One of the important parameters in packaging is the transparency, thus the light transmittance 3Oo.f2n .t1eh. eoO ffpitlthmiceas li wmParpos opmertreatainesstu ropefad trh aferm oNmeat ne7or0sc0o imntop p4oa0sc0ikt enasmg i n(Fgigisurteh e6)t.r Aans slpisaterden icny ,Ftighuurse t6hbe, ltihgeh atdtrdaitniosnm oitft ance of thenafinlomfilslewrsa ins tmhee amsautrriexd afffreocmted7 t0h0e ttroan4s0m0itntamnc(eF oifg tuhree fi6n)a.l nAasnolicsotmedpoisniteFsi,g duercere6absi,ntgh ietsa vdadluietiso n of One of the important parameters in packaging is the transparency, thus the light transmittance nanoogfifrl altehdreus afilinllym twhs ewithma sta hmter ieixansacurferfeemdcet enfrdto omtfh ec7h0ti0rta intno s n4ma0n0iot tcnarmnysc t(eaFloisgf uitnrh ete h6fie). n mAaalst nrliiaxsnt. eoHdc ooiwnm eFpvigeorus,i rtteeh se6, bmd, etahcteerr eiaaadslsdi niatgrioein tss toivlfl a lues gradnutraaanlnlosyfliulwlceeirtnsh ti,n ta htshe sehi mnocwartneri mxin ae Ffnfietgcuoterfdec 6thhaie.t i Ttnrhaenn astrmnaonitcstarmynicsttetaa onlfsc teih n(em tfihenaeaslum nraeadntro ficoxor.m sHppeoocswiimteeevsn,e dsr e,wctrhiethea smain taght iiectrks invaealsslus aeorsfe still transgalrupapcderuonaxtli,lmya sawtseihtlyho w0th.0ne7 i inmncmrFei)mg auetnr e6t 0o60fa n.chmTith iwne atnsra ananbosocmruytis t7tta2al%ns c infeo (rthm PeeL maAsa,u t6rri6ex%d. H ffooorrw sPepLveeAcr/,iC mth20ee.n 5m saanwtdeir ti6ha7la%s a thfroeir c sktthinlele ssof apprtoPrxaLniAms/lCuact12ee0nl.y5t, n0aa.s0n s7ohcmoowmmnp )oinas itFtei6gs0 u(0Freing 6muar.e wT 6hbae)s .t aTrabhnoessumet irt7etsa2un%lctesf o(inmrdePiacLsautAer e,tdh6 a6fto% trh sfepo terrcaPinmLsemAni/st tCwanitc0he. 5ais a tnhnoidctk a6nf7fee%scst eofdof r the 2 by the kind of modified CHNC. PLA/apCpro0x.5imnaatneloyc 0o.m07p mosmit)e sat( F6i0g0u nrme6 wb)a.sT ahbeosuet r7e2s%u ltfosri nPdLiAca, t6e6t%h aftort hPeLtAra/Cn2s0m.5i tatnadn c6e7%is nfoort tahfef ected 12 bythPeLkAin/Cd12o0.f5m naondoicfioemdpCoHsitNesC (F.igure 6b). These results indicate that the transmittance is not affected by the kind of modified CHNC. (a) (a) (b) Figure 6. Photographs of PLA, PLA/C20.5 and PLA/C120.5 nanocomposite films (a); and transmittance traces of PLA film and PLA/C2 and PLA/C12 nano(bco) mposite films (b). Figure 6. Photographs of PLA, PLA/C20.5 and PLA/C120.5 nanocomposite films (a); and transmittance Figure6.PhotographsofPLA,PLA/C 0.5andPLA/C 0.5nanocompositefilms(a);andtransmittance traces of PLA film and PLA/C2 and P2LA/C12 nanocomp1o2site films (b). tracesofPLAfilmandPLA/C andPLA/C nanocompositefilms(b). 2 12 Materials2017,10,546 10of16 Materials 2017, 10, 546 10 of 17 3.2.2. ThermalandMechanicalProperties 3.2.2. Thermal and Mechanical Properties LikeforthemodifiedCHNC,thethermalstabilityanddegradationprofilesofthePLAnanocomposite materialswLiekree afosrs etshsee dmboydtihfieerdm CogHrNavCim, tehtery t(hTearbmleal1 )s.taTbhielitTy GaAndp rdofeigleraodfatthioen PpLrAoffilielms oefx htihbei tsPaLAty pical singlenawnoecigomhtp-loossitse smteapte,riwalist hwearem asasxeismseudm byd theecrommopgroasvitimioentrrya (tTeaablte3 16).4 T◦hCe .TTGhAe ptrhoefirlem oaf lthdee PcLoAm fpiloms ition proceesxshiobfitas lal tthypeicPaLl Asin/gCle CwHeigNhCt-lo(sFsi gstuepre, w7ait)h aan mdaPxLimAu/mC deCcoHmNpoCsit(iFonig ruartee a7tb 3)6n4 a°nCo. cTohme tphoersmiteasl also 2 12 presednetecodmapsoinsigtiloenw epirgohcetslso sosfs tealpl ptrhoefi lPeL.AT/hCe2CthHeNrmCa l(Fstiagbuirlei ty7ap)r oafinlde sPoLfAal/lCs1a2CmHpNleCs w(eFrigeuvreer y7bsi)m ilar nanocomposites also presented a single weight loss step profile. The thermal stability profiles of all withoutimportantchanges,contrarilywithGuanetal. work[15]wherethethermaldegradationof samples were very similar without important changes, contrarily with Guan et al. work [15] where the PLAbionanocompositeswith2%ofchitinnanocrystalsdecreasedabout25◦C. thermal degradation of PLA bionanocomposites with 2% of chitin nanocrystals decreased about 25 °C. FigureFi7g.uTreh 7e.m Tohgemraovgimravetimricetarnica alynsailsysoifs: oPf:L PAL/AC/C2n naannooccoommppoossitiete (a()a;) a;nadn dPLPAL/AC1/2 Cnanoncaonmopcoosmitep (obs)i.t e(b). 2 12 The mechanical properties of PLA films and nanocomposite films were evaluated in terms of TYhouenmg’esc mhoadnuicluasl, pternospileer sttireesnogfthP aLnAd efilolmngsataionnd ant abnreoacko (mFigpuorsei t8e). fiTlhme sunwfiellreed ePvLaAlu fialmtesd shinowteerdm sof Youncgh’sarmacotedruisltuics ,vtaelnuseisl einst rYeonugntgh’sa nmdoedluolnugs aatito anroautnbdre 2a kG(PFaig, uinre t8en).siTleh esturennfigltlhe doPf L44A MfilPma sasnhdo wed charaecltoenrgisattiiconv aaltu berseaink oYfo 3u.1n%g’ [s6m0,6o1d].u Cluosntartarayr otou nprdev2ioGuPsa w,ionrktse n[2s0il,6e2s,6tr3e]n rgeltahteodf t4o4 thMe Pinacaonrpdoeralotinogn ation of CHNC into PLA matrices, in the present work, a minor loss in modulus and tensile strength was atbreakof3.1%[60,61]. Contrarytopreviousworks[20,62,63]relatedtotheincorporationofCHNC observed when the modified CHNC were incorporated in the PLA matrices. This effect was intoPLAmatrices,inthepresentwork,aminorlossinmodulusandtensilestrengthwasobserved associated to the C2CHNC and C12CHNC aggregation. However, PLA-based nanocomposites whenthemodifiedCHNCwereincorporatedinthePLAmatrices. Thiseffectwasassociatedtothe C2CH NCandC12CHNCaggregation. However,PLA-basednanocompositespreparedwithothertype ofnanofillers(i.e.,nanocrystallinecellulose,andnanoclays)alsoshowednosignificanteffectonthe
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