A new procedure based on column chromatography to purify bromelain by ion exchange plus gel filtration chromatographies.

ContentslistsavailableatScienceDirect

Industrial

Crops

and

Products

j ou rn a l h o m epa g e :w w w . e l s e v i e r . c o m / l o c a t e / i n d c r o p

A

new

procedure

based

on

column

chromatography

to

purify

bromelain

by

ion

exchange

plus

gel

filtration

chromatographies

Helber

B.

Costa

_,

_Patricia

_M.B.

_Fernandes

_,

_Wanderson

_Romão

_,

_José

_A.

_Ventura

b_{LaboratóriodePetroleômicaeQuímicaForense,DepartamentodeQuímica,UniversidadeFederaldoEspíritoSanto(UFES),AvenidaFernandoFerrari,514,}

Goiabeiras,Vitória,ES,CEP:29075-910,Brazil

c_{InstitutoFederaldeEducac¸ão,CiênciaeTecnologiadoEspíritoSanto,29040-780,VilaVelha,ES,Brazil} d_{InstitutoCapixabadePesquisaAssistênciaTécnicaeExtensãoRural-Incaper,29052-010,Vitória,ES,Brazil}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received13February2014

Receivedinrevisedform17April2014 Accepted23April2014

Availableonline2June2014 Keywords: Bromelain Enzyme Purification Chromatography

a

b

s

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Bioproductsseparationandpurificationprocessesareanimportantsegmentofthebiotechnicalindustry. Bromelainisanenzymewhichhasgreatcommercialvalueandisofwideinterestinthepharmaceutical, alimentary,andtextileindustries,amongothers.Thegoalofthisstudywastodevelopanewmethodfor bromelainpurificationfromthestemresiduesresultingfromagriculturalprocessingofthepineapple plant.Bromelainwaspurifiedusingtwoliquidchromatographysteps,ionexchangeplusgelfiltration chromatography.Useofthemethodologywhichwasdevelopedproducedanenzymewithamolecular weightof30kDa(confirmedbySDS-PAGE),highrecoveryofenzymaticactivity(89%),andwitha purifi-cationfactorof16.93,aresultsuperiortothemethodologiesdescribedintheliterature.HPLCshowed thepresenceoftwopeaksintheionexchangechromatogramandonlyoneproteininthegelfiltration chromatogram.Resultsindicatethat,dependingonthedestinationofthebromelain,theprocesscan bestoppedafterthefirstpurificationstep.TheMALDI-TOFMSprovidedthepeptidemassfingerprint ofbromelainandMALDI-MS/MSthefragmentationprofileandsequencingoftheionsofm/z951and 1584.Thus,theconnectivityandchemicalstructureofbromelainwasconfirmed.Moreover,besidesits superioritytoothermethodologies,itcanbeappliedtotakeadvantageoftheagriculturalandindustrial pineappleplantresidues.

©2014ElsevierB.V.Allrightsreserved.

1. Introduction

Theisolationandpurificationofbioproductsarevery impor-tantprocessesinthebiotechnologyindustry,representing80–90% oftotalproductioncosts.Furthermore,thedevelopmentofsimple, viablemethodsforproteinpurificationhasbeenanessential pre-requisiteformanyadvancesinbiotechnology(Harikrishnaetal., 2002;Costaetal.,2009).

Liquid chromatography (LC) is a technique commonly used forproteinpurification.However,otheranalyticaltechniquesalso couldbeusedsuchasliquid–liquidextractionbyaqueous two-phasesystems(Babuetal.,2008),reversemicellarsystems(Hebbar

∗ Correspondingauthorat:NúcleodeBiotecnologia,UniversidadeFederaldo EspíritoSanto,29040-090,Vitória,ES,Brazil.Tel.:+552731490833.

∗∗ Correspondingauthorat:InstitutoCapixabadePesquisaAssistênciaTécnicae ExtensãoRural-Incaper,29052-010,Vitória,ES,Brazil.Tel.:+552736369817.

E-mailaddresses:farmhel@gmail.com(H.B.Costa),

ventura@incaper.es.gov.br(J.A.Ventura).

etal.,2011),andseparationbymembraneandprecipitation(Doko etal.,2005;Silvesteetal.,2012).Inliquidchromatography, pro-teinsinsolution(mobilephase)areisolatedandpurifiedthrough theirinteractionwithastationaryphase (Cao,2005).Generally, theproductsobtainedbyLCpurificationareveryexpensiveand havehighaddedvalueduetothecostsofthematerialsusedinthe productionprocess.Thedevelopmentoflow-costLCpurification methodshasbeenchallenging(Costaetal.,2009).

Proteolytic enzymes, or proteases, are a class of hydrolytic enzymescapableofcleavingthepeptidebondsofproteinchains and are essential in physiological processes. In addition, pro-teasesareamongthemostrelevantenzymesfromanindustrial standpointbecauseoftheirinvolvementinseveraltechnological applications(Bonetal.,2008).

Bromelainis a proteolyticenzymebelongingtothecysteine peptidasefamily.Thisenzymecanbefoundinthetissuesofplants oftheBromeliaceaefamily,and pineapple(Ananascomosusvar. comosus)is its main source(Rowan et al.,1990; Hebbar etal., 2008).Bromelainhasanumberofbiotechnologicalapplications, especiallyinthefood,cosmeticsandpharmaceuticalindustries,as

http://dx.doi.org/10.1016/j.indcrop.2014.04.042

wellasseveralclinicalapplications,suchasanantitumoragent, immuneresponsemodulatorandanenhancerofantibioticeffects andmucolyticandgastrointestinalaction(Maurer,2001;Balaetal., 2012).Furthermore,theenzymehasaneffectoncardiovascular andcirculatorydiseasesandmayhavepotentialusesinsurgical proceduresandwoundcare(Costaetal.,2009).

Theworldwideapplicationofbromelainincreasesthe impor-tanceofdeterminingaviableextractionandpurificationmethod forthisenzyme.Such amethodmayhelptominimizelossesin thepineappleagribusinessbecausetheagriculturalandindustrial wastesof pineapple(stems,leavesand crowns)arerich in this enzyme.Usually,this residue isnot usedand hasno appropri-atedestination(Bardiyaetal.,1996;Costaetal.,2009).Therefore, thepresent studypresentsa new,viable methodfor the purifi-cationof bromelainfrompineapplestems usingLCasits main technique.

2. Methods

2.1. Materials

2.1.1. Pineapplewastes

Pineapplewastesfromthecv.Vitóriawereobtainedfromthe IncaperSooretamaExperimentalFarm,atSooretama-ES,Brazil.

2.1.2. Chemicals

Allchemicalsproductsusedinthisstudywerepurchasedfrom Sigma,MerckorGE(USA).HPLCgrade chemicalproductswere purchasedfromTediaBrazil.

2.2. Crudeextract

A known quantity (450g) of waste (stem of pineapple cv. Vitória)wascrushedforfiveminutesalongwith600mLof extrac-torsolution(0.4MH2SO4/2mMNa2SO4pH4.5at4◦C).Theextract

wasthenfilteredandcentrifuged(Eppendorfcentrifuge5804R, Germany)at 14,750×g for 20min at4◦C, andthe supernatant (crudeenzymeextract)obtainedwasusedforthefollowing exper-iments.Thecrudeextractwasstoredfrozenat−20◦_C.

2.3. Proteindetermination

The protein concentrations from enzymatic solutions were determinedaccordingtothemethoddescribedbyBradford(1976)

usingbovineserumalbuminasthestandard.

2.4. Bromelainactivity

Thebromelainactivitywasdeterminedaccordingtothecasein digestionunit(CDU)method,whichusescaseinasasubstrateinthe presenceofcysteineandEDTA(Murachi,1976withmodifications). Theassayswerebasedontheproteolytichydrolysisofthecasein substrate.

Theabsorbanceoftheclearfiltrate(solubilizedcasein)was mea-suredat280nmusingaspectrophotometer(ThermoSpectronic®

BIOMATE3,USA).Oneunitofbromelainactivitywasdefinedas 1␮goftyrosinereleasedin1minpermLofsamplewhencasein washydrolysedunderthestandardconditionsof37◦CandpH7.0 for10min.

Thesampleanalyseswereperformedagainsttheirrespective blanksolutions.Theproteinconcentrationreadingsweretakenin triplicate,andtheaveragevaluewasusedforthecalculationofthe

extractionefficiencies.Thespecificactivity,activityrecovery(%) andpurificationfoldwereestimatedbythefollowingequations:

Specificactivity=proteolytic_{proteincontent}activity_(mg/mL)(U/mL) (1) Bromelainrecovery(%)= bromelainactivityafterpurification

bromelainactivityofthecrudeextract

×100 (2)

Purificationfold= specificactivityafterpurification

specificactivityofthecrudeextract (3)

2.5. Ionexchangechromatography

A 25mm ID glass column, 110mm long was packed with caboxymethy-celulloseandequilibratedwithfourcolumnvolumes of5×10−3Macetatebuffer.Then10mLofthecrudeextractwas submittedandremainedincontactwiththeresinforonehour. After,sample waseluted usinga1MacetatebufferpH4.5 ata flowrateof0.5mL/min.Thefractionswerethencollectedin4mL aliquots.Thepresenceofproteinwasmonitoredusingrecording spectrophotometerat280nm(Biomate®_{),usingacetatebufferpH}

4.5asblank.Theproceduresfortheestimationofproteincontent andenzymeactivityaredescribedbelow.

Finally,thecolumnwasthenwashedwith2MNaClsolution untilnofurtherproteineluted.Allprocedurewasconductedina refrigeratedenvironmentat4◦C. Purifiedenzymewasstoredin frozenat−20◦_{C(Cabraletal.,2000;Hernándezetal.,2005with}

modifications).

2.6. Gelfiltrationchromatography

Aliquotsfromionexchangechromatographywerecollectedand submittedtogelfiltrationchromatography.Aglasscolumnwitha 17.5mmIDand200mmlongwaspackedwithSephadexG-50®_and

equilibratedwithoneandhalfcolumnvolumesofacetatebuffer. Thesamplewasappliedandleftincontactwiththeresinforone hour.Thesamplewasthenelutedwith1MacetatebufferpH4.5 atflowrateof0.7mL/min,and theeluentwascollectedas pre-viouslydescribed.Thecolumnwasthenwashedwith5×10−3M acetatebufferpH4.5solutionuntilnofurtherproteineluted.All procedurewasconductedin a refrigeratedenvironment at4◦C Purifiedenzymewasstoredinfrozenat−20◦_{C.(Cabraletal.,2000;}

Hernándezetal.,2005withmodifications).

2.7. SDS-PAGE

SDS-PAGEwasperformedaccordingtothemethodofLaemmli (1970)using15%(w/v)polyacrylamidegels.Thestainingofthegels wasperformedusingCoomassieBrilliantBlueG-250. Chromatog-raphymeasurementsandSDS-PAGEwereperformedintriplicate.

2.8. HPLC

A20␮Laliquotofthesampleobtainedfromtheionexchange andgelfiltrationchromatographywassubjectedtoanalyticalhigh performanceliquidchromatography(HPLC)performedona Shi-madzuProminence(Kyoto,Japan)apparatuswithaUV/visLC-20A detector.ThefractionswereelutedwithaShimadzuShimPackCLC (M)C18(4.6mmID×250mmlong)columnusingalineargradient betweenTFA/H2O(1:1000,v/v)andTFA/acetonitrile(1:1000,v/v)

over80minataflowrateof0.5mL/min,andtheabsorbancewere readat280nm(Hernándezetal.,2005withmodifications).

2.9. Massspectrometry

The protein spot of interest was excised out of the gel (Shevchenko et al., 1996). The spot was washed three times with a washing solution (1:1, v/v 25mM ammonium carbonate–acetonitrile)at25◦Cfor2h.Thewashingsolutionwas thenremovedandthespotwasnowcoveredwithacetonitrilefor 20minat25◦C.Finally,theacetonitrilewasremovedandtheexcess evaporated,thusproviding,thedryingofthespot.Thedehydrated gelparticleswerethenrehydratedfor10minwith10␮Lofdigest buffer(25mMammoniumcarbonate)containing20ngoftrypsin (sequencinggrademodifiedporcintrypsin,Promega,Madison,WI, USA).After,20␮Lofdigestbufferwere added,and thesample wasincubatedfor20hat37◦C.Theresultingtrypticpeptideswere twiceextractedwith50␮Lof0.1%trifluoroaceticacid(TFA)in50% (v/v)acetonitrilewith20minsonication.Theproductsfromthe twoextractswerecombined,vacuum-driedandthendissolvedin 0.1%TFAin50%(v/v)acetonitrile.

The matrix (10mg/mL CHCA (␣-cyano-4-hydroxycinnamic acid)in0.1%TFAand50%,v/vacetonitrile)andthesample(1:1,v/v) werespottedandcocrystallisedonatargetplate.Matrix-assisted laserdesorptionionizationtime-of-flight/time-of-flightmass spec-trometry(MALDI-TOF/TOFMS,model 4700ExplorerProteomics Analyser,AppliedBiosystem®_{,USA)hasbeenappliedtoPeptide}

MassFingerprint(PMF)andpeptidesequencing.ForMALDI-MS/MS measurements,N2wasusedascollisiongasinthecollisioncell at2.8×10−6Torr.Trypsinautolysispeptidesofmasses842.5and 2211.1andcalibrationmixtures1and2(SequazymePeptideMass Standardkit,PerSeptiveBiosystems®_{,FosterCity,CA,USA)were}

used,respectively,asinternalandexternalstandardsinboththe MALDIMSandMALDIMS/MSprocedures.

For database searching, ppw files were submitted to the Mascot search engine using Daemon 2.1.0 (Matrix Science:

http://www.matrixscience.com)onaMascotserverversion2.2.1. Thedataweresearched againstthelatestversionof thepublic non-redundantproteindatabaseoftheNationalCenterfor Biotech-nologyInformation(NCBI),downloadedonAugust2008,witha massaccuracyof15ppmfortheparention(MS)and0.2Daforthe fragmentions(MALDIMS/MS).

Thesearchwasconstrainedtotrypticpeptideswithamaximum ofonemissedcleavage.Thecarbamidomethylationofcysteinewas consideredafixedmodification,whereastheoxidationof methio-nineresidueswasconsideredasavariablemodification.Aninitial listofproteinswasgenerated and formedthebasis for further analysis.A“positivelist”wasgeneratedbyconsideringonlythose proteinsthatcontainedatleastoneuniquepeptide(minimum10 aa)witha Mascotscoreabove67(p-value<0.05).HPLCandMS experimentswereperformedinduplicate(Rodriguesetal.,2011).

3. Resultsanddiscussion

Thecrudeextractwasobtainedwith1.77mg/mLoftotalprotein, 40.84UmL−1 ofproteolyticactivityand23.07Umg−1 ofspecific activity(Table1).Afterthepurificationsteps,thecrudeextractwas appliedtotheionexchangechromatography.

3.1. Ionexchangechromatography

Bromelain purification with carboxymethylcellulose (CMC) exhibitedapurificationfactorof 3.01,with93% recoveryofthe proteolyticactivity.No activitywasdetectedafter thewashing flow.Theionexchangechromatographyofbromelainresultedin aprominentpeakinthesixthfraction(Fig.1).Thespecific activi-tiesoftheappliedcrudeextractandthesixthelutedfractionwere 23.07and69.50U/mgprotein,respectively.

Fig. 1.Ionexchange chromatographicpurification profile for bromelainfrom pineapplestems.

Fig. 2.Gel filtration chromatographicpurification profile for bromelain from pineapplestemsafterionexchangechromatography.

Weobservedahigherbromelainactivitythanthosereported forexistingmethodologiesofionexchangechromatography.This activity is due to theuse of 1Macetate bufferpH 4.5 in this step.Costa(2010),compareddifferentbuffersforthemaintenance of bromelain, and noted that this buffer atpH 4.5 contributed tobromelainactivity.Moreover,previousworkshave indicated thatdifferencesintheionchromatographyresinareimportantto bromelainactivity(Devakateetal.,2009;Costa,2010).

3.2. Gelfiltrationchromatography

Thefractionsobtainedbyionexchangechromatographywere pooled(fractions3–22)andsubjectedtomolecularexclusion chro-matographyusingSephadexG-50resin,andatotalof51fractions were collected (Fig. 2). The specific activity of bromelain was 390.75U/mg, whereas that the proteolytic activity was 89% of recoverywithapurificationfactorof16.93.

ThecouplingofCMCresinwiththeSephadex®_G-50resinisa

powerfulchromatographymethodtoobtainBromelaininitspure form.Suhet al.(1992) purified bromelainfrompineapplefruit andstemuntilhomogeneityusinggelfiltrationchromatography, obtainingayieldofonly23%ofactivity.

Hernández et al. (2005) reported a purification method for bromelainsimilartoourstudy.AuthorsconductedtheBromelain purificationhoweverusingoppositetreatment:theyperformedthe gelfiltrationfollowedbyionexchangechromatography.The gel-filtrationresinusedwasSephadex®_{G-100,yielding87.81%activity.}

ThesecondpurificationstepwasusedtheCMC,whichtheyieldof bromelainactivitydecreasedto41.15%.

TheSephadex®_{G-50ischeaperthanSephadex}®_G-100

(reduc-tionof50%ofcost),becomingthepurificationprocessofbromelain economically viable. Additionally, the purificationmethodology developedpresentsahigheryieldofproteolyticactivity.

Table1

Yieldintotalprotein,proteolyticactivity,specificactivityandpurificationfactorforeachbromelainpurificationstep.

Steps Fractionvolume(mL) Totalprotein(mg/mL) Proteolyticactivity(U/mL) Specificactivity(U/mg) PFa

Crudeextract 8 1.77 40.84 23.07 1

CMC 50 0.20 13.90 69.50 3.01

Sephadex 5 0.093 36.34 390.75 16.93

a_{PF:purificationfactor.}

Fig.2showsfractionswhichhaveproteolyticactivity(fraction 2–45).After51thfractionnonproteolyticactivitywasobserved correspondingtonon-targetsproteins.

3.3. SDS-PAGE

IntheSDS-PAGEanalysis,themolecularweightofbromelain wasestimatedas30kDa,andpredictedbyGautametal.(2010)

andotherpreviousstudies.Bromelainmaynotbecompletelypure whentheextractiselutedonlywithionexchange,assuggestedby thedoublegelbandinthe30kDaregion(Lane1–Fig.3).Theresult ofthegelfiltrationpurificationofbromelainwasconfirmedbythe SDS-PAGEresult,inwhichonlyonebandcanbeobservedinthe 30kDaregion(Lane2–Fig.3).

3.4. HPLC

Aliquotsfromthesamefractionsusedforthepreparation of theSDS-PAGEgelswerethenpreparedforHPLC.TheHPLC anal-ysiscorroboratedwiththeSDS-PAGEresults.Thereversedphase chromatographyconfirmedthepresenceoftwopeaksintheion exchangechromatogram(Fig.4a)andonlyoneprotein(Peak1) inthegelfiltrationchromatogram,whichwaselutedinthefirst 10min(Fig.4b).

Bromelainisaproteinwithpolarcharacteristics,asindicated bythepeakappearingduringthefirstfewminuteswhenwateris themainsolventinthenon-polarcolumn(C-18),causingittobe rapidlyeluted.Napperetal.(1994),inacomparisonofpineapple

Fig.3.SDS-PAGEanalysisofionexchange(Lane1)andgelfiltration(Lane2) chro-matography.(M–MolecularWeightMarker).

proteases,ascribedthepolarityofbromelainmainlytoitscontents oftheaminoacidslysineandarginine,whicharepresentinlarger amountsincomparisonwithotherproteases.

3.5. Massspectrometry

TheSDS-PAGEpreviouslyshown(Fig.3,lane2)wasexcisedand trypsinisedtoobtaina PMF.TheMALDI-TOF MSconfirmedthe purityofthebromelain(Fig.5a)fromidentificationof 10more abundantpeptides with m/z values of 951.44, 963.44,1000.49, 1016.48,1032.48,1068.52,1103.52,1526.70,1566.73and1584.75. Among signals identified, the ions of m/z 951 and 1584 were submittedtoMALDI-MS/MSexperimentsfordeterminationof frag-mentation profile and sequencing (Fig. 5b and c). The Mascot programhasbeenusedfortheMALDI-MS/MSdata,thus provid-ing,themostlikelysequenceforthepeptidesofm/z951(Probable sequence:WGEAGYIR) and 1584(Probable sequence: TNGVPN-SAYITGYAR).

TheNCBI(http://blast.ncbi.nlm.nih.gov)databaseanalysisofthe twopeptidesidentifiedaFBSBprecursor[Ananascomosus]withthe accesscodegi2463584,whichhasa95–100%identitywith brome-lain. Additionally, the SwissProt (http://blast.ncbi.nlm.nih.gov) database was also used, confirming again the purity of the

Fig.4.HPLCchromatogramsobtainedfromthefractionwiththehighest proteo-lyticactivityintheC18columnusingagradientof0–80%water–acetonitrileata flowof0.5mL/minfor80min(a)ionexchangechromatogramand(b)gelfiltration chromatogram.

Fig.5.(a)MALDI-TOFMSspectrumfrombromelainpurified,(b)MALDITOFMS/MSfrompeptidem/z951and(c)MALDITOFMS/MSfrompeptidem/z1584.

bromelainas anidentityof 95–100%between bromelainand a peptidewiththeaccesscodeBROM2ANACO.

TheMSDBdatabasewasalsousedtoanalyzethetwopeptides. Thepeptidewiththem/z951showedhighhomologywithananain, identifiedasanAN8precursor.

Rowan et al. (1990) described the presence of four main proteasesin pineapple (Ananascomosus):fruit bromelain,stem bromelain,ananainandcomosain.

Ananainshowsa77%aminoacidsequenceidentitywith brome-lain (Lee etal., 1997).Thispeptide (m/z951) is present at the

N-terminusregionofbromelain,whichisaportionidenticaltothat ofothercysteineproteases,asitcomprisesmainlytheregionof thecatalyticsite.Ifwealignonlyonefragmentofthetwoproteins (bromelainandananain–seebelow),thepeptidesdifferbyonly oneaminoacid:bromelainhasanalanine(A),whereastheananain hasaglycine(G).

Stembromelain WGEAGYIR

Ananain WGEGGYIR

Peptidem/z951 WGEAGYIR

Asproteasesofthesamefamily,theproteinswereexpected toshareseveralidenticalregionsintheirprimarysequences,and somepeptideswouldhaveahighidentitywithananainorother proteases.Themostlikelysequenceidentifiedforthepeptidemass ofm/z951isthebromelain.Moreover,Laroccaetal.(2010)also confirmedthechemicalidentityofpeptideofm/z951by MALDI-TOFMS.

3.6. Purificationprocess

Thebromelainpurificationmethodusing carboxymethylcellu-losewasabletopurifythecompoundapproximately3.01timesthe levelinthecrudeextract,andtheSephadex®_{columnpurifiedthe}

compoundapproximately16.93times(Table1).

Theresultsindicatethat,dependingonthedestinationofthe bromelain,theprocesscanbestoppedafterthefirstpurification stepusingionexchangechromatography.Thismethodispossible becausetheCMCpurifiedthecompound3.01timesthelevelinthe crudeextract(Table1),withthisresultconfirmedbytheSDS-PAGE andHPLCresults.

Inmanycases,thecommercialuseofbromelaininthefood, cosmetics,andnutritional,medicinalandpharmacological supple-mentindustries (Ketnawa etal., 2012)does notrequirea high puritybromelainenzyme preparation.The processdescribed in thepresentstudyenablesproducerstochoosethedesiredlevel ofbromelainpurity,therebyfurtherreducingthecostsofthe pro-ductionprocess.

Proteolyticactivityisthemainparameterforqualitycontrol, andthebromelainpurified bytheprocessdemonstrated inthe presentstudyhadaproteolyticactivityyieldof89%,whichishigher thanthatobtainedfromcurrentpurificationprocesses.

Finally,itisestimatedthatfrom1kgofstempineapple,itis possibletoobtainapproximately267mgofpre-purifiedbromelain and124mgofpurifiedbromelain.

4. Conclusions

The bromelain purification method of the present study employs two LC steps: ion exchange chromatography (car-boxymethylcellulose),followedbygelfiltrationchromatography (Sephadex®_{G-50).Thepurifiedbromelainhasamolecularweight}

ofapproximately30kDaandisof highpurity,andthe purifica-tionmethodyieldsahigherproteolyticactivityvalue(89%)ata lowercostthantheexistingmethodologies.Therefore,themethod demonstratedinthepresentstudycanbeusedtotransformthe agriculturalandindustrialwastesofpineapplecropintoaproduct withhighaddedvalueandsignificantbiotechnologicalinterest.

Acknowledgments

Theauthors thank Clair Barboza fromInstituto Capixaba de PesquisaAssistênciaTécnicaeExtensãoRural.Authorsthankstoo Dr.SilasRodriguesPessiniandInstitutodeBioquimicaMédicaof FederalUniversityofRiodeJaneiroandHemostaseandpoison labo-ratory(IBqM).ThisworkwassupportedbyFinanciadoradeEstudos eProjetos(FINEP),ConselhoNacionaldeDesenvolvimento Cien-tíficoeTecnológico(CNPq),Coordenac¸ãodeAperfeic¸oamentode

PessoaldeNívelSuperior(CAPES), Banco doNordestedo Brasil (BNB) and Fundacão de Amparo á Pesquisa do Espírito Santo (FAPES).

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