cellulose fi lm blends Effect of xanthan gum on the physical and mechanical properties ofgelatin-carboxymethyl Food Packaging and Shelf Life

Effect of xanthan gum on the physical and mechanical properties of gelatin-carboxymethyl cellulose fi lm blends

M.A.S.P. Nur Hazirah

^a

, M.I.N. Isa

^b

, N.M. Sarbon

^a,

*

aSchoolofFoodScienceandTechnology,UniversitiMalaysiaTerengganu,21030KualaTerengganu,Terengganu,Malaysia

bSchoolofFundamentalScience,UniversitiMalaysiaTerengganu,21030KualaTerengganu,Terengganu,Malaysia

ARTICLE INFO Articlehistory:

Received27August2015

Receivedinrevisedform4May2016 Accepted20May2016

Availableonline5June2016 Keywords:

Gelatin

Carboxymethylcellulose Xanthangum Blendedfilms Physicalproperties Mechanicalproperties

ABSTRACT

Theaimofthisstudyistodevelopcompositeediblefilmsfromthreedifferentpolymerstoinducecross- linkreactionsthatimprovedthequalityoffilmsmadefromtwopolymertypes.Gelatin-carboxymethyl cellulose (CMC)-xanthangumfilmswerepreparedbycastingto studyeffectsfromtheadditionof differentconcentrations(0,5,10,15,20and25%,w/wsolid)ofxanthangumtogelatin-CMCfilm.Physical andmechanicalpropertiesoftherespectivefilmswereevaluated.Theadditionofxanthangumincreased the thickness, moisture content and water vapour permeability of gelatin-CMC film (p<0.05).

Furthermore,Ultraviolet(UV)lightshieldingincreasedalongwithreducedvisiblelighttransparency (p<0.05)andincreasedthermalstability(Tg)(p<0.05).Nonewfunctionalgroupsformedalthough slightshiftsinintensityvaluesbyFourierTransformInfrared(FTIR)spectroscopywereobserved.X-ray diffraction(XRD)analysisshowedadiminishedcrystallinepeak.Theresultedfilmsalsodemonstrated lowertensilestrengthwithdiminishedelongationatthebreakpoint,aswellashigherpunctureforce andlowerpuncturedeformation,indicatinghigherpunctureresistancethancomparablegelatin-CMC film.Overall,gelatin-CMCfilmwithxanthangum(5%,w/wsolid)demonstratedimprovedphysicaland mechanicalpropertiesmorethanfilmspreparedfromcomparableformulations.

ã2016ElsevierLtd.Allrightsreserved.

1.Introduction

Food packaging is a crucial step in food manufacturing.

Packaging contains and protects foods from physical damage and also from biological and chemical deterioration while providingmultipleconveniencesforconsumers,includingbrand identityandcontents.(Kim,Min,&Kim,2014).

Presently, packaging films are used for perishable produce, meatandfish.Plasticfilmsmadefromsyntheticpolymershave beenincreasinglyusedforfoodpackagingduetotheirlowprice, easymouldingandsuperiormechanicalandbarrierproperties(Jia, Fang,&Yao,2009).However,theyarenon-degradableandnon- renewableandcauseseriousenvironmentalwasteandpollution (Mu,Guo,Li,Lin,&Li,2012).Consequently,biodegradableedible filmshavebeendevelopedasalternativepackagingmaterialsand areofgreatinterestformanyresearchers.Ediblefilmsaremade frompolysaccharidessuchascellulosederivatives,chitosan,starch and various vegetable and microbial gums; proteins such as

gelatin,cornzein,wheatglutenandsoyprotein;andlipidssuchas waxes,fattyacidsandresins(Bourtoom,2008).

Protein derivatives are the most attractive biopolymers for edible film formulations becausethey provide highnutritional value, superior mechanical properties and exhibit the most impressive O2 gas barrier (Ou, Kwok, & Kang, 2004). Gelatin, obtainedbythecontrolledhydrolysisofinsolublefibrouscollagen componentsof skin,bonesand connectivetissuesgenerated as wasteduringanimalslaughteringandprocessing(Guo,Ge,Li,Mu,

&Li,2014),ispresentlythemostpreferableproteinderivativeas thebasematerialforformulatingediblefilms.Thisisduetoits natural abundance, biodegradability, low cost and excellent functional and filmogenic properties (Arvanitoyannis, 2002).

Gelatin-basedfilmsarealsothin,flexibleand usefulforseveral food packaging applications, including drug delivery (Boanini, Rubini, Panzavolta,& Bigi, 2010). However, gelatin-basedfilms alone present several problems that limit food packaging applications because they are brittle, have poor water vapour resistance, poor thermal stability and absorb moisture (Bigi, Cojazzi, Panzavolta, Roveri, & Rubini, 2002). The addition of plasticizerssuchasglycerolandsorbitolhasbeenshowntoreduce brittleness but at the same time, increase water permeability (Sobral,Menegalli,Hubinger,&Roques,2001).

* Correspondingauthor.

E-mailaddresses:norizah@umt.edu.my,norizahsarbon@yahoo.com (N.M. Sarbon).

http://dx.doi.org/10.1016/j.fpsl.2016.05.008 2214-2894/ã2016ElsevierLtd.Allrightsreserved.

ContentslistsavailableatScienceDirect

Food Packaging and Shelf Life

j o u r n a l h o m e p a g e : h t t p : / / w w w . e l s ev i er . c o m / l o c a t e/ fp s l

Crosslinkingtechniquesintroducedinthepreparationoffilms forms intermolecular bonds between polymer chains (Hager, Vallons, & Arendt, 2012) which enhances water resistance, cohesion,rigidityandmechanicalstrength(Tropini,Lens,Mulder,

&Silvestre,2004).Theblendingofoneormorepolymertypescan enhancecrosslinking.Studieshaveshownimprovedgelatinfilm properties by blending with carboxymethyl cellulose (CMC) (Wiwatwongwana&Pattana,2010)orxanthangum(Guoetal., 2014).

CMCisawatersolublecellulosederivativewithanioniclinear

b

^-(1!4)-linkedglycopyranoseresidue(Su,Huang,Yuan,Wang,&

Li,2010)producedbythepartialsubstitutionof2,3,and6cellulose hydroxylgroups by carboxymethyl groups (Tong, Xiao, & Lim, 2008).Researchersarehighlymotivatedtoproduceediblefilms from CMC due to their ability to form a continuous matrix (Ghanbarzadeh&Almasi,2011).Incontrasttogelatinfilms,CMC- based films are also easily water soluble as it contains a hydrophobic polysaccharide backbone and many hydrophilic carboxylgroups(Suetal.,2010).CMCalsoimprovesproteinfilm mechanical properties by increasing thermal stability and the elasticitymodulus(Wiwatwongwana&Pattana,2010)asshown bytheblendingofsoyproteinisolatefilmswithCMC(Suetal., 2010).

Xanthangumisapentasaccharide.Itisaheteropolysaccharide which consists of D-glucose, D-mannose and D-glucuronic acid unitsthatisderivedfrombacteriaandfungi(Sworn,2000).Itis producedbysubmergedaerobicfermentationofa pureXantho- monas campestris culture after undergoing submerged aerobic fermentation (Guo et al., 2014). Xanthan gum’s film forming propertiescomprise pseudo-plastic rheological behaviourin an aqueousenvironmentthatisamenabletofilmfabricationbecause itisreadilydispersedincoldorhotwaterwithverylittleeffecton itsviscosityfromeithertemperatureorpH(Baldwin,Hagenmaier,

&Bai,2012).Gelatinfilmsblendedwithxanthangumproducea verytransparentfilmwithexcellentultraviolet light resistance, lowtotalsolublematterandmoisturecontent,lowwatervapour permeability, improved mechanical properties and thermal stability(Guoetal.,2014).

Gelatin based film generally hasgood functional properties, however it has several problems that limit application as packagingmaterialsas reported in several studies.It is brittle, haspoorwatervapourbarrier, thermalstabilityand canabsorb highmoisture(Bigietal.,2002).Besides,itsmechanicalstrengthis alsolowerthanthatofsyntheticpolymersfilm(Bourtoom,2008).

Addition of plasticizers like glycerol and sorbitol reduced the brittlenessoffilmsbuttheyincreasedwaterpermeabilityofthe film(Sobraletal.,2001).Inaddition,crosslinkingmethodthathas beenintroducedtoimprove thefunctional propertiesof edible filmsstillhassomelimitationswasreportedbyseveralresearchers

where; physical crosslinking is difficult to obtain the desired amount of crosslinking (Yao, Liu, Chang, Hsu, & Chen, 2004), chemicalcrosslinkingcanleadtotoxicityproblemthatmakethe film produced is no longer edible (Cao, Fu, & He, 2007) and enzymaticcrosslinkinghaslimitedavailabilityandhighproduc- tioncost(Galietta,diGioia,Guilbert,&Cuq,1998).Therefore,other typesofnaturalandbiodegradablecross-linkersthatarefreefrom theproblemsmentionedabovehavetobeusedasthealternative.

Xanthan gumwasreportedtobecompatible cross-linkertobe blendedwithvariousmaterials;itmaydissolvesdirectlyinmany highly acidic, alkaline, alcoholic systems containing different components. It is also compatible withcommercially available thickeners such as sodium alginate, carboxymethyl cellulose (CMC) and starch (Sharma, Naresh, Dhuldhoya, Merchant, &

Merchant,2006).Hence,thepresentworkusedxanthangumas a crosslinking agent to form a potentially new natural and biodegradablecompositefilmasanalternativematerialforfood packagingindustry.Therefore,theobjectivesofthisstudywereto formulategelatin-CMC-xanthangumblendedfilmsanddetermine thephysicalandmechanicalpropertiesatdifferentconcentrations ofxanthangumadded.

2.Materialandmethods

2.1.Materials

Bovine skin gelatin (Type B, 225 Bloom), carboxymethyl cellulose (CMC), xanthan gum and glycerol (plasticizer) were purchasedfromSigmaAldrich(St.Louis,MO,USA).

2.2.Methods

2.2.1.Filmpreparation

The film forming solutions were prepared according to the methoddescribedbyJahitetal.(2016),withsomemodification.

Thegelatin solutionwas preparedbydissolvinggelatin powder (80%,w/wsolid)indistilledwateratroomtemperaturefor30min followedbyheatingat50Cfor20minundercontinuousstirring.

TheCMCsolutionwaspreparedbydissolvingCMCpowder(20%, w/wsolid)indistilledwaterwhilestirringat50Cfor30min.The xanthangumsolutionwaspreparedbydissolvingxanthangum powder(5,10,15,20and25%,w/wsolid)indistilledwaterwhile stirringfor30minatroomtemperature(Arismendietal.,2013).

Thepreparedthreeseparate solutionsweremixedandblended togetherwithglycerol(30%,w/wsolid).Theblendingmechanism wasdemonstratedinTable1.Themixturewasconstantlystirredat 50Cfor20minforgelatinization.Approximately25mlofeach filmformingsolutionwascastonaPetridishandovendriedat 45Cfor48h.

Table1

FormulationoffilmformingsolutionofgelatinbasedfilmblendedwithCMCandxanthangum(gelatin/CMC/xanthangum).

Filmformulations Compositionoffilmformingsolution

Gelatin(g) CMC(g) Xanthangum(g) Glycerol(g) Water(ml)

Ctrl-control (80/20/0)

3.2 0.8 – 1.2 100

AXanthangum5%

(80/20/5)

3.2 0.8 0.2 1.2 100

BXanthangum10%

(80/20/10)

3.2 0.8 0.4 1.2 100

CXanthangum15%

(80/20/15)

3.2 0.8 0.6 1.2 100

DXanthangum20%

(80/20/20)

3.2 0.8 0.8 1.2 100

EXanthangum25%

(80/20/25)

3.2 0.8 1.0 1.2 100

2.2.2.Filmthicknessmeasurements

Filmthicknesswasmeasuredbydigitalmicrometer(Digimatic Micrometer 406-350, Mitutoyo Corp., Japan) to the nearest of 0.001mm.Fivemeasurementsweretakenforeachfilm:oneatthe sample’scentreandfourmoreatvariedperimetersites.Anaverage valuewascalculated.

2.2.3.Determinationofmoisturecontent

Astandardoven-drymethodwasusedtodeterminemoisture content.Approximately1cm²ofcutfilmwasweighed(0.0001g) andovendriedat105Cfor24handthenre-weighed(0.0001g).

Moisturecontent(MC)wascalculatedasthepercentageofdried weightlossasfollows:

MoistureContentð%Þ¼w1w2

w1 100 ð1Þ

wherew1wasinitialweight(g)andw2wassample’sdryweight.

All final determinations were recorded as the mean of three measurements.

2.2.4.Lighttransmissionandtransparency

Ultraviolet(UV)andvisible(vis)lightbarrierpropertieswere measuredusing a UV–vis spectrophotometer (50Probe, Cary¹, USA).Filmstripsof1cm4cmwerecutandplaceddirectlyintoa test cell. Transmittance at selected wavelengths (200–800nm) weremeasured.Anemptycelltestwasusedasreference.

Filmtransparencywascalculatedasfollows:

Transparency=logT/x (2)

whereTistransmission(%)at600nmandxisfilmthickness(mm) (Han &Floros,1997). Alldeterminations were recorded as the meanofthreemeasurements.

2.2.5.Determinationofwatervapourpermeability(WVP)

Water vapour permeability (WVP) was measured following ASTMmethod().Filmstripsof2.5cm2.5cmweremountedonto a clean dry plastic cup containing 10g of silica desiccant.The surface area of the film covering the cup and the weight (0.0001g) of each coveredcup weremeasured. Eachcovered cupwas storedina desiccatorwithdistilled water. Theweight (0.0001g)ofthecoveredcupwasmeasureddailyforsevendays.

WVPwascalculatedasfollows:

WVPðgmm=m²dPaÞ¼ wx

AtðP2P1Þ ð3Þ

where (w)is theweight gain of the cup (g); x is averagefilm thickness (mm); (A) is the film surface area exposed to the permeant (m²); (t) is the time of gain (d); and (P1–P2) is the differencebetweenthepartialatmosphericvapourpressurewith silicadesiccantand purewater (2800Paat 24C)(Pa).Allfinal determinations were recorded as the mean of three measure- ments.

2.2.6.Thermalanalysisbydifferentialscanningcalorimeter(DSC) Thermalproperties were determined byusing a differential scanning calorimeter (DSC) (DSC, TA Q2000 Instrument, USA).

Approximately5mg(0.001mg)offilmwascutintosmallpieces and sealed in an aluminium pan. Each sample was heated at 10Cmin¹from30to200C.Anemptyaluminiumpanwasused asreference.Theglasstransitiontemperature(Tg,C)wasobtained fromthethermogram.Alldeterminationswererecordedas the meanofthreemeasurements.

2.2.7.StructuralanalysisbyFouriertransforminfrared(FTIR) spectrometer

The secondary structures of the prepared films via the attenuatedtotalreflection(ATR)modeoftheFourierTransform Infrared (FTIR)spectrometer (Nicolet is10, ThermoNicolet380, USA), were examined. Filmstrips of approximately 1cm² were placed onthesampleholderoftheSmartiTRATR. FTIRspectra wererecordedfrom600to4000cm¹ataresolutionof4cm¹ withatotalof32scans.

2.2.8.X-Raydiffraction(XRD)analysis

X-raypatternsforeachfilmformulationwereanalyzedbyX-ray diffractometry with Cu K-

a

^{radiation (}

l

^{=1.54Å) at 30kV and}

15mA (MiniFlex II, Rigaku, Japan). Film samples (2cm²) were placedonthesampleholderandsecuredbytape.Eachsamplewas scannedbetween2

u

⁼³–60atascanningrateof2/min.

2.2.9.Tensilestrength(TS)andelongationatbreakpoint(EAB) Tensilestrength(TS)andelongationatbreakpoint(EAB)were determined with a texture analyzer (TA XT Plus, Stable Micro Systems,UK).Filmstripsof1cm7cmwereaffixedtoapairof gripson theAT/Gprobe. Initialgrip separation and cross-head speedweresetat50mmand1mm/s,respectively.

TS wascalculatedbydividingthemaximumload(N)bythe cross-sectionalarea(m²)asfollows:

TSðMPaÞ¼ P

ðbdÞ ð4Þ wherePismaximumload(N);bissamplewidth(mm);anddis filmthickness(mm).

ThepercentageofEABwascalculatedasfollows:

EABð%Þ¼lmax

lo 100 ð5Þ

wherel_maxisfilmelongation(mm)atthemomentofrupture;and lo is the initial grip length (mm) of each sample. All final determinations were recorded as the mean of three measure- ments.

2.2.10.Puncturetest

Punctureforceandpuncturedeformationforeachfilmsample weredeterminedbythetextureanalyzer(TAXTPlus,StableMicro Systems, UK) following the method described by Sobral et al.

(2001),withmodification.Eachfilmsamplewasfixedtoa50mm diameterannularspaceandperforatedbya3mmdiameterprobe movingat 1mm/s.Bothpunctureforce(N)and probedisplace- ment at breaking point (D) (mm) were determined. Puncture deformation(PD)wascalculatedasfollows:

PDð%Þ¼

ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi D²þl²_olo

q

lo 100 ð6Þ

whereloisconsideredthefilm’sinitiallength,equaltotheradiusof theannularspace(25mm).Allfinaldeterminationswererecorded asthemeanofthreemeasurements.

2.3.Statisticalanalysis

StatisticalanalysisforaCompletelyRandomizedDesign(CRD) with Analysis of Variance (ANOVA) utilizing the Minitab 14.0 (MinitabInc.,StateCollege,PA,USA)wereundertaken.Comparison ofmeanswasperformedbyFisher’sTestwithaconfidencelevelof p<0.05.

3.Resultsanddiscussion

3.1.Filmthickness

Table 2 shows thickness measurement results for different formulationsofgelatin-CMC-xanthangumcompositefilmsinthis study. Film thickness ranged from 0.09–0.14mm. The films producedweresufficientlythintoqualifyasfilm asdefinedby AmericanSociety for Testing and Materials (ASTM) (1985) (i.e.

0.25mm),aswellasbyEmbuscadoandHuber(2009)(<0.3mm).

Results showed significant difference (p<0.05) between thickness values, although the volume for each film forming solutioncastoneachPetridishwascontrolledduringthecasting processtoensuresimilarthicknessvalues.Thesedifferenceswere likelyduetothesolidcontentofeachfilmformingsolution,which influencesfilm thickness(Han &Krochta,1999).The significant thicknessincreaseswereobservedforgelatin-CMC(p<0.05)films castwithincreasingxanthangumcontent,whichappearedtohave producedamorecompactfilmnetwork.Asfilmthicknessdepends onfilm composition (Valenzuela, Abugoch, & Tapia, 2013), the observed thickness values increased as more component was addedtothefilm.Duetoitslowestsolidcontent(twopolymers:

gelatinand CMC),the controlsample had the lowestthickness value(0.09mm)comparedtoformulationsA,B,C,DandE(0.10, 0.12, 0.13, 0.13 and 0.14mm), respectively; each of which comprisedthreepolymers(gelatin,CMCandxanthangum).Film Ehadthehighestxanthangumconcentration(25%,w/wsolid)and thehighestthicknessvalue(0.14mm).Theseresultssuggestedthat xanthangumformeda compactfilm networkwithgelatin and CMCmoleculesduetocrosslinkingwithinthefilmmatrix,thus, resultinginincreasedthicknessvalue.Itappeared,therefore,that thehighestconcentrationofxanthangumdevelopedthehighest crosslinkingeffectwithinthefilmmatrix,andthus,helpedtoform themostcompactfilmnetwork.

3.2.Moisturecontent

The moisturecontentvaluebetweengelatin-CMCfilm blend (control)withgelatin-CMC-xanthangumfilmblendat5–20%(w/w solid) xanthan gum added (formulations –A through –D), respectively presented a significance difference (p<0.05) as showninTable2.Xanthan gummight interactwithgelatinvia hydrogen bonds that block hydroxyl positions capable of associatingwithwatertoreducemoisturecontentingelatinfilms (Guoetal.,2014),whereasthesamemechanismisalsoperformed byCMCin gelatin-CMCfilm(Jahit,Nazmi,Isa,&Sarbon,2016).

Thus, adding xanthan gum increased the capability to form hydrogen bonds within the polymer matrix. The insignificant differences(p>0.05)inmoisturecontentbetweenfilmsAthrough Dwaslikelyduetosimilarcapabilitiesinblockingthehydroxyl position of xanthan gum that may associate with water, at concentrationsfrom5to20%(w/wsolid)therefore,resultingin

formation ofhydrogenbond betweenxanthangumandgelatin matrix.

However, the addition of xanthan gum at the highest concentration (25%, w/w solid) in Formulation E, significantly increased(p<0.05)thefilm’smoisturecontent.Ingelatin-CMC- xanthangumfilms,itappearsthatCMCandxanthangumnotonly interactedwithgelatinmolecules,butalsowitheachother.The latter interactions in formulation Ecould have ledto a higher susceptibilitytowaterwithanincreasedaffinityforbindingwater molecules due totheexposure of a largernumber of hydroxyl groups present in both structures. This factor subsequently increasedmoisturecontentforthecompositefilmasalsoreported byMartinset al.(2012) in theirstudy, which blended

k

^-carra-

geenantolocustbeangumfilmsforaspecificformulation.

3.3.Lighttransmissionandtransparency

Table3showslighttransmissionandtransparencyvaluesfor eachfilms.Allformulationsshowedadecreaseinlighttransmis- sionwiththeincreasedconcentrationofxanthangumaddedat 200 and 280nm,respectively. Lower light transmission for the films indicated an excellent barrier for UV light which was in agreementwiththestudybyGuo etal.(2014).Theadditionof xanthangumsignificantlydecreased(p<0.05)lighttransmission wavelengthat200and280nm,respectively.Theincreasingfilm thicknessupontheincreasingcontentofxanthangumaddedinthe filmformulationpenetratedthetransmissionofUVlightthrough the film, so that thehigher the concentrationof xanthan gum added,thelowertheUVlighttransmission.However,theaddition ofxanthangumatconcentrationsupto20%didnotimprovethe UVlightbarrierpropertysincenosignificantdifference(p>0.05) wasobservedbetweenlighttransmissionsforfilmsBthroughEat both 200 and 280nm, respectively. These results suggest that xanthangumimprovesbarrierpropertiesofblendedfilmsagainst UV light by enhancingthe prevention of UV transmission. For visiblelighttransmission,assimilarwiththeUVlighttransmis- sion,theincreasingfilmthicknessupontheincreasingcontentof xanthanguminthefilmformulationloweredthetransmissionof visible light. No significant differences (p>0.05) were noted betweenFilmsDandEat 350–800nm, showingthatboth film formulationshadsimilardegreesofvisiblelightbarrierproperties.

Loweredlighttransmissionwithinvisiblerangesismostlikelydue toahigherlighttransmissionbarrierforhigherconcentrationsof xanthangum.

Transparencyat600nmfor thecontrolfilmwasrecorded at 1.99,higherthanthetransparencyindexforbovinehidegelatin film(0.58)obtainedbyMaetal.(2012).Thissuggeststhatgelatin- CMCfilmsarelesstransparentthanpuregelatinfilms.Increasing theconcentrationofxanthangumintheblendfrom5to25%(w/w solid) significantly elevated (p<0.05) the transparency value, stronglysuggesting that higher concentrationsof xanthan gum decreaseblendedfilmtransparency.Guoetal.(2014)foundthat Table2

Moisturecontent,watervapourpermeability,tensilestrength,elongationatbreak,punctureforceandpuncturedeformationvaluesforgelatin-CMC-xanthangumfilm formulations.

Film formulations

Thickness (mm)

Moisture content(%)

Watervapourpermeability(gmm/

m²dkPa)

Tensilestrength (MPa)

Elongationat break(%)

Punctureforce (N)

Puncturedeformation (%)

Control 0.090.001^c 21.970.06^ab 24.400.31^c 7.840.30^a 91.350.31^a 3.220.06^c 68.410.57^a A 0.100.011^bc 20.840.34^b 27.562.66^bc 4.880.02^b 76.130.95^b 4.240.04^ab 60.854.48^b B 0.120.009^b 21.370.24^b 29.780.41^b 2.680.16^e 65.090.52^c 4.680.04^a 47.743.06^c C 0.130.001^ab 20.601.15^b 33.121.12^ab 3.260.15^de 51.663.00^d 4.660.04^a 38.990.33^d D 0.130.002^ab 21.460.38^b 33.940.74^ab 3.410.08^d 50.641.24^d 3.850.13^b 23.902.11^e E 0.140.006^a 23.620.31^a 36.380.44^a 4.070.33^c 62.350.39^c 4.410.30^ab 22.320.35^e Filmformulations:Control(gelatin-CMC=G-CMC);A(G-CMC+xanthangum5%w/wsolid);B(G-CMC+XG10%);C(G-CMC+XG15%);D(G-CMC+XG20%);E(G-CMC+XG 25%).Resultsexpressedasmeansstandarddeviation.Meanvaluesinthesamecolumnwithdifferentsuperscriptletters(^a-b)aresignificantlydifferent(p<0.05).

crosslinkingreactionscausedadarkercolourationoffilmsintheir study.Thus,anincreaseinthetransparencyvalueofgelatin-CMC- xanthan gum films might directly result from darker colours caused bycrosslinking of xanthan gumwith gelatin-CMC. This resultalsoagreeswithvisualobservationofthefabricatedfilms, which appeared less transparent. There was no significant difference (p>0.05) between films B (4.50) and C (4.89) or betweenfilmsD(6.34)andE(6.50),withbothsetsshowingsimilar degreesoftransparency.Thisresultisalsosupportedbythefilm thickness, since the film with the increased thickness value showedthedecreasedtransparencyvalue.

3.4.Watervapourpermeability(WVP)results

Table2showswatervapourpermeability(WVP)valuesforthe different formulations of fabricated gelatin-CMC-xanthan gum filmsobservedinthisstudyweresignificantlyincreased(p<0.05) and graduated WVP values (p<0.05) were observed after the addition of xanthan gum. The significant differences (p<0.05) betweenWVPvaluesobtainedforthecontrolandfilmsA,B,C,D andEcorrespondwithastudybydeCarvalhoandGrosso(2004) where crosslinking reactions influenced the moisture diffusion coefficient within the gelatin’s network, indicating structural changesinthepolymericmatrixaftercrosslinkerswereintroduced togelatinfilms.Thus,inthepresentstudy,theadditionofxanthan gumtogelatin-CMCfilmappearstohavechangedthestructureof thepolymericmatrixaffectingthemoisturediffusioncoefficient within the gelatin-CMC network as influenced by crosslinking reactionswithinthecompositefilms.

FilmsCand Dwerenotsignificantlydifferent(p>0.05),thus indicatingboth of theformulationspossessedsimilardegreeof hydrophilicity.Theadditionof25%(w/wsolid)xanthangum(film E)exhibitedthemaximumWVPvalue,revealingthatthehighest concentrationofxanthangumcontributedtothehighestdegreeof hydrophilicity of the film. Moreover, films comprising three componentsmighthavehigherWVPvaluesthanthosewithonly two components.Thehigher WVP results obtainedfor gelatin- CMC-xanthan gum films, compared to gelatin-CMC films, also agree with a study conducted by Tong et al. (2008), where pullulan-alginate-CMC films demonstrated higher WVPs than pullulan-CMCandpullulan-alginatefilms.

Resultsfromthepresent studyrevealedthattheadditionof xanthan gum to gelatin-CMC films didnot improve thewater vapour barrier property but rather increased the film’s water vapourpermeability.Nevertheless,areducedwatervapourbarrier allows movement of water vapour across the film, which can preventcondensationwithinthepackagethatotherwise,servesto increase the microbial spoilage of packaged foods (Souza, Cerqueira, Teixera,& Vicente, 2010).Hence, suchfilmsmay be suitableasprimarypackagingwhere(i)awatervapourbarrieris notcriticalandwheresecondaryprotectioncanbeprovidedbyan

outerwrap,or(ii)forthepackagingofnon-water-sensitive-food products(Tongetal.,2008).

3.5.Thermalanalysisbydifferentialscanningcalorimeter(DSC) A differentialscanning calorimeter(DSC) isusedin orderto determine each fabricatedfilm’sthermal stability. Thethermo- gramsofeachfilmformulation’ssingleglasstransitiontempera- ture(Tg)andsinglemeltingpoint(Tm)wereobtained(Table4).The T_g forControl filmwas 56.36C, which ishigher thanpure fish gelatinfilmTg(29.8C;Hosseini,Rezaei,Zandi,&Ghavi,2012),or plasticized bovine-hide gelatin film Tg (41C; Gómez-Estaca, Montero,Fernández-Martín,&Gómez-Guillén,2009).Thisfinding demonstratedgreaterthermalstabilityforgelatin-CMCfilmthan for pure gelatin film. Tg values for CMC, pure CMC films and differentplasticizedCMCfilmswerereportedat99,75and69– 71C, respectively, (Ghanbarzadeh & Almasi, 2011). Theresults obtainedfrom this studyshowed that there was nosignificant difference(p>0.05)fromTgcontrolforthesmallestconcentration of xanthan gum (5%, w/w solid) in formulation A (Table 4).

However,significantTgvalueincreases(p<0.05)wereobserved for formulationswith10,15and 25%(w/w solid)xanthangum components,respectively.Besidesimprovingthermalstability,the additionofxanthangumatthreehigherconcentrationsappearsto havestimulatedinteractionsandincreasedenmeshmentbetween the film’s three components (Guo et al., 2014). Furthermore, xanthangumisahetero-polysaccharideofultra-highmolecular weight (1–2 million) (Sharma et al., 2006), which means its additiontogelatin-CMCfilmincreasedthefilm’smolecularweight andledtoanincreasedTgvalue.Thus,filmE’sdemonstrationofthe highestTgvaluewaslikelyduetoitshighermolecularweight.

In contrast to Tg results, insignificant differences (p>0.05) betweenTmvaluesindicate thatcrosslinking reactioncontribu- tionsfromtheadditionofxanthangum(allconcentrations)didnot affect Tm (Table 4). Crosslinking should increase the thermal stabilityofgelatinfilmsbyshiftingTmtowardsahighervalue.The Table3

Lighttransmissionandtransparencyvalueofgelatin-CMCxanthangumfilmsatdifferentformulationatselectedwavelengths.

Film formulations

Lighttransmissionatdifferentwavelengths(%) Transparencyvalueat

600nm

200nm 280nm 350nm 400nm 500nm 600nm 700nm 800nm

Control 0.090.002^a 2.390.326^a 31.910.068^a 50.950.899^a 59.220.398^a 65.521.676^a 68.260.287^a 70.840.241^a 1.990.135^d A 0.060.002^b 0.880.320^b 22.761.749^b 34.231.114^b 46.380.447^b 51.821.107^b 53.430.068^b 54.810.916^b 2.830.388^c B 0.040.005^c 0.780.432^b 12.380.011^c 19.721.705^c 26.090.407^c 30.291.824^c 32.022.208^c 32.922.221^c 4.500.132^b C 0.030.005^cd 0.410.008^b 9.100.197^d 15.941.150^d 18.640.030^d 24.221.187^d 26.112.737^d 27.143.126^d 4.890.223^b D 0.020.004^d 0.110.057^b 4.340.168^e 8.340.221^e 12.520.170^e 15.580.754^e 16.400.488^e 17.580.231^e 6.340.270^a E 0.020.001^d 0.090.012^b 3.420.168^e 7.820.009^e 11.560.243^e 12.820.878^e 15.130.044^e 15.700.270^e 6.500.083^a

Filmformulations:Control(Gelatin-CMC=G-CMC);A(G-CMC+xanthangum5%w/wsolid);B(G-CMC+XG10%);C(G-CMC+XG15%);D(G-CMC+XG20%);E(G-CMC+XG 25%).Resultsexpressedasmeansstandarddeviation.Meanvaluesinthesamecolumnwithdifferentsuperscriptletters(^a-c)aresignificantlydifferent(p<0.05).

Table4

Tgvaluesobtained fromDSCthermogramsforgelatin-CMC-xanthan gumfilm formulations.

Filmformulations Tg(C) Tm(C)

Control 56.360.55^b 138.765.68^a

A 57.630.38^b 154.533.08^a

B 60.070.38^ab 146.1414.98^a

C 60.482.02^ab 137.980.33^a

D 56.940.67^b 138.685.90^a

E 61.570.38^a 134.092.81^a

Filmformulations:Control(gelatin-CMC=G-CMC);A(G-CMC+xanthangum5%w/

wsolid);B(G-CMC+XG10%);C(G-CMC+XG15%);D(G-CMC+XG20%);E(G- CMC+XG25%).Resultsexpressedasmeansstandarddeviation.Meanvaluesin thesamecolumnwithdifferentsuperscriptletters(^a-b)aresignificantlydifferent (p<0.05).

compositefilmsproducedforthisstudydiddemonstrateimproved thermal stability as Tmvalues for all formulations werehigher comparedtobovinegelatinfilmTm(65.06C)asreportedbyde CarvalhoandGrosso(2004),and(66.2C)asreportedbyMaetal.

(2012). This finding was possibly due to the high Tm value attributedtomeltingcrystallineCMCdomains,sinceCMCfilms plasticized by glycerol showed Tm values between 153.5 and 170.7C(Ghanbarzadeh&Almasi,2011).Anotherfactoraffecting T_mincreaseisthatthehydroxylgroupsofCMCandxanthangum likelyinduceincreasedhydrogenbondswithinthefilm’smatrix.

3.6.StructuralanalysisbyFouriertransforminfrared(FTIR) spectrometry

Fig.1 showsFTIRspectraforgelatin,CMCandxanthangum powderswhile,Fig.2,bycomparison,showstheFTIRspectrafor thedifferentformulationsofgelatin-CMC-xanthangumblended filmsproduced inthis study.The spectrumfor gelatinin Fig.3 exhibitedfouramidebandsrepresentingvibrationalmodesofthe peptide bond. Bovine gelatin exhibits bands at 3276.99cm¹ corresponding with amide A; at 3077.08cm¹ representing characteristic amide B NH stretching coupled witha hydrogen bond; 1628.89cm¹ reflects the C¼^{O stretching of amide I or} hydrogenbondingcoupledtoCOO;1532.06cm¹indicatesamide II’sbendingvibrationsfromNHgroupsandstretchingvibrations of CN groups; bands at 1202.91cm¹ reflect vibrationsfrom amideIII’splaneof CN and NH groupsof boundamide,or vibrationsof glycine’s CH2 groups (Nur Hanani, Roos, &Kerry, 2014;Hashimetal.,2010).

The CMC spectrum showed transmission bands at 3262.61cm¹attributedtohydrogenbondingintheOHstretching region;bandsat2915.72cm¹representCHstretchingassoci- ated with the methane ring of hydrogen atoms; bands at 1586.07cm¹indicatethepresenceofCOOassignedtocarboxyl group stretching; bands at 1413.42cm¹ and 1323.15cm¹ are assigned to OH stretching in-plane and symmetrical CH

stretching; while bands at 1052cm¹ and 1021.28cm¹ might be characteristic of CO stretching on CMC’s polysaccharide skeleton(Chai&Isa,2013).

Thexanthangumspectrum displayedtransmissionbands at 3281.36cm¹attributedtotheaxialdeformationoftheOHgroup;

bandsat2903.36cm¹representtheaxialdeformationofCHOand CH (perhaps due to the absorption of symmetrical and asymmetrical CH3 or CH2 stretching); bands around 1700– 1800cm¹ indicate axial deformation of C¼^{O ester, carboxylic} acid, aldehydes and ketones; bands at 1604.41cm¹ can be assignedtotheaxialdeformationofC¼Ofromenols(-diketones);

bandsat1405.19cm¹mightcharacterizethedeflectionangleof C¼^H;andfinally,bandsat1021.28cm¹couldbecharacteristicof COaxialdeformation(Fariaetal.,2011).

ComparingtheFTIRspectrumofgelatin-CMCfilm(control)with spectrapresentedbygelatin-CMC-xanthanformulations,aslight shiftinintensityattributedtofunctionalgroupswasobserved.This shifting of transmission bands likely resulted from functional group interactions between polymers in the composite films, which indicatedexcellentmiscibility (Martinsetal., 2012).The shiftingofthecontrol’stransmissionbandfrom3281.23cm¹to higher values at 3281.42, 3281.45, 3281.56, 3281.54 and 3281.49cm¹ presented by filmsA, B, C, Dand E,respectively, revealedrobustinteractionsbetweenthehydroxylgroupsofCMC, xanthangumandglycerolastheybondedwiththegelatin’samino groups. These results corroborate similarfindings onchitosan- cassavastarch-gelatinfilmsbyZhongandXia(2008).Ofnoteis thatnoneofthecompositefilmsdisplayedgelatin’samide-Bgroup sincehydroxylgroupinteractionsmadeitundetectable,which,in turn,broadenedtheintensitypeak.

The most useful peak for infrared analysis of secondary structures in protein is the amide-I band between 1600 and 1700cm¹ (Pranoto, Lee, & Park, 2007). The range of intensity valuesweredetectedbyFTIRforallformulationswas1633.29– 1633.47cm¹,indicatinggelatin’s

b

^{-sheetstructurewaspresent}

throughout (Hashim et al., 2010). The control’s intensity value

Fig.1.FTIRspectraofgelatin,CMCandxanthangumfilms.

shiftedfrom1633.29cm¹to1633.32,1633.42,1633.47,1633.39 and1633.38cm¹,respectively,foreachfilm(A,B,C,DandE).This indicatedthatinteractionsbetweenthecarboxylicgroupsofCMC andxanthangumwithamide-I,aswellascrosslinkingenhance- mentfrom theadditionof xanthangum,had strengthened the

b

^{-sheetstructure ofallcomposite} films.Amide-IItransmissions

increasedfrom1552.13cm¹(control)to1522.18,1552.35, 1552.39, 1552.26and1552.45cm¹,respectively,forallcompositefilms(A, B, C, D and E). Amide-III transmissions also increased from 1240.02cm¹(control)to1240.35,1240.43,1240.38,1240.34and 1240.31cm¹,respectively,forallcompositefilms(A,B,C,DandE).

Furthermore,duetothepolyelectrolytecharacteristicofpolymer chainsinCMCandxanthangum,electrostaticinteractionsrelated toaminoandcarbonylmoietiesoccurredinallfilms(Kocherbitov, Ulvenlund, Briggner, Kober, & Arnebrant, 2010; Hosseini et al., 2012). Hence, increased transmissions related to amide-II and amide-IIIsuggestthattheadditionofxanthangumtogelatin-CMC filmstrengthenedelectrostaticinteractionsbetweengelatin and CMC.

3.7.X-raydiffraction(XRD)analysisresults

X-raydiffraction(XRD)patternsforallformulations(control,A, B,C,DandE)wereobtainedtoassessfilmstructure.Fig.3 lists resultsobtainedfrom2

u

^{intensityvaluesofXRDpatternsforeach}

filmformulation.Thecontrolfilmdemonstratedasemi-crystalline structurewithtwomajordiffractionpeaks:at2

u

^{=23.12,showing}

anamorphouspeak,andat2

u

^{=29.07,showinga}crystallinepeak.

AstudyconductedbyChaiandIsa(2013)reportedanamorphous peakat2

u

^=21.00forCMCfilms.Thepresentstudyaddedgelatin toCMCandobtainedaslightshift(to23.12)fromgelatinandCMC interactions.Theparticularlysharppeakobservedat29.07was likelyduetounorganizedmicrocrystallitemolecularresiduefrom CMC’s bulkier anionic side group activities, which disrupted crystallinelatticeformationduringfilmpreparation.Theseresults agreedwithasimilarreportfromMartinsetal.(2012)onblended Fig.2.FTIRspectrumofeachgelatin-CMC-xanthangumfilmformulation.

Filmformulations:Control(gelatin-CMC=G-CMC);A(G-CMC+xanthangum5%w/wsolid);B(G-CMC+XG10%);C(G-CMC+XG15%);D(G-CMC+XG20%);E(G- CMC+XG25%).

Fig.3.XRDpatternsofgelatin-CMC-xanthangumfilmformulationsdemonstrating amorphouscharacteristics.

Filmformulations:Control(gelatin-CMC=G-CMC);A(G-CMC+xanthangum5%w/

wsolid);B(G-CMC+XG10%);C(G-CMC+XG15%);D(G-CMC+XG20%);E(G- CMC+XG25%).

filmsmadeof

k

-carrageenanandlocustbeangum.Theadditionof xanthangumtogelatin-CMCfilms(A,B,C,DandE),appearstohave reduced thecontrol’scrystalline peak (2

u

^{=29.07).These latter}

filmsindividuallydisplayedonlyonediffractionpeak(2

u

^=20.13,

23.12,19.50,21.00 and20.81),respectively.Again,theresults agreedwithZhongandXia(2008)whoseincorporationofxanthan gum suppressed the crystalline peak of gelatin-CMC film and formedasingleamorphouspeakwhencassavastarchwasaddedto chitosanfilms.

Moreover,aslightshoulderatabout10wasobservedatapeak forXRDpatternofthecontrolfilm,whichwasalsoobservedby Martinsetal.(2012),indicatingaslightseparationphaseofthe film’s components (gelatin and CMC). Hence, the addition of xanthan gum to formulations A through E appears to have decreasedtheintensityofthisshoulderduetocrosslinking,which likelyreducedthephaseseparationobservedinthegelatin-CMC control.

TheamorphousstructuresdemonstratedbyAthroughEfilms werelikelytheresultofstructuralcontributionsfromtheprinciple components (xanthan gum, CMC and gelatin). The amorphous propertyofxanthangumwasestablishedbya broaddiffraction peak at 2

u

^{=23.12, probably the result of its double helix}

conformation(Guoetal.,2014).Furthermore,Kocherbitovetal.

(2010)foundxanthanguminaglassystate(amorphoussolid)at roomtemperature.AstudybyChaiandIsa(2013)establishedthe amorphous nature of CMC. Additionally, these XRD results supportedthehigherWVPofgelatin-CMC-xanthangumfilmsas theamorphous configurationcontributestoa permeablerather crystallineimpermeablestructure(Souzaetal.,2010).

3.8.Mechanicalpropertiesoffilms

3.8.1.Tensilestrength(TS)andelongationatbreakpoint(EAB) Theresultsobtainedfortensilestrength(TS)andelongationat break point (EAB) determinations for all film formulations is showninTable2.TSsignificantlydecreased(p<0.05)fromcontrol (7.84MPa)to4.88,2.68,3.26,3.41and4.07MPa,respectivelyfor films A through E. The decrease appeared consistent with increasing xanthan gum levels, indicating a weakening of intermolecularforces.Thepresence oftheOH group(water) in xanthan gum gave the highest intensity FTIR result and likely reducedthefilm’sstiffnessandresistancetoelasticforce,andthus, loweredtensile strength for thecomposites. This result corre- sponds with a study by Jia et al. (2009) which found that a reductioninTSoccurredwhenkonjacglucomannan-chitosanfilms wereaddedtosoyproteinisolate,whichweakenedintermolecular forcesbetweenfilmcomponents.

Theadditionofxanthangumfrom10%(w/wsolid)upto25%

(w/w)solidwhichrepresentedbyformulationsB-Esignificantly increased(p<0.05)duetothehighcrosslinkingreactionoffered bytheincreasingconcentrationofxanthangum.Xanthangumisa heteropolysaccharidewithahighmolecularweight(1-2million) (Sharma et al., 2006).Hence, the addition of xanthan gum to gelatin-CMCfilmincreasedthemolecularweightofthefilm.Due tothemolecularweightfactor,theincrementofTSvalueofFilms B-EcouldnotexceedTSvalueofFilmAsincetheconcentrationof xanthanguminformulationAwasthelowestandxanthangumat the higher concentration certainly provided higher molecular weighttothefilmswiththe higherdifficultytoinvolve inthe migration.

Similarly, as presentedin Table 2,xanthan gum addition to gelatin-CMCfilm (control) significantlydecreased (p<0.05)the EABvaluefrom91.35%to76.13, 65.09,51.66,50.66and 62.35%, respectively,forfilmsA,B,C,DandE.ThisdecreaseinEABvaluesis likely caused by gelatin cross-links with transglutaminase, formaldehyde or gloxal (de Carvalho & Grosso, 2004). The

increasedthicknessoffilmswithxanthangumadditioninfluenced theelasticpropertyofthefilms,whichresultedinthedecreasingof the elongation at the break point possessed by the films. In addition, there was no significant difference (p>0.05) in EAB valuesbetweenfilmsCandDwasnoted,indicatingthatincreasing xanthangumconcentrationfrom15to20%(w/wsolid)givesimilar effecttoEAB.However,formulationE(xanthangumat25%,w/w solid)significantlyincreasedtheEABvalue(p<0.05),likelydueto ahigherdegreeofcrosslinkingreactions.

Generally,increasedTSprecedesadecreaseinEAB(Tongetal., 2008), but resultsobtainedin this study showed decreased TS valuesprecedingdecreasedEABvalues,whichechoedastudyby Jiaetal.(2009).However,areductionofEABforthetwocomposite filmsdidoccurasexpectedduetotheincreasingdevelopmentof crosslinking within these films’ three components. This result closelyassociateswiththeamorphouspropertiesofformulationsA through E films demonstrated by XRD analyses, showing that mechanical properties of composite films withlow degrees of crystallinitywerenotsuperiortothecontrolfilm’ssemi-crystalline structure. This is especially so since organized structure in polymers with a high degree of crystallinity leads to higher- qualitymechanicalproperties(Souzaetal.,2010).

3.8.2.Puncturetest

Ahigherpunctureforceandlowerpuncturedeformationare desirable characteristics, as per the puncture test, indicating a film’shigherresistancetopuncture.Resultsofpuncturetestsfor punctureforceandpuncturedeformationofthecompositefilmsin thisstudyareshowninTable2.Theadditionofxanthangumto gelatin-CMCfilmsignificantlyincreased(p<0.05)punctureforce from 3.22N (control) to 4.24, 4.68, 4.66, 3.85 and 4.41N, respectively,forfilmsAthroughE.Theadditionofxanthangum appearstohaveincreasedthereinforcementofthefilm’smatrix viacrosslinkingreactions. In supportofthis view,Denaviet al.

(2009) determinedthat crosslinkingbetween film components caused a reinforcementof the matrix that increased thefilm’s breakingforce.Apartfromthat,theincreasedthicknessoffilms withtheadditionofxanthanguminthefilmformulationscaused thefilmsrequirelargerforcetobepunctured.Inthepresentwork, composites B and C showed thehighest reinforcement asthey exhibitedthehighestpunctureforce.

By contrast, the addition of xanthan gum significantly decreased(p<0.05)puncturedeformationfrom68.41%(control) to60.85,47.74,38.99,23.90and22.32%,respectively,forfilmsA throughE. Increasingconcentrations ofxanthan gumincreased proteincrosslinking,andthus,increasedreinforcementofthefilm, which allowed for film distortion and a lower degree of post- puncture deformation. This observed reduction agreed with a studybyDenavietal.(2009).Thefilms’thicknessalsoinfluenced thepercentageofdeformationpossessedbythefilmsafterbeing punctured,inwhichtheincreasingthicknessofthefilmsresulted intheincreasingpuncturedeformation.Nosignificantdifference (p>0.05)inpuncturedeformationvaluesbetweenfilmsDandE were observed, which indicated that increasing xanthan gum concentration up to 25% had no significant (further) effect on puncturedeformation.Theadditionof20%(w/wsolid)ofxanthan gumwas sufficienttooptimizereductionof thefilm’spuncture deformation.

The ranges for puncture force and deformation obtained correspondtovaluesreportedbyDenavietal.(2009):2–8Nfor puncture force, and 15–90% for puncture deformation. Since gelatin-CMC-xanthangumformulationsAthroughEshowedboth higherpunctureforceandlowerpuncturedeformationvaluesthan the gelatin-CMC control, xanthan gum addition undoubtedly improvedpunctureresistanceinthefilmsunderstudy.