Recovery of phenolics from apple peels using CO2+ethanol extraction: Kinetics and antioxidant activity of extracts

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The Journal of Supercritical Fluids

jou rn al h om ep a g e :w w w . e l s e v i e r . c o m / l o c a t e / s u p f l u

Recovery of phenolics from apple peels using CO ₂ + ethanol extraction: Kinetics and antioxidant activity of extracts

Audrey Massias

, Séverine Boisard

, Michel Baccaunaud

, Fernando Leal Calderon

, Pascale Subra-Paternault

aAgrotec,sited’Agropole,BP102,47931AgenCedex9,France

bCBMNUMRCNRS5248,UniversitéBordeaux,IPB,AlléeGeoffroySaintHilaire,Bat.14B,33600Pessac,France

cEA921SONAS/SFR4207QUASAV,Universitéd’Angers,16BoulevardDaviers,49045AngersCedex01,France

a r t i c l e i n f o

Articlehistory:

Received15September2014 Receivedinrevisedform 10December2014 Accepted10December2014 Availableonline18December2014

Keywords:

Applepeel Phenolics

Supercriticalextraction Antioxidant

a b s t r a c t

Subcritical extraction (SFE) of dry and ground Golden delicious peels (30g) was investigated at 25MPaand 50^◦C usingCO2 and ethanol (96%)in 75:25mol ratio.As forconventional ethanolor methanol/acetone/waterextraction,ninephenolicswereidentifiedinSFE-extractsincludingthesugar- basedphloridzinandquercetinderivatives.Extractionkineticsoftheninephenolicsandoftheglobal yieldweremonitoredviacollectionoffractionsthatwerealsocharacterizedfortheirantioxidantactivity (ABTSantiradicalactivity).Kineticsshowedaconstantextractionrateupto1.1kgoffluidandadecreasing rateafterwards,butthematrixwasnotexhaustedafter3hofextraction.Besidestheclassicalcontin- uousflowprotocol,SFEwasperformedbyintroducingstaticperiodsbetweenthedynamiccollectof fractions.Staticperiodsdidnotyieldsignificantimprovementintheoverallyieldandintheindividual yieldofmostphenolics.Increasingthematrixloadingdidnotimprovetherecoveryeither.Conversely, extractionsfrom15gprovidedthehighestphenolicsyieldof800mg/100gdrypeels.Forextractstestedfor antioxidantcapacity(30gloading),valuesupto5–6mgEquivalentAscorbicAcid/gextractwereobtained.

Activitieswerepositivelycorrelatedwithphenolicsconcentrationinfractionsonlyforstaticconditions.

©2014ElsevierB.V.Allrightsreserved.

1. Introduction

Adietrichin freshvegetablesand fruitsis generallyrecom- mendedforahealthylifestylebecausetheyconstituteimportant sourcesofnutrients[1]likeantioxidants[2],phytosterols[3]and fibre[4].Transformationofarawmaterialtofoodstuffcreatesa

‘waste’thatcomprisesaswelltheediblefoodmassthatislost,dis- carded,ordegradedinthedifferentstagesofthefoodsupplychain.

Otherremnantsarepeels,stems,cores,skins,seeds,husks,branor strawfromcereals,fishskin,headandbones,millwastewaters,etc.

Food‘wastes’havelongbeenconsideredasundesirablematerials thatweredisposedof,incostlymanners,viaanimalfeed,landfill orincineration,butnowadays,theyareconsideredaspromising sourcesofvaluablenutraceuticals[1,5].

Applesareoneofthemostconsumedfruitsworldwideandare amongthemajorsourcesofphytochemicalsandantioxidantsin thehumandiet.Approximately70milliontonnesofapplesarepro- ducedworldwide(http://www.wapa-association.org).Applefruits

∗Correspondingauthor.Tel.:+330540006832.

E-mailaddress:subra@enscbp.fr(P.Subra-Paternault).

haveavariedandwell-balancedcompositionwithalargediver- sityofvitamins,ahighcontentoffibrescomparedtootherfruits andaremoderatelyenergeticintermsofcaloricintake[6,7].Pre- ventionofvariouschronicdiseaseshasbeenassociatedwithapple consumption[8],inparticularcardiovasculardisease,inrelation withthemainbioactivecompoundsofapples,namelyfibreand polyphenols[9].Applescontainover60differentphenolic com- pounds[8].Thefourmajorphenolicgroupsarehydroxycinnamic acids(withchlorogenicacidasthemostabundantrepresentative), dihydrochalcone derivatives (specially phloridzin), flavan-3-ols (catechinasmonomersorprocyanidinsasoligomers)andflavonols (quercetinandquercetinglycosides)[10,11].Thedistributionand concentrationofpolyphenolsvarygreatlyamongapplecultivars (rangeof68–165mg/100gedibleportion[6])andwithintheapple fruit.Apple peelshavehigher levelsoftotal polyphenoliccom- pounds than flesh or core and concentrate specially quercetin glycosides,chlorogenicacidandphloridzin[11–15].Asexample, foreightapplecultivars,thetotalpolyphenolicsrangedfrom1.0 to2.3mg/goffreshweightinthepeel,tobecomparedwiththe 0.33–0.93mg/gof freshweightin flesh[13].Additionally, apple peelspolyphenolswereshowntohavebeneficialactionsonoxida- tivestressandinflammation[16].

http://dx.doi.org/10.1016/j.supflu.2014.12.007 0896-8446/©2014ElsevierB.V.Allrightsreserved.

InFrance,anaverageof20kgofapplespercapitaareconsumed [17]correspondingtoa20.4%marketshare,farmoreimportant thanthatofthesecondmostconsumedfruits,bananas(14%)and oranges(12%).AppleisthelargestfruitproducedinFrancewith 1.5milliontonnesin2013.Around30%oftheproductionistrans- formedinjuices,compotes,concentrates,generatinglargevolumes ofwastesincludingpeels.

Inthiswork,theuseofcompressedCO2 torecoverphenolic componentsfromapplepeelwasinvestigated. Sub-and super- criticalextractionoffunctionalingredientsfromnaturalsources wasreviewedbyHerreroin2006[18],whereasDiaz-Reinoso[19]

focusedoncompoundswithantioxidantactivitiesandMarostica [20]onphenolicsfromplantmaterials.Since2006,morethan70 papersabout‘polyphenols’and‘supercriticalextraction’havebeen published(scopussource).Withspecialmentiontofoodwastes, grape residues produced by thewine or distilling industry are amongthemostlargelystudiedby-products.Residuescomprised grapeskinorseeds[21,22],bagasse[23],marc[24,25]orpomace [26,27].As for otherby-products, colouring anthocyaninswere extractedfromeggplantpeels[28],phenolicantioxidantsfromsev- eralberriespressingwastes[29]oroilcontainingphenolicsfrom cherrykernels[30]orgrapeseeds[31].Fruitspulporleaveswere investigatedaswell,inparticularsweetcherries[32],jamunfruits [33],pitangaleaves[34]andarrabidaechicaleaves[35],forthe mostrecent studies.Tothebestofourknowledge,supercritical extractionfromappleswasonlyreportedonce[36].Theby-product wastheapplepomace,i.e.skinandpulpresiduesremainingafter pressing the fruits for juice production. One gram of pomace was extracted by CO₂+ethanol (14–20%) for 10–40min vary- ing pressure (20–60MPa) and temperature (40–60^◦C). Extracts werecharacterizedforthetotalphenoliccontentandantioxidant activity,butnoidentificationandquantificationoftheextracted phenolicspecieswerereported.

The aim of this work was to evaluate the potentialities of supercriticaltechnologytoextractphenoliccompoundsfromapple peels,focusingonthreeissues:

(1)Identificationandquantificationoftheextractedpolyphenols.

(2)Monitoringtheextractionkineticsofpolyphenolsandofthe totalextractedamount.

(3)Studyingtheimpactof processconditionsontokineticsand yields(implementationofstaticstepsduringextraction,varia- tionoftheamountofmatrixloadedinthevessel).

Inthiswork,dryapplepeelswereextractedbyCO2+25%mol cosolvent(ethanolat96%)at25MPaand50^◦Cduring3h.Beingan apolarfluid,CO₂hasalimitedcapacityfordissolvingpolypheno- licssoethanolisrequiredascosolventtoovercomethislimitation [37–39].Theuseofhighproportionofethanolwasjustifiedbythe factthatphloridzinandquercetinglycosides,abundantinapple peels,containa sugar moietythat is toopolartobesoluble in neatCO2.Theselectedtemperatureandpressureareintherange ofthoseusedforsupercriticalextractionofphenoliccompounds [20]andmoderatetemperatureandhighpressurearegenerally associatedwithlowthermaldegradationandhighsolubility.Due tothe25%ofcosolvent,extractionswereperformedinsubcriti- calconditionssince50^◦Cisbelowthecriticaltemperatureofthat CO2+cosolventmixture[36].Duringtheextractioncourse,frac- tionswereregularlycollectedinordertodescribeaccuratelythe extractionkinetics and possibly fractionate the phenolics pool.

Extractswerecharacterizedintermsofglobalyield,phenoliccom- positionandantioxidantactivity.Anewprocedureofextraction thatalternatedstaticanddynamicperiodswasalsoinvestigated withtheaimofgivinglongertimeformasstransferanddiffusion tooccur.

2. Materialsandmethods

2.1. Applepeelpreparationandchemicals

Golden delicious apples were purchased from a local con- ventional orchardand werestored at 1^◦C. Apples werepeeled mechanically(Kali,France)andtheobtainedpeelswereimmedi- atelypackedintopolyethylenebagsandfrozenat−18^◦Cfor24h.

Sampleswerethenfreeze-driedduring48h(HetoLabEquipment, HetoFD2.5,Denmark)andwerefurtherstoredinthedarkunder vacuumatroomtemperature.Themoisturecontentofthedried applepeelswasmeasuredbyweightlossat68^◦Cinovenunder vacuum(Multilab20,LeMatérielPhysico-Chimique,France)and wasintherangeof5–7%. Forextractions,thedriedapplepeels weregroundusingakitchen-typegrinder(Moulinex,France).The obtainedgroundmaterialwasnotsievedsothesamplesconsisted ingrainsofvarioussizesbelow1mm.

Carbondioxide(CO2,99.5wt.%,AirLiquide,France)andethanol (EtOH,96%,Xylab,France)wereusedforsupercriticalextractions.

SolventsforliquidchromatographywereofHPLCgrade(acetoni- trile,99.98%;aceticacid,99.5%)andwerepurchasedfromFisher Chemicaland AcrosOrganics,respectively. Severalstandardsof polyphenols(HPLC-grade,purityhigherthan97%)werepurchased fromSigma–AldrichandExtrasynthese(France):(+)-catechin,(−)- epicatechin,phloridzin,chlorogenicacid,quercetin-3-d-glucoside, quercitrin(quercetin-3-O-arabinoside),hyperoside(quercetin-3- O-galactoside).

2.2. Supercriticalfluidextraction

Extractionswereperformedusingahome-madesystemwhich consistsof anextractor vesselof490cm³ (lengthof 25cm and innerdiameterof5cm)heatedbyheatingmantle(Watlow)and twoGilsonpumpsforfluidsadmission(model305,headsof25SC and10SCforCO₂andcosolvent,respectively).TheCO₂andcosol- ventflow rateswerecheckedbya gas-meterandthecosolvent volumeconsumptionovertime,respectively.Multiplestopvalves (AutoclaveFrance)enabletobypasstheextractortostabilizethe extractingflux,andameteringvalveisusedtocontroltheover- all flow rate. The set-up comprises a pre-cooling unit (Julabo, Germany)forCO2,apre-heatercartridge(TOPIndustrie,France), areliefvalveandvariouspressureandtemperaturesensors.The extractionswereperformedat50^◦Cand25MPawhichiscloseto thepressurelimitoftheequipment.Theextractorwasfilledwith aweightedamountofdriedapplepeels,usingalternatedbedsof matrixand glassbeadsof2mmtoavoid caking.Theextracting CO2+cosolventmixturecirculatedfrombottomtotop.Dissolved specieswererecoveredinahome-madecycloniccollectorwhose bottomwasplungediniceandthatoperatedatatmosphericpres- sure.Fractionationofextractswasperformedbychangingregularly thecollectorbottom.

Extractionswerecarriedoutfollowingtwoprocedures.Ascom- monsteps,thevesselwaschargedwiththematrix,heated,and pressurizedwithCO2upto25MPa.Astaticperiodof20–30min wasappliedbeforestartingtheextractionbyactivatingtheCO₂ andcosolventpumps.Thefirstfractionwascollecteduntilacon- tinuousflowofethanolappearedinthecollector,typicallyafter 20–25min,foranoverallCO₂/EtOHflowof10g/min.Thisfraction, labelledF0,correspondstotheethanolbreakthroughandcontains mostlythematterthatwassolubilizedduringthestaticperiodin neatCO₂.Intheso-calleddynamicprocedure,theextractioncon- tinuedwithregularcollectsofextractsevery20min,i.e.forone breakthrough time,over 200min.Theextractor wassometimes subjectedto10minofstaticstepwhenrecoveringF0andF1in ordertowashthecollector.After200min,thecosolventpumpwas stoppedandneatCO2wasusedtoflushthematrixfromethanol

untilnosolventtracesweredetectedattheseparatorentrance,typ- icallyafter1h.Inthesecondprocedure,called‘static’,astaticperiod wasintroducedbeforecollectingeachfraction,i.e.attheendofa 20mindynamiccollection,thevesselwasisolatedandthefluxwas stoppedfor30min.Thestatic/dynamicalternationwascarriedout for4×20min,beforestoppingtheethanolflowandflushingthe matrixwithpureCO₂asinthedynamicprocedure.Extractionsby thetwoprocedureswererepeatedtwicetocheckreproducibility.

Thecollectedfractionswereanalyzedfortotalmassbygravime- try after evaporating ethanol under nitrogen flux. The total extracted mass was determined from the sum of the fraction masses collectedover theextraction time. Fractions wereana- lyzed for antioxidant activity via the Ascorbic Acid Equivalent AntioxidantCapacity(AAEAC)method.Phenolicswereidentified andquantified byliquid chromatography(HPLC). In Tables,the amountsofphenolics orofglobalextractsare expressedin mg orgper100g_drypeels,inwhichthesubscript‘drypeels’designates thelyophilizedapplepeelscontaining5–7wt%moisture.Infigures, kineticsdataaregivenascumulatedamountasafunctionofthe massofextractingfluid.Thelattervariablewaspreferredtotime sincetheamountoffluidusedtocollectafractionwasthesame whateverthenumberanddurationofstaticperiods.

2.3. Conventionalsolventextraction

Conventionalextractions were carried out on dry peels fol- lowing two different methods. In the first one, derived from Colin-Henrion [11] and named hereafter MeOH/Acetone, peels werefirstmixedwithmethanol(14mLfor0.6g).Afterfiltration undervacuum,peelswerere-suspendedinanacetone:watermix- ture70:30v:v(8mL)andfiltratedagain.Thetworesultingextracts werecombinedanddriedusingarotaryevaporatorundervacuum at30^◦C.Thefinalconcentratewasredissolvedin10mLmethanol forHPLCanalysis.Inthesecondmethod,peelsweremaceratedin ethanol(EtOH96%)at45^◦Cfor3hundermagneticstirring(100mL ofsolventper10gofpeels).Macerationwasperformedforthesake ofcomparisonwithsupercriticalextractionsandwasthuscarried outwithavolumeofethanolidenticaltothatpassedwith1.1kgof {CO₂+ethanol}mixture.Thesupernatantwasrecoveredbyfiltra- tionundervacuumanddirectlyinjectedinHPLC.

2.4. Analyticalmethods

Identification of polyphenols was performed by coupling chromatographicseparationandphotodiodearrayormassspec- trometry detectors [40]. Analytical HPLC was run on a 2695 Waters (Guyancourt,France) separation module equippedwith a diode array detector 2996 Waters. Separation was achieved on a Phenomenex (Le Pecq, France) Luna column 100 C18 (250mm×4.6mmi.d.,5␮m)protectedwithaPhenomenexcar- tridge(C18,4mm×3mmi.d.)atatemperatureof30^◦C.Themobile phaseconsistedof0.1%formicacidinwater(eluentA)andace- tonitrile(eluentB)andtheseparationwasperformedusingthe followinggradient:4–10%B(0–5min),10–45%B(5–40min),ata flowrateof1mL/min.Injectionvolumewas10␮LandUVdetection wasstudiedattwowavelengths:280and350nm.TheMSanalyses wereperformedonaBruker(Bremen,Germany)ESI/APCIion-trap Esquire3000+innegativemode,withthefollowingconditions:

collisiongas,He;collisionenergyamplitude,1.3V;nebulizerand dryinggas,N2,7L/min;pressureofnebulizergas,30psi;drytem- perature,340^◦C;flowrate,1.0mL/min;solventsplitratio1:9;scan range,m/z100–1000.Identificationofpolyphenolswasachieved bycomparisonofUVprofilesandmassspectrawithliteraturedata and/orusingpurestandards.

Routineanalysis of produced extracts was performed using anAccelaHPLCsystem(ThermoScientific,USA)consistingofan

Accela1250pumpwithadegasser,anautosampler,andaphoto- diodearraydetector.AC18pyramidcolumn(250mm×4.6mm, 5␮mparticlesize;Macherey-Nagel, France)wasusedforchro- matographicseparationsat30^◦C.Amobilephaseconstitutedby aceticacid(distilledwater:aceticacid97.5:2.5(v/v),eluentA)and acetonitrile(eluent B) was used witha discontinuousgradient of3–9%B(0–5min),9–16%B(5–15min),16–33%B(15–30min), 33–50%B(30–37min), 50–90%B(37–40min),at aflow rateof 1ml/min. Injection volume was 10␮L. Quantification was per- formedviacalibrationcurvesthatplotthepeakareaversusknown concentrationsofstandardsintherangeof0.05–1mg/mL.Calibra- tioncurvesderivedfor6dataminimumweresatisfactorilyfitted with linear regression equation (correlation coefficients larger than0.997).Standardsofcatechin,epicatechin,phloridzin,chloro- genicacid,andseveralquercetinderivatives(quercetin-glucoside, -arabinoside,-galactoside)wereusedforquantification.Quercetin xyloside and quercetin rhamnoside were quantified using the quercetin-arabinoside calibration curve, so data are expressed asarabinosideequivalent(mgEq.Arabi).Quantificationwasper- formedat=360nmforquercetinderivativesand=280nmfor otherphenolics.

Thedeterminationoftheextractsantioxidantcapacity(AAEAC) isbasedontheabilityofantioxidantstoscavengetheradicalcation of ABTS (2,2-azino-bis-3-ethylbenzothiazoline-6-sulfonate).The methodologyhereemployedfollowedthatdescribedbyReetal.

[41]withsomemodifications.Briefly,ABTSradicalcation(ABTS^+•) was produced by reacting 7mmol/L ABTS stock solution with 140mmol/L manganese dioxide (MnO2) in dark at room tem- peraturefor12h.TheABTS^+• solutionwasdilutedwithdistilled watertoanabsorbanceof0.70±0.02at=734nm.Aftertheaddi- tionof40␮Lofthesampletoanalyzeto1960␮Lofthediluted ABTS^+•solution,absorbancereadingsweretakenevery1minusing Helios␥spectrophotometer(ThermoElectronCorporation,USA).l- Ascorbicacid(AA,Sigma–Aldrich,USA)wasusedasreference.The percentageinhibitionofabsorbanceat734nmwascalculatedasa functionoftheconcentrationofextractsandascorbicacid.Results wereexpressedasmgofascorbicacidpergofextract.

3. Resultsanddiscussion

3.1. Polyphenolprofileofapplepeelsextracts

Chromatograms of apple peel extracts obtained by CO₂+25%EtOHextractionaregiveninFig.1.

HPLC analysisof extractsobtained by EtOHmaceration and MeOH/Acetone extractionareprovided as wellfor comparison.

PhenolicswereidentifiedbyUVspectra/massspectrometrydata and/or comparison with retention time of standards (Table1).

Whatevertheextract,theidentifiedphenolicswerecatechin,epi- catechin, chlorogenic acid, phloridzin and quercetin glycosides (quercetin-3-O-glucoside, -galactoside, -arabinoside,-xyloside,- rhamnoside)whicharethemajorphenolicsofapples[11,16,42].

ItisworthwhilenotingthatHPLCprofilesofsupercriticalextracts exhibitedallpeaksofphenolicsindicatingthattheconditionswere suitabletoextracteventhesugar-basedphloridzinandquercetin glycosides.

3.2. Supercriticalextraction:staticversusdynamicprocedure ExtractionswithCO₂+EtOHmixturewerecarriedoutbytwo methods,aconventionaldynamicandastepwiseprocedurethat involvedstaticperiodsbetweencollectingfractions.Theevolution oftheextractedamountineachfractionisillustratedFig.2forthe staticprocedure.TheF0 fractionrarelycontainedanyphenolics sinceitcollectedwhatwassolubilizedduringthestaticperiodin

Fig. 1.Phenolic profiles at =280nm of varioussamples: (A) SFE staticon 30g-loading,fractionF3; (B) SFEdynamic30g-loading,fractionF3; (C)EtOH maceration; (D) MeOH/acetone extraction. Peakattribution: (1) catechin; (2) chlorogenicacid; (3) epicatechin; (4) quercetin-3-O-glucoside; (5) quercetin- 3-O-galactoside; (6)quercetin-3-O-arabinoside;(7)quercetin-3-O-xyloside;(8) quercetin-3-O-rhamnoside;(9)phloridzin.

neatCO2.AllphenolicswereusuallypresentintheF1fractionindi- catingthatallcomponentswerereadilyaccessibleintheground applepeelsmatrix.TheirconcentrationincreaseduptoF2orF3 beforedecreasinginF4andeventuallybeingconstantwhenextrac- tionwascontinuedfor longerdurationas inthedynamic case.

CumulateddataaregiveninFig.3withdeviationbarsprovided byduplicationofextractionexperiments.

Thereproducibilitywascriticalindynamicconditionsforcat- echin, chlorogenic acid, quercetin-glucoside and -rhamnoside.

Catechinand chlorogenicacidareeluted inchromatography as smallpeaksamongotherpeaks,whichcouldcomplicatedetection

Fig.2.AmountofeachphenolicinfractionscollectedduringSFEat25MPa,50^◦C, andCO2+25%cosolvent(EtOH96%),staticprocedure,from30gofgroundapple peels,Ftotal=9.7±0.8g/min.E13labelisthesamplereferenceinourdatabase.

andthereforethequantificationincomplexextracts.Forchloro- genicacid[11,43,44]andcatechin[45],theirsusceptibilitytothe polyphenoloxidaseenzymecouldintroduceanadditionalsource ofdeviation.Noneofthehypothesisholdsforthetwoquercetin- derivatives.Besides,theextracting fluidis a ternarymixtureof CO₂/ethanol/water,whosecomponentscanabsorbseparatelyand differentlyontothesolidmatrixandinducethereforeadifferent partitioningofextractablespeciesbetweenthesolidandthefluid phasesalongtheextractorbed.Lookingcloserattheamountcol- lectedin eachindividualfraction, themaindeviations occurred infractions F1and F2,i.e. collectingwhat waselutedbya vol- umeoffluidcorrespondingtotwicetheethanolbreakthrough.The permanentregimeofflowandofthecomplexfluidcomposition mightbenotyetattained. Whentime wasgivenforconditions tosettleviastaticperiods,reproducibilitywasbetter.Regarding nowtheextractionprofiles,thecurvesshapewassimilarwhat- everthecompoundandproceededmostlyataconstantextraction rateupto1.1kgfluid. Notethat thelastpoint ofcurves corre- spondstothefractionobtainedduringthefinalflush withpure CO2,sothestrongerapparentfallingratediscernableattheend ofcurvesismorelikelyduetopoorersolubilizationratherthan tomatrixexhaustion.Therefore,theextractionofphenolicswas notfinishedafter2kgoffluid,i.e.afterafluid/matrixratioof67.

ThisisconsistentwithHasbayAdilresultsonapplepomacethat reportedthatavalueof80goffluid/gofmatrixwasnecessaryto reachaplateauonthetotalphenolicsextractioncurve[36].Taking reproducibilitydeviationsintoaccount,theintroductionofstatic periodsbetweenthedynamiccollectionsdidnotyieldappreciable improvementoftheextractedrate,exceptedforphloridzin.Static periodswereinvestigatedasawaytogivemoretimeforspecies todiffusewithinthepeelstructure,thematerialgrainsorinthe extractingfluid.Aninsignificantimpactindicatedthatmasstrans- ferwasnotasignificantlimitingmechanismfortheextractionof mostphenolics.

Cumulated amounts of phenolics extracted at 1.1kg of fluid from the 30g-loading are summarized in Table 2. For

Table1

PhenolicsidentifiedfromapplepeelsbydiodearrayandmassspectroscopydetectorscoupledtoHPLCseparation.tR:retentiontime,max:wavelengthofmaximalabsorption, [M−H]⁻:ionandfragmentinmassspectrometry,Mw:molecularweight.

Compounds tR(min) UVmax(nm) [M−H]⁻(m/z) Mw(g/mol)

Catechin(1) 14.40 280 nd 290

Chlorogenicacid(2) 15.37 298,325 353 354

Epicatechin(3) 17.29 280 289 290

Quercetin-3-O-glucoside(4) 22.50 256,355 463,frag301 464

Quercetin-3-O-galactoside(5) 22.76 256,355 463,frag301 464

Quercetin-3-O-arabinoside(6) 23.89 256,355 433,frag301 434

Quercetin-3-O-xyloside(7) 24.85 256,354 433 434

Quercetin-3-O-rhamnoside(8) 25.29 256,350 447,frag301 448

Phloridzin(9) 27.00 284 435,frag273 436

nd:notdetected.

Fig.3.Cumulatedamounts(inmg)ofphenolicsobtainedbySFEusingthedynamic(darksymbols)orthestatic(opensymbols)procedurefrom30gofgroundapplepeels.

Amountsofquercetin3-xylosideand3-rhamnosideareexpressedasequivalentquercetin3-arabinoside.

comparison with other extraction methods or literature, data are given for 100g of matrix. By the supercritical tech- nique, extracted amounts of phenolics were in the range 600mg/100g_drypeel in which quercetin glycosides grouprepre- sented77–78%.As individualcompounds,catechin, chlorogenic acid, phloridzin, quercetin-galactoside were extracted in the rangeof6–36mg/100g_drypeelwhereasepicatechinandtheother quercetinderivativeswerearound55–140mg/100g_drypeel.Com- pared to ethanol maceration, the ethanol+CO2 combination enhanced the extraction of all phenolics (except chlorogenic

acid),atrendthatwasevidencedaswellbyFarias-Compomanes et al. in case of grape bagasse extraction [23]. Total phenolics yield in the macerated sample was of 177mg/100g_drypeel and the glycosides-derivatives accounted for only 50% of the total.

Whenextractedwithmethanol/acetone/water,polyphenolstotal- ized792mg/100g_drypeelinwhichthepoolofquercetinglycosides accounted for 50%and chlorogenic acidfor 40%. Abundance of chlorogenic acid and quercetin-derivatives especially of galac- tosidemoiety was reportedin literature for Golden freshpeels extractedbymethanol[13],butepicatechinandphloridzinwere

Table2

Conditionsofextractionsandyieldofphenoliccompoundsextracted.EtOH:ethanol,MeOH:methanol,Ace:acetone;SFE:subcriticalfluidextraction.

Method SFEstatic-30g SFEdynamic-30g SFE-15g^c Maceration Solvent

Solvent(mol) CO2:EtOH:H2O75:22:3 CO2:EtOH:H2O75:22:3 CO2:EtOH:H2O75:22:3 EtOH:H2O88:12 MeOH+Ace/H2O

Solvent/peelsratio(wtbasis)^a 37 37 73 8 30

T/P 50^◦C/25MPa 50^◦C/25MPa 50^◦C/25MPa 45^◦C/0.1MPa 20^◦C/0.1MPa

Globalyielda(g/100gdry peel)^d

15.8±0.2 22.0±0.7 30.1±7 65±2 3.2

Phenolicsyield^a(mg/100gdrypeel^d)

Totalphenolics 550±115 603±182 800±25 177±32 792±26

Catechin 11.4±2.3 6.0±6.0 27.0±3.1 0 25.7±0.2

Chlorogenicacid 16.4±4.5 36.2±23.0 12.8±3.1 58.3±11.8 319.7±18.0

Epicatechin 71.6±3.4 78.7±11.4 118.3±4.8 28.5±4.6 43.5±1.9

Phloridzin 21.5±3.0 13.2±3.0 21.7±0.3 2.3±0.28 15.3±0.5

Quercetin-3-glucoside 81.2±23.0 113.7±60.4 147.1±4.5 26.1±4.9 86.6±2.3

Quercetin-3-galactoside 26.5±10.4 21.2±5.7 43.7±2.0 3.7±0.05 97.1±1.7

Quercetin-3-arabinoside 67.6±8.3 54.6±5.7 110.1±1.2 35.0±6.0 49.1±1.9

Quercetin-3-xyloside^b 144.5±27.5 139.8±14.2 183.5±4.6 9.6±2.8 69.8±2.6

Quercetin-3-rhamnoside^b 109.3±32.8 134.2±46.4 135.6±1.4 13.6±2.0 85.6±13.6

Totalphenolicsconcentration (gphenolics/kgextract)

34.7 27.4 26.7 2.5 247

a1.1kgofextractingfluidforsupercriticalextractions.

bExpressedasequivalentquercetin3-arabinoside.

c Datacorrespondtomeanvaluesbetweenstaticanddynamicresultssincetheyweresimilar.

d Drypeel=lyophilizedpeelsthatstillcontain5–7%humidity.

less abundant in oursample than usually reported [46–49]. In otherhand,compositionandconcentrationofphenolicsinfruits varywithfruitstype,growingseason,geographiclocation,genetic variation[48,50,51]andconditionsofextraction(sampletosol- vent ratio, time of exposure, temperature, etc.) influence the quantificationas well[52].Theparticularabundanceof chloro- genicacidinourextractpreparedbythemethanol/acetone/water methodcouldalsocomefromtheuseofacetonethatbyinhibit- ingthepolyphenoloxidaseenzymepreservestheoriginalcontent of the fruit [6]. Regarding quercetin-derivatives presence, the sum of total phenolics quantified by HPLC from a Gala apple pomace extracted by acetone/water, was 7.24g/kg dry matter, of which more than 50% was quercetin glycosides [42]. The quercetin-derivatives pool also represented the main phenolic constituents of methanol/waterextracts from 7 applevarieties peels,witha contentin therange of0.1–0.9g/kgfreshweight, which taking in account the peel humidity, corresponded to 40–360mg/100gofdrymatter[12].Thelevelofquercetinglyco- sidesinourvariousextractsandtheirhighcontributiontothetotal extractedphenolic pool are thereforeconsistentwithliterature data.

Regarding consistency with the only study of supercritical extractionfromapple[36],letfirstremindthatindividualpheno- licconcentrationswerenotmeasuredbyHPLCandthatthetotal phenoliccontentwasestimatedbytheFolin–Ciocalteumethod.

Expressed as gallic acid equivalent (GAE), contents of 171mg GAE/100g sample and 47mg GAE/100g sample was reported for ethanol and (CO2+20wt% EtOH) extractions, respectively, which is an opposite trend toour own findings. However,the Folin–Ciocalteureagentdoesnotonlymeasurephenolsandreact withanyreducingsubstance.Itthereforemeasuresthetotalreduc- ingcapacity ofa sample,not justphenolic compounds.Besides phenols,nitrogen-containingcompounds,triosesglyceraldehyde, vitaminderivativesorproteinsarereactivetowardsthisreagent [53]. For applepurees and juice, the contributionof the inter- feringsubstances canbebetween31%and 48%[36]. Moreover, HasbayHadilinvestigations[36]werecarriedoutonapplepomace (flesh+skin residues after pressing) from Starking and Amasya applevarieties,whereasourownstudyisperformedonfreshskin solelyandGoldenDeliciousvariety,twodifferencesthatinducefluc- tuationsinquantityofantioxidants[48].

Fig.4.GlobalamountsofextractsobtainedbySFEusingthedynamic(darksymbols) orthestatic(opensymbols)procedureon30gofgroundapplepeels.Deviationbars forthestaticcasearetoosmalltobeviewed.

Regardingglobalyields(Fig.4,Table2),theoverallextracted amountswereintherange ofgram,andtheextractionprofiles exhibited,asphenolics,alinearpartupto1.1kgoffluid.

Whentheprocesswasrunabove1.1kgfluidasfordynamic case,theextractioncontinuedatalowerrate.Itisinterestingto notethatthisfallingextractionrateoccursatthesamefluidcon- sumptionthantheonereportedbyFarias-Campomanes[23]for similarmatrixamountandvesselcapacity(20gofgrapebagasse, 415cm³ vessel).Theintroductionofstaticperiodsyieldsaslight decreaseoftheglobalextractedamount,intherangeof1.5gat 1.1kgof fluid (reproducibilitybars forstatic conditionsare too smalltoappearinthegraph).Inotherwords,givingtimefordif- fusionpenalizedtheoverallextractionwhereasithadalmostno impactforphenolics,maybebecausetheycontributedforonlyfew percentilestotheextract(seebelow).Lessextractedamountin case ofstaticperiodmeansa smallertransported concentration throughthebed.Thoughnotverypronounced,thebehavioursug- gestscompetitivereactionstoextraction,suchasdegradationof extractablespeciesinlesssolublecompoundsorare-absorptionof theextractablespecies.Becauseofthecomplexityoftheextracting mixture(CO₂:77mol%,ethanol:22mol%,water:3mol%),thefluid componentscanabsorbseparatelyandindifferentextendontothe solidmatrix,inducingadifferentpartitioningofextractablespecies betweenthesolidandthefluidphasesovertheextractionduration.

Whenexperimentswerecarriedoutinadynamicmode,therewas

Fig.5.EffectofthematrixloadingsontheglobalamountobtainedbySFEusing dynamicorstaticprocedure(15g,30gand55gofgroundapplepeels).

notenoughtimeforthecompetitiveredistributionordegradation tooccur,soextractableswereextracted.

Asmentionedbefore,therangeofextractedamountsofphen- olicsand global extractsis very different.More precisely, with valuesof27–35g/kgofextractat1.1kgoffluid(Table2),phenolics representonly3%oftheglobalextract.Althoughthequantifica- tiontookonlyinaccounttheidentifiedcompounds,thedifference istoolargetocomefromnon-identifiedpolyphenolssolely.The presenceofother compoundsinextractsis inherentoftheuse ofethanolthatis farfromselective.Similarrangevaryingfrom gramtotenofgramsperkgofextractwasreportedinliterature indicatingasubstantialextractionofmultiplecomponentsbesides thephenolicswhateverthematrix studied[23,29,32].Generally speaking,polyphenolsaresecondarymetabolitesofplantssothey arepresentatamuchlowerlevelthanconstitutivemoleculesof cellslikelipids,proteins,carbohydrates,and,asminorcompounds, minerals and vitamins. Among possible extracted components, lipidsand carbohydratesare potentialcandidatesalthough one cannotdiscardapossibleextractionbyleachingofanymolecules.

Lipids,including phospholipidsare wellextracted byCO2+25%

ethanol[54].Thesugarcontentofapplesisveryhigh(10gover 100goffreshedibleportionthatcontains83%ofwater)withd- fructoseandinlessextentd-glucoseaspredominantsugars[6].

ThoughscCO₂ is non-polarandhence notsuitablefor carbohy- dratesextraction,studieshaveshownthatusingapolarcosolvent as ethanol can increase notably their solubility: a solubility of 0.1–0.8mg/g inCO₂+21%ethanol(at96%)wasreportedforfour prebioticfructose-andgalactose-basedcarbohydrates[55].Com- paringscCO2+cosolventandsolventextractionsandcommenting respectiveglobalyields[56],Pineloetal.hypothesizedthatscCO₂ conditionscouldbeable topromote a breakdownof thesugar structure of the plant matrix and the consequent solubility of theresultingmonomeric and oligomeric polysaccharidesprevi- ouslyravelledinthecellwallframework.Moreover,applepeels CO₂-treatedinthisworkwereanalyzedforglucoseandfructose contents,andafterthesubcriticaltreatment,thelevelswerehalf theinitial values, which indicated that thesecompounds were indeedextracted.

3.3. Effectofapplepeelsloadings

SFEwasperformedbychargingvariousamountsofapplepeels intheextractor,15,30and55gforthestaticprocedure,and15 and30gforthedynamic.Increasingtheloadingdidnotyieldan enhancementoftheglobalextractedamountinthesamepropor- tionthantheloadingincrease(Fig.5).

Forthefirstfractions(upto0.6kgoffluid),theglobalextracted amountwasinthesamerangewhateverthemassloadedorthe procedure.Afterwards,theincreasefrom15to30gdidincreasethe extractedamountbutnotinatwo-foldfactor.Increasingfurtherthe

loadingfrom30to55g(incaseofstaticprocedure)didnotyield moreextractedamountsincecurveswerealmostsuperimposed.

Thepoorsensitivityoftheextractedamountwiththematrixweight mightcomefromapoorpackingofthesolidintheextractorand/or thesaturationofthefluid.Althoughthebedwasmadebyalternated layersofmatrixandglassbeads,thethicknessoftheapplelayers wereincreasedtoaccommodatetheincreaseofmatrix loading.

Whenchannelling occurs, only parts of thematrix arein con- tactwiththeextractingfluid;higherloadingscouldincreaseonly slightlythematrixincontactlettingalargerportionnotextracted.

Saturationofthefluid,especiallyattheearlytimesoftheextrac- tionandabove15gofmatrixcouldcontributeaswelltothepoor effectofloadings.Similarextractedamountmeanssimilartrans- portedconcentrationthroughthebed,e.g.afluidalreadyenriched withextractablescomingforinstancefromthefirst15gofmatrix atthebedbottomcannotcompletelysolubilizetheextractables availablefromtheother15gofthebedexit.Afteracertaintime, thenegativeeffectofsaturationprogressivelyvanishedallowing forothermolecules tobeextractedand forregainingeffects of operationalparameters.Asaturationthatpostponedextractionof othermoleculeswasencounteredbyUquicheetal.[57]dealing withextractionofoilandminorlipidsfromseedscakes.Specially, sterolsandcarotenoidsthatwerepresentatlowerconcentration incakeandwerelesssolublethanoilwereextractedinthelater stagesofextractionwhentherewasnotenoughoiltosaturatethe CO₂phase.Therefore,atimefractionationeffectbetweenthemajor oilandtheminorlipidswasobserved.

Forphenolics(Fig.6),itisworthwhilenotingthata15gofload- ingallowsforobtainingsimilarkineticsinstaticandindynamic modesandforattainingaplateau,visibleindynamicconditions sinceextractionwasperformedoverlongertime.Theextraction ratefelldownnotablyafter1.1kgfluidsothatat1.8kgoffluid,i.e.

forasolvent/matrixratioof120,theextractionofallphenolicswas almostfinished.

Theoverallamountofphenolicsextractedinthoseconditions wasintherange of140mgfor15goffeed.At1.1kgfluid,the amount extracted was 120mg, which gives the highest value of 0.8g of phenolics/100g_drypeel (Table 2). Whatever the pro- tocol, the extracts were mainly composed by epicatechin and quercetinderivatives(Table2),withthepoolofquercetinderiva- tivesaccountingfor76–79%.Asobservedfortheglobalextracted amount,anincrease of loadingdidnotyield a correspondingly increaseofextractedphenolics,exceptedforphloridzin.Allkinet- icswereinthesamerangewhatevertheprocedureandtheloading upto0.5–0.7kgfluid,whichisthesamebehaviourthanobserved foroverallextract.Beyondthatvalue,effectsofoperationalparam- eterswereregained,butphenolicsdidnotrespondin thesame extendtothevariationofloading.Forstaticconditionsthatwere morereproduciblethandynamicones,quercetine-glucosideand -galactoside were only slightly affected, whereas the extracted amountsofotherquercetinderivatives,epicatechinandphloridzin weremoreregularlyaugmented.Chlorogenicacidstillbehavedif- ferently,withanextractedamountthatwaslowerat60gthanfrom 30g.Indynamicconditionsandforcompoundsofhigherrepro- ducibledata(quercetine-xyloside,-arabinoside,-epicatechin),the extractedamountwasincreasedaswellprovidinghoweverthat morethan1.1kgoffluidwasused.

3.4. Antioxidantactivityofextracts

Theantioxidantcapacityofthevariousfractionscollecteddur- ingthestaticprocedure(30gloading)isillustratedFig.7.Deviation bars accounted for the analysis of the duplicated SFE experi- ments.TheF2andF3fractionsexhibitedthehighestantioxidant capacity (5–6mgAAEq./gextract)witha trendthat paralleledthe evolutionofthephenoliccontentinfractions(Fig.7).Lookingfora

Fig.6. EffectofthematrixloadingsonthephenolicamountobtainedbySFEusingdynamicorstaticprocedure(15g,30gand55gofgroundapplepeels).Amountsof quercetin3-xylosideand3-rhamnosideareexpressedasequivalentquercetin3-arabinoside.

correlation,theantioxidantactivitywasplotasfunctionofpheno- licsconcentrationinfractions(Fig.8),consideringdynamicresults obtainedat 30gloadingaswell.Staticresultsexhibiteda good trend (R²=0.916), whereas datafrom dynamic procedurewere morescattered(R²<0.15).Thetwodataoutofthetrendcontour fordynamicresultscorrespondedtofractionsF1andF2thatwere thoseattheearlystageoftheextraction.Thescatteringofdata andthetwodifferentcorrelationsdependingonprocedureseems

toindicatethateithernon-phenoliccompoundscontributetothe antioxidantactivity,oronlysomeofphenolicsareresponsiblefor itratherthanthewholepool.

Inliterature,antioxidantactivitiesdidnotcorrespondneces- sarilytohighcontentofphenols.Apositivecorrelationwasfound for56vegetablesbyDengetal.[58]whereasnorelationshipwas evidencedbyIsmailetal.for9typesofvegetables[59].Thedifferent methodsusedtoproduceextractsandtodeterminethephenolic

Fig.7. Ascorbicacidequivalentantioxidantcapacity(AAEAC,mg/gofdryextract) andtotalphenolicconcentration(mg/gofdryextract)offractionsproducedduring SFE(staticprocedure,30gofgroundapplepeels,duplicateextractions).

contentandtheantioxidantcapacitycouldhavegenerateddiffer- entselectivityandresponses:ethanolextraction,Folin–Ciocalteu reagent,coupledreactionoflinoleicacidand␤-caroteneinIsmail [59],twostepsextractionwithmethanol–aceticacid–watermix- tureforobtainingahydrophilicfraction,Folin–Ciocalteureagent, Troloxequivalentantioxidantcapacity in Deng[58].In another hand,allphenolicsdidnotexhibitthesameantioxidantcapacity.

Phenoliccompoundspossesschemicalstructuralrequirementsfor antioxidantactivitybecausetheyhavephenolichydroxylgroups abletodonateahydrogenatomoranelectrontoafreeradicaland extendedconjugatedaromaticsystemtodelocalizeanunpaired electron.AsdiscussedbyDaiandMumper[60],chlorogenicacid(an hydroxycinnamicacidderivative),isconsideredasapotentantiox- idantagent as it possesses a catechol moiety (ortho-dihydroxy structure)linkedtoaCH CH COO whichallowsthedelocaliza- tionofelectronbyresonance.Amongflavonoids,quercetinexhibits high antioxidant activity because it combines all the require- ments:acatecholmoietyintheBring,the2,3-doublebondwitha

Fig.8.Testofcorrelationbetweenantioxidantcapacityandconcentrationofphen- olics(mg/gofdryextract)infractionsobtainedduringSFEstaticanddynamic procedure(30gofgroundapplepeels,duplicateextractions).

4-oxofunctionintheCring,the3-and5-hydroxylgroupswith the4-oxofunctionin Aand Crings.Thefree 3-hydroxylgroup isimportantforantioxidantactivity.Evenifthe3-glycosylation may reducethe activity when compared with thecorrespond- ingaglycone[60],quercetinglycosylatedderivativesstillhavethe otherattributes.Whentested asinhibitorsoffishoiloxidation, quercetin-3-O-glucoside,-galactosideand-rhamnosideexhibited similarorslightlyhighereffectivenessthantheaglyconequercetin [61],whereas forphloridzin,its2-glycosylationyielded alower inhibitionthantheaglyconephloretin[62].Incaseofapples,an activityintherangeof820␮molTroloxeq./100goffruitweremea- suredforGoldenvarietybySerraetal.[63].Eightothervarieties ofapplewerestudiedaswell,andwhen submittingtheoverall resultstostatisticalanalysis,catechin,epicatechin,procyanidinB1 andquercetin-3-glucosidewereidentifiedasmajorcontributors totheantioxidantactivities(flesh+peelsextracts;ORAC,HORAC andLDLassays).Foranindustrialresiduecomingasapomaceof juice-squeezedFuji,GrannySmithandQinguanapplesmixture,the antioxidantactivity,expressedasABTSradicalinhibitionrate,was positivelycorrelatedwiththephenolicscontentofvariousfractions andspeciallytothepresenceofprocyanindinsB,chlorogenicacid andquercetin[64].Lookingforacorrelationwiththechlorogenic concentrationorquercetin-derivativepoolsinourextractswillbe investigatedinfuture.

4. Conclusions

Theaimofthisworkwastoevaluatethepotentialitiesofsuper- criticaltechnologytorecoverthevaluablepolyphenolicsfroma fruitwaste,namelytheapplepeels.Comparedtotheonlystudy reportedinliterature,thepresentworkidentifiedandquantified themajor extracted phenolics, monitoredtheextraction kinet- ics of the polyphenols and of the global extract amount, and investigatedseveralprocessparameters intheviewofscale-up (amount of matrix chargedin the vessel,introductionof static steps during extraction). All principal phenolics known to be presentinapplepeelswereextractedbytheconditionsused,i.e.

CO₂+25mol%cosolvent(EtOH96%),25MPa,50^◦C,includingthe polarsugar-based quercetin derivatives and phloridzin. Extrac- tionswerecarriedoutbycollectingfractionsoveratimerelated totheethanolbreakthrough,inordertoimprovetheaccuracyof kineticsandtheunderstandingofthepermanentregimeestablish- ment.Extractionofphenolicsstartedrapidlywithnodelaybetween components,indicatingthattheywereallreadilyaccessible.Over thedurationoftheextractions,theexhaustionofthematrixwas usuallynotattained,exceptforexperimentperformedona15g loadingtreatedwith1.8kgoffluid,i.e.forasolvent/feedratioof 120.Increasingthematrixloadingdidnotincreasetheextracted amountsinthesameratio,apoorefficiencythatcouldberelatedto thefluidsaturation,bedpacking,permanentregimeofextraction notestablishedyet,oracombinationofthosefactors.Introduction ofstaticperiodswasenvisagedaswaytogivetimesforspecies todiffusewithinthematerial.For phenolics,thedifferencewas notsignificant,whereasfor theoverallextract,staticconditions ledtoaslightlysmallerextractedamount.Althougheachpheno- licwasrespondingdiverselytotheeffect,thedifferenceswerenot significantenoughtoenvisageseparationbetweengroups.

Sofar,SCFtechnologywasefficienttorecoverthephenolicsata levelof120mgfrom15gofpeelswithonly1.1kgofextractingfluid.

Comparedtomethanol/acetone/waterorethanolextracts,contri- butionofquercetinderivativestothephenolicpoolinSCextracts wasparticularlyhighwithvalueintherangeof75–80wt%instead of50%.However,phenolicsconstitutedlessthan5%wtofthewhole extract,alowcontentinherenttotheuseofethanolthatisfarfroma selectivesolvent.Supercriticalextractswereexhibitingantioxidant