Assessment of the antioxidant and antibacterial activities of different olive processing wastewaters

16 Pages·2017·2.39 MB·English

RESEARCHARTICLE
Assessment of the antioxidant and
antibacterial activities of different olive
processing wastewaters
MajdoulineBelaqziz1*,ShiauPinTan2,AbdelilahEl-Abbassi3,HajarKiai3,
AbdellatifHafidi3,OrlaO’Donovan2,PeterMcLoughlin2
1 CenterofAnalysisandCharacterization,CadiAyyadUniversity,Marrakech,Morocco,2 The
Pharmaceutical&MolecularBiotechnologyResearchCentre(PMBRC),DepartmentofScience,Waterford
InstituteofTechnology,Waterford,Ireland,3 FoodScienceslaboratory,DepartmentofBiology,Facultyof
a1111111111
Sciences–Semlalia,CadiAyyadUniversity,Marrakech,Morocco
a1111111111
a1111111111 *[email protected]
a1111111111
a1111111111
Abstract
Oliveprocessingwastewaters(OPW),namelyolivemillwastewater(OMW)andtable-olive
wastewaters(TOW)wereevaluatedfortheirantibacterialactivityagainstfiveGram-positive
OPENACCESS
andtwoGram-negativebacteriausingthestandarddiscdiffusionandthinlayerchromatog-
Citation:BelaqzizM,TanSP,El-AbbassiA,KiaiH,
raphy(TLC)-bioautographyassays.DiscdiffusionscreeningandbioautographyofOMW
HafidiA,O’DonovanO,etal.(2017)Assessmentof
theantioxidantandantibacterialactivitiesof werecomparedtothephenolicextractsoftable-olivebrines.PositiveactivityagainstS.
differentoliveprocessingwastewaters.PLoSONE aureuswasdemonstrated.Theoptimizationofchromatographicseparationrevealedthat
12(9):e0182622.https://doi.org/10.1371/journal.
hexane/acetoneintheratioof4:6wasthemosteffectiveforphenoliccompoundssepara-
pone.0182622
tion.AHPLC-MSanalysiswasperformedshowingthatonlytwocompounds,hydroxytyrosol
Editor:CalogeroCaruso,UniversityofPalermo,
andtyrosol,werethepredominantphenoliccompoundsinallOPW.Thephenolicextractof
ITALY
OMWgeneratedbyasemi-modernprocessshowedthehighestfreeradical-scavenging
Received:October12,2016
activity(DPPHassay)comparedtotheotherphenolicextracts.Itisapparentfromthepres-
Accepted:July6,2017 entstudythatOPWarearichsourceofantioxidantssuitableforuseinfood,cosmeticor
Published:September5,2017 pharmaceuticalapplications.
Copyright:©2017Belaqzizetal.Thisisanopen
accessarticledistributedunderthetermsofthe
CreativeCommonsAttributionLicense,which
permitsunrestricteduse,distribution,and
reproductioninanymedium,providedtheoriginal
Introduction
authorandsourcearecredited.
DataAvailabilityStatement:Allrelevantdataare Olivefruitsareusedintheagro-industrymainlyforthepreparationoftableolivesandoil
withinthepaperanditsSupportingInformation extraction.Theoliveoilextractiongeneratesaneffluentformedbythecombinationofthe
files. watercontentoftheolivefruitwiththewatergeneratedfromthewashingandprocessingof
Funding:AuthorsacknowledgeAverroès theolives.Thiseffluentiscommonlyknownasolivemillwastewater(OMW).Thiseffluent
FellowshipProgram(fundedbytheEuropean isoneofthemostenvironmentallyconcerningfoodprocessingeffluentsintheMediterranean
CommissionwithintheframeworkofErasmus countriesduetoitsphytotoxicity[1].Virginoliveoilisextractedfromolivefruitusingmec-
Mundus)forfundingtheresearchstayofDr.
hanicalprocessesincludingthecrushingoftheolivefruitsfollowedbymalaxationstepwhich
MajdoulineBelaqzizinWaterford(Ireland).Aswell,
preparestheresultingpasteforsubsequentseparationoftheoil.Theoilyphaseisseparated
Prof.AbdelilahElAbbassigreatlythanksthe
throughpressureorcentrifugation.ThemainoliveoilextractionmethodusedinmanyMedi-
InternationalFoundationforSciences(IFS,
Sweden)fortheirfinancialsupportunderthe terraneancountriesisthecontinuouscentrifugationsystemknownasthree-phasesystem[2].
PLOSONE|https://doi.org/10.1371/journal.pone.0182622 September5,2017 1/16
Bioactivityofoliveprocessingwastewaters
grantsnumberW4749-1andW4749-2.The Thecentrifugaldecanterallowsfortheseparationofthreephases;theoliveoil,wastewater
fundershadnoroleinstudydesign,datacollection (OMW)andpomace(solidwaste).Besidethetraditionalpressextractionsystemandthetwo-
andanalysis,decisiontopublish,orpreparationof
phasecentrifugalsystem,theoliveoilextractionprocessesproducebetween10million[3]and
themanuscript.
30millionm3peryearofOMW[4].
Competinginterests:Theauthorshavedeclared Moreover,therearethreemaintradepreparationsoftableolives:a)thegreenSpanishstyle,
thatnocompetinginterestsexist.
b)Californianstyle(ripeolivesbyalkalineoxidation)andc)Greekstyle(naturallyblack
olives)[5,6].Asmentioned,tableolivepreparationgenerateslargevolumesofwastewaters.
Botheffluents,OMWandtable-olivewastewaters(TOW),couldbegroupedunderasingle
nameknownas“oliveprocessingwastewaters,OPW”.Thedisposalofthesewastewaterswith-
outpriortreatmenthasledtosevereproblemsforthewholeecosystem.OMWandtable-olive
brineshaveshowedtoxicitytosomeplantsandmicroorganismssincetheyexhibitasubstan-
tialconcentrationofpolyphenolsupto10g/L[7].Thephytotoxicandantibacterialproperties
ofOMWpolyphenolsweredemonstratedinarelativelyrecentstudyalongwiththenegative
effectstheyhaveonincreasingthesalinityandacidityofsoils[8].
Oliveoilcontainsonly2%ofthetotalphenoliccontentfromtheolivefruitandtheremain-
ing98%islostinOMWandpomace[9].Furthermore,duringtheprocessingoftableolives,
hydrolyzedpolyphenolsareliberatedintothebrines.Thus,OPWarepotentiallyarichsource
ofadiverserangeofpolyphenolswithalargespectrumofbiologicalactivities.Polyphenolsare
foundwidelyinavarietyofplantsincludingolivesandareinvolvedinmanyvitalfunctions
includingdefense[10].
Accordingtoseveralstudies,polyphenolsfromolivealsorepresentnaturalanti-inflamma-
toryagents[11]andexhibitawiderangeofinterestingbioactivitiessuchasantimicrobial,anti-
atherogenic,antitumoral,cytoprotectiveandcardioprotectiveproperties[12,13].Thus,they
mayhavesignificanthealthbenefits.Theycouldthereforebeusedtoreplacesyntheticdrugs
whichcancausesideeffects.
Studieshavealsodemonstratedthatphenoliccompoundsexhibitedbroadspectrumanti-
bacterialactivity[14].PhenoliccontentofOMWhasbeendemonstratedtohaveamollus-
cicidalactivity[15]inadditiontoantimicrobialactivity[3].Moststudiesofantimicrobial
activityhavefocusedonecologicalandenvironmentalconsequences[16]oronagricultural
applications[17].
Alargenumberofresearchpapershavebeenpublishedregardingthechemicalcomposi-
tionofolivesandoliveoil;but,onlyfewstudieshavefocusedonisolatingandidentifyingcom-
poundsfromtheOPW[18–20].Therecoveryofthesebioactivemetabolites,especially
hydroxytyrosol,aromaticacids,andconjugatedaromaticacidsfromOPW,isofparticular
interestsincetheypossessverypromisingbioactivitiesandhealthpromotingproperties[12].
AspartofacomprehensivestudyofthenatureandfunctionalityofOPWphenolicextracts,
weinvestigatedtheantioxidantandantibacterialactivityofOMWsamplesgeneratedbytwo
differentoliveoilprocessingtechniques,andTOWsamplesissuedfrombrinesoftableolives
withdifferentstagesofmaturity.Theantioxidantandantibacterialactivitywasstudiedusing
theDPPHandTLC-bioautographyassay,respectively.
Materialsandmethods
Oliveprocessingwastewatersamples
TensiftHydraulicBassinAgencyistheauthoritywhoisresponsibleforthemanagementof
waterresourcesinTensiftregioninasustainablemanner.ThesamplingoftheOlivemill
wastewaterandtheTableolivebrineswasconductedwiththeagreementoftheolivemilland
theolivemanufacturingcompanyowners.Nospecificpermissionwasrequiredfromthe
authoritytoproceedwiththesamplingoftheoliveprocessingwastewater.Ontheotherhand
PLOSONE|https://doi.org/10.1371/journal.pone.0182622 September5,2017 2/16
Bioactivityofoliveprocessingwastewaters
TheTensiftHydraulicBassinAgencypromotesthecollaborationwithCadiAyyadUniversity
toconductresearchstudiestooptimizethetreatmentandtherecoveryofoliveprocessing
wastewater.Olivemillwastewatersampleswerecollectedfromtwodifferentolivemillslocated
intheOudayaregion,intheWestofMarrakechcity(Morocco)duringtheseasonof2012/
2013.Thetwomillsusedifferentmillingtechniques,semi-modern(OMW1)andmodern
(OMW2)three-phaseprocesses.TheprocessedolivefruitsarefromtheMoroccanPicholine
variety.Table-olivebrineswerekindlyprovidedbyAgro-Hindtableolivemanufacturingcom-
pany(Marrakech,Morocco).Threedifferentsamplesoftable-olivewastewaterwerestudied,
green-olivebrine(GTOW),black-olivesbrine(BTOW)andpurpleolivebrine(PTOW).Sam-
plingwascarriedoutinamannertoinsurearepresentativesample.Allanalysesweredoneat
leasttwice.
Physicochemicalcharacterizationofsamples
ThephysicochemicalcharacterizationofOPWsampleswascarriedoutasfollow:Chemical
oxygendemand(COD)wasdeterminedbythedichromatemethodasusedbypreviousstudy
[19].Briefly,wastewatersamplewasdilutedupto100foldandintroducedintoalab-prepared
digestionsolutioncontainingpotassiumdichromate,mercuricsulfateandsulfuricacid.The
mixturewasincubatedfor120minat150˚CinaCODreactor(ModelWTWCR3000,Ger-
many).CODconcentrationwasthenmeasuredcolorimetricallyat600nmusingaMultiLab
P5(WTW,Germany).Standardsolutionsof1,2,3,and4gofO perliterwerepreparedusing
2
potassiumbiphthalate.Totalsuspendedsolidsweredeterminedafterfiltrationofagivenvol-
umeofolivemillwastewatersamplesthroughaWhatmanfilter(934-AH).Thedryresidue(g/
L)wasthendeterminedindirectlybydryingthepermeateat105˚Covernight.Theotherphysi-
cochemicalparameters(i.e.pH,sugar,acidity,sodiumandpotassium)wereanalyzedas
describedpreviouslybyKiaiandHafidi[21].Water-solublephenoliccompoundsofOPW
sampleswereextractedusingsolvent-solventextractionthreetimesfirstwithequalvolumeof
ethylacetatefollowedbyhalfvolumeofhexane.Afterevaporationoftheorganicphaseusinga
vacuumrotaryevaporator,theresiduewasdissolvedinpuremethanolandkeptat4˚Cuntil
use[22].
Totalphenoliccontent,flavonoids,flavanols,andproanthocyanidins
determination
Thetotalphenolicconcentration(TPC)inmethanolicextractsrecoveredafterextractionwith
ethylacetatewasdeterminedbytheFolin–Ciocalteaucolorimetricmethod[23].Fortotalfla-
vonoids,amodifiedmethodofKimetal.[24]wasused.Briefly,0.2mLaliquotofextractwas
mixedwith0.8mLofdistilledwaterina5mLassaytubefollowedby60μLof5%NaNO The
2.
mixturewasallowedtoreactfor5min.Followingthis,40μLof10%AlCl wasaddedandthe
3
mixturewasleftforafurther5minbeforeadding0.4mLof1MNa CO and0.5mLofdis-
2 3
tilledwatertothereactionmixture.Theabsorbancewasmeasuredat510nmagainstablank
preparedsimilarlyexceptthatdistilledwaterwasusedinsteadofextract.Totalflavonoidscon-
tentwascalculatedfromacalibrationcurveusingcatechinasastandard,andexpressedasmg
catechinequivalentsperliteroftheextract(CTE/L).
Flavanolsweredeterminedafterderivatizationwithp-(dimethylamino)-cinnamaldehyde
(DMACA),usingtheoptimizedprotocolestablishedbyNigelandGlories[25].Extract(0.2
mL),suitablydilutedwithmethanol,wasintroducedintoa5mLassaytubeand0.5mLHCl
(0.24Minmethanol)and0.5mLDMACAsolution(0.2%inmethanol)wereadded.Themix-
turewasallowedtoreactatroomtemperaturefor5min,andtheabsorbancewasmeasuredat
640nm.Controlwaspreparedbyreplacingsamplewithmethanol.Theconcentrationoftotal
PLOSONE|https://doi.org/10.1371/journal.pone.0182622 September5,2017 3/16
Bioactivityofoliveprocessingwastewaters
flavanolswascalculatedfromthecalibrationcurveobtainedusingcatechinasastandard.The
resultswereexpressedasmgofcatechinequivalentsperliteroftheextract(CTE/L).Proantho-
cyanidinswereanalyzedbythemethoddescribedbyWatermanandMole[26].Butanol
reagentwaspreparedbymixing70mgferroussulfate(FeSO )with5mLconcentratedHCl
4
andmadeupto100mLwithn-butanol.Analiquotof0.1mLsamplewasmixedvigorously
withbutanolreagent(1.4mL)andheatedfor45minat95˚Cwaterbath.Thesamplewasthan
cooledand0.5mLn-butanolwasaddedtoit,beforetheabsorbancewasreadat550nmwas
measured.Resultswereexpressedascyanidingequivalentsperliteroftheextract(CYE/L)
usingamolarextinctioncoefficientε=26,900withamolecularweightof449.2.
Antibacterialactivity
Discdiffusionassay. Antibacterialactivitywastestedagainstapanelofnonpathogenic
microorganismslistedbelow:Lactococcuslactis(HP),Lactobacillusbulgaricus(ATCC11842),
Staphylococcusaureus(DPC5246),Bacillussubtilis,Listeriainnocua(WIT361),Escherichiacoli
(DSMZ10720)andSalmonellatryphimurium(LT2).AllcultureswereobtainedfromthePhar-
maceuticalandMolecularBiotechnologyResearchCentre(PMBRC)atWaterfordInstituteof
Technology(Ireland).Thediscdiffusionmethod,knownastheKirby-Bauermethod,was
usedtodeterminetheantibacterialactivitiesofOPWphenolicextracts.Avolumeof5mLof
BHI(BrainHeartInfusion)brothwith50μLofS.aureus,B.subtilis,L.innocua,S.typhimur-
iumandE.colibacteriawasincubatedat37˚Covernight.B.subtiliswasincubatedat34˚C
whileshakingat200rpm.M17mediumwith0.5%lactosewasusedforL.innocuaincubated
at37˚CandMRSbroth(agarofMan,RogosaandSharpe)wasusedforL.bulgaricusandincu-
bationat37˚Cunderanaerobicconditions.1mLofeachculturewascentrifugedat13,000
rpmfor2min.Thesupernatantwasremovedandthepelletwasre-suspendedin1mLofster-
ilemaximumrecoverydiluent(MRD)vortexedfor~30sandthenre-centrifugedat13,000
rpmfor2min.Thesupernatantwasremovedforasecondtimeandthecellpelletre-sus-
pendedin1mLofsterileMRDandvortexedfor~30s.TheOPWphenolicextractswere
driedbynitrogenflux,weighedandreconstitutedinpuremethanoltogiveafinalconcentra-
tionof100mg/mL.Blankdiscs,6mmwereallowedtowarmtoroomtemperatureafter
removalfroma–20˚Cfreezerfor1handthenimpregnatedwith10μLofthephenolicextract
solutiontohavea1mgextract/discofeachsample.Thediscswerethenlefttodryforatleast
20minundersterileconditionstoallowevaporationofthesolvent.Thenegativecontrol
(10μLofmethanol)waspreparedinasimilarmannertothephenolicextractdiscs.Theposi-
tivecontrolwaschloramphenicol(10μg)antibioticdiscs.
TLCplates. Thephenoliccompoundsinthetestedextractswereseparatedonaluminum-
backedthinlayerchromatography(TLC)plateswithdifferentsolventsystemswidelyusedin
chromatography.TheTLCplatesweredevelopedundersaturatedconditionswitheachofthe
eluentsystems.Thedevelopedplateswerethendriedtoremovetracesofsolventontheplates.
TLC-bioautography. Thebioautographymethodallowsbothseparationandmicrobio-
logicaldetectiononthesameplate.Inthecurrentstudy,twobioautographicassayswereused.
TheTLCseparationwasdeveloped,optimizedandfullyvalidatedusingS.aureusasanindica-
torbacteriatodetectantibacterialactivity.Thefirstassaywasbasedontheoverlaymethodin
whichabacterialseededagarmediumwasappliedontheTLCplate.Thesecondassaywas
basedonsprayingthechromatogramswithbacterialsuspensionuntilalayerwasformed
homogenouslyonthesurface.Thisprocesswascarriedoutinthelaminarflowcabinetunder
sterileconditions.Thereafter,theplateswereincubatedovernightat37˚Cinthedark.The
producedinhibitionzonewasvisualizedbysprayingtheplatewitha2.5mg/mLsolutionof
MTTdye(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide)andfurther
PLOSONE|https://doi.org/10.1371/journal.pone.0182622 September5,2017 4/16
Bioactivityofoliveprocessingwastewaters
incubatedovernight.ClearbandsindicatedbacterialinhibitionastheMTTisnotreducedto
theredcolorformazan.LivebacterialcellswillconverttheMTTintoredcolorformazan.
Antioxidantactivity
Freeradicalscavengingactivity. Theantioxidantactivityoftheextractswasevaluated
basedonhydrogen-donatingorradical-scavengingabilityusingthestablefreeradical2,2-
diphenyl-1-picrylhydrazyl(DPPH).Anamount(0.1mL)ofphenolicextractswasaddedto3
mLof0.04%methanolicsolutionofDPPH[18].Themixturewasmixedthoroughlyandincu-
batedindarkatroomtemperaturefor60min.Thedecreaseinabsorbancewasthenmeasured
at517nm,againstmethanolasablank.Thecapacityofthetestedsamplestoscavengethe
DPPHradicalwascalculatedasapercentageofDPPHdiscolorationusingthefollowingequa-
tion:
%Inhibition¼f1(cid:0) ðA =A Þg(cid:2)100 Eqð1Þ
sample control
WhereA wasmeasuredastheabsorbanceofDPPHwithoutsample.Theextractconcen-
control
trationproviding50%inhibition(IC )wasdeterminedfromthegraphofpercentageofinhibi-
50
tionagainstphenolicextractconcentration[19].
TLCbioautographyassaywithDPPHreagent. Inordertoscreentheantioxidantactiv-
ityofthetestedphenolicextracts,aTLCbioautographymethodwasperformed[27].Aftersep-
arationonTLCplates,thecompoundswithfreeradicalscavengingactivityweredetermined
insituwithDPPHreagent[27].TheTLCplatewasobservedundervisiblelight.Areasproduc-
ingyellowishbandsagainstthepurplebackgroundwereconsideredasantioxidants.
HPLC-MSanalysisofphenolicextracts
HPLCanalysiswasperformedonanAgilent1200seriesequippedwithan210Agilent1200
seriesbinarypumpSL,anAgilent1200seriesG1316BSL211temperature-controlledcolumn
oven,amicrovacuumdegasserandaphotodiode212array(PDA)detector,controlledby
EZChromsoftware.SeparationwasachievedusingWaterssymmetryC185μm,3.9x150mm
columnwithagradientrunusingA=0.1%aqueousformicacid,B=89.5:9.5:1Methanol/
nitricacid/formicacid.Thegradientrunstartswith10%Bfor10min,30%Buntil20min,
maintaining30%Buntil25min,40%Buntil45min,thenuntil50minat50%B,until60min
at100%B,until65minat10%Bandfinallyuntil75minat10%B,makingthetotalruntime
of75min.Theusedflowratewas0.2mL/minand10μLinjectionvolume.Theextractswere
analyzedat240,280and365nm.Quantitativedeterminationswerecarriedoutusingexternal
standards.HPLCanalysiswasperformedfirstonthestandards,followedbytheOPWextracts,
andfinallyspikingthesampleswiththestandards.Theidentificationofphenoliccompounds
wasconfirmedusingLC-MSanalysis.LC-MSconditionsweresameasHPLCconditionsusing
negativemode,scanbetweenm/z15–500andtargetionatm/z153(hydroxytyrosol),m/z137
(tyrosol),atm/z540(oleuropein),m/z164(p-coumaricacid),andm/z193(ferulicacid).
Resultsanddiscussion
Physicochemicalcharacterizationofoliveprocessingwastewaters
Table1showstheresultsfromtheanalysisofcommonphysicochemicalparametersand
chemicalanalysisofthefivewastewatersfromtheoliveprocessingindustry.
OMWarerelativelydenseandacidicwithahighorganicloadthatreachesvaluesashighas
110g/LCOD(Table1).Itcontainslargeamountsofdryresidueuptoabout194g/L.OMW1,
whichisfromasemi-modernthree-phaseprocess,showshighsalinityandthereforehigher
PLOSONE|https://doi.org/10.1371/journal.pone.0182622 September5,2017 5/16
Bioactivityofoliveprocessingwastewaters
Table1. Physicochemicalcharacterizationofolivemillwastewaterandtable-olivebrinesamples.
Parameters Unit Olivemillwastewater Table-oliveswastewater
OMW1 OMW2 GTOW PTOW BTOW
pH - 5±0.10 5.10±0.10 4.5±0.1 4.5±0.1 5.1±0.1
EC mS/cm 56.30±0.50 11.01±0.60 76.2±0.4 83.8±0.6 106.4±0.5
Acidity g/L - - 6.6±0.63 5.81±0.63 -
Color - - - 0.75±0.01 0.8±0.05 20.6±1.1
TPC gTYE/L 8.5±0.4 6.46±0.8 3.67±0.4 4.5±0.1 2.6±0.1
Sugar g/L - - 1.6±0.12 6.9±1.53 8.5±1.8
COD gofO /L 110±4.9 50±5.4 3.26±0.1 3.12±0.15 12.6±3.33
2
Dryresidue g/L 194.2±11 132.7±7 84.7±2.05 101.5±1 275.4±4.8
TSS g/L 86±5 50±3.5 1.68±0.11 1.69±0.02 2.6±0.26
Sodium g/L 2.10±0.10 1.45±0.09 27.5±1.5 26.2±1 9.63±0.5
Potassium g/L 1.24±0.12 0.55±0.02 1.3±0.3 2.9±0.5 22±1.2
EC:electricalconductivity,TPC:totalphenoliccontent,COD:chemicaloxygendemand,TSS:totalsuspendedsolids,OMW1:semi-modernOMWphenolic
extract,OMW2:modernOMWphenolicextract,GTOW:greentable-olivewastewater,POTW:purpletable-olivewastewater,BTOW:blacktable-olive
wastewater.
https://doi.org/10.1371/journal.pone.0182622.t001
electricalconductivity(EC)comparedtoOMW2.Thisisduetothefactthatsomeproducers
usesaltforconservationofolivesuntilmilling.OMW2wasfoundtohavelowerphysicochem-
icalvaluesthanOMW1(Table1)whichcouldbeattributedtotherelativelylargervolumesof
waterusedinmodernolivemillingprocesses[19],resultinginrelativelydilutedOMW.The
BTOWexhibitsadarkcolorandhadthehighestorganiccontentbutthelowestphenoliccon-
tent(Table1).Amongstotherorganicconstituents,OPW(OMWandTOW)containhigh
concentrationofphenoliccompoundsrangingfrom2.6to8.5g/L,andhighECwhichexceeds
theenvironmentallegislationlimit,setat0.5mg/Lforphenoliccompounds[28]and3mS/cm
forEC[29].
Antibacterialactivity
Discdiffusionassay. Noantibacterialactivitywasobservedfornegativecontrolswhilst
thezoneofinhibitionforpositivecontrolswasbetween15.0±0.5mmand23.3±2.5mmfor
S.aureusandL.bulgaricus,respectively.AllphenolicextractstestedwereactiveagainstS.
aureus.NoneofthetestedsampleswereactiveagainstL.innocua.OMW1extract(semi-mod-
ernprocess)wasactiveagainstalltestedbacteriaexceptL.innocuawhileOMW2wasonly
activeagainstS.aureus,B.subtilisandL.bulgaricus.OMW1demonstratedthehighestantibac-
terialactivitiescomparedtootherphenolicextracts.Thisresultcanbeattributedpartiallyto
thefactthatOMW1exhibitsthehighestconcentrationofphenoliccompounds(Table1).
Inordertostudytheeffectofconcentrationofphenolicextractsontheirantibacterialactiv-
ity(againstS.aureus),differentconcentrationsofOMWphenolicextractsweretestedfortheir
antibacterialactivityandcomparedtoTOWphenolicextracts(Table2).Theantibacterial
activityincreasedwiththeincreaseinthephenoliccontentoftheextract.Thegreenbrinephe-
nolicextract(GTOW)demonstratedthehighestantibacterialactivitycomparedtotheother
TOWphenolicextracts(BTOWandPTOW).
Atconcentrationsof0.5,1.25and5mgtheGTOWextracthadhigherantibacterialactivity
againstS.aureuscomparedtotheOMWextracts.Theseresultsdemonstratedthattheantibac-
terialactivityofthephenolicextractsisnotonlycorrelatedtotheirconcentrationbutthekey
factorgoverningtheirantibacterialactivityistheirphenolicprofile.Duringthefermentation
PLOSONE|https://doi.org/10.1371/journal.pone.0182622 September5,2017 6/16
Bioactivityofoliveprocessingwastewaters
Table2. EffectofconcentrationofOMWandTOWphenolicextractsontheirantibacterialactivity
againstS.aureususingthediscdiffusionassay.
Testsubstance(amount/disc) ZoneofInhibition(mm)
OMW1(0.125mg) 6±0.5
OMW1(0.5mg) 7±0.5
OMW1(1.25mg) 8±0.5
OMW1(5mg) 11±0.5
PC 15.6±0.6
NC 0
OMW2(0.125mg) 6±0.5
OMW2(0.5mg) 7±0.5
OMW2(1.25mg) 8±0.5
OMW2(5mg) 9.3±0.6
PC 16±0.5
NC 0
PTOW(5mg) 10±1
BTOW(5mg) 9±0.5
GTOW(5mg) 12±1
PC 15±1
NC 0
GTOW(0.25mg) 6±0.5
GTOW(0.5mg) 8±0.5
GTOW(1.25mg) 9±0.5
GTOW(2.5mg) 11±0.5
PC 14.6±0.6
NC 0
PC:positivecontrol;NC:negativecontrol;OMW1:semimodernOMWphenolicextract;OMW2:modern
OMWphenolicextract;GTOW:greenbrinephenolicextract;BTOW:blackbrinephenolicextract;PTOW:
purplephenolicextract.
https://doi.org/10.1371/journal.pone.0182622.t002
oftableolives,thediffusionofphenoliccompoundsintothebrinedependsonseveralparame-
terssuchascultivarcharacteristics,fruitskinpermeability,typeofphenoliccompoundspres-
entinolivefleshandtheirabilitytodiffuseoutofthefruit[21].
Inasimilarstudy[30],hydroxytyrosolandoleuropeinwerefoundtobecytotoxictomany
clinicalbacterialstrainsalthoughtoalesserextentthantheATCCstrains.Theauthors
reportedthattheminimuminhibitoryconcentrationofhydroxytyrosolandoleuropeinagainst
S.aureuswas3.9–31.25mg/Land62.5–125mg/L,respectively.Inarelativelyrecentstudy,
hydroxytyrosol,tyrosolandoleuropeindidnotshowanybactericidalactivityataconcentra-
tionashighas(20mM)0.3g/L,0.27g/Land1g/L,respectively[31].Theseresultscontribute
tothedebateabouttheantimicrobialactivityoftheseolivecompounds,althoughthediffer-
encesinexperimentalconditionsandmicroorganismsusedtotesttheefficacyoftheseantimi-
crobialsmakeitdifficulttocomparetheireffectiveness.
Thinlayerchromatography(TLC)separation. Priortothebioautographyassayandin
ordertoseparatetheactivecomponentspresentinthecrudephenolicextracts,TLCwas
undertakenandthemostefficientsolventsystemwasdetermined.Ninemobilephasesystems
containingsolventsofdifferentpolarity(hexane:acetone(4/6);chloroform:methanol(varying
ratios)andothersolventsystemstoincludecombinationsofthesesolventsandethylacetate
and/ordichlorobenzenewereusedtodeterminetheappropriatesolventsystemforseparation
ofcompoundsinaparticularextractbyTLC.
PLOSONE|https://doi.org/10.1371/journal.pone.0182622 September5,2017 7/16
Bioactivityofoliveprocessingwastewaters
TheRf(retardationfactor)valuewhichistheratioofmovementofthesolutefromitsorigin
tothemovementofthesolventfromorigindeterminestheseparationofcompoundsonthe
solidphaseofTLCplate.Aratioof4:6,v/vhexane/acetonephasewasfoundtobethebestchro-
matographicsystemforachievingseparationofphenoliccompoundsfromamixturebasedon
theobservedspotsnumberandtheshapeofthespots(roundanddistinctfromeachother).
TLC-bioautography. TwoTLC-bioautographicassaymethodsweredeveloped,optimized
andfullyvalidatedinourlaboratoryusingS.aureusasanindicatorbacteriumtodetectanti-
bacterialactivity.Thefirstmethodwasagar-overlayorimmersionbioautography,wherethe
developedplatewasoverlaidwith15mLofmoltenagarseededwith150μLofbacterialcul-
ture.Afterovernightincubation,theinhibitionzonewasvisualizedbysprayingwithMTT
dye.Thebioactivecompoundsweretransferredbydiffusionfromthestationaryphasetothe
agarlayercontainingthebacteria.However,theuseofagargelasasupportmediumhassome
disadvantagesincludingslowdiffusionandbadcontrast.Inordertooptimizethevisualization
oftheinhibitionbands,asecondmethodwasusedusingTLCplatessprayedwithanovernight
cultureofS.aureus(50μLin5mLofBHI).Hence,athinnerlayerofagarwascoveringthe
TLCplate,allowingminimumdiffusion.
ThebioautographyscreeningofOMWandbrineextractsshowedpositiveactivityagainst
S.aureus.Bacterialinhibitionwasdenotedbyclearspotagainstaredpurplebackgroundon
theTLCplateaftersprayingwithMTT.MostofthecompoundsseparatedontheTLCplates
wereactiveagainstS.aureus.
Thebioautographytestconfirmedtheresultsofthediscdiffusionassaydemonstratingcon-
centrationdependentantibacterialactivity.ThehighestactivitywasregisteredbyGTOWphe-
nolicsamplecomparedtotheotherOPWphenolicsamples(Table2).
Whenallofthecompoundsthatshowedanactivitybasedonbioautographywereisolated
andcharacterizedtheyoftenhadamuchloweractivitythanexpected(datanotshown)indi-
catingsynergismcouldbeplayingasignificantrole[32].CompoundsfoundinOMWthat
werereportedtoexhibitantibacterialactivityaretyrosol,oleuropein,hydroxytyrosol,4-hydro-
xybenzoicacid,vanillicacid,andp-coumaricacid[33].Ithasbeenreportedthatolivepolyphe-
nolssuchashydroxytyrosolhaveinvitroantibacterialactivityagainstbothGram-negativeand
Gram-positivebacteriaresponsibleforintestinaltractandrespiratorytractinfections[34].
Antioxidantactivity
DPPHscavengingactivity. DPPHisacommonlyusedsubstrate(freeradical)forfast
andeasyevaluationoftheantioxidantactivityduetoitsstability,reliabilityandthesimplicity
oftheassay[35].Compoundswithantioxidantpropertieswouldchangethepurplecolorof
DPPHtoyellowastheradicalisquenchedbytheantioxidant[35].Thecolorchangecanbe
measuredquantitativelybyspectrophotometricabsorbanceat517nm.Caffeicacidwasused
asthereferenceantioxidantcompound.Caffeicacid(3,4-dihydroxycinnamicacid)isoneof
themajorhydroxycinnamicacidspresentinOMWandhasbeenidentifiedasoneofthemain
antioxidantsinthisstudy.TheIC ofcaffeicacidwasfoundtobe121±6μg/mL.OMW1and
50
OMW2exhibitedhighlyinterestingDPPHradicalscavengingabilitycomparedtothephenolic
extractsofdifferentbrines(Table3).DPPHradicalwaslesssensitivetotheconcentrationvari-
ationofbrineextractscomparedtothecaseofOMWextracts(Table3).Thisdifferencein
activityismainlyduetothephenoliccompositionofeachwastewatertype.OMW1showed
thehighestantioxidantactivity(thelowestIC Table3)followedbyOMW2.BothOMW
50,
extractsshowedconsiderablyhigherantioxidantactivitycomparedtocaffeicacid.PTOWphe-
nolicextractshowedanIC comparabletocaffeicacid,followedbyGTOWandfinallythe
50
BTOWwhichshowedthelowestradicalscavengingactivity(Table3).
PLOSONE|https://doi.org/10.1371/journal.pone.0182622 September5,2017 8/16
Bioactivityofoliveprocessingwastewaters
Table3. Totalphenoliccontent,phenolicconstituentsandantioxidantactivity(IC )ofdifferentoliveprocessingwastewaterssamples. Theval-
50
uesinthebracketsaretheproportionstothetotalphenoliccontent,TPC.
Sample TPC Flavonoids Flavanols Proanthocyanidins IC
50
TYEg/L CAEg/L CAEmg/L CYEmg/L mg/L
OMW1 8.5±0.4 5.74±0.36 2.63±0.12 19.32±2.03 15.83±1.9
(100%) (67.53%) (0.031%) (0.227%)
OMW2 6.46±0.8 2.85±0.24 2.1±0.09 14.10±1.53 32.32±4.7
(100%) (44.11%) (0.032%)) (0.218%)
GTOW 3.67±0.04 0.87±0.05 0.23±0.02 42.57±0.71 173±2.8
(100%) (23.71%) (0.006%)) (1.159%)
PTOW 4.5±0.01 1.22±0.09 0.17±0.03 533.39±20.51 126.3±3.9
(100%) (27.11%) (0.004%) (11.85%)
BTOW 2.6±0.01 0.67±0.05 4.17±0.14 399.83±40.31 261.3±4.8
(100%) (25.78%) (0.160%) (15.38%)
https://doi.org/10.1371/journal.pone.0182622.t003
ThedifferentreactionsofthephenolicextractsagainstDPPHradicaldependonthenature
oftheantioxidantsinvolvedinthereaction.Suchbehaviormustbetheresultofthedifferent
individualcontributionsofthephenoliccompoundspresentinthesamples.Furthermore,ina
previouswork,wehavefoundthattheantioxidantcapacityofdifferentOMWextractswas
directlycorrelatedtothepercentageoffreehydroxytyrosolandtheirantioxidantactivitywas
foundtobetheresultoftheirphenolicprofile(composition)ratherthantheirphenoliccon-
centration[19].Hydroxytyrosolisfoundinnature(inolivefruit,oliveoilandoliveleaf)inthe
formofitselenolicacidester:oleuropeinorintheformofhydroxytyrosylacetate.Owingto
thechemicalorenzymatichydrolysiswhichoccursduringstorage,freehydroxytyrosolisliber-
atedprogressivelyagainsttime[15].
TheantioxidantactivityofOPWandespeciallyOMWvarieswidelyfromstudytostudy.
ThevariationoftheOMWphenoliccontentanditsantioxidantpropertiesareaffectedby
manyfactorssuchasolivecultivar,theoliveoilextractionprocess,thephysicochemicalchar-
acteristicsofOMWsamples,thefungalandbacterialfloraexistinginOMW,andfinallythe
storageconditions.
PreviousstudiesonthedegradationofpolyphenolsinitsoriginalOMWmatrixduringthe
extractionprocessanduponstoragerevealedtheirpoorstabilityduetothecomplexandreac-
tivenatureofOMW,whereoxidation,condensation,polymerization,andenzymatichydroly-
siscanallpotentiallytakeplace[36,37].IthasbeenreportedpreviouslythatOMWfromsemi-
modernthree-phaseprocesshashigherphenoliccontentthanOMWfrommodernthree-
phaseprocess[19].Thisstudyconfirmsthesefindingsandhighlightstheeffectofmillingpro-
cessonthephenoliccontentandcompositionofthegeneratedOMW.
Antioxidantcompoundsarereducingagentswhichexhibitsitsantioxidantproperties
throughscavengingfreeradicals.Therefore,theyareabletoextendshelflifeoffoodand
pharmaceuticalproductsbydecreasingtheoxidationrateoftheproducts,hencepreventing
deteriorationoftheproductsduringprocessingandstorage.Heetal.[38]investigatedthe
antioxidantcapacityandstabilityofbioactivecompoundsinpurifiedoliveextract(POE)pre-
paredfromOMWbyadsorptionontoapolymerresin.Theirresultsshowedthatair/oxygen
wasthemainfactorthataffectsthestabilityofPOEduringstorageatlowtemperature,whereas
anincreaseintemperaturesignificantlydecreasedthetotalphenoliccontentoftheextract
(20–24%reduction)[38].
ManystudieshaveinvestigatedthephenoliccontentofOMW;however,thephenoliccom-
positionofbrinesisalsoaverypromisinglow-costsourceforhigh-addedvaluebioactive
PLOSONE|https://doi.org/10.1371/journal.pone.0182622 September5,2017 9/16
Bioactivityofoliveprocessingwastewaters
compounds,especiallyhydroxytyrosol.Tothebestofourknowledge,thisisthefirststudyto
comparethephenolicprofileandantioxidantpropertyofolivemilleffluentsandtable-olive
wastewaters.
Bioautographyassay. Bioautographyisalsoausefulmethodforseparationanddetection
oftheactiveantioxidantsinamixtureofcompounds[39].TheTLCbioautographyassayisthe
methodofchoiceinthescreeningofantioxidantsduetoadvantagessuchasitssimplicity,flex-
ibilityandhighthroughput.OntheTLC,antioxidantcompoundswouldbeseenasawhite/
yellowspotsonapurplebackground[27].Fig1showsaprofileofbrineandOMWphenolic
extractatdifferentconcentrationpresentingantioxidantactivitycomparedtostandardpheno-
liccompounds.HydroxytyrosolisthemostefficientantioxidantagentpresentinOPWex-
tractsasshownbyFig1,evenatalowconcentrationoftheextract(1mg/LforGTOW).The
absenceofantioxidantactivityinsomebandscouldbeduetotheevaporationoftheactive
compounds(highlyvolatilecompounds),photo-oxidationorduetothelowquantityofthe
activecompoundontheTLCplate[40].
ThedifferentdetectionsensitivitiesobservedinFig1areattributedtothediversenatureof
theoxidativecompounds.Thephenolicextractseparatedwith4:6,v/vhexane:acetoneshowed
severalTLCbandswithstrongantioxidantactivitywhichincreaseswiththeincreasingcon-
centrationofthephenolicextract.ResultsreportedinFig1suggestedthathydroxytyrosol
(HT),p-coumaricacid(PC)andcaffeicacid(CA)maybepresentinOMWandbrinephenolic
extractsasvisualizedbytheDPPH-TLCbands.Thepresenceofthesecompoundsincon-
firmedwithLC-MS.
Phenolicconstituentsoftheoliveprocessingwastewatersextracts
Flavonoids,themostdiverseandthelargestgroupofnaturalphenoliccompounds,areknownto
haveantioxidant,antiallergic,antimicrobial,anti-inflammatory,andanticarcinogenicproperties
[41].ThisphenolicgroupconstitutesthelargestpartofOMW1phenoliccontent(67.53%).How-
ever,flavonoidscorrespondonlyto44.11%ofOMW2phenoliccontent(Table3).Thethreestud-
iedtable-olivebrinescontainedbetween23%and27%flavonoidsoftheirtotalphenoliccontent.
Besidestheirantioxidantactivities,flavonoidswerefoundtoinhibitlipidperoxidation,platelet
aggregationandpossesschemopreventiveeffectsoncarcinogenesis[42].Themainflavonoid
subclassesinOMWareproanthocyanidinsandflavanols[19].Thesephenolicswerefoundto
decreasetheriskofischemia-reperfusiondamageofheartinratbyincreasingplasmaantioxidant
activity[43].Inthisstudy,fortheOPWsamples,flavanolsandproanthocyanidinswerefound
tohavealowerproportion,ofapproximately2%oftheirtotalphenoliccontent,exceptfor
BTOWwhichexhibitsthehighestproportionofproanthocyanidins(15.38%)followedbyPTOW
(11.85%)(Table3).Furthermore,theflavanolscontentvariedfrom0.17to4.17CAEmg/L(0.004
to0.16%)ofthetotalphenoliccontentofdifferentOPWsamples.
HPLC-MSanalysisofoliveprocessingwastewaters
HPLC-MSanalysiswasperformedtodetermine,compare,identifyandquantifythephenolic
compoundsinOPWsamples(Fig2andTable4).Compoundsfromthreemainphenolic
groupswereidentified,simplephenols(hydroxytyrosolandtyrosol),phenolicacids(p-couma-
ricacid,ferulicacidanditsderivative)andsecoiridoids(oleuropein).
ThetotalphenolicconcentrationasrevealedbytheHPLC-MSquantificationisnotcorre-
spondingtothedepictedspectrophotometricestimationinTable3.Previousstudieshigh-
lightedthatthedisadvantageoftheFolin-Ciocalteucolorimetricmethodisthatcomereducing
agentsmaybepresentintheextractcaninterfereintheanalysisandconsequentlyoverestimate
thetotalphenoliccontentoftheextract[44].Fromthecurrentstudy,hydroxytyrosoland
PLOSONE|https://doi.org/10.1371/journal.pone.0182622 September5,2017 10/16