Synthesis, characterization and antifungal activity of quaternary derivatives of chitosan on Aspergillus flavus

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MicrobiologicalResearch168 (2013) 50–55

ContentslistsavailableatSciVerseScienceDirect

Microbiological

Research

j o ur n a l ho me p ag e :w w w . e l s e v i e r . c o m / l o c a t e / m i c r e s

Synthesis,

characterization

and

antifungal

activity

of

quaternary

derivatives

of

chitosan

on

Aspergillus

flavus

Rafael

de

Oliveira

Pedro

a

_,

_Mirelle

_Takaki

a

_,

_Teresa

_Cristina

_Castilho

_Gorayeb

b

_,

_Vanildo

_Luiz

_Del

_Bianchi

b

_,

João

Cláudio

Thomeo

b

_,

_Marcio

_José

_Tiera

a

_,

_Vera

_Aparecida

_de

_Oliveira

_Tiera

a,∗

a_Department_of_Chemistry_and_{Environmental}_Sciences,_Institute_of_Biosciences,_Humanities_and_Exact_Sciences_–_IBILCE,_São_Paulo_State_University_–_UNESP,_São_José_do_Rio_Preto,

SãoPaulo,Brazil

b_Department_of_Technology_and_Food_Engineering,_Institute_of_Biosciences,_Humanities_and_Exact_Sciences_–_IBILCE,_São_Paulo_State_University_–_UNESP,_São_José_do_Rio_Preto,_São

Paulo,Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received29February2012

Receivedinrevisedform16June2012 Accepted23June2012

Keywords: Chitosan Derivatives Aspergillusflavus Antifungalactivity Mycotoxins

a

b

s

t

r

a

c

t

Twoseriesofnewchitosanderivativesweresynthesizedbyreactionofdeacetylatedchitosan(CH)with propyl(CH-Propyl)andpentyl(CH-Pentyl)trimethylammoniumbromidestoobtainderivativeswith increasingdegreesofsubstitution(DS).Thederivativeswerecharacterizedby1_H_NMR_and

potentiomet-rictitrationtechniquesandtheirantifungalactivitiesonthemycelialgrowthofAspergillusflavuswere investigatedinvitro.TheantifungalactivitiesincreasewithDSandthemoresubstitutedderivativesof bothseries,CH-PropylandCH-Pentyl,exhibitedantifungalactivitiesrespectivelythreeandsixtimes higherthanthoseobtainedwithcommercialanddeacetylatedchitosan.Theminimuminhibitory con-centrations(MIC)wereevaluatedat24,48and72hbyvaryingthepolymerconcentrationfrom0.5to 16g/Landtheresultsshowedthatthequaternaryderivativesinhibitedthefungusgrowthatpolymer concentrationsfourtimeslowerthanthatobtainedwithdeacetylatedchitosan(CH).Thechitosans mod-ifiedwithpentyltrimethylammoniumbromideexhibitedhigheractivityandresultsarediscussedtaking intoaccountthedegreeofsubstitution(DS).

1. Introduction

Chitosanisapolysaccharideusuallyobtainedfrom deacetyla-tionofchitin,whichaftercelluloseisthesecondmostabundant natural biopolymer foundin nature. It may be extracted from various sources, particularly from exoskeletons of arthropods ofcrustaceans, fungi,insects, annelids, mollusks and coelenter-ata. The structures of chitin and chitosan correspond to those of poly[␤(1→4)-2-acetamide-2-deoxy-d-glucopyranose] and

poly[␤(1→4)-2-amino-2-deoxy-d-glucopyranose], respectively

(Ravi Kumar 2000). The homopolymer is a weak base with a pKa valueofthed-glucosamineresidue ofabout6.2–7.0and is

therefore insolubleat neutral and alkaline pH values. In acidic mediums,theaminegroupswillbepositivelycharged,conferring tothepolysaccharidea highcharge density. Duetoits polyca-tionic nature, chitosan, after being dissolved in aqueous acid solutions,canbeeasilymoldedandusedasmembranes,beads, microparticlesandgels(Rinaudo 2006;RaviKumar2000).Also, itsfunctionalpropertiessuchasbiodegradabilityandlowtoxicity (Domard2011;KeanandThanou2010)havedriventheresearch

∗_{Corresponding}_author._Tel.:₊₅₅₁₇₃₂₂₁_2358;_fax:₊₅₅₁₇₃₂₂₁_2356.

E-mailaddress:verapoli@ibilce.unesp.br(V.A.deOliveiraTiera).

andapplicationsofchitosantomedicine(Rinaudo2006;Shietal. 2006;Jayakumar etal.2010), foodsadditivesand preservatives (Shahidietal.1999)aswellasinthepaperindustryandforthe treatmentofindustrialwastewater(NgahandTeong2011).

Oneofthemostattractivefeaturesofchitosanisitsantibacterial, antiviralandantifungalactivity(Zhangetal.2011;Jayakumaretal. 2011).Recentlytheutilizationofchitosanasafoodpreservative oradjuvantinagriculturetoprotectorstimulatethedefenseof differentcropshasincreased(Zhangetal.2011;Jayakumaretal. 2011).Itiswellknownthatpesticideresiduesaretoxicforhumans andanimalsandmanyofthemarenotbiodegradable,whatcan causeseriousenvironmentalproblemssuchascontaminationof thewaterandsoil(Satpathyetal.2011).Chitosananditsoligomers haveemergedasapromisingsourceformanyapplicationssinceit canbeusedtoproducebiodegradablefungicidestoregulatethe growingofplantsandtoprotectseeds(Alburquenqueetal.2010). Theantifungal activityof chitosan is believedto occurfrom the interaction between the cationic chain and the negatively chargedresiduesofmacromolecules exposedonthefungalcell surface,leadingtoleakageofintracellularelectrolytesandother constituents(Muzzarellietal.2001).Itisbelievedthatchitosan mayaffectthemorphogenisisofthecellwallinterferingdirectly ontheactivityofenzymesresponsibleforthegrowingofthefungi (ElGhaouthetal.1992).RecentlyLietal.,basedonconfocallaser

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subsequentaflatoxin productionbyinducing susceptibletissues toproducemorephenoliccompounds.Recently,Zhangetal. pre-paredchitosan-basedblendfilmsusingchitosan,soybeantrypsin inhibitorextractsandglycerolsolutions(Zhangetal.,2009).The authors showed that the germination and growth of A. flavus

werestronglyinhibitedbyfilmspreparedfromsoybeantrypsin inhibitorextract(STI)/wildsoybeantrypsininhibitorextract(WTI) andglycerol(Gly)solutions(chitosan-STI/WTI-Gly),indicatingthat thefilmscouldbeusefulaspotentialbio-controlpackagingagainst

A.flavusduringthepeanut’sandothercereal’sstorage.Thestudy ofnewbiomoleculeshavingantifungalactivitieshasemergedasa newsubjectandthemodificationofthechitosanstructureaiming toimproveitsactivityisapromisingwaytoachieveeffective bio-fungicides(Kenawyetal.2005;Mássonetal.2008)andbactericides (Xuetal.2011).

Theaimofthepaperistoreportasimpleandreliablemethodto preparechitosansderivativeswithimprovedcapabilitiesof inhib-itingthegrowthofA.flavus.Thesynthesis,characterizationand antifungal activityof chitosan derivativesagainst thefungus A. flavusaredescribed.Aseriesofchitosanderivativeswasobtained bythereactionoftheaminogroupsofchitosanwithpropyland pentyltrimethylammoniumbromides.Theresultsoftheantifungal activityofthesederivativeswerepresentedanddiscussed,taking intoaccountthedegreeofsubstitutionofthesubstituted deriva-tives.

2. Materialsandmethods

2.1. Materials

Chitosan(degree of deacetylation (DD) 85%) was purchased fromPolymarCo., Brazil,(3-bromopropyl) trimethylammonium bromide,(5-bromopentyl)trimethylammoniumbromide,sodium hydroxide,sodiumacetate,andaceticacidwerepurchasedfrom SigmaAldrichChemicalCo.,Brazil.Spectra/poremembranes (Spec-trum)were employedfor dialysis.All solventswere ofreagent gradeandusedasreceived.WaterwasdeionizedusingaGehaka waterpurificationsystem.

2.2. Instrumentation

1_H_NMR_spectra_were_recorded_on_a_Bruker_ARX-500₅₀₀_MHz

spectrometer.UV/VisspectraweremeasuredwithaCary100 spec-trophotometerequippedwithaPeltiersystem.pHvaluesofthe solutionsweredeterminedusinganDigimedpH-meter.

2.3. Preparationofthealkyltrimethylammonium-modified chitosans

Chitosan was deacetylated as described earlier (Tiera et al. 2006).Theresultingpolymerwaspurifiedbydialysisagainstwater for3daysandisolatedbylyophilization.Next,chitosanswith vary-ingamountofgraftedalkyltrimethylammoniumbromideswere preparedinaqueousNaOHsolutionsandtheprocedureisdescribed below(Fig.1).Thedegreeofsubstitutionwasvariedbysettingthe

for2days,thenagainstaqueousNaOH(0.05M)for1day,andfinally againstwaterfor2days.Theproductwasisolatedbylyophilization andcharacterizedby1_H_NMR_and_{potentiometry.}

2.4. Viscositymeasurements

Viscosity measurements were carried out in water ther-mostatedbathwithacapillarycalibratedviscosimeterfordilution Cannon-Ubbelohde9722M-50(CannonInstr.Co.)atpH4.5acetic acid(0.3M)/sodiumacetate(0.2M)buffer.Theviscosimeterwas immersed in a thermostatic bath at 298.15±0.05◦_C _and _the

samples were allowed to equilibrate for 10min in the bath beforemeasurements.Measurementsateachconcentrationwere repeated and the reproducibility was better than ±0.01s. The

resultsoftheviscositymeasurementswereexpressedasreduced viscosityvaluescalculatedfrom

=(t−to)/to

c

wheretisthemeasuredeffluxtimeofthepolymersolution,toisthe

effluxtimeofthepuresolvent,andcisthepolymerconcentration (g/L).Themeanviscosimetricmolecularweightofchitosanandits derivativesweredeterminedbyusingtheMark–Houwink equa-tion,withconstantsa=0.796,K=0.079mLg−1₍_Roberts_and_Wang 1996).

2.5. Pathogenandcultures

The microorganism chosen totest theantifungal activity of chitosanand itsderivativeswasA.flavus.Thestrainwaskindly providedbyBrazilianCollectionofMicroorganismsfromthe Envi-ronmentandIndustry–CBMAI,Campinas–SãoPaulo,Brazil,and it wasmaintained onpotato dextroseagar (PDA) (potato infu-sionfrom200g/L,20g/Ldextrose,and15g/Lagar)inthedarkat 25±2◦C.

2.6. Antifungalassays

Theantifungalactivityofdeacetylatedandcommercialchitosan wascompared tothoseofthealkyltrimethylammonium deriva-tivesmodifiedwithadegreeofsubstitutionvaryingfrom0.5to65%. ThepolymerssolutionwerepreparedatpH5.5inaceticacidand addedatconcentrationsof0.0(controlplate)0.1,0.5and1.0g/L tothemelted culturemedium, whichcontained10.0mLof10% potatodextrosebrothandthentransferredtoPetridishes.After solidification,themixtureswereinoculatedwitha1mmin diame-termyceliumfungusA.flavusatthecenterofPetridishesandthese wereincubatedinanovenfor7daysat25±2◦C.Inhibitionindexof

thefungusbythepolymerswasdeterminedbytheradialgrowthof thecolonywithacaliperonthe3rd,5thand7thdaysofcultivation, withtheresultofthe7thdayusedforcomparativepurposes.

Theantifungalindexwascalculatedasfollows:

Antifungalindex (%)=1−Da

Db

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52 168 (2013) 50–55

Fig.1.Schematicrepresentationofthesynthesisofthequaternaryderivativesofchitosan.

whereDaisadiameterofthegrowthzoneinthetestplatesandDb

isgrowthzoneinthecontrolplate,accordingtoGuoetal.(2006). Eachexperimentwasperformedinquadruplicate,andthedata wereaveraged.ThestudentT-testandtheKruskal–Wallistestwith Dunn’smultiplecomparisonwereusedtoevaluatethedifferences inantifungalindexinantifungaltests.ResultswithP<0.05were consideredstatisticallysignificant.

2.7. Minimuminhibitoryconcentrations

The minimum inhibitory concentrations (MICs) were deter-minedusingthemicrodilutionassayinpotatodextroseagar(PDA), followingthestandardmethodappliedtofilamentousfungi(CLSI 2008).Thechitosanderivativesweredissolvedinaqueousacetic acidatpH5.5toobtainstocksolutionsof16.0g/L.Thesesolutions weresubsequentlydilutedtoobtaindecreasingpolymer concen-trations(16.0,8.0,4.0,2.0,1.0,0.5,0.25g/L).Thereafter100␮Lof thepolymersolutionswastransferredon96-wellplatesand2␮L ofthefungussuspensionatadensityof106_conidia/mL_were

inoc-ulatedateachwell.Theexperimentswerecarriedoutintriplicate andtheMICsweredeterminedbyvisualinspectionofthewells. Thelowestconcentrationthatgavecompletegrowthinhibitionin allthreereplicateswasrecordedastheminimuminhibitory con-centration(MIC)after24,48and72h.

3. Resultsanddiscussion

3.1. Preparationoftheantifungalderivatives

Toachieve thefunctionalizationofchitosan withquaternary aminogroups,weusednucleophilicsubstitutionoftheC-2amine groups, thus leaving intact all thehydroxyl groups which play an important partin the biological activity of chitosan deriva-tives(Fig.1).Thesyntheticstrategywassuccessivelycarriedout byusingthealquiltrimethylammonium bromides.The transfor-mation was carried out starting from a deacetylated chitosan, obtainedviadeacetylationofacommercialchitosansamplewitha

nominaldegreeofdeacetylation(DD)of85%,followinga previ-ouslydescribedprocedure(Tieraetal. 2006).Thedeacetylation wascarriedoutundercontinuousbubblingofnitrogeninorderto preventdegradationofthepolymer.Thedegreeofdeacetylation, expressedin NH2mol%,determinedfromthe1HNMRspectraof

thestartingmaterialandthedeacetylatedsample,were86.1and 98.7mol%,respectively.Thesevalueswereobtainedfromtheareas ofthedoubletat5.5ppm,duetotheresonanceoftheanomeric pro-ton(H1)andofthesingletat2.7ppmattributedtotheacetamido methylprotons(Fig.2a).Potentiometrictitrationconductedona solutionofCHconfirmedthatthedeacetylationoccurredwithhigh efficiency,yieldingaDDvalueof98.5.

Thedegreeofsubstitutionwasvariedbysettingtheinitialmolar ratioofN-alkyltrimethylammoniumtoglucosamineunitsto val-uesrangingfrom1.2to8.0.The1_H_NMR_spectra_of_the_chitosan

derivativeobtainedfromreactionwith(3-bromopropyl) trimethy-lammoniumbromideexhibitedasingletat3.67ppm,whichwas attributedtotheresonanceofthetrimethylammoniumprotons of theN-alkyltrimethylammonium moiety and alsoexhibited a signalatı2.81ppmcorrespondingtotheresonanceofthe methy-lene protons CH2CH2CH2N+(CH3)3 (Fig. 2a). The attachment of

alkyltrimethylammoniumgroupstothechitosanframeworkbrings furtherchangestothe1_H_NMR_spectrum_of_chitosan,_most_notably

intheresonancesoftheprotonsincloseproximitytothe substitu-tionsite:theanomericprotonH-1andtheprotonH-2linkedtothe C2oftheglucosamineunit.Theanomericprotonsignalundergoes adownfieldshift,fromı5.40ppmtoı5.56ppm,whilethepeakat

ı3.67ppmshiftsto3.85ppm.Thesamepatternwasalsoobserved inthe1_H_NMR_spectra_of_the_{pentyltrimethylammonium}

deriva-tives,howeverthepentyltrimethylammoniumbromideexhibited atripletfrom2.00to2.50ppmattributedtothemethyleneprotons frompentylhydrocarbonchain.SimilardeshieldingofH-1andH-2 uponC-2derivatisationofchitosanhasbeenreportedinthecase ofchitosancarryingoligosaccharidebranches(Tømmeraasetal. 2002).

Thedegreeofsubstitution(DS)wasdeterminedfromtheareas ofthesignalatı2.81ppm,whichcorrespondstotheresonanceof

Table1

PropertiesandDS(degreeofsubstitution)ofthechitosanderivatives.

Derivative Molarratioa

R/NH2

DS(potentiometry) DS(1_H_NMR) _Mv_(kDa)

CH-C – 86.1% 85.9% 32.8

CHb _– _98.5% _98.7% _24.4

CH-Propyl-10 1.2 11.2% 10.7% 30.3

CH-Propyl-15 1.5 14.3% 14.7% 11.9

CH-Propyl-20 2.3 23.4% 18.1% 18.8

CH-Propyl-40 3.8 39.1% 38.4% –

CH-Pentyl-25 1.5 – 25.1% 17.4

CH-Pentyl-30 2.5 – 30.0% –

CH-Pentyl-65 8.0 – 64.9% 20.1

a_Molar_ratio_of_{alkyltrimethylammonium}_bromide_(R)_to_amino_groups_{( NH}

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Fig.2. 1_H_NMR_spectra_of_deacetylated_chitosan_and_their_(a) propyltrimethylam-moniumand(b)pentyltrimethylammoniumderivativesofchitosan.Theresonance ofthemethyleneprotons(greencolor)equidistantfromthenitrogenatomswas usedtodetermineDS.(Forinterpretationofthereferencestocolorinthisfigure legend,thereaderisreferredtothewebversionofthearticle.)

themethyleneprotonsCH2CH2CH2N+(CH3)3(Fig.2a)andfromthe

signalsduetotheanomericprotonsofpropyltrimethylammonium substitutedandunsubstitutedglucosamineresidues,H1sandH1,

respectively(Fig.2a)usingEq.(1):

DS= (1/2)ICH2

IH1+IH1s

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ThesameequationwasusedtodetermineDSforthederivatives obtainedwithpentyltrimethylammonium,howeverthesignalatı

2.05ppm,whichcorrespondstotheresonanceofthemethylene protonsCH2CH2CH2CH2 CH2N+(CH3)3,wasutilizedfor this

pur-pose(Fig.2b).Alldegreesofsubstitution(DS)areshowninTable1.

3.2. Antifungalactivityofcommercialchitosanandits deacetylatedandquaternaryderivatives

TheantifungalactivityagainstA.flavuswaspreviouslytested forcommercial(CH-C)anddeacetylated(CH)chitosans(Table1). Theconcentrationrangewasvariedfrom0.1to1.0g/Land inhi-bitionpercentageswerecalculated asdescribedin theprevious section(Section2.6).Theinhibition decreaseswithtime, which isdue tothe adaptationofthe fungustoitsnew environment, forinstancetheinhibitionindexobtainedwithdeacetylated chi-tosanis8.1,6.5and5.1inthe3rd,5thand7thdayrespectively

Fig.3.InhibitionpercentagesforCH-CandCHatdifferentpolymerconcentrations. ThedifferencesbetweenthegroupswerenotstatisticallysignificantwithP<0.05.

andthesamepatternwasobservedfortheotherderivatives.As canbeseenfromFig.3theinhibitionpercentageobtainedfor CH-CandCHremainedaround5%andincreasedslightlyfrom0.1to 1.0g/Lforthederivativesubstitutedwith10%ofpropyltrimethyl ammoniumgroups(CH-Propyl-10),which isduetothelowthe degreeofsubstitution.ThisresultissimilartothatobservedbyLi etal.investigatingtheantifungalactivitiesofchitosansonF.fulva. Theseauthorsshowedthatinhibitionpercentagesoflow molec-ularweightchitosans,measuredby themycelialradial growth, exhibitedmodestincreasesintheconcentrationrangefrom0.1to 1.0g/L(Lietal.2011).Althoughunexpected,inthisworkthefungal adaptationtothenewenvironmentwasrapidandasthetimeof incubationwas7daystheeffectofconcentrationwasnotclearly observed.However,thedeterminationoftheminimuminhibitory concentrations(MICs),discussedaheadinthepaper,makesclear thatderivativeshavingthehigherdegreesofsubstitutionexhibita significantinhibitionincrease.

Althoughincreasingconcentrations ofchitosanmayimprove theinhibitionpercentageonmanypathogensthistrenddepends onseveralfactorssuchasmolecularweightofchitosan,targeted pathogenandpH(Yangetal.2005).Theactivityofchitosanhas beenexplainedasbeingbasedontheelectrostaticinteractionof thechargedaminogroupsofchitosanwithnegativelychargedcell wallsurfaceofthetargetedmicroorganisms,whichcanleadtothe disruptionofthecellwallandthereforetothedeathofthecell. Anotherpossibilityforantifungalactivityofchitosanaccountsfor itschainstocrossthecellmembraneinhibitingthecellgrowing frominside(Guoetal.2007).However,fordeacetylatedchitosan (CH)andthederivativeshavinglow degreesofsubstitutionthe mechanismonA.flavusandtheinteractionwithcellsurfacemay formanimpermeablelayeraroundthecell,thusblockingthe trans-portofessentialsolutesintothecell(Eatonetal.2008).

Inthiswork,wehypothesizedthat,themodificationofchitosan by introducing permanently charged quaternary groups could improvetheantifungalactivityofchitosan.Theadditionof qua-ternizedchitosantotheBDAmediuminhibitedmycelialgrowth ofA.flavussignificantlyatallconcentrationstested.A representa-tiveexperimentcomparingtheeffectofCHandCH-Propyl-40is showninFig.4.AsshowninFig.5theresultsobtainedin microbi-ologicalassaysshowedthatthecapabilitytoinhibitfungusgrowth

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54 168 (2013) 50–55

Fig.4. Comparativeeffectofthequaternizationofchitosanontheinhibitionindexat0.5g/Lafter3daysofincubation:fromlefttorightBDA;CHandCH-Propyl-40.

significanceof0.05aimingtoverifytheexistenceofsignificant dif-ferencesbetweenresultsobtainedfortheinhibitionindex with 0.1and0.5g/L.Thedescriptivestatisticsshowedthatno signifi-cantdifferenceswereobservedregardingthe0.1and0.5g/L,since theobtainedPvalueswerealwayshigherthan0.5,whichconfirm that,inthisconcentrationrange,theinhibitiondoesnotchange. Acomparisonbetweentheinhibitionindexesofthedifferent chi-tosanderivativeswasalsoperformed.TheKruskal–Wallistestwith Dunn’smultiplecomparisonwasutilizedtothelevelofsignificance 0.05andthedescriptivestatisticsshowedthatPvaluesfoundfor 0.1g/Lweresmallerthan0.001,indicatingthatstructuralchanges onthechitosanchainsignificantlyincreasestheantifungalactivity. The most substituted derivative from CH-Propyl series (DS=38.5%)showedaninhibitionindexthreetimeshigherthan thatobservedwithdeaceylatedchitosan(CH).

However,thehighest inhibition indexwas obtainedfor CH-PentylderivativehavingwithDS65%,whichexhibitedaninhibition indexalmostsixtimeshigherthanCH.Asshownbyotherauthors, thisactivitycanbeattributedtoahighernumberofcationicgroups availableonthepolymerchain,whichallowsastronger interac-tionbetweenthepolycationchainsandthecellwall,increasing

the antifungal activity. However by comparingthe efficiencies of both series it becomes clear that the less substituted CH-Pentylderivative(DS=25%)exhibitedahigherinhibitionthanthat obtainedusingthemore substitutedderivativefromCH-Propyl series(DS=38.5%).Since boththeseriespresent thesame qua-ternary group,this higher activity for CH-Pentyl seriesmay be attributedtothelongerhydrocarbonchainofthegraftedpentyl groups,whichcouldfavorahydrophobicandstrongerinteraction withthecellmembrane.

3.3. Minimuminhibitoryconcentrations

TheresultsobtainedfromMICsdeterminationconfirmedthat thecationicderivativessynthesizedexhibitincreasingefficiencies withthedegreeofsubstitutionbythecationicgroups,withMICsin therangeof1.0–4.0g/L.AscanbeseenfromTable2inthefirst48h, thederivativesCH-Pentyl-30,CH-Propyl-40,CH-Pentyl-25and CH-Pentyl-65inhibitedcompletelythegrowthofthefungusat1.0g/L,

i.e.,aconcentrationfourtimessmallerthanthoseneededfor com-mercial(CH-C)anddeacetylatedchitosan(CH)(Table2).Moreover, after72hnogrowthwasobservedinthepresenceofCH-Pentyl-65

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CH-Pentyl-25 0.5 1.0 2.0

CH-Pentyl-30 0.5 1.0 2.0

CH-Pentyl-65 0.5 1.0 1.0

atconcentrationof2.0g/L.TheMICsforCH-CandCHwas4.0g/L andthisvalueiswithinoftherangereportedintheliterature,which variesfrom10to5000ppmdependingonmolecularweight,degree ofacetylationandtypeoffungi(Sajomsangetal.2012).Similar responseswereobservedbyotherauthorsandtheMICsof quater-nizedchitosansdecreasewithdegreeofsubstitution,whichcanbe explainedbasedontheinteractionofthesegroupswithnegatively chargedcellsurfacemembrane,preventingnutrientsfromentering thecell(Guoetal.2007;Yangetal.2005).

4. Conclusions

Thesynthesisandcharacterizationofnewquaternary deriva-tivesofchitosanwerecarriedoutandtheirantifungalactivities againstA.flavusweretested.Theresultshaveshownthatthe anti-fungalactivityofchitosancanbeimprovedbyincreasingthedegree of substitution(DS) of alkyltrimethylammoniumgroups onthe polymerchain.Theinhibitionindexesforbothsynthesizedseries (propyland pentyltrimethylammonium) increasedwith DS and themostsubstitutedderivatives(CH-Propyl-40andCH-Pentyl-65) exhibitedinhibitionvaluesthreeandsixtimesrespectivelyhigher thanthoseobtainedwithchitosan.Thehigheractivityexhibited byCH-Pentylderivativescanbeattributedtothelonger hydrocar-bonchainofthissubstituent,whichallowsastrongerinteraction withthe cellmembrane. Thesynthesis and characterization of newderivatives,havinglongerhydrocarbonchains,areongoingin ourlaboratoryaimingtoobtainderivativeswithhigherantifungal activities.

Acknowledgements

FinancialsupportbyFAPESP(GrantNo.2012/03619-9), FUN-DUNESP (Grant 430/09). R.O.P and M.T. thank FAPESP and PET-CAPESforundergraduatefellowship.

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