isolate/straw protein films composite Chemical characterization treatment and of soybean straw andsoybean Carbohydrate Polymers

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Carbohydrate Polymers

jo u r n al h om ep age :w w w . e l s e v i e r . c o m / l o c a t e / c a r b p o l

Chemical treatment and characterization of soybean straw and soybean protein isolate/straw composite films

Milena Martelli-Tosi

, Odílio B.G. Assis

, Natália C. Silva

, Bruno S. Esposto

, Maria Alice Martins

, Delia R. Tapia-Blácido

aDepartamentodeQuímica,FaculdadedeFilosofia,CiênciaseLetrasdeRibeirãoPreto,UniversidadedeSãoPaulo,BandeirantesAvenue3900,CEP 14040-901,RibeirãoPreto,SP,Brazil

bDepartamentodeEngenhariadeAlimentos,FaculdadedeZootecniaeEngenhariadeAlimentos,UniversidadedeSãoPaulo,RuaDuquedeCaxiasNorte 225,CEP13635-900,Pirassununga,SP,Brazil

cEmbrapaInstrumentac¸ão,NationalNanotechnologyLaboratoryforAgriculture,RuaXVdeNovembro,1452,CEP13561-206,SãoCarlos,SP,Brazil

a r t i c l e i n f o

Articlehistory:

Received27June2016 Receivedinrevisedform 20September2016 Accepted6October2016 Availableonline11October2016

Keywords:

Chemicaltreatment Soybeanstraw

Structurecharacterization Microfibermorphology Compositefilms Mechanicalproperties

a b s t r a c t

Thisworkinvestigatedchangesinthechemicalcompositionandstructureofsoybeanstraw(SS)treated withalkali(NaOH5%and17.5%)andbleachedwithhydrogenperoxide(H2O2)orsodiumhypochlorite (NaOCl).Removaloftheamorphousconstituentsincreasedthedegreeofcrystallinityandthecontentof cellulosefibersparticularlyafterreactionwithhighconcentrationsofalkali.TreatmentwithNaOH17.5%

contributedtotheallomorphtransitionfromcelluloseItoIIregardlessofthebleachingagent,butH2O2

asbleachingagentpromotedmoreeffectivedelignification.Thisworkalsoevaluatedthepotentialuseof treatedandnon-treatedSSasreinforcementfillerinsoyproteinisolatefilm(SPI).Filmsaddedwithtreated SSpresentedhighermechanicalresistance,lowerelongationatbreak,andlowersolubilityinwater.

Additionofnon-treatedSSdidnotaffectthepropertiesoftheSPIfilmsignificantly.Thelowsolubilityand thereasonablewatervaporpermeabilityofthecompositefilmsmakethemsuitablepackagingmaterials forfreshfruitandvegetables.

©2016ElsevierLtd.Allrightsreserved.

1. Introduction

SoybeanisasignificantagriculturalcommodityintheBrazilian economy(Milazzo,Spina,Cavallaro,&Bart,2013).The2015/2016 harvest placed Brazil as the second largest world producer of soybean (about 30% of the global production) (CONAB, 2015).

Unfortunately,grainthreshinggeneratesahugeamountofsoy- bean straw. According to Bose and Martins Filho (1984), the soybean/straw ratio statisticallyvaries from 1:1.2 to1:1.5t/ha, whichmeansthatabout104to130milliontons/yearofstraworig- inatesfromtheBraziliansoybeanharvestalone(FAOSTAT,2016).

Thesoybeanstrawconsistsofstems,leaves,andpods.Theaverage compositionofthis materialincludes35%cellulose,21%insolu- blelignin,17%hemicelluloses,11%ash,1%acidsolublelignin,and remainingconstituentssuchasprotein,pectin,andglucuronicacid

∗Correspondingauthorat:DepartamentodeEngenhariadeAlimentos,Faculdade deZootecniaeEngenhariadeAlimentos,UniversidadedeSãoPaulo,Av.Duquede CaxiasNorte225,CEP13635-900,Pirassununga,SP,Brazil.

E-mailaddress:[email protected](M.Martelli-Tosi).

substitutes(Cabreraetal.,2015;Wan,Zhou,&Li,2011).Soybean strawisusuallydisposedaswastethroughlandfilling,incineration, ordumping.

Somestudieshavesuggestedthatsoybeanstrawcouldbeused asrawmaterialtoproducepolymerandsolublesugars(Cabrera etal.,2015;Wanetal.,2011;Xu,Wang,Jiang,Yang,&Ji,2007)or asanaturalsourcetoobtainfibersviaalkaliandacidtreatments (Reddy&Yang,2009).

Various physical, chemical, and enzymatic treatments can enhancethebiodegradabilityand digestibilityof soybeanstraw (Cabreraetal.,2015;Khorvas,Kargar,Yalchi,&Ghorbani,2010).

Applicationofchemical treatmentsbeforeenzymatichydrolysis removes amorphous componentslike lignin and hemicellulose, thereby increasing the porosity of the material and facilitating furtherdefibrillationand extractionof fibers(Yueet al.,2015).

Chemical treatments include reactionswith ozone, alkali, acid, peroxide, or other organicsolvents (Oh et al., 2015).The pulp industryhastraditionallydelignifiedlignocelluloseviableaching withsodiumchlorite.However,environmentalconcernshaveled theindustrytoreplacebleachingwithsodiumchloritewithmore environmentallyfriendlymethodssuchasthermochemicalreac- http://dx.doi.org/10.1016/j.carbpol.2016.10.013

0144-8617/©2016ElsevierLtd.Allrightsreserved.

M.Martelli-Tosietal./CarbohydratePolymers157(2017)512–520 513

tionsthatuseoxygen(Kafleetal.,2015)andhydrogenperoxide (Andrade-Mahecha, Pelissari, Tapia-Blácido, & Menegalli, 2015;

Zeronian&Inglesby,1995).AccordingtoKhorvashetal.(2010), treatmentwithH₂O₂improvestheinvitrodigestibilityofsoybean strawascomparedtoNaClO₂-basedtreatments.Dependingonthe sizeofthegeneratedsoybeanfibers,theycanserveasreinforce- mentfillersduringformationofbiocompositematrixes.

Biomaterialsbasedonrenewableresources,suchasproteins and polysaccharides,canbepotentiallyprocessedintofilmsfor foodorbiomedicalapplications.Inparticular,soyproteinisolate (SPI), which can beproduced by casting (Denavi et al., 2009), extrusion,orinjectionmolding(Calabriaetal.,2012; Chan,Lim, Barbut,&Marcone,2014;Garrido,Pe ˜nalba,delaCaba,&Guerrero, 2016), is an attractive sustainable “green polymer” with good film-forming ability. Nevertheless, SPI films have low strength andabsorbahighamountofmoisture,whichlimitstheirappli- cations.Three methodscan help toovercometheselimitations andtoimprovethepropertiesofSPIfilms,namelycross-linking (González, Strumia, &AlvarezIgarzabal, 2011; Xuet al.,2015), blendingwithapolymertoformasoy-basedplastic(Calabriaetal., 2012;Kim&Netravali,2012;Saenghirunwattana,Noomhorm,&

Rungsardthong, 2014; Won, Lee,Jin,&Lee,2015), andaddition ofreinforcingfillers(Husseinsyah,Yeng,Kassim,Zakaria,&Ismail, 2014;Lodha&Netravali,2002;Siro&Plackett,2010).

Micro-and nanosizedfibershavebyfarbeenthemostcited reinforcement fillers in the literature (Chan et al., 2014; Chen, Zhang,Peng,&Liao,2006;Jensen,Lim,Barbut,&Marcone,2015;

Saenghirunwattanaetal.,2014;Satyanarayana,Arizaga,&Wypych, 2009;Wang,Cao,&Zhang,2006;Wei,Fan,Huang,&Chen,2006).

Hydrophobicandelectrostaticinteractionsarethedrivingforces behindtheestablishmentofanetworkbetweenfibersandproteins;

theseinteractionsimprovethemechanicaland waterresistance propertiesofcompositefilms(Jensenetal.,2015;Sun,Chen,Liu, Li,&Yu,2015;Saenghirunwattanaetal.,2014;Wangetal.,2006).

Therearefewreportsonthecharacterizationanduseofchemically treatedsoybeanstrawasreinforcingfillerinSPIfilms.

The present study aimedto evaluate(1) how four different sequencesofchemicaltreatmentwithalkaliandbleachingaffect thestructureandcompositionofthesoybeanstrawand(2)howthe incorporationoftreatedfibersasreinforcingfillersintosoyprotein filmsinfluencesthemechanicalandpermeabilitypropertiesofthe resultingfilm.

2. Materialsandmethods

2.1. Materials

SoybeanstrawwasprovidedbyEmbrapaSoja(Londrina,Brazil).

Theresidueswerewashedwithdistilledwateranddriedat50^◦C for72hinanovenwithforcedcirculation(Q314M,Quimis,Brazil).

ThedriedsampleswerethengroundinaknifemillSL31(Solab, Brazil)andsievedthrough100-meshsieves(Tylerseries,150␮m).

Solae^® (Brazil)suppliedthesoyproteinisolate(SPI).Glycerol was purchased from Sigma-Aldrich (Saint Louis, USP). Chem- icals were obtained from Labsynth (Diadema, Brazil) (NaOH, NaClO₂andH₂O₂)andDinâmica(Diadema,Brazil)(CH₃COOHand MgSO₄·7H₂O).

2.2. Chemicaltreatmentsofsoybeanstraw

Thesoybeanstrawparticlesunderwentfourdifferentchemical treatments,includingtreatmentwithalkalifollowedbyableaching step. The concentrations of alkali and H2O2 and the tempera- tureusedduringthetreatmentswerebasedonpreviousresults obtainedforthetreatmentofachirafibers(Andrade-Mahechaetal.,

2015).TheconditionsofthebleachingreactioninvolvingNaClO₂ werethesameastheconditionsdescribedbyCamposetal.(2013).

Fig.1summarizesthesequenceofthesetreatments.Accordingto theintensityofthereaction,thetreatmentsweredenotedsevere ormildasfollows:

(i)Severe:NaOHat5%(T1)or17.5%(T2)(w/v)at90^◦Cfor1h, repeatedtwice.Thesolutionwasbroughttoroomtemperature andrinsedtoneutralization.Thefiberswerethenbleachedwith aqueous0.7%acetic acidand 3.3%sodiumchlorite (NaClO2) solutionunderagitationat75^◦Cfor3h.

(ii)Mild:NaOHat5%(T3)or17.5%(T4)(w/v)at30^◦Cfor15h.After thisperiod,thesolutionwasbroughttoroomtemperatureand rinsedtoneutralization.Theresultingfiberswerebleachedin amixtureof4%H₂O₂(w/v)and2%NaOH(w/v)at90^◦Cfor3h.

MgSO₄.7H₂Oat0.3%(w/v)wasaddedasstabilizer.Thesolution wascooledtoroomtemperature,andthefiberswerefiltered andwashedwithdistilledwateruntilneutralpHwasachieved, followedbyrinsingwithethanolandacetone.Thefinalfibers weredriedat50^◦Cinanovenwithaircirculation.

2.3. Characterizationofsoybeanstraw 2.3.1. Chemicalcomposition

Analysis of the cellulose, holocellulose (cellu- lose+hemicelluloses),andlignincontentinthesoybeanstrawwas carriedoutbeforeandaftereachtreatment.Theprocedureswere performedaccordingtoTAPPIT19om-54(TAPPI,1991),TAPPIT 222om-22(TAPPI,1999),andthemethodologypresentedbySun (2004).

2.3.2. Particlesizedistribution

The particle size distribution of untreated and chemically treatedsoybeanstrawwasmeasuredwithaBeckmanCoulterLS 13320LaserDiffractionParticleSizeAnalyzer(BeckmanCoulter, Miami,FL,USA).Tothisend,thepowderedsamplesweresieved through100-meshsieves(Tylerseries,150␮m)anddispersedin ethanolundersonicationfor5min.Themeanparticlesizeandstan- darddeviation(SD)werecalculatedbyusingtheBeckmanCouter software.

2.3.3. Scanningelectronmicroscopy(SEM)

Soybeanstrawsamplesweremountedonaluminumstubsand coatedwithgoldinaBal-TecSCD050sputteringsystem(Balzers, Liechtenstein).ImageswereobtainedinaZeissEVO50(Cambridge, UK)scanning electron microscopeat anaccelerating voltageof 20kV.

2.3.4. X-raydiffraction(XRD)

Theeffectofchemicaltreatmentonthecrystallinityofsoybean strawwasdeterminedinaShimadzuX-rayDiffractometerXRD- 6000(Shimadzu,Japan)withCuk␣radiation,at30-kVvoltageand 30-mAtubecurrent.Themeasurementswerecarriedoutfor2␪ valuesrangingfrom5to40^◦,atascanrateof1^◦min⁻¹.Tocalculate thecrystallinityindex(I_cr)twodifferentmethodswereused.One methodconsistedofestimatingI_cr(S)accordingtotheSegal,Creely, Martin,andConrad(1959)relation:

Icr(S)= I002−Iam

I002 x100 (1)

whereI002istheintensityofthe002latticediffractionat2␪=22.8^◦ (crystalline contribution),andI_am istheintensityofthediffrac- tionat2␪=18^◦ consideringtheamorphousdiffraction.Theother methodusesacurve-fittingprocesstoestimateindividualcrys- talline peaks fromthe intensityof thediffraction profiles after subtractionofthebaselinespectrum.TheOrigin9.0softwarewas

Fig.1. Chemicalpre-treatmentsofthesoybeanstraw.

used,andGaussianfunctionswerefittedforeachpeak.Thebroad peakaround21.5^◦wasassignedtoamorphouscontribution(Park, Baker,Himmel,Parilla,&Johnson,2010).I_cr(D) wascalculatedby dividingtheareaunderthepeaksduetocrystallinecelluloseIand IIbythetotalareaoftheoriginaldiffractogram.

2.3.5. Thermogravimetricanalyzer(TGA)

Thethermalpropertiesofuntreatedandtreatedsoybeanstraw wereevaluatedonaThermogravimetricAnalyzerTGA-Q500(TA Instruments, USA). Approximately 10mg of each sample was weighed inplatinum pans. Thestudiedtemperature range was 25–700^◦C;theheatingratewas10^◦C/min,undernitrogenatmo- sphere.Masschangeswerecontinuouslyrecordedasafunctionof temperature.

2.3.6. AttenuatedtotalreflectionFouriertransforminfrared spectroscopy(ATR-FTIR)

Untreatedandtreatedsoybeanstrawsampleswereanalyzed byAttenuatedTotalReflectionFourierTransform InfraredSpec- troscopy(ATR-FTIR)(IRPrestige-21,Shimadzu,Japan)toidentify thefunctionalgroupsinthefiberandanychemicalchangesthat mayhaveoccurredinthepolymericstructure.Allthespectrawere

theaverageof40scansrecordedataresolutionof2cm⁻¹ from 4000cm⁻¹to400cm⁻¹.

2.4. PreparationofSPI/soybeanstrawcompositefilms

Untreated and treated soybean straw samples were sieved through100-meshsieves(Tylerseries,150␮m)andre-suspended in water24hbeforefilmprocessing. Thecomposite filmswere preparedfromanaqueoussoy proteinisolatesuspensionat5%

w/w,homogenizedinanUltra-Turraxhomogenizer(modelQ252- 28,Quimis,Brazil)for2min,andheatedat40^◦Cfor20minunder mechanicalagitationinathermostatic bath.Glycerol(25g)was added as plasticizer along with1g of untreated or chemically treatedsoybeanstraw asreinforcingfillertoevery100gofSPI.

ThepHwasadjustedto10.5withNaOH1M,andthefiberswere kept undermechanical agitation at 40^◦C for 10min. The solu- tionswereplacedinultrasonicbath(USC1400,Unique,Brazil)for 2min,pouredontheacrylicplates,and driedina BODincuba- tor(SL200/364U,SolabCientifica,Brazil)at30^◦Cand58%relative humidityfor24h.Beforecharacterization,allthefilmswerepre- conditionedforatleast72hindesiccatorscontainingsaturated NaBrsolution(58%RH).

M.Martelli-Tosietal./CarbohydratePolymers157(2017)512–520 515

Table1

Particlesizeandchemicalcompositionofuntreatedandchemicallytreatedsoybeanstraw.

Treatment ParticleSize(␮m) Cellulose(%) Hemicelluloses(%) InsolubleLignin(%) SolubleLignin(%)

Untreated 116±73^a 39.8±0.6^c 22.6±1.0^a 10.5±0.7^a 2.31±0.18^a

T1 108±78^a 62.9±0.6^b 11.1±0.1^b 4.9±0.2^b 1.42±0.09^b

T2 88±71^a 67.1±0.2^a 9.8±0.4^b 4.1±0.2^bc 1.51±0.04^b

T3 145±106^a 64.0±0.7^b 10.7±0.1^b 3.6±0.1^c 0.67±0.02^c

T4 122±79^a 66.2±0.5^a 9.5±1.1^b 3.5±0.1^c 0.73±0.09^c

a–dMeanswithdifferentsuperscriptlettersinthesamecolumnarestatisticallydifferentatp<0.05accordingtotheTukey’stest.

2.5. CharacterizationofSPI/soybeanstrawcompositefilms 2.5.1. Mechanicalproperties

ThemechanicaltestswereperformedonatextureanalyzerTA TXPlus(TAInstrument,England).The tensilestrength(TS)and elongation atbreak (EB)were obtainedaccording totheASTM D882-95method(ASTMD882-95,1995),takinganaverageoffive determinationsineachcase.Samplefilmswerecutinto2.54-cm- widestripswithalengthofatleast10cm.Theinitialgripseparation andthecrossheadspeedweresetat80mmand1.0mms⁻¹,respec- tively.Young’smodulus(YM)wascalculatedastheinclinationof theinitiallinearportionofthestressversusstraincurvebyusing theTextureExpertV.1.22(SMS)software.

2.5.2. Solubilityinwater,moisture,andwatervaporpermeability (WVP)

Thesolubilityinwaterwascalculatedasthepercentageofdry matterofthesolubilizedfilmafterimmersionofthreepreviously weighedsamples(discswith20mmofdiameter)for24hin50mL ofwaterat25±2^◦C,undermechanicalstirring.Thiswasfollowed byaproceduredescribedbyTapia-Blácido,Sobral,andMenegalli (2011).Sodiumazide(0.02%w/v)wasaddedtopreventmicrobial growth.Theremainingnon-solubblematterwasfilteredthrough qualitativefilterpaper(80gm⁻³)anddriedat50^◦Cuntilconstant weightwasachieved.Themoisturecontentinthefilmswasalso determinedbydryingthematerialsinanovenat105^◦Cuntilcon- stantweight(∼24h),andthewatervaporpermeability(WVP)test wasperformedbyusingamodifiedASTMStandardmethod(ASTM E96-95,1995)at25±2^◦C.Filmsamplesweresealedoverthecircu- laropeningofapermeationcellcontainingsilicagel,andthecells werethenplacedindesiccatorscontainingdistilledwater.After thesampleshadreachedsteady-stateconditions(∼20h),thecell wasweighedevery1hfor9honananalyticalscale.WVPwascal- culatedasWVP=w.x/t.A.P,where“x”wastheaveragethickness ofthefilms,“A”wasthepermeationarea(0.00196m²),“P”was thedifferencebetweenthepartialpressureoftheatmosphereover silicagelandoverpurewater(3.168kPa,at25^◦C),andtheterm w/twascalculatedbylinearregressionfromdataofweightgain asafunctionoftime.Thesolubilityinwater,WVP,andmoisture contentwereanalyzedintriplicate.

2.6. Statisticalanalyses

Experimental data were statistically evaluated by Fisher or Tukey’stestwithsignificancesetatp<0.05.ThesoftwareStatis- tica7.0(StatSoftInc,Tulsa,USA)wasemployed.

3. Resultsanddiscussion

3.1. Chemicaltreatmentofsoybeanstraw 3.1.1. Chemicalcomposition

Theuntreatedsoybean strawcontained39.8%cellulose,2.3%

solublelignin,10.5%insolublelignin,and22.6%polysaccharides, includinghemicelluloses,whichagreedwithvaluesreportedinthe

literature(Wang,Mo,Sun,&Wang,2007).Theligninandhemi- cellulosescontentsdecreasedandthecellulosefractionincreased afterthechemicaltreatments(Table1).

Treatments with high concentration of NaOH (17.5%) and bleachingwithNaClO2orH2O2(T2andT4)yieldedsampleswith highcelluloseandlowhemicellulosesandlignincontentregard- lessofthetreatmentconditions(severeormild).Severalauthors have reportedthatbasic conditions(alkalitreatment)favor the removalofhemicellulosesduetocleavageoftheester-linkedsub- stances(Deepaetal.,2011;Kalleletal.,2016;Xiao,Sun,&Sun, 2001).Kallelet al.(2016)also observedthatthelignin content in thefibers ofgarlic strawremained practicallyconstant after alkalitreatment.Toremovetheligninresidues,theauthorscon- ductedbleachingwithsodiumchloride,whichresultedinalmost purecellulosefibers.Inourstudy,bleachingwithH2O2(T3andT4) generatedfiberswithslightsuperiordelignificationascompared tobleachingwithsodiumchloride.Thefractionofacid-insoluble lignindecreased from10.5%to3.6and3.5%,respectively,while solubleligninreducedfrom2.3%toapproximately0.7%.Khorvash etal.(2010)observedthatH₂O₂removedacid-insolubleligninfrom soybeanstrawmoreeffectivelythantreatmentbasedonsodium hypochlorite,whichsuggestedthatbleachingwithH₂O₂improved theinvitrodigestibilityofsoybeanstraw.Songetal.(2016)also reportedthattheacid-insolublelignincontentdecreasedinbam- boopretreatedwithalkalihydrogenperoxide.Thisbehaviorwas attributedtotheoxidationofligninstructuresbyproductsfrom H₂O₂decomposition,suchashydroxylradicals(HO⁻)andsuper- oxideanionradicals(O2−•).

3.1.2. ParticlesizeandfibermorphologybySEM

Chemicaltreatmentremovedhemicellulosesand ligninfrom thefibers,resultinginparticleswithdifferentsizeandhighlyreac- tivesurfacesascomparedtountreatedsoybeanstraw(Table1).

Soybeanstraw(SS)treatedwithNaOHandH2O2tendedtopresent large particle size (T4=122␮m and T3=145␮m), whereas SS treatedwithNaOHandNaClO₂ hadsmalleraverageparticlesize (T2=88␮mandT1=108␮m).However,wedidnotverifysignifi- cantmorphologicaldifferences.Thisbehaviorisinagreementwith thebehaviordescribedbyBrígida,Calado,Gonc¸alves,andCoelho (2010)whentheytreatedcoconutfibers.Largerparticlesizecould indicateformationofagglomeratesduringmildtreatment, asin thecasewhenH₂O₂wasusedasreactivebleachingagent.Aspre- viouslydiscussed,NaOHassociatedwithH2O2removedligninthe mostefficiently.TheoxidationofligninstructuresbyH2O2yielded fibersinwhichmorehydroxylgroupswereavailableforhydrogen inter-chainbonding,sotheparticlesweremorepronetoagglom- eration.Thiswasmainlyaresultoftheirsoftercationicpotential (Brígidaetal.,2010;Songetal.,2016).

Fig.2showstheSEMmicrographsofuntreatedandchemically treated(T1,T2,T3,andT4)SS.Theuntreatedfibercontainedseveral cellulosemicrofibrilsorganizedinahighlyorderedparallelfashion featuringparticlesinatightlypackedconformation(Fig.2A).Non- treatedSSpossessedahigheraverageparticlediameter(∼100␮m) as compared tochemicallytreated SS,which inturnpresented isolatedmicrofibrilsinafreeconformationwithdifferentdimen-

Fig.2. SEMmicrographsof(A)untreatedsoybeanstrawandofsoybeanstrawafterthedifferentchemicaltreatments(B)T1,(C)T2,(D)T3,and(E)T4.

sions(diametersrangingfrom0.4to10␮mandseveralmicronsin length)(Figs.2B–E),aresultofthereducedligninandhemicellu- losescontentpromotedbythechemicaltreatments(Table1).The alkalitreatmentswithH2O2orNaClO2modifiedthemorphologyof thefiberbydestroyingsomecelltissuesandreducingtheconnec- tionsbetweenthecells,aconsequenceofthepartialremovalofthe externalnon-cellulosiclayercomposedofhemicelluloses,lignin, wax,andotherimpurities(Kalleletal.,2016).Thebleachingstep openedandsubsequentlydefibrillatedSS,whichhasatendencyto agglomerateintobundles.Thisconfirmedthelargerparticlesize measuredforthesamplesSS-T3andSS-T4.

3.1.3. XRDanalysisoffibercrystallinity

Fig.3presentsthediffractionpatternsofuntreatedandchem- ically treated SS. The patterns were typical of semicrystalline cellulosicmaterialswithanamorphousbroadbandanddefined crystallinepeaks.Inalloftheanalyzedsamples,thediffractogram profileindicatedthatcellulosetypeIpredominated,withamain diffractionintensityatabout2␪=23^◦(plane002)andidentifiable peaksat2␪=17^◦ (plane101)and34^◦ (plane004).In celluloseI, thecrystallinestructureconsistsoftwocoexistingcrystalphases:

celluloseI␣(triclinicunitcell)andcelluloseI␤(monoclinicunit cell), whicharearranged inparallel chains(Nishiyama,Langan,

&Chanzy,2002).Thecellulosemicrofibrilsimpartcrystallinityto thefibersbecausehemicellulosesandligninareamorphouscom- pounds.

Insamplestreatedwithhigherconcentrationsofalkali(T2and T4),theresultingcrystallinestructureconsistedof amixtureof polymorphsofcelluloseIandcelluloseII.Thetypicaldiffractions peaksat2␪=12^◦(plane101),20^◦(plane101),and22^◦(plane002)

Fig.3.X-raydiffractogramsandcrystallineindexofuntreatedSSandtreatedSS samples(T1,T2,T3,andT4)asestimatedbytheSegalequation-XRDpeakheight (Icr(S))andbytheXRDdeconvolutionmethod(Icr(D)).

confirmedthepresenceofcellulose typeII(Kafle,Greeson,Lee,

&Kim,2014;Yueetal.,2015).Themaindifferencebetweencel- luloseallomorphsisthestructure oftheirunit cell:incellulose typeI,thecellulosemoleculesarealignedinparallel,whilstthe crystalsarearrangedinanantiparallel fashionincellulosetype II.CelluloseIIhasamorestablestructureandcanoriginatefrom treatmentwithalkali(FlauzinoNeto,Silverio,Dantas,&Pasquini, 2013;Kafleetal.,2014;Yueetal.,2015)orfromre-precipitation afterhydrolysisinconcentratedsolutionofsulfuricacid(Flauzino Netoetal.,2013;Silva&D’Almeida,2009).Becausebleachingtreat- mentsalonecannotgeneratecellulosetypeII,modificationstothe

M.Martelli-Tosietal./CarbohydratePolymers157(2017)512–520 517

0 100 200 300 400 500 600

DT G (a .u. )

Temperature (

C)

0 100 200 300 400 500 600

0 20 40 60 80 100

M ass L o ss (%)

Temperature (

C)

(A)

(B)

50^oC 210^oC 330^oC

305^oC 440^oC

c

a e

d b

c

b e

d a

Ma ss (% )

Fig.4.(A)TGAand(B)DTGscansof(a)untreatedsoybeanstrawandsoybeanstraw afterchemicaltreatments(b)T1,(c)T2,(d)T3,and(e)T4,asdescribedinFig.1.

crystallinestructureofcellulosestemmainlyfromthehighercon- centrationsofalkali(Songetal.,2016).

Theresultingcrystallinityindexesarepresentedalongsidethe curves.Themethodsusedtoestimatethecrystallinityindex(I_cr(S) orI_cr(D))affordeddifferentvalues(Fig.3).Themethodbasedon theheightoftheXRDpeakheightgavehigherI_crthanthemethod basedondeconvolutionoftheXRDpeak.Groundsoybeanstraw presentedI_cr(S)of58%,whichagreedwithvaluesreportedinthe literature (Alemdar &Sain, 2008)for thecrystallinity of native celluloseestimatedbytheSegalequation(Parketal.,2010).Con- versely,themethodbasedondeconvolutionoftheXRDpeakhas beenwidelyusedtoanalyzethestructureofcelluloseIIstructure becausetheSegalrelationdoesnottakesomepeaks(12^◦and20^◦) intoaccount.TheXRDdeconvolutionmethodisthereforeconsid- eredmoreappropriatetocomparethetreatmentsappliedtoSS.

For the chemically treated samples, the crystalline fraction increased as the amorphous constituents were removed. I_cr(D) rangedfrom43.6%(untreatedsamples)upto56.4%(samplesT4).

Thedifferenttreatmentsgave I_cr(D) valuesthatdecreased inthe following order: T4≥T2>T3>T1>Untreated samples. The XRD resultsagreedwiththechemicalcomposition(Table1):samples withhighercelluloseandlowerhemicellulosecontents(T2and T4)hadhighercrystallinity.Thesesamplesweretreatedwithhigh concentrationofNaOH,andthebleachingagentapparentlydidnot affecttheseresults.

3.1.4. Thermalstabilityofthefiber

Fig.4A andBdisplaysthethermogravimetricanalysis(TGA) andthecorrespondingderivative(DTG)curvesofSS,respectively.

400035003000 2500 2000 1500 1000 500

965

Wavenumber (cm

)

Untreated T1 T2 T3 T4

3343 2906

17271598

1513 1420

1314 1162

1102 1927

895

Fig.5. ATR-FTIRspectraofuntreatedsoybeanstraw(untreated)andafterchemical treatmentsT1,T2,T3,andT4.

UntreatedSS presentedtwo distinctdecomposition events. The first,withmaximumintensityat50^◦C,masslossof7.6%,corre- spondedtodesorptionofphysicallyandchemicallyboundwater andvolatiles.Thesecondeventoccurredbetween230and350^◦C, withmaximum at305^◦C, andreferred tothethermo-oxidative reactionofthemainorganiccompounds(decompositionoflignin, hemicelluloses,andmainlycellulose)withoverallmasslossaround 60%.Theshoulderidentifiedat210^◦CintheDTGcurve(Fig.4B) wasconsidered thestartof hemicellulosedegradation(Antal&

Varhegyi,1995).Attemperatureshigherthan400^◦C,theweight droppedsignificantly,generatingsolidresiduessuchasashesand inorganicmaterials(around20%oftheinitialmass).

For thefirst massloss event,thetreated samplespresented similarmasslossvalues(7.3%),exceptforSStreatedwithNaOH 17.5%andH₂O₂(T4),whichexperiencedmasslossof10.1%.Higher exposureofcelluloseprobablyaccountedforthisobservation,as reportedbyBrígidaetal.(2010)andSilva,Souza,Machado,and Hourston(2000).Consequently,thesesamplespresentedcellulose withhighercrystallinity,asobservedbySEMandXRD.Moreover, thechemicaltreatmentschangedthethermalcharacteristicsand degradationofthelignocellulosematerials.Theinitialmassloss wasfollowedbyanarrowerandmoremarkedeventwithmaxi- mumcenteredaround330^◦C,primarilyattributedtodegradation ofcellulose(Rosaetal.,2009).Forthetreatedsamples,thismain decompositioneventshiftedtohighertemperaturesduetopre- viouschemicalremovalofpartofthehemicellulosespresentin thefibers(Table1), whichincreasedthethermalstabilityofthe fiber(Bismarcketal.,2001;Brígidaetal.,2010;Rosaetal.,2009).

Atthisstage,theaveragemasslosswas68%,whichwasascribed todegradationofhemicelluloseandcellulosegiventhatthisfig- urewasnotfarfromthemeasuredfractionofhemicellulosesplus cellulosefoundinthetreatedsamples(Table1).

Thethirdadditionalevent(notobservedforrawgroundmate- rials) occurredfrom380to500^◦C, withmaximumat 440^◦C. It wasascribedsolelytothedegradationoflignin(themoststableof thethreecompounds).Abovethistemperature,pyrolysisoccurred, whichwasassociatedwithreleaseofcarbondioxideandconse- quentreductionoftheresidualmass.

3.1.5. ATR-FTIRspectroscopy

Aspreviouslydiscussed,thechemicalcompositionofthefibers varieddependingontheappliedtreatment.Comparisonbetween theinfraredspectraofuntreatedandtreatedfibers(Fig.5)helped

Table2

Mechanicalproperties,moisture,solubilityinwater,andwatervaporpermeability(WVP)ofSPI/soybeanstrawcompositefilms.

Film^* Thickness

(␮m)

Tensilestrength (MPa)

Elongationat break(%)

Young’smodulus (MPa)

Moisture (%)

Solubilityin water(%)

WVP (10⁻¹⁰g/msPa)

SPI 55±11^c 5.6±0.7^cd 30±9^ab 298±52^c 15.3±0.5^a 63±5^a 12.2±0.2^a

SPI/soybeanstraw(untreated) 66±13^bc 4.6±0.4^d 35±12^b 270±31^c 15.9±0.4^a 61±9^a 9.8±1.2^a

SPI/soybeanstrawT1 63±11^bc 7.1±0.7^b 20±5^ac 392±32^b 13.2±0.2^bc 44±6^ab 10.9±1.5^a

SPI/soybeanstrawT2 81±17^ab 5.8±0.6^c 18±6^c 314±19^c 12.8±0.5^b 45±7^ab 9.9±1.6^a

SPI/soybeanstrawT3 83±16^ab 9.0±0.7^a 15±5^c 521±72^a 14.1±0.1^c 46±4^b 11.0±1.0^a

SPI/soybeanstrawT4 60±11^c 7.8±0.8^b 17±5^c 527±37^a 13.4±0.4^bc 36±3^b 12.8±2.1^a

a–eMeanswithdifferentsuperscriptlettersinthesamecolumnarestatisticallydifferent(Fisher,p<0.05).

*Allformulationscontained5%SPI(w/w),andtheaddeduntreatedsoybeanstraworchemicallytreatedsoybeanstraw(T1,T2,T3andT4)correspondedto1%(g/100gof SPI).

toidentifysomevariations.Allthespectrawererather similar, indicating that an analogous structure predominated in allthe samples.Ingeneral,thespectrawererepresentativeofthevibra- tionalpatternsofpolysaccharideswithbands typicallyassigned to cellulose and hemicelluloses and which have been widely interpretedintheliterature.Acommonbroadbandcenteredat 3343cm⁻¹correspondedtothestretchingvibrationof OHgroups, whichiscommoninhydrophilicmaterials.Thepeaksidentifiedat around2906cm⁻¹referredtosymmetricandasymmetricCHgroup stretching. According to Andrade-Mahecha et al. (2015), these peaksindicatethepresenceoflignin.Asmallbandat1727cm⁻¹ (bestobservedinthespectrumoftheuntreatedSS)wasattributed tothecarbonylgroupsoftheuronicestergroupsofhemicelluloses ortotheesterlinkageofthecarboxylicgroupoftheferulicand p-coumericacidsofligninand/orhemicelluloses(Alemdar&Sain, 2008;Andrade-Mahechaetal.,2015).Thisbanddiminishedoreven disappearedinthetreatedsamples,indicatingthatthechemical treatmentsremovedligninandhemicelluloses,inaccordance to thechemicalcompositionshowninTable1.Thepeakat1598cm⁻¹ wasstronglyassociatedwiththeCOstretchingmodeofthelignin aromaticring.Areductioninitsintensityand/oritsshifttohigher wavelengthswasrelatedtoscissionormodificationofthearomatic ringandcorrespondedtodelignification(Lietal.,2010).

Themultipleabsorbancepeaksbetween1420and1162cm⁻¹ wereduetotypicalvibrationsofprimaryandsecondaryhydroxyl bendingincellulose (Liu, Yu,&Huang,2005).Thepeaksinthe regionbetween1102and965cm⁻¹referredtoCH,HCH,andOCH deformation.Themostintensepeakcenteredat1927cm⁻¹ was assignedtoCHasymmetricvibrationofcellulose.Thispeakbecame sharperafterthetreatments,particularlyinthecaseofT2,which wasthetreatmentthatyieldedthemostcellulose.Themostread- ily identifiabledifferences among thesampleswasthebandat 1162cm⁻¹,correspondingtotheasymmetricstretchingoftheC- O-CglycosideandC-O-C␤-1,4glycosyllinkageofcellulose(Liu,Yu etal.,2005;Michell,1990).Thesedifferenceswereclearlyevident inthespectraofthefibersresultingfromthemoreseveretreat- ments(T1andT2).Additionally,thepeakcenteredat895cm⁻¹was ascribedtotheglycosidiclinkagesoftheglucoseringincellulose (Alriols,Blanco,Mondragon,&Labidi,2009;Chirayiletal.,2014).

Thispeakemergedinthespectraofallthesamples.Intheuntreated SS,thispeakoverlappedwiththebroadpeakduetoCHdeformation (centeredat1927cm⁻¹)ofthestructureof cellulose.Thetreat- mentsdefibrillatedcellulose.Consequently,thebandsnarrowed andbecamemoredefined,allowingforbettervisualizationofthe contributionoftheremaininghemicelluloses.

3.2. SPI/soybeanstrawcompositefilms:mechanicalandphysical properties

Table2summarizesthemechanicalproperties(tensilestrength, elongationat break,and Young’s modulus),moisture,solubility inwater,andwater vaporpermeability (WVP)ofSPIfilmscon-

taininguntreatedandchemicallytreatedSS.Thethicknessofthe SPI/soybeanstrawcompositefilmsrangedfrom60to83␮m.Addi- tionofuntreatedSStothesoyproteinfilmsdidnotimprovethe mechanicalpropertiesofthefilm.However,incorporationofchem- icallytreatedfibersintoSPIfilmspromotedhighertensilestrength (TS)and Young’s modulus (YM), butlower elongation atbreak (EB)ascomparedtothecontrolfilm.Thiseffectwasmoreevident whensoybeanstrawsubmittedtoT3wasaddedtothefilm.Inthis case, TSincreasedfrom5.6±0.7 to9.0±0.7MPa,YMrose from 298±52MPato521±72MPa,andelongationatbreakdecreased by50%(from30to15%).AdditionofSStreatedbyT1andT4had practicallythesameeffectonthemechanicalproperties,except thatT4improvedtheYMvaluesmoresignificantly,asreflectedon thestiffnessofthefilm.Fiberstreatedwithdifferentconcentra- tionsofNaOH(1%or2%)andpurecellulosefibersimprovedthe mechanicalpropertiesoffiber-reinforcedcompositessignificantly (Cho,Skrifvars,Hemanathan,Mahimaisenan,&Adekunle,2014).

Liu,Misra,Askeland,Drzal,andMohanty(2005)verifiedthesame resultwhentheyevaluatedIndiangrassfiberstreatedwithhigh concentrationofalkaliasfillerinsoy-basedbiocomposites.

The removal of hemicelluloses by alkali treatment and the removalofligninbybleachingreactioncanfavor theformation ofintermolecularhydrogenbondsbetweenthehydroxylandcar- bonylgroups oftheSPI matrixwiththehydroxylgroups ofthe treatedSS,toyieldanetworkwithbettermechanicalresistance (Husseinsyahetal.,2014).

IncorporationofuntreatedSSdidnotmodifythesolubilityor themoisturecontentofcomposite films.Comparedtothecon- trol,compositefilmscontainingchemicallytreatedSShadlower moistureandsolubility.Additionoftreatedfibers reducedboth thesolubilityandthemoisturecontentbyaround42%and13%, respectively,becausestrongerbondsprobablyemergedbetween thehydroxylandcarbonylgroupsoftheSPImatrixandthehydroxyl groupsoftreatedSS.SPIcontainsmanypolaraminoacids,which resultsinfilmsthataremoresusceptibletohighmoisture(Yarin, Pourdeyhimi,&Ramakrishna,2014).TheaddedtreatedSScould interactwiththesegroups,todecreasethewateraffinityofthe matrix.Watercanactasplasticizer,andthepresenceoffewerwater moleculesinaSPI/SScompositereducestheelongationatbreakof thesefilms,asobservedin Table2.Reductionintheamountof hydrophilicgroupsavailabletobindwiththewatermoleculesren- dersamorestablethree-dimensionalnetwork.AdditionofSS-T4 totheSPIfilmledtothelowestsolubilitybecausethissoybean strawsamplehadthelowestcontentofhemicellulosesandinsolu- blelignin(Table1).Kumar,Choudhary,Mishra,andVarma(2008) reportedsimilar resultsfor water affinityafter theyintroduced alkali-treatedbananafibersintoanSPImatrix.

Additionof1%(w/w)offibersasreinforcingfillerdidnotreduce WVPasexpected.Althoughthepresenceoffibersinthematrix usuallyincreasetortuosityandconsequentlyreducefilmperme- ability (Jensenet al.,2015), the amountof fibers addedherein didnotchangethepermeabilitysignificantlyasjudgedfromthe

M.Martelli-Tosietal./CarbohydratePolymers157(2017)512–520 519

resultsofTukey’stest(p>0.05)summarizedinTable2.Permeabil- itystronglydependsonfilmporosity.Theirregularincorporationof cellulosefibersintoproteinfilmscouldgenerateinternalcracksand pores,asreportedbyPereda,Amica,Rácz,andMarcovich(2011)for caseinate-basedfilmsreinforcedwithcellulosefibers.Duringthe dryingprocess,fiberscanactasnucleationsiteswherebubblescan grow,reducingthewatervaporbarrierproperties.

4. Conclusions

Chemicaltreatmentsofsoybeanstrawincludingreactionwith alkaliandbleachingyieldedmaterialswithhighercellulosecon- tentasconfirmedbyTGAandFTIRanalysis.Thecellulosecontent inthefiberswasattributedtoeffectiveremovalofhemicelluloses, lignin,andotherinertandinorganicmaterials.Thedegreeofcel- lulosecrystallinitywasincreasedastheamorphousconstituents wereremoved duringthe treatments.During thesetreatments, theuseof highconcentrationofNaOH contributedtotheallo- morphtransitionfromcelluloseItoII,regardlessofthebleaching agent.Thesespecificdifferencescaninfluencetheapplicationofthe resultingfibers.ThetreatedSSperformedbetterasreinforcement fillerinSPIfilms.Thefillerincreasedthemechanicalresistanceof thefilmbecauseit promotedstronger interactionsbetweenthe polargroupsintheproteinandthehydroxylfunctionalitiesinthe treatedfibers.Thesoybeanstrawtreatedwithhydrogenperoxide affectedboththemechanicalpropertiesandthesolubilityofthe film,mainlyduetotheremovalof lignin.Theadditionof1%of fibersdidnotmodifythewatervaporpermeabilityofthefilmsig- nificantly.Differentpercentagesoffillersshouldbeevaluatedfor aneffectivereductioninWVPvalues.

Acknowledgments

TheauthorsgratefullyacknowledgethefundingagencyFAPESP (SãoPaulo,Brazil)forthepostdoctoralfellowshipgrantedtoMilena Martelli Tosi (2012/22154-7) and CNPq for the IC fellowships grantedtoNataliaC.SilvaandBrunoS.ExpostoTheauthorswould liketoexpresstheirgratitudetoEMBRAPASOJAforprovidingsoy- beanstraw.

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