Determining Antioxidant Activities of Lactobacilli Cell-Free Supernatants by Cellular Antioxidant

16 Pages·2015·1.4 MB·English

RESEARCHARTICLE
Determining Antioxidant Activities of
Lactobacilli Cell-Free Supernatants by Cellular
Antioxidant Assay: A Comparison with
Traditional Methods
JialiXing1,GangWang1*,QiuxiangZhang1,XiaomingLiu1,ZhennanGu1,HaoZhang1,
YongQ.Chen1,WeiChen1,2*
1 StateKeyLaboratoryofFoodScienceandTechnology,SchoolofFoodScienceandTechnology,
JiangnanUniversity,Wuxi,China,2 SynergisticInnovationCenterforFoodSafetyandNutrition,Wuxi,
China
* [email protected](GW); [email protected](WC)
Abstract
OPENACCESS
Citation:XingJ,WangG,ZhangQ,LiuX,GuZ, Antioxidantactivityoflacticacidbacteriaisassociatedwithmultiplehealth-protectiveef-
ZhangH,etal.(2015)DeterminingAntioxidant fects.Traditionalindexesofchemicalantioxidantactivitiespoorlyreflecttheantioxidantef-
ActivitiesofLactobacilliCell-FreeSupernatantsby
fectsofthesebacteriainvivo.Cellularantioxidantactivity(CAA)assaywasusedinthis
CellularAntioxidantAssay:AComparisonwith
TraditionalMethods.PLoSONE10(3):e0119058. studytodeterminetheantioxidantactivityofcell-freesupernatants(CFSs)of10Lactobacil-
doi:10.1371/journal.pone.0119058 lusstrains.TheperformanceoftheCAAassaywascomparedwiththatoffourchemicalan-
AcademicEditor:KarlXChai,UniversityofCentral tioxidantactivityassays,namely,DPPHradicalscavenging,hydroxylradicalscavenging
Florida,UNITEDSTATES (HRS),reducingpower(RP),andinhibitionoflinoleicacidperoxidation(ILAP).Resultsof
Received:August6,2014 theCAAassaywereassociatedwiththoseofDPPHandILAPassays,butnotwiththoseof
RPandHRSassays.Theinter-andintra-specificantioxidantactivitiesofCFSwerecharac-
Accepted:January9,2015
terizedbychemicalandCAAassays.L.rhamnosusCCFM1107displayedahighantioxida-
Published:March19,2015
tiveeffectsimilartopositivecontrolL.rhamnosusGGATCC53103inalloftheassays.The
Copyright:©2015Xingetal.Thisisanopenaccess CAAassayisapotentialmethodforthedetectionofantioxidantactivitiesoflactobacilli
articledistributedunderthetermsoftheCreative
CFSs.
CommonsAttributionLicense,whichpermits
unrestricteduse,distribution,andreproductioninany
medium,providedtheoriginalauthorandsourceare
credited.
DataAvailabilityStatement:Allrelevantdataare
withinthepaperanditsSupportingInformationfiles.
Introduction
Funding:ThisworkwassupportedbytheNational
Increasingscientificevidencesuggeststhatoxidativestressisinvolvedinthepathogenesisof
HighTechnologyResearchandDevelopment
ProgramofChina(863ProgramNos. variousdisordersanddiseases,suchasalcohol-inducedliverinjury,non-alcoholicfattyliver
2013BAD18B01,2013BAD18B02,2012BAD28B07, disease,ageing,andcancer[1–4].Oxidativestressisaresultofanimbalancebetweenproduc-
2012BAD12B08),theNationalNaturalScience tionandeliminationofreactiveoxygenspecies(ROS)andfreeradicals,whichareprimarilyre-
FoundationofChina(Nos.31301407,31200691),the
movedbytheendogenousantioxidantdefensesystem[5].Consumptionofantioxidants,
NationalBasicResearchProgramofChina(973
whichcanquenchfreeradicalsandROS,maybebeneficialtohumanhealth.Syntheticantioxi-
ProgramNo.2012CB720802),keyprojectsinthe
dantsareeffectiveinslowingoxidation,butposeconcernsinregardtothesafetyandtoxicity
NationalScienceandTechnologyPillarProgram
duringthe12thFive-YearPlan(Nos. oftheantioxidants[6,7].
PLOSONE|DOI:10.1371/journal.pone.0119058 March19,2015 1/16
ACellularAssaytoAnalyzetheAntioxidantActivityofLactobacillus
2012BAD12B08,2012BAD28B07),the111project Lacticacidbacteria(LAB),whicharewidelyusedinthefoodindustry,aregenerallyrecog-
B07029,andaprojectfundedbythePriority nizedassafe[8].LAB,particularlylactobacilli,haverecentlyreceivedincreasingattentionbe-
AcademicProgramDevelopmentofJiangsuHigher
causeoftheirspecificroleinmaintaininghumanhealthandindecreasingtheriskofROS
EducationInstitutions.Thefundershadnorolein
accumulation[9,10].Thebenefitsoflactobacillihavebeenconsistentlysupportedbyepidemio-
studydesign,datacollectionandanalysis,decisionto
publish,orpreparationofthemanuscript. logicalreportswhichindicatethatlactobacilliareassociatedwithareducedriskofdeveloping
chronicdiseases[11,12].TheseabilitiesaremainlyfocusedonviableLABandcell-freeextracts
CompetingInterests:Theauthorshavedeclared
[13,14].Furthermore,cell-freesupernatant(CFS)isawell-knowngoodsourceofantioxidants
thatnocompetinginterestsexist.
[15,16].Bingetal.suggestedthatculturesupernatantofLactobacillusacidophiluscontains
anti-ulcerandanti-oxidativemetabolites[17].Wangetal.reportedthatLactobacillusrhamno-
susGGculturesupernatantamelioratedacutealcohol-inducedintestinalpermeabilityandliver
injury[18].Shenetal.foundthatCFSofBifidobacteriumanimalis01exhibitedstrongantioxi-
dantactivities(AAs)[19].Wangetal.discoveredthatCFSsfrom35LABexhibitstrongerAAs
thanbacterialsuspensionsandcell-freeextracts[20].
ChemicalAAassaysarecurrentlyavailabletostudyAAsofCFS.Commonlyusedchemical
assaysincludeDPPHradicalscavenging,hydroxylradicalscavenging(HRS)method,reducing
power(RP)andinhibitionoflinoleicacidperoxidation(ILAP)[19,21,22].Theseassaysdepend
onthecapacityofantioxidantstoquenchand/orreducefreeradicals.However,thesechemical
assayspresentseverallimitations.Forinstance,theDPPHassayisafastandsimplewaytode-
termineAA,however,thismethoddoesnotconsidercertainparametersincomplexcellenvi-
ronments,suchasbioavailabilityandmembranepermeability.Moreover,theDPPHassayis
affectedbylightintensity,oxygenconcentration,andsolventtype[23].Inaddition,themecha-
nismsofantioxidantsarenotonlybyscavengingfreeradicals,butalsobyinhibitingproduc-
tionoffreeradicalsandimprovinglevelsofendogenousantioxidants[24].Therefore,the
traditionalindexesofchemicalantioxidantcapacitypoorlyreflecttheantioxidanteffectsasso-
ciatedwithaparticularsampleinvivo[25].
Althoughstudiesinvolvinganimalmodelsandhumansaremoreappropriatethaninvitro
studies,theyareexpensiveandtimeconsuming[25].Meanwhile,cellularAA(CAA)assayis
anextremelyattractiveintermediatetestingmethodtosupportantioxidantresearchpriorto
animalstudiesandhumanclinicaltrials[26].Thismethodshowshighphysiologicalqualityin
AAmeasurement,thus,ithasbeenappliedtostudyseveralnaturalproductextracts,foods,
anddietarysupplements[25].Dataobtainedfromacell-basedassaymaybecomprehensively
interpretedgiventhemultiplehealth-protectiveeffectsoflactobacilliCFS.
Thepresentstudyaims(i)toevaluatethetoxicityofCFSusingHepG2cellline,(ii)toapply
CAAassaytodetectAAsofCFS,(iii)toanalyzethedifferenceofinterandintra-specificof
CFSbycomparingtheantioxidantactivitiesof10Lactobacillusstrainsfromsevenspecies
whichareusedasprobioticbacteria,and(iv)tostudythecorrelationsbetweenchemicaland
CAAassaystoprovidetheoreticalguidanceinrationallyscreeningLAB.
MaterialsandMethods
BacterialStrainsandCultureConditions
AllLactobacillusstrainsusedinthisstudyarelistedinTable1,includingL.rhamnosusGG
ATCC53103(LGG),whichwasusedinallofexperimentsasareferencestrain[12,27].L.aci-
dophilus,L.fermentum,L.rhamnosus,L.casei,L.plantarumandL.reuteriareknownspecies
thatdisplayAAs[12,28–32].L.farciminiswhichisrarelyreportedtoexhibitAAwasalso
adoptedasareferencestrain.Thesestrainsweremaintainedasfrozenstocks(−80°C)in
deMan,Rogosa,andSharpe(MRS)broth(HopebioCompany,Qingdao,China)supplemented
with30%(v/v)glycerol.Allstrainswereconsecutivelytransferredatleastthreetimesusing1%
(v/v)inoculuminMRSbrothat37°Cfor20hpriortouse.
PLOSONE|DOI:10.1371/journal.pone.0119058 March19,2015 2/16
ACellularAssaytoAnalyzetheAntioxidantActivityofLactobacillus
Table1.Lacticacidbacteriausedinthisstudy.
LactobacillusStrain Characteristics SourceorReference
L.rhamnosusGGATCC53103 Healthyhumanintestinalflora ValioLtd.,Helsinki,Finland
L.rhamnosusCCFM-JU1107 Pickles CCFM-JU*
L.rhamnosusCCFM-JU7469 Pickles CCFM-JU
L.casei2W Commercialyoghurt TCBD*
L.plantarumCCFM-JU8661 Pickles CCFM-JU
L.reuteriCCFM-JU14 Pickles CCFM-JU
L.acidophilusCCFM-JU137 Humanfeces CCFM-JU
L.farciminisCCFM—JU419 Fishtea CCFM-JU
L.fermentiCCFM-JU381 Oldleaveneddough CCFM-JU
L.fermentiCCFM-JU424 Acidkidneybean CCFM-JU
CCFM-JU*,CultureCollectionofFoodMicroorganismsofJiangnanUniversity(Wuxi,China);
TCBD*,TechnologyCenterofBrightDairyandFoodCo.,Ltd.(Shanghai,China).
doi:10.1371/journal.pone.0119058.t001
SolutionPreparation
A:Linoleicacidemulsion(20mL)waspreparedbymixing0.1mLoflinoleicacid(Sigma—
Aldrich,St.Louis,MO),0.2mLofTween20,and19.7mLofdeionizedwater,andtheemulsion
wasstoredinthedark.
B:A200mMstocksolutionof2’,7’-dichlorofluorescindiacetate(DCFH-DA)(Sigma—
Aldrich,St.Louis,MO)indimethylsulfoxide(Sigma—Aldrich,St.Louis,MO)wasprepared,
aliquoted,andstoredat-20°C.
C:A200mMstocksolutionof2,2-azobis(2-amidinopropane)dihydrochloride(ABAP)
(Sigma—Aldrich,St.Louis,MO)inHanks’BalancedSaltSolution(HBSS)(Sigma—Aldrich,
St.Louis,MO)waspreparedandstoredat-40°C.
PreparationoflactobacilliCFS
LactobacilliCFSswerepreparedasdescribedbyChenandWang[12,18]withslightmodifica-
tion.Culturesofthe10Lactobacillusstrainswereadjustedtoapproximately109CFU/mL.Sub-
sequently,thealiquotsofthecultureweretransferredto5mLpolypropylenetubesand
centrifuged(10,000g,10min,4°C).ThepHvalueofthesupernatantwascontinuouslyneutral-
izedwith1MNaOH[33].Theresultingsupernatantwasfiltered(0.22μmporesize).LGG
CFSwasusedasapositivecontrol.MRSbrothmedium(pH7.0,filteredwitha0.22μmpore
sizefilter)servedasanegativecontrol.
ChemicalAssaystoDetermineAAsofLactobacilliCFSs
DPPHRadicalScavengingActivityAssay. ThescavengingeffectoftheCFSsof10Lacto-
bacillusstrainsonthefreeradicalDPPHwasmeasuredinaccordancewiththeslightlymodi-
fiedmethodofLinandChang[28].ASample(CFS,MRSbroth,1mL)andafreshlyprepared
DPPHsolution(0.2mM,1mL,Sigma—Aldrich,St.Louis,MO)weremixed.Themixturewas
vigorouslyshakenandlefttoreactfor30mininthedarkatroomtemperature.Thecontrol
samplecontaineddeionizedwaterinsteadofthesamplesolution.ThescavengedDPPHwas
thenmonitoredbydeterminingtheabsorbanceat517nmusingSpectraMaxM5microplate
reader(MolecularDevices,SunnyvaleCA).Theradicalscavengingactivitywasquantifiedas
PLOSONE|DOI:10.1371/journal.pone.0119058 March19,2015 3/16
ACellularAssaytoAnalyzetheAntioxidantActivityofLactobacillus
units/mL(U/mL)byusingfollowingformula[34]:
DPPHactivityðU=mLÞ¼ðABS (cid:2)ABS Þ=S(cid:3)100
C S
whereABS andABS aretheabsorbanceofthecontrolandtestsamplesat517nm,respective-
C S
ly,andSisthevolume(mL)ofthesample.
HRSActivityofLactobacilliCFSs. TheHRSactivityoftheCFSswasanalyzedasprevi-
ouslydescribedmodifiedmethod[13,19].Asample(CFS,MRSbroth,1mL),1,10-phenan-
throline(2.5mM,1mL;Sigma—Aldrich,St.Louis,MO),PBS(pH7.4,1mL),andFeSO (2.5
4
mM,1mL)weremixed.ThereactionwasinitiatedbyaddingH O (20mM,1mL)andincu-
2 2
batingat37°Cfor90min.HRSactivitywasmonitoredbyidentifyingtheincreaseinabsor-
banceat536nmbyusinganSpectraMaxM5microplatereader(MolecularDevices,Sunnyvale
CA).HRSactivitywascalculatedusingthefollowingequation:
HRSactivityð%Þ¼ðA (cid:2)A Þ=ðA (cid:2)A Þ(cid:3)100%
S C b C
whereA istheabsorbanceofthesample,A istheabsorbanceofthecontrolsolution(deion-
S C
izedwaterwasusedinsteadofthesampleatthesameamount),andA istheabsorbanceofthe
b
solutionwithoutsamplesandH O .
2 2
RPofLactobacilliCFSs. TheCFSreducingactivityoftheCFSswasdeterminedasde-
scribedbyLinandYen[35]withslightmodification.Asample(CFS,MRSbroth,0.5mL)was
brieflymixedwithpotassiumferricyanide(1%,0.5mL)andPBS(pH6.6,0.5mL).Subsequent-
ly,themixturewasheatedat50°Cfor20minandallowedtocool.Uponcooling,0.5mLof
10%trichloroaceticacid(TCA)wasaddedtothemixtureandthencentrifugedat3000gfor
5min.Theupperlayer(1mL)wasmixedwithferricchloride(0.1%,1mL)andallowedto
reactfor10min.Theabsorbanceofthemixturewasobtainedat700nmbyusinganSpectra-
MaxM5microplatereader(MolecularDevices,SunnyvaleCA).Higherabsorbanceofthemix-
tureindicatedhigherreducingactivity.Thereducingactivityofcysteineservedasthestandard.
ILAPofLactobacilliCFSs. Theanti-lipidperoxidationactivityoftheCFSswasassessed
byusingthethiobarbituricacid(TBA)method[22]withslightmodifications.PBS(pH7.4,
0.5mL),linoleicacidemulsion(1mL),FeSO (0.01%w/v,0.2mL),ascorbicacid(0.02%,w/v,
4
0.2mL),andsample(CFS,MRSbroth,0.5mL)weremixedandthenincubatedat37°Cfor
12h.Subsequently,2.0mLofthereactionmixturewasmixedwithbutylatedhydroxytoluene
(0.4%w/v,0.2mL),TCA(4%w/v,0.2mL)andTBA(0.8%w/v,2mL).Themixturewasincu-
batedat100°Cfor30minandthenallowedtocool,2mLofchloroformwasthenaddedforex-
traction.Theupperextractwasobtainedandabsorbancewasdeterminedat532nmbyusing
anSpectraMaxM5microplatereader(MolecularDevices,SunnyvaleCA).Thesampleswere
substitutedwithdeionizedwaterinthecontrolgroup.Theinhibitionratewascalculatedby
usingthefollowingequation:
Inhibitioneffectð%Þ¼ð1(cid:2)A =A Þ(cid:3)100%
S C
whereA istheabsorbanceofthesample,andA istheabsorbanceofcontrolsolutionthat
S C
samplesolution,inwhichthesamplesolutionwasreplacedwithsameamountof
deionizedwater.
AssessmentofAntioxidantEffectsofLactobacilliCFSsusingCAA
Assay
CellCulture. HumanhepatocellularcarcinomaHepG2cells(CellBankoftheTypeCul-
tureCollectionoftheChineseAcademyofSciences,Shanghai,China)weregrowninHyClone
Dulbecco’smodifiedEagle’smedium(DMEM)withhighglucose(GEHealthcare,USA)
PLOSONE|DOI:10.1371/journal.pone.0119058 March19,2015 4/16
ACellularAssaytoAnalyzetheAntioxidantActivityofLactobacillus
supplementedwith10%fetalbovineserum(Gibco,GrandIsland,NY,USA),penicillin
(100U/mL),streptomycin(100μg/mL)(Sigma—Aldrich,St.Louis,MO),and10mM4(2-
hydroxyethyl)-1-piperazineethanesulfonicacid(HEPES)(Sigma—Aldrich,St.Louis,MO).
Thecellsweremaintainedat37°Cinanincubatorwith5%CO .Thecellsusedinthisstudy
2
wereatpassage10to20.
CellCytotoxicityAssay. CytotoxicityofthelactobacilliCFSswasmeasuredaccordingto
themodifiedmethyleneblueassay[36].Briefly,HepG2cellswereseededatadensityof4×104
cells/wellona96-wellmicroplatein100μLofDMEMfor24hat37°C.Afterwashingwith
PBS,HepG2cellsweretreatedwith100μLofthesample(CFS,MRS)ordeionizedwater(con-
trol)forupto24hat37°C.Toassesscellviability,thecellswerewashedwithPBSandthenin-
cubatedwith50μL/wellmethyleneblue(98%HBSS,0.67%glutaraldehyde,and0.6%
methyleneblue)for1hat37°C.Aftertheincubation,thecellswerewashedwithPBSuntilthe
PBSwasclear,and100μL/wellelution(49%PBS,50%ethanol,and1%aceticacid)wasthen
added,themicroplatewasthenplacedonatableoscillator(ThermomixerComfort,Eppendorf
AG,Hamburg,Germany)for20min.Theabsorbancewasobtainedat570nmbyusingan
SpectraMaxM5microplatereader(MolecularDevices,SunnyvaleCA).Differentsampleswere
comparedwiththecontrol.ACFSwithmorethan10%lessabsorbancethanthecontrol,was
consideredcytotoxic[37].
CAAAssay. CAAassaywasusedtoevaluatethelactobacilliCFSsaspreviouslydescribed
[26,38,39].Briefly,HepG2cells,themostcommonlyusedinCAAassay[26],wereseededata
densityof6×104cells/wellonablack96-wellmicroplate(withtransparentbottoms)in100μL
ofDMEMfor24hat37°C.AfterrinsingwithPBS,HepG2cellsweretreatedwith100μLofthe
sample(CFS,MRSbroth),whichincludes25μMofDCFH-DAforupto1hat37°C.Thecells
werewashedwithPBSandthentreatedwith100μLof600mMABAPsolution.Fluorescence
wasobtainedusinganSpectraMaxM5microplatereader(MolecularDevices,SunnyvaleCA)
for13cyclesat5minintervals(λex=485andλem=538).Afterblanksubtractionfromthe
fluorescentreadings,theareaunderthecurveoffluorescenceversustimewasintegratedtocal-
culatetheCAAvalueofeachsampleasfollows:
R R
CAAðunitÞ¼f1(cid:2)ð SA= CAÞg(cid:3)100
WhereSAistheareaofthesample,andCAistheintegratedareainthecontrolcurve.
StatisticalAnalysis
Allofthetestswereperformedintriplicate.Datawerepresentedasmean±standarddeviation
(SD).One-wayANOVAwasperformedwithSPSS(Version13.0,SPSSInc.,Chicago,IL),fol-
lowedbyFisher’sleastsignificantdifference,toverifysignificantdifferencesbetweensamples.
Theresultswereconsideredsignificantwhenp<0.05.ThePearsoncorrelationtestwascon-
ductedtodeterminecorrelationbetweenvariables.
ResultsandDiscussion
ChemicalAAAssayofLactobacilliCFSs
RadicalScavengingActivities. Resultsoftworadicalscavengingmethods(i.e.,HRSand
DPPH)showedthatthe10lactobacilliCFSscaninhibittheformationofthetworadicals
(Fig.1).TheCFSsofthe10lactobacilliCFSsshowedremarkablyweakerHRSactivitythan
DPPHradicalscavengingactivity.Furthermore,theAAsofthe10lactobacilliCFSswerehigher
thanthatofMRSbrothinscavengingtheDPPHradical.Thisfindingisconsistentwiththere-
portofShenetal.[19].Inaddition,theHRSmethodprovidedsimilarresultsontheAAsofthe
PLOSONE|DOI:10.1371/journal.pone.0119058 March19,2015 5/16
ACellularAssaytoAnalyzetheAntioxidantActivityofLactobacillus
Fig1.DPPHandHRSactivitiesoftheCFSs(mean±SD,n=3).MRSbrothcontrolsamplewithoutLactobacillus.Thelettersa,b,c,d,andeindicate
statisticallysignificantdifferenceatp<0.05withineachrowcomparisonbetweentheDPPHradicalscavenginggroups.Thelettersa’,b’,c’,d’,ande’
indicatestatisticallysignificantdifferenceatp<0.05withineachrowcomparisonbetweentheHRSgroups.Barswithnocommonlettersaresignificantly
different(p<0.05).
doi:10.1371/journal.pone.0119058.g001
CFSs(Fig.1).SignificantdifferencesinAAvalueswereobservedbetweenMRSbrothand
CFSs,exceptforCCFM8661,CCFM381,andCCFM424inscavenginghydroxylradicals
(Fig.1,p<0.05).BoththeHRSandDPPHmethodsindicatedthattheCFSsofCCFM1107
andCCFM7469exhibitedstrongAAs.
TheCFSsofCCFM8661exhibitedstrongDPPHradicalscavengingactivities(75.94±1.05
U/mL)thatwerenotsignificantlydifferentfromthatofthepositivecontrolLGG(77.29±1.51
U/mL)(p>0.05).However,CCFM8661demonstratedsignificantlyweakerHRSactivitythan
LGGinscavenginghydroxylradical(p<0.05).Furthermore,theeffectofL.casei2Won
DPPHandhydroxylradicalcontradictedthatofCCFM8661.Intactcellsandintracellularcell-
freeextractsfromL.caseisubsp.caseiSY13andL.delbrueckiisubsp.bulgaricusLJJalsodiffer
inDPPHandHRSactivities[13].Thedifferentmechanismsinvolvedintheradical-
antioxidantreactionsmayexplainthedifferentinscavengingpotentialsofthecompounds
[40].Moreover,theDPPHandhydroxylradicalactivitiesofbothL.rhamnosusCCFM1107
andCCFM7469weresignificantlystrongerthanthoseofL.fermentiCCFM381andCCFM
424(p<0.05).Liuetal.[8]reportedthat12Lactobacillusstrainsexhibitvaryingcapabilitiesin
DPPHradicalscavenging.Therefore,acertaindegreeofinter-specificdifferenceinradical
scavengingactivitiescouldexistamongthe10testedLactobacillusstrains.
RPofLactobacilliCFSs. AnearlierreportshowedthatAAsandRParedirectlyrelated
[41].TheRPoflactobacilliCFSisbasedonkineticsofthereductionofFe3+toFe2+toprevent
theoxidationreactionandcontroltransitionmetalions[41].TheRPactivitiesoflactobacilli
PLOSONE|DOI:10.1371/journal.pone.0119058 March19,2015 6/16
ACellularAssaytoAnalyzetheAntioxidantActivityofLactobacillus
CFSareshowninFig.2.TheCFSsofLGG,CCFM1107,CCFM7469,CCFM14,CCFM137,
CCFM419,CCFM381,andCCFM424showedsignificantlyhigherRPvaluesthanthatofMRS
broth(p<0.05).Meanwhile,theCFSsofL.casei2WandCCFM8661increased,butnotsig-
nificantly,comparedwiththatofMRSbroth(p>0.05).Inallofthetests,CCFM1107showed
ahighreducingactivitythatwasclosetothatofthepositivecontrolLGGandequivalentto
thatof62.68μML-cysteine.Theresultsareconsistentwiththefindingsofapreviousreport
[8],whichrevealedthat12strainsshowvariedabilitiesinreducingRPactivityandthatL.aci-
dophilusBCRC14079displayssix-foldstrongeractivitythanB.infantisBCRC14602.There-
fore,aninter-specificdifferenceexistsintheRPofthetestCFSs.
ILAPofLactobacilliCFSs. ILAPiscommonlyusedtoanalyzeAAs.Linoleicacidhas
beenusedasasourceofunsaturatedfattyacids[42].Inthepresentstudy,theAAsoflactoba-
cilliCFSsweremeasuredbyILAPassay.AsshowninFig.3,theinhibitoryratesoftestlactoba-
cilliCFSsonlinoleicacidperoxidationrangedfrom5.1±0.22%to67.93±1.22%(Fig.3).The
inhibitoryratesofthelactobacilliCFSsweresignificantlyhigherthanthoseofMRSbroth
(p<0.05),exceptforCCFM137,CCFM419,andCCFM424(p>0.05).TheCFSsof
CCFM1107,CCFM7469,andCCFM8661showedhighinhibitoryeffectsonILAPcompared
withthepositivecontrolLGG(p>0.05).Meanwhile,L.rhamnosusCCFM1107and
CCFM7469demonstratedsignificantlystrongeractivitiesonILAPthandidL.fermenti
CCFM381andCCFM424.Theseobservationsindicatedaninter-specificdifferenceinILAP
amongthelactobacilliCFSs.Moreover,thehigherinhibitoryeffectofCCFM381CFSthanof
CCFM424CFSindicatedanintra-specificdifferenceinILAPbetweenthesetwoCFSs.These
resultsagreewiththefindingsofanearlierreport[42],whichrevealedthattheinhibitoryrates
ofsixstrainsofL.acidophilusonILAPrangefrom34.9%to46.3%.
CorrelationsamongtheFourChemicalAAAssays. Resultsindicatedthatthelactobacilli
CFSs,whichweremeasuredbyusingchemicalmethods,exhibitedcertainantioxidantproper-
ties.AmongthetestedCFSs,LGGandCCFM1107hadthehighestAAinalloftheassays.In
addition,lactobacilliCFSsexerteddifferentAAsinvariouschemicalantioxidantmodels.For
example,theCFSofCCFM8661,CCFM7469,andL.casei2WdisplayeddifferentAAvaluesin
radicalscavenging,RP,andILAP.Inapreviousstudy,Zhuetal.[43]foundthatthewaterex-
tractsofokarakojiandthewaterextractofsoybeankojishowdifferentAAsaccordingtodif-
ferentinvitroantioxidantmodels.Inthiscase,fourchemicalassayswereusedandcompared
todeterminetheantioxidantcapacitiesoftheCFSs,andtheircorrelationsareshownin
Table2.AsignificantassociationwasfoundbetweenscavengingofDPPHandhydroxylradi-
cals(r=0.511,p<0.01,n=11).Meanwhile,theDPPHradicalscavengingoftheCFSscorre-
latedsignificantlywithILAP(r=0.604,p<0.01,n=11).HRSalsohadasignificant
associationwithRP(r=0.494,p<0.01,n=11).However,ILAPshowednosignificantcorrela-
tionwithHRSandRPbecauseofthediversechemicalaspectsofpotentialantioxidantcom-
pound(s)explored[25].Thelackofcorrelationbetweenresultsobtainedthroughthese
differentassayscanbeattributedtoreasonsrelatedtoinstrumentlimitations,mechanisms,
endpoint,quantificationmethod,andbiologicalrelevance[44].Therefore,theuseofasingle
chemicalmethodtoscreenstrainswithhighAAsisdifficultduetothedifferentmechanisms
ofCFSsinvivo.
CAAAssessmentoftheAAsofLactobacilliCFSs
CellCytotoxicity. Modifiedmethyleneblueassayisatypicallyfastandeasymethodofde-
tectingcelldeath.StudieshaveshownthatHepG2cellscouldbeusedtodemonstratecelltoxic-
ity[36,45].However,thetoxicityofCFSonHepG2cellshasnotbeenevaluated.Inthepresent
study,CFSandMRSbrothwereusedtoreactwithHepG2cellsandmethylenebluewasused
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ACellularAssaytoAnalyzetheAntioxidantActivityofLactobacillus
Fig2.RPvaluesoftheCFSs(mean±SD,n=3).MRScontrolsamplewithoutlactobacilli.RPvaluesareexpressedasμMofcysteine.Differentletters
indicatestatisticallysignificantdifferencesatp<0.05.Barswithnocommonlettersaresignificantlydifferent(p<0.05).
doi:10.1371/journal.pone.0119058.g002
tostaintheHepG2cells.ResultsshowedthatincubationwithCFSandMRSbrothinhibited
lessthan10%ofHepG2cells(Fig.4).ThisresultindicatesthatbothCFSandMRSbrothdo
notshowsignificantcytotoxicitytoHepG2cellsafter24hincubation[37].Previousresearch
showedthatfeijoadawholemeal,appleextracts,andvegetablesinhibitlessthan10%ofHepG2
cells[39,46,47].Therefore,wededucedthattheCFSsarenotcytotoxic,whichisatypicalobser-
vationforsuchfunctionalproducts.Moreover,Maudsdotteretal.[48]foundthattheCFSof
LGGcanreducecellcytotoxicitycausedbyStreptococcuspyogenesbyproducinglacticacid.Li
etal.[49]showedthattheCFSsofL.acidophilusnotonlydisplaynon-cytotoxicity,butalso
stimulateproliferationofembryonic,endothelial,andinflammatorycellsinvivo.Theseobser-
vationssuggestedthattheCFSsoftheLactobacillusstrainsarenon-cytotoxic.
CAAAssay. IntheCAAassaydevelopedbyWolfeandLiu[26],theABAP-inducedoxi-
dationofdyeDCFH-DAtofluorescentDCFandthefluorescenceintensityofDCFweremea-
suredtorepresenttheoxidationrate,therebyenablingtheCAAassaytomeasurethe
antioxidationcapacityoftheantioxidants[26,50].Inthepresentstudy,weappliedtheCAA
assaytodeterminetheAAsoflactobacilliCFSs.Thepositivecontrolshowedfluorescencein-
tensityofABAPoxidationovertime.Thenegativecontrolgroupcontainedthecellswithout
theadditionofABAP.Thesecellswereusedtoillustratetheconditionsdevoidofanyoxidation
inducer.TheCFSsofall10Lactobacillusstrainsinhibitedoxidation,withhigherfluorescence
intensitiesthanthenegativecontrol(p<0.05)(Fig.5A).However,theseintensitieswerelower
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ACellularAssaytoAnalyzetheAntioxidantActivityofLactobacillus
Fig3.ILAPoftheCFSs(mean±SD,n=3).MRScontrolsamplewithoutlactobacilli.Differentlettersmeanstatisticallysignificantdifferencesatp<0.05.
Barswithnocommonlettersaresignificantlydifferent(p<0.05).
doi:10.1371/journal.pone.0119058.g003
thanthatofthepositivecontrolovertime,exceptfortheMRSbroth,whichshowedsimilarin-
tensitiestothepositivecontrol.ThetrendsofinhibitingDCFH-DAtoDCFHofCFSaresimi-
lartothoseofpurecompounds,suchastheacrine,sugarcanemolasses,andCrataegusazarolus
[51–53].Puertollanoetal.previouslyreportedthatconcentratedsupernatantsfromL.plan-
tarumreduceROSaccumulationinHL-60cells[54].Inaddition,thepresentresultsshowed
thattheROSinHepG2cellscouldbereducedbylactobacilliCFSs.Similartoothermethods
usedforAAassessment,theCAAmethodrevealsthetotalantioxidativecapacityoftheana-
lyzedsampleratherthanthecapacityoftheindividualcomponentsofthesystem.However,
Table2.Correlationsamongthefourchemicalantioxidantactivityassays.
Correlationcoefficient DPPHradicalscavenging HRS RP ILAP
DPPHradicalscavenging 1
HRS 0.511** 1
RP 0.377 0.494** 1
ILAP 0.604** 0.273 −0.012 1
*p<0.05
**p<0.01
doi:10.1371/journal.pone.0119058.t002
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ACellularAssaytoAnalyzetheAntioxidantActivityofLactobacillus
Fig4.CytotoxicityoftheCFSsonhumanhepatocellularcarcinomaHepG2cells.Negativecontrolwasuntreatedcells(mean±SD,n=3).
doi:10.1371/journal.pone.0119058.g004
theCAAalsodiffersinotheraspects.Itistheonlymethodcapableofpredictingantioxidantre-
sponseatthecellularlevel.Italsoallowstheanalysisofsamplesactivitytochangetheredox
cellularstate.Moreover,theparticipationofdifferentcomponentcellsiscriticaltodevelopan
antioxidantresponse[25].
CAAunitswerecalculatedonthebasisoftheareaunderthecurveofthefluorescenceinten-
sitiesoftheCFSandABAP-treatedcellsovertime.AsmallerareadenoteshigherCAAunits
andhigherAAsofthesample[51].AllofthelactobacilliCFSspossessedsignificantlyhigher
CAAvaluesthantheMRSbroth(p<0.05)(Fig.5B).ThecellularAAofquercetinwasalsode-
terminedbyCAAassayinHepG2cells(S1Fig.)asastandard.ThecalculationresultsofCAA
values(units)oftheCFSsofLactobacillusstrainstotheequivalentamountofquercetinwith
thesameAA(μM)presentmoreintuitiveviewoftheAAoftheCFSsoftheLactobacillus
strainscomparedwiththisestablishedantioxidantwithknownclinicalefficacy(S1Table).The
CAAvaluesoftheCFSsrangedfrom69.14±4.87to95.55±2.99CAAunits.Thepositivecon-
trolLGGalsoexhibitedastrongantioxidantproperty(95.55±2.99CAAunits)intheCAA
assay,whichwasstrongerthanthatofCrataegusazarolusaqueousextract(79.62CAAunitsin
800μg/mL)[53].Thedifferenceessentiallydependsonthebioavailabilityofthespecificmix-
tureoftheavailablecompoundsandtheirsynergisticinteractionstoyieldfinalantioxidantre-
sponsesatthecellularlevel[25].CCFM1107,L.casei2W,andCCFM8661showedthehighest
AAs(95.40,94.06,and93.48CAAunits,respectively).Nosignificantdifferencewasfound
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