Effect of ultrasound on survival and growth of Escherichia coli in cactus pear juice during storage

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

Food

Microbiology

Effect

of

ultrasound

on

survival

and

growth

of

Escherichia

coli

in

cactus

pear

juice

during

storage

Nelly

del

Socorro

Cruz-Cansino

a

_,

_Isidro

_{Reyes-Hernández}

a

_,

_Luis

_{Delgado-Olivares}

a

_,

Diana

Pamela

Jaramillo-Bustos

b

_,

_José

_Alberto

_Ariza-Ortega

a

_,

_Esther

_{Ramírez-Moreno}

a,∗

a_{CentrodeInvestigaciónInterdisciplinario,ÁreaAcadémicadeNutrición,InstitutodeCienciasdelaSalud,UniversidadAutónomadel}

EstadodeHidalgo,SanAgustínTlaxiaca,Hidalgo,México,Mexico

b_{MikunaGroupErie,PA,USA}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received3September2014 Accepted12November2015 Availableonline2March2016 AssociateEditor:EduardoCesar Tondo

Keywords:

Ultrasound Growth

Escherichiacoli

Cactuspearjuice Storage

a

b

s

t

r

a

c

t

Theaimofthisstudywastoinvestigatetheeffectivenessofultrasoundasaconservation methodfortheinactivationofEscherichiacoliinoculatedintocactuspearjuices(greenand purple).Totalsolublesolids,pH,titratableacidity,andthekineticsofE.coliincactuspear juicestreatedbyultrasound(60%,70%,80%and90%amplitudelevelsfor1,3and5min) wereevaluatedover5days.Totalinactivationwasobservedinbothfruitjuicesafter5min ofultrasoundtreatmentatmostamplitudelevels(withtheexceptionof60%and80%).After oneandtwodaysofstorage,therecoveryofbacteriacountswasobservedinallcactuspear juices.Ultrasoundtreatmentat90%amplitudefor5minresultedinnon-detectablelevels ofE.coliincactuspearjuicefor2days.TheparametersofpH,titratableacidityandsoluble solidswereunaffected.

©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense

(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Healthconsciousconsumersaredemandingminimally pro-cessedfoods,whichhasstimulatedresearchonnon-thermal processing technologies.Pulsed electric fields, high hydro-staticpressure,shortwaveultravioletirradiation,and ultra-sound,usedaloneorcombined,areintendedtoachieve micro-bialandenzymaticinactivationwithsignificantlylessheat. Amongthese technologies, ultrasound processingfor food preservationpurposeshasreceivedincreasingattention.1

∗ _{Correspondingauthor.}

E-mail:rme1234@yahoo.com(E.Ramírez-Moreno).

Ultrasoundsappliedtoaliquidmediuminducecavitation bubbles,whichleadtothedisintegrationanddestructionof microorganisms.Thecollapseofbubblesresultsinanareaof hightemperatureandpressure,calledthe“hotspot”.2_During

ultrasound,twophasesaredistinguished: compressionand rarefaction.Inthefirstphase,wavemicrobubblesareformed atvariousnucleationsitesinthefluid.Inthesecondphase, thesebubblesgrowrapidlyandimplodeandcollapsewitha newcompressionphase,releasing ashock wavethat prop-agates throughthe liquid.1 _{These effects disrupt microbial}

structuresand inactivate anddecompose toxicchemicals.3

http://dx.doi.org/10.1016/j.bjm.2016.01.014

1517-8382/©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Variousstudiesaddressingtheeffectofultrasoundaloneor combined with other treatments on microbial inactivation havebeenpreviouslypublished.1,4

Ultrasoundisusedinavarietyofapplications,including foodprocessingandfoodanalysis.Twoapproachesare com-monlyused:low-intensity(highfrequencyof100kHzto1MHz andlowpower<1Wcm−2)andhigh-intensity(lowfrequency of16–100kHzandhighpowerof10–1000Wcm−2)ultrasound.5

Low-intensityultrasoundgenerateslowpowerlevelssuchthat thetreatedmaterialisnotphysicallyor chemicallyaltered. Generally,low-intensityultrasoundisanon-destructive treat-ment, which has been successfully used for non-invasive monitoringoffoodprocesses6_{andasananalyticaltechnique}

fordeterminingphysicochemicalfoodproperties(e.g.,texture, density,porosity,grainsize,etc.).Incontrast,high-intensity ultrasoundgeneratesphysicaldisruptionsandinduces chem-icalreactions onthe materialto whichit is applied.7 _This

ultrasoundapproachhasbeen usedinfood manufacturing forpeeling,celldisintegration,extractionofintracellular com-ponentsandenzymes,accelerationofenzymereactionsand microbialfermentation,dispersionofdrypowdersinliquids, emulsification,deactivationofenzymesandmicroorganisms, andotherprocesses.8,9

Cactuspear(Opuntiaficusindica)isacommonfruitin Mex-icoandvariousregionsofLatinAmerican,SouthAfrica,and the Mediterranean10 _{and is considereda nutraceuticaland}

functionalfood11_{becauseofitshighcontentsofvitaminC,}

flavonols,phenolicacidsandbetalains.12,13_Thisfruitis

clas-sifiedasalow-acidfood(pH>4.5)andcontainsahighcontent ofsoluble-solids,makingitsuitableforjuiceproduction14_but

alsosusceptibletomicrobialspoilageandashortshelflife.15

Escherichia coli is a fecal coliform bacteria, commonly found in the intestines ofanimals and humans. E. coli in waterand foodsisa strongindicationofrecent fecal con-tamination,and recognizedclasses ofenterovirulent E. coli

cause gastroenteritisin humans.16 _{E.colicells subjected to}

heat treatments exhibitvariable heat resistance17

depend-ing on the media, e.g., low pH and high acidity sensitizes cells to heat, whereas high sugar concentrations increase thermotolerance.18,19 _{Previous studies have demonstrated}

that ultrasound can inactivate E. coli in water and apple cider2,20 _{and that low pH can enhance this effect on the}

bacteria.21_{Thesestudieshaveevaluateddifferentultrasound}

conditionsbutthebehaviorofE.colipreviouslyinactivatedby ultrasoundhasnotbeenaddressedforotherfruitjuices,such ascactuspearjuiceduringstorage.Therefore,ouraimwas toevaluatetheeffectofultrasoundtreatmentofinoculated cactuspearjuices(greenandpurple)onthepH,solublesolids andsurvivalofE.colioverfivedaysofstorage.

Materials

and

methods

Greenandpurplecactuspearjuicepreparation

Greenandpurplecactuspearfruits(Opuntiaficusindica)were providedbytheMexicanassociation(CoMeNTuna,Actopan, Hidalgo,México)inthe springof2012.Fruitsfreeof exter-nalinjurieswereselected,washedandmanuallypeeled.To extractthe juices, the pulpwasstirredusing anindustrial

blender(38BL52(LBC10),WaringCommercial®_,USA)andthen

passedthroughaconventionalstrainertoremoveseeds. Sam-pleswerecentrifuged(BeckmanCoulter,Inc.,Allegra25R,CA, USA)at15,317×g,4◦C for25mintoclarifythe juices, and thenpasteurizedusingawater-jacket(400mLcapacity)ata controlledtemperatureof85◦Cfor25mintoeliminatenative microbiota. Juicesamples (100mL) were distributed asepti-callyintopreviouslysterilized250mLglassbottlesandthen stored at4◦Cuntil subsequentinoculation and ultrasound treatment. Afterheattreatment,the juicewasanalyzedby platingserialdilutionstoconfirmthesterilityofthejuice.

Bacteriastockcultures,inoculation

TheE. colistrain wasobtainedfrom the CultureCollection oftheLaboratoryofNutrigenomics(HealthScienceInstitute, AutonomousUniversityoftheStateofHidalgo,México)and maintainedinLB-Glycerol(Sigma–Aldrich,St.Louis,MO,USA). Stockcultureswerestoredat−80◦_{Cin0.7mLtrypticsoybroth}

(TSB:DifcoBectonDickinsonSparks,MD,USA).Cultureswere streakedontotrypticsoyagar(TSA;BDDifcoTM_,USA),

incu-bated at37◦C for24hand storedat 4◦C.Onecolony was inoculatedinTSBandincubatedwithshaking(S1600,Jeiotech, Co.,Ltd.,Korea)at37◦Cfor24h.Thefinalconcentrationof

E.coliintheinoculumwasdeterminedbyplatingserial dilu-tionsonTSBandincubatingat37◦Cfor24h.Pasteurizedjuice samples(100mL)placedinthesterileglassbottleswere inoc-ulatedwith100␮Loftheinoculumtoafinalconcentrationof 7logCFU/mLandallowedtoadaptfor20minpriorto ultra-soundtreatment.

Ultrasoundtreatment

Inoculated juiceswere treatedusinganultrasound genera-tor(VCX-1500,Sonics&Materials,Inc.Newtown,CT,USA)at 1500Wandaconstantfrequencyof20kHz,byapplying ampli-tudelevelsof60%,70%,80%and90%for1,3and5minwith pulsedurationsof2sonand4soff.Aliquotsof1mLofjuice weredistributedin1.5mLsterilizedmicrotubesandanalyzed formicrobialsurvivalimmediatelyafterultrasoundtreatment (day0).Anuntreatedinoculatedsamplewasusedasa con-trol.Sampleswerethenstoredat4◦Cuntilanalysisafter1,2, 3,4and5daysofstorage.Temperaturesbeforeandafterthe ultrasoundtreatmentwerealsomonitored(Table1).

pHandtotalsolublesolids(◦Brix)

ThepH wasmeasured usinga digital, calibratedpH-meter (HannaInstruments,pH210,USA)andthetotalsolublesolids weremeasuredusingarefractometer(Brix/ATCFG-113, Hang-zoung Chincan Trading Co.,Ltd., China)immediately after ultrasoundtreatment(day0)andattheendofstorage(day5).

Titratableacidity(TA)

Samplesof20mLwere placedin250mLglassbeakers,and 80mLofdistilledwaterwasadded.Thissolutionwastitrated againststandardized0.1NNaOH(Sigma–Aldrich,Dublin, Ire-land) to the phenolphthalein end point (pH 8.2±0.1). The volume ofNaOHwasconverted tograms ofcitricacid per

Table1–ConditionsofultrasoundtreatmentofgreenandpurplecactuspearjuicesinoculatedwithEscherichiacoli.

Treatment Temperature(◦C)

T1 _T2

Amplitude Time(min) Green Purple

60% 1 30 38.60±1.69 40.65±0.07 3 30 50.25±2.19 49.60±0.14 5 30 62.40±1.97 53.95±0.07 70% 1 30 40.25±0.50 40.60±2.97 3 30 53.85±0.50 55.05±2.47 5 30 66.10±0.56 64.30±1.13 80% 1 30 41.30±0.70 38.35±0.77 3 30 55.25±0.70 56.00±0.00 5 30 68.50±0.34 65.80±0.14 90% 1 30 34.75±5.16 23.70±4.95 3 30 48.20±0.57 34.20±7.07 5 30 62.55±4.03 62.45±4.31 T1_{,inlettemperature.} T2_{,outlettemperature.}

Temperatureinultrasoundtreatmentofgreenandpurplecactuspearjuice.

100mLofjuice.22_{TAwasmeasuredimmediatelyafter}

ultra-soundtreatment(day0)andattheendofstorage(day5),which wascalculatedusingthefollowingformula:

TA=mLbasetitrant×Normalityofbase×Acidfactor×100 Samplevolume(mL)

Microbiologicalanalysis

SerialdilutionsofjuiceswereperformedinTSBandplated onTSAforbacteriacountsand incubatedat37◦Cfor24h. Theresultswereexpressedaslogcolonyformingunitsper milliliter(CFU/mL) ofjuice, wherethe limit ofdetection is 1UFC/mL.

Statisticalanalysis

Data were obtained from three independent experiments. ANOVAwas performedtodeterminesignificantdifferences at the 5% probability level using the SPSS® _{System for}

WINTM _{(15.0.1 version) (SPSS Inc., Chicago, IL, USA). The}

Student–Neuman–Keuls(SNK)testwasusedforcomparison ofthedata.

Results

and

discussion

pH,totalsolublesolidsandtitratableacidity

ThemeanvaluesforpH,totalsolublesolidsandTAingreen and purple cactuspear juiceare shown inTables 2and 3, respectively.Solublesolidsand pHdeterminethe degreeof ripenessofthefruitandareinfluencedbyphysicalfactors, suchasplaceoforigin,species,maturity,andcultivar.12_The

resultsobtainedshowthatthepH,solublesolidsandTA dif-feredsignificantly(p<0.05)betweentreatments.Freshjuice (day1)showedvaluesofpHbetween4.68and 5.68,soluble solidscontentof12.78–13.33◦Brix,andTAof0.01,whichare similartovaluesreportedforultrasound-treatedcactuspear juices23,24_{andotherfruitjuices.}22,25_{After5daysofstorage,the}

pHandsolublesolidsvalueschangedtorangesof4.90–5.50

Table2–pH,solublesolidsandtitratableacidityvaluesofgreencactuspearjuiceinoculatedwithEscherichiacoliafter ultrasoundtreatmentand5daysofstorage.

Determination Days Treatment

Control 60%5min 70%5min 80%5min 90%5min

pH 0 5.68±0.01a _5.21±0.03b _4.68±0.00c _5.21±0.03b _5.28±0.10b 5 5.52±0.00a* _5.42±0.14a* _4.90±0.03d* _5.12±0.00b* _5.19±0.20b Solublesolids (◦Brix) 0 12.90±0.00b _13.20±0.00a _12.78±0.14c _13.00±0.00b _12.95±0.05b 5 13.81±0.04c* _16.30±0.10a* _12.93±0.08d _16.00±0.00b* _12.90±0.32d Titratableacidity (gcitricacid/mL) 0 0.01±0.00d _0.08±0.00c _0.15±0.01a _0.08±0.00c _0.14±0.02b 5 0.01±0.00c* _0.09±0.00c _0.16±0.01a _0.09±0.00c _0.12±0.04b a,b,c_{Differentlettersinthesamelineindicatesignificantdifferences(p<0.05).}

Table3–pH,solublesolidsandtitratableacidityvaluesofpurplecactuspearjuiceinoculatedwithEscherichiacoliafter ultrasoundtreatmentand5daysofstorage.

Determination Days Treatment

Control 60%5min 70%5min 80%5min 90%5min

pH 0 4.97±0.00b _5.50±0.01a _4.72±0.01c _5.50±0.00a _5.11±0.27b 5 5.52±0.00c* _5.07±0.00a* _4.90±0.10b* _5.07±0.00a* _5.00±0.12a Solublesolids (◦Brix) 0 13.33±0.00b _13.33±0.10a _12.90±0.24a _13.00±0.00a _13.33±0.73a 5 13.80±0.21b* _14.40±0.00a* _12.95±0.05c _14.00±0.00b* _12.90±0.32c Titratableacidity (gcitricacid/mL) 0 0.04±0.00d _0.10±0.00c _0.17±0.01a _0.10±0.00c _0.16±0.01b 5 0.01±0.00a* _0.10±0.00d _0.17±0.00b _0.10±0.00d _0.14±0.01c a,b,c_{Differentlettersinthesamelineindicatesignificantdifferences(p<0.05).}

∗ _{Significantdifferencesbetweendays0and5ofstorageforthesametreatment(p<0.05).}

and12.90–16.40◦Brix,respectively, andtheTAincreasedto 0.09–0.17.

Ultrasoundtreatmentcausesthereleaseofspecific com-pounds, such as sugars, phenolic compounds and organic acids.26–28_{Forinstance,releaseofcitricacidmayexplainthe}

increaseinTAaftertreatmentandstorage.Ultrasoundalso exertsamechanicaleffectthatincreasesthecontactsurface betweenthe solidand liquid, allowingforgreater penetra-tionofsolventintothematrixandthusgreaterdiffusionof materialintothemedium.29

During storage, significant differences were observed betweenultrasoundjuicesandthecontrol(p<0.05)forallof theseparameters,whereasexperimentalsamplesexhibited differences inpHand soluble solids, exceptat90%,which remainedunchanged.

SurvivalandgrowthofEscherichiacoli

E.colicountsafterultrasoundtreatmentandover5daysof juicestorageare shown inFigs.1and 2. Theinitial inocu-lumwas7logCFU/mL.Countsincreasedinthecontrolfrom day1tovalues>11logCFU/mLattheendofstorage,whereas ultrasound treatment reduced bacteria counts, particularly whenhigheramplitudesandlongertimes(3and5min)were applied.Itispossiblethatultrasoundappliedtothe micro-bialsuspensionsdispersesmicroorganism clumps,disrupts cells and modifies cellularactivity from the outside tothe insideofthestructures.30_{Theseeffectsresultfromthe}

com-binedphysicalandchemicalmechanismsthatoccurduring thecollapseofcavitationbubbles,theformationoffree radi-cals(e.g.,OH–),andthegenerationofhydrogenperoxide.31,32

Inaddition,duringultrasoundtreatment,microorganismsare alsosubjectedtomildtemperatures(>50◦C),whichincrease theweakeningofthebacteriamembraneandpossiblyfurther lysisattributedtocavitation.33–35_{Inourstudy,thetemperature}

increasedwithtreatmenttime(>3min),andmost ultrasoni-catedjuicesreachedtemperatures>50◦C(Table1).Although all samples subjected totreatment for 5min reachedhigh temperatures,treatedjuiceat90%for5minshowedthetotal inactivationreachingtemperaturesof62.5◦C.Weperformed additional experiments to prove the bactericidal effect at 62.5◦Ctodeterminethemicrobialinactivationatthis temper-ature.Theresultshowedreductiononlyof4.5±0.4logCFU/mL

and 4.6±0.2logCFU/mL for green and purple cactus pear juices,respectively,onday0(datanotshowed).Therefore,the combined effectsofultrasoundtreatmentand temperature mayexplaintheseresults.Similarobservationswerereported byHercegetal.36_{whofoundthatamplitude,time,and}

tem-perature duringultrasound treatmentofmilksubstantially affected the inactivation ofE.coli. These findingsreinforce thesuitabilityofthistypeofemergingtechnologytoprocess liquidswithoutaffectingtheirquality.23,24

For bothgreenand purple cactuspearjuice, ultrasound applied for 1min reduced bacteria counts by 1 and 3 log CFU/mL,whichincreased(3–4logCFU/mL)whentreatedfor

3min(Figs.1and2).InactivationofE.coliunderthedetection

limitwasobservedonlyinjuicestreatedfor5minand90% amplitude (Figs.1Dand2D);therefore,thehigh amplitudes andlongertreatmenttimesweremoreeffectiveformicrobial inactivation.

During storage, juice subjected to 5min and 60%, 70% and 80% amplitudes exhibited bacterialgrowth after

treat-ment (1 day) (Figs. 1A–C and 2A–C), whereas growth was

observed after 2days (Figs. 1Dand 2D) at90% amplitude. This delayed regrowth may result from the disruption of the lipid membraneoccurring athigher ultrasound ampli-tudes,whichimpairsbacteriagrowthandmayinduceartificial competence.30_{Thelethalandsublethaleffectsofultrasound}

treatmentsonmicrobialcellsarestronglyinfluencedbytime. Sublethaleffectsrefertoastageprevioustocelldeathwhere reversible damage occurs and the cell can recover if the effectceases underappropriatephysicalparameters.37

Cer-tainultrasoundprocessingconditionsseemtobeselectivein termsofexclusivelydestabilizingtheoutermembraneofE.coli

withoutseverelyaffectingthecytoplasmicmembrane.38_The

effectsofincreasingintensitiesofultrasoundoneukaryotic cellviabilityarewelldocumented.39,40 _{Studiesperformedby}

YeoandLiong38 _{withgram-negativebacteria,suchasE.coli}

andHaemophilusinfluenza,showedthatafter5minof sonica-tionat40kHz,thebacteriawerenearlyeliminated.However, Allison et al.41 _{reduced the viability without cell deathby}

applying20kHzsonication,whichsuggeststhatincreasingthe poweroutputlevelofultrasoundresultsinafastercelldeath rate.

The resultsobservedfor ultrasound-treated cactuspear juicesuggestthatregrowthofE.colioccurredduringstorage,

13 12 11 10 9 8 7 6 5 4 3 2 1 0 0 1 2 3 4 5 Days

A

B

C

D

0 1 2 3 4 5 Days 0 1 2 3 4 5 Days 0 1 2 3 4 5 Days Log CFU/ml Log CFU/ml Log CFU/ml Log CFU/ml 13 12 11 10 9 8 7 6 5 4 3 2 1 0 13 12 11 10 9 8 7 6 5 4 3 2 1 0 13 12 11 10 9 8 7 6 5 4 3 2 1 0

Fig.1–SurvivalandgrowthofEscherichiacoliduringstorageofgreencactuspearjuicetreatedbyultrasoundat(A)60%;(B)

70%;(C)80%and(D)90%amplitudelevelsfor1(),3()and5()minandcontrol(×),resultsofmicrobiologicalanalysis

realizedimmediatelyafterperformingultrasoundtreatment(---).

13 12 11 10 9 8 7 6 5 4 3 2 1 0

A

B

C

D

Log CFU/ml 13 12 11 10 9 8 7 6 5 4 3 2 1 0 Log CFU/ml 13 12 11 10 9 8 7 6 5 4 3 2 1 0 Log CFU/ml 13 12 11 10 9 8 7 6 5 4 3 2 1 0 Log CFU/ml 0 1 2 3 4 5 Days 0 1 2 3 4 5 Days 0 1 2 3 4 5 Days 0 1 2 3 4 5 Days

Fig.2–SurvivalandgrowthofEscherichiacoliduringstorageofpurplecactuspearjuicetreatedbyultrasoundat(A)60%;(B)

70%;(C)80%and(D)90%amplitudelevelsfor1(),3()and5()minandcontrol(×),resultsofmicrobiologicalanalysis

which may be attributed to reversible membrane perme-abilization formed upon treatment at low intensities. The permeabilitymayhaveincreasedthetransportofnutrients andothersubstancesintothecells,alleviatingcellmetabolism andsubsequentlyenhancingbacterialviability.42_Ultrasound

canalsoenhance thedisruption ofcell wallsand thusthe releaseoftheircontents,43_{makingthemavailableforbacterial}

growth.Forinstance,polysaccharidescanbereleasedbecause ofcavitation44,45 _{and carbohydrates are usedpreferentially}

byE. coli.46 _{Oncethe stress over the cells isremoved (e.g.,}

ultrasound), respiration and biosynthesisof carbohydrates, membranes,lipids,andproteinscanrecover,allowingforthe regenerationofthe cellmembraneand bacteriaphysiology andstructuralintegrity.47

Theseobservationsmayexplainthe resultsobtainedfor samplestreatedatamplitudes <90%,whichreached bacte-riacountssimilar tothose ofthe originalloadafter4days ofstorageat4◦C(Figs.1A–Cand2A–C).Otherauthorshave observed that refrigeration enhances survival of E. coli in anacidicenvironment,48–50 _{whichislikelyattributedtothe}

reducedpermeabilityofthecellmembranetoprotonsand/or areducedmetabolicactivity.51_{Althoughtreatmentat90%for}

5minexhibitedbacteriacounts<2logCFU/mLuntilthelast dayofstorage (Figs.1D and 2D), ultrasound atamplitudes of 70% and 90% for 1 and 3min only showed a bacterio-staticeffect(Figs.1B,Dand2B,D).Theresultsdemonstrated thattreatmentathigheramplitudes(90%)andlongertimes (5min) were effective in achieving a 5log reduction. This valuecomplieswiththeFDArequirement(<5logCFU)forfruit juices.

Conclusions

Theresultsfrom thisstudy revealed thatultrasound treat-mentat90%amplitudefor5minresultedinnon-detectable levels of E. coli in cactus pear juice for 2 days with no effectonpH,TAandsolublesolids.Inaddition,theseresults complied with the 5log reduction of E. coli recommended by the FDA guidelines for fruit juices. Under the evalu-ated conditions, ultrasound treatment can be considered analternative technologyforfruitjuice preservation. How-ever,furtherresearchisrequiredtoachieveconditionsthat preventre-growth ofbacteria, reach total inactivation dur-ing storage and confirm if re-growth results from injured cells.

Conflicts

of

interest

Theauthorsdeclarethatnoconflictsofinterestexist.

Acknowledgments

This work was financially supported by Programa Integral deFortalecimientoInstitucional(PIFI2012–2013).Theauthors acknowledgetheMexicanassociationCoMeNTuna(Hidalgo, México)forprovidingtheplantmaterials.

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