Zearalenone adsorption capacity of lactic acid bacteria isolated from pigs

h tt p : / / w w w . b j m i c r o b i o l . c o m . b r /

Food

Microbiology

Zearalenone

adsorption

capacity

of

lactic

acid

bacteria

isolated

from

pigs

María

F.

Vega

_,

_Susana

_N.

_Dieguez

_,

_Belén

_Riccio

_,

_Sandra

_Aranguren

_,

Antonio

Giordano

_,

_Laura

_Denzoin

_,

_Alejandro

_L.

_Soraci

_,

_María

_O.

_Tapia

_,

Romina

Ross

_,

_Ana

_Apás

_,

_Silvia

_N.

_González

a_{DepartamentodeTecnologíayCalidaddelosAlimentos,FacultaddeCienciasVeterinarias,UniversidadNacionaldelCentrodela}

ProvinciadeBuenosAires,Tandil,BuenosAires,Argentina

b_{LaboratoriodeToxicologíaCIVETAN–CONICET,FacultaddeCienciasVeterinarias,UniversidadNacionaldelCentrodelaProvinciade}

BuenosAires,Tandil,BuenosAires,Argentina

c_{DepartamentodeSaludPública,FacultaddeBioquímicaQuímicayFarmacia,UniversidadNacionaldeTucumán,SanMiguelde}

Tucumán,Tucumán,Argentina

d_{ComisióndeInvestigacionesCientíficasdelaProvinciadeBuenosAiresCICPBA}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received12November2014 Accepted17November2016 Availableonline27May2017 AssociateEditor:MarizaLandgraf

Keywords:

Zearalenone Adsorption Lacticacidbacteria

Lactobacillus

Pigs

a

b

s

t

r

a

c

t

The ability to adsorb zearalenone by fivestrain of lactic acid bacteria wasevaluated: fourstrainsofLactobacillusspp.isolatedfrompigrectalswabsandonecommercialstrain (Lactobacillusrhamnosus).Severalfactorsaffectingtheadsorptioncapacitywereevaluated in order to improve the adsorption ofthe mycotoxinby bacteria. The stability of the zearalenone–bacteriacomplexwasanalyzed.Ineverycase,bacterialadsorptioncapacity washigherthan40.0%.Thestrainshowingthehighestadsorption(68.2%)wasselectedfor thefollowingstepsofthisresearch.Theadsorptionpercentagesobtainedafterprocessing 6.5and7.5mLMRSbrothwere57.40%+3.53and64.46%+0.76,respectively.Thestability ofzearalenone–bacteriacomplexwasevaluatedbysuccessivelyrinsing.Inthefirst rins-ingstep42.26%+0.414wasstillbound.Inthesecondrinsingstep25.12%+0.664wasstill bound,whereas15.82%+0.675remainedinthepelletafterthethirdrinse.Resultsobtained demonstratedthatLacticAcidBacteriahascapacitytoadsorbzearalenone.Finally adsorp-tionwasincreasedusingahighervolumeofinitialbroth.Theseresultscouldbeusedto designanewlyophilizedpowderfordetoxification,usinglacticacidbacteriaaspotential zearalenoneadsorbents.

©2017SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/

licenses/by-nc-nd/4.0/).

∗ _{Correspondingauthor.}

E-mail:mfvega@vet.unicen.edu.ar(M.F.Vega).

http://dx.doi.org/10.1016/j.bjm.2017.05.001

1517-8382/©2017SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Zearalenone(ZEA)isaworldwidedistributed mycotoxin as indicatedbytheInternationalAgencyforResearchonCancer (IARC).1–3 _{Itstoxicityandincidencewasconfirmedbyrecent} reports.4,5_{Thisresorcyliclactoneisproducedmainlyby}

Fusa-riumgraminearumand isone ofthe mostimportanttoxins causingseriousreproductivefailuresinpigproduction,dueto itsabilitytocouple17-␤-estradiolreceptors.Thisinterference withcytosolicestrogenreceptorsoftargetcellsmakesitan endocrinedisruptor.Whenthedangerousnessofthis myco-toxinisconsidered,twofactorsareimportant:itsstabilityto heat,milling,storageandprocessingoffeedingstuffs,6_andthe severityoftheintoxication,eveniftheingestiontakesplace onlyforafewdaysasindicatedbyAnadónetal.7

Severalphysicochemicalmethodsfordetoxificationhave beenwidelyused,butthetendencyistousebiological meth-odsthat donot cause nutritional and palatability changes infeed.8 _{Thereare severalfoodadditivesfor} decontamina-tionand/or detoxificationofanimalfeedstuffs,suchasthe commercialadsorbentMycosorb® (AlltechInc.),inwhichthe structureofyeastglucanlayerismodifiedtoincreaseits affin-ityandbindingrateofmycotoxinsabovethatofthenative cellwallproduct.9 Inspiteofitsgreaterefficacy,theuseof Mycosorbislimitedbecauseofitshighprice.

InteractionsbetweenFusariummycotoxins,ZEA,its deriva-tive␣-zearalenol(␣-ZOL)andfood-gradestrainsof Lactobacil-lushavebeenreportedbyseveralresearchers.10–17_El-Nezami et al.13 _{demonstrated that, afterco-incubation of ZEA and}

Lactobacillus,aconsiderableproportion(38.0%–46.0%)ofthese mycotoxins were recovered from bacterial pellets. Bacteria showedcapacitytoadsorbtoxins and,asexpected, results demonstrated thatthe bindingdependedon bacterial con-centration. Co-incubation of ZEA and ␣-ZOL with bacteria significantly affected the percentage of toxin bound, sug-gestingthat thesetoxinsmay sharethe samebindingsite. RecentlyTinyiroetal.18_{usingsomeBacillusstrains,showed} thatthe adsorptionpercentagewashigh (78.0%and95.0%) andvariationdependedonthe strainused. ZEAadsorption abilitybyPlanococcusspphasalsobeendemonstratedbyLu etal.19

Two fundamental rules are considered when bacterial

strainsareisolated inorder tobeadministeredtoanimals. Thefirstisthehostspecificity,itisessentialtoachieveagood adaptation.20,21_{Thesecondistheproximityoftheecosystem,} itisvitalthatthemicroorganismsareisolatedinthesame placewhereitactsonthehost.22,23

Forallthesereasons,thepurposeofthepresentworkwas tostudytheZEAadsorptioncapacityoflacticacidbacteria iso-latedfrompigrectalswabsunderinvitroconditions,inorder tobroadenscientificknowledgeonfeedadditivesbasedonthe useoflacticacidbacteriaasZEAbinders.

Materials

and

methods

Bacterialstrainsandcultureconditions

Lactobacillus rhamnosus (R1) (Lyofast LR B Sacco) was pur-chasedfromSaccoSRL(Cadorago,Italy).Thisstrainwasused

as reference because of its demonstrated ability toadsorb ZEA.12,13,24

ThetestedLactobacillusstrains(L1,L2,L3,andL4)were iso-latedfrom7pigletskeptonasinglefarm.Thepiglets,aged 21–60days,wereclinicallyhealthyandnoantibioticshadbeen administered duringtheir lives.All sampleswere obtained withinthesameday.Thestrainswerecollectedwithakit con-tainingaswabandatransportmedium(LAPTgsemisolidagar withoutsugars). Afterbeingdeliveredtothelaboratory,the swabswerebrokenoffintopeptonewater,thedilutionswere made and inoculated onto solid medium: LAPTg Agar,25–27 thentheplateswereincubatedinmicroaerophilia(candlejar system)at37◦Cfor48h.

Strainsoflacticacidbacteria(LAB):L1,L2,L3andL4were isolatedatthe“LaboratoriodeToxicología”atthe“Universidad NacionaldelCentrodelaProvinciadeBuenosAires”Tandil, BuenosAires,Argentina.LABstrainswerekeptat−20◦_Cin

LAPTgbroth(1.5%peptone,1%tryptone,1%glucose,1%yeast extract,and0.1%Tween80),pH6.826_{containing15%glycerol} (v/v)untiluse.

Reactivationprocess

Thestrainsweredefrostedat37◦Cfor15min,thentheywere inoculated and incubated in LAPTg broth for24h at37◦C inmicroaerophiliabeforegenotypicanspeciesidentification. Beforeadsorptionprocesstworeactivationtimesweretested: 24h(t1)and72h(t2).

Genotypicidentification

ThestrainsisolatedforthisworkL1,L2,L3andL4,were char-acterizedaslactobacillibyPCR,accordingtothemethodology describedbyDubernetetal.28_{Industrialstrainsprovidedby} SaccoS.A.:L.rhamnosus(R1),Lactobacillusdelbrueckii(R2), Lacto-bacilluscasei(R3)andLactobacillushelveticus(R4)wereusedonly forgenotypicidentificationaspositivecontrol.Identification wasmadebyamplifyingspecificregionofDNA.The amplifi-cationproductswerevisualizedbyelectrophoresisinagarose gelsstainedwithethidiumbromide.29_{Generalprimersfor} lac-tobacilliLbLMA1-rev(5-CTCAAAACTTTCAAACAAAGT-3 andR16-15-CTTGTACACACCGCCCGTCA-3)wereused.28

Speciesidentification

Genotypicidentificationwascarriedoutbypartial16SrRNA

gene sequencing. Genomic DNA was extracted

accord-ing to Pospiech and Neumann.30 _{Oligonucleotide primers}

(PLB16, 5-AGAGTTTGATCCTGGCTCAG-3, and MLB16,

5-GGCTGCTGGCACGTAGTTAG-3) were used to amplify the

variable(V1)regionofthe16SribosomalRNAgeneaccording totheprotocol describedbyseveralauthors.31–33 _PCR prod-uctswereelectrophoresedin1%(wt/vol)agarosegels,stained and visualizedasdescribedabove. Ampliconswereexcised

from the gel and purified using a GFXPCR DNA gel band

purification kit (GE Healthcare, UK). Purified PCR products

were sequenced atCERELA-CONICET, Tucumán, Argentina,

byusing an ABI3130 DNAsequencer (Applied Biosystems,

Foster,CA).rRNAgenesequencealignmentswereperformed

identification querieswere fulfilled byaBLAST search36 _in GenBank(http://www.ncbi.nlm.nih.gov/GenBank/)andinthe RibosomalDatabaseProject.37

DeterminationofGrowthbyOpticalDensity(OD560nm)

Thequantifyingofthetotalnumberofcellsforeachstrain(R1, L1,L2,L3,andL4)wasmadeusingaturbidimetricmethod. TheOD560nmofthebacterialsuspensionincludeslivingcells,

deadcells,andnoncellularmaterialthatabsorbs/scattersthe lightandindicatesdetotalmassachieved.Thewavelengthof thespectrophotometer(UV-VisSpectrofotometerPharmacia LKBBiochromLimited,UltrospectIII,Cambridge,England)was setat560nm.Onemilliliterofthesterilebrothwhichwas usedtogrowbacteria(LAPTgorMRS)wasusedtocalibrate theequipmenttozeroabsorbanceand1mLofeachsample wasusedtomeasuretheOD560nm.

Pelletsadsorptionprocess

InfirstplaceastockZEAsolutionwaspreparedby dissolv-ing25mgZEA(99.5%purity,Sigma,St.Louis,USA)in125mL chloroform(HPLCgrade,Biopack,Argentina)toobtainafinal concentrationof200␮g/mL.Thissolutionwaskeptat−18◦_C

untilitsuse.95␮LofthestockZEAsolutionwereevaporated todrynessunderanN2currentat60◦C.Thedryresiduewas

resuspended in 50␮L ofmethanol (HPLC grade, Sintorgan, Argentina)and1500␮Lofa0.9%NaCl(Sigma,St.Louis,USA) (SP1)or0.9%CaCl2(Sigma,St.Louis,USA)(SP2)togiveaZEA

concentrationof12.3␮g/mLinbothsolutions.

Insecondplacetheadsorptionpelletswereprepared.LAB

wereactivatedandgrowninLAPTgmedium.Cultureswere

incubatedinmicroaerophilia(candlejarsystem)for72hat 37◦C.OD560nmwasmeasured,thenbacteriawereharvestedby

centrifugation(SigmaLaborzentrifugen1K15,St.Luis,United States)at5900×g,4◦C,15min.

FinallybacterialpelletswererinsedtwicewithNaCl0.9% solutionandthenresuspendedin1.5mLofSP1solutionorSP2 solutionaccordinglyandincubatedfor1hourat37◦C,with gentlestirringevery30minasindicatedbyBueno.11

After incubation, the solutions were centrifugated

(5900×g, 4◦C, 15min), then the supernatant was

fil-tered through a 0.22␮m nylon membrane and analyzed

byHPLC–UV.11,38

Asacontroltodemonstratethattheadsorptionprocess recoversallthemycotoxinreferencestrain(R1)wasused.ZEA inthesupernatantandinthepellet(extractedwithmethanol)

were quantified,thesetwoconcentrations wereadded and

comparedwiththeinitialmycotoxin.

ZEAdeterminationbyHPLC

TheHPLC system was equipped with a Gilson 151 UV-Vis

detector, and Gilson 712 software (Gilson, Inc., Middleton, USA).SeparationwasachievedbyHPLC,usingaC18 station-aryphase, 250mm×4.6mmi.d., 5␮mcolumn. Themobile phaseconsistedofacetonitrile(HPLCgrade,Sintorgan,Villa Marteli,Argentina):water(ultrapurified,Elga)mixture(55:45)

workingin isocratic mode at1.2mL/min.The columnwas

maintainedat30◦C.Theinjectionvolumewas20␮Landthe

chromatographicruntimewas20min.Detectionwascarried out at 236nm.39 _{To quantify ZEA using external standard} calibrationsevenconcentrations0.63,1.25,2.5,5,10,15and

20␮g/mL were prepared with standards (Sigma St. Louis,

USA), the responseswere foundtobelinear (coefficientof correlation>0.998).Recordedretentiontimesfor␤-ZOL,␣-ZOL andZEAwere6.70min;9.24min;and17.19minrespectively.

Adsorptionpercentage(ADS%)quantification

ADS%wasquantifiedbycomparingmycotoxinspeakareasin thesupernatantobtainedaftertheadsorptionprocesstopeak areasforSP1orSP2solutionswithoutbacterialaddition, rep-resentingpositivecontrols.TheADS%ofZEAwascalculated accordingtoEq.(1).38,40

ADS%=100−AUC2∗100

AUC1 (1)

Being:

ADS%:adsorptionpercentage.

AUC2:peakareaofthemycotoxininthesupernatantafter

theadsorptionprocess.

AUC1:peakareaofthemycotoxininthepositivecontrol

(SP1orSP2).

Strainselection

Two parameters were used for LAB strain selection: The

OD560nmachievedafterthereactivationprocess,usedas

indi-catorofmassproduction,andADS%.Weusedbothvariables followingEq.(2)becauseweconsiderthattheyhavethesame importanceforstrainselection.

X= OD560nm∗0.5+ADS%∗0.5

0.5+0.5 (2)

Being:

X=meanofOD560nmandADS%.

OD560nm=opticaldensity.

ADS%=adsorptionpercentage.

Optimizationofadsorptionprocessconditions

Withtheaimofincreasingadsorptionpercentage,usingthe selectedstrain,fourvariablesofthetechniquedevelopedby Bueno11_{wereevaluated.}

(i) Culturemedia usedin the reactivationprocess: bacte-rialgrowthwascomparedintwoculturemedia:MRSand LAPTg.FirstbacterialgrowthwasdeterminedbyOD560nm

andcellswereobservedbyscanningelectronmicroscopy (SEM)(JEOL35CFfromJEOL Ltd.,Tokyo, Japan).Finally bacteria were cultured until OD560nm=1 was reached.

Thisvaluewasstandardizedforeverystrain.The adsor-bentpelletswereobtainedforbothculturemediaandthe ADS%werecalculated.

(ii) Contactsolution:theeffectsofsolutionsSP1andSP2on adsorptionpercentagewerecompared.

(iii) Initialvolumeofculturemedia:theeffectsofaninitial volumeof7.5mLand6.5mLwerecompared.

(iv) Viable and non-viable strain: the effects of sterilizing (120◦C,15min)andnon-sterilizingstrainwerecompared.

Evaluationoftheselectedmethod

Using data from 3 samples on 4 separate days of assay

theselectedmethodwasalsoevaluated.Theprecisionwas expressedbytherelativestandarddeviation(RSD).Notethat theRSDiscalledassameasthecoefficientofvariation(CV) instatistics.Withindayprecision(repeatability) was calcu-lated by the mean coefficient ofvariation (CV) which was requiredtobelessthan15%inaccordancewiththeGuidance forIndustry–BioanalyticalMethodValidationpreparedbythe BiopharmaceuticsCoordinatingCommitteeintheCenterfor DrugEvaluationandResearch(CDER)incooperationwiththe CenterforVeterinaryMedicine(CVM) attheFoodandDrug Administration.41_{Betweendaysprecision(intermediate} pre-cision)wasexpressedasbetweendayscoefficientofvariation (CVbd),whichwascalculatedusingEq.(3).42

CVbd=

SDbd∗100

 (3)

Being:

=averagemedia.

SDbd=betweendaysstandarddeviation(calculatedasthe

squarerootofbetweendaysvariance).

Between daysvariance isobtainedaftersubtracting the contribution of within day variability, using the following Eq.(4) SD2_bd=SD2_()+(n−1) n ∗SD 2 wd (4) Being:

SD2_{()=varianceofeverydaymean.}

n=numberofobservationsperday.

SD2

wd=averagewithindayvariance.

SD2_wd=





D=totalnumberofdays.

n=totalnumberofreplicatesperday.

xdr=resultforreplicaterondayd.

¯xd=averageofallreplicatesondayd.

SD2 =







D=totalnumberofdays.

¯xd=averageofallreplicatesondayd.

¯¯x =averageofallresults.

Scanningelectronmicroscopy(SEM)

Thepelletsobtainedafterthereactivationprocessandafter

the adsorptionprocess were fixedwith amodifier

Karnof-sky’ssolution(1.6%glutaraldehyde+2.6%paraformaldehyde

in0.1Msodiumphosphatebuffer(pH7.2)).After24hat4◦C, they were dehydrated by successive passages in solutions (ethylalcohol–acetone)withgreatergraduationofethyl alco-holeachtime(30%,50%,70%,90%y100%),thelapseforeach passagewas10min.Afterdrying,thesamplesweregluedon stubs using carbon tape and coatedwith gold(ion sputter JFC-1100).BacteriawereanalyzedusingaJEOL35CFscanning electronmicroscope.

StabilityoftheZEA-selectedLactoctobacilluscomplex

UsingthestrainwiththehighestADS%,ZEAdissociationwas studybysuccessiverinses.Infirstplace,theselected adsorp-tion process was carried out,and then the pelletobtained wassuspendedin1.0mLofpurifiedwaterandmaintainedat roomtemperaturefor10min.Then,thesuspensionobtained wascentrifugedat5900×g,4◦C,15min,andfinallyZEA con-centrationwas analyzedinthesupernatant. Thisstepwas repeateduptothreetimes.Attheend,ZEAremainingbound wasrecoveredwith1.0mLofmethanol.ZEAquantificationin allsupernatantswasperformedbyHPLCasindicatedinZEA determination.ZEAboundtothebacterialpelletwas calcu-latedaccordingtoEq.(7).

ln(ZEAboundM)=ln



100



(7) Being:

%ADS=adsorptionpercentage.

Initial ZEA=␮M of Initial ZEA in the positive control SP1=38.64␮M.

BiotransformationofZEA

Themetabolization ofZEA into ␣-ZOLand ␤-ZOLwasalso

investigatedbecauseZEA concentrationcouldbereduceas aconsequenceofbiotransformationasindicatedbyBoswald etal.43_{andnotbecauseofZEA–bacteriacomplexformation.} HPLCanalysisofamixedstandardsolutioncontainingZEA and bothmetabolites wasusedtoidentifythe presenceor absenceofeachcompound.

Statisticalanalysis

All samples were analyzed in triplicate. Results were

expressedasmean±standarddeviation(SD).Datawere sta-tisticallyevaluatedusingtheInfoStatprogram,2010.(InfoStat groupFCA,UniversidadNacionaldeCórdoba,Argentina).A two-tailed Student’st-test forpaired samples(p<0.05) was usedtodeterminesignificantdifferencesbetweentreatments.

Results

and

discussion

Genotypicidentification

ResultsfromthePCRmoleculartechniqueareshowninFig.1. AllstrainsassayedandthereferencestrainsL.rhamnosus(R1),

L.delbrueckii(R2),L.casei(R3)andL.helveticus(R4)werepositive (amplified)withgeneralprimersforLactobacillus.Inthe nega-tivecontrol(Ct),asisexpected,noamplificationwasobserved.

L4 R4 L1 6 R1 8 7 5 R2 R3 C1 C2 C3 C4 L2 L3 Ct

Fig.1–PCRamplificationproductsforLactobacilliisolatedfrompigrectalswaps(L1–L2–L3–L4),andisolatedfromother sources(5–6–7–8–C1–C2–C3–C4)andreferencestrains(R1–R2–R3–R4)usinggeneralprimers:LbLMA1-revyR16-1.Ct,control (withoutanybacterialstrain).

Speciesidentification

ThecomparativeanalysisofthesequencesobtainedfromL4 strainwiththosedescribed invarious databases,showed a highidentitywithstrainsbelongingtoPhylum“Firmicutes” Class “Bacilli” Order “Lactobacillales” Family

“Lactobacil-laceae” Gender “Lactobacillus” and within this genus to

species“plantarum”and“pentosus”.Giventhephylogenetic proximityofthesespecies,additionalstudieswereperformed todifferentiatebetweenthem.L4straindidnotferment glyc-erolandD-xylose,whicharetypicallyfermentedbyL.pentosus

butnotbyL.plantarum.44,45 _{Theseresultsindicate thatthe} selectedstrain(L4)wasaL.plantarum.

Adsorptionprocess

When time t2 (72h) was used in the reactivation

pro-cess the reference strain R1 gave an adsorption value of 79.8%±6.8.Thisresultwassimilartotheresultsobtainedby El-Nezami12_{whennonviablebacteriawereused(80%).With} thisreactivation timeit wasprobablethatwaste and dead cellsbegantoaccumulateanditcausedsomechangesinthe adsorptionprocess.Inthenextassayreactivationtimet1(24h)

wasused.Thischangegaveanadsorptionvalueof30.5%±5.4, itwasclosethanvaluesreportedforviablecells.13

ForR1strain,thesumoftheZEApercentages,inthe super-natantandinthemethanolusedtoextractmycotoxininthe pelletwas97.73%.Thisvaluewas closeto100%mycotoxin whichindicatedthatthemethodachievedahighmycotoxin extraction.

Strainselection

The strains L1, L2,L3 and L4 showed adsorption percent-agesbetween46%and64%(Table1),thesewerehigherthan thoseshowedbyBueno11_{andsimilartothosedescribedby} El-Nezami.13_{ThesecondparameteranalyzedwasOD}

560nm,

asBueno11_{indicatedinordertoachieveagoodbacteria} pro-duction,thevalueshouldbe1orhigher.Onlyinonecase(L1 strain)thevaluewaslower.Accordingtoourresults,L4strain achievedthehighestaverage(X)betweenOD560nmandADS%,

soit wasdecidedtousethisstraininsubsequentresearch. Severalgeneraofbacteriahavebeenstudiedtoevaluatetheir

Table1–Resultsoftheparametersusedforstrain selection[OD560nm,%ADSandaverage(X)ofboth

values)].Reactivationtimeused24h.

Strain Adsorptionparameters OD560nm %ADS (X)average L4 1.062+0.085a _64.2+1.5c _32.618c L3 1.361+0.118b _47.6+1.5b _24.501b L2 1.426+0.143b _49.1+0.0b _25.278b L1 0.905+0.014a _46.5+5.8b _23.698b L.rhamnosus(R1) 1.129+0.019a _30.5+5.4a _15.825a Resultswererepresentedasmean±SD.Differentsuperscript let-tersinthesamecolumnindicatestatisticalsignificantdifferences (p<0.05).

zearalenoneadsorptionability.Bueno11 _{reportedadsorption} levels ranging from 0 to 36.44% for Bifidobacteria, 1.74 to 33.69%forLactobacilli,and0to59.09%forActinomycetes. El-Nezami13_{reportedtheZEAadsorptionpercentagesoftwofood} gradestrains(L.rhamnosusGGandL.rhamnosusLC705). Fur-thermore,Luetal19studiedZEAadsorptionbyPlanococcusspp,

achieving21.82%adsorptionforviablebacteriaand42.82%for inactivatedbacteria.

Resultswere representedasmean±SD.Different

super-script letters in the same column indicate statistical

significantdifferences(p<0.05).

Evaluationoftheadsorptionprocessconditions

FortheselectedLactobacillusstrainL4thechangeofvarious parametersinordertoimprovetheadsorptionprocesswas evaluated.

Culturemediacomparison,L4showedsignificantlyhigher OD560nm in MRS (1.707±0.16) than in LAPTg (1.101±0.15)

thesedifferenceswerestatisticallysignificant(p<0.05).With respecttoadsorptionpercentages,usingthesame OD560nm

(1) higher results were also obtained using MRS media

(57.38%±4.68)thanLAPTg(49.33%±4.10),butthesedifference werenotstatisticallysignificant(p>0.05).Takingintoaccount afutureindustrialuseandobservingthatMRSbrothachieved thehigherproductionitwasselectedasculturemedia.

In addition, scanning electron microscopy revealed few differencescomparing bacterialmorphologyduring incuba-tion inboth broths(Fig.2Aand B),bacillus seemed longer andthinnerwhenculturedinLAPTgthaninMRS(markedin

Fig.2–ScanningelectronmicrographsofL4strainin(A)LAPTgmedia,(B)MRSmedia,(C)aftertheadsorptionprocess.

Fig.2Awithasquare),thatshouldbestudiedinfuture investi-gations.Thesechangeswouldindicatedifferentmorphology ofthe cellular walland could explaindifferent adsorption percentageachievedwhenthisdifferentculturemediawere usedinthe reactivationprocess priorZEA adsorption. Fur-thermore,afterthe adsorptionprocess,L4 presentsseveral intercellularbonds(markedinFig.2Cwithcircles),thisbeing aninterestingstrainpropertythatshouldbestudiedinfuture investigations(Fig.2C).

Ahigher%ADSwasobtainedafterincubationwithSP1than withSP2 solution, (57.28%±1.85 and 49.33%±6.11, respec-tively). These differences were not statistically significant (p>0.05), whichindicatesthat achangeofcontactsolution doesnotproduceasignificantchangeintheadsorption.For thisreason,thetechniquewasnotchangedandSP1was main-tainedascontactsolution.

Adsorption percentages obtained after processing 6.5

and7.5mLMRSbrothwere57.40%±3.53and 64.46%±0.76, respectively. These differences were statisticallysignificant (p<0.05).Thereforetheuseof7.5mLofmediawasselected tocarryouttheadsorptionprocess.

Aftersterilizingculturemedia,theADS%was82.4%±3.3 and73.5%±1.0forviablestrainADS%.Thesedifferenceswere statisticallysignificant(p<0.05).Thesefindingscouldbe con-sideredinordertodecideifnon-viablebacteriamaybeused asmycotoxinsadsorbentwithhigherADS%orviablebacteria withlowerADS%butwithpossibleprobioticeffectatthesame time.Thesefindingsconfirmtheresultsofotherresearches.13

Table2showstheADS%obtainedbyprocessingthreedaily

samplesforfourdifferentdays, underidenticalconditions.

Relative standard deviations (%RSD) were calculated for

each day, these values 1.81 to 7.23 were similar withthat obtainedbyKolfk-Clauwetal.,39_{usingthesametechnology} fordeterminationofZEA(HPLCUV),andtheyneverexceed thevalueof15%thatwasourgoalproposal.Repeatability(Sr),

also known as within-run precision was calculated,under identicalconditionssuccessivemeasurementsobtainedwere close in a value of 6.00. Using the overall mean obtained (70.10)andtherelativestandarddeviationmaximumallowed (15%),themaximstandarddeviationclaimedwas10.52.This valuewasless thanSDachievedineach dayand lessthan therepeatabilityobtained.

Resultswere representedas mean±SD.Different super-scriptlettersinthesamerowindicatesignificantdifferences (p<0.05). 0 1 2 3 0.5 0.14 0.37 1.00 2.72 7.39 20.09 ln (Z EA b ound (uM)) Number of washes 26.3 6.1 9.7 16.3

Fig.3–ZEAremainingfromZEA–L4complexafter successiverinsing.

Withindayprecision(repeatability)andbetweenday pre-cisionwerecalculatedandasrequiredbothvalueswereless than15%.

StabilityoftheZEA–bacteriacomplex

UsingtheimprovedprocesswithaninitialZEAconcentration of38.7␮M,theinitialadsorptionpercentagewas68.20%±1.74 (ZEAbound:26.3␮M).Inthefirstrinsingstep42.26%±0.414 wasstillboundtothepellet(ZEAbound:16.3␮M).Inthe sec-ondrinsingstep,25.12%±0.664wasstillboundtothepellet (ZEAbound:9.7␮M),whereas15.82%±0.675remainedinthe pellet(ZEAbound6.1␮M)afterthethirdrinse.

Fig.3showsln(ZEAbound)vsnumberofwashes. Dissoci-ationofZEAwasrelativelylinear.Thedissociationconstant, whichcouldbeestimatedfromtheslope,washigherthanthat obtainedbyEl-Nezamietal.12_{Theseresultscouldprobablybe} duetotheuseofdifferentbacteriastrainsthanthoseusedby El-Nezamietal.,12_{specificallyintermsoftheirdifferencesin} thecellwall.Somecompoundsofthecellwallarereally impor-tantintheadsorptionprocessasindicatedbyYiannikouris etal.46,47

Table2–Adsorptionpercentage(%ADS)ofL4aftertheadsorptionprocessforthreesamplesinfourdifferentdays. Estimationofrepeatability(withindayprecision)andintermediateprecision(betweendayprecision).

Day1 Day2 Day3 Day4 Mean SD2

() SD2wd SD2bd

%ADS 64.46 65.30 75.61 75.01 70.095() 31.409 36.042 55.437

SD 1.17a _1.35a _5.47a _1.73a

%RSD(CV) 1.81 2.06 7.23 2.31

Resultswererepresentedasmean±SD.Differentsuperscriptlettersinthesamerowindicatesignificantdifferences(p<0.05).Withinday precision(repeatability)andbetweendayprecisionwerecalculatedandasrequiredbothvalueswerelessthan15%.

Within-dayprecision:3.36%.Between-dayprecision:10.62%.

B

β zearalenol α zearalenol

Zearalenone

Retention time Area% Compound 6.70 6 557 656 β zearalenol 9.24 8 125 206 α zearalenol 17.19 9 503 858 zearalenone

Retention time Area Area %

1.23 23312 6.387

1.86 268498 73.558

4.41 3780 1.036

18.14 69425 19.020

Retention time Area Area %

1.75 224226 17.961 1.95 161726 12.955 2.99 3086 0.247 3.51 5006 0.401 4.39 4599 0.368 6.27 16010 1.282 18.19 833743 66.785

A

C

D

Fig.4–Chromatographicprofiles.(A)Chromatographicprofileofstandards:␣zearalenol,␤zearalenolandzearalenona.(B) ChromatographicprofileofZEAsolution(SP1).(C)ChromatographicprofileofL4supernatantafterco-incubatingwithZEA (1h,37◦C).(D)ChromatographicprofileofZEAfromL4pelletaftersolubilizationwithmethanol.

ZearalenonedeterminationbyHPLCchromatography

ResultsfromamixedstandardsolutionofZEA,␣and␤ zear-alenolareshowedinFig.4A.ResultsfromSP1solutionwithout contactwithadsorbentare showedinFig.4B,peaks corre-sponding to␣and ␤ zearalenolwerenot observed.Results fromthesupernatantofL4strainafterincubation(37◦C,1h) withthemycotoxinareshowedinFig.4C,peaks correspond-ing to␣ and ␤ zearalenolwere notobserved. Resultsfrom L4 pellet after resuspension withmethanol are showed in

Fig.4D.Peakcorrespondingto␤zearalenolwithasmallArea %wasobserved,peakcorrespondingto␣zearalenolwasnot observed,itwasimportanttodemonstratedthatprobablythis

Lactobacillusdidnotmetabolizethemycotoxinasindicatedby El-Nezamietal.13_{.ThesumofZEAinsupernatantandin} pel-letwasalmosttheinitialZEAaddedinSP1solution,itshowed thattheinitialmycotoxinin(SP1)solutionwasalmost com-pletelyrecovered.

Conclusions

This work demonstrated that a Lactobacillus plantarum (L4 strain)isolatedfrompigshasgreatpotentialasadsorbentof themycotoxinzearalenone.Theresultsshowedthebest con-ditionsfortheadsorptionprocess.Theinitialculturemedium usedforbacterialreactivation,theviabilityofthecellsand

the amount ofbacterial mass measured by OD560nm were

stronglyinvolvedinthepercentageofadsorptionofL. plan-tarumtoZEA.Thestudiesshowedhighstabilityofthecomplex bacteria–mycotoxinand the absence ofmetabolites ofthe toxin.This study is the first stepin the developmentof a newZEAadsorbentbasedonalyophilizedpowderofalocally isolatedstrainand other componentsthathelptoimprove reproductivehealth.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

This work is part of a project entitled “Detoxificación de lamicotoxinazearalenona enalimentosparacerdos medi-antemétodosmicrobiológicos”whichisfinanciallysupported

by the “Comisión de Investigación Científica (CIC)” and

developedinthefollowinglaboratories:Departmentof

Pub-lic Health (FBQF – UNT – Tucuman, Tucuman, Argentina),

Laboratory of Toxicology (SNITV) (FCV – UNCPBA, Tandil,

Buenos Aires, Argentina), and Department of Technology

and Qualityof Food(FCV – UNCPBA,Tandil, Buenos Aires, Argentina).

We are grateful to Dr Peter Purslow from Canada,

Departamento de Calidad y Tecnología de los Alimentos,

UniversidadNacionaldelCentrode laProvinciadeBuenos Aires, Tandil, Argentina, for his valuable revision of the manuscript.

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