Presence of mycotoxins in sugar beet pulp silage collected in France

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Presence of mycotoxins in sugar beet pulp silage

collected in France

Hamid Boudra, Benoît Rouillé, Bernard Lyan, Diego Morgavi

To cite this version:

Hamid Boudra, Benoît Rouillé, Bernard Lyan, Diego Morgavi. Presence of mycotoxins in sugar beet

pulp silage collected in France. Animal Feed Science and Technology, Elsevier Masson, 2015, 205,

pp.131-135. �10.1016/j.anifeedsci.2015.04.010�. �hal-01901484�

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ContentslistsavailableatScienceDirect

Animal

Feed

Science

and

Technology

journalhomepage:www.elsevier.com/locate/anifeedsci

Short

communication

Presence

of

mycotoxins

in

sugar

beet

pulp

silage

collected

in

France

H. Boudra

a,∗

_,

_B.

_Rouillé

b

_,

_B.

_Lyan

c

_,

_D.P.

_Morgavi

a

a_INRA,_UMR1213_Herbivores,_F-63122,_{Saint-Genès-Champanelle}_and_Clermont_Université,_VetAgro_Sup,_UMR_Herbivores,_BP_10448,

F-63000Clermont-Ferrand,France

b_Institut_de_l’Elevage,_Monvoisin,_BP_85225,_F-35652_Le_Rheu_Cedex,_France

c_INRA,_UMR_1019,_Plateforme_{d’Exploration}_du_{Métabolisme,}_Nutrition_Humaine,_F-63122_{Clermont-Ferrand,}

Saint-Genès-Champanelle,France

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received14November2014 Receivedinrevisedform1April2015 Accepted9April2015

Keywords:

Sugarbeetpulpsilage Mycotoxins LC–ESI–MS/MS QuEChERS,Feedsafety

a

b

s

t

r

a

c

t

Sugarbeetpulp,amajorby-productofthesugarindustry,isacommonfeedcomponent incattledietsthatispreservedon-farmassilage.Thisstudywasdesignedtoinvestigateif sugarbeetpulpsilagecouldbeavehicleofcommonmycotoxinsfoundinsilagesandother regulatedmycotoxins.Samples(n=40)favouringmouldyspots,ifpresent,onthefront faceofopensilageswerecollectedin2011from5regionsrepresentingthemainFrench sugarbeetproducingareas.MycotoxinswereextractedbyQuEChERSprocedurewithout anyfurtherclean-upandanalyzedbyliquidchromatographycoupledwithelectrospray ionizationtandemmassspectrometry(LC–ESI–MS/MS).Themycotoxinsmonitoredwere: aflatoxinB1,deoxynivalenol,gliotoxin,ochratoxinA,mycophenolicacid,patulin, penicil-licacid,roquefortineCandzearalenone.Matrix-matchedcalibrationswereused,yielding acceptablelevelsofrecoveryrangingfrom64to168%,exceptforgliotoxinand roquefor-tineCforwhichrecoverywaslower(21and34%,respectively).Eightsamplesoutof40 (20%)werefoundtobepositive.Mycophenolicacidandzearalenonewerethemost pre-dominantofthemycotoxinsstudied.Mycophenolicacidwasfoundinfiveof40samples atlevelsrangingfromtracesupto1436␮g/kg.Zearalenonewasfoundinthreesamplesat concentrationsof1023,4862and6916␮g/kg.Thelast2sampleswereatconcentrations abovetherecommendedlimitof2000␮g/kg.OchratoxinAwasdetectedinonesample at15␮g/kg,whichisbelowtherecommendedEUlimitof250␮g/kg.RoquefortineCwas alsodetectedbutatlowlevels.Toourknowledge,thisstudyisthefirsttoreportonthe presenceofmycotoxinsinsugarbeetpulpsilage.Contaminationforthetestedmycotoxins waslowanddidnotseemtopresentahealthriskforanimalsorconsumersforthetested mycotoxins.

1. Introduction

Sugarbeet(BetavulgarisL.)isanimportantindustrialcropintemperateregionsthatprovidesaboutathirdofallsugar consumedintheworld.FranceisoneofthemainproducersofsugarbeetinEurope,with34milliontonnesharvested

in2009(http://www.labetterave.com).Duetoitsnutritionalvalueandavailability,thesugarbeetpulpby-productofthe

∗ Correspondingauthor.Tel.:+33473624104;fax:+33473624659. E-mailaddress:abdelhamid.boudra@clermont.inra.fr(H.Boudra). http://dx.doi.org/10.1016/j.anifeedsci.2015.04.010

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132 H.Boudraetal./AnimalFeedScienceandTechnology205(2015)131–135

sugarindustryiswidelyusedbyfarmersinproductionareas.Thesugarbeetpulpisensiledonthefarmandincorporated intocattlediets.Onecommonriskaffectingsilageconservationandqualityisthedevelopmentoffungiwhichreducesthe nutritivevalueoffeedsandanimalperformances(Morgavietal.,2008;Boudra,2009).Inaddition,fungaldevelopmentcan beaccompaniedbyproductionofmycotoxinsthatcanaffectanimalhealth.Themostcommoninfestationofsugarbeetin theﬁeldiscausedbyFusaria,andsomespeciesarecapableofproducingmycotoxinsintheﬁeld(Hanson,2006;Nitschke

etal.,2009;Hilletal.,2011)andinvitro(BoschandMirocha,1992;Burlakotietal.,2008;Christetal.,2011).Inaddition

toFusariumspp.thatcauselossesintheﬁeld,AspergillusandPenicilliumspeciescangrowwhensilageisnotappropriately conserved(Binderetal.,2007;Boudra,2009).Informationonsugarbeetcontaminationwithmycotoxinsissparse.Bosch

andMirocha(1992)reportedthat6outof25moldysugarbeetsamplescollectedintheﬁeldcontainedzearalenone(ZEA)in

concentrationsrangingbetween12and391␮g/kg.Zearalenonewasalsodetectedin31outof75sugarbeetfibersamples (13to47␮g/kg)(BoschandMirocha,1992).Toourknowledge,thereisjustasinglereportonfungalcontaminationofstored sugarbeetpulpsilage(Noutetal.,1993),butthepresenceofcorrespondingmycotoxinswasnotinvestigated.Theaimof thestudyreportedherewastoassesstheriskofmycotoxincontaminationinsugarbeetpulpsilagesamplescollectedfrom the5mainareasofsugarbeetproductioninFrance.Themethoddevelopedinthisstudytargetedmycotoxinsthatwere frequentlyfoundinsilages:patulin(PAT),penicillicacid(PENI),gliotoxin(GLIO),roquefortineC(ROQC),andmycophenolic acid(MYCO)(Auerbachetal.,1998;Richardetal.,2007;Mansfieldetal.,2008;Rasmussenetal.,2010;VanPameletal.,2011), inadditiontoaflatoxinB1(AFB1),ochratoxinA(OTA),deoxynivalenol(DON),andzearalenone(ZEA)thatareregulatedin feedsintheEuropeanUnion(EuropeanCommission,2006).

2. Materialsandmethods

2.1. Samplecollection

Sugarbeetpulpsilagesampleswerecollectedfrom5regions:Haute-Normandie(n=11),Ile-de-France(n=9),Picardie (n=7), Nord-Pas-de-Calais (n=8), and Centre (n=5), representing themain areas of sugar beet production in France

(http://agreste.agriculture.gouv.fr/).Silagesfromeachregionwerefromonesugarproductionplant.Allproductionplants

usedhighpressuretechnologytoextractsugarfrombeetrootandthepulpwastransported immediatelytofarmsfor ensilingwithinthesameday.Samplesweretakenfromsilosthatwereincurrentuseatﬁveplacesonthefrontface,ona diagonallinefromoneuppercornertotheoppositebottomcorner.Whilerespectingthissamplingprocedure,operators wereinstructedtofavourmoldyspotsifpresentonthefrontface.Thiswasdoneastheobjectivewastoassesstheriskof mycotoxinscontaminationfromthisfeedresource.Sampleswerethenpooledandasubsampleof150gwassenttothe laboratorywithin24hformycologicalandmycotoxinanalysis.Farmsincludedinthisstudyhadbunkersiloswithcapacities ofbetween100and750m3_and_did_not_use_{preservatives}_for_silage_making._The_dry_matter_(DM)_of_sugarbeet_silage_was

determinedintriplicatebydryingat105◦Cfor24h,andrangedfrom19to28%. 2.2. Mycologicalanalysis

Tengramsoffreshsampleweretransferredintoasterileplasticbag,suspendedin90mlofsterile0.05%Tween80,and homogenizedfor5mininalaboratoryStomacherblender(BayMixer400,Interscience,SaintNomlaBreteche,France).Serial dilutionsfrom10−2to10−5weremadeand0.1mlofeachwasinoculatedinduplicateintwodifferentculturemedia:2% Maltextractwithandwithoutsodiumchloride.Theplateswereincubatedat25◦Cinthedark,andmoldsandyeastswere countedafterthreedaysofincubation.Theresultswereexpressedascolony-formingunits(CFU)/gDM.Theremainderof thesamplewasdriedinaforceddraftovenat50◦Cfortwodays,groundthrougha1mmsieveandstoredat+4◦Cuntil mycotoxinanalysis.

2.3. Mycotoxinanalysis

ExtractionofselectedmycotoxinswasperformedbytheQuEChERS(Quick,Easy,Cheap,Effective,Rugged,andSafe) methodaccordingtotheoriginalWatersprocedurebasedontheAOACmethodintwodifferentsteps.Brieﬂy,sixgramsof silagewereplacedina50ml-DisQuETM_tube_(186004837,_Waters)_containing_trisodium_citrate_dihydrate₍₁_g),_disodium

hydrogencitratesesquihydrate(0.5g),NaCl(1g)andMgSO4(4g).Fivemilliliterofdistilledwatercontaining1%ofacetic

acidwereadded.Aftercompleteabsorptionofwater,10mlof1%ofaceticacidinacetonitrilewereaddedandtubeswere shakenvigorouslyfor1min,andthencentrifuged(2000g,5min).Onemlaliquotoftheupperphase(correspondingto0.4g ofsilage)wastransferredtoasecond2-ml-DisQuETM_tube_(186004837,_Waters)_containing_0.15_g_MgSO4_and_0.025_g_PSA

(primarysecondaryamine).Weadded7.5mgofGraphitizedCarbonBlack(DisQuETM_,_186004837,_Waters)_to_each_tube_in

ordertoremovepigmentssuchascarotenoidsandchlorophyllfromthesamples.Sampleswereshakenvigorouslyfor1min andthencentrifuged(2000g,5min).Theupperphasewasﬁlteredthrougha0.45_␮mﬁlter,and10_␮lwereinjectedinto theLCsystem.ThechromatographicsystemwasanAlliance2695module(WatersCorporation,St-Quentin-en-Yvelines, France).SeparationwasperformedatroomtemperatureonaC18RPcolumn(Luna,50×2mm,3␮m,Phenomenex,Paris,

France)usingagradientsolventsystem(solventA=0.1%formicacid–ammoniumacetate0.5mMadjustedtopH3.5,and solventB=Acetonitrile–0.1%formicacid).Thegradientconditionswereasfollows:theinitialpercentageofsolventBwas

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Table1

TransitionreactionsmonitoredbyLC–ESI–MS/MS,coneandcollisionvoltages.

Metabolites Tr Ionisationmode Precursorion Daughterion Conevoltage(V) Collisionenergy(eV)

Patulin 11.08 ESI− 153.0 81 15 13

109 15 9

Deoxynivalenol 2.28 ESI− 341.0 265 15 11

295.0 15 9

Penicillicacid 6.08 ESI− 169.0 93 20 19

110.0 20 9 Gliotoxin 9.55 ESI+ 327.1 245.29 15 17 263.241 15 9 AﬂatoxinB1 11.41 ESI+ 313.0 128 45 60 285.1 45 21 RoquefortineC 10.26 ESI+ 390.2 193.29 35 27 322.228 35 19

Mycophenolicacid 11.91 ESI+ 321.2 207 20 19

303.211 29 9

Zearalenone 13.23 ESI+ 319.2 97 20 17

301.294 20 9

OchratoxinA 13.43 ESI+ 404.1 239.19 25 23

358.19 25 13

Transitioninboldusedforquantiﬁcation.

Table2

Parametersofthemethodtestedforsugarbeetsamples.

Mycotoxins Parametersofmethod

SSE Rangeofcalibration

(%) (ngml−1) Recovery(%) AﬂatoxinB1 78 1.25–6.25 73±3 OchratoxinA 89 64±5 Zearalenone 187 137±43 Mycophenolicacid 96 168±21 Deoxynivalenol 4 45±18 RoquefortineC 62 50–250 34±2 Gliotoxin 77 21±3 Patulin 102 131±4 Penicillicacid 45 102±3

SSE,speciﬁcsignalsuppressionandenhancementwascalculatedaccordingtothefollowingequation:(Slopeofextract/Slopepurestandard)×100.

2%,heldat2%for4min,increasedto80%over12minandmaintainedfor8min.Itwasthenloweredtotheinitialpercentage in0.1min,andmaintainedfor6mintore-equilibratethecolumnpriortothenextinjection.Theflowratewas0.2mlmin−1. Undersuchconditions,allmycotoxinsandtheinternalstandard(IS)werewellseparated.Electrospraymassspectrometric (ESI–MS/MS)analyseswereperformedonaQuattroMicrotriplequadrupolemassspectrometer(WatersCorporation, St-Quentin-en-Yvelines,France)equippedwithanelectrospraysourceoperatinginpositiveandnegativeionmodes.Capillary voltagewassetat4kV,sourcetemperatureat120◦C,anddesolvatationtemperatureat450◦C.Theconeandnebulizationgas flows(bothnitrogen)weresetat50and500lh−1,respectively.Datawereacquiredusingthemultiplereactionmonitoring (MRM)scanningmode.Thevaluesofthetuneparameterswereseparatelyoptimizedbyinfusingasolutionofeachanalyte at10␮gml−1inmobilephaseataflowrateof10␮lmin−1.AllmycotoxinsselectedshowedgreatersensitivityinESI+, withtheexceptionofDON,PENIandPATwhichshowedgreatersensitivityinESI−.Runswerethereforeperformedin bothpositiveandnegativemodes.TheMRMtransitionsandconevoltagesandcollisionenergiesappliedaresummarized

inTable1.Themostintensetransitionreactionwasusedforquantiﬁcationpurposesandthesecondwasusedforanalyte

conﬁrmation.Thematrixeffect,evaluatedbydeterminationofenhancementofionsuppression,showedwidevariability. ThemassspectrometrysignalwasreducedforDONandPENI,andstronglyenhancedforZEA(Table2).Matrix-matched calibrationswerethereforeused,givingacceptableratesofrecoveryrangingfrom64to168,exceptforGLIOandROQ forwhichrecoverywaslower(21and34%,respectively).Theconcentrationofeachmycotoxinwascalculatedusingthe followingformula:Concentration (g/kg)=Mg/Ms,whereMgisthemassofmycotoxins(␮g),andMs themassofthe

sugarbeetpulpsilageinjected(kg)intotheLC–MS/MSsystem.

3. Resultsanddiscussion

FortyFrenchfarmsweresurveyedin2011forthepresenceofmoldsandmycotoxinsinsugarbeetpulpsilage.Asexpected fromthesamplingprotocol,mycologicalanalysisrevealedahighproportionofsampleswithcountsexceedingthestandard limitofhygienicqualityof5×104_CFU/g_(34/40,_85%)_according_to_Le_Bars₍₁₉₈₉₎₍_Table₃_)._This_high_level_of_{contamination}_is

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134 H.Boudraetal./AnimalFeedScienceandTechnology205(2015)131–135

Table3

Occurrenceofmolds,yeastsandmycotoxinsinsugarbeetsilagecollectedfromFrenchfarmsin2011.

Origin Samples analyzed

Mycotoxina_{concentration}_(␮g/kg)

Positive Molds Yeasts Mycotoxins OchratoxinA Zearalenone Mycophenolic acid

RoquefortineC

Ile-deFrance 9 7 3 4 1 1(15) trace

Haute-Normandie 11 11 7 9 5 1(1023) 4(trace,116, 224,1436) 2(23,264) Picardie 7 6 2 5 2 2(4862,6916) Nord-Pas-de-Calais 8 5 1 5 0 Centre 5 5 1 4 0 All 40 34 14 27 8

a_Mycotoxins_that_were_tested_with_no_positive_samples:_{deoxynivalenol,}_gliotoxin,_patulin,_penicillic_acid_and_aﬂatoxin.

certainlyduetosamplingofvisiblymoldysamples.In7samplestherewasco-occurrenceofyeastsandmolds.Theincidence ofyeastcontamination(68%)wasgreaterthanthatofmolds(35%)probablybecausesampledsilageshadarelativelylow DM(<28%).Aconditionthatfavorsyeastdevelopmentasreportedforothertypeofsilages(DriehuisandOudeElferink, 2000).Althoughyeastsdonotproducemycotoxins,theyindicatethequalityofsilage,includingtheriskofmycotoxin contamination.

ThepresenceofmycotoxinsisshowninTable3.Eightoutof40samples(20%)werecontaminatedwithfourmycotoxins (OTA,ZEA,MYCOandROQC).MYCOandZEAwerethemostpredominant;MYCOwasfoundinfiveout40samplesatlevels rangingfromtracesupto1436␮gkg−1whileZEAwasfoundinthreesamples,twoofthemexceedingtherecommendedlevel of2000␮gkg−1setbyEuropeanregulations(EuropeanCommission,2006).Thisdifferenceinmycotoxincontaminationwas probablyduetofarmmanagementpractices,especiallytheensilingprocessandtherateofsilageutilisation,bothofwhich wereundercontrolofthefarmer.OTAwasdetectedinonlyonesampleat15␮gkg−1,whichisbelowtherecommendedlimit of250_␮gkg−1.ZEAisproducedbyFusariumsppthatmainlycontaminatescropplantsinthefieldbeforeharvest.Several reportshaveshownthatFusariumspparethemaingenerainfestingsugarbeetinthefield(BoschandMirocha,1992;Nout

etal.,1993;Hanson,2006;Burlakotietal.,2008;Nitschkeetal.,2009;Christetal.,2011;Hilletal.,2011).Wecannotexclude

theproductionofZEAduringstoragebutthistoxin,thatwasalsodetectedinsugarbeetsamplescollectedintheﬁeld(Bosch

andMirocha,1992),isknowntobestableduringtheensilingprocess(KalacandWoollford,1982;Lepom,1988;Oldenburg,

1991;BoudraandMorgavi,2008).ThehighconcentrationofZEAfoundintwosamplescouldpotentiallyhaveanegative

effectonreproductiveefﬁciency.Thisisindependentofthemycotoxinoriginasmouldypartsofsilagearenotsystematically removed.InadditiontoZEA,therewerethreemycotoxinsthataretypicallyproducedduringstorage,i.e.OTA,MYCOand ROQC.MYCOandROQChavealreadybeenfoundinmaizeandgrasssilage(MüllerandAmend,1997;Auerbachetal.,

2000;Rasmussenetal.,2010)butthepresenceofOTAhasneverbeenreportedinsilage.Althoughwehavenottestedfor

thepresenceofmycotoxinsbeforeensiling,thetypesofmycotoxinsfoundsuggestthatcontaminationcouldbeoriginated bothintheﬁeldandduringstorage.Fieldcontaminationisdifﬁculttocontrolasitdependsonweatherconditions.Forthose mycotoxinsthatareproducedduringstorage,itisclearthatawarenessandtrainingforfarmersingoodensilingtechniques shouldreducetheriskofcontamination.

Inconclusion,mycotoxinsweredetectedinsugarbeetpulpsilageinFrance.However,theprevalenceandtypesof mycotoxinsfoundinthisparticularsurveydidnotsuggestahighriskforcattleandconsumerhealth.Complementary studiesunderdifferentconditionsandcoveringothermycotoxinsareneededtoconﬁrmthelow-riskmycotoxinstatusof sugarbeetpulpsilage.

Conﬂictofintereststatement

Thereisnoconﬂictofinterestinthiswork.

Acknowledgements

PartofthisworkwassupportedﬁnanciallybytheComitéNationaldesCoproduits(Paris,France).WethankSergeHamelin from“ContrôleLaitierd’Ile-de-France”forhisassistanceinprovidingthesilagesamples,andDelphineDelabrefromINRA (Clermont-Theix)forhertechnicalassistance.

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